DOI: https://doi.org/10.21203/rs.3.rs-2829365/v1
Several collagen types are important for maintaining skin structure and function. Previous reports show that L-hydroxyproline (Hyp), N-acetyl-L-hydroxyproline (AHyp), and L-alanyl-L-glutamine (Aln-Gln) are biological active substances with collagen synthesis-promoting effects. In this study, we combined the promotive effects of pulsed electrical stimulation (PES) with three amino acid derivatives (AADs) in human dermal fibroblasts. Fibroblasts were exposed to PES with a 4,800 Hz pulse frequency and a voltage at 1 V or 5 V for 15 minutes. The gene expression of type I and Ⅲ collagen (fibrillar collagen), type Ⅳ and Ⅶ collagen (basement membrane collagen and anchoring fibril collagen) were measured by RT-PCR 48 hours after PES. PES alone promoted the expression of COL1A1 and COL3A1 at 5 V but did not alter that of COL4A1 and COL7A1. Each AAD and the AAD mixture promoted the expression of COL4A1 and COL7A1 but either repressed, or did not alter, that of COL1A1 and COL3A1. Compared to treatment with each AAD, PES at 5 V with Hyp promoted the expression of COL1A1 and COL3A1, enhanced COL3A1 expression with AHyp, and stimulated COL3A1 expression with Aln-Gln, while COL4A1 and COL7A1 expressions were not affected. PES and the AAD mixture significantly promoted COL4A1 expression in a voltage-dependent manner, and COL1A1 and COL3A1 demonstrated a similar but nonsignificant trend, whereas COL7A1 expression was not affected. The combination of PES with each AAD or the AAD mixture may improve skin structure and function by increasing the expression of basement membrane collagen and dermal fibrillar collagen.
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).
We purchased normal human dermal fibroblasts from the skin of a 27-year-old (HF27) from Biopredic International (Saint-Grégoire, France). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich Co. LLC, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Nichirei Bio, Tokyo, Japan) and 1.0% penicillin–streptomycin–neomycin (Invitrogen, Carlsbad, CA, USA). All cultures were maintained for 6 days in a humidified atmosphere of 5.0% CO2 at 37°C.
L-Hydroxyproline (Hyp) and L-alanyl-L-glutamine (Aln-Gln) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). N-acetyl-L-hydroxyproline (AHyp) was purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). Individual stocks of Hyp, AHyp, and Aln-Glu were dissolved in 0.2% FBS-DMEM to final concentrations of 0.3%, 0.1%, and 0.3%, respectively. The amino acid derivative (AAD) mixture had a final concentration of 0.3% Hyp, 0.1% AHyp, and 0.3% Aln-Glu in in 0.2% FBS-DMEM.
For all experiments, we used PES equipment and a carbon electrode for a 24-well plate originally created at Homer Ion Laboratories (Tokyo, Japan) (Fig. 1) (Hori Y et al. 2009; Arai KY et al. 2013). We measured the waveform and frequency using a digital oscilloscope (Iwatsu Electric Co., Ltd., Tokyo, Japan) at three voltages: 0 (electrodes immersed in culture medium without ES), 1, and 5 V. The PES exposure time was 15 min. For all experiments, we used a 4,800 Hz frequency, single-phase DC (also called the Gunpatsu pulse) in the positive direction (Urabe H et al. 2021).
We seeded HF27 cells cultured in 10% FBS-DMEM onto a 24-well plate at a density of ~ 30,000–50,000 cells/well. After 1 day of culture, the medium was replaced with 0.2% FBS-DMEM alone, supplemented with each AAD solution, or supplemented with the triple AAD mixture. After 1 day incubation period, PES was performed on the cultures. The cells were cultured in 0.2% FBS-DMEM with or without AAD supplementation for an additional 48 hours. The control culture (N) did not undergo PES (no electrode immersion) or receive AAD supplementation.
RNA extraction and purification,
We collected cell lysates at 48 hours post-PES and fully homogenized them using a QIAshredder (QIAGEN, Hilden, Germany). Next, we extracted total RNA using a RNeasy Mini Kit (QIAGEN) and purified it using a RNeasy MinElute Cleanup Kit (QIAGEN). Finally, we measured the total RNA concentration and purity on a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA).
Using a random primer (Promega, Madison, WI, USA) and Moloney murine leukemia virus (M-MLV) Reverse Transcriptase (Promega), we reverse-transcribed the purified total RNA for 10 min at 25°C, 50 min at 37°C, and 15 min at 70°C, according to the manufacturer’s instructions. We measured the concentration and purity of the complementary DNA (cDNA) obtained on a NanoDrop 2000.
Using TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA), we performed a gene expression analysis by reverse transcription polymerase chain reaction (RT-PCR) with the StepOne Real-Time PCR System (Applied Biosystems). The PCR program was 20 sec at 95°C, followed by 1 sec at 95°C and 20 sec at 60°C for 40 cycles. We analyzed the data with the ΔCt method. We prepared COL1A1, COL3A1, COL4A1, and COL7A1 TaqMan probes, with MRPL19 as an internal standard. The results were representative of four independent experiments performed in quadruplicate.
Numerical values are the mean ± standard deviation. Data were analyzed using the t-test for unidirectional variance. A P value less than 0.05 was considered statistically significant.
To determine the effect of PES on collagen gene expression, we measured the gene expression of type I and III collagen (fibrillar collagen; COL1A1 and COL3A1) as well as type IV and VII collagen (basement membrane collagen and anchoring fibril collagen; COL4A1 and COL7A1). Human dermal fibroblasts were cultured for 24 hours in 0.2% FBS-DMEM and stimulated with PES for 15 min at a pulse frequency of 4,800Hz and voltage at 0, 1, and 5 V. The fibroblasts were then cultured for an additional 48 hours at which point total RNA was isolated. RT-PCR results showed that PES significantly promoted the expression of COL1A1 and COL3A1 at 5 V as well as COL4A1 at 1V; it did not have an effect on COL4A1 at 5 V or COL7A1 at either 1 or 5 V (Fig. 2). These data indicate that PES has differential effects on collagen gene expression.
To examine the effect of AAD supplementation alone on collagen gene expression, we measured the gene expression of type I and III collagen (COL1A1 and COL3A1) as well as type IV and VII collagen (COL4A1 and COL7A1). Human dermal fibroblasts were treated with each AAD as well as the triple AAD mixture for 72 hours in 0.2% FBS-DMEM. In our data, the N column (open column) is the negative control, an experimental system without PES or AAD supplementation while the 0 V column is a negative control for PES and indicates the effect of each AAD on fibroblast gene expression (closed column).
Supplementation with 0.3% Hyp or 0.1% AHyp significantly promoted the expression of COL4A1 and COL7A1 but suppressed the expression of COL1A1 and COL3A1 (Figs. 3 and 4, respectively). Supplementation with 0.3% Aln-Gln increased the expression of COL4A1 in a small but significant manner but did not affect the expression of COL1A1, COL3A1, or COL7A1 (Fig. 5). The triple AAD mixture significantly promoted the expression of COL4A1 and COL7A1 but did not affect the expression of COL1A1 or COL3A1 (Fig. 6). These results suggest that the effect of the triple AAD mixture on collagen gene expression may be additive, combining the promoting effects of each AAD.
To determine the effect of PES in combination with each AAD on collagen gene expression, we performed the following experiment. Human dermal fibroblasts were incubated with each AAD for 24 hours in 0.2% FBS-DMEM prior to PES (0, 1, and 5 V; 4,800 Hz) for 15 minutes. The fibroblasts were then incubated with each AAD for an additional 48 hours. Compared to the AAD controls (0 V PES), 5 V PES with Hyp supplementation promoted the expression of COL1A1 and COL3A1 (Fig. 3), enhanced COL3A1 expression with AHyp (Fig. 4), and stimulated COL3A1 expression while slightly repressing COL4A1 expression with Aln-Gln (Fig. 5). These data indicate that PES in combination with each AAD significantly
promoted the fibroblast expression of COL3A1 but only at 5 V. The expression of COL4A1 and COL7A1 were not affected. Together these data suggest that PES alters the impact of individual AADs on collagen gene expression.
We next examined the effect of combining PES and the triple AAD mixture on collagen expression. Human dermal fibroblasts were treated with the triple AAD mixture for 24 hours in 0.2% FBS-DMEM, exposed to PES (0, 1, and 5 V; 4,800 Hz) for 15 minutes, and then incubated for an additional 48 hours. Compared to the control culture (N column, Fig. 6), the triple AAD mixture alone significantly promoted the expression of COL4A1 and COL7A1, whereas treating with PES and the mixture together significantly increased the expression of COL1A1, COL3A1, COL4A1, and COL7A1. In particular, the expression of COL4A1 was significantly enhanced in a voltage-dependent manner when PES occurred in the presence of the triple AAD mixture. The expression of COL1A1 and COL3A1 also trended upward in a, nonsignificant, voltage-dependent manner. However, the expression of COL7A1, which was enhanced by the triple AAD mixture, was not affected by PES. These results suggest that combining PES and the triple AAD mixture may improve skin structure and function due to the increased expression of both basement membrane and dermal fibrillar collagen.
In this study, we examined that the effects on collagen gene expression in human dermal fibroblasts of three AADs (Hyp, AHyp or Aln-Gln) and a mixture of the three as biologically active substances in combination with PES. The triple AAD mixture seemed to have an additive effect on the action of each AAD. PES alone promoted the expression of certain collagen genes, and the effect of PES with each AAD was additive. We found that the combination of PES and the triple AAD mixture significantly increased 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 as basement membrane components).
In particular, type IV collagen, had increased gene expression in an electrical intensity-dependent manner (i.e., voltage-dependent manner). Type I and III collagen did not have a significant difference in gene expression, but there was an upward trend that depended on the electrical intensity. Conversely, type VII collagen expression was not PES dependent but instead increased by supplementation with AADs.
In a previous study, we reported that the PES of fibroblasts induced cell proliferation in an electrical intensity-dependent manner and increased the expression of at least three growth factors (PDGF, FGF, and TGFβ). In particular, we reported that the enhancement of PDGF gene expression contributed to cell proliferation in early stages of growth based on the suppression of cell proliferation by PDGFR inhibition of PDGFR signaling (Urabe H et al. 2021). Furthermore, PDGF activates intracellular signaling systems cell membrane-bound PDGFR, and PDGFR inhibition decreased αSMA, type I, and type III collagen gene expression (LeBleu VS et al. 2011). The direct or indirect (via TGF-β1) effect of PDGF on multiple cell systems promotes the transcription and translation of collagen types I, III, IV, and VII (Hänsch GM et al. 1995; Yamabe H et al. 2000; Wang XT et al. 2004; Mu E et al. 2011; Amano S et al. 2007; Pierce GF et al. 1989). There are also multiple reports that the ES of fibroblasts promotes FGF and TGFβ production as well as that of αSMA, type I and type III collagen (Golberg A et al. 2015; Rouabhia M et al. 2013; Wang Y et al. 2017; Kim TH et al. 2014). Finally, αSMA is a marker of myofibroblast differentiation, and myofibroblasts are reported to promote the production of collagen types I, III, IV, V, and VI (Klingberg F et al. 2013). Therefore, the promotion of type I, III, and IV collagen gene expression by PES and AADs may be due to (a) the effect of each AAD on cells as previously reported (Aoki M et al. 2012; Rong H et al. 2019; Molimard R et al. 1972; Pasolini G et al. 1998; Krüger K et al. 2007; Makimoto Y et al. 2000; Bellon G et al. 1995; Karna E et al. 2001; Pithon-Curi TC et al. 2006; Albers S et al. 1988) and (b) the promotion of PDGF production in fibroblasts by PES, which accelerated myofibroblast differentiation and the expression of several collagen genes (Urabe H et al. 2021; LeBleu VS et al. 2011).
In wound healing, where collagen metabolism is an important process, the production of αSMA promotes fibroblast differentiation into myofibroblasts during the inflammatory phase. The production of type I and type III collagen forms cell scaffolds and granulation tissue during the proliferative phase, and the production of type IV and type VII collagen is promoted after the transition to the remodeling phase (Tracy LE et al. 2016). We believe that the stimulation of αSMA production by PES promoted the differentiation of fibroblasts into myofibroblasts, which, combined with the stimulation of type I and III collagen production by PES and AADs, may have begun transitioning the fibroblast culture environment to the remodeling phase of wound healing. This transition promotes the gene expression of type IV collagen, a component of the basement membrane.
Type VII collagen is a major component of anchoring fibrils in the epidermal-dermal basement membrane. Chen et al. reported that type VII collagen gene expression was reduced in fibroblasts in an age-dependent manner due to a reduced response to pro-inflammatory cytokines (IL-1β and TNF-α) (Chen YQ et al. 1994). The studies suggest that gene expression of type VII collagen is regulated by a cytokine network that may modulate anchoring fibril construction. The effect of cytokines on type I and type VII collagen gene expression in cultured dermal fibroblasts have also been reported (Mauviel A et al. 1994). A study of the pro-inflammatory cytokines interleukin-1 alpha (IL-1α), interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and leukoregulin (LR) showed that multiple pathways are involved in cytokine regulation of type I and type VII collagen gene expression. Thus, the regulatory process of type VII collagen gene expression may differ from that of type I collagen (and likely type III and IV collagens also). Therefore, PES might not affect the regulatory process in which type VII collagen gene expression was promoted by the triple AAD mixture.
There are several ES parameters such as voltage, current, duration, frequency, etc. There is also a parameter called duty ratio in electrical engineering, which is the ratio of the ES duration to one cycle of electrical output. Previous reports show that the degree of cell proliferation changes when the ES duty ratio differs (Yoshikawa Y et al. 2016). Reports also suggest that the duty ratio affects myofibroblast differentiation (Uemura M et al. 2021). Thus, there are several electrical condition parameters, and the cellular response may vary greatly. Against this background, it is possible that our results with a duty ratio of 50:50 were due to a cellular response specific to the ES conditions, coupled with the action of AADs. There may be other electric stimulation conditions with more useful results than ours. Further investigations of ES technology will likely lead to its application in health science as well as in medicine.
This study suggests that combining PES with each AAD or the triple AAD mixture may improve skin structure and function by increasing the expression of basement membrane collagen and dermal fibrillar collagen. However, there is a possibility that there are more useful ES conditions than those used here, due to the countless number of electric current parameters. The advancement of electrical stimulation technology is expected to lead to its application in the field of health science as well as in medicine.
Competing Interests: All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.