基于１６ＳｒＤＮＡ高通量测序研究针灸对克罗恩病大鼠肠道菌群的调节作用
钟蕊１ 林亚莹２ 朱毅２ 吴焕淦２，３ 孙可鑫３ *艳２ 祁琴２ 吴璐一２，３
（１上海中医药大学上海市气功研究所，上海，２０００３０；２上海市针灸经络研究所，上海，２０００３０；３上海中医药大学附医院，上海，２００４３７）
目的：采用16SrDNA高通量测序技术分析针灸对克罗恩病（CD）大鼠肠道菌群物种相对丰度和多样性的影响，探讨针灸对克罗恩病的作用机制。方法：将40只SD大鼠随机分为正常组、模型组、隔药饼灸组、电针组和西药组，每组8只。采用2,4,6-三硝基苯磺酸溶液灌肠4周构建CD大鼠模型，隔药饼灸组和电针组选取天枢（双侧）、气海穴进行干预，西药组采用柳氮磺吡啶溶液灌胃干预。干预结束后取结肠组织进行HE染色，收取大鼠粪便进行16SrDNA高通量测序并对进行物种相对丰度、alpha多样性和基因功能代谢预测分析等。结果：隔药饼灸组和电针组大鼠结肠组织病理学评分显著低于模型组（P＜0.01）。与正常组比较，模型组拟杆菌门、放线菌门、拟杆菌纲、拟杆菌目、紫单胞菌科、普雷沃菌科、拟杆菌科、普雷沃菌属等的相对丰度升高，厚壁菌门、变形菌门、梭菌纲、梭菌目、毛螺菌科、瘤胃菌科、乳酸杆菌属等的相对丰度降低；在经过隔药饼灸和电针治疗后，上述大多数微生物相对丰度均有不同程度改善。与正常组比较，模型组肠道菌群的Chao1ObservedspeciesShannonSimpson等alpha多样性指数均显著下降（P＜0.05）；与模型组比较，隔药饼灸组和电针组上述指数均显著升高（P＜0.05）。功能预测分析发现隔药饼灸和电针均能调节参与碳水化合物代谢、氨基酸代谢等的功能基因。结论：CD与肠道菌群结构变化有关，针灸能够调节CD大鼠肠道菌群物种相对丰度和菌群多样性。
克罗恩病；针灸；肠道菌群；高通量测序；功能预测
RegulatoryEffectofAcupunctureandMoxibustiononGutMicrobiotaofRatswithCrohn′sDiseaseBasedon16SrDNAHigh-throughputSequencing
ZHONGRui1,LINYaying2,ZHUYi2,WUHuangan2,3,SUNKexin3,HUANGYan2,QIQin2,WULuyi2,3
(1ShanghaiQigongResearchInstitute,ShanghaiUniversityofTraditionalChineseMedicine,Shanghai,China;2ShanghaiResearchInstituteofAcupunctureandMeridian,Shanghai,China;3YueyangHospitalofIntegratedTraditionalChineseandWesternMedicine,ShanghaiUniversityofTraditionalChineseMedicine,Shanghai,China)
Abstract
Objective:ToanalyzetheeffectsofacupunctureandmoxibustionontherelativeabundanceanddiversityofgutmicrobiotainratswithCrohn′sdisease(CD)by16SrDNAhigh-throughputsequencingandexplorethemechanismofacupunctureandmoxibustiononCD.Methods:FortySDratswererandomlydividedintoanormalgroup,amodelgroup,anindirectmoxibustiongroup,anelectroacupuncture(EA)group,andawesternmedicinegroup,witheightratsineachgroup.TheCDmodelwasinducedinratsbyenemawith2,4,6-trinitrobenzenesulfonicacidforfourweeks.Thebilateral“Tianshu”(ST25)and“Qihai”(CV6)wereselectedforinterventionintheindirectmoxibustiongroupandtheEAgroup,andtheratsinthewesternmedicinegroupreceivedsulfasalazinesolutionbygavage.Aftertheintervention,thecolontissuesandratfeceswerecollectedforHEstainingand16SrDNAhigh-throughputsequencing,respectively.Thesequencingresultswereanalyzedforspeciesrelativeabundance,alphadiversity,andfunctionalgeneprediction.Results:ThehistopathologicalscoresofcolontissuesofratsintheindirectmoxibustiongroupandtheEAgroupweresignificantlylowerthanthoseinthemodelgroup(P0.01).Comparedwiththenormalgroup,themodelgroupshowedtheincreasedrelativeabundanceofBacteroidetes,Actinobacteria,Bacteroidia,Bacteroidales,Porphyromonadaceae,Prevotellaceae,Bacteroidaceae,andPrevotella,andthedecreasedrelativeabundanceofFirmicutes,Proteobacteria,Clostrida,Clostridiales,Lachnospiraceae,Ruminococcaceae,andLactobacillus.AfterindirectmoxibustionandEAtreatment,therelativeabundanceofthemajorityoftheabove-mentionedmicroorganismsimprovedtovaryingdegrees.Comparedwiththenormalgroup,themodelgroupdisplayeddecreasedalphadiversityindexesofthegutmicrobiota,suchasChao1,Observedspecies,Shannon,andSimpson(P0.05).ThoseindexeswereelevatedintheindirectmoxibustiongroupandtheEAgroup(P0.05)as