Polypeptide From Chlamys Farreri Inhibits UVA Plus UVB-induced Apoptosis In HaCaT Cells Via ROS/JNK/caspase-3 Pathway

AIM To establish UVA plus UVB-induced apoptotic model of HaCaT cells and investigate themechanism of polypeptide from Chlamys farreri (PCF)protecting HaCaT cells from apoptosis inducedby mimicking the action of environmental UV irradiation on human skin, through ROS, JNK pathwayand caspase-3. MATHODS An apoptotic model of UV irradiation-induced HaCaT cells wasestablished by orthogonal design and apoptosis rate was determined by flow cytometry PI staining.The viability of HaCaT cells was determined by MTT. The activities of superoxide dismutase (SOD),glutathione peroxidase (GSH-Px) and Catalase (CAT), total antioxidative capacity (T-AOC), andmalondialdehyde (MDA) formation were measured by biochemical methods; Using flow cytometry(FCM), the production of ROS in HaCaT cells was tested by 2', 7'-Dichlorofluorescin diacetate(DCFH-DA). The effects of PCF and caspase-3 inhibitor Ac-DEVD-CHO on UVA plus UVB inducedapoptosis was analyzed by agarose gel electrophoresis and Hoechst 33258 staining. JNK,Phosphorylated JNK, activated caspases-3, and cytochrome c were investigated by western blotting.RESULTS Results of orthogonal experiment suggest that 4 J/cm~2 UVA plus 10 mJ/cm~2 UVB and 12hours incubation after radiation make up the best apoptotic model. PCF inhibited UVirradiation-induced apoptosis of HaCaT cells. PCF strongly reduced the intracellular ROS level,enhanced activities of SOD, GSH-px, and CAT; increased T-AOC, decreased the formation of MDA inHaCaT cells following UV irradiation. Furthermore, we found that 1.42～5.68 mmol/L PCF couldinhibit the phosphorylation of JNK, the activity of caspases-3, and the release of cytochrome c in aconcentration-dependent manner. The scavenger of ROS can inhibit the phosphorylation of JNK, andthe JNK inhibitor can decrease the expression of cleaved caspases-3 protein, at the same time, thecaspases-3 inhibitor can block the apoptosis of HaCaT cells. CONCLUSION PCF protected HaCaTcells from apoptosis induced by UVA plus UVB mainly through decreasing intracellular ROS level andincreasing the activities of antioxidative enzymes to block the ROS-JNK-caspase-3-apoptosissignaling pathway.