黄芩汤通过Nrf2信号通路对Caco-2细胞抗氧化应激作用的机制

Anti-oxidative Stress Effect and Mechanism of Huangqintang on Caco-2 Cells Through Nrf2 Signaling Pathway

目的:以Caco-2细胞为载体,结合基因干扰(RNAi)技术,通过体外实验进一步验证黄芩汤基于核因子E2相关因子2(Nrf2)通路发挥的抗氧化应激作用。方法:将处于对数生长期的Caco-2细胞,经siRNA转染,构建siRNA Caco-2细胞;将正常Caco-2细胞和siRNA Caco-2细胞与不同浓度的黄芩汤共同孵育后,提取RNA和蛋白,采用实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)技术检测血红素氧合酶-1(HO-1)、醌氧化还原酶1(NQO1)、谷胱甘肽巯基转移酶(GST)、接头蛋白Kelch样ECH相关蛋白1(Keap1)及Nrf2 mRNA和蛋白的表达;同时,采用比色法和探针法检测各组细胞中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的活力及丙二醛(MDA)、活性氧(ROS)的表达水平。结果:与空白组比较,仅有黄芩汤400 mg·L^(-1)组与萝卜硫素(SFN)组可降低正常Caco-2细胞内ROS、MDA含量(P<0.01);而黄芩汤各剂量组与SFN组细胞内SOD与GSH-Px活力均有增加的趋势,且黄芩汤400 mg·L^(-1)组和SFN组与空白组比较差异有显著统计学意义(P<0.01);同时,各组细胞HO-1、GST、Keap1、NQO1和Nrf2蛋白及mRNA的表达量均有明显升高的趋势(P<0.05,P<0.01)。经转染后,与空白组比较,模型组细胞内MDA和ROS的含量升高,而GSH-Px和SOD的活力及HO-1、GST、Keap1和NQO1蛋白及mRNA的表达量均有不同程度地降低(P<0.05,P<0.01),与药物孵育后,与模型组比较,SFN组细胞内SOD活力和SFN组、黄芩汤各给药组GSH-Px活力显著升高(P<0.01),黄芩汤400 mg·L^(-1)组、200 mg·L^(-1)组和SFN组细胞内SOD和GSH-Px活力均有升高的趋势(P<0.01),黄芩汤400 mg·L^(-1)组和SFN组MDA有降低的趋势,且各给药组ROS均有降低(P<0.01);HO-1、GST、Keap1、NQO1和Nrf2的蛋白及mRNA表达均有不同程度增加(P<0.05,P<0.01)。结论:黄芩汤可以通过调控Nrf2通路发挥抗氧化应激的作用。

Objective:To verify the anti-oxidative stress effect of Huangqintang based on the nuclear factor E2-related factor 2(Nrf2)signaling pathway by using Caco-2 cells as a carrier and RNA interference(RNAi)technology with in vitro experiments.Method:The Caco-2 cells in the logarithmic growth phase were transfected with siRNA to construct siRNA Caco-2 cells.After normal Caco-2 cells and siRNA Caco-2 cells were incubated with Huangqintang of different doses,RNA and protein were extracted.Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)and Western blot were used to detect the mRNA and protein expression of heme oxygenase-1(HO-1),NAD(P)H quinone oxidoreductase 1(NQO1),glutathione Stransferase(GST),Kelch-like ECH-associated protein 1(Keap1),and Nrf2.Meanwhile,the activities of superoxide dismutase(SOD)and GSH-Px,as well as the expression levels of malondialdehyde(MDA)and reactive oxygen species(ROS),were detected by the colorimetric method and the probe method.Result:Compared with the results in the normal group,only the 400 mg·L^(-1)Huangqintang group and the sulforaphane(SFN)group could reduce the content of ROS and MDA in Caco-2 cells(P<0.01),while the activities of SOD and GSH-Px in the cells of the Huangqintang groups and the SFN group showed an upward trend.Furthermore,there were significant differences in the 400 mg·L^(-1)Huangqintang group/the SFN group and the normal group(P<0.01).Meanwhile,the protein and mRNA expression levels of HO-1,GST,Keap1,NQO1,and Nrf2showed an upward trend in all groups(P<0.05,P<0.01).After transfection,compared with the normal group,the model group showed increased content of MDA and ROS,blunted activities of GSH-Px and SOD,and reduced protein and mRNA expression of HO-1,GST,Keap1,and NQO1(P<0.05,P<0.01).After drug incubation,compared with the model group,the SFN group showed potentiated SOD activity,and the SFN group and the Huangqintang groups showed enhanced GSH-Px activity(P<0.01).Moreover,the activities of SOD and GSH-Px in the 400 and 200 mg·L^(-1)Huangqintang groups and the SFN group showed an upward trend(P<0.01),and the content of MDA in the 400 mg·L^(-1)Huangqintang group and the SFN group showed a downward trend.ROS decreased in all groups with drug intervention(P<0.01),and the protein and mRNA expression of HO-1,GST,Keap1,NQO1,and Nrf2 increased to varying degrees(P<0.05,P<0.01).Conclusion:Huangqintang can play an anti-oxidative stress role by regulating the Nrf2 pathway.