ABSTRACT
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 (0.1 μmol/L, 1 μmol/L, 5 μmol/L, 10 μmol/L, 20 μmol/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 (0.1 μmol/L, 0.75±0.05, t=7.65, P<0.001; 5 μmol/L, 0.50±0.05, t=14.25, P<0.001; 10 μmol/L, 0.26±0.05, t=20.67, P<0.001) and invasion (0.1 μmol/L, 0.75±0.11, t=4.93, P<0.001; 5 μmol/L, 0.49±0.06, t=10.32, P<0.001; 10 μmol/L, 0.26±0.07 , t=14.96, P<0.001) of RA-FLs, reduced the levels of MMP-9 (0.1 μmol/L, 0.72±0.10, t=5.60, P<0.001; 5 μmol/L, 0.48±0.08, t=11.03, P<0.001; 10 μmol/L, 0.27±0.06, t=15.94, P<0.001) and MMP-13 (0.1 μmol/L, 0.77±0.06, t=8.66, P<0.001; 5 μmol/L, 0.58±0.06, t=11.03, P<0.001; 10 μmol/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.