布拉氏酵母菌通过抑制TLR-4/NF-κB炎症信号通路预防新生鼠坏死性小肠结肠炎

Prevention of Saccharomyces boulardii on neonatal mice with necrotizing enterocolltis by inhibiting TLR-4/NF-κB inflammation signaling pathways

目的探讨布拉氏酵母菌预防新生鼠坏死性小肠结肠炎(NEC)的保护机制。方法64只新生小鼠随机分为4组,每组16只,正常对照组给予母鼠喂养,人工喂养组仅给予代乳品人工喂养,NEC模型组通过人工喂养+缺氧复氧冷刺激+脂多糖方法制备NEC模型,布拉氏酵母菌干预组在NEC模型基础上加布拉氏酵母菌,800mg·kg^-1·d^-1,每日灌胃1次,连用3d。HE染色、病理评分检测肠组织损伤程度;气相色谱法检测结肠粪便中短链脂肪酸(SCFAs)含量;pH计检测结肠粪便中pH值;实时荧光定量PCR法(Real-time PCR)法检测肠组织中Toll样受体4(TLR-4)、核因子(NF)-κB mRNA表达水平;蛋白质印迹法(Western blot)检测肠组织TLR-4、NF-κB蛋白表达水平。结果与正常对照组比较,人工喂养组体重稍下降,病理评分略升高,差异无统计学意义(P>0.05);NEC模型组体重下降明显,病理评分升高,差异有统计学意义(P<0.05);布拉氏酵母菌干预组体重较NEC组上升,病理评分降低(P<0.05),各种指标接近于或稍好于人工喂养组。与正常对照组比较,NEC模型组结肠粪便中乙酸、丁酸含量降低,pH值增高(P<0.05);与NEC模型组相比,布拉氏酵母菌干预组结肠粪便中乙酸、丁酸含量增加(P<0.05),pH值降低(P<0.05)。与正常对照组比较,NEC模型组中TLR-4和NF-κB mRNA及蛋白表达水平均升高(P<0.05,P<0.01);与NEC模型组相比,布拉氏酵母菌干预组TLR-4和NF-κB mRNA及蛋白表达水平均下降,差异具有统计学意义(P<0.05,P<0.01)。结论布拉氏酵母菌可以保护新生鼠NEC,其作用可能与提高肠道中SCFAs的含量、降低肠道pH值,最终抑制TLR-4/NF-κB炎症有关。

Objective To investigate the protective mechanism of Saccharomyces boulardii in preventing necrotizing enterocolltis(NEC) in neonatal mice.Methods Sixty-four neonatal mice were randomly divided into 4 groups,16 in each group.Breast feeding was given in normal control group.The artificial feeding of dairy substitute was performed in NEC model group.The artificial feeding + hypoxia-reoxygenation cold stimulation + lipopolysaccharide were used to prepare NEC model.On the basis of NEC model group,Saccharomyces boulardii (800 mg·kg^-1·d^-1 ) was fed once a day for 3 days in Saccharomyces boulardii intervention group.HE staining and pathological score were used to detect the degree of intestinal tissue damage;gas chromatography was used to detect the content of short-chain fatty acids (SCFAs) in colonic feces;pH meter was used to detect the pH value in colonic feces;real-time PCR was used to detect the expression of Toll-like receptor 4 (TLR-4) and nuclear factor(NF)-κB mRNA in intestinal tissues;Western blot was used to detect the levels of TLR-4 and NF-κB protein in intestinal tissues.Results Compared with the normal control group,the body weight decreased slightly,and the pathological score increased slightly in the artificial feeding group (P > 0.05),but the body weight decreased significantly,and the pathological score increased significantly in the NEC model group(P < 0.05).Compared with the NEC model group,the body weight increased significantly,and the pathological score decreased significantly in the Saccharomyces boulardii intervention group(P < 0.05).The various indicators in the Saccharomyces boulardii intervention group were close to or slightly better than the artificial feeding group.Compared with the normal control group,the content of acetic acid and butyric acid in feces decreased significantly,and the pH value of feces increased significantly in the NEC model group (P < 0.05 ).Compared with the NEC model group,the content of acetic acid and butyric acid in feces increased significantly and the pH value of feces decreased significantly in the Saccharomyces boulardii intervention group (P < 0.05).Compared with the normal control group,the expression levels of TLR-4 and NF-κB mRNA and protein increased significantly in the NEC model group (P < 0.05,P < 0.01).Compared with the NEC model group,the expression levels of TLR-4 and NF-κB mRNA and protein decreased significantly in the Saccharomyces boulardii intervention group (P < 0.05,P < 0.01).Conclusion Saccharomyces boulardii can prevent necrotizing enteritis in neonatal mice,which may be related to increasing the content of SCFAs and decreasing pH in the intestine,and ultimately inhibiting TLR-4 /NF-κB inflammation.