大黄素诱导人肾上皮HK-2细胞凋亡及内质网应激的介导作用

Induction of apoptosis in human renal tubular epithelial HK-2 cells by emodin and mediation of endoplasmic reticulum stress

目的研究大黄素诱导人肾小管上皮HK-2细胞凋亡的作用及其机制是否与内质网应激有关。方法不同浓度大黄素处理HK-2细胞不同时间。MTT法检测细胞存活率,实时荧光定量PCR(RT-PCR)检测葡萄糖调节蛋白78(GRP78)、CCAAT/增强子结合蛋白同源蛋白(CHOP)、转录激活因子3(ATF3)和X盒结合蛋白1(XBP1)的基因表达,Western blotting法检测caspase-3、GRP78、CHOP、真核生物启动因子2α(eIF2α)的蛋白表达。结果大黄素以浓度相关方式降低HK-2细胞存活率,诱导caspase-3剪切。RT-PCR检测表明,大黄素能诱导内质网相关基因GRP78、CHOP、ATF3基因表达,Westernblottinh检测结果进一步证实大黄素能诱导GRP78、CHOP的蛋白表达。大黄素还明显诱导eIF2α磷酸化和XBP1 mRNA剪切。在大黄素给药之前给予内质网应激干扰药4-苯基丁酸和salubrinal,能增加HK-2细胞的存活率。结论大黄素能诱导HK-2凋亡,内质网应激参与了大黄素诱导HK-2细胞凋亡的作用。

Objective To investigate the induetion of apoptosis in human renal tubular epithelial HK-2 cells by emodin and whether endoplasmic reticulum（ER） stress is involved in its mechanism.Methods HK-2 cells were cultured and treated with various concentration of emodin at different time points.The cell viability was determined by MTT assay.The gene expression of glucose-regulated protein 78（GRP78）,CCAAT/enhancer-binding protein-homologous protein（CHOP）,activating transcription factor 3（ATF3）,and X-box binding protein 1 splicing（XBP1s） was evaluated by quantitative real-time PCR.The protein expression of caspase-3,GRP78,CHOP,and eukaryotic initiation factor 2 alpha（eIF2α） was detected by Western blotting.Results The viability of HK-2 cells was decreased by emodin in a dose-dependent manner,and the apoptosis of the cells was associated with caspase-3 shear activation.The treatment with emodin in HK-2 cells caused the increase in eIF2α phosphorylation,XBP1 mRNA splicing,the gene expression of GRP78,CHOP,and ATF3,and the protein expression of GRP78 and CHOP.The pretreatment with 4-phenylbutyric acid and salubrinal significantly increased the viability of HK-2 cells,indicating the role of ER stress in emodin-induced apoptosis.Conclusion Emodin induces the apoptosis in HK-2 cells and ER stress is involved in emodin-induced apoptosis.