DataSheet1_Anti-rheumatoid arthritis effects of traditional Chinese medicine Fufang Xiaohuoluo pill on collagen-induced arthritis rats and MH7A cells.docx

BackgroundFufang Xiaohuoluo pill (FFXHL) is a commonly used prescription in clinical practice for treating rheumatoid arthritis in China, yet its specific mechanism remains unclear. This study aims to elucidate the pharmacological mechanisms of FFXHL using both in vivo and in vitro experiments.
MethodsThe collagen-induced arthritis (CIA) rat model was established to evaluate FFXHL’s therapeutic impact. Parameters that include paw swelling, arthritis scores, and inflammatory markers were examined to assess the anti-inflammatory and analgesic effects of FFXHL. Human fibroblast-like synoviocytes (MH7A cells) is activated by tumour necrosis factor-alpha (TNF-α) were used to explore the anti-inflammatory mechanism on FFXHL.
ResultsOur findings indicate that FFXHL effectively reduced paw swelling, joint pain, arthritis scores, and synovial pannus hyperplasia. It also lowered serum levels of TNF-α, interleukin-1β (IL1β), and interleukin-6 (IL-6). Immunohistochemical analysis revealed decreased expression of nuclear factor-kappa B (NF-κB) p65 in FFXHL-treated CIA rat joints. In vitro experiments demonstrated FFXHL’s ability to decrease protein secretion of IL-1β and IL-6, suppress mRNA expression of matrix metalloproteinases (MMP) −3, −9, and −13, reduce reactive oxygen species (ROS) levels, and inhibit NF-κB p65 translocation in TNF-α stimulated MH7A cells. FFXHL also suppressed protein levels of extracellular signal-regulated kinase (ERK), c-Jun Nterminal kinase (JNK), p38 MAP kinase (p38), protein kinase B (Akt), p65, inhibitor of kappa B kinase α/β (IKKα/β), Toll-like receptor 4 (TLR4), and myeloid differentiation primary response 88 (MyD88) induced by TNF-α in MH7A cells.
ConclusionThe findings imply that FFXHL exhibits significant anti-inflammatory and antiarthritic effects in both CIA rat models and TNF-α-induced MH7A cells. The potential mechanism involves the inactivation of TLR4/MyD88, mitogen-activated protein kinases (MAPKs), NF-κB, and Akt pathways by FFXHL.

背景：复方红火落丸（FFXHL）是我国临床治疗类风湿关节炎的常用方剂，但其具体作用机制尚未阐明。本研究旨在通过体内外实验阐明FFXHL的药理学作用机制。
方法：本研究建立胶原诱导性关节炎（collagen-induced arthritis, CIA）大鼠模型，以评估FFXHL的治疗作用。通过检测足肿胀度、关节炎评分及炎症标志物水平，评价FFXHL的抗炎与镇痛效果。采用肿瘤坏死因子-α（tumour necrosis factor-alpha, TNF-α）刺激人成纤维样滑膜细胞（MH7A细胞），探究FFXHL的抗炎作用机制。
结果：本研究结果显示，FFXHL可有效减轻足肿胀、缓解关节疼痛、降低关节炎评分，并抑制滑膜血管翳增生。同时可降低血清中TNF-α、白细胞介素-1β（IL-1β）及白细胞介素-6（IL-6）的水平。免疫组化分析表明，经FFXHL干预的CIA大鼠关节组织中，核因子-κB（nuclear factor-kappa B, NF-κB）p65的表达量显著下调。体外实验证实，FFXHL可减少TNF-α诱导的MH7A细胞中IL-1β与IL-6的蛋白分泌量，抑制基质金属蛋白酶（matrix metalloproteinases, MMP）-3、-9及-13的mRNA表达，降低活性氧（reactive oxygen species, ROS）水平，并阻滞NF-κB p65的核转位。此外，FFXHL可抑制TNF-α诱导的MH7A细胞中细胞外信号调节激酶（extracellular signal-regulated kinase, ERK）、c-Jun氨基末端激酶（c-Jun N-terminal kinase, JNK）、p38丝裂原活化蛋白激酶（p38 MAP kinase, p38）、蛋白激酶B（protein kinase B, Akt）、p65、κB激酶抑制剂α/β（inhibitor of kappa B kinase α/β, IKKα/β）、Toll样受体4（Toll-like receptor 4, TLR4）及髓系分化初级应答基因88（myeloid differentiation primary response 88, MyD88）的蛋白表达水平。
结论：本研究结果表明，FFXHL在CIA大鼠模型及TNF-α诱导的MH7A细胞中均具有显著的抗炎与抗关节炎活性。其潜在作用机制可能为通过灭活TLR4/MyD88、丝裂原活化蛋白激酶（mitogen-activated protein kinases, MAPKs）、NF-κB及Akt信号通路实现。