抗生素预处理对丁酸梭菌治疗葡聚糖硫酸钠诱导结肠炎疗效和肠道菌群的影响

Antibiotic pretreatment on the efficacy of Clostridium butyricum in the treatment of dextran sulfate sodium induced colitis and the influence of intestinal microbiota

目的探讨在有无抗生素预处理条件下,丁酸梭菌对小鼠结肠炎的疗效和肠道菌群的影响。方法将30只清洁级BALB/c小鼠随机分为空白对照组、葡聚糖硫酸钠(DSS)组、抗生素+DSS组、丁酸梭菌+DSS组、抗生素+丁酸梭菌+DSS组,每组6只。采用四联抗生素(氨苄西林1 g/L、新霉素1 g/L、甲硝唑1 g/L、万古霉素0.5 g/L)加正常饮用水循环预处理30 d后,用DSS诱导小鼠结肠炎模型,同时给予丁酸梭菌+DSS组、抗生素+丁酸梭菌+DSS组小鼠1×106 CFU丁酸梭菌灌胃。通过小鼠疾病活动指数(DAI)、结肠长度和组织学病理评分评估丁酸梭菌对结肠炎小鼠的作用,采用ELISA法检测血清炎症因子,通过粪便16S rRNA测序检测丁酸梭菌对小鼠肠道菌群的影响。统计学方法采用两样本t检验。结果空白对照组小鼠一般情况良好,DAI在0分上下波动;DSS组小鼠给予DSS饮用水4 d时出现体重下降、粪便不成形、肉眼血便等结肠炎表现;在DSS干预4 d时,丁酸梭菌+DSS组小鼠DAI低于DSS组[(0.000±0.000)分比(0.444±0.111)分],差异有统计学意义(t=4.000,P=0.016 1);在DSS干预10和12 d时,抗生素+丁酸梭菌+DSS组小鼠DAI均低于抗生素+DSS组[(0.000±0.000)分比(1.111±0.222)分、(0.667±0.000)分比(1.889±0.222)分],差异均有统计学意义(t=5.000、5.500,P均<0.05)。丁酸梭菌+DSS组小鼠结肠病理组织学评分低于DSS组[(2.50±1.73)分比(5.50±1.00)分],抗生素+丁酸梭菌+DSS组小鼠结肠病理组织学评分低于抗生素+DSS组[(1.25±0.96)分比(5.00±0.82)分],差异均有统计学意义(t=3.000、5.960,P均<0.05)。丁酸梭菌+DSS组IL-1β水平高于空白对照组[(4.464±0.075) ng/L比(3.907±0.080) ng/L],丁酸梭菌+DSS组和抗生素+丁酸梭菌+DSS组TNF-α、IL-6和IL-1β水平均低于DSS组[(2.402±0.383) ng/L、(1.845±0.345) ng/L比(6.958±1.084)ng/L,(1.752±0.146)ng/L、(1.307±0.048) ng/L比(3.537±0.608)ng/L,(4.464±0.075)ng/L、(4.066±0.190) ng/L比(7.477±0.339)ng/L],差异均有统计学意义(t=5.055、3.964、4.495、4.693、6.294、8.674、8.774,P均<0.05)。16S rRNA测序结果显示,丁酸梭菌干预小鼠肠道菌群中出现了大量差异有统计学意义的抑炎或产短链脂肪酸细菌,其中丁酸梭菌+DSS组优势菌有小螺旋菌属[线性判别式分析(LDA)评分=3.667 log10,P=0.004]和寡养单胞菌属(LDA评分=2.778 log10,P=0.044),抗生素+丁酸梭菌+DSS组优势菌属有消化球菌属(LDA评分=2.685 log10,P=0.018)、丁酸单胞菌属(LDA评分=2.712 log10,P=0.011)、嗜胆菌属(LDA评分=3.204 log10,P=0.014)、肠道单胞菌属(LDA评分=3.346 log10,P=0.010)、萨克候选菌(LDA评分=3.363 log10,P=0.029)、脱硫弧菌属(LDA评分=3.402 log10,P=0.025)、颤杆菌属(LDA评分=2.870 log10,P=0.019)和阿克曼氏菌属(LDA评分=4.031 log10,P=0.005)。结论丁酸梭菌可有效改善小鼠结肠炎,调整小鼠肠道菌群结构,而抗生素预处理可强化其对肠道菌群的调节作用并增强丁酸梭菌的疗效。

Objective To investigate the effects of Clostridium butyricum on colitis and intestinal microbiota in mice with or without antibiotic pretreatment.Methods Thirty specific pathogen free BALB/c mice were randomly divided into the blank control group,dextran sulfate sodium(DSS)group,antibiotic+DSS group,Clostridium butyricum+DSS group and antibiotic+Clostridium butyricum+DSS group,with 6 mice in each group.After the mice were pretreated with quadruple antibiotics(ampicillin 1 g/L,neomycin 1 g/L,metronidazole 1 g/L,and vancomycin 0.5 g/L)in normal drinking water for 30 d,the mice colitis model was induced with DSS.At the same time,the mice in Clostridium butyricum+DSS group and antibiotics+Clostridium butyricum+DSS group were given 1×106colony-forming unit(CFU)Clostridium butyricum by gavage.The effect of Clostridium butyricum on mice with colitis was evaluated by disease activity index(DAI),colon length and histopathological score.The level of serum inflammatory factors was detected by enxyme linked immunosorbent assay,and the effect of Clostridium butyricum on gut microbita in mice was determined by fecal 16S rRNA sequencing.Results The general condition of mice of the blank control group were good,and their DAI scores fluctuated around 0.Since the fourth day after DSS drinking water was given,the mice of the DSS group showed signs of colitis such as weight loss,unformed stools and bloody stools.On the fourth day after intervention,the DAI score of Clostridium butyricum+DSS group was lower than that of DSS group(0.000±0.000 vs.0.444±0.111),and the difference was statistically significant(t=4.000,P=0.0161).On the tenth and twelfth day after the intervention,the DAI scores of antibiotic+Clostridium butyricum+DSS group were both lower than those of antibiotic+DSS group(0.000±0.000 vs.1.111±0.222,0.667±0.000 vs.1.889±0.222),and the differences were statistically significant(t=5.000 and 5.500,both P<0.05).The histopathological score of mice colon tissue of Clostridium butyricum+DSS group was lower than that of DSS group(2.50±1.73 vs.5.50±1.00),and the histopathological score of mice colon tissue of antibiotic+Clostridium butyricum+DSS group was lower than that of antibiotic+DSS group(1.25±0.96 vs.5.00±0.82),and the differences were statistically significant(t=3.000 and 5.960,both P<0.05).The serum level of interleukin(IL)-1βClostridium butyricum+DSS group was higher than that of blank control group((4.464±0.075)ng/L vs.(3.907±0.080)ng/L),the serum levels of tumor necrosis factor-α,IL-6 and IL-1βof Clostridium butyricum+DSS group and antibiotic+Clostridium butyricum+DSS group were all lower than those of DSS group((2.402±0.383)ng/L,(1.845±0.345)ng/L vs.(6.958±1.084)ng/L,(1.752±0.146)ng/L,(1.307±0.048)ng/L vs.(3.537±0.608)ng/L,(4.464±0.075)ng/L,(4.066±0.190)ng/L vs.(7.477±0.339)ng/L),and the differences were statistically significant(t=5.005,3.964,4.495,4.693,6.294,8.674 and 8.774,all P<0.05).The results of 16S rRNA sequencing showed that there were a significantly large number of anti-inflammatory or short-chain fatty acid producing bacteria in the gut microbiota of mice intervened by Clostridium butyricum,among which the dominant bacteria genus in Clostridium butyricum+DSS group and antibiotic+Colstridium butyicum+DSS group were Mucispirillum(linear discriminant analysis(LDA)=3.667 log10,P=0.004)and Stenotrophomonas(LDA=2.778 log10,P=0.044).In the antibiotic+Clostridium butyricum+DSS group,the dominant bacteria genus were Peptococcus(LDA=2.685 log10,P=0.018),Butyricimonas(LDA=2.712 log10,P=0.011),Bilophila(LDA=3.204 log10,P=0.014),Intestinimonas(LDA=3.346 log10,P=0.010),Candidatus-Saccharimonas(LDA=3.363 log10,P=0.029),Desulfovibrio(LDA=3.402 log10,P=0.025),Oscillibacter(LDA=2.870 log10,P=0.019)and Akkermansia(LDA=4.031 log10,P=0.005).Conclusions Clostridium butyricum can effectively improve colitis in mice and regulate the intestinal microbial structure of mice,whlie antibiotic pretreatment can strengthen its regulation of intestinal microbiota to and enhance the efficacy of Clostridium butyricum.