【作者】 周婷婷；

【导师】 李世刚；

【作者基本信息】 三峡大学 ， 药学（专业学位）， 2021， 硕士

【摘要】 研究背景:随着肥胖症和代谢综合征在全球的流行,非酒精性脂肪肝疾病(NAFLD)已成为全球慢性肝病之一,且发病率逐年上升,严重影响人类的健康生活。目的:通过肠道菌群—胆汁酸—肝代谢轴与NAFLD的相关性研究青砖茶(QZT)对NAFLD的作用。方法:C57BL/6小鼠随机分为5组,即正常对照组(NC),模型对照组(MC),阳性药物对照组(PC)以及青砖茶水提物低剂量组(LD)、高剂量组(HD),以高脂饲料喂养小鼠14周建立NAFLD模型,同时预防性给予低、高剂量青砖茶水提物和阳性药物血脂康,(1)常规指标检测小鼠的体重、摄食量、食物利用率、肝重和肝指数;(2)HE染色和油红O染色观察肝组织病理切片;(3)生化分析检测TC、LDL-C、HDL-C、ALT含量;(4)小鼠粪便16S rDNA V3-V4区高通量测序分析肠道菌群变化;(5)LC/MS-MS靶向代谢组学方法检测血清胆汁酸变化;(6)Spearman相关性分析方法分析肠道菌群、胆汁酸、NAFLD三者之间的相关性;(7)Western blot检测肝组织TGR5、NLRP3、Pro-caspase-1、Caspase-1 p10蛋白表达水平;(8)ELISA法检测肝组织IL-1β、IL-18含量变化。结果:(1)各组小鼠平均摄食量无明显差异,与正常对照组比较,模型组体重、食物利用率、肝重和肝指数均显著增加,与模型组比较,青砖茶水提物干预组上述指标均显著降低。(2)肝组织病理切片结果显示,模型组小鼠肝组织出现脂肪堆积和炎症,青砖茶水提物干预组肝脏病变程度有所改善。(3)生化分析结果显示,与正常对照组比较,模型组小鼠TC、LDL-C/HDL-C、ALT含量均显著增高,与模型组比较,青砖茶水提物干预组上述指标均显著降低。(4)肠道菌群分析结果显示,模型组小鼠肠道菌群整体结构和丰度与正常对照组有明显区别,线性判别分析显示5种差异菌群,其中,与正常对照组比较,模型组小鼠Bacteroides丰度增加,Lactobacillus、Alloprevotella、Saccharibacteria＿genera＿incertae＿sedis、Ruminococcaceae丰度降低;与模型组比较,青砖茶水提物干预组小鼠Bacteroides丰度降低,Lactobacillus、Alloprevotella、Saccharibacteria＿genera＿incertae＿sedis丰度增加。(5)胆汁酸分析结果显示,共鉴定了23种胆汁酸,其中初级胆汁酸10种,次级胆汁酸13种,筛选得到17种差异胆汁酸,与正常对照组比较,模型组的差异胆汁酸含量相对减少,与模型组比较,青砖茶水提物干预组的差异胆汁酸含量有不同程度的增加。(6)相关性分析结果显示肠道菌群及其代谢产物胆汁酸与NAFLD具有相关性,其中相关性最强的菌群为Bacteroides和Lactobacillus,相关性最强的代谢物为DCA和ωMCA。(7)Western blot实验结果显示,与正常对照组比较,模型组小鼠肝组织TGR5蛋白表达显著降低,NLRP3、Pro-caspase-1、Caspase-1 p10蛋白表达显著增加;与模型组比较,青砖茶水提物干预组TGR5蛋白表达显著增加,NLRP3、Pro-caspase-1、Caspase-1 p10蛋白表达显著降低。(8)ELISA结果显示,与正常对照组比较,模型组IL-1β、IL-18含量均显著增高;与模型组比较,青砖茶水提物干预组IL-1β、IL-18含量均显著降低。结论:(1)经过14周高脂饮食及青砖茶水提物干预,常规指标、组织形态学和生化分析指标结果说明青砖茶对小鼠NAFLD模型有疗效。(2)肠道菌群及其代谢产物胆汁酸与NAFLD具有相关性,其中相关性最强的菌群为Bacteroides和Lactobacillus,相关性最强的代谢物为DCA和ωMCA,胆汁酸通过结合其肝受体TGR5,负向调控NLRP3炎症小体,从而达到减轻NAFLD的效果。(3)青砖茶通过影响肠道菌群—胆汁酸—肝脏NLRP3炎症小体轴产生抗NAFLD的作用。更多还原

【Abstract】 Background With the global prevalence of obesity and metabolic syndrome,non-alcoholic fatty liver disease(NAFLD)has become one of the global chronic liver diseases,and the incidence is increasing year by year,seriously affecting the healthy life of human beings.Objective To study the effect of Qingzhuan tea(QZT)on NAFLD through the correlation between gut microbiota-bile acid-liver metabolism axis and NAFLD.Methods C57BL/6 mice were randomly divided into 5 groups,normal control group(NC),model control group(MC),positive drug control group(PC),low-dose group(LD)and high-dose group(HD)of QZT water extract.A NAFLD model was established by feeding mice with high-fat diet for 14 weeks,and supplemented with low and high doses of QZT water extract and positive drug(Xuezhikang)preventively.(1)Routine indicators including the body weight,food intake,food utilization efficiency,liver weight and liver index of mice were determined.(2)Liver tissue pathological sections were observed by HE staining and Oil Red O staining.(3)Biochemical analysis indicators including TC,LDL-C,HDL-C,ALT content were detected.(4)The changes of gut microbiota were analyzed by high-throughput sequencing in 16 S rDNA V3-V4 region of mice feces.(5)The changes of serum bile acid were determined by LC/MS-MS targeted metabolomics method.(6)Spearman’s correlation analysis was used to analyze the correlation between gut microbiota,bile acid and NAFLD.(7)The expression of liver tissue TGR5,NLRP3,Pro-caspase-1,Caspase-1 p10 protein were detected by Western blot.(8)ELISA method was used to detect changes in liver tissue IL-1βand IL-18.Results(1)There was no significant difference in the average food intake of mice in each group.Compared with normal control group,the body weight,food utilization efficiency,liver weight and liver index of model group increased significantly.Compared with model group,the above indicators of QZT water extract intervention groups were significantly reduced.(2)The liver tissue pathological section results showed that the liver tissues of mice in model group had fat accumulation and inflammation,and the degree of liver disease in QZT water extract intervention groups was improved.(3)The results of biochemical analysis showed that compared with normal control group,the contents of TC,LDL-C/HDL-C and ALT in model group were significantly increased.Compared with model group,the above indicators of QZT water extract intervention groups were significantly reduced.(4)The results of the gut microbiota analysis showed that the overall structure and abundance of the gut microbiota of model group were significantly different from those of the normal control group.LEf Se result showed five different floras.Among them,compared with normal control group,the abundance of Bacteroides in model group increased,and the abundance of Lactobacillus,Alloprevotella,Saccharibacteria＿genera＿incertae＿sedis,and Ruminococcaceae in model group decreased.Compared with model group,the abundance of Bacteroides in QZT water extract intervention groups decreased,and the abundance of Lactobacillus,Alloprevotella,and Saccharibacteria＿genera＿incertae＿sedis in QZT water extract intervention groups increased.(5)The results of bile acids analysis showed that a total of 23 bile acids were identified,including 10 primary bile acids and 13 secondary bile acids.17 differential bile acids were screened.Compared with normal control group,the differential bile acids abundance of model group relatively decrease,compared with model group,the abundance of the differential bile acids in QZT water extract intervention groups increased to varying degrees.(6)Correlation analysis results showed that the gut microbiota and bile acids were related to NAFLD,among which the most correlated floras were Bacteroides and Lactobacillus,and the most correlated metabolites were DCA and ωMCA.(7)Western blot results showed that compared with normal control group,the expression of TGR5 protein in the liver tissue of model group was significantly reduced,and the expression of NLRP3,Pro-caspase-1,and Caspase-1 p10 protein were significantly increased;compared with model group,the expression of TGR5 protein of QZT water extract intervention groups increased significantly,and the expression of NLRP3,Pro-caspase-1,and Caspase-1 p10 protein of QZT water extract intervention groups decreased significantly.(8)ELISA results showed that compared with normal control group,IL-1β and IL-18 in model group were significantly increased;compared with model group,IL-1β and IL-18 in QZT water extract intervention groups were significantly decreased.Conclusion(1)After 14 weeks of high-fat diet and QZT water extract intervention,the results of conventional indicators,histomorphology and biochemical analysis indicate that QZT has curative effect on the mouse NAFLD model.(2)Gut microbiota and its metabolites bile acids are related to NAFLD.Among them,the most correlated gut microbiota are Bacteroides and Lactobacillus,and the most correlated metabolites are DCA and ωMCA.Bile acids negatively regulate the NLRP3 inflammasome by binding to its liver receptor TGR5,so as to achieve the effect of reducing NAFLD.(3)QZT has an anti-NAFLD effect by regulating the gut microbiota-bile acid-liver axis.更多还原