Polypeptide From Chlamys Farreri Inhibits UVB-induced Apoptosis In HaCaT Cells Via ROS/Fas/Caspase-8 Pathway

OBJECTIVE To establish UVB-induced apoptotic model of HaCaT cells and and investigate the mechanism of Polypeptide from Chlamys farreri(PCF) protecting HaCaT cells from UVB-induced apoptosis through ROS,apoptosis-related surface molecule Fas(CD95),Fas-associated protein with death domain(FADD) and caspase-8.METHODS UVB-induced apoptotic model of HaCaT cells was established by orthogonal design and apoptotic rate was determined by flow cytometry PI staining. Cells were divided into six groups:control group,UVB model group,UVB±5.68 mmol·L^-1 vitamine C positive control group,UVB+5.69 mmol·L^-1 PCF group,UVB+2.84 mmol·L^-1 PCF group,UVB+1.42 mmol·L^-1 PCF group.Effect of PCF on UVB-induced production of ROS was detected by 2', 7'-Dichlorofluorescin diacetate(DCFH-DA).SiRNA for Fas inhibited Fas expression of UVB-induced HaCaT cells.Using fluorescence staining(Hoechst 33258),the effects of PCF on UVB-induced apoptosis of HaCaT cells were investigated.Using agarose gel Electrophoresis,the effects of Fas siRNA,ROS scavenger NAC and caspase-8 inhibitor z-IETD-fmk on UVB-induced apoptosis of HaCaT cells were investigated.Expression levels of Fas(CD95) mRNA were detected by RT-PCR. Expression levels of cytochrome c,FADD,caspase-8,caspase-3 and poly-(ADP-ribose) polymerase (PARP) were determined by Western blot analysis.RESULTS Results of orthogonal experiment suggest that 20mJ·cm²UVB and 18 hours incubation after radiation make up the best apoptotic model. PCF significantly inhibited UVB-induced apoptosis and ROS accumulation.Fas siRNA,NAC and z-IETD-fmk had inhibitory effects on UVB-induced apoptosis of HaCaT cells.1.42～5.69 mmol·L^-1 PCF dose-dependently attenuated UVB-induced Fas,FADD activation,cytochrome c release and caspase-8,caspase-3 and PARP activation.CONCLUSION PCF could protect HaCaT cells from UVB-induced apoptosis.Its inhibitory effect on apoptosis may attributes to inhibition of production of ROS,release of cytochrome c,activation of Fas-FADD,caspase-8,caspase-3 and PARP.