低氧训练对大鼠小肠微生物的影响

Effects of Hypoxic Training on Intestinal Microorganisms of Rats

目的:研究低氧应激及低氧和运动复合应激对大鼠肠道微生物的影响。方法:雄性8周龄SD大鼠120只,适应性饲养1周后,用分层随机抽样法按体重随机分为常氧安静组(normal oxygen sedentary,NOS组)、常氧运动组(normal oxygen exercise,NOE组)、低氧安静组(low oxygen sedentary,LOS组)、低氧运动组(low oxygen exercise,LOE组),每组30只。递增强度适应性训练1周后开始正式干预,低氧组大鼠入驻常压低氧动物房(氧浓度12.7%,对应4000 m海拔)进行低氧干预,运动组大鼠进行每天24 m/min、45 min的中等强度跑台运动干预。分别在开始干预后第3、6、9天的末次训练后24 h取材,收集大鼠空肠内容物,提取全基因组DNA,用NanoDrop2000进行DNA质控,质控合格样品用Illumina HiSeq 2500型测序仪对16SrRNA的V3-V4可变区进行测序,得到结果用生物信息学手段进行微生物多样性分析。结果:(1)测序总共得到2697529条16SrRNA序列(Reads),双端Reads拼接、过滤后共产生2206002条高质量测序标签(Clean tags),平均每个样品产生73533条Clean tags。(2)在门分类学水平上,优势物种分别是,厚壁菌门、变形菌门、放线菌门;同时,属水平上具有较高丰度的物种主要集中于厚壁菌门、放线菌门及变形菌门中。厚壁菌门中,属水平上丰度最高的是乳酸菌属,变形菌门中Pannonibacter菌属、假单细胞菌属丰度较高;放线菌门中,节细菌属相对丰度较高。(3)干预后3天时,NOS组厚壁菌门相对丰度显著低于LOS组(P<0.05),NOS组假单胞菌属相对丰度显著高于LOE组(P<0.01);干预后第6天时,NOS组厚壁菌门相对丰度仍显著低于LOS组(P<0.05),NOS组变形菌门及放线菌门相对丰富显著低于LOE组(P<0.05,P<0.01),NOS组节杆菌属相对丰度显著低于LOE组(P<0.05);干预后9天时,NOS组假单胞菌属相对丰度显著高于LOS组(P<0.05)。结论:低氧或低氧训练作为唯一变量,导致大鼠肠道微生物构成发生显著变化。低氧干预导致大鼠肠道微生物中厚壁菌门相对丰度显著升高,低氧训练导致大鼠肠道微生物中放线菌门、变形菌门相对丰度显著下降;这可能导致肠道微生物失调,宿主肠黏膜屏障功能受损。

Objective To investigate the microbial changes in rats’small intestine during hypoxia exposure and hypoxic training.Methods One hundred and twenty male Sprague-Dawley rats aged 8 weeks were given adaptive breeding for one week.Then they were randomly divided into a normal-oxygen stationary(NOS)group,a normal-oxygen exercise(NOE)group,a hypoxia stationary(HS)group,and a hypoxia exercise(HE)group,each of 30,using the tratified random sampling method.Rats in the hypoxia group were housed in a normal-pressure hypoxic animal room(oxygen concentration was12.7%,corresponding to that at an altitude of 4000 m)and subjected to the hypoxic intervention.The exercise groupwas subjected to a moderate intensity exercise model of 40 minutes at 24 m/min.The materials were collected 24 h after the last training on the 3 rd,6 th,and 9 th days,and the contents of the jejunum were collected and the whole genome DNA was extracted.The DNA quality control was performed using NanoDrop2000.Then samples were sequenced on an Illumina HiSeq 2500 Machine,and given the microbial diversity analysis using bioinformatics methods.Results(1)A total of 2,697,529 16 S rRNA reads were obtained after the sequencing.After the two-end reads were spliced and filtered,a total of 2,220,002 clean tags were generated.On average,73,033 clean tags were generated for each sample.(2)The dominant phylum were the Firmicutes,Proteobacteria,and Actinomycetes;at the same time,species with higher abundance at the level were mainly concentrated on Firmicutes,Proteobacteria,and Actinomycetes.In the Firmicutes,the Lactobacillus was the most abundant at the level,the Pannobacteria and Pseudomonas were the most abundant in the Proteobacteria,and the Arthrobacter Conn and Dimmick was the most abundance in the Actinomycetes.(3)On the3 rd day after the intervention,the relative abundance of Firmicutes of the NOS group was significantly lower than the LOS group(P<0.05),the relative abundance of pseudomonas in the NOS group was significantly higher than the LOE group(P<0.01).On the 6 th day after the intervention,the relative abundance of firmicutes of the NOS group was still significantly lower than the LOS group(P<0.05),while that of proteobacteria and actinomycetes in the NOS group were significantly lower than the LOE group(P<0.01).On the 9 th day after the intervention,the relative abundance of pseudomonas of the NOS group was significantly higher than the LOS group(P<0.05).Conclusion Hypoxia or hypoxia exercise as the unique variable,causes a significant change in the composition of the gut microbiota in rats.The hypoxia training results in a significant decrease in the relative abundance of actinomycetes and proteobacteria in the intestinal microbes of rats,which may lead to microbial disorders in the intestinal tract,and impair host intestinal mucosal barrier function.