基于细胞焦亡探讨琴叶榕提取物对急性酒精性肝损伤的保护机制

Protective Effect of Ficus pandurata Extract on Alcohol-induced Acute Liver Injury Base on Pyroptosis

目的:研究细胞焦亡在琴叶榕根的不同溶剂提取物保护急性酒精性肝损伤中的作用。方法:56只雄性C57BL/6小鼠随机分为正常组,模型组,水飞蓟宾组(60 mg·kg^-1),鲜药水提物组(48 g·kg^-1),干药水提物组(48 g·kg^-1),干药50%醇提物组(48 g·kg^-1),干药95%醇提物组(48 g·kg^-1),每组8只。其中水飞蓟宾和不同溶剂的琴叶榕提取物,各组小鼠灌胃给药18 d,正常组与模型组小鼠灌胃给予等体积的纯水。灌胃给药15 d后,模型组、水飞蓟宾组和各个琴叶榕根提取物组小鼠给予50%乙醇(12 mL·kg^-1)灌胃,建立急性酒精性肝损伤模型。末次造模结束后禁食14 h后摘眼球取血,开腹取肝脏,计算肝脏指数,苏木素-伊红(HE)染色观察肝脏组织病理学变化;试剂盒检测小鼠血清中丙氨酸氨基转移酶(ALT),天门冬氨酸氨基转移酶(AST)活性;硫代巴比妥酸(TBA)法检测肝脏丙二醛(MDA)含量,微板法检测乳酸脱氢酶(LDH)水平;肝脏组织切片末端标记法(TUNEL)染色检测肝细胞焦亡;蛋白免疫印迹法(Western blot)检测细胞焦亡相关蛋白。结果:与正常组比较,模型组ALT,AST,MDA,LDH水平显著升高,肝脏指数显著升高,TUNEL染色阳性、炎症因子、焦亡相关蛋白表达显著升高(P<0.05);与模型组比较,各个琴叶榕根提取物给药干预组小鼠ALT,AST,MDA,LDH水平均明显下降(P<0.05),肝脏指数有不同程度的下降,TUNEL染色阳性、炎症因子、焦亡相关蛋白表达水提物处理组明显下降(P<0.05);肝脏病理切片表明,琴叶榕根提取物能改善小鼠急性酒精肝损伤肝组织病理学变化。结论:琴叶榕根提取物对急性酒精性肝损伤具有一定的保护作用,且其水提物作用机制可能与焦亡通路相关。

Objective:To study the protective effect of Ficus pandurata extract on acute alcoholic liver injury based on pyroptosis mechanism. Method: The 56 male C57 BL/6 mice were randomly divided into normal control group,model control group,positive control group(60 mg·kg^-1),fresh medicine water extract group(48 g·kg^-1),dry drug water extract group(48 g·kg^-1),dry drug 50% alcohol extract group(48 g·kg^-1)and dry drug 95% alcohol extract group(48 g·kg^-1),8 mice in each group. Positive control and different solvent extract groups of Ficus tenuifolia were intragastrically administrated for 18 days,once a day,while normal group and model group were given the same volume of pure water intragastrically. After 15 days of continuous gavage,mice received 50% ethanol(12 mL·kg^-1)intragastrically for 3 days to induce acute alcoholic liver injury model except for the normal control group. At 14 h after the last treatment,serum and liver samples were obtained,the serum content of alanine aminotransferase(ALT) and aspartate transaminase(AST) were determined,the histopathologic changes of the hepatic tissues were observed by hematoxylin ecosin(HE)staining. The content of malondialdehyde(MDA) in liver was determined by thiobarbituric acid(TBA) and the content of lactate dehydrogenase(LDH)was determined by microplate method. Western blot and TUNEL assay kit was used to detect the cell pyroptosis rate. Result: Compared with normal group,ALT,AST,MDA and LDH levels in the model group were significantly increased,liver index was significantly increased,TUNEL staining positive,inflammatory factors and pyroptosis related protein expressions were significantly increased(P<0. 05).Compared with model control group,the ALT,AST,MDA and LDH of the drug intervention group decreased significantly(P<0. 05). The liver index decreased in different degrees,and the expression of inflammatory factors and pyroptosis related protein in the water extract treatment group decreased significantly(P<0. 05).Conclusion: The root extract of Ficus pandurata Hance has protective effect on acute alcoholic liver injury,and the mechanism of water extract might relate to inhibiting hepatocyte pyroptosis.