蒙药古古勒-4味汤对原代肝细胞氧化应激损伤的保护作用及机制

Protective Effect and Mechanism of Mongolian Medicine Gugule-4 Decoction on Primary Hepatocytes Injury by Oxidative Stress

目的:研究古古勒-4味汤(黑云香四味汤散)对H2O2所致的原代肝细胞氧化应激损伤的保护作用及其作用机制.方法:取新生乳鼠肝脏,加入0.1%Ⅱ型胶原酶以及0.1%胰蛋白酶混合液分离培养原代肝细胞,加入不同剂量(100μmol/L、200μmol/L、400μmol/L)H2O2建立原代肝细胞氧化应激损伤模型.原代肝细胞氧化应激损伤模型建立成功后,加入0.5 mg/mL、l mg/mL及1.5 mg/mL古古勒-4味汤确定古古勒-4味汤最佳剂量.MTT检测H2O2和古古勒-4味汤最佳剂量及肝细胞活性;收集细胞,采用ELISA检测不同组细胞中GSH-Px和SOD的表达情况;采用real-time PCR法检测各组肝细胞Nrf2、Keapl的mRNA表达情况.结果:对数生长期的原代肝细胞加人终浓度为200μmol/L H2O2培养6个小时后,给予1mg/mL古古勒-4味汤处理肝细胞后,肝细胞活力较模型组明显上升;古古勒-4味汤处理后,细胞中S0D、GSH-Px活性增强;古古勒-4味汤处理后,肝细胞活性氧明显降低;古古勒-4味汤处理后,肝细胞Nrf2、Keapl的mRNA水平下调.结论:古古勒-4味汤通过影响Keapl-Nrf2/ARE信号通路,保护由H2O2所致的原代肝细胞氧化应激损伤.

Objective:To investigate the protective effect and mechanism of Gugule-4 soup(Heiyunxiang-4 decoction)on oxidative stress injury induced by H2O2 in primary hepatocytes.Methods:The hepatocytes were iso-lated and cultured from neonatal suckling mice using 0.1%collagenase Ⅱ and 0.1%trypsin mixture.The oxidative stress injury model was established by using the doses of 100,200,and 400μmol/L H2O2.Thereafter,0.5,1 and 1.5 mg/mL Gugule-4 decoction were used to determine the optimal dosage.MTT was used to determine the optimal dose of H2O2 and Gugule-4 soup.The primary hepatocytes were collected and the expressions of GSH-Px and SOD in different groups were detected by ELISA.The mRNA expression of Nr￡2 and Keapl in hepatocytes was detected by real-time PCR.Results:The primary hepatocytes at logarithmic growth phase were cultured for 6 hours with a final concentration of 200μmol/L H2O2.Compared with the model group,the cell hepatocyte viability was significantly higher after treatment with 1 mg/mL Gugule-4;the activity of SOD and GSH-Px in cells increased after treatment with Gugule-4 decoction,the reactive oxygen species in hepatocytes decreased significantly;and the mRNA expres-sion level of Nrf2 and Keapl was down-regulated in primary hepatocytes.Conclusion:The protective effect of Gu-gule-4 soup on oxidative stress injury induced by H2O2 in primary hepatocytes was found to be related to Keapl-Nrf2/ARE signaling pathway.