五味温通除痹胶囊促进佐剂性关节炎大鼠滑膜组织细胞自噬活性及机制

Wuwei Wentong Chubi Capsule promotes the autophagy in synovial tissue of adjuvant-induced arthritis rats and its mechanism

目的观察五味温通除痹胶囊对佐剂性关节炎(AA)大鼠滑膜组织中磷脂酰肌醇3激酶/蛋白激酶B/哺乳动物雷帕霉素白蛋白(PI3K/AKT/mTOR)信号通路关键基因表达的影响,探讨其防治类风湿性关节炎的可能机制。方法SD大鼠,随机分为正常组、模型组、(0.80、1.60、3.20)g/kg五味温通除痹胶囊治疗组、40mg/kg雷公藤多甙片治疗组,每组10只。除正常组外,采用Freund完全佐剂诱导AA大鼠模型。致炎后第12天灌胃给药,每天1次,连续12d。实验结束后,取非致炎侧踝关节,HE染色观察病理组织学改变,实时定量荧光PCR测定滑膜组织中PI3K、AKT、mTOR、p70s6、beclin1mRNA的水平,免疫荧光组织化学染色和Westernblot法检测滑膜组织中PI3K、AKT、磷酸化的AKT(p-AKT)、mTOR、p-mTOR、p70s6、p-p70s6、beclin1蛋白的表达。结果与模型组相比,(1.60、3.20)g/kg五味温通除痹胶囊治疗组不仅可减轻AA大鼠踝关节病理组织学损伤程度,还可降低PI3K、AKT、p-AKT、mTOR、p-mTOR、p70s6、p-p70s6的水平,提高beclin1的水平。结论五味温通除痹胶囊可抑制PI3K/AKT/mTOR信号通路,上调AA大鼠滑膜细胞自噬活性,减轻关节软骨损伤。

Objective To evaluate the effect of Wuwei Wentong Chubi （VVW3/VTCB） Capsule on the PI3K/AKT/mTOR signaling pathway in the synovial tissues of adjuvant-induced arthritis （AA） rats, and investigate its potential pharmacological mechanisms of treating rheumatoid arthritis. Methods Sixty Sprague Dawley （SD） rats were randomly assigned into six groups evenly： normal group, model group, WWWTCB groups at 0.80, 1.60, 3.20 g/kg body mass, and tripterygium glycosides tablet （TPT） group at 40 mg/kg body mass. Except for the normal group, the other five groups were induced into AA models with Complete Freund＇s Adjuvant （CFA）. The WWWTCB or TPT, was administrated from day 12 after injection of CFA by gavage, once a day for 12 days. After that, unaffected ankle-joint tissues from the AA rats were collected for histopathological examination. The mRNA levels of PI3K, AKT, mTOR, p70s6 and beclinl in the synovial tissue were detected by real-time quantitative PCR. Meanwhile, the protein levels of PI3K, AKT, p-AKT, roTOR, p-roTOR, p70s6, p-p70s6 and beclinl were determined by immunofluorescence histochemical staining and/or Western blotting. Results Compared with the model group, WWVVTCB （1.60, 3.20 g/kg body mass） groups showed less ankle-joint injury and decreased proliferation of synovial cells in the ankle-joint tissues. In addition, the administration of WWWTCB decreased the mRNA and protein levels of PI3K, AKr, p-AKT, roTOR, p-mTOR, p70s6 and p-p70s6, while increased the level of beclint. Coaclusion WWWTCB ameliorated AA in rats. The improvement might be closely related to the inhibitory effect of WWWTCB on the PI3K/AKT/ mTOR signaling pathway and its promoting effect on the autophagy activity of synovial cells.