Inhibition Of UVA-induced Apoptosis By The Polypeptide From Chlamys Farreri Via Regulation Of ASMase/JNK/COX-2 Pathway In HaCaT Cells

OBJECTIVE To repeat UVA-induced apoptotic model of HaCaT keratinocytes and investigate the mechanism of Polypeptide from Chlamys farreri(PCF) protecting HaCaT cells from UVA-induced apoptosis through acid sphingomyelinase(ASMase),c-Jun NH₂-terminal kinase(JNK) and cyclooxygenase-2(COX-2).METHODS UVA-induced optimal apoptotic model of HaCaT cells was established and cells were divided into five groups:control group,UVA model group,5.68 mmol·L^-1 vitamine C positive control group(as control only for detecting apoptosis of cells),PCF groups(5.68 mmol·L^-1 PCF,2.84 mmol·L^-1 PCF,1.42 mmol·L^-1 PCF) and the corresponding inhibitor groups(25μmol·L Desipramine,20μmol·L SP600125,1μmol·L celecoxib).Using agarose gel electrophoresis, the effects of PCF,ASMase inhibitor Desipramine,JNK inhibitor SP600125 and COX-2 inhibitor celecoxib on UVA-induced apoptosis were investigated.Morphology alteration influenced by UVA and PCF were observed by Hoechst 33258 fluorescent staing.Protein expression levels of JNK, phosphorylated JNK were determined by Western blot analysis.Expression of c-jun mRNA was examined by Perfect Real Time PCR.Expression of ASMase and COX-2 were assayed by RT-PCR and Western blot analysis.RESULTS 1.42～5.68 mmol·L^-1 PCF significantly inhibited UVA-induced apoptosis(P＜0.05).PCF attenuated apoptotic morphology alteration caused by UVA.Desipramine, SP600125 and celecoxib had inhibitory effects on UVA-induced apoptosis of HaCaT cells.1.42～5.68 mmol·L^-1 PCF dose-dependently attenuated UVA-induced activation of JNK,expression of ASMase, c-jun and COX-2(P＜0.05,P＜0.01).CONCLUSION PCF could protect HaCaT cells from UVA-induced apoptosis.Its inhibitory effect on apoptosis may attributes to inhibition of activation of ASMase---JNK/c-jun---COX-2 pathway induced by UVA.