CRISPR/Cas9沉默Fas可抑制急性肝衰竭细胞线粒体自噬及减轻线粒体损伤

CRISPR/Cas9 silencing Fas inhibits autophagy formation and mitochondrial damage in acute liver failure cells

目的 探讨凋亡相关因子(Fas/CD95)在急性肝衰竭疾病进展中的作用及机制。方法 构建刀豆蛋白A诱导的急性肝衰竭小鼠动物模型,通过透射电子显微镜及免疫印迹检测了急性肝衰竭肝细胞内的线粒体自噬发生;在小鼠尾静脉输注靶向Fas基因的CRISPR/Cas9质粒,随后予以刀豆蛋白A诱发急性肝衰竭,检测小鼠肝组织急性损伤情况及肝细胞内线粒体自噬及线粒体损伤的变化,同时体外转染靶向Fas的CRISPR/Cas质粒到细胞内,刀豆蛋白A诱导HepG2肝细胞损伤,验证Fas敲除后对线粒体自噬和线粒体损伤的影响。结果 刀豆蛋白A可诱导小鼠急性肝衰竭,透射电镜观察发现肝细胞中发生了线粒体自噬,肝细胞内p62蛋白水平降低,PINK1/Parkin的蛋白上调,Fas的表达上调;小鼠肝组织和体外肝细胞先予以PX330Fas质粒转染,再予以刀豆蛋白A诱导肝损伤后,LC3II/LC3I表达比值减低,p62蛋白的表达得到恢复,线粒体蛋白COX4I1和线粒体DNA含量的水平下降,Fas表达下调,与转染空白质粒的对照组相比,差异均有统计学意义。结论 CRISPR/Cas9沉默Fas抑制急性肝衰竭进展及肝细胞内线粒体自噬的发生和线粒体损伤,从而减轻急性肝衰竭的细胞死亡,为临床开展急性肝衰竭的新治疗策略提供了新依据和新靶点。

Objective In order to explore the role and mechanism of the death receptor Fas/CD95in the progression of acute liver failure.Method We first established,a mouse animal model of ALF induced by concanavalin A(Con A).The formation of mitophagy in hepatocyte with ALF was detected by Western blot and transmission electron microscopy.Then,Knocking out Fas by administering CRISPR/Cas9 plasmid through the tail vein of mice,and then acute liver failure of mouse was induced by Con A,liver tissue injury,the formation of mitophagy and mitochondrial damage were detected.We treated HepG2 that knocked out Fas with Con A in vitro to detect the expression of mitochondrial autophagy related proteins and mitochondrial damage.Result The results show that after acute liver failure induced by concanavalin A in mice,transmission electron microscopy observation revealed mitochondrial autophagy in liver cells,a decrease in p62 protein levels,upregulation of PINK1/Parkin protein,and upregulation of Fas expression.After transfection with PX330Fas plasmid,the expression ratio of LC3II/LC3I in mouse liver tissue and in vitro liver cells decreased,the expression of p62 protein was restored,the levels of mitochondrial protein COX4I1 and mitochondrial DNA content decreased,and Fas expression was downregulated,with significant statistical significance.Conclusion In this study,we found that CRISPR/Cas9 silencing Fas inhibits the progression of ALF,the formation of mitochondrial autophagy and mitochondrial damage in hepatocytes.