基于网络药理学探究苗药“四大血”对类风湿性关节炎自噬的影响

Study on the Effect of Miao Medicine"Sidaxue"on Autophagy in Rheumatoid Arthritis Based on Network Pharmacology

本研究皆在探究苗药“四大血”(Sidaxue,SX)调控类风湿性关节炎(rheumatoid arthritis,RA)中细胞自噬的分子机制。首先,构建胶原诱导型关节炎(collagen-induced arthritis,CIA)大鼠模型,SX给药21 d后计算大鼠胸腺指数和脾脏指数,观察大鼠膝关节滑膜组织病理改变,并检测大鼠血清白细胞介素-6(interleukin-6,IL-6)和白细胞介素-37(interleukin-37,IL-37)水平。进一步利用网络药理学分析SX调控RA中细胞自噬的可能分子机制,并借助Autodock vina软件进行分子对接验证,同时通过在线数据库预测SX主要活性成分的药学性质,结合分子对接评分初筛效应成分。最后,利用RT-qPCR和Western blot分别检测SX对CIA大鼠滑膜组织中磷脂酰肌醇3-激酶(phosphoinositide-3 kinase,PI3K)、蛋白激酶B(protein kinase B,AKT)、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)及微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)mRNA和蛋白表达的影响。动物实验结果表明,SX能有效缓解CIA大鼠关节肿胀,缓解免疫器官的过度反应,改善滑膜组织病理改变,降低炎症因子IL-6、IL-37水平(P<0.05)。网络药理学结果表明,AKT1、CASP3、BCL2L1等靶点较为关键,动物自噬、凋亡及PI3K/AKT信号通路等途径与RA相关且显著性较高,分子对接结果显示PI3K/AKT信号通路关键靶点与SX主要活性成分稳定结合,β-谷甾醇、卡亚宁、乌索酸和胡萝卜苷可能是SX调控自噬的较优效应成分。RT-qPCR和Western blot实验结果显示,SX呈剂量依赖性下调PI3K、AKT、mTOR mRNA(P<0.05)和蛋白(P<0.001)的表达,上调自噬标志物LC3 mRNA表达水平(P<0.05),促进LC3-Ⅰ转变为LC3-Ⅱ(P<0.01)。综上所述,SX具有改善RA的作用,其机制可能是抑制PI3K/AKT/mTOR信号通路,进而促进滑膜细胞发生自噬性细胞死亡以拮抗滑膜细胞的异常增殖。

This study aims to explore the molecular mechanism of cellular autophagy in rheumatoid arthritis(RA)regulated by the Sidaxue(SX).Firstly,a collagen-induced arthritis(CIA)rat model was constructed,and after 21 days of SX administration,the thymus and spleen indices of rats were calculated,the histopathological changes of rat knee synovial joint were observed,and the serum interleukin-6(IL-6)and interleukin-37(IL-37)levels of rats were measured.Further,the possible molecular mechanism of SX regulating cellular autophagy in RA was analyzed using network pharmacology and validated by molecular docking with the help of Autodock vina software,while the pharmacological properties of the main active components of SX were predicted by online database,combined with molecular docking score for primary screening of effector components.Finally,RT-qPCR and Western blot were used to detect the effects of SX on phosphatidylinositol 3-kinase(PI3K),protein kinase B(AKT),mammalian target of rapamycin(mTOR)and microtubule-associated protein 1 light chain 3(LC3)mRNA and protein expression in the synovial tissue of CIA rats.The results of animal experiments showed that SX could effectively relieve joint swelling,alleviate the overreaction of immune organs,improve synovial histopathological changes and reduce the levels of inflammatory factors IL-6 and IL-37 in CIA rats(P<0.05).Network pharmacology results showed that AKT1,CASP3,BCL2L1 and other targets were more critical,and animal autophagy,apoptosis and PI3K/AKT signaling pathways were associated with RA and had high significance.Molecular docking results showed that key targets of PI3K/AKT signaling pathway stably bound to the main active components of SX,β-sitosterol,Cajinin,ursolic acid,and eleutheroside A may be the superior effector components of SX in regulating autophagy.RT-qPCR and Western blot experimental results showed that SX dose-dependently downregulated PI3K,AKT,mTOR mRNA(P<0.05)and protein(P<0.001)expression of PI3K AKT and mTOR,upregulated the expression level of autophagy marker LC3 mRNA(P<0.05)and promoted the conversion of LC3-Ⅰto LC3-Ⅱ(P<0.01).In conclusion,SX has an ameliorative effect on RA,and its mechanism may be the inhibition of PI3K/AKT/mTOR signaling pathway,which promotes autophagic cell death of synovial cells to inhibit abnormal proliferation of synovial membrane.