基于AMPK/SIRT1通路探究祛风明目丸对实验性自身免疫性葡萄膜炎大鼠的作用机制

Exploration on the action mechanism of Qufeng Mingmu Pills on autoimmune uveitis rats based on the AMPK/SIRT1 pathway

目的:研究祛风明目丸对实验性自身免疫性葡萄膜炎(EAU)大鼠腺苷酸活化蛋白激酶(AMPK)/SIRT1通路及Th1/Th17细胞的作用机制。方法:SPF级雄性Lewis大鼠70只,随机取19只作为空白组,剩余51只建立EAU模型,造模后行炎症评分。造模成功后随机分为模型组、中药常量组、中药高倍组。中药常量组、中药高倍组每天分别以2.4、4.8 g/kg祛风明目丸药液灌胃,空白组、模型组每天予等量蒸馏水灌胃。14 d后ELISA检测血清γ干扰素(IFN-γ)、白细胞介素(IL)-6、IL-17A、IL-22水平,流式细胞数检测外周Th1/Th17细胞比例,qRT-PCR定量检测视网膜中CCL20、CXCL9、CXCL10、CCR6和CXCR3表达,Western Blot检测视网膜AMPK、P-AMPK、SIRT1蛋白表达。结果:Caspi评分示:自造模后第3天起模型组EAU大鼠眼前节炎症评分显著高于空白组(P<0.01)。ELISA示:与空白组比较,模型组IFN-γ、IL-6、IL-17A、IL-22水平显著升高(P<0.05);与模型组比较,中药常量组、中药高倍组炎症因子表达显著下降(P<0.05)。qRT-PCR示:与空白组比较,模型组CXCL9、CXCL10、CCL20、CCR6和CXCR3水平显著升高(P<0.05);与模型组比较,中药高倍组趋化因子及受体的表达显著下调(P<0.05),中药常量组CXCL10、CCL20、CCR6和CXCR3的表达显著降低(P<0.05)。流式细胞数示:与空白组比较,模型组Th1和Th17细胞亚群比例显著升高(P<0.05);与模型组比较,中药高倍组Th1和Th17细胞比例显著下降(P<0.05),中药常量组Th1细胞比例显著下降(P<0.05)。Western Blot示:与空白组比较,模型组P-AMPK、P-AMPK/AMPK、SIRT1表达显著下调;与模型组比较,中药常量组与高倍组表达显著上调(P<0.05)。结论:祛风明目丸减轻EAU大鼠炎症、抑制Th1/Th17细胞的作用机制可能与AMPK/SIRT1通路激活相关。

Objective:To study the mechanism of action of Qufeng Mingmu Pills on the AMPK/SIRT1 pathway and Th1/Th17 cells in experimental autoimmune uveitis(EAU)rats.Methods:Seventy SPF grade male Lewis rats were randomly selected,with 19 rats as the blank group,and the remaining 51 rats were used to establish an EAU model.After modeling,inflammation scores were performed.After successful modeling,they were randomly divided into model group,Chinese medicine constant group,and Chinese medicine high magnification group.The Chinese medicine constant group and Chinese medicine high magnification group were orally administered with 2.4,4.8 g/kg Qufeng Mingmu Pills solution every day,while the blank group and model group were orally administered with equal amounts of distilled water every day.ELISA detection of serum IFN-γ,IL-6,IL-17A,IL-22 levels after 14 days,flow cytometry detection of peripheral Th1/Th17 cell ratio,qRT-PCR quantitative detection of CCL20,CXCL9,CXCL10,CCR6,and CXCR3 expression in the retina,Western Blot detection of retinal AMPK,P-AMPK,and SIRTI protein expression.Results:Caspi score showed that from the third day after self modeling,the inflammation score of the anterior segment of EAU rats in the model group was significantly higher than that in the blank group(P<0.01).ELISA showed that compared with the blank group,the levels of IFN-γ,IL-6,IL-17A,and IL-22 of the model group,significantly increased(P<0.05);Compared with the model group,the expression of inflammatory factors in the constant and high magnification groups of traditional Chinese medicine decreased significantly(P<0.05).qRT-PCR showed that compared with the blank group,the levels of CXCL9,CXCL10,CCL20,CCR6,and CXCR3 in the model group were significantly increased(P<0.05).Compared with the model group,the expression of chemokines and receptors in the high magnification group of traditional Chinese medicine was significantly downregulated(P<0.05),while the expression of CXCL10,CCL20,CCR6,and CXCR3 in the constant Chinese medicine group was significantly reduced(P<0.05).Flow cytometry analysis showed that compared with the blank group,the proportion of Th1 and Th17 cell subsets in the model group significantly increased(P<0.05).Compared with the model group,the proportion of Th1 and Th17 cells in the high magnification group of traditional Chinese medicine was significantly down regulated(P<0.05),while the proportion of Th1 cells in the constant dosage group of traditional Chinese medicine was significantly down regulated(P<0.05).Western Blot showed that compared with the blank group,the expression of P-AMPK/AMPK and SIRTI in the model group was significantly reduced(P<0.05).Compared with the model group,the expression of traditional Chinese medicine constant group and high magnification group significantly increased(P<0.05).Conclusion:The mechanism of Qufeng Mingmu Pills in reducing inflammation and inhibiting Th1/Th17 cells in EAU rats may be related to the activation of the AMPK/SIRT1 pathway.