Department of Gastroenterology and Hepatology, Austin Hospital, Heidelberg, Victoria, Australia.
Department of Medicine, Austin Hospital, University of Melbourne, Heidelberg, Victoria, Australia.
J Gen Virol. 2014 Oct;95(Pt 10):2204-2215. doi: 10.1099/vir.0.065862-0. Epub 2014 Jun 27.
Chronic hepatitis C virus (HCV) infection results in progressive liver fibrosis leading to cirrhosis and liver cancer. The mechanism for this remains unclear but hepatocyte apoptosis is thought to play a major role. Hepatocyte apoptosis in human liver tissue was determined by immunohistochemistry for cytokeratin 18 (M30 CytoDEATH) and cleaved poly(ADP-ribose) polymerase (PARP). In vitro studies were performed with replication-defective recombinant adenoviruses expressing HCV proteins (rAdHCV) to study the effects of HCV on cell death in Huh7 cells, primary mouse hepatocytes (PMoHs) and primary human hepatocytes (PHHs). Cell viability and apoptosis were studied using crystal violet assays and Western blots probed for cleaved caspase-3 and cleaved PARP, with and without treatment with the pan-caspase inhibitor Q-VD-OPh and necrostatin-1. Liver tissue of HCV-infected patients expressed elevated levels of apoptotic markers compared with HCV-negative patients. rAdHCV infection reduced cell viability compared with uninfected controls and cells infected with control virus (rAdGFP). Huh7, PMoHs and PHHs infected with rAdHCV showed significantly increased levels of apoptotic markers compared with uninfected controls and rAdGFP-infected cells. In rAdHCV-infected Huh7, treatment with Q-VD-OPh and necrostatin-1 both improved cell viability. Q-VD-Oph also reduced cleaved PARP in rAdHCV-infected Huh7 and PMoHs. Hepatocyte apoptosis is known to be increased in the livers of HCV-infected patients. HCV promoted cell death in primary and immortalized hepatocytes, and this was inhibited by Q-VD-OPh and necrostatin-1. These findings indicate that HCV-induced cell death occurs by both apoptosis and necroptosis, and provide new insights into the mechanisms of HCV-induced liver injury.
慢性丙型肝炎病毒（HCV）感染可导致进行性肝纤维化，进而发展为肝硬化和肝癌。其机制尚不清楚，但肝细胞凋亡被认为起主要作用。通过细胞角蛋白 18（M30 CytoDEATH）和聚（ADP-核糖）聚合酶（PARP）的免疫组织化学法测定人肝组织中的肝细胞凋亡。使用表达 HCV 蛋白的复制缺陷型重组腺病毒（rAdHCV）进行体外研究，以研究 HCV 对 Huh7 细胞、原代小鼠肝细胞（PMoHs）和原代人肝细胞（PHHs）中细胞死亡的影响。使用结晶紫测定法和 Western blot 检测 caspase-3 裂解和 PARP 裂解来研究细胞活力和凋亡，同时用泛半胱天冬酶抑制剂 Q-VD-OPh 和 necrostatin-1 进行处理。与 HCV 阴性患者相比，HCV 感染患者的肝组织表达更高水平的凋亡标志物。与未感染对照和感染对照病毒（rAdGFP）的细胞相比，rAdHCV 感染降低了细胞活力。与未感染对照和 rAdGFP 感染的细胞相比，rAdHCV 感染的 Huh7、PMoHs 和 PHHs 显示出明显增加的凋亡标志物水平。在 rAdHCV 感染的 Huh7 中，用 Q-VD-OPh 和 necrostatin-1 处理均可改善细胞活力。Q-VD-OPh 还降低了 rAdHCV 感染的 Huh7 和 PMoHs 中的裂解 PARP。已知 HCV 感染患者的肝内肝细胞凋亡增加。HCV 在原代和永生化肝细胞中促进细胞死亡，并且这种作用被 Q-VD-OPh 和 necrostatin-1 抑制。这些发现表明，HCV 诱导的细胞死亡既通过凋亡也通过坏死发生，并为 HCV 诱导的肝损伤的机制提供了新的见解。
