扇贝多肽对UVB照射HaCaT细胞后NO释放及热休克蛋白70表达的影响

摘要

Aim To observe dynamic changes of nitric oxide( NO )release and inducible nitric oxide synthase ( iNOS )expression in HaCaT cells after UVB radiation ,and to investigate the effect of Polypeptide from Chlamys farreri( PCF )on UVB-induced NO release and heat shock protein 70( HSP70 )expression.Methods The release of NO was assayed by electron spin resonance ( ESR ).Western blot analysis was used to determine the protein levels of iNOS and HSP70.Results NO release was significantly increased after 20 mJ· cm-2 UVB radiation and there were two peaks at 3 h and 24 h respectively.PCF inhibited the release of NO at each time point.iNOS protein expressed six hours after UVB radiation and increased gradually , then reached to peak 18～ 24 h later.iNOS inhibitor reduced the release of NO at 24 h after UVB radiation, but had no effect at 3 h.PCF enhanced HSP70 expression in a dose-dependent manner.Conclusions UVB exposure to HaCaT cells induced expression of iNOS and then the release of NO increased, but in early stages of exposure, NO can also be generated by non-iNOS pathway.PCF may enhance the expression of HSP70.thereby inhibit iNOS expression and reduce the generation of NO, thus protect HaCaT cells against UVB radiation.%目的 观察UVB诱导的HaCaT细胞一氧化氮(nitric oxide,NO)释放及诱导型一氧化氮合酶(iNOS)表达的动态变化,探究扇贝多肽(polypeptide from Chlamys farreri,PCF)对UVB照射后NO释放、热休克蛋白70(heat shock protein 70,HSP70)表达的影响.方法 以电子自旋共振(electron spin resonance,ESR)技术检测NO的释放量;蛋白质印迹法检测iNOS、HSP70蛋白表达.结果 20 mJ·cm-2 UVB照射HaCaT细胞后NO释放明显增多,有3、24 h两个释放高峰期;PCF对各时间点NO的释放均有抑制作用;iNOS蛋白表达在UVB照射后6 h开始逐渐升高,18～24 h达到高峰;iNOS抑制剂可抑制UVB照射后24 h时NO的释放,对3 h时NO的释放无影响;PCF可剂量依赖性增强UVB照射后HSP70的蛋白表达.结论 UVB照射HaCaT细胞诱导iNOS高表达从而引起NO产生增加,但在照射前期也可通过非iNOS途径生成NO;PCF可能通过增强UVB照射后HSP70的表达,进而抑制iNOS蛋白表达、减少NO的生成而保护HaCaT细胞免受UVB辐射损伤.