基于AMPK-FoxO3a-MnSOD信号通路探讨类风湿关节炎患者氧化应激的机制及黄芩清热除痹胶囊对其影响

Effects of Huangqin Qingre Chubi capsule on oxidative stress in rheumatoid arthritis patients and its mechanism based on AMPK-FoxO3a-MnSOD signaling pathway

目的基于AMPK-FoxO3a-MnSOD信号通路研究黄芩清热除痹胶囊(HQC)改善类风湿关节炎患者氧化应激的机制。方法将50例RA患者按随机数字表法分为2组:HQC组(3粒/次,每日3次)和来氟米特(LEF)组(10 mg/次,每日1次),每组25例,连续治疗3个月。评定2组中医证候临床疗效和脾虚症状评分;魏氏法测ESR;日立7060型全自动生化分析仪检测Hs-CRP、RF、CCP-AB;ELISA检测血清中IL-1β、TNF-α、IL-4、IL-6、IL-10、IL-35、SOD、TAOC、MDA、LPO;FCM法检测单个核细胞CD36水平;RT-PCR法检测PPAR-γ、AMPKα1、FoxO3a mRNA表达;Western blot法检测PPAR-γ、p-FoXO3a、FoXO3a、AMPKα1、MnSOD蛋白表达。采用Spearman方法对RA患者氧化/抗氧化指标、信号通路指标与细胞因子、中医脾虚症状、CD36之间进行相关性分析。结果 HQC组总有效率为88%,LEF组总有效率为92%,2组总有效率比较差异无统计学意义(P>0.05)。与治疗前比较,2组中医脾虚各个症状评分均降低,Hs-CRP、RF、CCP-AB、IL-1β、TNF-α、IL-6等指标均显著下降,IL-4、IL-10显著升高,SOD、TAOC均显著升高,LPO显著降低,AMPK-α1、PPAR-γ、p-FoxO3a蛋白水平升高,CD36表达水平明显升高,PPAR-γmRNA表达升高。且HQC组IL-35明显升高,MDA显著降低,FoxO3a、MnSOD蛋白水平显著升高,AMPK-α1 mRNA、FoxO3a mRNA表达明显升高。且HQC组食欲减退、少气懒言、食后腹胀、大便稀溏、关节重着等评分、LPO治疗后较LEF组降低明显,IL-35、FoxO3a、p-FoxO3a、MnSOD蛋白水平、CD36表达、AMPK-α1 mRNA、FoxO3a mRNA表达治疗后较LEF组升高明显。相关性分析显示,RA患者氧化/抗氧化指标、信号通路指标与细胞因子、中医脾虚症状、CD36存在一定相关性。结论 RA患者氧化应激的发生机制及黄芩清热除痹胶囊改善氧化应激状态可能是与AMPK-FoxO3a-MnSOD信号通路密切相关。

This study was designed to evaluate the mechanism of Huangqin Qingre Chubi capsule(HQC) on relieving oxidative stress in rheumatoid arthritis(RA) patients, based on the AMPK-FoxO3 aMnSOD signaling pathway. Total of 50 cases of RA patients were divided into 2 groups according to the random number table method: group HQC(3 tables/time, 3 times a day for 3 months) and leflunomide(LEF) group(10 mg/time, 1 time a day for 3 months), with 25 cases in each group. The clinical curative effect and spleen deficiency symptom score of the two groups were evaluated. ESR was measured by Wei’s method, while the levels of Hs-CRP, RF and CCP-AB were detected by Hitachi 7060 automatic biochemical analyzer;ELISA was used to detect the levels of IL-1 beta, TNF-α, IL-4, IL-6, IL-10, IL-35, SOD, IL-10, CCP-AB in the serum of patients, while flow cytometry was used to detect CD36 level in mononuclear cell. The mRNA expression of PPAR-γ, AMPK 1 and FoxO3 a were detected by RT-PCR, while the protein expression of PPAR-γ, p-FoXO3 a, FoXO3 a, AMPK 1 and MnSOD were detected by Western blotting.Spearman method was used to analyze the correlation of oxidation/antioxidant index or signal pathway indicators with cytokines, spleen deficiency symptoms or CD36 in RA patients. Data showed that the total effective rate of group HQC and group LEF were 88% and 92%, which demonstrated no significant difference(P>0.05). Compared with before treatment, the scores of TCM spleen deficiency of the two groups were all decreased, the levels of Hs-CRP,RF, CCP-AB, IL-1 beta, TNF-alpha and IL-6 were decreased significantly, the levels of IL-4 and IL-10 were increased significantly, the levels of SOD and TAOC were increased significantly, LPO was decreased significantly,the protein levels of AMPK-α1, PPAR-γ, p-FoxO3 a was increased, CD36 expression was increased, and the mRNA expression of PPAR-γ was increased significantly. Furthermore, HQC group demonstrated more improvement in some indicators mentioned above, less scores of loss of appetite, breath and laziness, post-feeding abdominal distention, stool loose stool, and so on, as compared with LEF group. The levels of IL-35, FoxO3 a, p-FoxO3 a,MnSOD protein, CD36 expression, AMPK-alpha 1 mRNA and FoxO3 a mRNA in HQC were significantly higher than those of the LEF group after LPO treatment. Correlation analysis showed that oxidative/antioxidant indexes and signal pathway indicators in RA patients were correlated with cytokines, spleen deficiency symptoms and CD36. The mechanism of oxidative stress in RA patients and the therapeutic mechanism of Huangqin Qingre Chubi capsule on oxidative stress may closely relate to the AMPK-FoxO3 a-MnSOD signaling pathway.