N-乙酰半胱氨酸通过阻断Nrf2/Keap1通路抑制(H_(2)O_(2))处理的佐剂性关节炎成纤维细胞样滑膜细胞增殖  

N-acetylcysteine inhibits the proliferation of hydrogen peroxide treated fibroblast-like synoviocytes in rats with adjuvant arthritis(AA) via blocking Nrf2/Keap1pathway

作　　者：杨珺[1] 黄焱平 刘梅梅[1] 汪陶荣 张晔 周文瀚 瞿子庭 陈晓宇[2] YANG Jun;HUANG Yanping;LIU Meimei;WANG Taorong;ZHANG Ye;ZHOU Wenhan;QU Ziting;CHEN Xiaoyu(Experimental Center of Morphology,Anhui Medical College,Hefei 230061;Experimental Center of Morphology,Anhui Medical University,Anhui Medical University,Hefei 230032,China;Clinical Medical College,Anhui Medical University,Hefei 230032,China)

机构地区：[1]安徽医学高等专科学校形态学实验中心,安徽合肥230061 [2]安徽医科大学形态学实验中心,安徽合肥230032 [3]安徽医科大学临床医学院,安徽合肥230032

出　　处：《细胞与分子免疫学杂志》2021年第8期687-692,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81373421);安徽省高校自然科学研究重点项目(KJ2019A1100);安徽省高校优秀青年人才支持计划(gxyq2019190);大学生创新创业训练计划(201910366008,S201910366002)。

摘　　要：目的探讨N-乙酰半胱氨酸(NAC)对低浓度过氧化氢(H_(2)O_(2))处理的佐剂性关节炎(AA)大鼠成纤维细胞样滑膜细胞(AA-FLS)增殖的影响及机制。方法SD大鼠分为正常对照组和模型组(均为10只),模型组采用足趾皮下注射Freund完全佐剂造模,28 d后处死大鼠。硫代巴比妥酸法检测血清丙二醛(MDA)含量、羟胺法测定超氧化物歧化酶(SOD)活性,比色法测定谷胱甘肽过氧化物酶(GSH-Px)活性,免疫组织化学染色法检测踝关节滑膜组织中核因子E2相关因子2(Nrf2)、Kelch样ECH相关蛋白1(Keap1)蛋白表达;体外培养和鉴定AA-FLS,CCK-8法检测(0、0.3、0.9、3、10、30、90、180)μmol/L NAC对5μmol/L H_(2)O_(2)处理的AA-FLS活力的影响,采用MitoSOX线粒体活性氧荧光探针检测AA-FLS中线粒体活性氧(ROS)含量,Western blot法检测(0、3、10、30)μmol/L NAC对低浓度H_(2)O_(2)处理AA-FLS的Nrf2、Keap1蛋白表达的影响。结果与正常对照组比较,AA模型大鼠血清MDA水平增加、SOD、GSH-Px水平降低,滑膜组织中Nrf2表达升高、Keap1表达降低;免疫细胞化学染色证实分离培养的细胞是AA-FLS,NAC可浓度依赖性抑制H_(2)O_(2)处理的AA-FLS增殖,且线粒体ROS含量下降,Nrf2、Keap1蛋白表达降低。结论NAC可抑制H_(2)O_(2)处理后的AA-FLS增殖,其机制可能与阻断Nrf2/Keap1通路有关。Objective To investigate the effect of N-acetylcysteine(NAC) on the proliferation of fibroblast-like synoviocytes(FLS) treated with low concentration of hydrogen peroxide(H_(2)O_(2)) in rats with adjuvant arthritis(AA) and its mechanism. Methods Twenty SD rats were divided into a normal group and a model group(10 rats in each group). The model group was established by subcutaneous injection of Freund’s complete adjuvant into the toe of rats, and the rats were sacrificed 28 days later. The contents of serum malondialdehyde(MDA) were detected by thiobarbituric acid method;the activities of superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px) were determined by hydroxylamine method and colorimetry respectively;and Nrf2 and Keap1 proteins in ankle synovial tissues of AA rats were detected by immunohistochemistry. AA-FLS were isolated, cultured, and identified by digestion of ankle joint slides of AA rats in vitro. The effects of NAC at different concentrations(final concentration 0, 0.3, 0.9, 3, 10, 30, 90, 180 μmol/L) on the activity of AA-FLS treated with H_(2)O_(2) atlow concentration(5 μmol/L) were detected by CCK-8 assay. The content of mitochondrial reactive oxygen species(ROS) in AA-FLS was detected by MitoSOX fluorescent probe. The effects of NAC(final concentration 0, 3, 10, 30 μmol/L) on Nrf2 and Keap1 protein expressions in AA-FLS treated with H_(2)O_(2) at low concentration were detected by Western blotting. ResultsCompared with those in the control group, in AA model, the MDA level increased and SOD and GSH-Px levels decreased in serum, and the Nrf2 protein increased and the Keap1 protein decreased in synovial tissue. Immunocytochemical staining confirmed that the isolated and cultured cells were AA-FLS;NAC inhibited the proliferation of AA-FLS treated with H_(2)O_(2) in a concentration-dependent manner, and the mitochondrial ROS content and the protein expressions of Nrf2 and Keap1 decreased. Conclusion NAC can inhibit the proliferation of AA-FLS treated with H_(2)O_(2), which may be relate

关 键 词：N-乙酰半胱氨酸(NAC) 核因子E2相关因子2(Nrf2) Kelch样ECH相关蛋白1(Keap1) 氧化应激 佐剂性关节炎(AA) 成纤维细胞样滑膜细胞(FLS) 

分 类 号：R593.22[医药卫生—内科学] R684.2[医药卫生—临床医学]