基于微生物-脑-肠轴探讨益气解毒方抗缺血性脑卒中作用机制  

Mechanism of Yiqi Jiedu Formula Against Ischemic Stroke Based on Microbial-gut-brain Axis

作　　者：杨家霖 蔡冰洁 戴玉豪 李淑婷 张可可 高艳华 张琪曼 张滢 沈建英[1] 李韶菁[1] YANG Jialin;CAI Bingjie;DAI Yuhao;LI Shuting;ZHANG Keke;GAO Yanhua;ZHANG Qiman;ZHANG Ying;SHEN Jianying;LI Shaojing(Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;Anhui University of Chinese Medicine,Hefei 230000,China;Changsha College of Health Professions,Changsha 410100,China;Beijing City University,Beijing 100083,China)

机构地区：[1]中国中医科学院中药研究所,北京100700 [2]安徽中医药大学,合肥230000 [3]长沙卫生职业学院,长沙410100 [4]北京城市学院,北京100083

出　　处：《中国实验方剂学杂志》2024年第6期91-100,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家科技重大专项(2019ZX09201-004);中国中医科学院科技创新工程项目(CI2021A02001,CI2021A05110);中国中医科学院中药研究所自选课题(ZXKT20006)。

摘　　要：目的:从微生物-脑-肠轴(MGBA)角度探讨益气解毒方(YQ)治疗缺血性脑卒中(IS)的可能作用机制。方法:将大鼠随机分为5组,每组6只,分别为假手术组、模型组、YQ低、中、高剂量组(1、5、25 mg˙kg^(-1)),除假手术组外,其余各组采用线栓法建立大鼠大脑中动脉阻塞(MCAO)模型。首先采用神经行为学评分判断是否建模成功并对YQ的缺血性脑卒中保护作用进行评价,再通过16S rDNA测序技术比较大鼠MCAO造模前后及YQ给药后肠道菌群的变化,并分析该方作用可能相关的生物学通路。最后通过酶联免疫吸附测定法(ELISA)检测血清中炎症因子白细胞介素-6(IL-6)、白细胞介素-17A(IL-17A)和白细胞介素-10(IL-10)的表达,通过蛋白免疫印迹法(Western blot)检测紧密连接蛋白ZO-1和Occludin在脑和肠组织中的表达,苏木素-伊红(HE)染色观察脑皮层区和结肠的病理变化,对可能的作用机制进行验证。结果:YQ显著提高了MCAO大鼠的神经行为学评分(P<0.01),对在急性期由于富集病原体和机会致病菌引发的肠道微生物紊乱起到了较好调节作用,其中显著变化的微生物包括摩根氏菌属、埃希氏-志贺氏菌属、Adlercreutzia和安德克氏菌属等,并通过生物信息学分析发现这些菌可能与脑内炎症调节密切相关。与假手术组比较,血清中炎症因子检测发现IS模型大鼠血清中炎症因子IL-6与IL-17A显著升高(P<0.01),抑炎因子IL-10的含量显著降低(P<0.01);与模型组比较,给药组血清中炎症因子IL-6与IL-17A的含量明显降低(P<0.05),抑炎因子IL-10的含量显著增加(P<0.01)。屏障蛋白ZO-1和Occludin的表达结果表明IS模型大鼠在脑、结肠组织中两者的表达量均明显降低(P<0.05),给药组两者的表达量均明显增加(P<0.05)。结论:急性脑缺血可导致肠道微生物群失调,破坏肠道屏障,增加肠道通透性。YQ可调节脑缺血引起的肠道菌群失调、抑制全身炎症反应和改善�Objective:To explore the possible mechanism of the Yiqi Jiedu formula(YQ)in treating ischemic stroke(IS)from the perspective of the microbial-gut-brain axis(MGBA).Method:Rats were randomly divided into five groups,with six in each group,including sham surgery group,model group,and low,medium,and high dose YQ groups(1,5,and 25 mg·kg^(-1)).Except for the sham surgery group,all other groups were established with a middle cerebral artery occlusion(MCAO)model using the thread occlusion method.The success of modeling was determined through neurobehavioral scoring,and the protective effect of YQ on IS was evaluated.Then,the changes in gut microbiota before and after MCAO modeling and YQ administration were compared using 16S rDNA sequencing technology,and the possible biological pathways related to the effect of this formula were analyzed.The expression of inflammatory factors such as interleukin-6(IL-6),interleukin-17A(IL-17A),and interleukin-10(IL-10)in serum was detected by enzyme-linked immunosorbent assay(ELISA).Western blot was used to detect the expression of tight junction proteins ZO-1 and Occludin in brain and intestinal tissue,and hematoxylin-eosin staining(HE)was used to observe pathological changes in the cerebral cortex and colon,so as to validate the possible mechanism of action.Result:YQ significantly improved the neurobehavioral score of MCAO rats(P<0.01)and played a good regulatory role in intestinal microbial disorders caused by enriched pathogens and opportunistic pathogens during the acute phase.Among them,significantly changed microorganisms include Morgentia,Escherichia Shigella,Adlercreutzia,and Androbacter.Bioinformatics analysis found that these bacteria may be related to the regulation of inflammation in the brain.Compared with the blank group,the detection of inflammatory factors in the serum of IS model rats showed an increase in inflammatory factors IL-6 and IL-17A(P<0.01)and a decrease in the content of anti-inflammatory factor IL-10(P<0.01).Compared with the model group,the content of in

关 键 词：益气解毒方 肠道菌群 微生物-脑-肠轴 缺血性脑卒中 16S rDNA 

分 类 号：R284[医药卫生—中药学] R285[医药卫生—中医学] R289R287R22R2-031R33R24