基于mTOR信号通路研究黄芩苷对对乙酰氨基酚诱导肝损伤后肝修复作用

Effect of baicalin on liver repair after acetaminophen-induced liver injury based on mTOR signaling pathway

目的探讨黄芩苷对对乙酰氨基酚(acetaminophen,APAP)诱导的肝损伤后肝再生的作用及机制。方法C57BL/6小鼠随机分成对照组、模型组和黄芩苷(40、80 mg/kg)组,小鼠ip APAP(300 mg/kg)1 h后ig黄芩苷,ip APAP 24 h或48 h后检测血清中天冬氨酸氨基转移酶(aspartate aminotransferase,AST)、丙氨酸氨基转移酶(alanine aminotransferase,ALT)活性;通过苏木素-伊红(HE)染色法观察肝脏的病理变化;采用q RT-PCR检测肝脏组织中肝细胞生长因子(hepatocyte growth factor,HGF)和表皮生长因子(epidermal growth factor,EGF)mRNA表达;通过BeyoClick^(TM)EdU法检测肝脏组织细胞增殖情况;通过免疫组化染色法观察肝脏组织中增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)阳性细胞数量;通过Western blotting检测肝脏组织中PCNA、细胞周期蛋白D1(Cyclin D1)、p62、微管相关蛋白1轻链3(microtubuleassociated protein 1 light chain,LC3B)、磷酸化哺乳动物雷帕霉素靶蛋白(phosphorylated mammalian target of rapamycin,pm TOR)和p-S6蛋白的表达。给予mTOR抑制剂雷帕霉素后,观察雷帕霉素对黄芩苷促进肝修复作用的影响。结果黄芩苷可以显著减少APAP诱导的急性肝损伤小鼠的肝坏死面积(P<0.05、0.01),上调肝脏HGF、EGF mRNA表达和PCNA、Cyclin D1、p62、p-S6、p-mTOR蛋白表达(P<0.05、0.01),下调LC3B蛋白表达(P<0.05、0.01),从而激活mTOR信号通路,促进APAP诱导的肝损伤后肝再生。此外,黄芩苷对肝再生的促进作用在给予m TOR抑制剂雷帕霉素后减弱(P<0.05)。结论黄芩苷能够通过激活mTOR信号通路,促进肝细胞增殖,并抑制其介导的自噬,从而促进肝修复。

Objective To explore the effect and mechanism of baicalin on liver regeneration after acetaminophen(APAP)-induced liver injury.Methods C57BL/6 mice were randomly divided into control group,model group and baicalin(40,80 mg/kg)groups.After 1 h of ip APAP(300 mg/kg),mice were given baicalin,activities of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)in serum were measured after 24 h or 48 h of ip APAP;The pathological changes of liver tissue was observed using hematoxylin eosin(HE)staining method;qRT-PCR was used to detect hepatocyte growth factor(HGF)and epidermal growth factor(EGF)mRNA expressions in liver tissue;BeyoClick^(TM)EdU method was used to detect the cell proliferation in liver tissue;The proliferating cell nuclear antigen(PCNA)positive cells in liver tissue was observed using immunohistochemistry staining method;The expressions of PCNA,Cyclin D1,p62,microtubule associated protein 1 light chain 3(LC3B),phosphorylated mammalian target of rapamycin(p-mTOR)and p-S6 proteins in liver tissue were detected using Western blotting.After administering mTOR inhibitor rapamycin,the effect of rapamycin on the promotion of liver repair by baicalin was observed.Results Baicalin could significantly reduce the area of liver necrosis in APAP-induced acute liver injury mice(P<0.05,0.01),up-regulate HGF,EGF mRNA expressions and PCNA,Cyclin D1,p62,p-S6 and p-mTOR protein expressions in liver tissue(P<0.05,0.01),down-regulate LC3B protein expression(P<0.05,0.01),thereby activating the mTOR signaling pathway and promoting liver regeneration after APAP-induced liver injury.In addition,the promoting effect of baicalin on liver regeneration was weakened after administration of mTOR inhibitor rapamycin(P<0.05).Conclusion Baicalin can promote liver cell proliferation and inhibit its mediated autophagy by activating mTOR signaling pathway,thereby promoting liver repair.