蛇床子素改善小鼠实验性肝损伤机制的研究

Study on mechanism of osthole in improving experimental liver injury in mice

目的探讨蛇床子素对四氯化碳(CCl_(4))诱导的实验性小鼠肝损伤的作用及其相关机制。方法18只C57BL/6小鼠随机分成3组:①空白对照组:小鼠每天给予80μL玉米油腹腔注射;②CCl_(4)造模组:给小鼠腹腔注射CCl_(4)(0.07 g/kg),1周3次,共持续3周,诱发肝组织损伤;③蛇床子素组:在小鼠腹腔注射CCl_(4)(0.07 g/kg)3周后,提前24 h给予小鼠腹腔注射蛇床子素(0.1 g/kg),共1次。摘取小鼠眼球提取血浆,测定转氨酶指标谷丙转氨酶(ALT)、谷草转氨酶(AST)的表达水平,了解肝脏损伤情况;提取小鼠肝组织,检测丙二醛(MDA)、谷胱甘肽(GSH)氧化应激指标的变化情况;采用实时荧光定量聚合酶链式反应(real-time PCR)检查小鼠肝组织炎症指标:白介素-1β(interleukin,IL-1β)、IL-6以及肿瘤坏死因子α(tumor necrosis factor-alpha,TNF-α)mRNA的表达情况;利用蛋白质免疫印迹(Western blot)技术观测肝组织AMPK蛋白磷酸化水平情况。结果CCl_(4)作用后小鼠转氨酶指标AST和ALT明显升高(P<0.01),氧化应激MDA水平显著上调(P<0.01),GSH生成受到抑制(P<0.01),炎症因子IL-1β、IL-6和TNF-αmRNA的表达水平升高(P<0.01),蛇床子素处理后这些指标明显逆转(P<0.05或P<0.01),表明肝功能明显改善。CCl_(4)作用后肝组织AMPK磷酸化显著增强,蛇床子素可以抑制该蛋白的磷酸化(P<0.05)。结论蛇床子素可以改善CCl_(4)引起的肝损伤:具体原因可能与减轻氧化应激,下调炎症因子表达有关,相关信号通路可能与其抑制AMPK的磷酸化有关。

Objective To investigate the effects of osthole on liver injury induced by carbon tetrachloride(CCl_(4))in mice and its related mechanism.Methods Eighteen C57BL/6 mice were randomly divided into 3 groups:①Blank control group:mice were intraperitoneally injected with 80μL corn oil every day;②CCl_(4) model group:mice were intraperitoneally injected with CCl_(4)(0.07 g/kg)three times a week for 3 weeks to induce liver tissue injury;③Osthole group:After three-week intraperitoneal injection with CCl_(4)(0.07 g/kg),mice were intraperitoneally injected with osthole(0.1 g/kg)24 h in advance,once in total.And then the eyeballs of the mice were extracted for plasma extraction,and the expression levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were measured to understand the liver injury.Mice liver tissues were extracted to detect the changes of oxidative stress indexes,malondialdehyde(MDA)and glutathione(GSH).Real time fluorescence quantitative polymerase chain reaction(real-time PCR)was used to detect the inflammatory indexes of mice liver tissues:the mRNA expression of interleukin-1β(IL-1β),IL-6 and tumor necrosis factor-α(TNF-α).The phosphorylation of AMPK protein in liver tissues was detected by Western blot.Results After CCl_(4) treatment,AST and ALT significantly increased(P<0.01);MDA level in oxidative stress significantly increased(P<0.01);GSH production was inhibited(P<0.01),and the mRNA expression levels of inflammatory factors IL-1β,IL-6 and TNF-αincreased(P<0.01).These indexes were significantly reversed after osthole treatment(P<0.05 or P<0.01),indicating that liver function was significantly improved.The phosphorylation of AMPK in liver tissue was significantly enhanced after CCl_(4) treatment,and osthole could inhibit the phosphorylation of AMPK protein(P<0.05).Conclusion Osthole can reduce CCl_(4) induced liver injury.The specific reasons may be related to reducing oxidative stress,down-regulating the expression of inflammatory factors,and associated signaling pathways may be related to its inhibition of AMPK phosphorylation.