Fas途径参与大黄酸诱导HK-2细胞凋亡

Involvement of Fas-dependent pathway in rhein-induced apoptosis of HK-2 cells

探讨大黄酸对人肾小管上皮细胞系HK-2的毒性作用及毒性作用机制。以Hg Cl2为阳性对照,通过MTT法检测细胞活力,LDH释放实验检测细胞毒性,AnnexinⅤ-FITC/PI双染法检测细胞凋亡率,Real-Time q PCR检测Fas,Fas L,FADD,caspase-3,caspase-8的mRNA表达,Western blot检测Fas,Fas L,胞浆Cyt-c,caspase-3,caspase-8,caspase-9蛋白表达。实验结果显示:大黄酸可剂量依赖性地抑制HK-2细胞活力,增加LDH释放和细胞凋亡率,使Fas,Fas L,FADD,caspase-3,caspase-8的mRNA表达显著性上调,Fas,Fas L,胞浆Cyt-c蛋白表达量显著升高,caspase-8原型表达显著降低,caspase-3,caspase-8裂解片段表达显著增加,caspae-9表达无变化。结果证明:大黄酸体外对HK-2细胞具有毒性作用,其毒性作用机制可能是通过Fas途径诱导HK-2细胞凋亡。

To investigate the effect and mechanism of cytotoxicity by rhein 2 ceils, HgC12 was choosen as positive control. Cell viability was determined used to evaluate cell membrane damage. The activity of caspase-3, and -8 qPCR was employed to determine the mRNA expressions of Fas, FasL, in human renal tubular epithelial HK- by MTI＂ assay. LDH release assay was was measured by assay kit. Real-Time FADD, caspase-3 and caspase-8. The protein expressions of Fas, FasL, cytoplasmic cytochrome C, caspase-3, caspase-8, caspase-9 were detected by Western blot. The results demonstrated that rhein inhibited cell viability and increased LDH release in a dose- dependent manner. The activity of caspase-3 and caspase-8 was significantly enhanced by rhein. The mRNA expression of Fas, FasL, FADD, caspase-3, caspase-8 was remarkably up-regulated by rhein. Rhein also elevated protein expressions of Fas, FasL, cytoplasmic cytochrome C, cleaved caspase-3, caspase-8 and reduced expressios of Pro caspase-8. There was no significant difference in caspase-9 expression. These results indicate that rhein has a cytotoxic effect and apoptosis-inducing effect in HK-2 cells. The Fas-dependent pathway is involved in rhein- induced apoptosis.