川芎嗪通过AMPK/SIRT1通路介导的自噬在急性肝衰竭中的作用及其机制研究

Effect and mechanism of ligustrazine in acute liver failure mediated by AMPK/SIRT1 pathway

目的 探讨川芎嗪对脂多糖(LPS)/D-氨基半乳糖(D-GalN)诱导的急性肝衰竭小鼠的作用及其机制。方法 将C57BL/6J小鼠随机分对照组、模型组、川芎嗪组、川芎嗪联合SIRT1抑制剂(EX527)组,每组10只。通过ip LPS/D-GalN构建急性肝衰竭小鼠模型,检测血清中天冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)的水平;苏木精–伊红(HE)染色检测肝组织病理学变化;实时荧光定量PCR(RT-qPCR)检测肝组织中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、IL-1β和环氧化酶-2(COX-2)mRNA水平;检测肝组织中氧化应激因子谷胱甘肽(GSH)、丙二醛(MDA)和超氧化物歧化酶(SOD)含量;Western blotting检测肝组织中沉默调节蛋白1(SIRT1)、磷酸化单磷酸腺苷活化蛋白激酶α(p-AMPK)α/AMPKα蛋白和自噬相关蛋白人微管相关蛋白1轻链3(LC3)-II/LC3-I、p62的水平。结果 与模型组比较,川芎嗪组小鼠血清ALT和AST水平均显著降低,肝细胞坏死区域以及炎症细胞浸润明显减少,肝组织中MDA、TNF-α、IL-6、IL-1β、COX-2 mRNA和p62蛋白水平均显著降低,SOD、GSH、SIRT1、p-AMPKα/AMPKα、LC3-II/LC3-I蛋白水平显著升高(P<0.05)。EX527处理后显著逆转了川芎嗪对急性肝衰竭小鼠的作用。结论 川芎嗪通过抑制LPS/D-GalN诱导的急性肝衰竭小鼠炎症反应发挥肝脏保护作用,其机制可能与AMPK/SIRT1信号通路和自噬有关。

Objective To investigate the effect of ligustrazine on lipopolysaccharide(LPS)/D-galactose(D-GalN)-induced acute liver failure in mice, and its mechanism. Methods C57BL/6J mice were randomly divided into control group, model group, ligustrazine group, and ligustrazine + SIRT1 inhibitor(EX527) group, each group had 10 mice. The mice model of acute liver failure was established by ip LPS/D-GalN, and the serum levels of aspartate aminotransferase(AST) and alanine aminotransferase(ALT) were detected in each group. Hematoxylin and eosin(HE) staining was used to detect the pathological changes of liver tissue in each group. mRNA levels of tumor necrosis factor-α(TNF-α), interleukin(IL)-6, IL-1β, and cyclooxygenase-2(COX-2) in liver tissue of each group were detected by real-time fluorescent quantitative PCR(RT-qPCR). Contents of oxidative stress factors glutathione(GSH),malondialdehyde(MDA), and superoxide dismutase(SOD) in liver tissue were detected. Western blotting was used to detect the levels of silencing regulatory protein 1(SIRT1), phosphorylated adenosine monophosphate activated protein kinase(p-AMPK) α/AMPKαprotein and autophagy-related proteins human microtubule-associated protein 1 light chain 3(LC3)-II/LC3-I and p62 in liver tissue of mice in each group. Results Compared with the model group, the serum levels of ALT and AST in ligustrazine group were significantly decreased, the necrosis area of hepatocytes and the infiltration of inflammatory cells were significantly decreased, and the mRNA and protein levels of MDA, TNF-α, IL-6, IL-1β, and COX-2 in liver tissue were significantly decreased. Protein levels of SOD, GSH, SIRT1, P-AMPKα/AMPKα, and LC3-II/LC3-I were significantly increased(P < 0.05). EX527 treatment significantly reversed the effect of ligustrazine on acute liver failure mice. Conclusion Ligustrazine exerts hepatic protection by inhibiting the inflammatory response in acute liver failure mice induced by LPS/D-GalN, and its mechanism may be related to AMPK/SIRT1signaling pathway and autophagy.