UNITED STATES PATENT:
Non-Toxic Skin Cancer Therapy
with Copper Peptides (2017)
GHK and DNA: Resetting the
Human Genome to Health
BioMed Research International (2014)
The Effect of Human Peptide
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and Cognitive Decline
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Oncotarget (2016)
New Data of the Cosmeceutical
and TriPeptide GHK
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Scientific Reports (2016)

GHK-Cu May Prevent
Oxidative Stress in Skin
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in Neuroblastoma Cells
Biotech & Biomaterials (2012)
GHK Peptide as a
Natural Modulator of
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in Skin Regeneration (2015)
Emphysema-Related
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Expression and DNA Repair Analytical Oncology (2014)
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The Effect of the Human Peptide GHK on Gene Expression Relevant to Nervous System Function and Cognitive Decline

Brain Sciences article: Loren Pickart, Jessica Michelle Vasquez-Soltero and Anna Margolina (Published February 2017)

Abstract: Neurodegeneration, the progressive death of neurons, loss of brain function, and cognitive decline is an increasing problem for senior populations. Its causes are poorly understood and therapies are largely ineffective. Neurons, with high energy and oxygen requirements, are especially vulnerable to detrimental factors, including age-related dysregulation of biochemical pathways caused by altered expression of multiple genes. GHK (glycyl-L-histidyl-L-lysine) is a human copper-binding peptide with biological actions that appear to counter aging-associated diseases and conditions. GHK, which declines with age, has health promoting effects on many tissues such as chondrocytes, liver cells and human fibroblasts, improves wound healing and tissue regeneration (skin, hair follicles, stomach and intestinal linings, boney tissue), increases collagen, decorin, angiogenesis, and nerve outgrowth, possesses anti-oxidant, anti-inflammatory, anti-pain and anti-anxiety effects, increases cellular stemness and the secretion of trophic factors by mesenchymal stem cells. Studies using the Broad Institute Connectivity Map show that GHK peptide modulates expression of multiple genes, resetting pathological gene expression patterns back to health. GHK has been recommended as a treatment for metastatic cancer, Chronic Obstructive Lung Disease, inflammation, acute lung injury, activating stem cells, pain, and anxiety. Here, we present GHK’s effects on gene expression relevant to the nervous system health and function.

Keywords: GHK; copper; dementia; Alzheimer’s disease; Parkinson’s disease; neurons; glial cells; DNA repair; anti-oxidant; anti-anxiety; anti-pain

1. Introduction

Age-related cognitive decline is a common problem for many elderly people, yet its cause is poorly understood. Over 99% of investigational drugs, participating in over 200 clinical trials, failed to receive approval for the treatment of Alzheimer’s disease [1]. Even the success of a few approved drugs provides only minimal patient improvement. There is a need for new, safe, and effective therapeutics with extensive safety and efficacy data that can be developed for use in humans within the next few years.

GHK (glycyl-L-histidyl-L-lysine) is a human plasma copper-binding peptide with a stunning array of actions that appear to counter aging-associated diseases and conditions. GHK was isolated in 1973 as an activity bound to human albumin that caused aged human liver tissue to synthesize proteins like younger tissue [2]. It has a strong affinity for copper and readily forms the complex GHK-Cu. It was first proposed that GHK-Cu functions by modulating copper intake into cells [3]. Since then, it has been established that the GHK peptide has stimulating and growth-promoting effects on many cells and tissues such as chondrocytes [4], liver cells and human fibroblasts [5]. It increases stemness and stimulates integrin secretion in human epidermal basal keratinocytes [6], as well as has a strong wound-healing and tissue-repairing effect [7]. GHK has also been shown to improve wound healing in controlled experiments using animals, such as rats, dogs, and rabbits [8–10].

In 2010, Hong et al. using the Broad Institute’s Connectivity Map (cMap), a compendium of transcriptional responses to compounds, identified GHK as the most active of 1309 bioactive substances, uniquely capable of reversing the expression of 54 genes in a metastatic-prone signature for aggressive early stage mismatch-repair colorectal cancer. GHK was active at a very low concentration of 1 μM [11].

Another study, which also used the cMap to identify genes affected by GHK, was conducted in 2012 when Campbell et al. identified 127 genes whose expression levels were associated with regional severity of chronic obstructive pulmonary disease (COPD). Emphysema and chronic bronchitis, the two main conditions of COPD, cause both small airway obstruction and significant loss of lung function over time. The cMap predicted that GHK would reverse the aberrant gene-expression signature associated with emphysematous destruction and induce expression patterns consistent with healing and repair. These finding were supported by laboratory experiments. GHK, at 10 nM, added to cultured fibroblasts from the affected lung areas of patients, changed gene expression patterns from tissue destruction to tissue repair. This led to the organization of the actin cytoskeleton, elevated the expression of integrin beta 1, and restored collagen contraction [12].

In addition to topping the list of 1309 biologically active molecules as the computer-recommended treatment for both human COPD (chronic obstructive pulmonary disease) and aggressive metastatic colon cancer, GHK has been recommended as a treatment for inflammations, acute lung injury, activation of stem cells, regeneration of aged skin, wound healing and tissue regeneration (skin, hair follicles, stomach and intestinal linings, hair growth, and boney tissue). It is also widely used in anti-aging skin products [13].

Even though it is not always possible to link gene expression data to biological actions, it is important to notice that GHK is highest in very healthy young people. Unfortunately, GHK declines with age. In studies at the University of California at San Francisco, young (age 20–25), male medical students were found to have about 200 nanograms/mL of GHK in their blood plasma, while the healthy, male medical school faculty (average age of 60) had only 80 nanograms/mL [7].

Our previous publication reviewed the biological effects of GHK relevant to neurodegeneration and cognitive health [14]. This paper will discuss the effect of GHK on gene expression relevant to nervous system functions and cognitive decline as well as review genetic and laboratory data relevant to nerve outgrowth, copper transport into cells, anxiety and pain, DNA repair, the ubiquitin proteasome system, the anti-oxidant system, changes in gene expression for glial cells, astrocytes, brain cells, dendrites, ganglia, motor neurons, Schwann cells, and sensory cells. It will also present possible methods for the use of therapeutic GHK in the treatment of nerve diseases.

2. Materials and Methods

The cMap was used to acquire the gene expression data. It is a large database that contains more than 7000 gene expression profiles of 5 human cell lines treated with 1309 distinct small molecules. Three GHK profiles are contained in this repository. The profiles are the result of cell lines treated with GHK at 1 micromolar which is around the concentration where many of GHK’s cellular effects occur [15]. These profiles were created using the GeneChip HT Human Genome U133A Array. Among the 5 cell lines used by the Connectivity Map only 2 were treated with GHK. Two of the profiles were created using the PC3 cell line - human prostate cancer cells, while the third used the MCF7 cell line – human breast cancer cells. Our studies utilized all three gene expression profiles.

GenePattern, a publicly available computational biology open-source software package developed for the analysis of genomic data, was used to analyze the gene data obtained from the cMap. The CEL files (data files used by Affymetrix software, used by the Broad Institute) were processed with MAS5 (Microarray Analysis Suite 5 software, Affymetrix, Santa Clara, CA, USA) and background correction. Files were then uploaded to the ComparativeMarkerSelectionViewer module in order to view fold changes for each probe set. Gene abbreviations appearing throughout the paper are consistent with the NCBI Gene database [16].

Due to multiple probe sets mapping to the same gene, the fold changes in m-RNA production produced by GenePattern were converted to percentages, and then all probe sets representing the same gene were averaged. It was determined that the 22,277 probe sets in the Broad data represent 13,424 genes. This ratio (1.66) was used to calculate the overall number of genes that affect GHK at various cutoff points (rather than probe sets).

The percentage of genes stimulated or suppressed by GHK with a change greater than or equal to 50% was estimated to be 31.2% [17]. Listed in the article are the gene expression effects of GHK on over 700 human genes associated with various nerve cell types. For well-defined systems where animal and cell cultures exist, such as anti-pain and anti-oxidation, relevant genes were manually chosen. For other systems, each gene’s Gene Ontology description was searched, using terms such as “neuron” or “glial”. The Gene Ontology consortium provides controlled vocabularies for the description of the molecular function, biological process, and cellular component of gene products. [18]. For most systems, gene expression numbers were given from 100% + or − and larger.

The cMap data was proven to be predictive of biological actions in most cases. In 2010, cMap predicted the anti-cancer actions of GHK. Subsequent work found GHK at 1 to 10 nanomolar reset the programmed cell death system on human nerve cancer cells and inhibited their growth in culture, while having the same effect on sarcoma cell growth in mice; it changed the gene expression of over 80 genes in an anti-growth manner [17]. Data from cMap also led to experiments that found GHK at 10 nanomolar caused human COPD-afflicted lung cells to switch cell expression from tissue destruction to repair and remodeling. For anti-oxidant actions, cMap has been very predictive of actions in mammals. However, gene expression numbers can vary widely at times and are not always predictable. For example, the cMap gives NGF (nerve growth factor) as a −243% decrease, yet in vivo rat studies have found NGF to be increased and two in vitro cell culture studies have found GHK to increase nerve outgrowth, an effect usually attributed to NGF.

Below, we cover GHK’s relationship with the following.

1. Nerve Outgrowth

2. Copper Lack in Nerve Diseases

3. Anti-Anxiety and Anti-Pain

4. Anti-Oxidant Biological and Gene Expression Data

5. DNA Repair Data and Gene Expression DNA Repair

6. Restoring Regeneration after Cortisone Treatment

7. Data and Gene Expression - Mitochondria

8. Gene Expression - Clearing Damaged Protein - UPS system

9. Gene Expression - Neurons

10. Gene Expression - Motor neurons

11. Gene Expression - Glial cells

12. Gene Expression - Astrocytes

13. Gene Expression - Schwann

14. Gene Expression - Myelin

15. Gene Expression - Dendrites

16. Gene Expression - Oligodendrocyte cells

17. Gene Expression - Schwann cells

18. Gene Expression - Spinal

19. Possible methods of therapeutic use of GHK for nerve disease

 

3. Results

3.1. Nerve Outgrowth

The lack of nerve outgrowth growth is considered a major factor in dementia [19–21]. Brain Sci. 2017, 7, 20 4 of 34 GHK was discovered in 1973 as a growth factor for cultured hepatocytes. In 1975, Sensenbrenner GHK was discovered in 1973 as a growth factor for cultured hepatocytes. In 1975, Sensenbrenner and colleagues reported that GHK induced the formation chick embryonic neurons while suppressing and colleagues reported that GHK induced the formation chick embryonic neurons while glial cells. See Figure 1 [22].

GHK Nerve Research

Figure 1. (Top)—Control; (Bottom)—Addition of 200 ng/mL of GHK to culture media
(Phase contrast ×250, photo micrographs used with permission of John Wiley and Sons).

Lindner and colleagues found that explants from chick embryo PNS (ganglion trigeminale) and from CNS of embryonal rats (hippocampus) and dissociated cells from chick embryo cerebral hemispheres that 0.01 microgram GHK per ml of medium stimulated the outgrowth of neuronal processes. Again, GHK promoted neuronal growth but not glial cells [21]⁠.

In studies of rats, severed sciatic nerves (axotomy) were inserted into a collagen prosthesis to which GHK was bonded. These were re-inserted into the rat, then removed after 10 days. GHK enhanced the production of trophic factors (Nerve Growth Factor, Neurotrophins 3 and 4) and recruited hematogenous cells and Schwann cells, which in turn help in the secretion of certain vital trophic and tropic factors essential for early regeneration. This improved nerve regeneration following axotomy [22]⁠. Surprisingly, GHK's gene expression data gives suppression of NGF (-243%) and NGFR (nerve growth factor receptor) (-132%). Thus, the biological system within wounded rat's nervous tissue is more complex and probably due to other nerve stimulatory molecules.

3.2. Copper Deficiency, Dementia, and Nerve Dysfunction

Copper is an essential component of important anti-oxidant proteins such as SOD (copper zinc superoxide dismutase), ceruloplasmin, and Atox1 (human antioxidant protein 1). Copper deficiency has been postulated as a causative factor in a variety of gene diseases such as Alzehiemer's [23–28]⁠, myelopathy [29]⁠, motor neuron diseases and amyotrophic lateral sclerosis [30]⁠, Huntington’s [31]⁠, Lewy bodies and Creutzfeldt Jakob diseases [32]⁠.

More importantly, analysis of actual human brains from deceased patients with dementia has found the damaged areas to have very little cellular copper. In plaques from persons with Alzheimer's disease, iron and aluminum appear to cause plaque formation while copper and zinc may be protective [24–26,33–35]⁠.

Copper deficiency caused by bariatric surgery or gastrointestinal bleeding led to myelopathy (human swayback), paralysis, blindness and behavioral and cognitive changes. Mice born and maintained on a copper deficient diet had 80% reduction in brain copper level at 6-8 weeks and had neuronal and glial changes typical for neurodegenerative disorders [23,29,36,37]⁠.

3.2.1 Supplying Copper to Nerve Cells

Though copper deficiency appears linked to major nerve diseases, the use of copper supplements as a treatment has been disappointing. A placebo-controlled study of 68 Alzheimer's patients (34 control, 34 copper) with a treatment of 8 mgs of daily copper (a high level) for 1 year produced no negative findings. This seems to rule out excessive copper levels as a causative agent for the development of Alzheimer's. The predictive protein marker, CSF Abeta42, is lower in persons developing Alzheimer's. Subjects given extra copper supplementation maintained this protein at a higher level, a possible positive effect, but there was minimal improvement in the disease [38]⁠.

One small copper complex chelator, CuATSM (diacetylbis(4-methylthiosemicarbazonato)copper 2+), has given indications of ameliorating the effects of ALS (familial amyotrophic lateral sclerosis) in a strain of genetically modified mice that develop a form of ALS. CuATSM extends life in such mice by up to 25%. The motor neuron disease can be restarted and then stopped by controlling CuATSM treatment. The treatment increases the amount of active superoxide dismutase in the mice [39]⁠. The safety of CuATSM is largely unknown. The safety data sheet states the following: “Material may be irritating to the mucous membranes and upper respiratory tract. May be harmful by inhalation, ingestion, or skin absorption. May cause eye, skin, or respiratory system irritation. To the best of our knowledge, the toxicological properties have not been thoroughly investigated.”

GHK-Copper 2+ increased superoxide dismutase (SOD) activity in mice as detailed below in section 4 [40]⁠.

3.2.2 Albumin, GHK and Copper Transport

Both albumin and GHK transport copper 2+ to cells and tissues. However, in human blood, there are 700 albumin molecules for each GHK molecule, so albumin is the major source of copper for tissue use. GHK and albumin have high and very similar binding constants for copper 2+ (Albumin = pK binding log 10 |16.2|; GHK = pK binding log 10 |16.4|). Human plasma contains about 15 micromolar copper and 12% (1.8 micromolar) of this is bound to albumin. But GHK-Cu is maximally active on most cells around one nanomolar or less. Aqueous dialysis studies established that GHK can obtain copper 2+ from albumin. We assume that this also occurs in cell culture and within mammals and that GHK has adequate copper for biological actions.

Our studies over the past 39 years have indicated that virtually all biological GHK effects require the presence of copper 2+ chelated to the tripeptide. Strong copper chelators such as bathocuproine abolish GHK actions. GHK alone is often effective in murine wound healing or hair growth models, but GHK-Cu always produced much stronger responses. GHK attached to radioactive copper-64 increases copper uptake into cultured hepatoma cells [7]⁠.

The intravenous injection of tritiated copper-free GHK into mice was found, after 4 hours, to concentrate most densely within the animals' kidneys and brain. See Figure 2 [43]⁠.

GHK Nerve Research

 

Figure 2. Uptake of glycyl-L-histidyl-L-lysine (GHK) into various mouse tissues. (Reprinted from Pickart, L. [43]).

The best evidence that GHK can obtain copper 2+ from body fluids was from a study that used biotinylated GHK bound to collagen films placed over wounds in rats. The GHK pads raised the copper concentration by ninefold at the wound site when compared to non-GHK collagen films. Such biotinylated GHK collagen files also increased wound healing, cell proliferation, and increased the expression of antioxidant enzymes in the treated group [9]⁠.

Most importantly, GHK activates numerous regenerative and protective genes. Albumin will not mimic the GHK activated systems. So GHK must act through a separate pathway, not the albumin pathway. Albumin's copper feeds cells; GHK's copper activates regenerative and protective genes.

GHK-Cu's regenerative and protective actions on tissue are very similar to those found by John R Sorenson throughout his 33 years of work on various copper salicylates. See Table 1. It appears that GHK-Copper and Sorenson's diisopropylsalicylate-copper both activate the same pathway, a pathway strongly associated with tissue health and repair. GHK-copper 2+ (molecular weight 404) and Sorenson's diisopropylsalicylate-copper 2+ (molecular weight 506) are both very small molecules while albumin is much larger (molecular weight 64,000). Hence, they are likely to use different cell receptor systems [42–47]. See Figure 3.

Table 1. Similarity of Actions of GHK-Copper and Diisopropylsalicylate-Copper

Action

GHK-Copper 2+

Diisopropylsalicylate-Copper 2+

Wound Healing

Yes

Yes

Inhibit Cancer Growth

Yes

Yes

Anti-Ulcer

Yes

Yes

Anti-Pain

Yes

Yes

Improve Recovery After Radiation

Yes

Yes

Increase Stem Cell Activity

Yes

Yes


GHK Nerve Research

Figure 3. Proposed cell receptor for GHK-Cu.

 

3.3. Anti-Anxiety (Anxiolytic) and Anti-Pain

Anxiety and pain are serious issues in patients with dementia and other disabling mental conditions. Opiate peptides often possess both anti-pain and wound healing properties [48]⁠. When healthy human males were fed a low copper diet (1 mg/day of copper) for 11 weeks, their plasma opiate levels dropped by 80%. As soon as copper was restored (with a diet containing 3 mg/day of copper), the levels returned to normal [49]⁠.

GHK has been found to possess analgesic and anxiolytic effects (anti-anxiety) in animal experiments. GHK reduced pain after thermal injury to rats at a dose of 0.5 milligrams/kg. Within 12 minutes after intraperitoneal injection, it also increased the amount of time the rats spent exploring more open areas of the maze and decreased time spent immobile (the freeze reaction), which indicated reduction of fear and anxiety. These effects were observed at 0.5 micrograms/kg [50,51]⁠. We have been informed that Cage Fighters (Mixed Martial Arts) inject themselves intravenously with one milligram of GHK before fights to gain more confidence. This information is in agreement with the anxiolytic actions of GHK observed in rats [52]⁠.

These effects also prove that GHK rapidly affects the brain perception and function. This is an area where GHK could be used on patients today.

A manual search of genes affected by GHK found that seven anti-pain genes increased and two genes decreased. See Table 2 and 3.

Table 2. Distribution of Genes Effected by GHK and Associated with Pain

Percent Change in Gene Expression

Genes UP

Genes DOWN

50 - 99%

0

0

100 – 199%

5

2

200 - 299%

1

0

300 -399%

0

0

400 - 499%

0

0

500% +

1

0

Total

7

2

Table 3. GHK and Genes Associated with Pain

UP

Gene

Percent Change in Gene Expression

Comments

1

OPRMI

1294

Opioid mu 1-High Affinity for enkephalins and beta-endorphins

2

OPRL1

246

Receptor for neuropeptide nociceptin

3

CCKAR

190

Cholecystokinin, affects satiety, release of beta-endorphin and dopamine

4

CNR1

172

Cannabinoid receptor, pain-reducing

5

SIGMAR1

155

Non-opioid receptor

6

PNOC

150

Prepronociceptin, complex interactions with pain and anxiety induction

7

OXT

136

Ocytocin, bonding protein – gene also increases human chorionic gonadotropin

DOWN

Gene

Percent Change in Gene Expression

Comments

1

AMPA 3/GRIA3

-126.00%

Glutamate receptor, retrograde endocannaboid signaling, nervous system

 

3.4. Antioxidant Activity of the GHK Peptide

High metabolic activity found in the brains of both humans and animals results in elevated oxygen consumption and constant production of reactive oxygen species (ROS) in mitochondria. At the same time, the brain tissue is rich in unsaturated fatty acids and transition metal ions, yet has relatively fewer antioxidants compared to other organs, creating favorable conditions for oxidative damage. Since the blood-brain barrier prevents many dietary antioxidants from entering the brain, it largely relays on endogenous antioxidants such as Cu and Zn dependent superoxide dismutase (Cu, Zn SOD1). This enzyme requires the metal ions copper and zinc in order to be active. Hence, copper deficiency can lead to reduced SOD activity and increased oxidative brain damage. When pregnant rats were fed a copper deficient diet, the embryos displayed low SOD activity, increased super oxide anion radical level, and higher incidence of DNA damage and malformations [53]⁠.

GHK has broad and powerful anti-oxidation properties in both mammals and cell culture, and it is known to increases anti-oxidant gene expression. Tissue oxidation has been postulated as a causative factor of various nerve diseases of aging [54–58]⁠.

Diminished copper has been found in cells expressing SOD1 mutations postulated to cause ALS in mice and increase memory loss [59,60]⁠.

A peptidomimetic inhibitor (P6), based on GHK, interacts with amyloid beta (Aβ) peptide and its aggregates. P6 prevents the formation of toxic Aβ oligomeric species, fibrillar aggregates and DNA damage. It is a potential therapeutic candidate to ameliorate the multifaceted Aβ toxicity in Alzheimer's [61]⁠.

3.4.1 GHK's Anti-Oxidant Effects in Mammals and Cell Culture

The use of GHK-Cu in mice protected their lung tissue from lipopolysaccharide-induced acute lung injury (ALI). When GHK-Cu was used by mice with LPS-induced ALI, it attenuated related histological alterations in the lungs and suppressed the infiltration of inflammatory cells into the lung parenchyma. The GHK-Cu also increased superoxide dismutase (SOD) activity while decreasing TNF-α and IL-6 production through the suppression of the phosphorylation of NF-κB p65 and p38 MAPK in the nucleus of lung cells [40]⁠.

P38 mitogen-activated protein kinases are responsive to stress stimuli, such as cytokines, ultraviolet irradiation, heat shock, and osmotic shock, and are involved in cell differentiation, apoptosis, and autophagy. NF-κB/RELA p65 activation has been found to be correlated with cancer development, suggesting the potential of RELA as a cancer biomarker. Specific modification patterns of RELA have also been observed in many cancer types.

Multiple antioxidant actions of GHK have been demonstrated in vitro and in animal wound healing studies. They include inhibiting the formation of reactive carbonyl species (RCS), detoxifying toxic products of lipid peroxidation such as acrolein, protecting keratinocytes from lethal UVB radiation, and preventing hepatic damage by dichloromethane radicals.

The ability of GHK to prevent oxidative stress was tested in vitro using Cu(2+)-dependent oxidation of low-density lipoproteins (LDL). LDL were treated with 5 μM Cu(2+) for 18 h in either phosphate buffered saline (PBS) or Ham’s F-10 medium. There was increased production of thiobarbituric acid reactive substances (TBARSs), which indicated increased oxidation. GHK and histidine “entirely blocked” the in vitro Cu(2+)-dependent oxidation of low-density lipoproteins (LDL). In comparison, superoxide dismutase (SOD1) provided only 20% reduction of oxidation [62]⁠.

Acrolein, a well-known carbonyl toxin, is produced by lipid peroxidation of polyunsaturated fatty acids. GHK effectively reduces the formation of both acrolein and another product of oxidation, 4-hydroxynonenal. GHK also blocks lethal ultraviolet radiation damage to cultured skin keratinocytes by binding and inactivating reactive carbonyl species such as 4-hydroxynoneal, acrolein, malondialdehyde, and glyoxal [63–65]⁠.

The intraperitoneal injection of 1.5 mg/kg of GHK into rats for five days before dichloromethane poisoning and five days thereafter provided protection of the functional activity of hepatocytes and immunological responsiveness. Dichloromethane is toxic to hepatic tissue via the formation of a dichloromethane free radical that induces acute toxic damage [66]⁠.

In rats with experimental bone fractures peptides, GHK (0.5 μg/kg), dalargin (1.2 μg/kg), and thymogen (0.5 μg/kg) were injected intraperitoneally. Within 10 days, there was a decrease of malonic dialdehyde and an increase of catalase activity in blood. There was also a marked increase of reparative activity. Each combination of peptides was more potent than any of the studied peptides injected separately. The synergetic action of peptides Gly-His-Lys, thymogen, and dalargin was proposed for stimulation of reparative osteogenesis [67]⁠.

GHK-Cu reduced iron release from ferritin by 87%. Iron has also been shown to have a direct role in the initiation of lipid peroxidation. An Fe(2+)/Fe(3+) complex can serve as an initiator of lipid oxidation. In addition, many iron complexes can catalyze the decomposition of lipid hydroperoxides to the corresponding lipid alkoxy radicals. The major storage site for iron in serum and tissue is ferritin. Ferritin in blood plasma can store up to 4500 atoms of iron per protein molecule, and superoxide anions can promote the mobilization of iron from ferritin. This free iron may then catalyze lipid peroxidation and the conversion of a superoxide anion to the more damaging hydroxyl radical [68]⁠.

3.4.2. Synthesis of GHK-Cu Analogs with Higher Anti-ROS Activity

GHK-Cu has, on a molar basis, about 1% to 3% of the activity of the Cu, Zn superoxide dismutase protein. By simple modifications to the peptide, it is possible to raise the SOD-mimetic activity up 223-fold. Given the broad range of the antioxidant actions of GHK, it is likely that modifications will increase its countering reactive species such as RCS and dichloromethane radicals. See Table 4 [69]⁠.

Table 4. Superoxide Dismutase Mimetic Activity of GHK and Analogs

Molecule

Superoxide dismutase mimetic activity

GHK:Cu(2+)

100

KHG-Amide:Cu(2+)

21

GHKAFA:Cu(2+)

561

AHK:Cu(2+)

563

GHK-Octyl Ester:Cu(2+)

810

GHCaprolactam:Cu(2+)

4500

HGK:Cu(2+)

22300


3.4.3. Antioxidant Gene Expression Analysis

A manual search of antioxidant associated genes effected by GHK yielded 18 genes with significant antioxidant activity. See Table 5 and 6.

Table 5. Distribution of Genes Effected by GHK with Antioxidant Activity

Percent Change in Gene Expression

Genes UP

Genes DOWN

50 - 99%

2

0

100 – 199%

7

1

200 - 299%

2

0

300 -399%

1

0

400 - 499%

1

0

500% +

3

1

Total

16

2


Table 6. GHK and Genes Associate with Antioxidant Activity

UP

Genes

Percent Change in Gene Expression

Comments

1

TLE1

762

Inhibits the oxidative/inflammatory gene NF-κB [79]⁠.

2

SPRR2C

721

This proline-rich, antioxidant protein protects outer skin cells from oxidative damage from ROS. When the ROS level is low, the protein remains in the outer cell membrane, but when the ROS level is high, the protein clusters around the cell’s DNA to protect it [81,82]⁠.

3

ITGB4

609

Up-regulation of ITGB4 promotes wound repair ability and antioxidative ability [83]⁠.

4

APOM

403

Binds oxidized phospholipids and increases the antioxidant effect of high-density lipoproteins (HDL) [84].

5

PON3

319

Absence of PON3 (paraoxonase 3) in mice resulted in increased rates of early fetal and neonatal death. Knockdown of PON3 in human cells reduced cell proliferation and total antioxidant capacity [85]⁠.

6

IL18BP

295

The protein encoded by this gene is an inhibitor of the pro-inflammatory cytokine IL18. IL18BP abolished IL18 induction of interferon-gamma (IFN gamma), IL8, and activation of NF-κB in vitro. Blocks neutrophil oxidase activity [86]⁠.

7

HEPH

217

Inhibits the conversion of Fe(2+) to Fe(3+). HEPH increases iron efflux, lowers cellular iron levels, suppresses reactive oxygen species production, and restores mitochondrial transmembrane potential [87]⁠.

8

GPSM3

193

Acts as a direct negative regulator of NLRP3. NLRP3 triggers the maturation of the pro-inflammatory cytokines IL-1β and IL-18 [88]⁠.

9

FABP1

186

Reduces intracellular ROS level. Plays a significant role in reduction of oxidative stress [70,71]⁠.

10

AGTR2

171

AT(2)R exerts an anti-inflammatory response in macrophages via enhanced IL-10 production and ERK1/2 phosphorylation, which may have protective roles in hypertension and associated tissue injury [72]⁠.

11

PON1

149

PON1 (paraoxonase 1) is a potent antioxidant and a major
anti-atherosclerotic component of HDL [73]⁠.

12

MT3

142

Metallothioneins (MTs) display in vitro free radical scavenging capacity, suggesting that they may specifically neutralize hydroxyl radicals. Metallothioneins and metallothionein-like proteins isolated from mouse brain act as neuroprotective agents by scavenging superoxide radicals [74,75]⁠.

13

PTGS2

120

Produces cyclooxygenase-II (COX-II), which has antioxidant activities [76]⁠.

14

SLC2A9

117

The p53-SLC2A9 pathway is a novel antioxidant mechanism. During oxidative stress, SLC2A9 undergoes p53-dependent induction, and functions as an antioxidant by suppressing ROS, DNA damage, and cell death [77]⁠.

15

NFE2L2

56

Nuclear respiratory factor 2 helps activate antioxidant responsive
element-regulated genes which contribute to the regulation of the cellular antioxidant defense systems [78]⁠.

16

PTGS1

50

Produces cyclooxygenase-I (COX-I), which has antioxidant activity [76]⁠.

DOWN

Genes

Percent Change in Gene Expression*

Comments

1

IL17A

−1018

This cytokine can stimulate the expression of IL6 and cyclooxygenase-2 (PTGS2/COX-2), as well as enhance the production of nitric oxide (NO). High levels of this cytokine are associated with several chronic inflammatory diseases including rheumatoid arthritis, psoriasis, and multiple sclerosis (http://www.ncbi.nlm.nih.gov/gene/3605).

2

TNF

−115

GHK suppresses this pro-oxidant TNF gene [80]⁠.


3.5. DNA Repair, Cell Culture, and Gene Expression

A lack of adequate DNA repair may be related to neurological degeneration in the aging population [89–92]⁠.

DNA damage is a major problem in the life cycle of biological cells. Normal cellular metabolism releases compounds that damage DNA such as reactive oxygen species, reactive nitrogen species, reactive carbonyl species, lipid peroxidation products and alkylating agents, among others, while hydrolysis cleaves chemical bonds in DNA. It is estimated that each normally functioning cell in the human body suffers at least 10,000 DNA damaging incidents daily [93]⁠.

Radiation therapy is believed to stop cell replication by damaging cellular DNA. A study of cultured primary human dermal fibroblast cell lines from patients who had undergone radiation therapy for head and neck cancer found that the procedure slowed the population doubling times for the cells. But treatment with one nanomolar GHK-Cu restored population doubling times to normal. Irradiated cells treated with GHK-Cu also produced significantly more basic fibroblast growth factor and vascular endothelial growth factor than untreated irradiated cells [94]⁠.

GHK is primarily stimulatory for gene expression of DNA Repair genes (47 UP, 5 DOWN), suggesting an increased DNA repair activity. Here we searched the Gene Ontology descriptions for “DNA Repair”. See Table 7 and 8.

Table 7. Distribution of Genes Effected by GHK and Associated with DNA Repair

Percent Change in Gene Expression

Genes UP

Genes DOWN

50% - 100%

41

4

100% - 150%

2

1

150% - 200%

1

0

200% - 250%

2

0

250% - 300%

1

0

Total

47

5

Table 8. GHK and Genes Associate with DNA Repair

UP

Gene Title

Percent Change in
Gene Expression

1

poly (ADP-ribose) polymerase family, member 3, PARP3

253

2

polymerase (DNA directed), mu, POLM

225

3

MRE11 meiotic recombination 11 homolog A MRE11A

212

4

RAD50 homolog (S. cerevisiae), RAD50

175

5

eyes absent homolog 3 (Drosophila), EYA3

128

6

retinoic acid receptor, alpha, RARA

123

DOWN

Gene Title

Percent Change in
Gene Expression

1

cholinergic receptor, nicotinic, alpha 4, CHRNA4

-105


3.6. Restoring Regeneration After Cortisone Treatment

Steroid dementia syndrome describes the signs and symptoms of hippocampal and prefrontal cortical dysfunction, such as deficits in memory, attention, and executive function, induced by glucocorticoids. Dementia-like symptoms have been found in some individuals who have been exposed to glucocorticoid medication, often dispensed in the form of asthma, arthritis, and anti-inflammatory steroid medications. The condition reverses, but not always completely, within months after steroid treatment is stopped [95]⁠.

In the human body, cortisone and cortisol are easily interconvertible and have similar anti-inflammatory actions. They also profoundly inhibit tissue regeneration, such as wound repair. DHEA (dehydroepiandrosterone) is an androgenic hormone. It is a precursor for testosterone and the estrogens. DHEA antagonizes the effects of cortisol but decreases about 80% from age 20 to age 80 while cortisone/cortisol levels remain high. It has been proposed that many of the deleterious effects of aging are due to excessive cortisol that is not balanced by DHEA.

GHK-Cu, when administered systemically to mice, rats, and pigs, counters the wound healing inhibition of cortisone throughout the animal [96]⁠.

3.7. GHK and Mitochondria Gene Expression

Mitochondrial bioenergetic deficits have been suggested to contribute to the onset of dementia and various neurodegenerative disorders of aging as nerve cells have high energy needs. The enzymes of the citric acid cycle are located in the mitochondria where the citric acid cycle oxidizes the acetyl-CoA to carbon dioxide and energy exchanging molecules such as GTP and ATP. Mitochondria affected by oxidative stress produce increased hydrogen peroxide [97–100]⁠.

GHK increases mitochondrial gene expression in 207 genes and decreases it in 59. Here we searched the Gene Ontology descriptions for “Mitochondria”. See Table 9 and 10.

Table 9. Distribution of Genes Effected by GHK and Associated with Mitochondria

Percent Change
in Gene Expression

Genes UP

Genes DOWN

50 - 99%

144

29

100 – 199%

39

16

200 - 299%

12

8

300 -399%

5

3

400 - 499%

2

1

500% +

5

2

Total

207

59

 

Table 10. GHK and Genes Associated with Mitochondria

UP

Gene Titie

Percent Change

1

acyl-CoA synthetase medium-chain family member 2A /// acyl-CoA synthetase medium-chain family member 2B, ACSM2A /// ACSM2B

992

2

tumor protein p73, TP73

938

3

myostatin, MSTN

829

4

alanine-glyoxylate aminotransferase 2-like 1, AGXT2L1

675

5

trimethyllysine hydroxylase, epsilon, TMLHE

597

6

glycine-N-acyltransferase, GLYAT

418

7

makorin ring finger protein 2, MKRN2

402

8

caspase 8, apoptosis-related cysteine peptidase, CASP8

399

9

gamma-aminobutyric acid (GABA) A receptor, alpha 5 /// gamma-aminobutyric acid receptor subunit alpha-5-like, GABRA5 /// LOC100509612

392

10

translocase of inner mitochondrial membrane 17 homolog A (yeast), TIMM17A

377

11

protein phosphatase 2, regulatory subunit B, beta, PPP2R2B

325

12

v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian), SRC

308

13

voltage-dependent anion channel 3, VDAC3

287

14

T cell receptor delta variable 3, TRDV3

279

15

dickkopf-like 1, DKKL1

277

16

FK506 binding protein 8, 38kDa, FKBP8

272

17

caspase 1, apoptosis-related cysteine peptidase (interleukin 1, beta, convertase), CASP1

257

18

nitric oxide synthase 1 (neuronal), NOS1

240

19

sirtuin 4, SIRT4

235

20

dystrophia myotonica-protein kinase, DMPK

230

21

family with sequence similarity 162, member A, FAM162A

228

22

enolase 1, (alpha), ENO1

218

23

solute carrier family 25 (mitochondrial oxodicarboxylate carrier), member 21, SLC25A21

214

24

dihydrolipoamide branched chain transacylase E2, DBT

213

25

carbohydrate (chondroitin 6) sulfotransferase 3, CHST3

192

26

A kinase (PRKA) anchor protein 1, AKAP1

190

27

fatty acid binding protein 1, liver, FABP1

186

28

fumarate hydratase, FH

185

29

hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1, HSD3B1

184

30

polymerase (RNA) mitochondrial (DNA directed), POLRMT

180

31

acetyl-CoA acyltransferase 2, ACAA2

178

32

solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 31, SLC25A31

175

33

parkinson protein 2, E3 ubiquitin protein ligase (parkin), PARK2

169

34

dual specificity phosphatase 21, DUSP21

165

35

N-myristoyltransferase 1, NMT1

156

36

activating transcription factor 2, ATF2

156

37

GEM interacting protein, GMIP

150

38

peptidylprolyl isomerase F, PPIF

149

39

polyamine oxidase (exo-N4-amino), PAOX

146

40

cytochrome b5 type A (microsomal), CYB5A

142

41

metallothionein 3, MT3

142

42

endo/exonuclease (5'-3'), endonuclease G-like, EXOG

138

43

ataxin 3, ATXN3

136

44

protein (peptidylprolyl cis/trans isomerase) NIMA-interacting, 4 (parvulin), PIN4

135

45

lipin 1, LPIN1

135

46

acyl-CoA dehydrogenase, long chain, ACADL

134

47

Sirtuin (silent mating type information regulation 2 homolog) 5 (S. cerevisiae), SIRT5

134

48

acyl-CoA synthetase medium-chain family member 3, ACSM3

133

49

peroxisome proliferator-activated receptor gamma, coactivator 1 alpha, PPARGC1A

126

50

v-erb-a erythroblastic leukemia viral oncogene homolog 4 (avian), ERBB4

125

51

cell death-inducing DFFA-like effector b, CIDEB

121

52

isovaleryl-CoA dehydrogenase, IVD

120

53

voltage-dependent anion channel 1, VDAC1

120

54

interferon-induced protein with tetratricopeptide repeats 2, IFIT2

117

55

5',3'-nucleotidase, mitochondrial, NT5M

113

56

transcription factor Dp-1, TFDP1

112

57

keratin 6A /// keratin 6B /// keratin 6C, KRT6A /// KRT6B /// KRT6C

112

58

non-SMC condensin II complex, subunit H2, NCAPH2

112

59

MLX interacting protein, MLXIP

109

60

arginyl-tRNA synthetase 2, mitochondrial, RARS2

103

61

proline dehydrogenase (oxidase) 1, PRODH

102

62

steroidogenic acute regulatory protein, STAR

101

63

TBC1 domain family, member 5, TBC1D5

100

64

aldehyde dehydrogenase 18 family, member A1, ALDH18A1

99

65

LSM4 homolog, U6 small nuclear RNA associated (S. cerevisiae), LSM4

98

66

DEAH (Asp-Glu-Ala-His) box polypeptide 30, DHX30

97

67

presenilin 1, PSEN1

95

68

tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide, YWHAZ

95

69

adenylate kinase 2, AK2

94

70

carbonic anhydrase VB, mitochondrial, CA5B

94

71

phosphatidylinositol 4-kinase type 2 alpha, PI4K2A

93

72

stomatin, STOM

92

73

solute carrier family 5 (sodium/myo-inositol cotransporter), member 3, SLC5A3

90

74

mitochondrial ribosomal protein S18C, MRPS18C

90

75

ATP synthase, H+ transporting, mitochondrial Fo complex, subunit s (factor B), ATP5S

89

76

tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide, YWHAE

87

77

purinergic receptor P2X, ligand-gated ion channel, 7, P2RX7

87

78

hepatocyte growth factor (hepapoietin A; scatter factor), HGF

85

79

arrestin, beta 2, ARRB2

85

80

ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C2 (subunit 9), ATP5G2

85

81

solute carrier family 6 (neurotransmitter transporter, GABA), member 1, SLC6A1

83

82

succinate dehydrogenase complex, subunit C, integral membrane protein, 15kDa, SDHC

83

83

CD24 molecule, CD24

82

84

inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon, IKBKE

82

85

mitogen-activated protein kinase 8, MAPK8

80

86

glycogen synthase kinase 3 alpha, GSK3A

80

87

myosin, heavy chain 11, smooth muscle, MYH11

79

88

solute carrier family 25 (mitochondrial carrier; oxoglutarate carrier), member 11, SLC25A11

79

89

RAP1, GTP-GDP dissociation stimulator 1, RAP1GDS1

79

90

ATP-binding cassette, sub-family A (ABC1), member 8, ABCA8

78

91

valosin-containing protein, VCP

77

92

biphenyl hydrolase-like (serine hydrolase), BPHL

76

93

DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked, DDX3X

76

94

phospholipase A2, group VI (cytosolic, calcium-independent), PLA2G6

76

95

calcitonin-related polypeptide beta, CALCB

75

96

taxilin alpha, TXLNA

75

97

adenylate kinase 4, AK4

75

98

Mitochondrial translational release factor 1-like, MTRF1L

74

99

coenzyme Q7 homolog, ubiquinone (yeast), COQ7

74

100

chromosome 2 open reading frame 18, C2orf18

73

101

aldehyde dehydrogenase 7 family, member A1, ALDH7A1

73

102

ACN9 homolog (S. cerevisiae), ACN9

73

103

F-box and leucine-rich repeat protein 4, FBXL4

73

104

isocitrate dehydrogenase 3 (NAD+) beta, IDH3B

72

105

mitochondrial ribosomal protein S18A, MRPS18A

72

106

ATG3 autophagy related 3 homolog (S. cerevisiae), ATG3

72

107

v-akt murine thymoma viral oncogene homolog 1, AKT1

71

108

Fc fragment of IgG, low affinity IIIa, receptor (CD16a) /// Fc fragment of IgG, low affinity IIIb, receptor (CD16b), FCGR3A /// FCGR3B

71

109

GUF1 GTPase homolog (S. cerevisiae), GUF1

71

110

hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), alpha subunit, HADHA

70

111

sequestosome 1, SQSTM1

70

112

aldehyde dehydrogenase 1 family, member B1, ALDH1B1

70

113

acyl-CoA thioesterase 1 /// acyl-CoA thioesterase 2, ACOT1 /// ACOT2

69

114

phosphoinositide-3-kinase, catalytic, alpha polypeptide, PIK3CA

68

115

small nuclear ribonucleoprotein polypeptides B and B1, SNRPB

68

116

cell division cycle 37 homolog (S. cerevisiae), CDC37

68

117

ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit homolog (E. coli), CLPP

68

118

methylcrotonoyl-CoA carboxylase 2 (beta), MCCC2

67

119

mitochondrial ribosomal protein L17, MRPL17

67

120

glutamyl-tRNA(Gln) amidotransferase, subunit C homolog (bacterial), GATC

67

121

superoxide dismutase 2, mitochondrial, SOD2

67

122

translocase of inner mitochondrial membrane 23 homolog (yeast) /// translocase of inner mitochondrial membrane 23 homolog B (yeast), TIMM23 /// TIMM23B

67

123

translocase of inner mitochondrial membrane 17 homolog B (yeast), TIMM17B

66

124

acyl-CoA synthetase medium-chain family member 1, ACSM1

66

125

tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide, YWHAH

66

126

isocitrate dehydrogenase 3 (NAD+) gamma, IDH3G

66

127

BCL2-associated agonist of cell death, BAD

66

128

ubiquinol-cytochrome c reductase core protein II, UQCRC2

66

129

branched chain keto acid dehydrogenase E1, beta polypeptide, BCKDHB

65

130

myeloid cell leukemia sequence 1 (BCL2-related), MCL1

65

131

caspase 3, apoptosis-related cysteine peptidase, CASP3

65

132

translocase of outer mitochondrial membrane 20 homolog (yeast), TOMM20

65

133

4-aminobutyrate aminotransferase, ABAT

65

134

malic enzyme 2, NAD(+)-dependent, mitochondrial, ME2

65

135

nudix (nucleoside diphosphate linked moiety X)-type motif 2, NUDT2

64

136

nipsnap homolog 1 (C. elegans), NIPSNAP1

64

137

glycerol kinase 3 pseudogene, GK3P

64

138

aldehyde dehydrogenase 6 family, member A1, ALDH6A1

62

139

chromosome 2 open reading frame 56, C2orf56

62

140

mitochondrial ribosomal protein L20, MRPL20

62

141

acyl-CoA dehydrogenase, short/branched chain, ACADSB

61

142

slowmo homolog 1 (Drosophila), SLMO1

61

143

creatine kinase, mitochondrial 2 (sarcomeric), CKMT2

60

144

carnitine palmitoyltransferase 2, CPT2

60

145

mitochondrial ribosomal protein L15, MRPL15

60

146

glutamate dehydrogenase 1, GLUD1

60

147

succinate-CoA ligase, ADP-forming, beta subunit, SUCLA2

60

148

growth arrest and DNA-damage-inducible, gamma interacting protein 1, GADD45GIP1

60

149

ADP-ribosylation factor-like 2, ARL2

60

150

mitogen-activated protein kinase 9, MAPK9

60

151

neurofilament, medium polypeptide, NEFM

60

152

deoxyguanosine kinase, DGUOK

60

153

mitochondrial ribosomal protein S12, MRPS12

59

154

nicotinamide nucleotide transhydrogenase, NNT

58

155

APEX nuclease (apurinic/apyrimidinic endonuclease) 2, APEX2

58

156

prion protein, PRNP

58

157

glycine amidinotransferase (L-arginine:glycine amidinotransferase), GATM

58

158

mitochondrial ribosomal protein 63, MRP63

57

159

tafazzin, TAZ

57

160

chromosome 1 open reading frame 163, C1orf163

57

161

ras homolog gene family, member T2, RHOT2

57

162

nucleolar protein 3 (apoptosis repressor with CARD domain), NOL3

57

163

biotinidase, BTD

57

164

solute carrier family 25, member 37, SLC25A37

57

165

cytochrome c-1, CYC1

56

166

lysine-rich coiled-coil 1, KRCC1

56

167

chromosome 12 open reading frame 5, C12orf5

56

168

cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4), CDKN2A

56

169

calcium/calmodulin-dependent protein kinase II alpha, CAMK2A

56

170

solute carrier family 11 (proton-coupled divalent metal ion transporters), member 2, SLC11A2

55

171

hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2, HSD3B2

55

172

tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, beta polypeptide, YWHAB

55

173

ribosomal protein S6 kinase, 70kDa, polypeptide 1, RPS6KB1

55

174

solute carrier family 25, member 28, SLC25A28

55

175

protein kinase C, alpha, PRKCA

55

176

serine hydroxymethyltransferase 2 (mitochondrial), SHMT2

54

177

nucleotide binding protein-like, NUBPL

54

178

hydroxyacyl-CoA dehydrogenase, HADH

54

179

leucyl-tRNA synthetase 2, mitochondrial, LARS2

54

180

mitochondrial ribosomal protein L39, MRPL39

54

181

ATP-binding cassette, sub-family B (MDR/TAP), member 7, ABCB7

54

182

sideroflexin 1, SFXN1

53

183

phosphatidylinositol 4-kinase, catalytic, beta, PI4KB

53

184

aldehyde dehydrogenase 4 family, member A1, ALDH4A1

53

185

heat shock 60kDa protein 1 (chaperonin), HSPD1

53

186

3-hydroxybutyrate dehydrogenase, type 1, BDH1

53

187

pyruvate dehydrogenase (lipoamide) alpha 2, PDHA2

53

188

coenzyme Q9 homolog (S. cerevisiae), COQ9

53

189

pyruvate dehydrogenase phosphatase regulatory subunit, PDPR

52

190

acylglycerol kinase, AGK

52

191

acyl-CoA synthetase long-chain family member 1, ACSL1

52

192

arginase, type II, ARG2

52

193

meningioma expressed antigen 5 (hyaluronidase), MGEA5

52

194

eyes absent homolog 2 (Drosophila), EYA2

52

195

peroxisomal biogenesis factor 3, PEX3

52

196

acyl-CoA thioesterase 9, ACOT9

51

197

peroxisomal biogenesis factor 13, PEX13

51

198

K(lysine) acetyltransferase 2A, KAT2A

51

199

golgi phosphoprotein 3 (coat-protein), GOLPH3

51

200

prohibitin, PHB

51

201

glutathione peroxidase 4 (phospholipid hydroperoxidase), GPX4

51

202

histone deacetylase 6, HDAC6

51

203

DDHD domain containing 2, DDHD2

51

204

proteasome (prosome, macropain) 26S subunit, non-ATPase, 10, PSMD10

50

205

BCL2 binding component 3, BBC3

50

206

mitochondrial ribosomal protein S15, MRPS15

50

207

slowmo homolog 2 (Drosophila), SLMO2

50

DOWN

Gene Title

Percent Change

1

solute carrier family 22 (extraneuronal monoamine transporter), member 3, SLC22A3

-524

2

insulin-like growth factor 1 (somatomedin C), IGF1

-522

3

haptoglobin /// haptoglobin-related protein, HP /// HPR

-444

4

cytochrome P450, family 11, subfamily A, polypeptide 1, CYP11A1

-393

5

3-hydroxy-3-methylglutaryl-CoA synthase 2 (mitochondrial), HMGCS2

-342

6

ornithine carbamoyltransferase, OTC

-331

7

ATPase, Ca++ transporting, cardiac muscle, fast twitch 1, ATP2A1

-299

8

mitogen-activated protein kinase 8 interacting protein 1, MAPK8IP1

-289

9

harakiri, BCL2 interacting protein (contains only BH3 domain), HRK

-248

10

serpin peptidase inhibitor, clade B (ovalbumin), member 10, SERPINB10

-247

11

Acyl-CoA synthetase long-chain family member 6, ACSL6

-226

12

sperm mitochondria-associated cysteine-rich protein, SMCP

-207

13

polymerase (DNA directed), gamma, POLG

-204

14

CD93 molecule, CD93

-200

15

vesicle-associated membrane protein 1 (synaptobrevin 1), VAMP1

-193

16

uncoupling protein 3 (mitochondrial, proton carrier), UCP3

-192

17

T cell receptor delta locus, TRD@

-156

18

nerve growth factor receptor, NGFR

-132

19

DEAD (Asp-Glu-Ala-Asp) box polypeptide 28, DDX28

-126

20

phospholamban, PLN

-120

21

Phenylalanyl-tRNA synthetase 2, mitochondrial, FARS2

-120

22

acyl-CoA synthetase medium-chain family member 5, ACSM5

-116

23

hypothetical LOC100506517, LOC100506517

-112

24

NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 2, 8kDa, NDUFA2

-109

25

telomerase reverse transcriptase, TERT

-108

26

obscurin, cytoskeletal calmodulin and titin-interacting RhoGEF, OBSCN

-106

27

acetyl-CoA carboxylase beta, ACACB

-104

28

ADAM metallopeptidase with thrombospondin type 1 motif, 7, ADAMTS7

-104

29

BCL2-like 10 (apoptosis facilitator), BCL2L10

-103

30

potassium channel, subfamily K, member 3, KCNK3

-102

31

potassium channel tetramerisation domain containing 14, KCTD14

-98

32

ataxin 3-like, ATXN3L

-89

33

interferon, alpha-inducible protein 27, IFI27

-87

34

malic enzyme 3, NADP(+)-dependent, mitochondrial, ME3

-85

35

phosphoglycerate kinase 2, PGK2

-82

36

ligase III, DNA, ATP-dependent, LIG3

-81

37

aminoadipate-semialdehyde synthase, AASS

-76

38

tumor necrosis factor (ligand) superfamily, member 10, TNFSF10

-74

39

loss of heterozygosity, 3, chromosomal region 2, gene A, LOH3CR2A

-72

40

carnitine palmitoyltransferase 1A (liver), CPT1A

-71

41

Solute carrier family 25, member 42, SLC25A42

-70

42

chromosome 1 open reading frame 69, C1orf69

-69

43

carbonic anhydrase VII, CA7

-69

44

D4, zinc and double PHD fingers, family 3, DPF3

-68

45

mitogen-activated protein kinase kinase kinase 12, MAP3K12

-66

46

signal transducer and activator of transcription 2, 113kDa, STAT2

-65

47

radical S-adenosyl methionine domain containing 2, RSAD2

-64

48

cytochrome P450, family 27, subfamily A, polypeptide 1, CYP27A1

-63

49

aminolevulinate, delta-, synthase 2, ALAS2

-63

50

cytochrome c oxidase subunit VIIa polypeptide 1 (muscle), COX7A1

-62

51

cholecystokinin, CCK

-59

52

choline kinase-like, carnitine palmitoyltransferase 1B (muscle) transcription unit /// carnitine palmitoyltransferase 1B (muscle), CHKB-CPT1B /// CPT1B

-57

53

microtubule-associated protein 1A, MAP1A

-56

54

sarcosine dehydrogenase, SARDH

-55

55

B-cell CLL/lymphoma 2, BCL2

-53

56

thymidine kinase 2, mitochondrial, TK2

-52

57

G protein-coupled estrogen receptor 1, GPER

-52

58

pyruvate dehydrogenase kinase, isozyme 4, PDK4

-52

59

cell death-inducing DFFA-like effector a, CIDEA

-50

3.8. Gene Expression - Clearing Damaged Protein – Ubiquitin Proteasome System

The ubiquitin proteasome system (UPS) clears damaged proteins. Insufficient activity of this system is postulated to produce an accumulation of toxic protein oligomers which start the neurodegenerative process. During aging, there is decreased activity of the ubiquitin proteasome system. To date, no effective therapies have been developed that can specifically increase the UPS activity [101–104]⁠.

GHK strongly stimulates the gene expression of the UPS system with 41 genes increased and 1 gene suppressed. Here we searched gene title for “ubiquitin” or “proteasome”. See Table 11 and 12.

Table 11. Distribution of Genes Effected by GHK and Associated with the Ubiquitin Proteasome System

Percent Change in Gene Expression

Genes UP

Genes DOWN

50 - 99%

31

1

100 – 199%

7

0

200 - 299%

0

0

300 -399%

1

0

400 - 499%

1

0

500% +

1

0

Total

41

1


Table 12. GHK and Genes Associated with the Ubiquitin Proteasome System

UP

Gene Title

Percent Change

1

ubiquitin specific peptidase 29, USP29

1056

2

ubiquitin protein ligase E3 component n-recognin 2, UBR2

455

3

gamma-aminobutyric acid (GABA) B receptor, 1 /// ubiquitin D, GABBR1 /// UBD

310

4

ubiquitin specific peptidase 34, USP34

195

5

parkinson protein 2, E3 ubiquitin protein ligase (parkin), PARK2

169

6

ubiquitin-conjugating enzyme E2I (UBC9 homolog, yeast), UBE2I

150

7

ubiquitin protein ligase E3 component n-recognin 4, UBR4

146

8

ubiquitin protein ligase E3B, UBE3B

116

9

ubiquitin specific peptidase 2, USP2

104

10

ubiquitin-like modifier activating enzyme 6, UBA6

104

11

ubiquitination factor E4B (UFD2 homolog, yeast), UBE4B

99

12

ubiquitin-conjugating enzyme E2M (UBC12 homolog, yeast), UBE2M

92

13

ubiquitin-like modifier activating enzyme 7, UBA7

88

14

HECT, C2 and WW domain containing E3 ubiquitin protein ligase 1, HECW1

81

15

proteasome (prosome, macropain) 26S subunit, ATPase, 3, PSMC3

81

16

ubiquitin-conjugating enzyme E2D 1 (UBC4/5 homolog, yeast), UBE2D1

79

17

proteasome (prosome, macropain) subunit, beta type, 2, PSMB2

79

18

ubiquitin protein ligase E3 component n-recognin 5, UBR5

77

19

ubiquitin specific peptidase 21, USP21

76

20

OTU domain, ubiquitin aldehyde binding 2, OTUB2

76

21

proteasome (prosome, macropain) inhibitor subunit 1 (PI31), PSMF1

75

22

ubiquitin-conjugating enzyme E2H (UBC8 homolog, yeast), UBE2H

73

23

ubiquitin-conjugating enzyme E2N (UBC13 homolog, yeast), UBE2N

72

24

ubiquitin carboxyl-terminal hydrolase L5, UCHL5

71

25

proteasome (prosome, macropain) 26S subunit, non-ATPase, 13, PSMD13

70

26

ubiquitin associated protein 1, UBAP1

70

27

ubiquitin-conjugating enzyme E2B (RAD6 homolog), UBE2B

69

28

TMEM189-UBE2V1 readthrough /// ubiquitin-conjugating enzyme E2 variant 1, TMEM189-UBE2V1 /// UBE2V1

67

29

proteasome (prosome, macropain) 26S subunit, non-ATPase, 1, PSMD1

64

30

proteasome (prosome, macropain) 26S subunit, non-ATPase, 3, PSMD3

64

31

ariadne homolog, ubiquitin-conjugating enzyme E2 binding protein, 1 (Drosophila), ARIH1

61

32

BRCA1 associated protein-1 (ubiquitin carboxy-terminal hydrolase), BAP1

60

33

ubiquitin interaction motif containing 1, UIMC1

60

34

ubiquitin associated protein 2-like, UBAP2L

57

35

ubiquitin protein ligase E3 component n-recognin 7 (putative), UBR7

56

36

ubiquitin-conjugating enzyme E2G 1 (UBC7 homolog, yeast), UBE2G1

54

37

itchy E3 ubiquitin protein ligase homolog (mouse), ITCH

54

38

ubiquitin-conjugating enzyme E2D 4 (putative), UBE2D4

51

39

proteasome (prosome, macropain) 26S subunit, non-ATPase, 10, PSMD10

50

40

WW domain containing E3 ubiquitin protein ligase 1, WWP1

50

41

ubiquitin-like 3, UBL3

50

DOWN

Gene Title

Percent Change

1

ubiquitin associated and SH3 domain containing A, UBASH3A

-89

3.9. Gene Expression – Neurons

Neurons are cells that carry messages between the brain and other parts of the body; they are the basic units of the nervous system.

GHK is primarily stimulatory for gene expression of neuron related genes. Here we searched the Gene Ontology descriptions for “Neuron”. See Table 13 and 14.

Table 13. Distribution of Genes Effected by GHK and Associated with Neurons

Percent Change
in Gene Expression

Genes UP

Genes DOWN

50 - 99%

230

80

100 – 199%

99

80

200 - 299%

45

35

300 -399%

19

14

400 - 499%

9

10

500% +

6

11

Total

408

230

3.10. Motor Neurons

Motor neurons are nerve cells forming part of a pathway along which impulses pass from the brain or spinal cord to a muscle or gland.

Here we searched Gene Ontology descriptions for “motor neuron”. See Table 15 and 16.

Table 15. Distribution of Genes Effected by GHK and Associated with Motor Neurons

Percent Change
in Gene Expression

Genes UP

Genes DOWN

50 - 99%

9

5

100 – 199%

2

0

200 - 299%

2

1

300 -399%

0

0

400 - 499%

0

2

500% +

0

1

Total

13

9


Table 16. GHK and Genes Associate with Motor Neurons

UP

Gene Title

Percent Change

1

calcium channel, voltage-dependent, P/Q type, alpha 1A subunit, CACNA1A

286

2

plexin C1, PLXNC1

282

3

GLI family zinc finger 2, GLI2

183

4

NK2 homeobox 2, NKX2-2

181

5

sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3A, SEMA3A

82

6

neuropilin 1, NRP1

73

7

slit homolog 2 (Drosophila), SLIT2

69

8

cytokine receptor-like factor 1, CRLF1

68

9

BCL2-associated athanogene, BAG1

63

10

GRB10 interacting GYF protein 2, GIGYF2

62

11

chemokine (C-X-C motif) ligand 12, CXCL12

52

12

zinc finger protein 259 /// zinc finger protein 259 pseudogene 1, ZNF259 /// ZNF259P1

52

13

chemokine (C-X-C motif) ligand 12, CXCL12

50

DOWN

Gene Title

Percent Change

1

slit homolog 1 (Drosophila), SLIT1

-553

2

chemokine (C-X-C motif) receptor 4, CXCR4

-496

3

EPH receptor A3, EPHA3

-485

4

sonic hedgehog, SHH

-220

5

homeobox D10, HOXD10

-65

6

NK6 homeobox 1, NKX6-1

-63

7

LIM homeobox 1, LHX1

-60

8

dicer 1, ribonuclease type III, DICER1

-60

9

ladybird homeobox 1, LBX1

-54

 

3.11. Gene Expression - Glial Cells

Glial cells are non-neuronal cells that maintain homeostasis, form myelin, and provide support and protection for neurons in the central and peripheral nervous systems.

Here we searched Gene Ontology descriptions for “glial”. See Table 17 and 18.

Table 17. Distribution of Genes Effected by GHK and Associated with Glial Cells

Percent Change
in Gene Expression

Genes UP

Genes DOWN

50 - 99%

11

4

100 – 199%

7

3

200 - 299%

4

4

300 -399%

2

1

400 - 499%

0

1

500% +

0

2

Total

24

15


Table 18. GHK and Genes Associated with Glial Cells

UP

Gene Title

Percent Change

1

neurogenic differentiation 4, NEUROD4

362

2

growth associated protein 43, GAP43

305

3

nuclear factor I/B, NFIB

279

4

caspase 1, apoptosis-related cysteine peptidase (interleukin 1, beta, convertase), CASP1

257

5

Kruppel-like factor 15, KLF15

238

6

adenylate cyclase activating polypeptide 1 (pituitary), ADCYAP1

215

7

neuregulin 1, NRG1

164

8

versican, VCAN

134

9

protein kinase C, eta, PRKCH

124

10

SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4, SMARCA4

107

11

chemokine (C-X3-C motif) receptor 1, CX3CR1

104

12

achaete-scute complex homolog 1 (Drosophila), ASCL1

103

13

neurofibromin 1, NF1

102

14

toll-like receptor 4, TLR4

86

15

forkhead box D1, FOXD1

77

16

glial fibrillary acidic protein, GFAP

77

17

transforming growth factor, beta 2, TGFB2

77

18

paired-like homeobox 2b, PHOX2B

74

19

sulfatase 1, SULF1

73

20

GDNF family receptor alpha 1, GFRA1

71

21

ets variant 5, ETV5

71

22

protein kinase C, delta, PRKCD

61

23

protein kinase C, alpha, PRKCA

55

24

PTK2 protein tyrosine kinase 2, PTK2

51

DOWN

Gene Title

Percent Change

1

necdin homolog (mouse), NDN

-729

2

insulin-like growth factor 1 (somatomedin C), IGF1

-522

3

forkhead box D4 /// forkhead box D4-like 1, FOXD4 /// FOXD4L1

-498

4

PTK2B protein tyrosine kinase 2 beta, PTK2B

-348

5

pleiomorphic adenoma gene 1, PLAG1

-276

6

lin-28 homolog A (C. elegans), LIN28A

-259

7

sonic hedgehog, SHH

-220

8

forkhead box E1 (thyroid transcription factor 2), FOXE1

-204

9

allograft inflammatory factor 1, AIF1

-144

10

GDNF family receptor alpha 2, GFRA2

-141

11

chondroitin sulfate proteoglycan 4, CSPG4

-113

12

CD34 molecule, CD34

-85

13

forkhead box D2, FOXD2

-69

14

NK6 homeobox 1, NKX6-1

-63

15

SRY (sex determining region Y)-box 2, SOX2

-58


3.11. Astrocyte

Astrocytes are characteristic star-shaped glial cells in the brain and spinal cord. The astrocyte proportion varies by region and ranges from 20% to 40% of all glial cells. They perform many functions, including biochemical support of endothelial cells that form the blood–brain barrier, provision of nutrients to the nervous tissue, maintenance of extracellular ion balance, and a role in the repair and scarring process of the brain and spinal cord following traumatic injuries.

Here we searched Gene Ontology descriptions for “astrocyte”. See Table 19 and 20.

Table 19. Distribution of Gene Effected by GHK and Associated with Astrocytes

Percent Change
in Gene Expression

Genes UP

Genes DOWN

50 - 99%

8

3

100 – 199%

5

2

200 - 299%

2

1

300 -399%

0

0

400 - 499%

0

0

500% +

0

0

Total

15

6

Table 20. GHK and Genes Associated with Astrocytes

UP

Gene Title

Percent Change

1

chemokine (C-C motif) ligand 3 /// chemokine (C-C motif) ligand 3-like 1 /// chemokine (C-C motif) ligand 3-like 3, CCL3 /// CCL3L1 /// CCL3L3

228

2

inhibitor of DNA binding 4, dominant negative helix-loop-helix protein, ID4

203

3

NK2 homeobox 2, NKX2-2

181

4

metallothionein 3, MT3

142

5

bridging integrator 1, BIN1

130

6

matrix metallopeptidase 14 (membrane-inserted), MMP14

114

7

neurofibromin 1, NF1

102

8

v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian), MYCN

98

9

activating transcription factor 5, ATF5

84

10

glutamate receptor, metabotropic 3, GRM3

81

11

S100 calcium binding protein B, S100B

81

12

glial fibrillary acidic protein, GFAP

77

13

T-cell acute lymphocytic leukemia 1, TAL1

68

14

chemokine (C-C motif) receptor 2, CCR2

63

15

glial cells missing homolog 1 (Drosophila), GCM1

50

DOWN

Gene Title

Percent Change

1

neurotrophic tyrosine kinase, receptor, type 3, NTRK3

-230

2

contactin 2 (axonal), CNTN2

-180

3

bone morphogenetic protein 2, BMP2

-159

4

glutamate receptor, metabotropic 5, GRM5

-85

5

fibroblast growth factor receptor 3, FGFR3

-71

6

coagulation factor II (thrombin), F2

-62


3.13. Schwann Cells

Schwann cells are cells of the peripheral nervous system that wrap around a nerve fiber, jelly-roll fashion, forming the myelin sheath.

Here we searched Gene Ontology descriptions for “Schwann”. See Table 21 and 22.

Table 21. Distribution of Genes Effected by GHK and Associated with Schwann Cells

Percent Change
in Gene Expression

Genes UP

Genes DOWN

50 - 99%

5

1

100 – 199%

2

0

200 - 299%

0

0

300 -399%

1

1

400 - 499%

0

0

500% +

0

0

Total

8

2

Table 22. GHK and Genes Associated with Schwann Cells

UP

Gene Title

Percent Change

1

Mediator complex subunit 12, MED12

393

2

neurofibromin 2 (merlin), NF2

105

3

neurofibromin 1, NF1

102

4

POU class 3 homeobox 1, POU3F1

78

5

glial fibrillary acidic protein, GFAP

77

6

ring finger protein 10, RNF10

62

7

cyclin-dependent kinase 1, CDK1

58

8

glypican 1, GPC1

50

DOWN

Gene Title

Percent Change

1

cytochrome P450, family 11, subfamily A, polypeptide 1, CYP11A1

-393

2

dicer 1, ribonuclease type III, DICER1

-60

3.14. Myelin

Myelin is a mixture of proteins and phospholipids that form a whitish insulating sheath around many nerve fibers, increasing the speed at which impulses are conducted.

Here we searched Gene Ontology descriptions for “myelin”. See Table 23 and 24.

Table 23. Distribution of Genes Effected by GHK and Associated with Myelin

Percent Change in Gene Expression

Genes UP

Genes DOWN

50 - 99%

24

5

100 – 199%

8

8

200 - 299%

4

0

300 -399%

0

3

400 - 499%

0

2

500% +

0

0

Total

36

18

Table 24. GHK and Genes Associated with Myelin

UP

Gene Title

Percent Change

1

inositol 1,4,5-triphosphate receptor, type 3, ITPR3

298

2

sodium channel, voltage-gated, type II, alpha subunit, SCN2A

264

3

myelin associated glycoprotein, MAG

229

4

inhibitor of DNA binding 4, dominant negative helix-loop-helix protein, ID4

203

5

aspartoacylase, ASPA

195

6

probable transcription factor PML-like /// promyelocytic leukemia, LOC652346 /// PML

144

7

retinoic acid receptor, beta, RARB

142

8

retinoic acid receptor, alpha, RARA

123

9

myosin VA (heavy chain 12, myoxin), MYO5A

122

10

neurofibromin 1, NF1

102

11

histamine receptor H3, HRH3

101

12

transforming growth factor, beta 1, TGFB1

100

13

transcription factor 7-like 2 (T-cell specific, HMG-box), TCF7L2

92

14

claudin 5, CLDN5

84

15

serine palmitoyltransferase, long chain base subunit 2, SPTLC2

81

16

POU class 3 homeobox 1, POU3F1

78

17

membrane protein, palmitoylated 5 (MAGUK p55 subfamily member 5), MPP5

73

18

serine incorporator 5, SERINC5

73

19

v-akt murine thymoma viral oncogene homolog 1, AKT1

71

20

FIG4 homolog, SAC1 lipid phosphatase domain containing (S. cerevisiae), FIG4

70

21

ATPase, Na+/K+ transporting, alpha 3 polypeptide, ATP1A3

67

22

discs, large homolog 1 (Drosophila), DLG1

67

23

ring finger protein 10, RNF10

62

24

protein kinase C, delta, PRKCD

61

25

Rho guanine nucleotide exchange factor (GEF) 10, ARHGEF10

60

26

CD59 molecule, complement regulatory protein, CD59

60

27

neurofascin, NFASC

60

28

zinc finger protein 24, ZNF24

57

29

myotubularin related protein 2, MTMR2

54

30

attractin, ATRN

54

31

erythrocyte membrane protein band 4.1-like 3, EPB41L3

53

32

peptidylprolyl isomerase (cyclophilin)-like 2, PPIL2

52

33

zinc finger protein 259 /// zinc finger protein 259 pseudogene 1, ZNF259 /// ZNF259P1

52

34

interferon, gamma, IFNG

52

35

sphingomyelin phosphodiesterase, acid-like 3A, SMPDL3A

52

36

glypican 1, GPC1

50

DOWN

Gene Title

Percent Change

1

chemokine (C-X-C motif) receptor 4, CXCR4

-496

2

gap junction protein, gamma 2, 47kDa, GJC2

-428

3

lethal giant larvae homolog 1 (Drosophila), LLGL1

-393

4

myelin basic protein, MBP

-361

5

chromosome 11 open reading frame 9, C11orf9

-342

6

promyelocytic leukemia, PML

-196

7

myelin protein zero, MPZ

-180

8

contactin 2 (axonal), CNTN2

-180

9

toll-like receptor 2, TLR2

-169

10

laminin, alpha 2, LAMA2

-150

11

retinoid X receptor, gamma, RXRG

-110

12

integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12), ITGB1

-107

13

thyroglobulin, TG

-100

14

Hexosaminidase A (alpha polypeptide), HEXA

-74

15

fibroblast growth factor receptor 3, FGFR3

-71

16

dicer 1, ribonuclease type III, DICER1

-60

17

quaking homolog, KH domain RNA binding (mouse), QKI

-53

18

B-cell CLL/lymphoma 2, BCL2

-53

3.15. Gene Expression – Dendrites

Dendrites are short branched extensions of a nerve cell, along which impulses received from other cells at synapses are transmitted to the cell body.

Here we searched Gene Ontology descriptions for “dendrite”. See Table 25 and 26.

Table 25. Distribution of Genes Effected by GHK and Associated with Dendrites

Percent Change in Gene Expression

Genes UP

Genes DOWN

50 - 99%

47

14

100 – 199%

19

31

200 - 299%

11

15

300 -399%

8

3

400 - 499%

0

3

500% +

2

2

Total

87

68


Table 26. GHK and Genes Associated with Dendrites

UP

Gene Title

Percent Change

1

potassium voltage-gated channel, Shal-related subfamily, member 1, KCND1

845

2

contactin associated protein-like 2, CNTNAP2

581

3

leukocyte specific transcript 1, LST1

395

4

gamma-aminobutyric acid (GABA) A receptor, alpha 5 /// gamma-aminobutyric acid receptor subunit alpha-5-like, GABRA5 /// LOC100509612

392

5

chemokine (C-C motif) ligand 19, CCL19

378

6

doublecortin-like kinase 1, DCLK1

365

7

p21 protein (Cdc42/Rac)-activated kinase 1, PAK1

363

8

potassium voltage-gated channel, Shaw-related subfamily, member 3, KCNC3

332

9

EPH receptor B1, EPHB1

330

10

gamma-aminobutyric acid (GABA) B receptor, 1 /// ubiquitin D, GABBR1 /// UBD

310

11

calcium channel, voltage-dependent, P/Q type, alpha 1A subunit, CACNA1A

286

12

nephroblastoma overexpressed gene, NOV

275

13

obscurin-like 1, OBSL1

263

14

neuroligin 1, NLGN1

261

15

low density lipoprotein receptor-related protein 1, LRP1

249

16

glutamate receptor, ionotropic, kainate 3, GRIK3

246

17

RNA binding protein, fox-1 homolog (C. elegans) 2, RBFOX2

245

18

glutamate receptor, metabotropic 1, GRM1

231

19

glutamate receptor interacting protein 1, GRIP1

230

20

glutamate receptor, ionotropic, N-methyl D-aspartate 1, GRIN1

216

21

MCF.2 cell line derived transforming sequence, MCF2

202

22

purinergic receptor P2X, ligand-gated ion channel, 4, P2RX4

180

23

synapsin I, SYN1

170

24

Abl-interactor 2, ABI2

168

25

calcium channel, voltage-dependent, L type, alpha 1F subunit, CACNA1F

168

26

membrane associated guanylate kinase, WW and PDZ domain containing 2, MAGI2

155

27

ubiquitin-conjugating enzyme E2I (UBC9 homolog, yeast), UBE2I

150

28

nuclear mitotic apparatus protein 1, NUMA1

147

29

glutamate receptor, ionotropic, N-methyl D-aspartate 2C, GRIN2C

146

30

probable transcription factor PML-like /// promyelocytic leukemia, LOC652346 /// PML

144

31

fasciculation and elongation protein zeta 1 (zygin I), FEZ1

143

32

glutamate receptor, metabotropic 7, GRM7

140

33

acetylcholinesterase, ACHE

131

34

retinoic acid receptor, alpha, RARA

123

35

misshapen-like kinase 1, MINK1

119

36

kelch-like 1 (Drosophila), KLHL1

119

37

neuralized homolog (Drosophila), NEURL

115

38

protein kinase C, gamma, PRKCG

106

39

drebrin 1, DBN1

103

40

neurofibromin 1, NF1

102

41

complexin 2, CPLX2

97

42

presenilin 1, PSEN1

95

43

bone morphogenetic protein 7, BMP7

93

44

phosphatidylinositol 4-kinase type 2 alpha, PI4K2A

93

45

MYC associated factor X, MAX

89

46

urocortin, UCN

83

47

sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3A, SEMA3A

82

48

transmembrane protein 90B, TMEM90B

80

49

deleted in colorectal carcinoma, DCC

77

50

phosphatidylinositol binding clathrin assembly protein, PICALM

75

51

potassium voltage-gated channel, Shaw-related subfamily, member 1, KCNC1

75

52

KIAA0319, KIAA0319

73

53

neuropilin 1, NRP1

73

54

lysosomal-associated membrane protein 1, LAMP1

72

55

Kruppel-like factor 7 (ubiquitous), KLF7

72

56

mitogen-activated protein kinase 1, MAPK1

72

57

Bardet-Biedl syndrome 4, BBS4

71

58

cell cycle associated protein 1, CAPRIN1

70

59

TAO kinase 2, TAOK2

70

60

adenylate cyclase 9, ADCY9

69

61

bone morphogenetic protein receptor, type IA, BMPR1A

69

62

EPH receptor A5, EPHA5

68

63

interferon gamma receptor 1, IFNGR1

68

64

EPH receptor B2, EPHB2

67

65

tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide, YWHAH

66

66

bone morphogenetic protein 5, BMP5

64

67

T-cell leukemia homeobox 2, TLX2

63

68

chemokine (C-C motif) receptor 2, CCR2

63

69

activin A receptor type II-like 1, ACVRL1

63

70

gamma-aminobutyric acid (GABA) A receptor, beta 1, GABRB1

62

71

RAB27A, member RAS oncogene family, RAB27A

58

72

fragile X mental retardation 1, FMR1

57

73

neural precursor cell expressed, developmentally down-regulated 4, NEDD4

57

74

GNAS complex locus, GNAS

57

75

chemokine (C-C motif) receptor 7, CCR7

57

76

solute carrier family 11 (proton-coupled divalent metal ion transporters), member 2, SLC11A2

55

77

neurotrophic tyrosine kinase, receptor, type 2, NTRK2

55

78

protein kinase C, alpha, PRKCA

55

79

myotubularin related protein 2, MTMR2

54

80

cullin 7, CUL7

53

81

striatin, calmodulin binding protein 3, STRN3

51

82

staufen, RNA binding protein, homolog 1 (Drosophila), STAU1

51

83

Down syndrome cell adhesion molecule, DSCAM

51

84

histone deacetylase 6, HDAC6

51

85

ras homolog gene family, member A, RHOA

50

86

frizzled homolog 3 (Drosophila), FZD3

50

87

tuberous sclerosis 2, TSC2

50

DOWN

Gene Title

Percent Change

1

bassoon (presynaptic cytomatrix protein), BSN

-563

2

membrane metallo-endopeptidase, MME

-540

3

adenylate cyclase 10 (soluble), ADCY10

-460

4

discs, large homolog 4 (Drosophila), DLG4

-452

5

Kv channel interacting protein 1, KCNIP1

-413

6

EPH receptor A7, EPHA7

-365

7

PTK2B protein tyrosine kinase 2 beta, PTK2B

-348

8

sterile alpha motif domain containing 4A, SAMD4A

-315

9

dopamine receptor D4, DRD4

-296

10

FEZ family zinc finger 2, FEZF2

-295

11

calcium channel, voltage-dependent, N type, alpha 1B subunit, CACNA1B

-290

12

mitogen-activated protein kinase 8 interacting protein 1, MAPK8IP1

-289

13

regulator of G-protein signaling 11, RGS11

-266

14

cyclin-dependent kinase 5, regulatory subunit 1 (p35), CDK5R1

-260

15

glutamate receptor, ionotropic, kainate 1, GRIK1

-254

16

thyroid hormone receptor, alpha (erythroblastic leukemia viral (v-erb-a) oncogene homolog, avian), THRA

-253

17

cyclic nucleotide gated channel alpha 3, CNGA3

-249

18

adenylate cyclase 2 (brain), ADCY2

-247

19

proprotein convertase subtilisin/kexin type 2, PCSK2

-242

20

Rho guanine nucleotide exchange factor (GEF) 15, ARHGEF15

-230

21

potassium voltage-gated channel, Shal-related subfamily, member 3, KCND3

-224

22

protein tyrosine phosphatase, receptor type, D, PTPRD

-221

23

cytochrome b-245, beta polypeptide, CYBB

-217

24

GABA(A) receptors associated protein like 3, pseudogene, GABARAPL3

-197

25

neutrophil cytosolic factor 1C pseudogene, NCF1C

-196

26

promyelocytic leukemia, PML

-196

27

C-reactive protein, pentraxin-related, CRP

-182

28

glutamate receptor, ionotropic, N-methyl D-aspartate 2A, GRIN2A

-180

29

tubby like protein 1, TULP1

-176

30

Mitogen-activated protein kinase 8 interacting protein 3, MAPK8IP3

-174

31

cell adhesion molecule with homology to L1CAM (close homolog of L1), CHL1

-171

32

choline O-acetyltransferase, CHAT

-160

33

glutamate receptor, ionotropic, kainate 5, GRIK5

-159

34

glutamate receptor, ionotropic, kainate 4, GRIK4

-155

35

5-hydroxytryptamine (serotonin) receptor 6, HTR6

-150

36

tachykinin receptor 3, TACR3

-150

37

5-hydroxytryptamine (serotonin) receptor 5A, HTR5A

-149

38

protease, serine, 12 (neurotrypsin, motopsin), PRSS12

-141

39

cholinergic receptor, nicotinic, alpha 4, CHRNA4

-139

40

5-hydroxytryptamine (serotonin) receptor 2A, HTR2A

-135

41

leucine zipper, putative tumor suppressor 1, LZTS1

-130

42

neuroligin 4, X-linked, NLGN4X

-128

43

glutamate receptor, ionotrophic, AMPA 3, GRIA3

-126

44

glutamate receptor, metabotropic 6, GRM6

-120

45

paralemmin, PALM

-115

46

copine VI (neuronal), CPNE6

-114

47

cytoplasmic polyadenylation element binding protein 3, CPEB3

-112

48

corticotropin releasing hormone receptor 1, CRHR1

-109

49

doublecortin, DCX

-108

50

regulator of G-protein signaling 14, RGS14

-108

51

apolipoprotein E, APOE

-107

52

calcium binding protein 1, CABP1

-106

53

mitogen-activated protein kinase 8 interacting protein 2, MAPK8IP2

-103

54

neurochondrin, NCDN

-102

55

plexin domain containing 1, PLXDC1

-86

56

kinase D-interacting substrate, 220kDa, KIDINS220

-83

57

chemokine (C-C motif) ligand 21, CCL21

-77

58

sortilin 1, SORT1

-71

59

cholinergic receptor, nicotinic, alpha 3, CHRNA3

-69

60

glutamate receptor, ionotropic, N-methyl D-aspartate 2B, GRIN2B

-65

61

neurotrophic tyrosine kinase, receptor, type 1, NTRK1

-65

62

cholinergic receptor, nicotinic, beta 2 (neuronal), CHRNB2

-62

63

dicer 1, ribonuclease type III, DICER1

-60

64

cholecystokinin, CCK

-59

65

glutamate receptor, ionotropic, AMPA 2, GRIA2

-58

66

TRAF2 and NCK interacting kinase, TNIK

-57

67

G protein-coupled receptor kinase 4, GRK4

-57

68

G protein-coupled estrogen receptor 1, GPER

-52

3.16. Gene Expression - Oligodendrocytes

Oligodendrocytes are glial cells similar to astrocytes, but with fewer protuberances, which are concerned with the production of myelin in the central nervous system.

Here we searched Gene Ontology descriptions for “oligodendrocyte”. See Table 27 and 28.

Table 27. Distribution of Genes Effected by GHK and Associated with Oligodendrocytes

Percent Change
in Gene Expression

Genes UP

Genes DOWN

50 - 99%

6

4

100 – 199%

6

3

200 - 299%

3

1

300 -399%

0

1

400 - 499%

0

1

500% +

1

0

Total

16

10


Table 28. GHK and Genes Associated with Oligodendrocytes

UP

Gene Title

Percent Change

1

tumor protein p73, TP73

938

2

adenylate cyclase activating polypeptide 1 (pituitary), ADCYAP1

215

3

gelsolin, GSN

214

4

inhibitor of DNA binding 4, dominant negative helix-loop-helix protein, ID4

203

5

aspartoacylase, ASPA

195

6

NK2 homeobox 2, NKX2-2

181

7

dopamine receptor D3, DRD3

164

8

histone deacetylase 11, HDAC11

105

9

achaete-scute complex homolog 1 (Drosophila), ASCL1

103

10

neurofibromin 1, NF1

102

11

NK2 homeobox 1, NKX2-1

98

12

transcription factor 7-like 2 (T-cell specific, HMG-box), TCF7L2

92

13

bone morphogenetic protein 4, BMP4

67

14

met proto-oncogene (hepatocyte growth factor receptor), MET

57

15

neurotrophic tyrosine kinase, receptor, type 2, NTRK2

55

16

SRY (sex determining region Y)-box 11, SOX11

42

DOWN

Gene Title

Percent Change

1

chemokine (C-X-C motif) receptor 4, CXCR4

-496

2

chromosome 11 open reading frame 9, C11orf9

-342

3

sonic hedgehog, SHH

-220

4

zinc finger protein 287, ZNF287

-143

5

early growth response 1, EGR1

-121

6

apolipoprotein E, APOE

-107

7

distal-less homeobox 2, DLX2

-81

8

fibroblast growth factor receptor 3, FGFR3

-71

9

NK6 homeobox 1, NKX6-1

-63

10

dicer 1, ribonuclease type III, DICER1

-60

3.17. Gene Expression - Sensory Nerve cells

Sensory neurons are nerves that transmit sensory information (sight, sound, feeling, etc.). They are activated by sensory input and send projections to other elements of the nervous system, ultimately conveying sensory information to the brain or spinal cord.

Here we searched Gene Ontology descriptions for “sensory”. See Table 29 and 30.

Table 29. Distribution of Genes Effected by GHK and Associated with Sensory Nerve Cells

Percent Change
in Gene Expression

Genes UP

Genes DOWN

50 - 99%

45

25

100 – 199%

24

36

200 - 299%

18

6

300 -399%

7

1

400 - 499%

1

3

500% +

2

4

Total

97

75


Table 30. GHK and Gene Associate with Sensory Nerve Cells

UP

Gene Title

Percent Change

1

opioid receptor, mu 1, OPRM1

1294

2

T-box 1, TBX1

553

3

adrenergic, beta-1-, receptor, ADRB1

477

4

gamma-aminobutyric acid (GABA) A receptor, alpha 5 /// gamma-aminobutyric acid receptor subunit alpha-5-like, GABRA5 /// LOC100509612

392

5

calcium channel, voltage-dependent, L type, alpha 1D subunit, CACNA1D

372

6

olfactory receptor, family 2, subfamily W, member 1, OR2W1

370

7

guanine nucleotide binding protein (G protein), alpha activating activity polypeptide, olfactory type, GNAL

366

8

olfactory receptor, family 2, subfamily B, member 6, OR2B6

345

9

cyclic nucleotide gated channel beta 1, CNGB1

330

10

EPH receptor B1, EPHB1

330

11

inositol 1,4,5-triphosphate receptor, type 3, ITPR3

298

12

olfactory receptor, family 7, subfamily A, member 17, OR7A17

285

13

nuclear factor I/B, NFIB

279

14

islet amyloid polypeptide, IAPP

276

15

opiate receptor-like 1, OPRL1

246

16

potassium voltage-gated channel, KQT-like subfamily, member 4, KCNQ4

245

17

myosin, heavy chain 14, non-muscle, MYH14

243

18

taste receptor, type 2, member 13, TAS2R13

237

19

olfactory receptor, family 2, subfamily F, member 2, OR2F2

232

20

glutamate receptor, metabotropic 1, GRM1

231

21

chemokine (C-C motif) ligand 3 /// chemokine (C-C motif) ligand 3-like 1 /// chemokine (C-C motif) ligand 3-like 3, CCL3 /// CCL3L1 /// CCL3L3

228

22

polycystic kidney disease 2-like 1, PKD2L1

225

23

glutamate receptor, ionotropic, N-methyl D-aspartate 1, GRIN1

216

24

adenylate cyclase activating polypeptide 1 (pituitary), ADCYAP1

215

25

ATPase, Ca++ transporting, plasma membrane 2, ATP2B2

214

26

olfactory receptor, family 7, subfamily C, member 1, OR7C1

207

27

purinergic receptor P2X, ligand-gated ion channel, 3, P2RX3

207

28

neuropeptide Y receptor Y1, NPY1R

201

29

family with sequence similarity 38, member B, FAM38B

193

30

olfactory receptor, family 1, subfamily A, member 1, OR1A1

189

31

taste receptor, type 2, member 14, TAS2R14

181

32

purinergic receptor P2X, ligand-gated ion channel, 4, P2RX4

180

33

receptor accessory protein 2, REEP2

174

34

endothelin receptor type A, EDNRA

173

35

cannabinoid receptor 1 (brain), CNR1

172

36

melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor), MC1R

164

37

olfactory receptor, family 12, subfamily D, member 3 /// olfactory receptor, family 5, subfamily V, member 1, OR12D3 /// OR5V1

163

38

odorant binding protein 2A /// odorant binding protein 2B, OBP2A /// OBP2B

162

39

prepronociceptin, PNOC

150

40

phospholipase C, beta 2, PLCB2

148

41

glutamate receptor, metabotropic 7, GRM7

140

42

oxytocin, prepropeptide, OXT

136

43

WD repeat domain 1, WDR1

127

44

olfactory receptor, family 1, subfamily D, member 4 (gene/pseudogene) /// olfactory receptor, family 1, subfamily D, member 5, OR1D4 /// OR1D5

125

45

UDP glucuronosyltransferase 2 family, polypeptide A1 /// UDP glucuronosyltransferase 2 family, polypeptide A2, UGT2A1 /// UGT2A2

121

46

prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase), PTGS2

120

47

taste receptor, type 2, member 4, TAS2R4

118

48

lysozyme, LYZ

111

49

protein kinase C, gamma, PRKCG

106

50

collagen, type XI, alpha 1, COL11A1

103

51

POU class 4 homeobox 3, POU4F3

102

52

nuclear receptor subfamily 2, group F, member 6, NR2F6

100

53

POU class 4 homeobox 1, POU4F1

99

54

cystatin SA, CST2

97

55

solute carrier family 6 (neurotransmitter transporter, dopamine), member 3, SLC6A3

92

56

echinoderm microtubule associated protein like 2, EML2

91

57

aquaporin 4, AQP4

91

58

potassium voltage-gated channel, KQT-like subfamily, member 1, KCNQ1

88

59

purinergic receptor P2X, ligand-gated ion channel, 7, P2RX7

87

60

arrestin, beta 2, ARRB2

85

61

sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3A, SEMA3A

82

62

solute carrier family 26, member 4, SLC26A4

80

63

olfactory receptor, family 2, subfamily C, member 1, OR2C1

80

64

myosin IA, MYO1A

79

65

chloride intracellular channel 5, CLIC5

78

66

collagen, type II, alpha 1, COL2A1

77

67

sedoheptulokinase /// transient receptor potential cation channel, subfamily V, member 1, SHPK /// TRPV1

77

68

Usher syndrome 1C (autosomal recessive, severe), USH1C

75

69

sodium channel, voltage-gated, type III, beta, SCN3B

75

70

otoraplin, OTOR

75

71

transcription factor AP-2 alpha (activating enhancer binding protein 2 alpha), TFAP2A

74

72

aldehyde dehydrogenase 7 family, member A1, ALDH7A1

73

73

ets variant 1, ETV1

72

74

mitogen-activated protein kinase 1, MAPK1

72

75

collagen, type I, alpha 1, COL1A1

71

76

Bardet-Biedl syndrome 4, BBS4

71

77

acid phosphatase, prostate, ACPP

68

78

snail homolog 2 (Drosophila), SNAI2

67

79

zinc finger protein 354A, ZNF354A

67

80

integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor), ITGA2

66

81

caspase 3, apoptosis-related cysteine peptidase, CASP3

65

82

taste receptor, type 2, member 1, TAS2R1

65

83

diaphanous homolog 1 (Drosophila), DIAPH1

64

84

nipsnap homolog 1 (C. elegans), NIPSNAP1

64

85

olfactory receptor, family 2, subfamily F, member 1, OR2F1

63

86

spectrin, beta, non-erythrocytic 4, SPTBN4

63

87

olfactory receptor, family 12, subfamily D, member 2, OR12D2

61

88

proline rich, lacrimal 1, PROL1

61

89

vomeronasal 1 receptor 1, VN1R1

60

90

v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog, KIT

57

91

GNAS complex locus, GNAS

57

92

olfactory receptor, family 52, subfamily A, member 1, OR52A1

54

93

F-box protein 11, FBXO11

54

94

transducin (beta)-like 1X-linked, TBL1X

54

95

cyclin-dependent kinase inhibitor 1B (p27, Kip1), CDKN1B

53

96

latexin, LXN

50

97

adenylate cyclase 3, ADCY3

50

DOWN

Gene Title

Percent Change

1

taste receptor, type 2, member 9, TAS2R9

-1494

2

endothelin receptor type B, EDNRB

-768

3

necdin homolog (mouse), NDN

-729

4

membrane metallo-endopeptidase, MME

-540

5

EPH receptor A3, EPHA3

-485

6

arachidonate lipoxygenase 3, ALOXE3

-461

7

bradykinin receptor B1, BDKRB1

-426

8

gap junction protein, beta 4, 30.3kDa, GJB4

-317

9

nerve growth factor (beta polypeptide), NGF

-243

10

guanine nucleotide binding protein (G protein), alpha transducing activity polypeptide 1, GNAT1

-242

11

olfactory receptor, family 3, subfamily A, member 1, OR3A1

-234

12

apelin receptor, APLNR

-230

13

olfactory receptor, family 2, subfamily F, member 1 /// olfactory receptor, family 2, subfamily F, member 2, OR2F1 /// OR2F2

-212

14

olfactory receptor, family 12, subfamily D, member 3, OR12D3

-201

15

olfactory receptor, family 6, subfamily A, member 2, OR6A2

-199

16

cholecystokinin B receptor, CCKBR

-198

17

carbonic anhydrase VI, CA6

-192

18

olfactory receptor, family 5, subfamily I, member 1, OR5I1

-191

19

collagen, type XI, alpha 2, COL11A2

-186

20

olfactory receptor, family 10, subfamily H, member 3, OR10H3

-182

21

glutamate receptor, ionotropic, N-methyl D-aspartate 2A, GRIN2A

-180

22

protein phosphatase, EF-hand calcium binding domain 2, PPEF2

-178

23

sodium channel, nonvoltage-gated 1 alpha, SCNN1A

-175

24

trace amine associated receptor 5, TAAR5

-168

25

gastric inhibitory polypeptide, GIP

-164

26

olfactory receptor, family 2, subfamily H, member 1, OR2H1

-156

27

olfactory receptor, family 2, subfamily J, member 2, OR2J2

-155

28

otoferlin, OTOF

-155

29

discs, large homolog 2 (Drosophila), DLG2

-142

30

cholinergic receptor, nicotinic, alpha 4, CHRNA4

-139

31

5-hydroxytryptamine (serotonin) receptor 2A, HTR2A

-135

32

tectorin alpha, TECTA

-126

33

sodium channel, voltage-gated, type XI, alpha subunit, SCN11A

-124

34

olfactory receptor, family 7, subfamily C, member 2, OR7C2

-120

35

taste receptor, type 2, member 16, TAS2R16

-120

36

glutamate receptor, metabotropic 6, GRM6

-120

37

opioid receptor, kappa 1, OPRK1

-119

38

ATPase, H+ transporting, lysosomal 56/58kDa, V1 subunit B1, ATP6V1B1

-118

39

olfactory marker protein, OMP

-118

40

contactin 5, CNTN5

-116

41

cysteinyl leukotriene receptor 2, CYSLTR2

-113

42

olfactory receptor, family 2, subfamily H, member 2, OR2H2

-110

43

rhodopsin, RHO

-108

44

interleukin 10, IL10

-107

45

olfactory receptor, family 11, subfamily A, member 1, OR11A1

-107

46

polymeric immunoglobulin receptor, PIGR

-107

47

guanine nucleotide binding protein (G protein), gamma 13, GNG13

-106

48

tubby homolog (mouse), TUB

-101

49

glutamate receptor, metabotropic 8, GRM8

-101

50

cystatin S, CST4

-101

51

olfactory receptor, family 1, subfamily F, member 2, OR1F2P

-99

52

espin, ESPN

-98

53

POU class 4 homeobox 2, POU4F2

-91

54

Norrie disease (pseudoglioma), NDP

-87

55

calcitonin-related polypeptide alpha, CALCA

-82

56

bestrophin 2, BEST2

-81

57

taste receptor, type 2, member 10, TAS2R10

-78

58

sodium channel, nonvoltage-gated 1, gamma, SCNN1G

-76

59

Hexosaminidase A (alpha polypeptide), HEXA

-74

60

cystatin SN, CST1

-70

61

glutamate receptor, ionotropic, N-methyl D-aspartate 2B, GRIN2B

-65

62

neurotrophic tyrosine kinase, receptor, type 1, NTRK1

-65

63

POU class 3 homeobox 4, POU3F4

-63

64

transient receptor potential cation channel, subfamily A, member 1, TRPA1

-62

65

trace amine associated receptor 3 (gene/pseudogene), TAAR3

-62

66

cholinergic receptor, nicotinic, beta 2 (neuronal), CHRNB2

-62

67

calcium channel, voltage-dependent, R type, alpha 1E subunit, CACNA1E

-62

68

growth factor independent 1 transcription repressor, GFI1

-62

69

lactoperoxidase, LPO

-59

70

amiloride-sensitive cation channel 2, neuronal, ACCN2

-59

71

cholecystokinin, CCK

-59

72

SRY (sex determining region Y)-box 2, SOX2

-58

73

transient receptor potential cation channel, subfamily V, member 4, TRPV4

-56

74

microtubule-associated protein 1A, MAP1A

-56

75

olfactory receptor, family 7, subfamily E, member 24, OR7E24

-51

3.18. Spinal Nerve Cells

Spinal nerve cells transfer information, which travels down the spinal cord, as a conduit for sensory information in the reverse direction, and finally as a center for coordinating certain reflexes.

Here we searched Gene Ontology descriptions for “spinal”. See Table 31 and 32.

Table 31. Distribution of Genes Effected by GHK and Associated with Spinal Nerve Cells

Percent Change in Gene Expression

Genes UP

Genes DOWN

50 - 99%

8

6

100 – 199%

9

3

200 - 299%

1

2

300 -399%

0

1

400 - 499%

1

0

500% +

1

1

Total

20

13

Table 32. GHK and Genes Associated with Spinal Nerve Cells

UP

Gene Title

Percent Change

1

tumor protein p73, TP73

938

2

smoothened homolog (Drosophila), SMO

415

3

calcium channel, voltage-dependent, P/Q type, alpha 1A subunit, CACNA1A

286

4

GATA binding protein 2, GATA2

193

5

GLI family zinc finger 2, GLI2

183

6

NK2 homeobox 2, NKX2-2

181

7

dopamine receptor D3, DRD3

164

8

paired box 7, PAX7

161

9

slit homolog 3 (Drosophila), SLIT3

154

10

polycystic kidney disease 1 (autosomal dominant), PKD1

137

11

achaete-scute complex homolog 1 (Drosophila), ASCL1

103

12

neurofibromin 1, NF1

102

13

deleted in colorectal carcinoma, DCC

77

14

slit homolog 2 (Drosophila), SLIT2

69

15

T-cell acute lymphocytic leukemia 1, TAL1

68

16

GRB10 interacting GYF protein 2, GIGYF2

62

17

aggrecan, ACAN

61

18

forkhead box B1, FOXB1

57

19

Zic family member 1 (odd-paired homolog, Drosophila), ZIC1

57

20

pre-B-cell leukemia homeobox 3, PBX3

53

DOWN

Gene Title

Percent Change

1

slit homolog 1 (Drosophila), SLIT1

-553

2

SRY (sex determining region Y)-box 1, SOX1

-337

3

growth differentiation factor 11, GDF11

-221

4

sonic hedgehog, SHH

-220

5

glutamate receptor, ionotropic, N-methyl D-aspartate 2A, GRIN2A

-180

6

even-skipped homeobox 1, EVX1

-110

7

aquaporin 1 (Colton blood group), AQP1

-101

8

plexin domain containing 1, PLXDC1

-86

9

homeobox D10, HOXD10

-65

10

NK6 homeobox 1, NKX6-1

-63

11

LIM homeobox 1, LHX1

-60

12

dicer 1, ribonuclease type III, DICER1

-60

13

ladybird homeobox 1, LBX1

-54

4. Possible Methods of Therapeutic Use of GHK for Nerve Diseases

4.1. Mode of Administering GHK-Cu to Patients

As a Skin Cream or Patch

GHK-Cu has an unexpectedly rapid passage through skin's stratum corneum. When tested by Howard Maibach's group (Univerisity of California at San Francisco), 0.68% GHK-Cu was applied to dermatomed skin. Over 48 hours, 136 micrograms of GHK-Cu passed through the skin per centimeter squared. This is a significant amount of GHK-Cu, and a transdermal patch of a several centimeters squared may pass therapeutically effective amounts throughout the human body [105]⁠.

Russian studies reported that 0.5 micrograms/kg reduced anxiety in rats. Scaled up for a human weight of 70 kg, this would be 35 micrograms in a human [50]⁠. Our studies on activation of systemic healing in mice, rats, and pigs suggest that about 50 milligrams of GHK-Cu would be effective throughout the human body, although dose-ranging to determine the minimum active dosage was never performed.


As a Liposomal Encapsulated Oral Tablet

Alternately, the use of encapsulated liposomal GHK-Cu would allow its oral administration at relatively high dosages. Some sellers of an encapsulated liposomal tripeptide glutathione claim that 60% of the orally administrated peptide enters the human blood stream [106]⁠. Direct administration in a regular pill form is unlikely to work because of GHK's extreme sensitivity to breakdown by intestinal carboxypeptidase [107]⁠.

GHK-Cu costs about $8/gram in kilogram amounts. For a 50 mg dosage, the GHK-Cu would cost about $0.40. It is possible that GHK alone would be effective in humans and be able to obtain sufficient amounts of copper 2+ from albumin. If so, this would simplify its therapeutic use. The minimum effective dosage of GHK-Cu for various uses is unknown since such studies were never performed.

GHK-Cu does lower blood pressure, but the LD50 (Lethal Dose for 50% of mice) for such effects would be about a single dosage of 23,000 mgs of GHK-Cu in a 70 Kg human. In GHK-Cu's long history of use in cosmetics, no health issues have ever arisen. We were never able to find an LD 50 for GHK without copper.

In our studies, equimolar mixtures of GHK-Cu and GHK (no copper) are often used to avoid any release of loosely bound copper. Also, copper chelators such as penicillamine have been reported to cause psychosis in humans [108]⁠. We have been informed of a study in which a young, male medical student was infused intravenously with one gram of the histidine, a copper-binding amino acid. This caused severe psychosis and retention of the student in a locked ward for a week, after which, he fully recovered. Thus, any intravenous infusion of GHK or GHK-copper 2+ should be approached with caution [109]⁠.

5. Conclusions

Given all the failed attempts to develop effective treatment methods for nerve degeneration, it is suggested that researchers must take a very broad view of the possible factors causing neurodegenerative diseases and not focus on limited possible causes. It is sensible to concentrate research efforts on the reversion of affected tissues to a healthier condition more characteristic of younger humans. GHK gene studies have increasingly led to the conclusion that the conditions and diseases of aging cannot be scientifically treated without understanding the extensive changes in overall gene activity during aging.

There are three sources of evidence on GHK actions:

A. The best data is in vivo mammalian data, including human clinical studies. As reviewed in this paper, these studies give overwhelming evidence of GHK’s effects on cells and tissue growth, as well as anti-cancer, anti-oxidant, wound-healing, anti-inflammation, anti-pain, anti-anxiety and skin regeneration actions.

B. A second form of data is in vitro cell culture and organ culture results. Culture results give evidence about the effect of GHK on cellular production of collagen and other structural proteins, the effect on stem cell function, the recovery of cellular function after anticancer radiation or ultraviolet radiation, and sensitivity of cells to oxidative molecules.

C. A third source of data is in Human Gene expression. Data analysis found that GHK induces a 50% or greater (plus or minus) change of expression in 31.2% of human genes, affecting genes linked to multiple biochemical pathways in many organs and tissue, including the nervous system.

Many studies highlight gene expression effects of various molecules. Given today’s advances in computer modeling, it is not that difficult to find substances which affect gene expression in one way or another. However, in most cases, computer-based predictions do not have the same supporting evidence of in vivo and in vitro laboratory data as GHK has. Also, in many cases, the safety and cost of the proposed treatments are a big concern. GHK is safe, inexpensive, and can be used in humans today.

The best administration method, in our opinion, would be GHK-Cu incorporated into liposomes, then administered as an enteric capsule for oral use. A dosage of 10 mgs per dose would be a good starting point, at least for safety studies, but inducing positive actions will most likely require a higher dosage.

Acknowledgements

We would like to thank Germaine Emilie Pugh and Cassia McClain for their invaluable work in the manuscript preparation.

Author Contributions

The authors have equally contributed to the writing and revision of this article.

Conflicts of Interest

The authors declare no conflict of interest.


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Nerve Regeneration

When wound healing is inadequate, the healed area is often devoid of sensory abilities.

 

GHK tripeptide is suggested as a possible therapeutic agent against age-associated neurodegeneration and cognitive decline.

Oxid Med Cell Longev. 2012;2012:324832. Epub 2012 May 10.

The Human Tripeptide GHK-Cu in Prevention of Oxidative Stress and Degenerative Conditions of Aging: Implications for Cognitive Health.

Pickart L, Vasquez-Soltero JM, Margolina A.

Skin Biology, Research & Development Department, 4122 Factoria Boulevard SE-Suite No. 200, Bellevue, WA 98006, USA.

Abstract

Oxidative stress, disrupted copper homeostasis, and neuroinflammation due to overproduction of proinflammatory cytokines are considered leading causative factors in development of age-associated neurodegenerative conditions. Recently, a new mechanism of aging-detrimental epigenetic modifications-has emerged. Thus, compounds that possess antioxidant, anti-inflammatory activity as well as compounds capable of restoring copper balance and proper gene functioning may be able to prevent age-associated cognitive decline and ward off many common neurodegenerative conditions. The aim of this paper is to bring attention to a compound with a long history of safe use in wound healing and antiaging skin care. The human tripeptide GHK was discovered in 1973 as an activity in human albumin that caused old human liver tissue to synthesize proteins like younger tissue. It has high affinity for copper ions and easily forms a copper complex or GHK-Cu. In addition, GHK possesses a plethora of other regenerative and protective actions including antioxidant, anti-inflammatory, and wound healing properties. Recent studies revealed its ability to up- and downregulate a large number of human genes including those that are critical for neuronal development and maintenance. We propose GHK tripeptide as a possible therapeutic agent against age-associated neurodegeneration and cognitive decline.

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Studied the effect of GHK on nerve growth. Gly-His-Lys supported chick neuron differentiation and viability in cell culture and increased nerve outgrowth. The optimum concentrations of GHK for neuron function were 100-400 ng/ml. 

Neurobiology. 1975 Aug;5(4):207-13.

Effects of Synthetic Tripeptide on the Differentiation of Dissociated Cerebral Hemisphere Nerve Cells in Culturen

Sensenbrenner M, Jaros GG, Moonen G, Mandel P.

University of Strausbourg, France

Abstract
Dissociated cells from 7-day-old chick embryo cerebral hemispheres were cultivated on collagen in Falcon Petri dishes in the presence of various concentrations of fetal calf serum and of a chemically synthesized tripeptide Gly-His-Lys. Four different culture conditions were employed in the composition of the nutrient medium in which the cells were cultivated: a low serum concentration of 1, 2 or 5% (group A), a low serum concentration with 200 ng/ml tripeptide (group B), a serum concentration of 10 to 20% (group C) and a serum concentration of 10 or 20% with 200 ng/ml tripeptide (group D). Within the first 24 h of cultivation the cells settled on the collagen substrate and outgrowth of neuronal processes started in all four culture conditions. After 48 h in culture, differences between the groups became evident. In group A most isolated nerve cells had disappeared and glial cells proliferated from the remaining clumps. In group B the neurons had differentiated in absence of glial cells, the proliferation of which was greatly suppressed. In group C and D a differentiation of neurons occurred in a similar way to group B, but in addition the glial cells had proliferated. After 7-8 days in culture the cells in group A and B suddenly degenerated. In group C and D the nerve cells maintained for up to 3 weeks. The optimum concentrations of tripeptide in which the neuroblasts grew fibers and maintained in culture during 7-8 days were in the range of 100-400 ng/ml. The role of the tripeptide in the differentiation and maintenance of nerve cells is discussed.

Studied the effect of GHK on nerve growth. Gly-His-Lys supported chick neuron differentiation and viability in cell culture of various neurons -  chick embryo PNS (ganglion trigeminale) and from CNS of embryonal rats (hippocampus) and dissociated cells from chick embryo cerebral tissue.  The optimum concentrations of GHK for neuron outgrowth was 10 ng/ml. GHK increased the ratio of neurons to glial cell in culture. 

Z Mikrosk Anat Forsch. 1979;93(5):820-8.


Uber die Wirkung eines synthetischen Tripeptids auf  in vitro kultiviertes Nervengewebe [The effect of a synthetic tripeptide nervous tissue cultured in vitro].
[Article in German]

Lindner G, Grosse G, Halle W, Henklein P.

Abstract

Explants from chick embryo PNS (ganglion trigeminale) and from CNS of embryonal rats (hippocampus) and dissociated cells from chick embryo cerebral hemispheres were cultivated in maximow chambers in the presence of various concentrations of placental serum and of a chemically synthesized tripeptide Gly-His-Lys. 1. The presence of tripeptide in the nutrient medium with a low concentration of serum did not compensate the outgrowth of nerve fibers, that take place in the growth medium. 2. In the presence of tripeptide in the nutrient medium with low concentration of serum the index of growth area increased significantly. 3. Within the first days in cell cultures 0,01 microgram tripeptide pro ml medium stimulated the outgrowth of neuronal processes. 4. The experiments indicated, that the tripeptide did not replace the serum. The possible role of tripeptide as a system in controlling neuron-glial ratio in vitro is discussed.

Severed nerves are placed in a collagen tube impregnated with GHK. This caused an increase in the productino of Nerve Growth Factor and the neurotrophins NT-3 and NT-4..

J Peripher Nerv Syst. 2005 Mar;10(1):17-30.

Initial upregulation of growth factors and inflammatory mediators during nerve regeneration in the presence of cell adhesive peptide-incorporated collagen tubes.

Ahmed MR, Basha SH, Gopinath D, Muthusamy R, Jayakumar R.

Bio-organic and Neurochemistry Laboratory, Central Leather Research Institute, Adyar, Chennai, India.

Abstract

Neurotrophic factors play an important modulatory role in axonal sprouting during nerve regeneration involving the proliferation of hematogenous and Schwann cells in damaged tissue. We have exposed lesioned sciatic nerves to a collagen prosthesis with covalently bonded small cell adhesive peptides Arg-Gly-Asp-Ser (RGDS), Lys-Arg-Asp-Ser (KRDS), and Gly-His-Lys (GHK) to study local production of growth factors and cytokines in the regenerating tissues. Western/enzyme-linked immunosorbent assay (ELISA) studies were performed after 10 days of regeneration, when the tubular prosthesis is filled with fibrous matrix infiltrated by hematogenous cells and proliferating Schwann cells with growth factors produced locally. Regeneration was also analyzed by morphometrical methods after 30 days. The quantification of growth factors and proteins by ELISA revealed that there was an enhanced expression of the neurotrophic factors nerve growth factor (NGF) and neurotrophins (NT-3 and NT-4) in the regenerating tissues. This was further established by Western blot to qualitatively analyze the presence of the antigens in the regenerating tissues. Schwann cells were localized in the regenerating tissues using antibodies against S-100 protein. Other growth factors including growth-associated protein 43 (GAP-43), apolipoprotein E (Apo E), and pro-inflammatory cytokine like interleukin-1alpha (IL-1alpha) expression in the peptide groups were evaluated by ELISA and confirmed by Western blotting. Cell adhesive integrins in the proliferating cells were localized using integrin-alpha V. The combined results suggest that the early phase of regeneration of peripheral nerves in the presence of peptide-incorporated collagen tubes results in the enhanced production of trophic factors by the recruited hematogenous cells and Schwann cells, which in turn help in the secretion of certain vital trophic and tropic factors essential for early regeneration. Furthermore, hematogenous cells recruited within the first 10 days of regeneration help in the production of inflammatory mediators like interleukins that in turn stimulate Schwann cells to produce NGF for axonal growth.

 


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