基于Ras/Raf/Mek/Erk信号通路探讨化湿定痛汤减轻膝骨关节炎大鼠滑膜炎症的机制

Mechanism of Huashi Dingtong Decoction in alleviating inflammation of synovial in knee osteoarthritis rats based on Ras/Raf/Mek/Erk signaling pathway

目的:基于Ras/Raf/Mek/Erk信号通路探讨化湿定痛汤对膝骨关节炎(KOA)大鼠滑膜炎症的可能作用机制。方法:采用膝关节腔注射Ⅱ型胶原酶建立KOA模型,将38只大鼠随机分为空白组、模型组、化湿定痛汤组及Mek抑制剂组。空白组和模型组给予0.9%氯化钠溶液灌胃,化湿定痛汤组给予化湿定痛汤4.68 mg·kg^(-1)·d^(-1)灌胃,Mek抑制剂组给予腹腔注射PD98059 1 mg·kg^(-1)·d^(-1),连续28 d。观察大鼠膝关节直径变化,MRI观察大鼠膝关节影像学变化,HE染色观察大鼠关节软骨及滑膜病理改变,Western Blot检测大鼠滑膜中Ras、Raf、p-Mek、p-Erk蛋白表达,RT-qPCR检测大鼠滑膜中Ras、Raf、Mek、Erk的mRNA表达。结果:与空白组比较,模型组大鼠膝关节核磁共振评分(MOAKS)显著升高(P<0.01)。与模型组比较,化湿定痛汤组及Mek抑制剂组大鼠第35天膝关节直径显著减小(P<0.01,P<0.05),滑膜组织炎症浸润减轻,Ras、Mek、Erk mRNA和Ras、Raf、p-Mek、p-Erk蛋白表达显著降低(P<0.01,P<0.05)。结论:化湿定痛汤可能通过调节大鼠膝关节滑膜中Ras/Raf/Mek/Erk信号通路改善炎症损伤。

Objective:To investigate the effect of Huashi Dingtong Decoction on expression of Ras/Raf/Mek/Erk signaling pathway in rats with knee osteoarthritis(KOA).Methods:A total of 38 SD rats were randomly divided into control group,model group,Huashi Dingtong Decoction group,Mek inhibitors group.KOA was induced by injection of collagenase Ⅱ into the left knee joint of rats.Control group and model group were given 0.9% sodium chloride solution by intragastric administration,Huashi Dingtong Decoction group was given Huashi Dingtong Decoction(4.68 mg·kg^(-1)·d^(-1)) intragastrically,Mek inhibitors group was injected with PD98059(1 mg·kg^(-1)·d^(-1)) intraperitoneally,the drug was administered continuously for 28 days.The color of skin and diameter was used to observed the macroscopical change of knee.MRI was conducted to verify the imageological change of knee.HE staining was used to observe the pathological morphology of chondrocyte and synovium.Western Blot was performed to detect the protein expressions of Ras,Raf,p-Mek and p-Erk in the synovium of rats.RT-qPCR was performed to detect the mRNA expressions of Ras,Raf,Mek and Erk in the synovium of rats.Result:Compared with the control group,MOAKS of the model group was significantly increased(P<0.01).Compared with the model group,the diameter of knee joint in Huashi Dingtong Decoction group and Mek inhibitor group decreased significantly(P<0.01,P<0.05);the inflammatory of synovial tissue was relieved;the mRNA expressions of Ras,Mek,Erk mRNA,and the protein expressions of Ras,Raf,p-Mek,p-Erk decreased significantly(P<0.01,P<0.05).Conclusion:Huashi Dingtong Decoction can significantly inhibit inflammation in KOA rats and down regulate the overexpression of Ras/Raf/Mek/Erk signaling pathway related factors.