二甲双胍通过腺苷酸活化蛋白酶-哺乳动物雷帕霉素靶蛋白信号通路调节胶原诱导性关节炎大鼠辅助性T细胞17／调节性T细胞的分化及细胞因子的表达

Metformin regulate the balance of Th17/regulatory T cells and expression of serum cytokines through AMPK-mTOR signaling pathway in collagen-induced arthritis

目的以胶原诱导性关节炎（CIA）大鼠为模型，观察二甲双胍对CIA大鼠血清细胞因子、脾脏辅助性T细胞17（Th17）及调节性T细胞分化的影响和脾脏腺苷酸活化蛋白酶-哺乳动物雷帕霉素靶蛋白（AMPK—mTOR）的表达，初步探讨二甲双弧治疗RA的可能机制。方法使用牛Ⅱ型胶原建立CIA大鼠模型，将大鼠随机分为模型组、二甲双胍-30mg／kg（Met-30mg／kg）组、二甲双胍-100mg／kg（Met-100mg／kg）组、二甲双胍-300mg／kg（Met-300mg／kg）组及对照组，分别给予相应的处理。记录大鼠足趾容积，每周1次，初次免疫后第35天取得标本，采用ELISA检测各组大鼠血清TNF－α、IL-6、IL-1β、IL-17，采用流式细胞术分析各组大鼠脾脏Th17、调节性T细胞表达，采用蛋白印迹法检测各组大鼠脾脏AMPK、mTOR及p-AMPK、p—roTOR的表达。采用单因素方差分析进行统计分析。结果足趾容积在初次免疫后第35天时二甲双胍-100mg／kg组[（2．43±0．37）ml，t=2．97，P〈0.05]、二甲双胍-300mg／kg组[（2．58±0．21）ml，t=2．96，P〈0．05]明显低于模型组（2．97±0．23）ml。初次免疫后第35天，各治疗组的血清细胞因子[二甲双胍-300mg／kg组TNF－α（104±8）pg／ml，t=42．77，P〈0．05；IL—1β（183±24）pg／ml，t=60．457，P〈0．05；IL-6（19.3±2．8）pg／ml，t=53．62，P〈0．05；IL-17（64．5±6．7）pg／ml，t=47．92，P〈0．05]均明显低于模型组[TNF－α（246±8）pg／ml；IL-1β（1336±40）pg／ml；IL-6（87．0±5．1）pg／ml；IL-17（282．3±6．8）pg／m1]，二甲双胍-300mg／kg组脾脏Th17[（6．57±0．39）％，t=8．74，P〈0．05]较模型组[（9．89±0．53）％]明显减少、调节性T细胞明显增多[（7．60±0．45）％与（3．94±0．61）％，t=8．37，P〈0．05]、Th17／调节性T细胞比值明显下降（0．87±0．10与2．55±0．47，t=6．98，P〈0．05）且与对照组相近（0．67±0．04，t=1．08，P〉0．05），予二甲双胍治疗组的脾脏p-AMPK、p—roTOR水平均与模型组差异有统计学意义（均P〈0．05）。结论二甲双胍可有效减轻CIA病情，可能通过AMPK—roTOR信号通路使脾脏Th17表达减少、调节性T细胞表达增多，调节Th17，调节性T细胞比例，并减少血清促炎细胞因子IL-17、IL-1β、IL-6、TNF-α的表达，达到治疗CIA的作用。

Objective To evaluate therapeutic effects of metformin on the expression of serum cytokines, balance of splenic Th17/regulatory T cells （Treg） and expression of splenic AMPK-mTOR in collagen-induced arthritis （CIA） rats, and the mechanism thereof. Methods The rat model of CIA was established by injecting bovine type Ⅱ collagen. Forty rats were randomly divided into five groups： the CIA model group（sterile water by gavage）, Met-30 mg/kg group （mefformin 30 mg·kg^-1·d^-1 by gavage）, Met- 100 mg/kg group（metformin 100 mg·kg^-1·d^-1 by gavage）, Met-300 mg/kg group（metformin 300 mg·kg^-1·d^-1 by gavage） and control group （sterile water by gavage）. The hind paw volume was recorded once a week. The serum level of cytokines, tumor necrosis factor （TNF）-α, interleukin （IL）-1β, IL-6, and IL-17, were measured by enzyme linked immunosorbent assay （ELISA） 35 days after the initial immunization. The quantity of Th17 and Treg cells were examined by flow eytometry. The expression of AMPK, p-AMPK, mTOR and p-roTOR were examined by western blotting. One-way analysis of variance was used to evaluate the experimental data. Results The values of hind paw volume were decreased on the 35 d of the initial immunization in the Met- 100 mg/kg [（2.43±0.37） ml, t=2.97, P〈0.05] and Met-300 mg/kg groups [（2.58±0.21） ml, t=2.96, P〈0.05] than those of CIA model group （2.97±0.23） ml. The serum levels of TNF-α, IL-1β, IL-6 and IL-17 on the 35-d after the initial immunization were significantly lower in the metformin therapeutic groups [Met-300 mg/kg group TNF-α （104±8） pg/ml, t=42.77, P〈0.05; IL-1β （183±24） pg/ml, t=60.457, P〈0.05; IL-6 （19.3±2.8） pg/ml, t= 53.62, P〈0.05; IL-17 （64.5＋6.7） pg/ml, t=47.92, P〈0.05] than those of the CIA model group [TNF-α （246±8） pg/ml; IL-1β （1 336±40） pg/ml; IL-6 （87.0±5.1） pg/ml; IL-17 （282.3±6.8） pg/ml]. The quantity of Th17 and Treg cells were significantly different in the Met-300 mg/kg group than those of the CIA model group 35 d after the initial immunization [Th17（6.57±0.39） vs （9.89±0.53）, t=8.74, P〈0.05; Treg （7.60±0.45） vs （3.94±0.61）, t=8.37, P〈0.05]. The expression of p-AMPK on the 35 d after the initial immunization in the metformin therapeutic groups was significantly higher than in the CIA model group （P〈0.05）, and the expression of p-mTOR was significantly lower （P〈0.05）. Conclusion Metformin can significantly inhibit the serum levels of TNF-α, IL-1β, IL-6 and IL-17 in CIA model rats, and regulate the balance of Th17/Treg through AMPK-mTOR signaling pathway.