基于miR-92a-3p調(diào)控緊密連接探討24-乙酰澤瀉醇A改善腦微血管內(nèi)皮細(xì)胞OGD-R損傷摘要：本文旨在探討miR-92a-3p在腦微血管內(nèi)皮細(xì)胞缺血再灌注損傷（OGD/R）過程中的作用以及24-乙酰澤瀉醇A（24-epiAA）對其的影響。通過原代培養(yǎng)的腦微血管內(nèi)皮細(xì)胞模型，我們發(fā)現(xiàn)miR-92a-3p在OGD/R過程中明顯上調(diào)。而24-epiAA可以降低miR-92a-3p的表達(dá)，同時(shí)也能夠減輕OGD/R引起的細(xì)胞凋亡和線粒體損傷。我們還發(fā)現(xiàn)，miR-92a-3p的上調(diào)可以通過抑制緊密連接蛋白CLDN5的表達(dá)來導(dǎo)致腦微血管內(nèi)皮細(xì)胞通透性的增加。而24-epiAA的作用可以通過維持CLDN5的表達(dá)水平來維持腦微血管內(nèi)皮細(xì)胞的屏障功能。綜上所述，這些結(jié)果表明了miR-92a-3p和CLDN5在腦微血管內(nèi)皮細(xì)胞OGD/R損傷過程中的關(guān)鍵作用，同時(shí)也證明了24-epiAA作為潛在的神經(jīng)保護(hù)劑的作用。

關(guān)鍵詞：miR-92a-3p、24-epiAA、OGD/R、緊密連接蛋白、腦微血管內(nèi)皮細(xì)胞、通透性、細(xì)胞凋亡、線粒體損傷、神經(jīng)保護(hù)劑。

Abstract:ThisarticleaimstoexploretheroleofmiR-92a-3pand24-epiAAintheprocessofischemia-reperfusioninjuryinbrainmicrovascularendothelialcells(BMECs).OurstudyrevealedthatmiR-92a-3pwassignificantlyup-regulatedinBMECssubjectedtooxygenandglucosedeprivation/reoxygenation(OGD/R)treatment.Incontrast,24-epiAAtreatmentnotonlydecreasedtheexpressionofmiR-92a-3p,butalsoalleviatedOGD/R-inducedcellapoptosisandmitochondrialdysfunction.Furthermore,wefoundthatmiR-92a-3pupregulationincreasedthepermeabilityofBMECsbyinhibitingtheexpressionoftightjunctionproteinCLDN5,while24-epiAAtreatmentmaintainedtheexpressionofCLDN5andthereforemaintainedthebarrierfunctionofBMECs.Takentogether,theseresultsdemonstratethecriticalroleofmiR-92a-3pandCLDN5intheprocessofOGD/RinjuryinBMECswhilealsoindicatingthepotentialof24-epiAAasaneuroprotectiveagent.

Keywords:miR-92a-3p,24-epiAA,OGD/R,tightjunctionprotein,brainmicrovascularendothelialcells,permeability,cellapoptosis,mitochondrialdysfunction,neuroprotectiveagentInadditiontomaintainingtheexpressionofCLDN5,4-epiAAtreatmentalsodecreasedcellapoptosisandmitochondrialdysfunctioninBMECssubjectedtoOGD/R.Thesefindingssuggestthat4-epiAAmayhavemultiplemechanismsofneuroprotectioninthecontextofischemicstroke.

PreviousstudieshaveshownthepotentialofmiR-92a-3pinhibitionasatherapeuticstrategyforstroke.Forexample,onestudyfoundthatdownregulatingmiR-92a-3pexpressioninstrokeratsimprovedneurologicalfunctionanddecreasedinfarctsize(Wangetal.,2019).Similarly,anotherstudydemonstratedthatsilencingmiR-92a-3pexpressionincerebralmicrovascularendothelialcellsdecreasedpermeabilityandprotectedagainstneuronalinjuryinvitro(Zengetal.,2021).OurstudyfurthersupportsthepotentialofmiR-92a-3pinhibitionasatherapeuticapproachbyspecificallytargetingthetightjunctionproteinCLDN5.

Inconclusion,ourstudyprovidesnovelinsightsintothemechanismsunderlyingOGD/RinjuryinBMECsandidentifiesmiR-92a-3panditstargetCLDN5ascriticalregulatorsoftheblood-brainbarrier.Additionally,ourfindingsdemonstratetheneuroprotectivepotentialof24-epiAAandsuggestthatthiscompoundmaybeapromisingtherapeuticagentforischemicstroke.Futurestudiesarewarrantedtofurtherexploretheefficacyandsafetyof24-epiAAtreatmentinanimalmodelsofstrokeandultimatelyinclinicaltrialsInconclusion,ischemicstrokeisadevastatingconditionthataffectsmillionsofpeopleworldwideeveryyear.Thedisruptionoftheblood-brainbarrierandsubsequentbrainedemaandneuronalcelldeatharemajorhallmarksofischemicstroke.Despiteextensiveresearch,treatmentoptionsforischemicstrokearestilllimited.

OurstudyprovidesnovelinsightsintothemechanismsunderlyingOGD/RinjuryinBMECsandidentifiesmiR-92a-3pandCLDN5ascriticalregulatorsoftheblood-brainbarrier.Wehavealsoshownthattreatmentwith24-epiAAcanattenuatethedamagecausedbyOGD/RbyincreasingtheexpressionofCLDN5andimprovingtheintegrityoftheblood-brainbarrier.

Thesignificanceofourfindingsliesinthepotentialforthedevelopmentofanoveltherapeuticoptionforischemicstroke.Futurestudieswillberequiredtofurtherexploretheefficacyandsafetyof24-epiAAtreatmentinanimalmodelsofstrokeandultimatelyinclinicaltrials.

Insummary,ourstudyhighlightsthecriticalroleofmiR-92a-3pandCLDN5inthepathogenesisofischemicstrokeandunderscoresthepotentialof24-epiAAasatherapeuticoptionforthisdevastatingcondition.Withthecontinuedsupportofresearchinthisarea,wemayfinallybeabletodevelopeffectivetreatmentsforischemicstrokethatcanimproveoutcomesformillionsofpeopleaffectedbythisconditionAlthoughourstudyprovidespromisingresults,therearestillmanystepsthatneedtobetakenbeforeourfindingscanbetranslatedintoaviabletreatmentoptionforischemicstroke.Oneofthemostpressingissuesistheneedforfurtheranimalstudiestoconfirmthesafetyandefficacyofthe24-epiAAtreatment.Itisimportanttodeterminetheoptimaldoseanddurationoftreatmenttoachievethebestresultswithoutcausingunwantedsideeffects.

Inaddition,ourstudyfocusedontheacutephaseofischemicstroke,whichoccurswithinhoursordaysafterthestrokeevent.However,theeffectsof24-epiAAmayvarydependingonthedurationandseverityoftheischemia,aswellastheindividualpatientfactors.Therefore,itisnecessarytoinvestigatethelong-termeffectsof24-epiAAonstrokerecoveryandneurologicalfunctioninanimalmodelsandinclinicaltrials.

Furthermore,itisimportanttoidentifybiomarkersorimagingtechniquesthatcanaccuratelymeasuretheextentofbraindamageandmonitorthetreatmentresponse.Thiscanhelptoselecttheappropriatepatientsforthetreatmentandassessthetherapeuticefficacyinclinicalsettings.

Finally,thedevelopmentofanewdrugrequiressignificantfinancialandregulatoryresources,andtheprocesscantakeseveralyearsorevendecades.Therefore,itiscrucialtosecurefundingandpartnershipswithpharmaceuticalcompanies,regulatoryagencies,andclinicalresearchorganizationstoacceleratethetranslationofourfindingsintoclinicalpractice.

Inconclusion,ourstudyprovidesnewinsightsintothemolecularmechanismsofischemicstrokeandidentifiesapotentialtherapeutictargetanddrugcandidate.However,furtherresearchisneededtoconfirmthesafetyandefficacyofthetreatmentandtoovercomethechallengesoftranslatingbasicresearchintoclinicalpractice.Withperseverance,collaboration,andinnovation,wecanachievethegoalof