Department of Rheumatology and Immunology, Nanfang Hospital, Southern Medical University, Guangzhou, PR China; Department of Traditional Chinese Internal Medicine, School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, PR China.
Department of Traditional Chinese Internal Medicine, School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, PR China.
Phytomedicine. 2024 Sep;132:155762. doi: 10.1016/j.phymed.2024.155762. Epub 2024 May 25.
BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by exacerbated synovial inflammation and joint destruction. Recent studies suggest toll-like receptor 4 (TLR4) internalization facilitate inflammatory response of macrophage. The role of TLR4 internalization in the pathogenesis of RA is unknown. PURPOSE: To investigate the role and mechanism of TLR4 internalization in macrophage inflammatory response of RA and explore whether TLR4 internalization mediates the anti-arthritic effect of Xiaowugui (XWG) decoction, a patented herbal formula used in China. METHODS: The co-expression of TLR4 and the internalization marker, early endosome antigen 1 (EEA1), in the synovial samples of RA patients and joint tissue of collagen-induced arthritis (CIA) mice, were evaluated using immunofluorescence. The effect of Rab5a-mediated early internalization of TLR4 on the activation induced by lipopolysaccharide (LPS) in RAW264.7 cells was investigated using small interfering RNAs that act against Rab5a. CIA was induced in Rab5a mice to evaluate the role of Rab5a in vivo. The disease progression and expression of Rab5a and TLR4 in the joint tissue were evaluated in CIA mice treated with XWG. Inflammatory factors production, TLR4 internalization, and activation of downstream signaling pathways were examined in RAW264.7 cells treated with XWG in vitro. RESULTS: The co-expression and co-localization of TLR4 and EEA1 were elevated in the synovial samples of RA patients and joint tissue of CIA mice. Pharmaceutical inhibition of TLR4 internalization reduced macrophages inflammatory responses induced by LPS. The co-expression and co-localization of Rab5a and TLR4 were significantly increased in macrophages treated with LPS. Silencing Rab5a reduced LPS-induced TLR4 internalization, inflammatory factors production, and phosphorylation of Jun N-terminal kinases (JNK) and p65. Genetic deletion of Rab5a inhibited TLR4 internalization and the development of arthritis in vivo. The co-expression of TLR4 and Rab5a was also elevated in the synovial samples of RA patients. XWG treatment of mice with CIA alleviated arthritis and reduced the co-expression of Rab5a and TLR4 in the joint tissue. XWG treatment of macrophage inhibited LPS-induced IL-6 and TNF-α production, co-expression of Rab5a and TLR4, and phosphorylation of JNK and p65. CONCLUSIONS: Our findings highlight the pathogenic role of TLR4 internalization in patients with RA and identify a novel Rab5a-dependent internalization pathway that promotes macrophage inflammatory response. XWG treatment demonstrated outstanding therapeutic effects in experimental arthritis, and targeting the Rab5a-mediated internalization of TLR4 may be the main underlying mechanism.
背景：类风湿关节炎（RA）是一种慢性自身免疫性疾病，其特征为滑膜炎症加剧和关节破坏。最近的研究表明，Toll 样受体 4（TLR4）内化有助于巨噬细胞的炎症反应。TLR4 内化在 RA 发病机制中的作用尚不清楚。
目的：探讨 TLR4 内化在 RA 巨噬细胞炎症反应中的作用及其机制，并探讨 TLR4 内化是否介导了中国专利草药配方小续命汤（XWG）对关节炎的抗风湿作用。
方法：采用免疫荧光法检测 RA 患者滑膜样本和胶原诱导关节炎（CIA）小鼠关节组织中 TLR4 与早期内体标志物早期内体抗原 1（EEA1）的共表达。用针对 Rab5a 的小干扰 RNA 研究 Rab5a 介导的 TLR4 早期内化对脂多糖（LPS）诱导的 RAW264.7 细胞激活的影响。在 Rab5a 小鼠中诱导 CIA 以评估 Rab5a 在体内的作用。在 CIA 小鼠中给予 XWG 治疗，评估 XWG 对关节组织中疾病进展和 Rab5a、TLR4 表达的影响。体外用 XWG 处理 RAW264.7 细胞，检测细胞炎性因子产生、TLR4 内化及下游信号通路激活情况。
结果：RA 患者滑膜样本和 CIA 小鼠关节组织中 TLR4 和 EEA1 的共表达和共定位增加。TLR4 内化的药物抑制减少了 LPS 诱导的巨噬细胞炎症反应。用 LPS 处理的巨噬细胞中 Rab5a 和 TLR4 的共表达和共定位明显增加。沉默 Rab5a 减少了 LPS 诱导的 TLR4 内化、炎性因子产生以及 Jun N-末端激酶（JNK）和 p65 的磷酸化。Rab5a 基因缺失抑制了体内 TLR4 内化和关节炎的发展。RA 患者滑膜样本中 TLR4 和 Rab5a 的共表达也升高。XWG 治疗 CIA 小鼠减轻了关节炎并降低了关节组织中 Rab5a 和 TLR4 的共表达。XWG 处理巨噬细胞抑制了 LPS 诱导的 IL-6 和 TNF-α产生、Rab5a 和 TLR4 的共表达以及 JNK 和 p65 的磷酸化。
结论：本研究结果强调了 TLR4 内化在 RA 患者中的致病作用，并确定了一种新的 Rab5a 依赖性内化途径，该途径促进了巨噬细胞炎症反应。XWG 治疗在实验性关节炎中表现出出色的治疗效果，而靶向 TLR4 介导的 Rab5a 内化可能是其主要潜在机制。
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