针灸对克罗恩病大鼠低氧环境下结肠DNA甲基转移酶的影响
祁 琴1 王晓梅1 吴焕淦1,2 包春辉1 马晓秡1,2 赵 琛3
朱 毅1 刘慧荣1,2 施 茵1 * 艳1 刘雅楠1
(1上海市针灸经络研究所,上海,200030;2上海中医药大学附医院,上海,200437;3上海中医药大学针灸推拿学院,上海,201203)
目的:观察针灸对克罗恩病(CD)大鼠结肠组织DNA甲基转移酶的影响,初步探讨针灸对CD的表观遗传调控机制。方法:将SD大鼠随机分为空白组、模型组、隔药饼灸组、电针组和柳氮磺吡啶组。采用5%2,4,6-三硝基苯磺酸灌肠的方法制备CD大鼠模型。隔药饼灸组和电针组均采用天枢(双侧)和气海穴进行干预,柳氮磺吡啶组采用柳氮磺吡啶肠溶片进行灌胃干预。治疗结束后,采用ELISA检测血清中C反应蛋白(CRP)、IL-6、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)的含量;实时荧光定量PCR(RT-qPCR)检测结肠组织中缺氧诱导因子-1α(HIF-1α)的mRNA表达;WesternBlotting检测结肠组织中DNMT1、DNMT3a、DNMT3b蛋白的表达。结果:与正常组比较,模型组血清中CRP、IL-6、IL-1β、TNF-α的蛋白含量均显著升高(P<0.01);与模型组比较,隔药饼灸组、电针组和柳氮磺吡啶组上述炎症介质的蛋白含量均显著降低(P<0.01)。与正常组比较,模型组结肠组织中HIF-1αmRNA和DNMT1、DNMT3a、DNMT3b蛋白表达均显著升高(P<0.01);与模型组比较,隔药饼灸组和柳氮磺吡啶组HIF-1αmRNA和DNMT1、DNMT3a、DNMT3b蛋白表达均显著降低(P<0.05或P<0.01),电针组HIF-1αmRNA和DNMT1蛋白表达均显著降低(P<0.05或P<0.01)。结论:隔药饼灸和电针均能抑制CD大鼠CRP、IL-6、IL-1β、TNF-α等炎症介质以及HIF-1α、DNA甲基转移酶DNMT1的表达,其中隔药饼灸还能抑制DNMT3a、DNMT3b表达,可能是针灸减轻CD肠道炎症的机制之一。
克罗恩病;针灸;DNA甲基转移酶;表观调控;缺氧诱导因子
cupunctureandMoxibustiononColonicDNAMethyltransferaseUnder
HypoxicEnvironmentinRatswithCrohn′sDisease
QIQin1,WANGXiaomei1,WUHuangan1,2,BAOChunhui1,MAXiaopeng1,2,ZHAOChen3,ZHUYi1,LIUHuirong1,2,SHIYin1,HUANGYan1,LIUYanan1
(1ShanghaiResearchInstituteofAcupunctureandMeridian,Shanghai,China;2YueyangHospitalofIntegratedTraditionalChineseandWesternMedicine,ShanghaiUniversityofTraditionalChineseMedicine,Shanghai,China;3SchoolofAcupuncture-MoxibustionandTuina,ShanghaiUniversityofTraditionalChineseMedicine,Shanghai,China)
Abstract
Objective:ToobservetheeffectsofacupunctureandmoxibustiononDNAmethyltransferaseinthecolontissuesofratswithCrohn′sdisease(CD)andexplorethemechanismofacupunctureandmoxibustionintheepigeneticregulationofCD.Methods:SDratswererandomlydividedintoanormalgroup,amodelgroup,anindirectmoxibustiongroup,anelectroacupuncture(EA)group,andasulfasalazinegroup.TheCDmodelwasinducedbyenemawith5%2,4,6-trinitrobenzenesulfonicacidinrats.TheratsintheindirectmoxibustiongroupandtheEAgroupweretreatedcorrespondinglyatbilateral“Tianshu”(ST25)and“Qihai”(CV6),andthoseinthesulfasalazinegroupreceivedsulfasalazineenteric-coatedtabletsbygavage.ELISAwasusedtodetecttheproteincontentofCRP,IL-6,IL-1β,andTNF-αintheserum.RT-qPCRwasusedtodetecttheexpressionofHIF-1αmRNAinthecolontissues.WesternBlottingwasusedtodetecttheproteinexpressionofDNMT1,DNMT3a,andDNMT3binthecolontissues.Results:Comparedwiththenormalgroup,themodelgroupshowedincreasedproteincontentofCRP,IL-6,IL-1β,andTNF-αintheserum(P0.01).Comparedwiththemodelgroup,theindirectmoxibustiongroup,theEAgroup,andthesulfasalazinegroupshoweddecreasedproteincontentoftheabove-mentionedinflammatoryfactors(P0.01).Comparedwiththenormalgroup,theindirectmoxibustiongroupandthesulfasalazinegroupexhibiteddecreasedexpressionofHIF-1αmRNAandDNMT1,DNMT3a,andDNMT3bproteins(P0.05orP0.01),andtheEAgroupshowedreducedexpressionofHIF-1αmRNAandDNMT1protein(P0.05orP0.01).Conclusion:TheindirectmoxibustionandEAcanbothinhibittheexpressionofinflammatoryfactorssuchasCRP,IL-6,IL-1β,TNF-α,aswellasHIF-1αandDNMT1inCDrats,andtheindirectmoxibustioncanalsoinhibittheexpressionofDNMT3aandDNMT3b,whichmaybeoneofthemechanismsofacupunctureandmoxibustioninreducingintestinalinflammationinCD.
Keywords
Crohn′sdisease;Acupunctureandmoxibustion;DNAmethyltransferase;Epigeneticregulation;Hypoxia-induciblefactor