Chen Jianhai, Chen Jingqin, Tan Jianwei, Li Jian, Cheng Wenxiang, Ke Liqing, Wang Qijing, Wang Anqiao, Lin Sien, Li Gang, Zhang Peng, Wang Benguo
Rehabilitation Department of The Second Affiliated Hospital, School of Medicine, The Chinese University of Hong Kong, Shenzhen & Longgang District People's Hospital of Shenzhen, Shenzhen, Guangdong, 518172, China.
Center for Translational Medicine Research and Development, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China.
J Orthop Translat. 2023 Jun 6;40:49-57. doi: 10.1016/j.jot.2023.05.004. eCollection 2023 May.
The purpose of this work is to investigate how the Rho family of GTPases A (RhoA) mediates the pathogenesis of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS).
The expression of RhoA in the synovial tissues of RA and Healthy people (Control) was detected using immunohistochemistry methods. The expression of RhoA and hypoxia-inducible factor-1α (HIF-1α) is inhibited by small interfering RNAs (siRNAs). The inhibition effect on RA-FLS migration was further investigated. The protein expression level of HIF-1α, RhoA, focal adhesion kinase (FAK), and myosin light chain (MLC) was also analysed using western blotting (WB). DBA1 mice were immunised with the mixture of bovine type II collagen and Freund's adjuvant to establish collagen induced arthritis (CIA) mouse model. Lip-siRhoA is administered through joint injection every two days. Micro-computed tomography (micro-CT) was used to detect mouse ankle joint destruction and evaluate the bone loss of the periarticular side. Destruction of the ankle articular cartilage was tested by histology. Expressions of P-RhoA, P-FAK and P-MLC in the ankle joint was detected by immunohistochemistry assay.
The expression level of RhoA in the synovial tissues of RA patients was significantly higher than that in control group. Hypoxia was able to up-regulate the expression of RhoA. Whereas, HIF-1α siRNA (siHIF-1α) could down-regulate the expression of RhoA. Additionally, both of siHIF-1α and RhoA siRNA (siRhoA) delivered by liposome (Lip-siHIF-1α and Lip-siRhoA) were found to suppress FAK and MLC phosphorylation in vitro. In CIA mouse model, Lip-siRhoA was demonstrated to ameliorate the destruction of ankle joint and reduce the severity of ankle joint cartilage damage by micro-CT and histological staining, respectively. Therefore, inhibition of FLS cell migration can protect articular bone from destruction. Furthermore, the expression of P-RhoA, P-FAK and P-MLC was evaluated and found to be down-regulated by Lip-siRhoA in vivo.
The results demonstrated that under hypoxic environment, HIF-1α dependent RhoA pathway played an important role on cytoskeleton remodelling and RA-FLS migration. Through down-regulating RhoA expression, it could effectively treat RA in vitro and in vivo.
Our study provides new evidence for the potential clinical application of RhoA as a candidate for the treatment of RA.
本研究旨在探讨Rho家族鸟苷三磷酸酶A（RhoA）如何介导类风湿关节炎成纤维样滑膜细胞（RA-FLS）的发病机制。
采用免疫组化方法检测类风湿关节炎（RA）患者和健康对照者滑膜组织中RhoA的表达。小干扰RNA（siRNA）抑制RhoA和缺氧诱导因子-1α（HIF-1α）的表达。进一步研究其对RA-FLS迁移的抑制作用。同时采用蛋白质免疫印迹法（WB）分析HIF-1α、RhoA、黏着斑激酶（FAK）和肌球蛋白轻链（MLC）的蛋白表达水平。用牛II型胶原与弗氏佐剂混合物免疫DBA1小鼠，建立胶原诱导性关节炎（CIA）小鼠模型。每两天通过关节注射给予脂质体包裹的RhoA siRNA（Lip-siRhoA）。采用微型计算机断层扫描（micro-CT）检测小鼠踝关节破坏情况，并评估关节周围骨丢失情况。通过组织学检测踝关节软骨破坏情况。采用免疫组化法检测踝关节中磷酸化RhoA（P-RhoA）、磷酸化FAK（P-FAK）和磷酸化MLC（P-MLC）的表达。
RA患者滑膜组织中RhoA表达水平显著高于对照组。缺氧能够上调RhoA的表达。而HIF-1α siRNA（siHIF-1α）能够下调RhoA的表达。此外，脂质体包裹的siHIF-α和RhoA siRNA（Lip-siHIF-1α和Lip-siRhoA）在体外均能抑制FAK和MLC的磷酸化。在CIA小鼠模型中，通过micro-CT和组织学染色分别证实Lip-siRhoA可改善踝关节破坏并减轻踝关节软骨损伤的严重程度。因此，抑制FLS细胞迁移可保护关节骨免受破坏。此外，体内实验发现Lip-siRhoA可下调踝关节中P-RhoA、P-FAK和P-MLC的表达。
结果表明，在缺氧环境下，HIF-1α依赖的RhoA信号通路在细胞骨架重塑和RA-FLS迁移中起重要作用。通过下调RhoA表达，可在体外和体内有效治疗RA。
我们的研究为RhoA作为RA治疗候选药物的潜在临床应用提供了新证据。
