扇贝多肽经由EGFR-CDK-4通路抑制UVA诱导的HaCaT细胞凋亡

Inhibitory effect of polypeptide from Chlamys farreri on UVA-induced apoptosis of HaCaT cells via EGFR-CDK-4 signaling pathway

目的建立8J.cm-2紫外线A(UVA)辐射损伤永生化的人角质形成细胞株(HaCaT)细胞的病理模型,经由信号通路的表皮生长因子受体(EGFR),磷脂酰肌醇-3激酶(AKT),细胞周期蛋白(CyclinD1)至周期蛋白依赖激酶4(CDK-4)角度研究扇贝多肽(Polypeptide from Chlamys farreri,PCF)抑制UVA诱导HaCaT细胞凋亡的分子机制。方法采用琼脂糖凝胶电泳检测细胞凋亡;RT-PCR和DNA测序法检测胞内表皮生长因子受体EGFR的mRNA表达及基因变化;蛋白印迹法检测AKT,p-AKT,CyclinD1及CDK-4的蛋白表达水平。结果EGFR抑制剂AG1478和AKT抑制剂PHZ1023均可阻断UVA引起的细胞凋亡;1.42～5.68mmol.L-1范围内的PCF可抑制UVA辐射后细胞内EGFR的表达量。预先加入AG1478和PHZ1023则分别抑制UVA引起的AKT及CyclinD1,CDK-4蛋白水平的表达。结论PCF可以通过阻断EGFR-CDK-4通路来抑制UVA诱导的HaCaT细胞凋亡。

Objective To investigate whether polypeptide from Chlamys farreri （PCF） protects HaCaT cells from apoptosis induced by 8 J.cm-2UVA irradiation and explore related molecular mechanisms from EGFR to CDK-4 signaling pathway.Methods The apoptosis was analyzed by agarose gel electrophoresis.A DNA fragmentation assay for genetic transformation,detection of EGFR by RT-PCR.AKT,phosphorylated AKT,activated CyclinD1,and CDK-4 were investigated by western blotting.Results The results showed that EGFR inhibitor AG1478 and AKT inhibitor PHZ1023,corresponding the inhibitor of EGFR and AKT could significantly prevent UVA-induced apoptosis of HaCaT cells.PCF not only strongly reduced the AKT production,but also diminished expression of phosphorylated AKT,CyclinD1 and CDK4 in HaCaT cells radiated by UVA in a dose-dependent manner.Pretreatment with AG1478 PHZ1023 was found to effectively prohibit UVA-induced apoptosis,and AG1478 markedly blocked phosphorylation of AKT.Conclution PCF obviously protects HaCaT cells from apoptosis induced by UVA and protective effects may attribute to decreasing intracellular EGFR level and blocking EGFR/CDK-4 apoptotic signaling pathway.