葛黄颗粒含药血清对乙醇诱导L-02肝细胞损伤的保护作用及其机制研究

The protective effect of Gehuang granules medicated serum on the injured L-02 hepatocytes induced by ethanol and its mechanism

目的观察葛黄颗粒含药血清对乙醇诱导的L-02肝细胞损伤的保护作用并探讨其分子机制。方法将SD大鼠分3组:空白对照组、葛黄颗粒组(3.1g·100g^(-1)·d^(-1))、美他多辛组(0.1g·100g^(-1)·d^(-1)),每组20只,每日灌胃给药2次,连续给药5d后,取血制备含药血清。构建3%乙醇干预24h诱导的体外肝细胞损伤模型。L-02肝细胞分为6组,包括正常组、模型组、葛黄颗粒高、中、低剂量组及阳性对照组,L-02肝细胞接种培养24h后,正常组加入含10%正常大鼠血清的培养基,模型组在正常组基础上同时加入3%乙醇,葛黄颗粒高、中、低剂量组则加入3%乙醇及不同体积比的葛黄颗粒含药血清,阳性对照组加入3%乙醇和美他多辛含药血清,干预24h后,收集上清液及L-02肝细胞。采用全自动生化分析仪检测上清液天门冬氨酸氨基转移酶(AST)、乳酸脱氢酶(LDH),流式细胞仪检测细胞凋亡,反转录-聚合酶链反应(RT-PCR)检测Fas、FasL、caspase3、caspase8mRNA的表达。结果在模型组中,细胞上清液AST、LDH的水平,L-02细胞的凋亡率及Fas、FasL、caspase3、caspase8mRNA的表达水平均较正常组明显增高(P<0.05),经相应干预后,上述各项检测指标在葛黄颗粒中、高剂量组和阳性对照组中的表达水平均显著下降(P<0.05);中剂量组和阳性对照组比较差异有统计学意义(P<0.05),高剂量组和阳性对照组比较差异无统计学意义(P>0.05)。结论葛黄颗粒含药血清可降低细胞上清液中AST、LDH的水平,下调Fas、FasL、caspase3、caspase8mRNA的表达,减少凋亡率,对酒精性肝病的保护作用可能与抑制细胞凋亡有关。

Objective To observe the protective effect of Gehuang granules medicated serum on the ethanol-induced injured L-02 hepatocytes and explore its mechanism.Methods The SD rats were divided into three groups with 20 cases in each group:the blank control group,the Gehuang granules group(3.1 g·100 g-1·d-1),and the metadoxine group(0.1 g·100 g-1·d-1),fed twice a day for five consecutive days,then collected aortic blood serum.Constructed hepatocytes injury model in vitro induced by 3%ethanol for 24 h.The L-02 hepatocytes were divided into six groups:the normal group,the model group,the high,middle and low dose Gehuang granules groups and the positive control group.After L-02 hepatocytes inoculation and cultivation for 24 h,the normal group was supplemented with medium containing 10%normal rat serum,the model group was supplemented with 3%ethanol on the basis of the normal group,the high,middle and low dose Gehuang granules groups were added 3%ethanol and different doses of Gehuang granules,the positive control group was added 3%ethanol and metadoxine medicated serum,after 24-hour training,collected supernatants and L-02 hepatocytes for subsequent experiments.The aspartate aminotransferase(AST)and lactate dehydrogenase(LDH)were detected by automatic biochemical analyzer,and apoptosis was detected by flow cytometry.Reverse transcription-polymerase chain reaction(RT-PCR)was used to detect the mRNA level of Fas,FasL,caspase3 and caspase8.Results The levels of AST,LDH,apoptosis rate,Fas,FasL,caspase3 and caspase8 mRNA in the model group significantly increased(P<0.05);after intervention,the expressions of above indexes in the middle dose group,the high dose group and the control group decreased(P<0.05),there was statistically significant difference between the middle dose group and the positive control group(P<0.05),but there was no significant difference between the high dose group and the positive control group(P>0.05).Conclusion Gehuang granules medicated serum can decrease the levels of AST and LDH,down-regulate the mRNA expression of Fas,FasL,caspase3 and caspase8,reduce the apoptosis rate.The protective effect may be related to the inhibition of apoptosis.