摘要
背景溃疡性结肠炎被世界卫生组织列为现代难治性疾病之一。目前治疗技术均有相应的不足之处,粪菌移植(FMT)治疗溃疡性结肠炎的有效性已被证实,但疗效参差不齐。目的比较FMT治疗小鼠普通型溃疡性结肠炎模型(CUCM)与湿热型溃疡性结肠炎模型(DUCM)的效果及FMT(新金汁)的中药性味。方法于2019年12月9—28日,将35只雄性SPF级C57BL/6小鼠分为7组,分别为正常对照组(Control组)、CUCM组、CUCM+FMT组、CUCM+5-氨基水杨酸(5-ASA)组、DUCM组、DUCM+FMT组、DUCM+5-ASA组,每组各5只小鼠。Control组不做任何干预处理;CUCM组和DUCM组根据造模需求分别制备模型;CUCM+FMT组和DUCM+FMT组于造模成功后,给予制备的粪菌液0.2 ml对小鼠进行灌肠;CUCM+5-ASA组和DUCM+5-ASA组于造模成功后,给予0.0195 g/ml 5-ASA对小鼠进行灌肠。通过HE染色观察各组肠组织的变化,透射电镜观察组织的超微结构变化,流式细胞检测辅助性T细胞1(Th1)、Th2细胞含量,血常规检测血液中白细胞计数(WBC)、红细胞计数(RBC)、血小板计数(PLT)和血红蛋白(HGB),尼莫地平法评估干预前后疗效指数,16S rRNA高通量测序技术检测肠内容物中的菌群分布。结果各组小鼠均造模成功。肠组织HE染色显示:CUCM组与DUCM组肠黏膜表面出现不同程度的缺损或脱落坏死;CUCM+FMT组、CUCM+5-ASA组、DUCM+FMT组、DUCM+5-ASA组肠黏膜基本完整;并且DUCM+FMT组与CUCM+FMT组相比,前者腺体较后者排列整齐并紧密。肠组织透射电镜超微结构显示:CUCM组与DUCM组上皮细胞表面微绒毛稀疏,杯状细胞减少;CUCM+FMT组、CUCM+5-ASA组、DUCM+FMT组、DUCM+5-ASA组微绒毛较为致密,杯状细胞数目较多;并且DUCM+FMT组与CUCM+FMT组相比,前者微绒毛较后者紧密,杯状细胞也较后者多。各组小鼠Th1、Th2细胞含量、WBC、RBC、PLT、HGB比较,差异均有统计学意义(P<0.001)。CUCM+5-ASA组、DUCM+FMT组、DUCM+5-ASA组疗效指数高于CUCM+FMT组(P<0.05)。菌群分析显示CUCM组与DUCM组中丰度显著降低的属是Ruminococcus,显著升高的属是Akkermansia,经过FMT治疗后,CUCM+FMT组、DUCM+FMT组有逐步向Control组靠拢的趋势,表明肠道菌群正在改善。结论FMT可治疗溃疡性结肠炎模型小鼠,且针对DUCM疗效更佳,FMT(新金汁)的中药性味苦寒,推测其发挥功效的途径可能是通过调节肠道菌群进而改善Th1/Th2细胞平衡,达到治疗的目的。
Background As an intractable disease recognized by the WHO,current available treatments have limitations for ulcerative colitis(UC).Although the effectiveness of fecal microbiota transplantation(FMT)has been confirmed,the reported efficacies are various.Objective To compare the effect of FMT between a mouse model of common UC and a mouse model of damp-heat UC,and the medicinal flavor of fecal microbiota(new jinzhi).Methods This experiment was implemented from December 9th to 28th,2019.Thirty-five male SPF C57BL/6 mice were equally divided into seven groups:normal control group(no intervention),common UC model(CUCM)group(received interventions to be a CUCM according to the requirements),CUCM+FMT group(received interventions to be a CUCM successfully,then received an enema of 0.2 ml prepared fecal microbiota solution),CUCM+5-aminosalicylic acid(5-ASA)group(received interventions to be a CUCM successfully,then received an enema of 0.0195 g/ml 5-ASA),damp-heat UC model(DUCM)group(received interventions to be a DUCM according to the requirements),DUCM+FMT group(received interventions to be a DUCM successfully,then received an enema of 0.2 ml prepared fecal microbiota solution),and DUCM+5-ASA group(received interventions to be a DUCM successfully,then received an enema of 0.0195 g/ml 5-ASA).The intestinal tissues of each group of mice were taken out when the experiment ended,and were stained with H&E for observing the morphology,and ultrastructure of them was observed using transmission electron microscope,Th1 and Th2 cells in them were detected using flow cytometry,and microbial community in them were detected using high-throughput 16S rRNA gene sequencing.Routine blood test was conducted to measure the levels of white blood cell count(WBC),red blood cell count(RBC),platelet count(PLT)and hemoglobin(HGB).Nimodipine method was used to evaluate the efficacy index before and after intervention.Results The mice in each group were successfully established.The morphology of intestinal tissues stained with H&E:the intestinal mucosal surface of CUCM group and DUCM group had different degrees of defect or shedding and necrosis,while the intestinal mucosa was basically intact in CUCM+FMT group,CUCM+5-ASA group,DUCM+FMT group and DUCM+5-ASA group.The DUCM+FMT group had more neatly arranged and compacted glands than did CUCM+FMT group.The ultrastructure of intestinal tissue observed using transmission electron microscope:the surface microvilli of epithelial cells in CUCM and DUCM groups were sparse with decreased goblet cells.The CUCM+FMT,CUCM+5-ASA,DUCM+FMT and DUCM+5-ASA groups had denser microvilli with more goblet cells.Compared with the CUCM+FMT group,the DUCM+FMT group had denser microvilli and more goblet cells.Th1 and Th2 cell contents:there were significant differences in Th1 and Th2 cell contents,WBC,RBC,PLT and HGB across all groups of mice(P<0.001).Efficacy analysis:the efficacy index of CUCM+5-ASA group,DUCM+FMT group or DUCM+5-ASA group was better than that of CUCM+FMT group(P<0.05).Microbial community analysis:the genus with significantly decreased abundance in CUCM and DUCM groups was Ruminococcus,and the increased abundance was Akkermansia.The CUCM+FMT and DUCM+FMT groups had a tendency of gradually increasing similarities to the control group,indicating that the intestinal flora of these two groups were improving.Conclusion FMT were effective for mice models of UC,especially for the mice model of damp-heat UC.The new jinzhi has a bitter and cold flavor,which may work by regulating the intestinal flora to improve the Th1/Th2 balance for therapeutic purposes.
作者
马晓飞
胡家丽
崔曼曼
葛巍
杨苏琴
丁成华
张磊昌
MA Xiaofei;HU Jiali;CUI Manman;GE Wei;YANG Suqin;DING Chenghua;ZHANG Leichang(Graduate School,Jiangxi University of Chinese Medicine,Nanchang 330004,China;Affiliated Hospital of Jiangxi University of Chinese Medicine,Nanchang 330046,China;School of Basic Medical Sciences,Jiangxi University of Chinese Medicine,Nanchang 330004,China)
出处
《中国全科医学》
CAS
北大核心
2022年第15期1875-1882,共8页
Chinese General Practice
基金
国家自然科学基金资助项目(81760871)
江西省教育厅课题(GJJ190682)
江西省自然科学基金资助项目(2020BABL206135)。