基于自噬探讨健脾益肠散对溃疡性结肠炎模型大鼠肠黏膜保护的作用机制  

The Mechanism of Protective Effect of Jianpi Yichang Powder on Intestinal Mucosa of Rats with Ulcerative Colitis Based on Autophagy

作　　者：刘琴 周赛男[1] 刘杰民[2] 蔺晓源[1] LIU Qin;ZHOU Sainan;LIU Jiemin;LIN Xiaoyuan(The First Hospital of Hunan University of Chinese Medicine,Changsha 410007 Hunan,China;Guizhou Provincial People’s Hospital,Guiyang 550002 Guizhou,China)

机构地区：[1]湖南中医药大学第一附属医院,湖南长沙410007 [2]贵州省人民医院,贵州贵阳550002

出　　处：《中药新药与临床药理》2024年第6期814-821,共8页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金项目(82060850);贵州省高层次创新型人才项目(黔科合平台人才[2020]6016-2)。

摘　　要：目的通过观察健脾益肠散对溃疡性结肠炎模型大鼠结肠自噬相关分子表达的影响,探讨其肠黏膜保护的作用机制。方法将SD大鼠随机分为正常组、模型组、健脾益肠散组和柳氮磺吡啶组。采用2,4,6-三硝基苯磺酸(TNBS)-乙醇溶液复合法建立溃疡性结肠炎大鼠模型;模型复制成功后灌胃给药,每日1次,持续14 d。观察大鼠一般状况,计算疾病活动指数(DAI)评分;苏木素-伊红(HE)染色法观察结肠组织病理形态;透射电镜观察结肠上皮细胞自噬情况;免疫荧光染色法、蛋白免疫印迹法(Western Blot)检测结肠组织肌球蛋白样BCL2结合蛋白(Beclin1)、微管相关蛋白1轻链3(LC3)蛋白表达;实时荧光定量聚合酶链反应(Real-time PCR)检测自噬蛋白5(Atg5)、自噬蛋白7(Atg7)、泛素结合蛋白62(p62)mRNA表达。结果与正常组比较,模型组一般情况较差,DAI评分明显升高(P<0.0001);结肠组织出现明显的上皮细胞损伤,电镜下自噬体的数量明显减少;结肠组织Beclin1、LC3-II/LC3-I蛋白及Atg5、Atg7 mRNA表达明显降低(P<0.0001),p62 mRNA表达明显升高(P<0.0001)。与模型组比较,健脾益肠散组大鼠一般情况明显恢复,DAI评分明显降低(P<0.0001);结肠组织病理损伤有不同程度的改善,电镜下自噬体的数量明显增多;结肠组织Beclin1、LC3-II/LC3-I蛋白及Atg5、Atg7 mRNA表达明显升高(P<0.01,P<0.001,P<0.0001),p62 mRNA表达明显降低(P<0.01)。结论健脾益肠散可通过上调自噬相关分子Beclin1、LC3、Atg5及Atg7的表达,下调p62的表达,增强自噬以达到保护溃疡性结肠炎模型大鼠结肠黏膜损伤的作用。Objective To explore the mechanism of protective effect of Jianpi Yichang Powder on intestinal mucosa by observing its effect on the expression of autophagy-related molecules in colon of ulcerative colitis(UC)model rats.Methods SD rats were randomly divided into normal group,model group,Jianpi Yichang Powder group and sulfasalazine group.UC rat model was established by 2,4,6-trinitrobenzene sulfonic acid/ethanol solution.After the model was successfully reproduced,the rats were given drugs orally once a day for 14 days.The general condition of rats was observed and the disease activity index(DAI)score was calculated.The pathological morphology of colonic tissue in all groups was observed by hematoxylin eosin(HE)staining.Autophagy of colonic epithelial cells was examined by transmission electron microscope.The expressions of myosin-like BCL2 interacting protein(Beclin1)and microtubule-associated protein 1 light chain 3(LC3)in colonic tissue were detected by immunofluorescence staining and Western Blot.The mRNA expressions of autophagy protein 5(Atg5),autophagy protein 7(Atg7)and ubiquitin-binding protein 62(p62)were detected by real-time quantitative polymerase chain reaction(Real-time PCR).Results Compared with the normal group,the general situation of the model group was worse,and the DAI score increased significantly(P<0.0001).There was obvious epithelial cell damage in colonic tissue,and the number of autophagosomes decreased obviously under electron microscope.The expressions of Beclin1 and LC3-II/LC3-I protein as well as Atg5 and Atg7 mRNA in colonic tissue decreased significantly(P<0.0001),while the expression of p62 mRNA increased significantly(P<0.0001).Compared with the model group,the general condition of rats in Jianpi Yichang Powder group had recovered significantly,and the DAI score decreased significantly(P<0.0001).Pathological damage of colonic tissue has been improved in different degrees,and the number of autophagosomes increased significantly under electron microscope.The expressions of Beclin1

关 键 词：健脾益肠散 溃疡性结肠炎 自噬 BECLIN1 LC3 Atg5 Atg7 P62 大鼠 

分 类 号：R285.5[医药卫生—中药学]