The Third Unit, The Department of Hepatology, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China.
Beijing Institute of Hepatology, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China.
Int J Mol Sci. 2023 Jun 2;24(11):9681. doi: 10.3390/ijms24119681.
Patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 polymorphism (I148M) is strongly associated with non-alcoholic steatohepatitis and advanced fibrosis; however, the underlying mechanisms remain largely unknown. In this study, we investigated the effect of PNPLA3-I148M on the activation of hepatic stellate cell line LX-2 and the progression of liver fibrosis. Immunofluorescence staining and enzyme-linked immunosorbent assay were used to detect lipid accumulation. The expression levels of fibrosis, cholesterol metabolism, and mitochondria-related markers were measured via real-time PCR or western blotting. Electron microscopy was applied to analyze the ultrastructure of the mitochondria. Mitochondrial respiration was measured by a Seahorse XFe96 analyzer. PNPLA3-I148M significantly promoted intracellular free cholesterol aggregation in LX-2 cells by decreasing cholesterol efflux protein (ABCG1) expression; it subsequently induced mitochondrial dysfunction characterized by attenuated ATP production and mitochondrial membrane potential, elevated ROS levels, caused mitochondrial structural damage, altered the oxygen consumption rate, and decreased the expression of mitochondrial-function-related proteins. Our results demonstrated for the first time that PNPLA3-I148M causes mitochondrial dysfunction of LX-2 cells through the accumulation of free cholesterol, thereby promoting the activation of LX-2 cells and the development of liver fibrosis.
载脂蛋白样磷脂酶域蛋白 3（PNPLA3）rs738409 多态性（I148M）与非酒精性脂肪性肝炎和肝纤维化进展密切相关；然而，其潜在机制在很大程度上仍不清楚。在这项研究中，我们研究了 PNPLA3-I148M 对肝星状细胞系 LX-2 激活和肝纤维化进展的影响。免疫荧光染色和酶联免疫吸附试验用于检测脂质积累。通过实时 PCR 或 Western blot 测量纤维化、胆固醇代谢和线粒体相关标志物的表达水平。电子显微镜用于分析线粒体的超微结构。通过 Seahorse XFe96 分析仪测量线粒体呼吸。PNPLA3-I148M 通过降低胆固醇外排蛋白（ABCG1）的表达，显著促进 LX-2 细胞内游离胆固醇聚集，随后诱导线粒体功能障碍，表现为 ATP 产生和线粒体膜电位减弱、ROS 水平升高、线粒体结构损伤、耗氧率改变以及线粒体功能相关蛋白表达降低。我们的研究结果首次表明，PNPLA3-I148M 通过游离胆固醇的积累导致 LX-2 细胞的线粒体功能障碍，从而促进 LX-2 细胞的激活和肝纤维化的发展。
