【作者】 王琳；

【导师】 黄卫华；

【作者基本信息】 中南大学 ， 药理学， 2024， 硕士

【摘要】 背景:炎症性肠病(Inflammatory Bowel Disease,IBD)是一种病因与发病机制不明的非特异性慢性、渐进性、复发性胃肠道疾病,严重威胁人体生命健康。研究表明IBD与遗传及微生物引起的肠道固有免疫失调密切相关,但确切机制仍未研究清楚。IBD患者肠道菌群和胆汁酸代谢失调,研究显示肠道菌群和胆汁酸在免疫调控方面扮演关键角色。前期研究也证实了三七皂苷可调节肠道菌群调控辅助性T细胞17/调节性T细胞(Th17/Treg)平衡,缓解IBD,但三七皂苷对胆汁酸的调控及胆汁酸如何影响炎症性肠病尚未有研究报道。目的:本研究旨在探究三七皂苷调节肠道菌群-胆汁酸代谢调控Th17/Treg平衡缓解IBD作用。方法:(1)采用葡聚糖硫酸钠(DSS)构建IBD小鼠模型。通过疾病活动指数(DAI)评分、组织病理学分析、检测结直肠长度、脾脏重量、炎症因子和肠道屏障表达水平等评价PNS抗IBD疗效;通过16S r RNA测序筛选三七皂苷调控的肠道菌群;通过LC-MS/MS检测并筛选血浆差异胆汁酸,最后关联分析差异菌和差异胆汁酸。(2)移植HPNS组与模型组小鼠肠道菌群至IBD受体小鼠,通过16S r RNA测序进行菌群定殖分析,观察三七皂苷重塑菌群对IBD的影响;通过血浆胆汁酸测定,比较菌群移植对胆汁酸代谢影响。(3)采用牛磺胆酸(TCA)、牛磺鹅去氧胆酸(TCDCA)治疗IBD,并以5-氨基水杨酸(5-ASA)作为阳性药物,观察其是否通过调控Th17/Treg平衡,发挥抗炎症性肠病的作用。(4)体外胆汁酸与脾脏Na(?)ve T细胞共孵育,RT-q PCR检测调控Treg和Th17细胞分化相关的细胞因子表达。结果:(1)三七皂苷调节肠道菌群的组成及丰度,显著降低Allobaculum、Dubosiella、Muribaculum和Alistipes的相对丰度;并上调结合型胆汁酸,尤其是TCA、TCDCA等胆汁酸相对含量,下调多数游离胆汁酸如胆酸(CA)、石胆酸(LCA)等相对含量;关联分析结果显示Allobaculum、Dubosiella、Muribaculum和Alistipes与TCA、TCDCA显著负相关。(2)粪菌移植实验结果显示,三七皂苷重塑的肠道菌群对胆汁酸代谢具有调控作用,上调TCA、TCDCA等结合型胆汁酸的相对含量。(3)在体内,TCA、TCDCA通过增加Treg细胞比例,改善肠道屏障功能,从而缓解IBD。(4)在体外,TCA、TCDCA可促进Il-2、Il-10和Foxp3的表达,抑制Il-6、Il-Iβ和Il-17的表达。结论:三七皂苷调控肠道群平衡,上调结合型胆汁酸TCA、TCDCA的表达,调节Th17/Treg平衡,发挥抗炎症性肠病的作用。图35幅,表8个,参考文献81篇更多还原

【Abstract】 Background:Inflammatory Bowel Disease(IBD)is a non-specific,chronic,progressive,recurrent gastrointestinal disease of unknown etiology and pathogenesis.It is generally believed that IBD is closely related to genetic factors and microbial-induced aberrant immunity,but the exact mechanism has not yet been investigated.Imbalance of gut microbiota causes bile acid metabolism dysregulation,and IBD patients have dysregulated gut microbiota and bile acid metabolism,and several studies have reported that gut microbiota and bile acid play a key role in immune regulation.The previous group also demonstrated that Panax notoginseng saponins(PNS)could regulate the gut microbiota and Th17/Treg balance to alleviate IBD,but the regulation of bile acids by PNS and how bile acids affect inflammatory bowel disease has not been reported.Objective:The aim of this study was to investigate the anti-IBD effect of PNS regulating gut microbiota-bile acid metabolism to modulate Th17/Treg balance.Methods:(1)Inflammatory bowel disease(IBD)mouse model was established using dextran sulfate sodium(DSS).The anti-IBD efficacy of PNS was evaluated by Disease activity index(DAI)score,histopathological analysis,detection of colorectal length,spleen weight,inflammatory factors and intestinal barrier expression levels;PNS-regulated gut microbiota were screened by 16S r RNA sequencing;detecting and screening of plasma differential bile acids by LC-MS/MS.and finally differential microbiota and differential bile acids were analyzed by correlation.(2)Transplantation of gut microbiota from HPNS group and model group mice to IBD recipient mice,and analysis of microbiota colonization by 16S r RNA sequencing,to observe the effect of PNS remodeling of the microbiota on IBD;and measurement of plasma bile acids,to compare the effect of the transplantation of the microbiota on the metabolism of bile acids.(3)IBD was treated with taurocholic acid(TCA),taurochlorodeoxycholic acid(TCDCA),and 5-aminosalicylic acid(5-ASA)as a positive drug to observe whether they exerted anti-inflammatory bowel disease effects by affecting the balance of T helper 17/regulatory T cells(Th17/Treg).(4)Co-incubate bile acids with splenic Na(?)ve T cells in vitro to detect the expression of cytokines related to the regulation of Treg and Th17 cell differentiation by RT-q PCR.Results:(1)PNS regulated the composition and abundance of gut microbiota,significantly decreased the relative abundance of Allobaculum,Dubosiella,Muribaculum and Alistipes;and up-regulated the relative content of conjugated bile acids,especially the bile acids such as TCA,TCDCA,etc.,and down-regulated the relative content of most of free bile acids,such as CA,LCA,etc.;The results of correlation analysis showed that Allobaculum,Dubosiella,Muribaculum and Alistipes were significantly negatively correlated with TCA and TCDCA.(2)The results of FMT showed that the gut microbiota remodeled by PNS had a regulatory effect on bile acid metabolism,and up-regulated the relative contents of TCA,TCDCA and other conjugated bile acids.(3)TCA and TCDCA alleviated IBD by increasing the proportion of Treg cells and improving intestinal barrier function in vivo.(4)TCA and TCDCA promoted the expression of Il-2,Il-10 and Foxp3,and inhibited the expression of Il-6,Il-Iβand Il-17 in vitro.Conclusion:Panax notoginseng saponins could regulate the balance of gut microbiota,up-regulate the expression of TCA and TCDCA,regulate the balance of Th17/Treg,and exert anti-inflammatory bowel disease effects.更多还原