In the dermis of the skin, collagen, elastin, hyaluronic acid, and proteoglycan constitute the extracellular matrix structure and are produced by fibroblasts scattered throughout the dermis. The normal functions of fibroblasts include the synthesis, degradation, and structural formation of the extracellular matrix in the skin as well as to maintain skin morphology (such as wrinkles), firmness, and elasticity (Gilchrest 1989; Naylor et al. 2011; Watson et al. 2014; Tracy et al. 2016). The epidermal basement membrane between the epidermis and dermis also plays an important role in maintaining the structure and function of the skin (Amano et al. 2000; Amano et al. 2001; Inomata et al. 2003; Matsuura-Hachiya et al. 2016). Several extracellular matrix components that constitute the basement membrane are produced by both epidermal keratinocytes and dermal fibroblasts (Marinkovich et al. 1993; Nishiyama et al. 1998). For example, type Ⅳ collagen (basement membrane collagen), type Ⅶ collagen (anchoring fibril collagen), and laminins were reported to be produced by both cell types. Therefore, controlling the production of extracellular matrix by dermal fibroblasts is necessary to maintain the homeostasis of skin structure and function. In particular, the regulation of collagen production is important because several collagen types comprise the foundation of dermal fibrillar structures as well as the network structures and anchoring fibrils in the basement membrane.
Many studies have investigated bioactive substances, organic synthetic compounds, and natural compounds that promote collagen production (collagen synthesis and gene expression) in cell-culture systems (Amano et al. 2007; Kishimoto et al. 2013; Sivasubramanian et al. 2017; Shen et al. 2018). Ascorbic acid, retinoic acid, and cellular growth factors [TGF-β, platelet-derived growth factor (PDGF), etc.] promote the gene expression and synthesis of several collagen types. It has also been reported that several compounds, such as natural compounds, promote collagen production in collagen-producing cells like fibroblasts. For example, two amino acids, L-hydroxyproline (Hyp) and N-acetyl-L-hydroxyproline (AHyp) as well as a dipeptide, L-alanyl-L-glutamine (Aln-Glu) promote collagen production and are often used as ingredients in cosmetics.
Several studies explored the effects of administering both amino acids Hyp and AHyp to living organisms. The oral ingestion of Hyp increases the amount of collagen in rat skin (Aoki et al. 2012) and promotes the collagen accumulation in swim bladders to an extent that may involve promoting the expression of type I collagen genes (Rong et al. 2019). AHyp, a Hyp derivative, is used medicinally in Europe (France and Germany). Orally administered AHyp increases the collagen synthesis of burn-injured skin (Molimard et al. 1972), improves wound healing in patients with skin ulcers (Pasolini G et al. 1988), and improves symptomatic knee osteoarthritis (Krüger et al. 2007). Both amino acids have also been reported to promote collagen production in human neonatal dermal fibroblasts (Makimoto Y et al. 2000). These studies suggest that both amino acids regulate the biological response by promoting collagen production.
In collagen-producing cells like fibroblasts, glutamine is an important stimulator of collagen biosynthesis (Bellon G et al. 1995; Karna E et al. 2001; Pithon-Curi TC et al. 2006). However, glutamine is unstable and quickly degrades in aqueous solutions. Aln-Glu is a stable glutamine covalent source used in amino acid infusions and cell-culture media to replace glutamine. Aln-Glu is rapidly, and quantitatively, hydrolyzed as a source of free glutamine (Albers S et al. 1988). The stability of Aln-Glu as a glutamine suggests that it also promotes collagen production.
The bioelectric potential of the skin affects skin structure, function, and cellular activities (Messerli MA et al. 2011; Ud-Din S et al. 2014; Golberg A et al. 2015). For example, in skin wound healing, the difference in electrical potential at the wound site promotes the migration of dermal fibroblasts, which aggregate at the wound site (Guo A et al. 2010; Kim MS et al. 2015; Snyder S et al. 2017; Lee GS et al. 2019). Previous reports describe the electrical stimulation (ES) of cultured fibroblasts with a pulsed current, which promoted the expression of collagen and elastin, the secretion of fibroblast growth factors, the differentiation of fibroblasts into myofibroblasts, and the enrichment of collagen fibrils (Nguyen EB et al. 2018; Rouabhia M et al. 2013; Wang Y et al. 2017). These findings indicate that external ES greatly influences the biological response of skin cells; that it may also affect the skin must also be considered.
We previously reported several effects of pulsed electrical stimulation (PES, at 4,800 Hz and 1–5 V) on the skin and cultured cells. We found that PES (a) promotes the effect of vitamin C introduction on collagen production in rat skin (Hori Y et al. 2009); (b) induces human keratinocyte differentiation (Arai KY et al. 2013); (c) promotes the gene expression of PDGF, fibroblast growth factor 2 (FGF2), and transforming growth factor beta (TGF-β1) in human fibroblasts (Urabe H et al. 2021); and (d) stimulates fibroblast proliferation by enhancing PDGF expression (Urabe H et al. 2021). In addition, our results suggest that PES may promote gene expression for several collagen types and collagen production in human dermal fibroblasts. To further clarify the biological effects of PES on human dermal fibroblasts, we investigated the effects of Hyp, AHyp, and the dipeptide Aln-Glu, in combination with PES, on the gene expression of type I and III collagen (fibrillar collagen as dermal collagen fibrils) as well as type IV and VII collagen (basement membrane collagen and anchoring fibril collagen).