Abstract
The human peptide GHK (glycyl-l-histidyl-l-lysine) has multiple biological actions, all of which, according to our current knowledge, appear to be health positive. It stimulates blood vessel and nerve outgrowth, increases collagen, elastin, and glycosaminoglycan synthesis, as well as supports the function of dermal fibroblasts. GHK’s ability to improve tissue repair has been demonstrated for skin, lung connective tissue, boney tissue, liver, and stomach lining. GHK has also been found to possess powerful cell protective actions, such as multiple anti-cancer activities and anti-inflammatory actions, lung protection and restoration of chronic obstructive pulmonary disease (COPD) fibroblasts, suppression of molecules thought to accelerate the diseases of aging such as NFκB, anti-anxiety, anti-pain and anti-aggression activities, DNA repair, and activation of cell cleansing via the proteasome system. Recent genetic data may explain such diverse protective and healing actions of one molecule, revealing multiple biochemical pathways regulated by GHK.
Keywords: GHK, GHK-Cu, gene profiling, wound healing, COPD, skin regeneration, anti-oxidant, fibrinogen
1. Introduction
The human copper-binding peptide GHK-Cu (glycyl-l-histidyl-l-lysine) is a small, naturally occurring tri-peptide present in human plasma that also can be released from tissues in case of an injury. Since its discovery in 1973, GHK-Cu established itself as a powerful protective and regenerative ingredient, which is currently widely used in skin and hair products [1].
Up-to-date, it is established that GHK-Cu is able to:
- -Tighten loose skin and reverse thinning of aged skin
- -Repair protective skin barrier proteins
- -Improve skin firmness, elasticity, and clarity
- -Reduce fine lines, depth of wrinkles, and improve structure of aged skin
- -Smooth rough skin
- -Reduce photodamage, mottled hyperpigmentation, skin spots and lesions
- -Improve overall skin appearance
- -Stimulate wound healing
- -Protect skin cells from UV radiation
- -Reduce inflammation and free radical damage
- -Increase hair growth and thickness, enlarge hair follicle size
Most authors would attribute effects of GHK to its ability to bind copper(II) ions. It was proposed that because of the GHK’s small size and its ability to bind copper, it can play a crucial part in copper metabolism [2]. However, since 2010, a new mechanism has started to emerge. The Broad Institute of MIT and Harvard (Cambridge, MA, USA) has created the Connectivity Map—a publicly available library of transcriptional responses to known perturbagens, substances that modulate gene expression [3]. This tool allowed researchers to investigate genome-wide effects of GHK and establish that GHK-Cu is able to up- and down-regulate a significant number of human genes. Today, it has become possible to connect biological effects of GHK-Cu and its effects on gene expression, to develop a more comprehensive view on GHK’s mechanism of action [4].
The present paper reviews protective and regenerative actions of the GHK-Cu peptide in human skin, as well as new gene data, revealing possible mechanisms behind these actions.
2. GHK and Gene Expression
The number of human genes stimulated or suppressed by GHK with a change greater than or equal to 50% is 31.2%. GHK increases gene expression in 59% of the genes, while suppressing it in 41%. For our studies, we used the gene expression results from 50% . This gave the best correlation with our biological data. Table 1 presents an estimate of the number of genes affected by GHK at various cutoff points.
Table 1.
Estimate of number of genes affected by glycyl-l-histidyl-l-lysine (GHK) [5].
Percent ChangeGenes StimulatedGenes Suppressed50–99%1569583100–199%646469200–299%227196300–599%196207600–899%3947900–1199%871200% or more24
2.1. GHK Improves Skin Regeneration
Skin’s ability to withstand damage and repair itself is highest in children and young individuals because of well-functioning repair and protective mechanisms. However, with age, skin’s ability to repair damage declines. GHK content is highest in the plasma of young, healthy individuals. At age 20, the plasma level of GHK is about 200 ng/mL (10−7 M), and by the age of 60, it declines to 80 ng/mL. In the experiment that led to discovery of GHK, plasma from young individuals added to liver tissue obtained from older individuals, caused old liver tissue to produce proteins more characteristic of younger individuals [6].
In the 1980s, Maquart et al. proposed that GHK may be an early signal for skin repair. The GHK amino acid sequence is present in the alpha 2(I) chain of type I collagen, and when damage activates proteolytic enzymes, GHK is released into the site of an injury [7]. A number of experiments established that GHK stimulates synthesis of collagen, selected glycosaminoglycans and small proteoglycan decorin [8,9]. It also modulates activity of key metalloproteinases, which are enzymes that facilitate breakdown of proteins of extracellular matrix, as well as activity of anti-proteases. This suggests a general regulatory effect on protein breakdown in skin, helping to prevent both buildup of damaged proteins and excessive proteolysis [10,11]. Since excessive breakdown of the dermal matrix as well as inadequate removal of damaged proteins can negatively affect skin’s health and appearance, GHK’s ability to regulate both metalloproteinases and their inhibitors can support skin regeneration and improve its appearance.
GHK also demonstrated beneficial effects on skin fibroblasts, which are considered key cells in the skin regeneration process. Fibroblasts not only synthesize structural elements of the dermal matrix but also produce a wide range of growth factors essential for skin repair. GHK, in combination with LED irradiation (light emitting diode irradiation, 625–635 nm), compared with the LED irradiation alone increased: cell viability 12.5-fold, production of the basic fibroblast growth factor (bFGF), 230%, and collagen synthesis, 70% [12].
GHK-Cu has been found to stimulate epidermal basal cells, markedly increasing integrins and p63 expression. The cells’ shape became more cuboidal, which indicates an increase in their stemness [13].
2.2. Cosmetic Use of GHK-Cu
A number of clinical studies confirmed GHK-Cu’s ability to improve appearance of aging skin. A facial cream containing GHK-Cu applied for 12 weeks to the facial skin of 71 women with mild to advanced signs of photoaging increased skin density and thickness, reduced laxity, improved clarity, reduced fine lines and the depth of wrinkles [14].
A GHK-Cu eye cream applied for 12 weeks to around-the-eye area of 41 women with mild to advanced photodamage performed better than placebo and vitamin K cream. It reduced lines and wrinkles, improved overall appearance, and increased skin density and thickness [15].
GHK-Cu applied to thigh skin for 12 weeks improved collagen production in 70% of the women treated, in contrast to 50% treated with the vitamin C cream, and 40% treated with retinoic acid [16]. In addition to improving skin laxity, clarity, firmness and appearance, reducing fine lines, coarse wrinkles and mottled pigmentation, and increasing skin density and thickness, GHK-Cu cream applied twice daily for 12 weeks also strongly stimulated dermal keratinocyte proliferation [17].
With their pilot study for topical application of copper tripeptide complexes in aged skin, Krüger et al. confirmed an increase in skin thickness in the range of the epidermis and dermis, improved skin hydration, a significant smoothing of the skin by stimulating collagen synthesis, increased skin elasticity, a significant improvement in skin contrast and an increased production of collagen I [18,19].
GHK-Cu at 0.01, 1 and 100 nM incubated with human adult dermal fibroblasts increased production of elastin and collagen. GHK also increased gene expression of MMP1 and MMP2 at the 0.01 nM. All concentrations increased TIMP1. The effects of GHK-Cu were also investigated in a randomised, double–blind clinical trial. Female volunteers applied GHK-Cu, encapsulated in nano-lipid carrier twice a day in the course of 8 weeks using either carrier alone or the commercially available peptide Matrixyl® 3000 as controls. Compared to Matrixyl® 3000, GHK-Cu produced a 31.6% reduction of wrinkle volume. Compared to control serum, GHK-Cu reduced wrinkle volume 55.8% and wrinkle depth 32.8% [20].
2.3. Animal Studies Confirm Wound Healing Activity of GHK
Multiple animal studies have established the wound healing activity of GHK. It appears that GHK stimulates wound healing through a variety of mechanisms. In rabbit experimental wounds, GHK alone or in combination with high dose helium–neon laser improved wound contraction and formation of granular tissue, as well as increasing activity of antioxidant enzymes and stimulating blood vessel growth [21,22]. Collagen dressing with incorporated GHK (PIC-Peptide Incorporated Collagen) accelerated healing of wounds in healthy and diabetic rats. The treated group displayed higher glutathione (GSH) and ascorbic acid levels, better epithelialization, as well as increased synthesis of collagen and activation of fibroblasts and mast cells in wounds. In healthy rats, treatment of wounds with PIC increased collagen 9-fold [23,24]. GHK-Cu improved healing of ischemic open wounds in rats. Wounds displayed faster healing, decreased concentration of metalloproteinases 2 and 9 as well as of TNF-β (a major inflammatory cytokine) compared with vehicle alone or with untreated wounds [25].
One problem with GHK-Cu is that it is very sensitive to breakdown by carboxypeptidase enzymes. Wounds such as diabetic skin ulcers or bedsores usually develop a “wound serum”, thought to be generated by airborne bacteria settling on the wound. The “serum” rapidly breaks down GHK and probably other growth factors such as TGF (Transforming Growth Factor) and PDGF (Platelet Derived Growth Factor).
2.4. Stimulation of Blood Vessel and Nerve Growth
Nerve and blood vessel growth is an important factor in skin healing and regeneration.
Sage et al. observed that GHK and related peptides are produced in the course of protein breakdown after an injury from a SPARC protein. SPARC (Secreted Protein Acidic and Rich in Cysteine) is a glycoprotein, mostly expressed in embryonic tissues and in tissues undergoing repair and remodeling. At initial stages of tissue repair, GHK and other peptides containing the GHK sequence (such as KGHK), which are released from SPARC in the course of proteolysis, stimulate new vessels growth. Later in the healing process, GHK and GHK-related peptides inhibit blood vessel growth [26].
Promotion of Nerve Outgrowth
When skin healing is inadequate, the healed area is often devoid of sensory abilities. In cell cultures, both Monique Sensenbrenner’s lab (France) and Gertrude Lindler’s lab (Germany) found that GHK stimulates nerve outgrowth, an essential attribute of skin repair. GHK helps restore skin’s innervation through increased production of neurotrophic factors [27,28].
Ahmed and colleagues at the Neurochemistry Lab in Chennai, India wrote that when severed nerves within a rat are placed in a collagen tube impregnated with GHK, there is an increased nerve outgrowth. GHK-Cu increased production of nerve growth factor and the neurotrophins NT-3 and NT-4, sped up the regeneration of nerve fibers from nerve stubs placed in a collagen tube, and increased axon count and proliferation of Schwann cells compared to the control group [29].
When we searched for GHK’s gene activation effects on the Gene Ontology for neurons, we came up with 408 genes up and 230 genes down. So GHK has a significant effect on neurons, but we don’t know exactly what this means. With time, we will be able to analyze the huge amount of data. Table 2 presents the top 10 genes upregulated by GHK and the top 10 downregulated [30].
%20-%201.png)