Polypeptides From Chlamys Farreri Regulates Hsf1/Hsp70/ROS、NO、JNK Pathway Inhibiting UVA-induced Apoptosis In HaCaT Cells

OBJECTIVE To copy 8J/cm2 UVA-induced apoptotic model of HaCaT cells and to establish iNOS transfected HaCaT cells to investigate the mechanism of Polypeptide from Chlamys farreri (PCF) protecting HaCaT cells from UVA-induced apoptosis through HSF1/HSP70/ROS、NO、JNK pathway.METHODS 8J/cm2 UVA-induced apoptotic model of HaCaT cells was replicated and iNOS transfected model of HaCaT cells was established using of cationic oiposomes(LipefectimeTM2000) method. Cells were divided as designed:control group, UVA model group, UVA+1 mol/L iNOS inhibitor SMT group,UVA+0.5 mmol/L ROS inhibitor NAC group, UVA+50μmol/L HSP70 transcriptic inhibitor quercetin group、UVA+100μmol/L XO inhibitor Oxy group、UVA+5.69 mmol/L PCF group, UVA+2.84 mmol/L PCF group and UVA+1.42 mmol/L PCF group. Apoptotic rate of cells was determined by Hoechst 33258 fluorescent staining; Protein expression levels of p-HSF1、HSP70、iNOS after UVA irradiation at different time point(1、3、6、12、18、24 h) and JNK、p-JNK after 6 h were detected by Western blot analysis; The mRNA expression of HSF1、HSP70、XO were assayed by RT-PCR; The time-dependent release of NO and ROS were determined by ESR,as well as the effect of Oxy, SMT and PCF on the release of NO. RESULTS 8J/cm2 UVA-induced apoptotic model of HaCaT cells was successfully replicated determined by Hoechst 33258 fluorescent staining; 1.42～5.69 mmol/L PCF、1mmol/L SMT and 0.5 mmol/L NAC all could inhibit HaCaT cells apoptosis induced by UVA radiation (P<0.05).Western blot results showed p-HSF1 protein reached a peak at 3 h(P<0.05) after 8J/cm2UVA radiation and HSP70 at 6 h(P<0.05).Pretrating with quercetion before UVA radiation increased the expression of p-JNK、iNOS、XO and the release of NO、ROS causing increased cells apoptosis rate(P<0.05).1.42～5.69 mmol/L PCF could improve transcriptional activity of HSF1、protein and mRNA expression of HSP70,however inhibit the release of ROS NO and the activity of JNK(P<0.05, P<0.01)in a dose dependent. NO generation showed two peaks at 3 h and 8 h after UVA radiation in HaCaT cells.CONCLUSION PCF could protect HaCaT cells from 8J/cm2 UVA-induced apoptosis.The mechanism is that it can improve the transcriptional activity of HSF1 then increasing the expression of HSP70,which making it as free radical scavengers to inhibit the release of ROS and NO,what’s more,through HSF1/HSP70 pathway it can reduced the activity of JNK.NO and ROS all has two expression peaks after UVA radiation.The first peak of NO is catalyzed by XO and the second one relate with iNOS.