调气汤对大鼠类风湿性关节炎的治疗作用和机制研究

Study on the Efficacy and Mechanism of Tiaoqi Decoction on the Treatment of Rheumatoid Arthritis in Rats

目的:研究调气汤对类风湿性关节炎的治疗作用和机制。方法:体外培养胶原诱导型关节炎大鼠成纤维样滑膜(CIA-FLS)细胞,将细胞分为对照组、TNF-α组、调气汤40,50和60μg/mL组,TNF-α(10 ng/mL)处理细胞30 min,然后用调气汤处理细胞48 h。检测细胞活力、细胞凋亡和细胞中Atg5、Beclin-1、LC3-Ⅰ/Ⅱ、PI3K、p-AKT、AKT、p-mTOR的蛋白表达,以及细胞上清中TNF-α、IL-6、TGF-β、IL-17、IFN-γ和IL-10含量。SPF级SD大鼠(雄性,30只)随机分为对照组、模型组、低剂量组(25 mg/kg调气汤的佐剂诱导型关节炎(AIA)大鼠)、中剂量组(50 mg/kg调气汤的AIA大鼠)、高剂量组(100 mg/kg调气汤的AIA大鼠),建立胶原诱导型关节炎(CIA)大鼠模型。采用苏木精-伊红染色观察AIA大鼠的组织病理变化,采用ELISA法测量大鼠血清中TNF-α、IL-6、IL-1β含量。结果:与TNF-α组比较,调气汤各剂量组细胞活力下降、细胞凋亡增加,且细胞中Atg5、Beclin-1、LC3-Ⅰ/Ⅱ、PI3K、p-mTOR蛋白表达下调,同时细胞上清中TNF-α、IL-6、TGF-β、IL-17、IFN-γ含量减少,IL-10含量增加,差异有统计学意义(P<0.05)。与模型组比较,调气汤低剂量组的组织病理图像中观察到轻度骨侵蚀;调气汤中剂量和高剂量组的组织病理图像显示为正常的关节腔,没有骨侵蚀和滑膜增生;血清上清中TNF-α、IL-6和IL-1β含量均明显下降,差异有统计学意义(P<0.05)。结论:调气汤通过下调PI3K/AKT/mTOR信号通路抑制CIA-FLS细胞增殖和自噬,并促进细胞凋亡和发挥抗炎活性,对类风湿性关节炎具有潜在的治疗活性。

Objective:To investigate the effect and mechanism of Tiaoqi decoction on the treatment of rheumatoid arthritis.Methods:Collagen-induced arthritis fibroblast-like synovial(CIA-FLS)cells of collagen-induced arthritis rats were cultured in vitro.The cells were divided into control group,TNF-αgroup,Tiaoqi decoction 40μg/mL,50μg/mL and 60μg/mL groups.TNF-α(10 ng/mL)was used to treat the cells for 30 min,and then cells were treated with Tiaoqi decoction for 48 h.Cell viability,apoptosis,protein expression of Atg5,Beclin-1,LC3-Ⅰ/Ⅱ,PI3K,p-AKT,AKT,p-mTOR in cells was detected.The content of TNF-α,IL-6,TGF-β,IL-17,IFN-γand IL-10 in the cell supernatant was measured.30 male SPF SD rats were randomly divided into control group,model group,low-dose group(25 mg/kg Tiaoqi decoction adjuvant induced arthritis(AIA)rats),middle-dose group(50 mg/kg Tiaoqi decoction AIA rats),high-dose group(100 mg/kg Tiaoqi decoction AIA rats).A collagen-induced arthritis(CIA)rat model was established.Hematoxylin-eosin staining was used to observe the pathological changes of AIA rats,and ELISA was used to measure the contents of TNF-α,IL-6,and IL-1βin rat serum.Results:Compared with TNF-αgroup,the cell viability was decreased and the apoptosis was increased in Ttiaoqi decoction group.The protein expression of Atg5,Beclin-1,LC3-Ⅰ/Ⅱ,PI3K and p-mTOR was down-regulated by Tiaoqi decoction.The content of TNF-α,IL-6,TGF-β,IL-17 and IFN-γwas decreased and the content of IL-10 was increased in the cell supernatant of Tiaoqi decoction group.Compared with the model group,mild bone erosion was observed in the pathological images of the Tiaoqi decoction low-dose group,the histological study of the Tiaoqi decoction medium-dose and high-dose groups showed normal joint cavity without bone erosion and slippage,the levels of TNF-α,IL-6 and IL-1βin the serum supernatant were all significantly decreased,the difference was statistically significant(P<0.05).Conclusion:Tiaoqi decoction can inhibit the proliferation and autophagy of CIA-FLS cells by down-regulating PI3K/AKT/mTOR signaling pathway,and promote cell apoptosis and exert anti-inflammatory activity,which has potential therapeutic activity on rheumatoid arthritis.