前言
糖尿病通过不完全了解的机制加剧了心肌缺血/再灌注（MI/R）损伤。脂肪细胞功能障碍导致远端器官损伤。然而，将功能异常的脂肪细胞与增加的MI/R损伤联系起来的分子机制仍未确定。1月10日托马斯·杰斐逊大学急诊医医院团队在Circulation查看期刊详情上发表了“SmallExtracellularMicrovesiclesMediatedPathologicalCommunicationsbetweenDysfunctionalAdipocytesandCardiomyocytesasaNovelMechanismsExacerbatingIschemia/ReperfusionInjuryinDiabeticMice”，当前研究试图阐明小细胞外囊泡（sEV）是否以及如何介导糖尿病脂肪细胞与心肌细胞之间的病理学通讯，从而加剧MI/R损伤。在非糖尿病性心脏内心肌内注射糖尿病血清sEV显着加重了MI/R损伤，这可通过心功能恢复较差，梗塞面积更大和心肌细胞凋亡更大来证明。同样，心肌内或全身给予糖尿病脂肪细胞sEV或HG/HL挑战的非糖尿病脂肪细胞sEV会显着加重MI/R损伤。糖尿病附睾脂肪移植显着增加了非糖尿病小鼠的MI/R损伤，而sEV生物发生抑制剂的给药显着减轻了糖尿病小鼠的MI/R损伤。机理研究发现，miR-b-3p是糖尿病血清sEV，糖尿病脂肪细胞sEV和HG/HL挑战的非糖尿病脂肪细胞sEV中显着增加的常见分子。在接受糖尿病sEV注射治疗的糖尿病和非糖尿病心脏中，成熟（而非原发性）miR-b-3p显着增加。心肌内注射miR-b-3p模拟物可显着加重非糖尿病小鼠的MI/R损伤，而miR-b-3p抑制剂可显着减轻糖尿病小鼠的MI/R损伤。分子研究确定AMPKα1/α2，Birc6和Ucp3是miR-b-3p的直接下游靶标。这些分子的过度表达逆转了miR-b-3p诱导的促凋亡/心脏有害作用。最后，来自2型糖尿病患者的血浆sEV中miR-b-3p水平显着增加。糖尿病患者sEV孵育心肌细胞会严重加剧缺血性损伤，miR-b-3p抑制剂可阻断这种作用。文章首次证明miR-b-3p在功能异常的脂肪细胞衍生的sEV中的富集及其对心肌细胞中多种抗凋亡/心脏保护分子的抑制，是加剧糖尿病心脏MI/R损伤的新机制。靶向功能障碍的脂肪细胞和心肌细胞之间的miR-b-3p介导的病理学交流可能是减轻MI/R损伤的糖尿病恶化的新策略。
研究思路
Figure1.DeleteriouseffectsofadipocytesEVderivedfromobese/diabeticmiceinMI/Rinjury.48hoursaftersEVintramyocardialinjection,miceweresubjectedtoMI/R.(A,D,G)24hoursafterreperfusion,cardiacfunctionwasevaluatedbyhemodynamictesting.(B,E,H)CardiacinjurywasidentifiedbymyocardialEvansblue/TTCdoublestain.(C,F,I)3hoursafterreperfusion,cardiomyocyteapoptosiswasdeterminedbyTUNELassay.
Figure2.ThecontentshiftofHFDadipocytesEVwasresponsibleforMI/Rinjuryexacerbation.48hoursaftertantamountsEVintramyocardialinjection,miceweresubjectedtoMI/R.24hoursafterreperfusion.(A)cardiacfunctionwasevaluatedbyhemodynamictesting.(B)CardiacinjurywasidentifiedbymyocardialEvansblue/TTCdoublestain.(C,D)3hourafterreperfusion,cardiomyocyteapoptosiswasdeterminedby(C)TUNELassayand(D)cleavedcaspase-3assaybyWesternblot.(E)InvitroadipocytesEVuptakenanalysis.PKH67-labledorPKH26-labledadipocytesEVwereharvestedfromprimaryadipocyteculturemedium,andsubsequentlyincubatedwithH9c2cells,humancardiomyocytes,andadultmousecardiomyocytesfor6hours.(F)Neonatalratventricularmyocyte(NRVM)cellviabilitywasevaluatedbyMTTassayinvitrowithadipocytesEVplussimulatedischemia/reoxygenation(SI/R)administration.(G)CelldeathwasdeterminedbyLDHrelease.(H)Cellapoptosiswasdeterminedbycleavedcaspase-3assaybyWesternblot.
Figure3.DeleteriouseffectsofdysfunctionaladiposetissueandsystemicsEVfromHFDfedmiceinMI/R-injury.(A-D)Theepididymaladiposetissues(eWAT)wereisolatedfromHFDmiceorNDdonormice,andtransplantedinto8-week-oldmalenon-diabeticrecipientmiceremovedepidydimalfatdepots.7daysaftereWATtransplantation,MI/Rwasperformed.(E-H)TheHFD-fedmicewereintraperitonealinjectedGW(2mg/kg)orvehicle3timesaweekfor12weeks.Then,MI/Rwasperformed.(A,E)Cardiacfunctionwasevaluatedbyhemodynamictesting.(B,F)CardiacinjurywasidentifiedbymyocardialEvansblue/TTCdoublestain.Cardiomyocyteapoptosiswasdeterminedby(C,G)TUNELassayand(D,H)cleavedcaspase-3assaybyWesternblot.
Figure4.IncreasedmiR-b-3ppresentinHFDadipocytesEV.(A,B)DifferentiallyexpressedmiRNAswereselectedfromsequencingdataofserumsEVmiRNAs,shownin(A)and(B).(C)VennanalysisselectedthreecandidatemiRNAssatisfyingfourconditions.(D,E)TherelativeexpressionofthreematuremiRNAsinequalnumbersofserumsEVandeWATadipocytesEV.(F)TherelativeexpressionofthreematuremiRNAs,inadipocytesEVderivedfromthesamenumberofadipocytes.(G,H)Therelativeexpressionofpri-miR-bandmaturemiR-b-3pineWATfromND-fedandHFDfedmice(12weeks),analyzedbyreal-timePCR.(I,J)Therelativeexpressionofpri-miR-bandmaturemiR-b-3pineWATadipocytesfromND-fedandHFD-fedmice,orNDadipocytestreatedwithHG/HL,analyzedbyreal-timePCR.(K,L)Therelativeexpressionofpri-miR-bandmaturemiR-b-3pinhearttissuefromND-fedandHFD-fedmice,analyzedbyreal-timePCR.(M)TherelativeexpressionofmiR-b-3pincardiomyocytesandadipocytes.(N,O)Therelativeexpressionofpri-miR-bandmaturemiR-b-3pincardiactissue+/-adipocytesEVintramyocardialinjection,analyzedbyreal-timePCR.
Figure5.DeleteriouseffectsofmiR-b-3puponMI/R-injuryinobese/diabeticmiceandinculturedcells.(A-D)miR-b-3pincreasedMI/R-injuryinnon-diabeticmice.48hoursaftermiR-b-3pmimicandNCmimictransfectionofthemyocardiumofnon-diabeticmice,MI/R