基于AMPK/ULK1自噬通路探讨人参败毒散对溃疡性结肠炎黏膜屏障的干预机制  

Renshen Baidusan Protects Mucosal Barrier in Ulcerative Colitis via AMPK/ULK1 Autophagy Pathway

作　　者：熊珮宇 钟春 张培旭 刘兴隆[1] 陈旭 贾波[1] XIONG Peiyu;ZHONG Chun;ZHANG Peixu;LIU Xinglong;CHEN Xu;JIA Bo(School of Basic Medical Sciences,Chengdu University of Traditional Chinese Medicine(TCM),Chengdu 610075,China;Sichuan Second Hospital of TCM,Chengdu 610014,China)

机构地区：[1]成都中医药大学基础医学院,成都610075 [2]四川省第二中医医院,成都610014

出　　处：《中国实验方剂学杂志》2023年第19期52-59,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(82074327);四川省科学技术项目(2018JY0446)。

摘　　要：目的:研究人参败毒散调控腺苷酸活化蛋白激酶/Unc-51样激酶1(AMPK/ULK1)自噬通路抑制溃疡性结肠炎小鼠黏膜屏障损伤的作用机制。方法:将60只SD大鼠随机分为6组,分为正常组,模型组,柳氮磺胺吡啶肠溶片组(西药组),人参败毒散高、中、低剂量组。通过2,4,6-三硝基苯磺酸(TNBS)/50%乙醇诱导UC模型,西药组(0.3125 g·kg^(-1))、人参败毒散高、中、低剂量组(31.2、15.6、7.8 g·kg^(-1))灌胃2周后,检测结肠组织病理学改变;实时荧光定量聚合酶链式反应(Real-time PCR)检测腺苷酸活化蛋白(AMPKα)mRNA表达水平;蛋白免疫印迹法(Western blot)检测紧密连接蛋白中闭合蛋白(Occludin)、紧密连接蛋白蛋白-2(Claudin-2)、自噬标志蛋白p62、微管相关蛋白1轻链3B(LC3B)及AMPK/ULK1通路磷酸化蛋白p-AMPK、p-ULK1的表达水平。结果:与正常组比较,模型组结肠损伤评分明显上调(P<0.05),AMPKαmRNA表达明显下调(P<0.05),p-AMPK、p-ULK1、Occludin蛋白水平及LC3Ⅱ/Ⅰ明显下调(P<0.05),而p62、Claudin-2蛋白水平明显上调(P<0.05);与模型组比较,人参败毒散高、中、低剂量组的结肠损伤评分下降,AMPKαmRNA明显上调,p-AMPK、p-ULK1、Occludin蛋白水平及LC3Ⅱ/Ⅰ上升,而p62、Claudin-2蛋白表达下降,以人参败毒散中剂量组干预效应最明显(P<0.05)。结论:人参败毒散可抗肠道黏膜屏障损伤,以人参败毒散中剂量组疗效最佳,其机制可能与激活AMPK/ULK1自噬通路有关,通过加速LC3Ⅰ向LC3Ⅱ转化,促进p62降解,从而改善紧密连接蛋白Occludin、Claudin-2功能,修复肠道机械屏障损伤。Objective:To study the mechanism of Renshen Baidusan in regulating adenylate-activated protein kinase(AMPK)/Unc-51-like kinase 1(ULK1)autophagy pathway to inhibit mucosal barrier damage in the mouse model of ulcerative colitis(UC).Method:Sixty SD rats were randomized into normal,model,sulfasalazine enteric-coated tablets(0.3125 g·kg^(-1),western medicine),and high-,medium-,and low-dose(31.2,15.6,7.8 g·kg^(-1),respectively)Renshen Baidusan groups.The UC model was induced by 2,4,6-trinitrobenzenesulfonic acid(TNBS)/50%ethanol.The drugs were administrated by gavage for 2 weeks,and then the histopathological changes of the colon were examined.Real-time quantitative polymerase chain reaction was conducted to measure the mRNA level of AMP-activated protein kinase subunit alpha(AMPKα).Western blot was employed to determine the protein levels of closure protein(Occludin),compact linking protein-2(Claudin-2),autophagy marker p62,microtubule-associated protein 1 light chain 3B(LC3B),phosphorylated AMPK(p-AMPK),and phosphorylated ULK1(p-ULK1).Result:Compared with the normal group,the model group showed increased colon injury score(P<0.05),down-regulated mRNA level of AMPKα(P<0.05)and protein levels of p-AMPK,p-ULK1,and Occludin,decreased LC3Ⅱ/Ⅰratio(P<0.05),and up-regulated protein levels of p62 and Claudin-2(P<0.05).Compared with the model group,all the doses of Renshen Baidusan lowered the colon injury score,up-regulated the mRNA level of AMPKαand the protein levels of p-AMPK,p-ULK1,and Occluding,increased LC3Ⅱ/Ⅰratio,and down-regulated the protein levels of p62 and Claudin-2.Moreover,the medium-dose group showed a significant intervention effect(P<0.05).Conclusion:Renshen Baidusan can protect the intestinal mucosal barrier from damage,and the medium dose showed the best efficacy.It may activate the AMPK/ULK1 pathway to accelerate the transformation of LC3Ⅰto LC3Ⅱand promote the degradation of p62,so as to improve the function of Occludin and Claudin-2 and repair the mechanical damage of the intestinal ba

关 键 词：人参败毒散 溃疡性结肠炎 腺苷酸活化蛋白激酶/Unc-51样激酶1(AMPK/ULK1)通路 自噬 肠黏膜屏障 

分 类 号：R2-0[医药卫生—中医学] R33R289R318.14