新风胶囊通过调节Keapl-Nrf2/ARE信号通路改善佐剂型关节炎大鼠肺功能研究

Research of Xinfeng Capsule on improving pulmonary function via Keap1/Nrf2-ARE signal pathway in adjuvant arthritis rats

目的:观察新风胶囊对佐剂型关节炎(AA)大鼠肺功能、肺组织氧化应激状态及Keap1-Nrf2/ARE信号通路表达的影响。方法:48只雄性SD大鼠按随机数字表随机分为正常组(NC),模型组(MC),新风胶囊治疗组(XFC),来氟米特对照组(LEF),每组12只。除正常组外,采用右后足跖皮内注射弗氏完全佐剂法制成AA大鼠模型,致炎后第19天开始给药,共给药30d。检测大鼠体质量、足跖肿胀度、关节炎指数;小动物肺功能仪检测大鼠肺功能;免疫组化法检测肺组织ROS、RNS、GSH、TRX;RT-PCR法检测肺组织Keap1、maf、Nrf2m RNA的表达,Western blot法检测肺组织Keap1、maf、Nrf2蛋白表达;ELISA法检测大鼠外周血IL-4、TNF-α。结果:与正常组比较,模型组大鼠体质量与肺功能参数降低;足跖肿胀度、关节炎指数升高;肺组织ROS、RNS蛋白表达升高,GSH、TRX蛋白表达降低;Keap1 m RNA表达增多,Maf,Nrf2 m RNA表达减少,Keap1、maf、Nrf2蛋白表达均升高(P<0.01,P<0.05);与模型组比较,各治疗组大鼠体质量、肺功能参数升高;ROS、RNS蛋白表达降低,GSH、TRX蛋白表达升高;Keap1,Maf,Nrf2 m RNA表达升高,Keap1、maf、Nrf2蛋白表达均降低(P<0.01,P<0.05);与来氟米特组比较,新风胶囊组肺功能参数FEF50、PEF较高;ROS、RNS明显较低,TRX明显较高;Keap1,Nrf2 m RNA表达较低,maf的m RNA和蛋白表达均较高,Keap1蛋白表达较低(P<0.01,P<0.05)。相关性分析显示,肺功能参数与AI呈负相关,ROS、RNS、TNF-α与AI、足跖肿胀度呈正相关;GSH、TRX、IL-4与AI、足跖肿胀度呈明显负相关(P<0.01,P<0.05)。结论:AA大鼠除关节滑膜病变还存在肺功能损伤。新风胶囊能够在改善关节病变的同时,改善肺功能,其机制与调节异常激活的Keapl-Nrf2/ARE通路,抑制氧化应激反应有关。

Objective： To observe the effects of Xinfeng Capsule（XFC） on the pulmonary function, oxidative stress of lung tissue and Keap1/Nrf2-ARE signal pathway in adjuvant arthritis（AA） rats. Methods： Forty-eight male rats were randomly divided into the normal control（NC） group, model control（MC） group, leflunomide（LEF） group, and XFC group, with 12 rats for each group. The AA rat model was induced by a subcutaneous injection of Freund＇s complete adjuvant in right hindpaw, then administration for 30 d from the 19 th day after modeling. The body weight, paw swelling and arthritic index（AI） were measured. The pulmonary function was measured by small animal lung function instrument. The proteins of ROS, RNS, GSH and TRX were detected by immunocytochemistry. The m RNA expression of Keap1, Maf and Nrf2 were detected by real-time PCR. The proteins of Keap1, Maf and Nrf2 in pulmonary tissues were detected by Western blotting. ELISA assay was used to detect the level of IL-4 and TNF-α in peripheral blood. Results： 1 Compared with NC group, the paw swelling and AI in MC group were increased, while the body weight and pulmonary function parameters were decreased; the expression of ROS, RNS and Keap1 in MC group were increased, while GSH, TRX, Maf and Nrf2 were decreased（P〈0.01, P〈0.05）. 2 Compared with MC group, the pulmonary function parameters and body weight in the two treatment groups were increased; the expression of ROS, RNS, Keap1, Maf and Nrf2 in the treatment groups were decreased, while the expression of GSH and TRX were increased（P〈0.01, P〈0.05）. Compared with the LEF group, the expression of Keap1 and Nrf2 in the XFC group were decreased, while the expression of Maf and pulmonary function parameter were increased（P〈0.01, P〈0.05）. Additionally, compared with the LEF group, the expression of RNS and ROS in the XFC group were decreased, but TRX was increased（P〈0.01, P〈0.05）. But, there was no significant difference in the expression of GSH between the LEF group and XFC group. 3 Correlation analysis showed that pulmonary function parameters were positively correlated with Keap1, antioxidant indicators, and anti-inflammation cytokines, and negatively correlated with Nrf2, oxidation indicators, REDOX product, pro-inflammation cytokines, and inflammation indictors（P〈0.01, P〈0.05）. Conclusion： AA rat showed lung function damage besides joint synovium lesions. XFC can improve lung function while improving joint disease, whose mechanism may relate to regulate abnormal activation of Keapl-Nrf2-ARE pathway and inhibit oxidative stress reaction.