Protective Effect Of Polypeptide From Chlamys Farreri On Human Dermal Fibroblasts In Vitro And The Skin Of Hairless Mice In Vivo Irradiated By Ultraviolet A

Background Polypeptide from Chlamys farreri (PCF), M₁=912, is a novel marine active product isolated from gonochoric Chinese scallop Chlamys farreri which has been served as sea food for several thousand years. The extraction yield of PCF was 1.14 g.kg^-1 Chlamys farreri by our techniques. As an octapeptide, PCF consists of Pro, Asn, Ser, Thr, Arg and Cys. However, the primary structure of PCF is kept secret. Previous work in our laboratory has shown that PCF exhibited direct reactive oxygen scavenging activity and oxidation protective effects on HeLa cells and hairless mice skin exposed to ultraviolet A. It implied a potential clinical usage of preventing skin damage induced by UV or solar light. Objective To further elucidate a possible role for PCF on UVA-damaged normal human cells and the skin of hairless mice, we established the oxidative damage models of normal human dermal fibroblasts (NHDF) and hairless mice exposed to ultraviolet A (cells: 1.905 J/cm²;mice: 4556.4J/cm²) to study the protective effect of PCF on human dermis fibroblasts and the skin of hairless mice. Methods The cells were randomly divided into six groups: control group, UVA model group, UVA+PCF1 group (UVA+2.74mM PCF), UVA+PCF2 group(UVA+5.48mM PCF), UVA+PCF3 group (UVA+10.96mM PCF) and UVA+VitC group(UVA+5.68mM Vit C). MTT method was used to detect the viability of NHDF. The intracellular SOD, GSH-px, CAT, XOD, MDA, ROS, T-AOC, and A-ASC were measured. The effect of PCF on UVA-induced apoptosis were investigated by Annexin V-FITC assay. Intracellur calcium was determined with the calcium-sensitive fluorochrome Fluo-3, and mitochondrial transmembrane potential with rhodamine 123. Comet assay was employed to detect the UVA-induced DNA damage. The ultrastructure of NHDF was observed under transmissionelectron microscope. Themice were randomly divided into I'iu1 groups: control group. UVA model group. UVA+5 %PCF group, UVA+20 %PCF group, and UVA+IO ck Vit C group. Imnuinohistochemical methods were used to examined the expression of mutant p53 gene, EGFR, and substance P. Results The results indicated that PCF could greatly enhance the viability of NHDF and markedly promote SOD, GSH-px, T-AOC, and A-ASC, while the amounts of MDA and ROS, the activity of XOD were decreased. PCF could inhibit UVA-induced apoptosis and DNA damage in NHDF. The concentration of cellular free calcium and the mitochondnal transmembrane potential were increased by PCF. In ultrastructure of NHDF, PCF could greatly decrease /UVA-induced damage, especially membrane. Immunohistochemistry showed that PCF could also inhibit the expression of p53 gene, EGFR and substance P. Conclusion PCF can inhibit UVA-induced oxidative damage on normal human dermal fibroblasts. Its mechanism is due to its abilities of scavenging oxygen free radical, inhibiting lipid peroxidation, increasing anti-oxidative enzymes, decreasing intracellular calcium and protection of membrane structure. The protective effect of PCF on the hairless mice skin against UVA-induced damage is associated with its ability of decreasing the overexpression of p53 gene, EGFR, and substance P.