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目的：探究利福平（Rifampicin,RFP）调节NRF2/NQO1通路对小鼠肝细胞AML12造成的影响。方法：培养正常的小鼠肝细胞系AML12。采用不同浓度的利福平对细胞进行染毒，CCK8法检测细胞活力，根据结果选取适用于本研究的染毒浓度（50、100、200μmol/L）。采用微孔板法检测AML12细胞内过氧化氢酶（Catalase,CAT）、超氧化物歧化酶（Superoxidedismutase,SOD）活力以及丙二醛（Malondialdehyde,MDA）的含量；采用流式细胞仪检测细胞内活性氧（Reactive oxygen species,ROS）含量以及通过检测JC-1荧光探针分析线粒体内膜电位变化；采用qRT-PCR方法测定NQO1、SOD、NRF2基因的mRNA表达水平，Western Blot法检测NRF2、NQO1的蛋白表达水平。结果：随着利福平染毒浓度的升高，AML12细胞活力也随之减少。与对照组相比，利福平染毒处理组中AML12细胞的MDA含量、CAT活力、ROS水平均升高，且随着染毒浓度的增加线粒体膜电位也随之升高，且差异均具有统计学意义（P<0.05）。与对照组相比，SOD活性以及NQO1、SOD、NRF2三个基因的mRNA表达水平随利福平染毒浓度上升有所降低，且差异具有统计学意义（P<0.05）。NQO1、NRF2的蛋白表达水平随利福平染毒浓度上升降低。结论：利福平可能通过NRF2/NQO1通路调节ROS对小鼠肝细胞AML12造成氧化损伤，且具有浓度依赖性。
Abstract：Objective: To explore the effect of rifampicin regulating NRF2/NQO1 pathway on AML12 in mouse hepatocytes.Methods: Normal mouse liver cell line AML12 cells were cultured in this study. The cells were treated with various concentrations of rifampicin, and cell viability was assessed by CCK8 method. Based on the CCK8 results, suitable concentration of rifampicin were determined to be 50, 100 and 200 μmol/L. The activity of catalase, superoxide dismutase(SOD) and the levels of malondialdehyde were detected using the microporous plate method. The levels of intracellular reactive oxygen species and the mitochondrial membrane potential were assessed by DCFH-DA and JC-1 fluorescence probe respectively using flow cytometry. The m RNA expression levels of NQO1, SOD and NRF2 genes were determined by q RT-PCR, and the protein expression levels of NRF2 and NQO1 were detected by Western Blot.Results: The activity of AML12 cells decreased with the increase of rifampicin concentration. Compared with the control group, the malondialdehyde content, catalase activity and reactive oxygen species levels of AML12 cells were increased in treated groups, as well as the mitochondrial membrane potential(P<0.05). The SOD activity and the m RNA expression levels of NQO1, SOD and NRF2 genes all demonstrated a significant decrease in response to increasing concentrations of Rifampicin(P<0.05). Consequently, the expression levels of NQO1 and NRF2 exhibited a similar trend.Conclusion: Rifampicin can cause oxidative damages through NRF2/NQO1 pathway by a concentration-dependent manner in mouse hepatocyte AML12 cell line.
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基本信息:
DOI：10.13210/j.cnki.jhmu.20240914.001
中图分类号:R52;R575
引用信息:
[1]李亚奇,郑志坚,张美诗,等.利福平通过NRF2/NQO1通路对小鼠肝细胞造成氧化损伤[J].海南医科大学学报,2025,31(04):248-253.DOI:10.13210/j.cnki.jhmu.20240914.001.
基金信息:
河北省高等学校基本科研业务费（JQN2021032）~~
2024-09-14
2024-09-14
2024-09-14