Table1_Identification of Active Compounds From Yi Nationality Herbal Formula Wosi Influencing COX-2 and VCAM-1 Signaling.docx

The Yi nationality herbal formula Wosi is used in China as a folk medicine to treat arthritis and related diseases. Despite its widespread use, the active ingredients, and pharmacological mechanisms are not performed. This is the first time to identify the active compounds from Wosi with the aim at providing the potential effect of Wosi and exploring its underlying anti-inflammatory mechanism in monosodium urate crystals (MSU)-induced arthritis rats. In this study, anti-hyperuricemia effect was assessed by reducing the serum uric acid levels and increasing uric acid excretion in the urine for the hyperuricemia rat model. Wosi significantly suppressed the degree of joint swelling and improved the symptoms of inflammation induced by MSU crystals. The inhibition of IL-2, IL-1β, IFN-γ, and IL-6 secretion and IL-10 increase in the serum were also observed. This study also focuses on the screening of the main compounds from Wosi against cyclooxygenase for anti-inflammatory properties using molecular docking. The result showed 3-O-[α-L-pyran rhamnose(1-3)-β-D-pyran glucuronic acid]- oleanolic acid, 3-O-(β-D-pyran glucuronic acid)-oleanolic acid-28-O-β-D-pyran glucoside, and 3-O-[α-L-pyran rhamnose(1-3)-β-D-pyran glucuronic acid]-oleanolic acid-28-O-β-D-pyran glucoside with a higher binding affinity for COX-2 than COX-1 which indicated relatively higher interaction than COX-1. The preferential selectivity toward inhibiting COX-2 enzyme over COX-1 of three compounds from Wosi were evaluated using in-vitro cyclooxygenases 1 and 2 (COX-1/2) inhibition assays. Meanwhile, the down-regulated protein expression of COX-2 and VCAM-1 in synovial tissue sections from ankle joints of experiments rats were confirmed by immunohistochemistry analysis after the Wosi treatment. In conclusion, three oleanolic acid glycosides were implied as mainly efficient compounds in Yi nationality herbal formula Wosi for arthritis therapy via selectively influencing COX-2 and VCAM-1 signaling.

彝族(Yi nationality)草药方剂沃司(Wosi)在中国作为民间药物用于治疗关节炎及相关疾病，尽管其应用广泛，但其活性成分与药理机制尚未得到系统研究与阐明。本研究首次从沃司中鉴定活性化合物，旨在明确沃司的潜在药效，并探讨其在尿酸钠结晶(monosodium urate crystals, MSU)诱导的关节炎大鼠模型中的抗炎机制。本研究通过检测高尿酸血症大鼠模型的血清尿酸水平降低幅度与尿液尿酸排泄量提升情况，评估沃司的抗高尿酸血症(anti-hyperuricemia)活性，结果显示沃司可显著抑制关节肿胀程度，改善尿酸钠结晶(MSU)诱导的炎症症状，同时还观察到沃司可抑制血清中白细胞介素2(IL-2)、白细胞介素1β(IL-1β)、干扰素γ(IFN-γ)及白细胞介素6(IL-6)的分泌，并促进白细胞介素10(IL-10)的表达上调。本研究同时采用分子对接(molecular docking)技术，筛选沃司中针对环氧合酶(cyclooxygenase, COX)具有抗炎活性的主要成分，结果表明3-O-[α-L-吡喃鼠李糖(1→3)-β-D-吡喃葡萄糖醛酸]-齐墩果酸、3-O-(β-D-吡喃葡萄糖醛酸)-齐墩果酸-28-O-β-D-吡喃葡萄糖苷以及3-O-[α-L-吡喃鼠李糖(1→3)-β-D-吡喃葡萄糖醛酸]-齐墩果酸-28-O-β-D-吡喃葡萄糖苷对环氧合酶2(COX-2)的结合亲和力高于环氧合酶1(COX-1)，提示其与COX-2的相互作用更强。随后通过体外环氧合酶1/2(COX-1/2)抑制实验，进一步验证了沃司中这三种化合物对COX-2的抑制选择性优于COX-1，同时经免疫组织化学(immunohistochemistry)分析证实，沃司治疗后实验大鼠踝关节滑膜组织中的COX-2与血管细胞黏附分子1(VCAM-1)蛋白表达水平显著下调。综上，三种齐墩果酸苷类化合物被认定为彝族草药方剂沃司治疗关节炎的核心有效成分，其作用机制为选择性调控COX-2与VCAM-1信号通路。