ATPR通过促进自噬缓解脂多糖诱导的小鼠急性肝损伤

ATPR alleviates lipopolysaccharide-induced acute liver injury in mice by promoting autophagy

目的研究4-氨基-2-三氟甲基苯基维甲酸酯(ATPR)对脂多糖(LPS)诱导C57BL/6小鼠急性肝损伤的影响及相关机制。方法6周龄的雄性C57BL/6品系小鼠15只随机均分为正常组、模型组和ATPR组。ATPR组小鼠腹腔注射ATPR[15 mg/(kg·d)],正常组和模型组给予溶剂,持续给药1周后,模型组和ATPR组腹腔注射LPS(6 mg/kg),6 h后处死所有小鼠。检测小鼠血清中谷丙转氨酶(ALT)和谷草转氨酶(AST)含量;qPCR检测肝脏组织白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)的mRNA水平;苏木精-伊红(HE)染色观察小鼠肝脏组织形态学变化;透射电子显微镜(TEM)观察小鼠肝细胞超微结构变化;Western blot检测线粒体损伤相关蛋白FUNDC1、OPA1和自噬相关蛋白LC3B、P62、Beclin1、ATG5的表达水平。结果与正常组相比,模型组小鼠血清中ALT、AST含量上升,肝脏组织IL-6、TNF-α的mRNA水平升高,ATPR组逆转这一变化。HE染色显示正常组小鼠肝小叶结构正常,肝索呈放射状排列,无充血和炎性细胞浸润,肝细胞边界清晰;模型组小鼠肝脏细胞间隙增大,肝索排列发生紊乱,出现炎性细胞浸润;ATPR组肝脏细胞间隙、肝索结构有所恢复,炎性细胞浸润较少。TEM显示与正常组相比,模型组小鼠肝细胞中受损线粒体和脂滴堆积,ATPR组小鼠肝细胞中线粒体形态和数量、脂滴数量趋于正常。Western blot显示与正常组相比,模型组小鼠肝脏组织中FUNDC1蛋白表达增多,OPA1蛋白表达减少,LC3BⅡ和LC3BⅠ的比值(LC3BⅡ/Ⅰ)下降,P62蛋白表达增多,Beclin1和ATG5蛋白表达减少,ATPR组逆转以上变化。结论ATPR通过促进自噬缓解脂多糖引起的小鼠急性肝损伤。

Objective To investigate the effect of 4-amino-2-trifluoromethyl-phenyl retinate(ATPR) on acute liver injury induced by lipopolysaccharide(LPS) in C57BL/6 mice and its related mechanism.Methods Fifteen 6-week-old male C57BL/6 strain mice were randomly divided into normal group,model group and ATPR group,with 5 mice in each group.Mice in the ATPR group were intraperitoneally injected with ATPR(15 mg/kg·d),and normal group and model group were given solvent.After continuous administration for one week,model group and ATPR group were intraperitoneally injected with LPS(6 mg/kg),and all mice were sacrificed 6 hours later.The contents of Alanine aminotransferase(ALT) and Aspartate aminotransferase(AST) in serum of mice were detected.The mRNA levels of Interleukin-6(IL-6) and Tumor necrosis factor-alpha(TNF-α) were detected by qPCR.Hematoxylin-eosin(H&E) staining was used to observe the histopathological changes of liver in mice.The ultrastructural changes of mouse hepatocytes were observed by Transmission electron microscope(TEM).The expression levels of mitochondrial damage-related proteins FUNDC1 and OPA1 and autophagy related proteins LC3B,P62,Beclin1 and ATG5 were detected by Western blot.Results Compared with the normal group,the content of ALT and AST in serum and the mRNA levels of IL-6 and TNF-α in liver tissue increased in the model group,and the changes were reversed in the ATPR group.H&E staining showed that the hepatic lobule structure was normal in the normal group,the hepatic cords were arranged radially,there was no hyperemia and inflammatory cell infiltration,and the hepatocyte boundary was clear.In the model group,the intercellular space of liver was enlarged,the arrangement of hepatic cords was disordered,and inflammatory cells infiltrated.In the ATPR group,the intercellular space of liver and the structure of hepatic cords were restored,and the inflammatory cell infiltration was less.TEM showed that the damaged mitochondria and lipid droplet accumulation in the hepatocytes of mice in the model group were compared with that in the normal group,and the morphology and quantity of mitochondria and lipid droplet in the hepatocytes of mice in the ATPR group tended to be normal.Western blot showed that compared with the normal group,the expression of FUNDC1 protein in the liver tissues of mice in the model group increased,the expression of OPA1 protein decreased,the ratio of LC3BⅡ to LC3BⅠ decreased,the expression of P62 protein increased,the expression of Beclin1 and ATG5 protein decreased,and the above changes were reversed in the ATPR group.Conclusion ATPR alleviates acute liver injury induced by lipopolysaccharide in mice by promoting autophagy.