益肾通痹汤通过抑制 SLC3A2/整合素 β3 信号通路对实验性类风湿关节炎的抗增殖和抗迁移作用。
Anti-proliferation and anti-migration effects of Yishen Tongbi decoction in experimental rheumatoid arthritis by suppressing SLC3A2/integrin β3 signaling pathways.
机构信息
First Clinical Medical School, Guangzhou University of Chinese Medicine, Guangzhou, China.
Department of Rheumatology, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, China.
出版信息
Phytomedicine. 2023 Jun;114:154741. doi: 10.1016/j.phymed.2023.154741. Epub 2023 Mar 6.
BACKGROUND
Yishen 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.
PURPOSE
Based 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 DESIGN
The 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.
METHODS
The 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.
RESULTS
IHC 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.
CONCLUSION
YSTB inhibits the proliferation and migration of synovial fibroblasts in RA by downregulating the SLC3A2/integrin β3 pathways.
背景
益肾通痹（YSTB）汤是一种专利草药配方，用于治疗类风湿关节炎（RA）；然而，其抗滑膜增生疗效的确切机制尚未完全阐明。
目的
基于我们之前的蛋白质组学研究，我们旨在揭示 YSTB 是否通过体内和体外 SLC3A2/整合素 β3 途径抑制 RA-FLS 的增殖和迁移。
研究设计
该研究设计包括三个部分，比较 RA 和 OA 患者滑膜组织中 SLC3A2 和整合素 β3 的表达；验证 YSTB 药效的动物实验和阐明 YSTB 具体机制的体外实验。
方法
通过免疫组织化学（IHC）检测 RA 和骨关节炎（OA）患者滑膜组织中 SLC3A2 和整合素 β3 的表达。体外，首先通过 EdU 染色和划痕愈合实验分别比较 HFLS（人成纤维样滑膜细胞）和 HFLS-RA（人成纤维样滑膜细胞-RA）细胞的增殖和迁移能力，并通过 IF 和 WB 检测 SLC3A2、整合素 β3、p-FAK 和 p-Src 在 HFLS 和 HFLS-RA 细胞中的表达和定位差异。体内，DBA/1 小鼠注射牛胶原蛋白 II 构建 CIA 小鼠模型。爪肿胀、体重和关节炎指数（AI）作为 YSTB 的基本治疗评估指标。还对膝关节和踝关节进行了 micro-CT 和组织病理学分析。此外，通过 IHC 检测小鼠滑膜组织中 SLC3A2、整合素 β3、p-FAK 和 p-Src 的表达。随后，通过 CCK-8 筛选出适合用于 HFLS-RA 细胞的 YSTB 浓度。通过 EdU 染色和 Transwell 迁移实验评估 YSTB 对细胞增殖和迁移的抑制作用，并通过 WB 评估 YSTB 是否通过下调 SLC3A2/整合素 β3 途径抑制 HFLS-RA 迁移。
结果
IHC 显示 RA 滑膜组织中 SLC3A2 和整合素 β3 的表达高于 OA 组织。体内实验表明，YSTB 抑制滑膜增生，防止骨破坏，降低 SLC3A2、整合素 β3、p-FAK 和 p-Src 的表达。体外实验表明，YSTB 通过抑制 SLC3A2/整合素 β3 及其下游信号分子的表达抑制 HFLS-RA 的迁移和增殖。
结论
YSTB 通过下调 SLC3A2/整合素 β3 途径抑制 RA 滑膜成纤维细胞的增殖和迁移。
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