基于PI3K/Akt/mTOR信号通路调控自噬探讨当归四逆汤对痛风性关节炎大鼠的影响

Danggui Sinitang Mitigates Gouty Arthritis in Rats by Regulating Autophagy via PI3K/Akt/mTOR Signaling Pathway

目的:基于磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(m TOR)信号通路,探讨当归四逆汤对痛风性关节炎(GA)大鼠自噬水平的影响。方法:60只雄性SD大鼠随机分为正常组、模型组、秋水仙碱组(0.3 mg·kg^(-1))和当归四逆汤低、中、高剂量组(6.54、13.08、26.16 g·kg^(-1)),每组10只,并分别给予相应药物灌胃,正常组、模型组给予等体积生理盐水灌胃,连续7 d。于第5天给药1 h后向各组(正常组除外)大鼠右侧踝关节注射尿酸钠混悬液(50 g·L^(-1))建立GA模型,正常组注射等体积的无菌生理盐水。观察大鼠踝关节肿胀情况;测定血清中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、IL-1β水平;观察踝关节病理形态变化;蛋白免疫印迹法(Western blot)检测滑膜PI3K、磷酸化(p)-PI3K、Akt、p-Akt、m TOR、p-m TOR、微管相关蛋白1轻链3Ⅱ/Ⅰ(LC3Ⅱ/Ⅰ)、自噬效应蛋白(Beclin-1)、泛素结合蛋白(p62)表达水平;实时荧光定量聚合酶链式反应(Real-time PCR)检测PI3K、Akt、m TOR、LC3、Beclin-1、p62 m RNA表达水平。结果:与正常组比较,模型组大鼠关节肿胀指数显著升高(P<0.01),血清TNF-α、IL-6、IL-1β水平升高(P<0.01),踝关节滑膜组织可见明显炎性细胞浸润和纤维组织增生,滑膜组织PI3K、p-PI3K、Akt、p-Akt、m TOR、p-m TOR、p62蛋白表达显著升高(P<0.01),PI3K、Akt、m TOR、p62 m RNA表达显著升高(P<0.01),LC3Ⅱ/Ⅰ、Beclin-1蛋白和LC3、Beclin-1 m RNA表达降低(P<0.01)。与模型组比较,当归四逆汤中、高剂量组大鼠关节肿胀明显减轻(P<0.05);血清中TNF-α、IL-6、IL-1β表达量显著明显降低(P<0.05);踝关节滑膜组织未见明显炎性细胞浸润,纤维组织增生减轻;滑膜组织PI3K、p-PI3K、Akt、p-Akt、m TOR、p-m TOR、p62蛋白表达量均明显降低(P<0.05),PI3K、Akt、m TOR、p62 m RNA表达量亦明显降低(P<0.05),而LC3Ⅱ/Ⅰ、Beclin-1蛋白和LC3、Beclin-1 m RNA表达量均明显上升(P<0.05)。结论:当归四逆汤可以抑制PI3K/Akt/m TOR信号通路,提高大鼠滑膜组织自噬水平,改善痛风性关节炎,并且以高剂量效果最佳。

Objective:To study the mechanism of Danggui Sinitang in mitigating gouty arthritis (GA) in rats by regulating autophagy via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (m TOR) signaling pathway.Method:Sixty male SD rats were randomly assigned into normal,model,colchicine (0.3 mg·kg^(-1)),and low-,medium-,and high-dose Danggui Sinitang (6.54,13.08,and 26.16 g·kg^(-1)) groups (n=10) and administrated with corresponding drugs by gavage.The rats in the normal group and model group were administrated with equal volume of normal saline by gavage for 7 days.One hour after administration on day 5,the GA model was established by injecting sodium urate suspension (50 g·L^(-1)) into the right ankle joint of rats in other groups except the normal group,and the rats in the normal group were injected with sterile normal saline of the same volume.The swelling and pathological changes of the ankle joint were observed.The serum levels of tumor necrosis factor-α (TNF-α),interleukin-6 (IL-6),and IL-1β were determined.Western blot was employed to determine the protein levels of PI3K,phosphorylated PI3K (p-PI3K),protein kinase B (Akt),phosphorylated Akt (p-Akt),m TOR,phosphorylated m TOR(p-m TOR),microtubule-associated protein 1 light chain 3Ⅱ/Ⅰ(LC3Ⅱ/Ⅰ),autophagy effector Beclin-1,and ubiquitin-binding protein p62 in the synovial tissue.Real-time fluorescent quantitative PCR (Real-time PCR) was employed to determine the m RNA levels of PI3K,Akt,m TOR,LC3,Beclin-1 and p62.Result:Compared with the normal control,the model group showed increased joint swelling index (P<0.01),elevated serum levels of TNF-α,IL-6,and IL-1β,inflammatory cell infiltration,and fibrous tissue hyperplasia.In addition,the model group showed up-regulated protein levels of PI3K,p-PI3K,Akt,p-Akt,m TOR,p-m TOR,and p62 and m RNA levels of PI3K,Akt,m TOR,and p62 in the synovial tissue,while it showed downregulated protein levels of LC3Ⅱ/Ⅰand Beclin-1 and m RNA levels of LC3 and Beclin-1 (P<0.01).Compared with the model group,medium-and high-dose Danggui Sinitang alleviated the joint swelling (P<0.01),lowered the serum levels of TNF-α,IL-6,and IL-1β (P<0.05),and relieved the inflammatory cell infiltration in the synovial tissue of the ankle joint and the fibrous tissue hyperplasia.Moreover,they down-regulated the protein levels of PI3K,p-PI3K,Akt,p-Akt,m TOR,p-m TOR,and p62 and the m RNA levels of PI3K,Akt,m TOR,and p62 in the synovial tissue (P<0.05),while they up-regulated the protein levels of LC3Ⅱ/Ⅰand Beclin-1and the m RNA levels of LC3 and Beclin-1 (P<0.05).Conclusion:Danggui Sinitang,especially at a high dose,can inhibit PI3K/Akt/m TOR signaling pathway to improve autophagy in the synovial tissue,thereby mitigating GA.