丹酚酸C通过上调转录因子-E2相关因子信号通路抑制类风湿关节炎成纤维样滑膜细胞炎症及促进凋亡

Salvianolic acid C inhibits inflammation and induce apoptosis by regulating nuclear factor-erythroid 2-related factor 2 pathway in rheumatoid arthritis-fibroblast-like synoviocytes cells

目的探讨丹酚酸C(SalC)对RA成纤维样滑膜细胞(FLSs)的作用,及转录因子-E2相关因子(Nrf2)信号通路在其中所扮演的角色。方法用丹酚酸C 0.1、1、5、10、20μmol/L分别处理RA-FLSs 24~72 h,之后用CCK8检测细胞活性。根据半抑制浓度(IC50)值,选取实验所需的时间和剂量处理RA-FLSs。用伤口划痕实验和Transwell小室技术检测细胞迁移及侵袭能力。ELISA检测TNF-α,IL-1β及IL-6水平,蛋白质印迹实验检测MMP-9和MMP-13的表达及细胞凋亡相关蛋白及Nrf2及介导的相关基因的表达。用ML385干扰Nrf2,实验分成3组RA-FLSs、RA-FLSs+丹酚酸C(10μmol/L)、RA-FLSs+丹酚酸C(10μmol/L)+ML385(2μmol/L),测量迁移及侵袭能力,并检测凋亡、炎性及Nrf2信号通路相关蛋白的表达。统计学分析采用方差分析,两两比较采用Turkey检验。结果与对照组相比,丹酚酸C处理后RA-FLS的细胞迁移水平(丹酚酸C 0.1μmol/L,0.75±0.05;丹酚酸C 5μmol/L,0.50±0.05;丹酚酸C 10μmol/L,0.26±0.05)显著性减少(t=7.65,P<0.001;t=14.25,P<0.001;t=20.67,P<0.001)和侵袭能力(丹酚酸C 0.1μmol/L,0.75±0.11;丹酚酸C 5μmol/L,0.49±0.06;丹酚酸C 10μmol/L,0.26±0.07)显著性降低(t=4.93,P<0.001;t=10.32,P<0.001;t=14.96,P<0.001),MMP-9(丹酚酸C 0.1μmol/L,0.72±0.10;丹酚酸C 5μmol/L,0.48±0.08;丹酚酸C 10μmol/L,0.27±0.06)水平显著性降低(t=5.60,P<0.001;t=11.03,P<0.001;t=5.94,P<0.001)及MMP-13(丹酚酸C 0.1μmol/L,0.77±0.06;丹酚酸C 5 mol/L,0.58±0.06;丹酚酸C 10μmol/L,0.32±0.13)水平显著性减少(t=8.66,P<0.001;t=11.03,P<0.001;t=14.22,P<0.001),促进细胞凋亡。丹酚酸C显著性降低促炎性细胞因子的水平(P<0.001),激活Nrf2信号通路蛋白Nrf2,过氧化氢酶(CAT),醌NADH脱氢酶1(NQO1)、超氧化物歧化酶1(SOD1)及谷胱甘肽合成酶(GSS)的表达(P<0.001)。用ML385干扰Nrf2后,显著性逆转丹酚酸C对Nrf2通路蛋白Nrf2(0.68±0.06;t=5.08,P<0.001),CAT(1.44±0.12;t=4.77,P<0.001),NQO1(0.65±0.12;t=5.04,P<0.001),SOD1(1.43±0.10;t=6.36,P<0.001)及GSS(1.42±0.10;t=7.60,P<0.001)的水平,及TNF-α[(260±22)pg/ml;t=13.75,P<0.001],IL-1β[(701±30)pg/ml;t=12.98,P<0.001],IL-6[(180.3±10.2)pg/ml;t=16.38,P<0.001]炎症因子的水平。除此之外ML385阻碍丹酚酸C对细胞迁移和侵袭的抑制作用(0.70±0.09;t=11.24,P<0.001;0.64±0.04;t=8.03,P<0.001)及对凋亡的诱导作用(24.4±1.8;t=23.02,P<0.001)。结论丹酚酸C可能通过上调Nrf2信号通路,抑制细胞活力及炎症反应,促进凋亡。丹酚酸C是治疗RA的有效潜在药物,但有待进一步动物及临床深入研究。

Objective To investigate the effect of salvianolic acid C(SalC)on fibroblast-like synoviocytes and through the role of nuclear factor-erythroid 2-related factor 2(Nrf2)pathway.Methods Rheumatoid arthritis-fibroblast-like synoviocytes(RA-FLSs)were exposed to different concentrations of SalC(0.1μmol/L,1μmol/L,5μmol/L,10μmol/L,20μmol/L)for 24-72 h and measured for viability,proliferation,migration and invasion by Cell counting kit 8(CCK-8)assay,wound-healing and transwell assay.The levels of Tumor Necrosis Factor-α(TNF-α),Interleukin-1 beta(IL-1β)and IL-6 were measured by enzyme linked immunosorbent assay(ELISA).Western blot was used to detect the expression of matrix metallopeptidase(MMP)-9,MMP-13,apoptosis-related proteins and Nrf2 mediated gene.Then we used ML385 to inhibit Nrf2 signaling pathway.RA-FLSs were measured for migration and invasion,and the expression of proteins related to apoptosis,inflammation and Nrf2 pathway.Results Compared with the control group,SalC inhibited the cell migration significantly(0.1μmol/L,0.75±0.05,t=7.65,P<0.001;5μmol/L,0.50±0.05,t=14.25,P<0.001;10μmol/L,0.26±0.05,t=20.67,P<0.001)and invasion(0.1μmol/L,0.75±0.11,t=4.93,P<0.001;5μmol/L,0.49±0.06,t=10.32,P<0.001;10μmol/L,0.26±0.07,t=14.96,P<0.001)of RA-FLs,reduced the levels of MMP-9(0.1μmol/L,0.72±0.10,t=5.60,P<0.001;5μmol/L,0.48±0.08,t=11.03,P<0.001;10μmol/L,0.27±0.06,t=15.94,P<0.001)and MMP-13(0.1μmol/L,0.77±0.06,t=8.66,P<0.001;5μmol/L,0.58±0.06,t=11.03,P<0.001;10μmol/L,0.32±0.13,t=14.22,P<0.001),and promoted apoptosis.SalC reduced the level of pro-inflammatory cytokines significantly(P<0.001)and activated the expression of Nrf2 signaling pathway proteins Nrf2,CAT,NQO1,SOD1 and GSS(P<0.001).After ML385 was used to interfere Nrf2,the levels of SalC on Nrf2 pathway proteins,such as Nrf2(0.68±0.06,t=5.08,P<0.001),CAT(1.44±0.12,t=4.77,P<0.001),NQO1(0.65±0.12,t=5.04,P<0.001),SOD1(1.43±0.10,t=6.36,P<0.001)and GSS(1.42±0.10,t=7.60,P<0.001),as well as the levels of TNF-α[(260±22)pg/ml,t=13.75,P<0.001],IL-1β[(701±30)pg/ml,t=12.98,P<0.001],IL-6[(180±10)pg/ml,t=16.38,P<0.001]were significantly reduced.In addition,ML385 inhibited the inhibition of SalC on cell migration and invasion(0.70±0.09,t=11.24,P<0.001;0.64±0.04,t=8.03,P<0.001)and induction of apoptosis(24.4±1.8,t=23.02,P<0.001).Conclusion SalC may inhibit cell activity and inflammatory response,promote apoptosis via the upregulation of Nrf2 signaling pathway.SalC may have therapeutic potential in RA.However,further investigation are needed in animal models and human.