.ros/nf-¦Êb/cox-2 Pcf Protection Of Uvb-induced Apoptosis Of Hacat Cells

OBJECTIVE To repeat UVB-induced apoptotic model of HaCaT keratinocytes and investigate the mechanisms of Polypeptide from Chlamys farreri(PCF) protecting HaCaT cells from UVB-induced apoptosis through reactive oxygen species(ROS),nuclear factor-KappaB(NF-κB) and cyclooxygenase-2(COX-2) signaling pathway.METHODS UVB-induced optimal apoptotic model of HaCaT cells was established and cells were divided into four groups:control group,UVB model group, PCF groups(5.68,2.84,and 1.42 mmol·L^-1PCF) and the corresponding inhibitor groups (5mmol·L^-1NAC,0.2umol·L^-1 MG132,1μmol·L^-1celecoxib).Using Hoechst 33258 fluorescent staining and agarose gel electrophoresis to investigate UVB influenced morphologic and biochemical alteration and the effects of PCF,ROS scavenger NAC,NF-kB inhibitor MG132 and COX-2 inhibitor celecoxib on UVB-induced apoptosis.The generation of ROS was detected by Electron spin resonance spectrometry(ESR),Protein expression levels of cytoplasmic superoxide dismutase(Cu,Zn-SOD), NF-κB/p65,phosphorylated NF-κB/ p65,IκBα,phosphorylated IκBα,IKKαand COX-2 were determined by Western blot analysis.MRNA expressions of glutathione peroxidase(GPx),Catalase (CAT),IκBαand COX-2 were assayed by RT-PCR.The transolation of NF-κB/p65 was measured by immunofluorescent staining.The interaction of IKKαand IκBαwere measured by immunoprecipitation.RESULTS UVB irradiation induced pyknosis and formation of DNA Ladder; the ROS levels were increased during 48h,peaked in 3h and 24h,especially in 3h meanwhile the expressions of Cu,Zn-SOD,CAT and GPx were decreased(P<0.01).UVB irradiation induced transcriptional activation and translocation of NF-κB/p65 to nucleus,which was mediated through increasing the phosphorylation /degradation of IκBα,activation of IKKαand enhancing the integration of IKKαand IκBαand also increased the activation of COX-2(P<0.01).1.42·5.68mmol·L^-1 PCF and the corresponding inhibitor groups significantly inhibited UVB induced apoptosis and attenuated apoptotic morphology alteration caused by UVB(P<0.05),meanwhile increased the expression of CuZn-SOD,CAT,GPx and COX-2 dose-dependently(P<0.05).1.42～5.68mmol·L^-1 PCF also inhibited the integration of IKKαand IκBα,blocked activation of IKKαand inhibited the phosphorylation /degradation of IκBαso as to inhibit UVB-induced activation of NF-κB/p65(P<0.05).2.84 mmol·L^-1 PCF significantly deceased the generation of ROS at any of the evaluated times espescially in 3h post-irradiation,the inhibitory effect could last 48 hours(P<0.05).CONCLUSION The activation of IKKα/IκBα/NF-κB/COX-2 signal pathway played an important role in UVB-induced apoptosis, and PCF likely exerted its anti-apoptotic effect in HaCaT cells through scavenging ROS,increasing the expression of antioxidative enzymes and modulating the NF-κB and COX-2 signaling pathway.