肝再生增强因子调控线粒体功能稳态在非酒精性脂肪肝炎中的作用研究

目的：研究非酒精性脂肪性肝炎 (non-alcoholic steatohepatitis, NASH) 中线粒体内肝再生增强因子（Augmenter of liver regeneration, ALR）与线粒体功能障碍之间的关系.方法：以不含脂肪酸的BSA处理作为对照组（BSA组），利用0.2mM棕榈酸（Palmitic acid, PA）体外刺激正常人肝细胞系L-O2 24h诱导NASH模型（PA组），通过油红染色、检测细胞活力、乳酸脱氢酶（Lactate dehydrogenase，LDH）释放等指标验证细胞模型。通过质粒转染的方式构建肝再生增强因子过表达的细胞(ALR-OE组)以及空白对照组细胞(Vector组),两组细胞分别接受BSA处理和PA处理。CCK8法检测细胞活力、乳酸脱氢酶检测试剂盒检测脂毒性、JC-1染色分析线粒体膜电位、流式凋亡检测分析细胞凋亡比例以及Western Bolt检测ALR、Bax、Bcl-2、Cytochrome C等蛋白表达水平。结果：与BSA组相比, PA处理24h后细胞内脂滴堆积明显增多、细胞活力下降约50%、乳酸脱氢酶释放也增加了13倍，与此同时线粒体内ALR的表达则显著降低。此外，在BSA处理条件下Vector组和ALR-OE组脂毒性、线粒体功能以及凋亡等方面均无明显差异。而在PA刺激条件下，ALR-OE组相较于Vector组表现出对PA更强的耐受性:细胞活力增加了约16%，LDH释放则减少了约40%，另外受损的线粒体膜电位也明显恢复、细胞色素C的释放减少，同时细胞凋亡比例由19%下降至11.4%。结论：ALR参与了PA诱导的NASH的发生的过程，靶向调控线粒体内ALR的表达可以阻止NASH的发生，其作用的机制可能是通过恢复线粒体功能稳态进而抑制线粒体依赖性的凋亡而实现的。

The role of Augmenter of liver regeneration mediating regulation of mitochondrial homeostasis in nonalcoholic steatohepatitis

Objective: To study the relationship between mitochondrial liver regeneration (ALR) and mitochondrial dysfunction in non-alcoholic steatohepatitis (NASH).Method: The fatty acid-free BSA treatment was used as a control group (BSA group), and the normal human liver cell line L-O2 was induced by 0.2 mM palmitic acid (PA) to induce the NASH model (PA group) in vitro. Cell models were validated by Oil red staining, cell viability measuring, Lactate dehydrogenase (LDH) release and other indicators. The cells overexpressing the liver regeneration enhancing factor (ALR-OE group) and the blank control cells (Vector group) were constructed by plasmid transfection, and the two groups of cells were subjected to BSA treatment and PA treatment, respectively. CCK8 assay detects cell viability, lactate dehydrogenase assay kit detects the lipotoxicity, JC-1 staining assays mitochondrial membrane potential, flow apoptosis assays the apoptosis ratio, and Western Bolt for ALR, Bax, Bcl-2, Cytochrome C The level of protein expression. Results: Compared with the BSA group, the intracellular lipid droplet accumulation increased significantly, the cell viability decreased by about 50%, and the lactate dehydrogenase release increased by 13 times compared with the BSA group. At the same time, the expression of ALR in the mitochondria was significantly decreased. In addition, there were no significant differences in lipid toxicity, mitochondrial function, and apoptosis between the Vector group and the ALR-OE group under BSA treatment conditions. Under PA stimulation conditions, the ALR-OE group showed stronger tolerance to PA than the Vector group: cell viability increased by about 16%, LDH release decreased by about 40%, and damaged mitochondrial membrane. The potential was also significantly restored, the release of cytochrome C was reduced, and the proportion of apoptosis decreased from 19% to 11.4%. Conclusion: ALR is involved in the process of PA-induced NASH, and targeting the regulation of ALR expression in mitochondria can prevent the occurrence of NASH. The mechanism may be achieved by restoring mitochondrial homeostasis and inhibiting mitochondria-dependent apoptosis.