丁酸梭菌对慢性肾衰竭犬肠道菌群及血清小分子尿毒素含量的影响

本试验旨在研究丁酸梭菌对慢性肾衰竭(CRF)犬肠道菌群及血清小分子尿毒素含量的影响。选取20条2岁左右的雄性贵宾犬,随机分成4组:假手术对照组(CL组)、模型组(MD组)、低剂量丁酸梭菌组(LCB组)和高剂量丁酸梭菌组(HCB组),每组5只。CL组只做腹腔打开手术,其余3组肾动脉结扎制作CRF模型。造模成功后,LCB组和HCB组分别按每天0.5、1.0 g/kg BW的剂量灌喂丁酸梭菌,进行为期4周的干预。检测血清和粪便尿素氮(UN)、肌酐(Cr)和硫酸吲哚酚(IS)含量,并对肠道菌群16S rDNA进行高通量测序。结果表明:1)试验结束时(第4周),LCB组和HCB组的血清UN、Cr和IS含量极显著低于MD组(P<0.01),但极显著高于CL组(P<0.01);与LCB组比较,HCB组的血清UN、IS含量显著降低(P<0.05),血清Cr含量极显著降低(P<0.01)。试验结束时,HCB组的粪便UN、Cr含量均显著低于MD组(P<0.05),CL组的粪便UN、Cr含量均显著低于MD组、LCB组和HCB组(P<0.05)。2)饲喂丁酸梭菌均降低CRF犬肠道菌群的α多样性,HCB组的Chao1指数、Shannon指数显著低于MD组(P<0.05)。主坐标分析(PCoA)结果显示4组样本间发生明显聚类。3)在门水平上,MD组的肠道放线菌门(Actinobacteria)和变形菌门(Proteobacteria)的相对丰度显著高于CL组(P<0.05),LCB组和HCB组的肠道Actinobacteria、Proteobacteria的相对丰度均显著低于MD组(P<0.05);在属水平上,饲喂丁酸梭菌可降低CRF犬肠道变形杆菌属(Proteus)、埃希氏菌属(Esche-richia)、肠杆菌属(Enterobacter)、普氏菌属(Prevotella)、放线菌属(Actinomyces)和涅斯捷连科氏菌属(Nesterenkonia)的相对丰度。LEfSe分析发现,CRF犬肠道菌群有19个具有差异的生物标志物,主要集中于Actinobacteria、Proteobacteria,饲喂丁酸梭菌可减少CRF犬生物标志物的差异。4)Spearman相关性分析显示,血清UN、Cr和IS含量与Enterobacter、Proteus和Escherichia相对丰度存在较强的正相关关系,与乳杆菌属(Lactobacillus)、双歧杆菌属(Bifidobacterium)相对丰度存在较强的负相关关系。由此可见,CRF犬肠道菌群紊乱与血清小分子尿毒素含量之间存在相关性,丁酸梭菌可通过调节CRF犬肠道菌群结构,促进血清小分子尿毒素的清除,以每天1.0 g/kg BW添加量为宜。

Effects of Clostridium butyricum on Intestinal Flora and Serum Micromolecule Urotoxin Contents of Dogs with Chronic Renal Failure

This experiment was conducted to study the effects of Clostridium butyricum on intestinal flora and serum micromolecule urotoxin contents of dogs with chronic renal failure(CRF).Twenty 2-year-old male poodles were randomly divided into four groups:sham operation control group(CL group),model group(MD group),low dose Clostridium butyricum group(LCB group)and high dose Clostridium butyricum group(HCB group),each group contained 5 dogs.Dogs in CL group were operated to only open abdominal cavity,while dogs in other 3 groups were established CRF model by ligation of renal artery.After successful modeling,LCB group and HCB group were intervened with 0.5 and 1.0 g/kg BW Clostridium butyricum per day for 4 weeks,respectively.The contents of urea nitrogen(UN),creatinine(Cr)and indolephenol sulfate(IS)in serum and feces were detected,and the intestinal flora 16S rDNA was detected by high throughput sequencing.The results showed as follows:1)at the end of the experiment(week 4),the contents of UN,Cr and IS in serum of LCB group and HCB group were significantly lower than those of MD group(P<0.01),but significantly higher than CL group(P<0.01);compared with the LCB group,the contents of UN and IS in serum of HCB group were significantly decreased(P<0.05),and the serum Cr content was significantly decreased(P<0.01).At the end of the experiment,the contents of UN and Cr in feces of HCB group were significantly lower than those of MD group(P<0.05),the contents of UN and Cr in feces of CL group were significantly lower than those of MD group,LCB group and HCB group(P<0.05).2)Feeding Clostridium butyricum could decreased the α diversity of intestinal flora of dogs with CRF,the Chao1 index and Shannon index of HCB group were significantly lower than those of MD group(P<0.05).The Principal coordinate analysis(PCoA)results showed that samples of 4 groups had obvious clustering.3)At the phylum level,the relative abundances of Actinobacteria and Proteobacteria in intestine of MD group were significantly higher than those of CL group(P<0.05),and the relative abundances of Actinobacteria and Proteobacteria in intestine of LCB group and HCB group were significantly lower than those of MD group(P<0.05);at the genus level,deeding Clostridium butyricum could decreased the relative abundances of Proteus,Escherichia,Enterobacter,Prevotella,Actinomyces and Nesterenkonia in intestine of dogs with CRF.LEfSe analysis showed that there were 19 difference biomarkers in intestinal flora of dogs with CRF,were mainly concentrated in Proteobacteria and Actinobacteria,and feeding Clostridium butyricum could decreased the difference of biomarkers of dogs with CRF.4)Spearman rank correlation analysis showed that the contents of UN,Cr and IS were strongly positively correlated with the relative abundances of Enterobacter,Proteus and Escherichia,and were strongly negatively correlated with the relative abundances of Lactobacillus and Bifidobacterium.In summary,intestinal flora disorder is correlated with serum micromolecule urotoxin contents,and Clostridium butyricum can promote the clearance of micromolecule urotoxin in serum by regulating the intestinal flora structure of dogs with CRF,and the appropriate dosage is 1.0 g/kg BW per day.