竹节参皂苷成分对对乙酰氨基酚诱导急性肝损伤的保护作用

Protective effects of total saponins from Panax japonicus C.A.Mey.on acetaminophen-induced liver injury

目的:观察竹节参总皂苷提取物(PJST)对对乙酰氨基酚(APAP)诱导急性肝损伤的预防保护作用.方法:构建APAP诱导HepG2细胞损伤的体外模型,采用MTT法评价PJST对APAP诱导HepG2细胞死亡的预保护作用,并观察细胞形态变化,采用Hoechst 33258荧光染色法研究PJST对细胞凋亡形态的影响.取40只昆明种小鼠构建体内模型,小鼠随机分为空白组、模型组和PJST低、高剂量组(50、100 mg·kg^(−1)),腹腔单次注射APAP溶液(400 mg·kg^(−1))以构建肝损伤模型.给药造模结束后,处死小鼠并收集血清及肝脏组织.采取生化试剂盒检测血清中ALT、AST和LDH水平,ELISA法检测肝脏组织TNF-α、IL^(-1)β和IL-6水平,HE染色法观察肝脏组织显微结构变化情况,免疫组化法检测肝组织Bax和Bcl-2蛋白的表达差异,免疫印迹检测肝组织Bax、Bcl-2、P-NF-κB p65和核内NF-κB p65的蛋白表达水平.结果:PJST可以有效干预APAP诱导的HepG2细胞凋亡;体内实验中PJST组小鼠血清ALT、AST和LDH水平均显著低于模型组,且肝脏显微结构变化也较模型组有所恢复.进一步研究结果显示:PJST可以显著改善由APAP诱导的肝脏细胞凋亡,Bax和Bcl-2蛋白表达的异常,NF-κF p65的异常磷酸化和入核,以及抑制肝脏内炎症因子TNF-α、IL^(-1)β和IL-6的过量产生与释放.结论:PJST可以明显缓解APAP诱导的急性肝损伤,其机制可能与抑制肝细胞凋亡和炎症反应有关.

Objective:To observe the protective effect of total saponins from Panax japonicus C.A.Mey.(PJST)on acetaminophen(APAP)-induced acute liver injury.Methods:The model of HepG2 cells induced by APAP was established in vitro.The preprotective effect of PJST on the death of HepG2 cells induced by APAP was evaluated by MTT method,and the morphological changes of HepG2 cells were monitored.The effect of PJST on the morphology of apoptotic cells was studied by Hoechst 33258 fluorescence staining.Forty Kunming mice were selected to construct the in vivo model.Mice were randomly divided into four groups,i.e.,normal group,APAP group,PJST low-dosage group and PJST high-dosage groups(50,100 mg·kg^(−1)).Liver injury model in mice was constructed by a single intraperitoneal injection of APAP(400 mg·kg^(−1)).The mice were killed after drug administration,and their serum and liver were collected.The levels of ALT,AST and LDH in serum were detected by biochemical kit.The levels of TNF-α,IL^(-1)βand IL-6 in liver tissues were determined by ELISA.The changes of liver microstructure were observed by HE staining.Expression of Bax and Bcl-2 proteins in liver tissue was detected by immunohistochemistry staining assay.Protein expression of P-NF-κB p65,NF-κB p65 in the nucleus and Bax and Bcl-2 were detected by immunoblotting.Results:PJST could effectively interfere with APAP-induced apoptosis in HepG2 cells.The serum ALT,AST and LDH levels of mice in the PJST group were significantly lower than in the model group.The microstructural changes in the liver were also restored compared with the model group.Further investigations revealed that PJST could significantly relieve APAP-induced apoptosis,abnormal Bax and Bcl-2 protein expression,abnormal phosphorylation and nucleation of NF-κB p65,and inhibit the overproduction and release of inflammatory factors TNF-α,IL^(-1)βand IL-6 in the liver.Conclusion:PJST could significantly alleviate APAP-induced acute liver injury,and the mechanism may be related to the inhibition of hepatocyte apoptosis and inflammatory response.