扇贝多肽经由死亡受体和线粒体通路抑制UVB诱导的HaCaT细胞凋亡

Polypeptide from Chlamys farreri prevents UVB-induced apoptosis in the HaCaT cells through death receptor pathway and mitochondrial pathway

目的从凋亡相关分子Fas（CD95）、半胱天冬酶-3（caspase-3）和活性氧（ROS）、细胞色素C的角度,研究扇贝多肽（polypeptide fromChlamys farreri,PCF）抑制UVB引起的人角质HaCaT细胞凋亡的分子机制。方法实验设计为5组：对照组、UVB模型组、UVB＋5.69mmol.L-1PCF组、UVB＋2.84mmol.L-1PCF组、UVB＋1.42mmol·L-1PCF组。应用小干扰RNA（siRNA）干扰UVB辐射的HaCaT细胞的Fas表达;琼脂糖凝胶电泳分析Fas siRNA及ROS清除剂NAC对细胞凋亡的影响;以DCFH-DA为荧光探针,检测细胞内ROS的含量;免疫印迹法检测细胞色素C及用RNAi干扰降低Fas表达后caspase-3的表达。结果干扰Fas后,UVB辐射的HaCaT细胞凋亡受到明显抑制及caspase-3的表达降低;NAC对UVB诱导的HaCaT细胞凋亡有抑制作用;1.42~5.69mmol.L-1剂量范围内的PCF可剂量依赖性抑制UVB引起的ROS的生成及细胞色素C的释放。结论PCF可抑制UVB诱导的HaCaT细胞凋亡,其作用机制与抑制Fas-caspase-3及ROS-细胞色素C通路有关。

Aim To investigate whether the polypep- tide from Chlamys farreri（PCF） protected HaCaT cells from UVB-induced apoptosis through Fas-caspase-3 and ROS-cytochrome C. Methods Experiment designs were divided into five groups：control group, UVB model 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. SiRNA for Fas inhibited Fas expression of UVB-induced HaCaT cells. Using agarose gel electro- phoresis, the effects of Fas siRNA and ROS scavenger NAC on UVB-induced apoptosis of HaCaT cells were investigated. Expression levels of cytochrome C and caspase-3 after inhibitory Fas were determined by Western blot analysis. Intracellular ROS was detected by means of an oxidation-sensitive fluorescent probe （DCFH-DA）. Results SiRNA for Fas had inhibitory effects on UVB-induced apoptosis of HaCaT cells and caspase-3 expression. NAC had inhibitory effects on UVB-induced apoptosis of HaCaT cells. PCF inhibited UVB-induced generation of ROS and cytochrome C re- lease dose-dependently. Conclusions PCF protected HaCaT cells from UVB-induced apoptosis. Its inhibitory effect on apoptosis could be attributed to inhibition of Fas-caspase-3 and ROS-cytochrome C pathways.