摘要
背景：与内质网（ER）应激相关的细胞凋亡涉及多种肝脏疾病，包括肝纤维化、非酒精性脂肪肝和肝硬化。肝细胞通过引发未折叠蛋白反应（UPR）和增强自噬来应对ER应激。自噬是维持ER正常功能的关键机制，它通过降解受损的ER片段和清除ER腔中的异常蛋白质聚集体来实现。如果不能通过自噬恢复ER平衡，就会对肝细胞造成危害，并导致与ER应激相关的细胞凋亡。最近的研究结果表明，C/EBP同源蛋白（CHOP）可通过下调自噬作用加剧ER应激相关的细胞凋亡，但其潜在机制仍难以确定。目的：研究CHOP对ER应激诱导的大鼠肝细胞凋亡的影响及其潜在的分子机制：方法：用雷帕霉素（RAP）和 3-甲基腺嘌呤预处理 BRL-3A 细胞，然后用二硫苏糖醇（DTT）处理。分别使用实时细胞分析（RTCA）和流式细胞仪检测细胞的生长率和凋亡率。ER应激相关分子水平通过Western印迹法测定。使用 CHOP、小干扰 RNA 和慢病毒载体系统转染 BRL-3A 细胞，观察 CHOP 基因沉默或过表达对自噬和细胞凋亡的影响。染色质免疫共沉淀（ChIP）被用来确认CHOP是否直接与ER应激下的ATG12、ATG5和LC3启动子区域结合：结果：ER应激相关分子在BRL-3A肝细胞中急剧上调，肝细胞凋亡增加。RAP 预处理明显降低了 DTT 诱导的 ER 应激相关分子的表达；相反，3-MA 预处理促进了 DTT 诱导的 ER 应激相关凋亡分子的水平。随着肝细胞中 CHOP 表达的减少，自噬相关分子的水平急剧上升，DTT 诱导的肝细胞凋亡也随之减少。然而，在过表达 CHOP 的细胞中却观察到了相反的趋势。通过 ChIP 检测发现，DTT 处理后，CHOP 对 BRL-3A 细胞中的 ATG12、ATG5 和 LC3 等自噬相关分子有负向调节作用：结论：CHOP在ER应激过程中的增强抑制了自噬，促进了肝细胞凋亡；然而，CHOP基因表达的减少可减轻DTT诱导的肝细胞凋亡。过表达 CHOP 会加重 DTT 诱导的肝细胞凋亡。Background: Endoplasmic reticulum (ER) stress-associated apoptosis is involved in various liver diseases, including liver fibrosis, nonalcoholic fatty liver disease, and cirrhosis. Hepatocytes respond to ER stress by eliciting unfolded protein response (UPR) and enhancing autophagy. Autophagy is a pivotal mechanism for sustaining normal ER function, through degradation of damaged ER fragments and removal of abnormal protein aggregates in the ER lumen. Failure to restore ER homeostasis via autophagy is harmful to hepatocytes and contributes to ER stress-associated apoptosis. Recent findings indicated that C/EBP homologous protein (CHOP) could exacerbate ER stress-related apoptosis by down-regulating autophagy, but the underlying mechanism remains elusive.
Aim: To investigate the impact of CHOP on ER stress-induced apoptosis in rat hepatocytes, and the potential molecular mechanisms.
Methods: BRL-3A cells were pre-treated with rapamycin (RAP) and 3-methyladenine, then treated with dithiothreitol (DTT). Growth and apoptotic rates were detected using real-time cellular analysis (RTCA) and flow cytometry, respectively. ER stress-associated molecule levels were determined via western blotting. CHOP, small interfering RNA, and the lentivirus vector system were used to transfect BRL-3A cells and observe the impact of CHOP gene silencing or overexpression on autophagy and apoptosis. Chromatin immunoprecipitation (ChIP) was used to confirm whether CHOP binds directly to ATG12, ATG5, and LC3 promotor regions undergoing ER stress.
Results: ER stress-associated molecules were dramatically upregulated in BRL-3A hepatocytes and hepatocyte apoptosis was augmented. RAP pre-treatment significantly reduced DTT-induced expression of ER stress-associated molecules; conversely, 3-MA pre-treatment promoted DTT-induced levels of ER stress-associated apoptotic molecules. Following the decreased CHOP expression in hepatocytes, the level of autophagy-associated molecules dramatically increased, and DTT-induced hepatocyte apoptosis decreased. However, opposite trends were observed in CHOP overexpression cells. A negative regulation of CHOP on autophagy-associated molecules including ATG12, ATG5, and LC3 in BRL-3A cells upon DTT treatment was detected via ChIP.
Conclusion: CHOP enhancement during ER stress inhibits autophagy and promotes hepatocyte apoptosis; however, the decreased CHOP gene expression could attenuate DTT-induced hepatocyte apoptosis. Overexpression of CHOP could aggravate DTT-induced hepatocyte apoptosis.
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