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

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(1 μmol/L，2 μmol/L，4 μmol/L，8 μmol/L，16 μmol/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。

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

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 (1 μmol/L, 2 μmol/L, 4 μmol/L, 8 μmol/L, 16 μmol/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.