Fermentation Production of Ganoderma lucidum by Bacillus subtilis Ameliorated Ceftriaxone-induced Intestinal Dysbiosis and Improved Intestinal Mucosal Barrier Function in Mice  

枯草芽孢杆菌-灵芝子实体发酵液改善头孢曲松钠诱导的小鼠肠道菌群失调及肠黏膜屏障功能受损

作　　者：LIU He FAN Qing-Jie LIU Yin-Hui LI Ming YUAN Jie-Li 刘赫;范青杰;刘银辉;李明;袁杰力(大连医科大学基础医学院,辽宁大连116044)

机构地区：[1]College of Basic Medical Science,Dalian Medical University,Dalian,Liaoning 116044,China

出　　处：《Digital Chinese Medicine》2020年第3期199-212,共14页数字中医药（英文）

基　　金：We thank for the funding support from the National Natural Science Foundation of China(No.31900920);the Nutrition and Care of Maternal&Child Research Fund Project of Guangzhou Biostime Institute of Nutrition&Care(No.2019BINCMCF02);the Liaoning Provincial Program for Top Discipline of Basic Medical Sciences,China.

摘　　要：Objective This study aimed to develop a type of Ganoderma lucidum(G.lucidum)-probiotic fermentation broth that can effectively improve the intestinal mucosal barrier function of mice with ceftriaxone-induced intestinal dysbiosis.Methods By means of absorbance of optical density(OD)value and phenol-concentrated sulfuric acid measurement of polysaccharide content,the probiotic species can grow on the medium of G.lucidum were screened out,and the concentration of the medium of G.lucidum was determined,and the fermentation broth was prepared for subsequent experiments.Thirty-two SPF grade male BALB/c mice were randomly divided into four groups(eight mice in each group),namely control group(CON),intestinal mucosal barrier damage model group(CS),fermentation broth intervention group(FT)and G.lucidum medium intervention group(GL),respectively.The intestinal dysregulation model was induced by intra-gastric administration of 0.2 mL ceftriaxone sodium(twice a day for seven consecutive days).From day 8,the FT group and GL group were gavage with 0.2 mL fermentation broth and G.lucidum medium,respectively.On day 15,all mice were sacrificed.To draw the weight curve and measure the cecal index;pathological examination of colon tissues with HE staining;serum levels of LPS,IL-10,TNF and IL-6 were detected by ELISA.Flow cytometry was used to analyze the changes of CD3+T cells,CD4+T cells,CD8+T cells and macrophages in spleen.16S rRNA sequencing was performed to detect the intestinal microbiota structure of mice.Results Bacillus subtilis can decompose and utilize G.lucidum fruiting body medium,and the suitable concentration of G.lucidum fruiting body medium is 33.2 mg/mL.The effect of Bacillus subtilis-G.lucidum fermentation broth on the damage of intestinal mucosal barrier caused by ceftriaxone sodium was reduced,the body weight of mice recovered and colon swelling improved,colon histopathological injury was alleviated,inflammatory cell infiltration was alleviated,serum IL-10 increased significantly,LPS、TNF-αand IL-6 decreased 目的本实验旨在筛选出能分解利用灵芝子实体培养基的益生菌菌种,并探究用该益生菌-枯草芽孢杆菌发酵的灵芝子实体发酵液对头孢曲松钠诱导的肠黏膜屏障受损模型小鼠的影响。方法通过吸光度测量OD值、苯酚-浓硫酸测量多糖含量的方法筛选出能在灵芝子实体培养基上生长的益生菌菌种,并确定灵芝子实体培养基浓度,制备发酵液用于后续实验。采用32只健康SPF级雄性BALB/c小鼠,将小鼠随机分为4组(每组8只),分别为对照组(CON)、肠黏膜屏障受损模型组(CS)、发酵液干预组(FT)、灵芝培养基干预组(GL)。0.2 mL头孢曲松钠灌胃诱导肠道失调模型(每天两次,连续7天),从第8天开始,FT组和GL组分别灌胃益生菌-灵芝发酵液和灵芝培养基0.2 mL。第15天,处死所有小鼠,并收集血、盲肠、结肠、脾脏、粪便样本。绘制体重曲线、测量盲肠指数;结肠组织HE染色病理学检测;ELISA法检测小鼠血清中LPS、IL-10、TNF-α、IL-6水平;流式细胞仪分析脾脏CD3+T细胞、CD4+T细胞、CD8+T细胞、巨噬细胞变化情况;16S rRNA测序检测小鼠肠道菌群结构。结果枯草芽孢杆菌可以分解、利用灵芝子实体培养基,且灵芝子实体培养基的适宜浓度为33.2 mg/mL。枯草芽孢杆菌-灵芝子实体发酵液对头孢曲松钠引起的小鼠肠黏膜屏障受损有一定的缓解作用,其表现是发酵液治疗后小鼠体重恢复,结肠肿胀情况有所好转;结肠组织病理学损伤减轻,炎细胞浸润明显缓解;血清IL-10较模型组显著上升,LPS、TNF-α、IL-6较模型组显著下降;改善了头孢曲松导致的T细胞比例的过度上调和肠道菌群失调。结论实验结果提示枯草芽孢杆菌-灵芝子实体发酵液可以有效改善抗生素过量所致的肠黏膜屏障功能受损、免疫功能紊乱和肠道菌群失调,对肠黏膜屏障功能有一定调节作用。

关 键 词：Ganoderma lucidum Bacillus subtilis Traditional Chinese medicine fermentation Ganoderma lucidum acid Intestinal mucosal barrier function Intestinal dysbiosis Regulate Tcell responses 

分 类 号：R285.5[医药卫生—中药学]