Anti-proliferation and anti-migration effects of Yishen Tongbi decoction in experimental rheumatoid arthritis by suppressing SLC3A2/integrin β3 signaling pathways

Abstract

BackgroundYishen Tongbi (YSTB) decoction is a patented herbal formula that is used in China to treat rheumatoid arthritis (RA); however, the exact mechanism of its anti-synovial hyperplasia efficacy has not been fully elucidated. PurposeBased on our previous proteomics study, we aimed to reveal whether YSTB inhibits the proliferation and migration of RA-FLSs through the SLC3A2/integrin β3 pathway in vivo and in vitro. Study designThe study design consists of three parts, a comparison of the expression of SLC3A2 and integrin β3 in synovial tissues of RA and OA patients; an animal experiment to verify the pharmacodynamic effect of YSTB, and in vitro experiment to elucidate the specific mechanism of YSTB. MethodsThe expression of SLC3A2 and integrin β3 in the synovial tissues of patients with RA and osteoarthritis (OA) patients were detected by immunohistochemistry (IHC). In vitro, firstly, the proliferation and migration abilities of HFLS (human fibroblast-like synoviocytes) and HFLS-RA (human fibroblast-like synoviocytes-RA) cells were compared by EdU staining and wound healing assays, respectively, and the differences in the expression and localization of SLC3A2, integrin β3, p-FAK and p-Src between HFLS and HFLS-RA cells were detected by IF and WB. In vivo, DBA/1 mice were injected with bovine collagen II to construct a CIA mouse model. Paw swelling, body weight and the arthritis index (AI) were used as basic treatment evaluation indicators for YSTB. Micro-CT and histopathological analyses of the knee and ankle joints were also performed. In addition, the expression of SLC3A2, integrin β3, p-FAK and p-Src in the synovial tissue of mice was detected by IHC. Subsequently, CCK-8 was used to screen for suitable concentrations of YSTB for use in HFLS-RA cells. EdU staining and transwell migration assays were performed to evaluate the inhibitory effect of YSTB on cell proliferation and migration, and WB was conducted to assess whether YSTB inhibited HFLS-RA migration through downregulation of the SLC3A2/integrin β3 pathways. ResultsIHC showed that the expression of SLC3A2 and integrin β3 was higher in RA synovial tissues than in OA tissues. In vivo experiments showed that YSTB inhibited synovial hyperplasia, prevented bone destruction, and reduced the expression of SLC3A2, integrin β3, p-FAK and p-Src. In vitro experiments showed that YSTB inhibited HFLS-RA migration and proliferation by inhibiting the expression of SLC3A2/integrin β3 and downstream signaling molecules. ConclusionYSTB inhibits the proliferation and migration of synovial fibroblasts in RA by downregulating the SLC3A2/integrin β3 pathways.