内质网应激通过C/EBP同源蛋白调控死亡受体5对肝星状细胞凋亡的影响

Endoplasmic reticulum stress-induced apoptosis up-regulation of DR5 via a CHOP-dependent mechanism in HSC-T6 cells

目的:研究内质网应激(ERS)与肿瘤坏死因子相关凋亡诱导配体(TRAIL)对肝星状细胞凋亡的影响以及在凋亡过程中二者相互关系。方法:以大鼠肝星状细胞HSC-T6为研究对象,使用毒胡萝卜素作为内质网应激诱导剂,熊去氧胆酸作为内质网应激抑制剂,SP600125作为c-Jun氨基末端激酶(JNK)抑制剂,将HSC-T6分成正常组、DMSO组、TRAIL组、毒胡萝卜素组、熊去氧胆酸组、siCHOP组及SP600125组。利用流式法检测毒胡萝卜素诱导HSC-T6凋亡程度;应用小分子RNA干扰技术沉默CHOP基因;免疫组化法检测Caspase-8表达;RT-PCR与Western blot法检测ERS标志性蛋白C/EBP同源蛋白(CHOP)及肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis inducing ligand,TRAIL)受体死亡受体5(DR5)的表达。结果:随着毒胡萝卜素浓度增加(1μmol/L,2μmol/L,4μmol/L,8μmol/L,16μmol/L),可诱导HSC-T6发生不同程度的凋亡。RT-PCR与Western blot结果表明ERS标志性蛋白CHOP可诱导TRAIL受体DR5及Caspase-8表达上调;同时,siCHOP及JNK抑制剂SP600125的应用,均可使HSC细胞中DR5及下游Caspase-8的表达随之减少。结论:CHOP和JNK可能是调节DR5表达的潜在因子,两者在诱导肝星状细胞凋亡的过程中发挥着重要的作用。

AIM:To investigate the inhibitory effects of endoplasmic reticulum stress(ERS)and TRAIL on hepatic stellate cells in vitro and how their interaction affect the apoptosis of hepatic stellate cells.METHODS:Take thapsigargin(TG)as the endoplasmic reticulum stress-inducing agents,ursodeoxycholic acid(UDCA)for the endoplasmic reticulum stress inhibitors,SP600125 as a c-Jun N-terminal kinase(JNK)inhibitor,HSC-T6 cells were divided into normal control group,DMSO group,TRAIL group,TG group,UDCA group,siCHOP group and SP600125 group.The apoptosis rate of HSC-T6 cell was detected by flow cytometry.Small interference RNA was applied to silence C/EBP homologous protein(CHOP)gene.The protein expression levels of Caspase-8 were detected by immunohistochemistry method.The ERS marker protein CHOP and TRAIL receptor DR5 expression levels were determined by RT-PCR and Western blot.RESULTS:TG(1μmol/L,2μmol/L,4μmol/L,8μmol/L,16μmol/L)increased cell apoptosis rate of HSC-T6.RT-PCR and Western blot showed that the endoplasmic reticulum stress protein marker CHOP could induce the upregulation of TRAIL receptor DR5 and Caspase-8.Moreover,siCHOP and the JNK inhibitor SP600125 could reduce the expression of DR5 and Caspase-8 in HSC cells.CONCLUSION:These results indicated that CHOP and JNK may be a potential factor regulating DR5 expression,and play an important role in the process of apoptosis of hepatic stellate cells.