毛果芸香碱减轻对乙酰氨基酚所致肝损伤的作用及机制研究

Effect and mechanism of pilocarpinein alleviating liver injury induced by acetaminophen(APAP)

目的 研究毛果芸香碱减轻对乙酰氨基酚(Acetaminophen, APAP)所致肝损伤的作用及机制。方法 采用小鼠腹腔注射APAP建立急性肝损伤模型,将18只C57BL/6小鼠随机分为空白对照组、APAP模型组、毛果芸香碱组,每组6只。毛果芸香碱组小鼠腹腔给药毛果芸香碱(2.5 mg/kg体重)7 d,末次给药1 h后,APAP模型组和毛果芸香碱组腹腔注射APAP(400 mg/kg体重),空白对照组腹腔注射等量生理盐水,APAP注射后12 h处死所有小鼠,采集血液和肝脏等组织样本。血清生化检测仪检测血清肝功指标天门冬氨酸氨基转移酶(Aspartate aminotransferase, AST)、丙氨酸氨基转移酶(Alanine aminotransferase, ALT),采用HE染色方法检测肝脏病理评估毛果芸香碱对APAP诱导肝损伤的药效作用;Western blot方法检测肝脏增殖细胞核抗原(Proliferating cell nuclear antigen, PCNA)及细胞外调节蛋白激酶(Extracellular regulated protein kinases1/2, ERK1/2)蛋白表达,探索肝细胞增殖作用及其可能的机制。结果 与空白对照组比较,APAP模型组小鼠的ALT和AST含量均显著升高(P<0.001),表明APAP模型建立成功;与APAP模型组比较,毛果芸香碱组小鼠血清AST含量显著降低(P<0.01),ALT含量无统计学差异(P>0.05)。HE染色病理学结果显示,与空白对照组比较,APAP模型组小鼠肝脏出现肝小叶中心坏死、肝内出血和细胞核消失或固缩;与APAP模型组比较,毛果芸香碱组小鼠改善了肝小叶中心坏死、肝内出血和细胞核固缩。Western blot结果显示,与空白对照组和APAP模型组比较,毛果芸香碱组小鼠肝脏PCNA蛋白表达水平升高,差异有统计学意义(P<0.01);与空白对照组和APAP模型组比较,毛果芸香碱组小鼠p-ERK/ERK蛋白表达水平升高,差异有统计学意义(P<0.01)。结论 毛果芸香碱能减轻APAP所致肝损伤,其分子机制可能是毛果芸香碱通过激活肝细胞中m3AchR-ERK1/2信号通路进而促进肝细胞增殖来发挥作用。

Objective To investigate the efficacy and mechanism of pilocarpine in alleviating liver injury induced byacetaminophen(APAP).Methods In this study,an acute liver damage model was established u-sing mice that received intraperitoneal injections of APAP.A total of 18 C57BL/6 mice were randomly as-signed to a blank control group,an APAP model group,and a pilocarpine group,with 6 mice in each group.After intraperitoneal administration of pilocarpine(2.5 mg/kg)for 7 days,an APAP acute liver injury model(400 mg/kg)was established 1 hour after the last administration.After 12 hours,blood and liver tissue samples were collected from mice.Evaluate the protective effect of pilocarpine on APAP in-duced liver injury by detecting serum liver function indicators such as aspartate aminotransferase(AST),alanine aminotransferase(ALT),using HE staining method to detect liver pathology and evaluate the pharmacological effect of pilocarpine on APAP induced liver injury.To explore the possible mechanism of liver cell proliferation by detecting proliferating cell nuclear antigen(PCNA)and extracellular protein ki-nase ERK1/2 signaling pathway.Results Compared withblank control group,the levels of ALT and AST in APAP model group mice were significantly increased(P<0.001),indicating the successful establish-ment of the APAP model.Compared with APAP model group,the serum AST content of mice in the pilo-carpine group was significantly reduced(P<0.01),while there was no statistically significant difference in ALT content(P>0.05).The pathological results of HE staining showed that pilocarpine group mice reduced central necrosis of hepatic lobules,intrahepatic hemorrhage,and nuclear pyknosis compared to APAP model group.Western blot results showed that the expression of PCNA protein in liver of mice trea-ted with pilocarpine increased,with a statistically significant difference(P<0.01).The expression of p-ERK/ERK proteins in the pilocarpine group mice wereincreased,and the difference was statistically signif-icant(P<0.01).Conclusion Pilocarpine can alleviate liver damage caused by APAP,and its molecular mechanism may be that pilocarpine promotes liver cell proliferation by activating the m3AchR-ERK1/2 signaling pathway in liver cells.