白藜芦醇通过下调MnSOD诱导类风湿性关节炎成纤维样滑膜细胞凋亡
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摘要
目的探讨锰超氧化物歧化酶(MnSOD)蛋白表达改变在白藜芦醇(Res)介导的类风湿性关节炎成纤维样滑膜细胞(RA-FLSs)的增殖和凋亡中的作用。方法 Res处理RA-FLSs后,CCK-8法检测细胞增殖,Western blot检测MnSOD蛋白表达水平;慢病毒感染RA-FLSs后,8 mg·L~(-1)的嘌呤霉素筛选感染后细胞,以此建立3种稳定细胞系:MnSOD过表达、MnSOD干扰、MnSOD对照;激光共聚焦显微镜检测每种细胞系在5μmol·L~(-1)过氧化氢(H_2O_2)和200μmol·L~(-1) Res处理后线粒体活性氧(mtROS)水平;流式细胞术检测细胞凋亡水平。结果 Res抑制RA-FLSs增殖,降低细胞内MnSOD表达;荧光倒置显微镜与Western blot检测证实慢病毒可高效感染RA-FLSs,使MnSOD表达上调和抑制。与对照组相比,在同种因素处理下,MnSOD过表达组mtROS水平降低,凋亡细胞减少。而MnSOD干扰组则呈现相反的结果。结论 Res可通过抑制MnSOD表达来上调mtROS水平,从而促进RA-FLSs凋亡。
Aim To investigate the role of manganese superoxide dismutase(MSOD) protein expression in the proliferation and apoptosis of fibroblast-like syno-viocyte(RA-FLS) in rheumatoid arthritis mediated by resveratrol( Res). Methods After RA-FLSs was treated with Res,the expression of MnSOD protein was detected by CCK-8 method,and the expression of MnSOD protein was detected by, Western blot. After lentivirus infection with RA-FLSs,8 mg · L~(-1) purine mycin was used to screen the infected cells,and three stable cell lines were established: MnSOD overexpression,Mn SOD interference and MnSOD control. The mitochondria reactive oxygen species( mtROS) levels of each cell line treated with 5 μmol·L~(-1) hydrogen peroxide( H_2O_2) and 200 mol·L~(-1) Res were detected by laser confocal microscopy,and cell apoptosis was detected by flow cytometry. Results Res could inhibit the proliferation and the expression of MnSOD in RAFLSs. The lentivirus-mediated MnSOD regulation could significantly increase or decrease the expression of MnSOD in RA-FLSs. MnSOD over expression could decrease the level of mtROS and apoptosis of the RAFLSs treated with 5 μmol·L~(-1) H_2O_2 and 200 μmol·L~(-1) Res. While the MnSOD RNAi showed the opposite results. Conclusions Res may up-regulate mtROS levels by inhibiting the expression of Mn SOD,thus promoting the apoptosis of RA-FLSs.
Aim To investigate the role of manganese superoxide dismutase(MSOD) protein expression in the proliferation and apoptosis of fibroblast-like syno-viocyte(RA-FLS) in rheumatoid arthritis mediated by resveratrol( Res). Methods After RA-FLSs was treated with Res,the expression of MnSOD protein was detected by CCK-8 method,and the expression of MnSOD protein was detected by, Western blot. After lentivirus infection with RA-FLSs,8 mg · L~(-1) purine mycin was used to screen the infected cells,and three stable cell lines were established: MnSOD overexpression,Mn SOD interference and MnSOD control. The mitochondria reactive oxygen species( mtROS) levels of each cell line treated with 5 μmol·L~(-1) hydrogen peroxide( H_2O_2) and 200 mol·L~(-1) Res were detected by laser confocal microscopy,and cell apoptosis was detected by flow cytometry. Results Res could inhibit the proliferation and the expression of MnSOD in RAFLSs. The lentivirus-mediated MnSOD regulation could significantly increase or decrease the expression of MnSOD in RA-FLSs. MnSOD over expression could decrease the level of mtROS and apoptosis of the RAFLSs treated with 5 μmol·L~(-1) H_2O_2 and 200 μmol·L~(-1) Res. While the MnSOD RNAi showed the opposite results. Conclusions Res may up-regulate mtROS levels by inhibiting the expression of Mn SOD,thus promoting the apoptosis of RA-FLSs.
引文
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[10] Ye T H, Zhu S R, Zhu Y X, et al. Cryptotanshinone induces melanoma cancer cells apoptosis via ROS-mitochondrial apoptotic pathway and impairs cell migration and invasion[J]. Biomed Pharmacother, 2016(82):319-26.
[2] Murphy D, Hutchhinson D. Is male rheumatoid arthritis an occupational disease? A review[J]. Open Rheumatol J, 2017, (11):88-105.
[3] Wu W J, Jia W W, Liu X H, et al. S-propargyl-cysteine attenuates inflammatory response in rheumatoid arthritis by modulating the Nrf2-ARE signaling pathway[J]. Redox Biol, 2016, (10):157-67.
[4] Somaiya M, Shagufta M,Abdul Q K,et al. Increased reactive oxygen species formation and oxidative stress in rheumatoid arthritis[J]. PLoS One, 2016, 11(4):e0152925.
[5] Fu B, Zhao J, Peng W, et al. Resveratrol rescues cadmium-induced mitochondrial injury by enhancing transcriptional regulation of PGC-1alpha and SOD2 via the Sirt3/FoxO3a pathway in TCMK-1 cells[J]. Biochem Biophys Res Commun, 2017, 486(1): 198-204.
[6] 朱茄慧,曾于晔, 王晗, 等. 白藜芦醇抑制人结肠癌细胞增殖与骨形态发生蛋白9及其受体表达的关系研究[J]. 中国药理学通报, 2016,32(12):1705-11.[6] Zhu Q H,Zeng Y Y, Wang H, et al. Study on relationship between anti-proliferation effect of resveratrol and resveratrol-induced bone morphogenetic protein 9 and its receptors in colon cancer cells[J]. Chin Pharm Bull, 2016,32(12):1705-11.
[7] Zhang J, Song X, Cao W, et al. Autophagy and mitochondrial dysfunction in adjuvant-arthritis rats treatment with resveratrol[J]. Sci Rep, 2016,6:32928.
[8] He B, Li X, Hu T, et al. Construction of a lentiviral vector containing shRNA targeting ADAM17 and its role in attenuating endotoxemia in mice[J]. Mol Med Rep, 2017, 16(5):6013-9.
[9] Beatrix B, Gary S F. Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis[J]. Immunol Rev, 2010, 233(1):233-55.
[10] Ye T H, Zhu S R, Zhu Y X, et al. Cryptotanshinone induces melanoma cancer cells apoptosis via ROS-mitochondrial apoptotic pathway and impairs cell migration and invasion[J]. Biomed Pharmacother, 2016(82):319-26.