New-Aaron Moses, Ganesan Murali, Dagur Raghubendra Singh, Kharbanda Kusum K, Poluektova Larisa Y, Osna Natalia A
Department of Environmental, Agriculture and Occupational Health, College of Public Health, University of Nebraska Medical Center, Omaha, NE 68105, United States.
Research Service, Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, NE 68105, United States.
World J Hepatol. 2020 Nov 27;12(11):965-975. doi: 10.4254/wjh.v12.i11.965.
The morbidity and mortality of human immunodeficiency virus (HIV)-infection is often associated with liver disease, which progresses slowly into severe liver dysfunction. There are multiple insults which exacerbate HIV-related liver injury, including HIV-associated dysregulation of lipid metabolism and fat turnover, co-infections with hepatotropic viruses and alcohol abuse. As we reported before, exposure of hepatocytes to HIV and alcohol metabolites causes high oxidative stress, impairs proteasomal and lysosomal functions leading to accumulation of HIV in these cells, which end-ups with apoptotic cell death and finally promotes development of liver fibrosis.
To study whether obeticholic acid (OCA) prevents HIV/ethanol metabolism-induced hepatotoxicity and subsequent activation of hepatic stellate cells (HSC) by HIV apoptotic hepatocyte engulfment.
Huh7.5-CYP (RLW) cells were exposed to HIV and acetaldehyde-generating system (AGS) in the presence or absence of OCA. In the cells, we measured the expression of HIV-related markers: HIVgagRNA-by real-time polymerase chain reaction (PCR), p24- by western blot, HIV DNA-by semi-nested PCR, integrated HIV DNA-by ddPCR. Lysosomal and proteasomal activities were measured using fluorometrically-labeled substrates. For hepatocyte apoptosis, cleaved caspase 3 and cleaved PARP were visualized by western blot and cytokeratin 18- by M30 ELISA-in supernatants. Apoptotic bodies were generated from untreated and HIV-treated RLW cells exposed to UV light. Pro-fibrotic activation of HSC was characterized by Col1A1 and transforming growth factor-β mRNAs, while inflammasome activation- by NLRP3, caspase 1, interleukin (IL)-6, IL-1β mRNA levels.
In RLW cells, OCA treatment attenuated HIV-AGS-induced accumulation of HIVgagRNA, HIV DNA and p24. OCA suppressed reactive oxygen species production and restored chymotrypsin-like proteasome activity as well as cathepsin B lysosome activity. OCA also decreased HIV-AGS-triggered apoptosis in RLW cells. Exposure of HIV-containing apoptotic hepatocytes to HSC prevented activation of inflammasome and induced pro-fibrotic activation in these cells.
We conclude that by suppressing oxidative stress and restoring proteasomal and lysosomal functions impaired by HIV and ethanol metabolism, OCA decreases accumulation of HIV in hepatocytes, leading to down-regulation of apoptosis in these cells. In addition, OCA reverses pro-fibrotic and inflammasome-related activation of HSC triggered by engulfment of HIV-containing apoptotic hepatocytes, potentially contributing to suppression of liver fibrosis development.
人类免疫缺陷病毒（HIV）感染的发病率和死亡率通常与肝脏疾病相关，肝脏疾病会缓慢进展为严重的肝功能障碍。有多种因素会加剧与HIV相关的肝损伤，包括HIV相关的脂质代谢和脂肪周转失调、与嗜肝病毒的合并感染以及酒精滥用。正如我们之前报道的，肝细胞暴露于HIV和酒精代谢产物会导致高氧化应激，损害蛋白酶体和溶酶体功能，导致HIV在这些细胞中积累，最终导致细胞凋亡死亡，并最终促进肝纤维化的发展。
研究奥贝胆酸（OCA）是否能预防HIV/乙醇代谢诱导的肝毒性以及随后因HIV凋亡肝细胞被吞噬而导致的肝星状细胞（HSC）激活。
在有或没有OCA的情况下，将Huh7.5-CYP（RLW）细胞暴露于HIV和乙醛生成系统（AGS）。在这些细胞中,我们检测了HIV相关标志物的表达：通过实时聚合酶链反应（PCR）检测HIVgagRNA、通过蛋白质印迹法检测p24、通过半巢式PCR检测HIV DNA、通过数字滴度PCR检测整合的HIV DNA。使用荧光标记的底物测量溶酶体和蛋白酶体活性。对于肝细胞凋亡，通过蛋白质印迹法观察裂解的半胱天冬酶3和裂解的聚（ADP-核糖）聚合酶，并通过M30 ELISA检测上清液中的细胞角蛋白18。从未经处理和经HIV处理的RLW细胞暴露于紫外线下产生凋亡小体。HSC的促纤维化激活通过Col1A1和转化生长因子-β mRNA来表征，而炎性小体激活则通过NLRP3、半胱天冬酶1、白细胞介素（IL）-6、IL-1β mRNA水平来表征。
在RLW细胞中，OCA处理减弱了HIV-AGS诱导的HIVgagRNA、HIV DNA和p24的积累。OCA抑制了活性氧的产生，并恢复了类胰凝乳蛋白酶样蛋白酶体活性以及组织蛋白酶B溶酶体活性。OCA还减少了HIV-AGS触发的RLW细胞凋亡。将含有HIV的凋亡肝细胞暴露于HSC可防止炎性小体激活，并诱导这些细胞中的促纤维化激活。
我们得出结论，通过抑制氧化应激并恢复因HIV和乙醇代谢而受损的蛋白酶体和溶酶体功能，OCA减少了HIV在肝细胞中的积累，从而导致这些细胞中凋亡的下调。此外，OCA逆转了因吞噬含有HIV的凋亡肝细胞而触发的HSC的促纤维化和炎性小体相关激活，这可能有助于抑制肝纤维化的发展。
