基于肠道菌群和代谢组学研究黄连素和黄芩苷配比对湿热内蕴型2型糖尿病粪菌移植小鼠的影响  

Effect of Berberine-Baicalin Combination on Fecal Microbiota Transplantation-induced Type 2 Diabetes Mellitus Due to Internal Accumulation of Dampness-heat in Mice from Perspectives of Gut Microbiota and Metabolomics

作　　者：陈梦婕 刘艺敏 周云 余柯铭 夏敏 刘红宁[1] 吉燕华 曾治君[1] CHEN Mengjie;LIU Yimin;ZHOU Yun;YU Keming;XIA Min;LIU Hongning;JI Yanhua;ZENG Zhijun(Research Center for Differentiation and Development of Traditional Chinese Medicine(TCM)Basic Theory,Jiangxi Province Key Laboratory of TCM Etiopathogenisis,Jiangxi University of Chinese Medicine,Nanchang 330004,China)

机构地区：[1]江西中医药大学中医基础理论分化发展研究中心,江西省中医病因生物学重点实验室,南昌330004

出　　处：《中国实验方剂学杂志》2025年第5期52-64,共13页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81760787,82460801,82460857);江西省中医药中青年骨干人才项目(赣中医药科教字〔2021〕2号);江西省中医药管理局科技计划项目(2023Z002);江西中医药大学校级科技创新团队发展计划项目(CXTD22007);江西中医药大学校级创新专项(JZYC22S76)。

摘　　要：目的:从肠道菌群和代谢组学角度探讨黄连素(BBR)和黄芩苷(BAI)配比改善湿热内蕴型2型糖尿病(T2DM)的作用机制。方法:用抗生素诱导小鼠为“伪无菌鼠”,然后将30只“伪无菌鼠”随机分为正常粪菌移植组(n=10)和湿热内蕴型T2DM粪菌移植组(n=20),分别灌胃健康人粪菌悬液和湿热内蕴型T2DM患者粪菌悬液,每只小鼠灌胃200μL,隔日灌胃,共15次,以此重塑小鼠肠道微生物群。将造模后的T2DM小鼠随机分为模型组(n=8)和BBR-BAI给药组(n=11)。BBR给药剂量为200 mg·kg^(-1),根据前期对BBR和BAI配比研究结果,按照BBR-BAI 10∶1加入BAI溶液后进行灌胃,连续给药8周。通过检测空腹血糖(FBG)、糖化血红蛋白(HbA1c)、胰岛素(INS)、甘油三酯(TG)、总胆固醇(CHOL)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)、天冬氨酸氨基转移酶(AST)和丙氨酸氨基转移酶(ALT)水平,评估BBR-BAI配比对T2DM小鼠糖脂代谢和肝功能的影响;应用苏木素-伊红(HE)染色观察小鼠结肠组织病理变化;蛋白免疫印迹法(Western blot)检测结肠组织紧密连接蛋白封密蛋白-1(Claudin-1)、闭锁小带蛋白-1(ZO-1)、闭合蛋白(Occludin)的表达;实时荧光定量聚合酶链式反应(Real-time PCR)检测结肠组织中肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β和IL-6的水平;16S rRNA测序和超高效液相色谱-四极杆-飞行时间串联质谱法(UPLC-Q-TOF-MS)分别检测小鼠粪便肠道菌群和差异代谢物。结果:BBR-BAI干预明显降低T2DM小鼠FBG、HbA1c及INS水平(P<0.05,P<0.01),改善胰岛素抵抗(P<0.01);此外BBR-BAI明显增加紧密连接蛋白ZO-1、Occludin、Claudin-1含量(P<0.05,P<0.01),明显降低结肠炎症因子TNF-α、IL-1β和IL-6表达水平(P<0.05,P<0.01);16S rRNA测序结果发现BBR-BAI可明显升高Ligilactobacillus、Phascolarctobacterium、Akkermansia等菌属的相对丰度(P<0.05),明显降低Alistipes、Odoribacter、Colidextribacter等菌属的相对丰度(Objective:To investigate the mechanisms by which the combination of berberine(BBR)and baicalin(BAI)ameliorates type 2 diabetes mellitus(T2DM)due to internal accumulation of dampness-heat from the perspectives of gut microbiota and metabolomics.Methods:Antibiotics were used to induce pseudo-sterile mice.Thirty pseudo-sterile mice were randomized into a normal fecal microbiota transplantation group(n=10)and a T2DM(syndrome of internal accumulation of dampness-heat)fecal microbiota transplantation group(n=20).The mice were then administrated with suspensions of fecal microbiota from healthy volunteers and a patient with T2DM due to internal accumulation of dampness-heat by gavage,respectively.Each mouse received 200μL suspension every other day for a total of 15 times to reshape the gut microbiota.The T2DM model mice were then assigned into a model group(n=8)and a BBR-BAI group(n=11).BBR was administrated at a dose of 200 mg·kg^(-1),and BAI was administrated in a ratio of BBR-BAI 10∶1 based on preliminary research findings.The administration lasted for 8 consecutive weeks.Fasting blood glucose(FBG),glycated hemoglobin(HbA1c),insulin(INS),triglycerides(TG),total cholesterol(CHOL),low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C),aspartate aminotransferase(AST),and alanine aminotransferase(ALT)levels were measured to evaluate the effects of the BBR-BAI combination on glucose and lipid metabolism and liver function in T2DM mice.Hematoxylin-eosin staining was employed to observe pathological changes in the colon tissue.The expression of claudin-1,zonula occludens-1(ZO-1),and occludin in the colon tissue was determined by Western blot.Real-time quantitative polymerase chain reaction(Real-time PCR)was employed to assess the levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and interleukin-6(IL-6)in the colon tissue.The fecal microbiota composition and differential metabolites were analyzed by 16S rRNA sequencing and ultra-high performance liquid chromatography-quad

关 键 词：粪菌移植 湿热内蕴 2型糖尿病 黄连素 黄芩苷 肠道菌群 代谢组学 

分 类 号：R284.2[医药卫生—中药学] R285[医药卫生—中医学] R289