| Objective To investigate the protective effect of PCF on the oxidative damages in HaCaT keratinocytes and the skin of hairless mice induced by single UVA.Methods Polypeptide from Chlamys farreri (PCF,Mr=879) was isolated from Chla-mys farreri using enzymetically engineering technique. The cells were randomly divided into seven groups: control group, UVA model group, UVA+0. 125%PCF group, UVA+ 0. 25%PCF group, UVA + 0. 5%PCF group, UVA + 1%PCF group and UVB + 0. 1 % VitC group. MTT method was used to detect the viability of the cells. The intracellular SOD, GSH-px, MDA, ROS, T-AOC, and A-ASC were measured by biochemical methods. The effects of PCF on UVA-induced apoptosis, intracellur calcium, and mitochondrial membrane potential were investigated by flow cytometer. UVA-induced DNA damage was detected by comet assay. The ultrastructures of HaCaT keratinocytes were observed under transmission electron microscope. In situ hybridization was used to examine the changes of p21 mRNA expression. The mice were randomly divided into five groups: control group, UVA model group, UVA+ 5% PCF group, UVA+ 20 % PCF group, and UVA+10 % Vit C group. The expression of mutant p53 protein, EGFR, substance P, collegen type I and type IV were examined by immunohistochemical methods.Results PCF greatly enhanced the viability of HaCaT keratinocytes and markedly increased the activities of SOD and GSH-px, the contents of T-AOC and A-ASC, while decreased the amounts of MDA and ROS (all P<0.05 compared PCF groups with model group). UVA-induced apoptosis and DNA damage in HaCaT keratinocytes were inhibited by PCF. The concentration of cellular free calcium decreased and the mitochondrial membrane potential were increased by PCF (P<0. 05). In ultrastructure, PCF greatly decreased UVA-induced damage, especially membrane injuries. p21mRNA expression was inhibited in PCF groups. All results were in a dose-dependent manner. Immunohistochem-istry showed that PCF also inhibited the expression of p53 protein, EGFR, substance P and collegen type I (P<0. 05), but there was no statistical significance in the expression of collegen type W(P>0. 05).Conclusion PCF can inhibit UVA-induced oxidative damage on HaCaT keratino-cytes. The mechanism were due to its abilities to scavenge oxygen free radical, inhibit lipid peroxidation, increase anti-oxidarvt enzymes, decrease intracellular calcium, protect the membrane structure, and block the expression of p21mRNA. 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 protein, EGFR, substance P and collegen type I. |
