基于Nrf2/TXNIP信号通路探讨葛根芩连汤含药血清对非酒精性脂肪性肝炎的影响  

Effect of Gegen Qinliantang-medicated Serum on Nonalcoholic Steatohepatitis Based on Nrf2/TXNIP Signaling Pathway

作　　者：魏悦 盛军庆[2] 程子文 罗小泉 宋针珍 吴爱兰 曹岚[1] 章常华[1] WEI Yue;SHENG Junqing;CHENG Ziwen;LUO Xiaoquan;SONG Zhenzhen;WU Ailan;CAO Lan;ZHANG Changhua(Research Center for Traditional Chinese Medicine Resources and Ethnic Minority Medicine,Laboratory Animal Science and Technology Center,School of Pharmacy,Jiangxi University of Chinese Medicine,Nanchang330004,China;School of Life Sciences,Nanchang University,Nanchang330031,China)

机构地区：[1]江西中医药大学药学院实验动物科技中心中药资源与民族药研究中心,南昌330004 [2]南昌大学生命科学学院,南昌330031

出　　处：《中国实验方剂学杂志》2022年第20期8-16,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81774194);江西省自然科学基金重点项目(20192ACBL20027);江西省教育厅科技计划重点项目(GJJ190636);江西省教育厅科技计划一般项目(GJJ213111)。

摘　　要：目的:探讨葛根芩连汤(GGQLT)含药血清对游离脂肪酸(FFA)诱导人肝癌细胞HepG2非酒精性脂肪性肝炎(NASH)体外模型的影响。方法:建立HepG2细胞NASH体外模型,使用不同体积分数的GGQLT含药血清及白藜芦醇干预细胞。利用油红O染色检测各组细胞内脂质沉积;采用流式细胞术检测各组细胞内活性氧(ROS)水平;通过试剂盒检测各组细胞内谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)活性和甘油三酯(TG)、丙二醛(MDA)含量。采用实时荧光定量聚合酶链式反应(Real-time PCR)检测各组HepG2细胞内核转录因子(NF)E2相关因子2(Nrf2)、血红素加氧酶-1(HO-1)、醌氧化还原酶1(NQO1)、Kelch样环氧氯丙烷相关蛋白1(Keap1)、NF-κB、白细胞介素-1β(IL-1β)、硫氧还蛋白相互作用蛋白(TXNIP)的mRNA表达情况。运用蛋白免疫印迹法(Western blot)检测各组细胞Nrf2、TXNIP的蛋白表达情况。结果:FFA诱导引起细胞内脂质大量堆积。与正常组比较,模型组抗氧化酶GSH-Px和SOD的活性显著降低(P<0.01),TG、ROS和MDA含量明显升高(P<0.05,P<0.01)。与模型组比较,GGQLT各剂量组和白藜芦醇组均可不同程度地升高细胞内SOD的活性(P<0.05,P<0.01),明显降低细胞内ROS、MDA水平(P<0.05,P<0.01);GGQLD高、中剂量组和白藜芦醇组可显著升高GSH-Px的活性(P<0.01),GGQLD中、低剂量组和白藜芦醇组明显降低TG的含量(P<0.05,P<0.01)。与模型组比较,GGQLT高、中剂量组和白藜芦醇组可显著上调Nrf2、HO-1、NQO1的mRNA表达水平(P<0.01),GGQLT各剂量组、白藜芦醇组可明显下调TXNIP蛋白表达水平和Keap1、NF-κB的mRNA表达水平(P<0.05,P<0.01)。Nrf2-小分子干扰核糖核酸(siRNA)转染细胞后发现,与相应剂量药物的阴性对照(NC)-siRNA组比较,其Nrf2-siRNA组细胞内Nrf2表达显著下调(P<0.01);GGQLT和白藜芦醇对TXNIP、IL-1β的抑制作用被减弱。结论:FFA诱导HepG2细胞内产生ROS和炎症因子,GGQLT可提高细胞的抗炎Objective:To investigate the effect of Gegen Qinliantang(GGQLT)-medicated serum on free fatty acid(FFA)-induced nonalcoholic steatohepatitis(NASH)in vitro model of human hepatoma cells HepG2.Method:NASH model of HepG2 cells was established in vitro,and the cells were intervened with different volume fractions of GGQLT-medicated serum and resveratrol.Intracellular lipid deposition in each group was detected by oil red O staining,the level of reactive oxygen species(ROS)in each group were detected by flow cytometry,the levels of glutathione peroxidase(GSH-Px),superoxide dismutase(SOD),triglyceride(TG)and malondialdehyde(MDA)in each group were detected by kits.Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was used to measure the mRNA expression levels of nuclear transcription factor(NF)E2-related factor 2(Nrf2),heme oxygenase-1(HO-1),quinone oxidoreductase 1(NQO1),Kelch-like epichlorohydrin-associated protein-1(Keap1),NF-κB,thioredoxin interacting protein(TXNIP),interleukin-1β(IL-1β)in HepG2 cells of each group.The protein expression of Nrf2,TXNIP in cells of each group was detected by Western blot.Result:FFA induced large accumulation of intracellular lipids.Compared with the normal group,the activities of GSH-Px and SOD were significantly decreased(P<0.01)and the contents of TG,ROS and MDA were significantly increased(P<0.05,P<0.01)in the model group.Compared with the model group,all GGQLT groups and resveratrol group could elevate intracellular SOD activity to different degrees(P<0.05,P<0.01)and significantly reduce the levels of intracellular ROS and MDA(P<0.05,P<0.01),GGQLD high-and medium-dose groups and resveratrol group significantly elevated GSH-Px activity(P<0.01),GGQLD medium-and low-dose groups and resveratrol group significantly decreased TG content(P<0.05,P<0.01).Compared with the model group,GGQLT high-and medium-dose groups and resveratrol group could significantly upregulate the mRNA expression levels of Nrf2,HO-1 and NQO1(P<0.01),all GGQLT groups and resveratrol gro

关 键 词：葛根芩连汤 含药血清 非酒精性脂肪性肝炎(NASH) 氧化应激 炎症因子 核转录因子(NF) 硫氧还蛋白相互作用蛋白(TXNIP) 

分 类 号：R22[医药卫生—中医基础理论] R28[医药卫生—中医学] R96[轻工技术与工程—纺织化学与染整工程] TS193[轻工技术与工程—纺织科学与工程]