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

Background Polypeptide from Chlamys farreri (PCF, Mr=800-1000) is a novel marine polypeptide which was isolated from Chlamys farreri using enzymetically engineering technique. Previous studies from our laboratory indicated that PCF could inhibit the oxidative damage of ultraviolet A on HeLa cells in vitro and the skin of hairless mice in vivo. To further elucidate a possible role for PCF on UVB-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 B (cells:1.176 X 10-4J/cm2; mice: 154.5 J/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, UVB model group, UVB+0.25 %PCF group, UVB+0.5%PCF group, UVB+1 %PCF group and UVB+0.1 %VitC group. 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 UVB -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 UVB -induced DNA damage. The ultrastructure of NHDF was observed under transmissionelectron microscope. The mice were randomly divided into five groups: control group, UVB model group, UVB+5 %PCF group, UVB+20 %PCF group, and UVB+10 % Vit C group. Immunohistochemical 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 UVB-induced apoptosis and DNA damage in NHDF. The concentration of cellular free calcium and the mitochondrial transmembrane potential were increased by PCF. In ultrastructure of NHDF, PCF could greatly decrease UVB-induced damage, especially membrane. Immunohistochemistry showed that PCF could also inhibit the expression of p53 gene, EGFR and substance P. Conclusion PCF can inhibit UVB-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 UVB-induced damage is associated with its ability of decreasing the overexpression of p53 gene, EGFR, and substance P.