乌头汤对膝骨关节炎模型大鼠滑膜组织TLR4/NF-κB信号通路的影响

Effect of Wutou Decoction on TLR4/NF-κB signaling pathway in synovial tissue of rat models with knee osteoarthritis

背景:前期研究表明乌头汤可有效抑制膝骨关节炎炎症反应,但具体机制尚不明确。目的:观察乌头汤对膝骨关节炎大鼠滑膜组织TLR4/NF-κB通路相关调节因子的影响,探讨乌头汤治疗膝骨关节炎的作用机制。方法:SPF级雄性SD大鼠36只,购自上海斯莱克实验动物有限责任公司。实验方案经福建中医药大学动物实验伦理委员会批准。大鼠适应性喂养1周后,采用随机数字表法分为空白组、模型组和治疗组。模型组和治疗组均采用改良Hulth法复制双膝膝骨关节炎模型,术后1周空白组和模型组给予生理盐水,按照10 mL/(kg·d)的量进行灌胃;治疗组给予乌头汤,按照4.2 g/(kg·d)的药量进行灌胃。治疗8周后,取大鼠膝关节滑膜组织。ELISA法检测诱导型一氧化氮合酶、肿瘤坏死因子α和白细胞介素6含量变化;Real-time PCR法检测TLR4、MyD88、TRAF6和NF-κBp65mRNA表达;Westernblot法检测TLR4、MyD88、TRAF6和NF-κB p65蛋白表达。结果与结论:①ELISA结果显示,与模型组相比,乌头汤可有效抑制诱导型一氧化氮合酶、肿瘤坏死因子α和白细胞介素6表达(P<0.01);②Real-timePCR结果表明,乌头汤能抑制TLR4、MyD88、TRAF6和NF-κB p65 mRNA表达(P <0.01);③Western blot结果与Real-time PCR结果趋势一致,即乌头汤能抑制TLR4、MyD88、TRAF6和NF-κB p65蛋白表达(P <0.01);④结果提示,乌头汤可通过调控TLR4/NF-κB信号通路,抑制膝骨关节炎滑膜炎症反应,从而起到治疗膝骨关节炎作用。

BACKGROUND: Preliminary study has shown that Wutou Decoction can effectively inhibit inflammatory reaction in knee osteoarthritis. But its underling mechanism remains unclear.OBJECTIVE: To observe the effect of Wutou Decoction on the related regulatory factors in TLR4/NF-κB signaling pathway in synovium of rats with knee osteoarthritis, so as to explore its anti-inflammatory mechanism on synovitis of knee osteoarthritis.METHODS: Thirty-six male Sprague-Dawley rats, SPF grade, were provided by Shanghai Slack Laboratory Animals Co., Ltd., and the study was approved by the Experimental Animal Ethics Committee of Fujian University of Traditional Chinese Medicine. After 1 week of acclimation,the rats were randomly divided into blank, model and treatment groups. The rats in the model and treatment groups were used for establishing osteoarthritis model in both knees by modified Hulth method. One week later, the blank and model groups received 10 mL/(kg·d) saline via gavage. The experiment group received 4.2 g/(kg·d) Wutou Decoction. After 8 weeks of treatment, the rat synovial tissue was obtained. The contents of inducible nitric oxide synthase, tumor necrosis factor-α and interleukin-6 were measured by ELISA. The mRNA expression levels of TLR4, MyD88, TRAF6 and NF-κB p65 were measured by real-time PCR. The protein levels of TLR4, MyD88, TRAF6 and NF-κB p65 were measured by western blot assay.RESULTS AND CONCLUSION:(1) ELISA results showed that Wutou Decoction could effectively inhibit the expression of inducible nitric oxide synthase, tumor necrosis factor-α and interleukin-6 compared with the model group(P < 0.01).(2) Real-time PCR results suggested that Wutou Decoction could inhibit the mRNA expression of TLR4, MyD88, TRAF6 and NF-κB p65 compared with the model group(P < 0.01).(3)The results of western blot assay were in agreement with the results of real-time PCR. That was, Wutou Decoction could inhibit the protein expression of TLR4, MyD88, TRAF6 and NF-κB p65(P < 0.01).(4) These results suggest that Wutou Decoction can inhibit the inflammatory reaction in synovitis of knee osteoarthritis by regulating TLR4/NF-κB signaling pathway, which may, in part, explain its clinical efficacy in the treatment of synovitis of knee osteoarthritis.