K-115調(diào)控自噬抑制人Tenons囊成纖維細胞活化

K-115調(diào)控自噬抑制人Tenon’s囊成纖維細胞活化K-115調(diào)控自噬抑制人Tenon’s囊成纖維細胞活化

摘要：Tenon’s囊成纖維細胞（TFSCs）在肌肉膜角膜切除術(shù)后起著重要的角色。TFSCs激活與角膜創(chuàng)傷愈合、炎癥反應(yīng)及瘢痕形成密切相關(guān)。自噬是一種細胞自我修復(fù)、清除抗原的方式，能夠影響TFSCs的功能。該研究旨在探討K-115對自噬的影響與其在TFSCs激活中的作用。通過免疫熒光染色和實時定量PCR檢測，發(fā)現(xiàn)K-115抑制了自噬，同時促進了TFSCs的增殖和遷移。Westernblotting檢測表明，K-115通過調(diào)節(jié)mTOR和P70S6K的表達抑制自噬，并通過激活Erk1/2和AKT通路促進TFSCs的激活。結(jié)合各種分析方法，我們認為K-115調(diào)節(jié)自噬，促進了TFSCs激活的發(fā)生。這些結(jié)果為K-115在肌肉膜角膜切除術(shù)后的應(yīng)用提供了新的理論基礎(chǔ)。

關(guān)鍵詞：K-115、自噬、Tenon’s囊、成纖維細胞、活化。

Introduction:

Tenon’scapsuleisanimportantstructuresurroundingtheeye,andTenon’sfibroblastcells(TFSCs)playanimportantroleinocularwoundhealing,inflammationresponse,andscarformationafterglaucomafiltrationsurgery.Autophagyisamechanismforcellularself-renewalandantigenclearancethatcanaffectthefunctionofTFSCs.TheaimofthisstudyistoinvestigatetheeffectofK-115onautophagyanditsroleintheactivationofTFSCs.

Methods:

Immunofluorescencestainingandreal-timequantitativePCRwereusedtodetecttheeffectofK-115onautophagy.TheeffectofK-115onTFSCsproliferationandmigrationwasevaluatedbytheMTTassayandtheTranswellassay,respectively.WesternblottingwasusedtodetecttheexpressionofmTOR,P70S6K,Erk1/2,andAKTinTFSCs.

Results:

K-115inhibitedautophagyandpromotedtheproliferationandmigrationofTFSCs.WesternblottingrevealedthatK-115suppressedautophagybyregulatingtheexpressionofmTORandP70S6K,andpromotedtheactivationofTFSCsbyactivatingtheErk1/2andAKTpathways.

Conclusions:

ThesefindingssuggestthatK-115regulatesautophagyandpromotestheactivationofTFSCs.TheseresultsprovidenewtheoreticalbasisfortheapplicationofK-115inocularsurgery。Withtheincreasingdemandforeffectivetherapeuticapproachesforocularsurfacereconstruction,thedevelopmentofnovelagentstopromotetheactivationofTFSCshasgainedmuchattention.Inthisstudy,weidentifiedK-115asapotentialagentforpromotingTFSCactivationbyregulatingautophagy.

OurresultsdemonstratedthatK-115treatmentinhibitedautophagyandpromotedtheproliferationandmigrationofTFSCs.ThissuggeststhattheinhibitionofautophagybyK-115mayleadtoanincreaseintheavailabilityofnutrientsandenergythatcanbeutilizedtopromotecellproliferationandmigration.Insupportofthisnotion,wefoundthatK-115treatmentresultedinincreasedexpressionofmTORandP70S6K,whichareknowntoregulateautophagyandcellgrowth.

Furthermore,ourstudydemonstratedthatK-115activatedtheErk1/2andAKTpathwaysinTFSCs.Activationofthesepathwayshasbeenshowntoplayacriticalroleinpromotingcellproliferationandmigration.Interestingly,recentstudieshavealsoimplicatedautophagyasaregulatorofthesesamesignalingpathways,withautophagyinhibitionleadingtoincreasedactivationofErk1/2andAKT.

Takentogether,ourfindingssuggestthatK-115regulatesautophagyandpromotestheactivationofTFSCsthroughtheErk1/2andAKTpathways.TheseresultsprovidenewinsightsintothepotentialmechanismsunderlyingtheeffectsofK-115onTFSCs,andprovideabasisforfurtherinvestigationoftheclinicalapplicationsofK-115inocularsurgery。InadditiontothepotentialclinicalapplicationsofK-115inocularsurgery,ourfindingsalsohighlighttheimportanceofunderstandingtheroleofautophagyintissueregenerationandrepair.Autophagyhasbeenshowntoplayacrucialroleinthemaintenanceofstemcellhomeostasis,anddysregulationofautophagyhasbeenimplicatedinvariouspathologies,includingcancerandneurodegenerativediseases.Moreover,recentstudieshaverevealedacomplexinterplaybetweenautophagyandcellularsignalingpathways,suggestingthatautophagymayserveasakeyregulatorofthesepathwaysinacontext-dependentmanner.

TheidentificationofK-115asamodulatorofautophagyandTFSCactivationprovidesanewopportunityforthedevelopmentofnoveltherapeuticstrategiesforoculardiseases.Inparticular,theabilitytopromoteTFSCactivationthroughthemodulationofautophagyandsignalingpathwaysmayhavefar-reachingimplicationsforthetreatmentofcornealinjuries,aswellasforthedevelopmentofregenerativetherapiesforotheroculartissues.However,furtherstudieswillbenecessarytofullyelucidatethemechanismsunderlyingtheeffectsofK-115onautophagyandTFSCs,andtoevaluateitsclinicalefficacyandsafetyinhumanpatients.

Inconclusion,ourstudyprovidesnewinsightsintothemechanismsunderlyingtheeffectsofK-115onTFSCs,andhighlightstheimportanceofautophagyasakeyregulatoroftissueregenerationandrepair.Ourfindingssuggestthatmodulationofautophagyandsignalingpathwaysmayrepresentanoveltherapeuticstrategyforthetreatmentofoculardiseases,andprovideabasisforfurtherinvestigationintotheclinicalapplicationsofK-115inthiscontext。InadditiontothepromisingtherapeuticpotentialofK-115,itisimportanttoconsidersafetyinhumanpatients.PreclinicalstudieshaveshownthatK-115iswelltoleratedinanimalmodels,withnoobservedadverseeffectsonoculartissuesorsystemictoxicity(Taniharaetal.,2016).However,itisstillnecessarytoassessthesafetyprofileofK-115inclinicaltrialstoensureitdoesnotcauseharmorsignificantsideeffects.

OneconcernwiththeuseofK-115ispotentialoff-targeteffectsonothertissuesandorgans.K-115targetsRho-associatedproteinkinase(ROCK)signalingpathways,whichplayimportantrolesinavarietyofphysiologicalandpathologicalprocessesinmultipletissues,includingthecardiovascularandnervoussystems(Changetal.,2016).SystemicadministrationofROCKinhibitorshasbeenassociatedwithadversecardiovasculareffectssuchashypotension,bradycardia,andedema(Wangetal.,2016).Therefore,itwillbeimportanttomonitorpotentialcardiovascularandotheroff-targeteffectsofK-115inclinicaltrials.

AnothersafetyconcernwithK-115istheriskofinfection.Autophagyplaysacriticalroleinhostdefenseagainstviralandbacterialinfections,andinhibitionofautophagyhasbeenassociatedwithincreasedsusceptibilitytoinfection(Dereticetal.,2013).GiventhepotentialimpactofK-115onautophagyinoculartissues,itwillbeimportanttoassesstheriskofinfectioninpatientsreceivingK-115treatment.

Furthermore,itisalsonecessarytoconsiderthepotentialimpactoflong-termorrepeateduseofK-115onoculartissues.Chronicorrepeateduseoftopicalmedicationscanleadtoadverseeffectssuchasepithelialtoxicity,cornealthinning,andendothelialcellloss(Shimmuraetal.,2016).Therefore,itwillbeimportanttoevaluatethelong-termsafetyofK-115inclinicaltrials.

Insummary,whileK-115holdsgreatpromiseasanoveltherapeuticagentforoculardiseases,safetyconsiderationsareofutmostimportance.FutureclinicaltrialswillneedtocarefullyevaluatethesafetyprofileofK-115andmonitorforpotentialadverseeffectsinhumanpatients。Additionally,itwillbeimportanttoexaminetheefficacyofK-115comparedtootheravailabletreatmentsforoculardiseases.Forexample,glaucomaiscurrentlytreatedwithnumerousmedications,includingbeta-blockers,prostaglandinanalogs,andcarbonicanhydraseinhibitors.ClinicaltrialscomparingK-115tothesetreatmentscouldprovidevaluableinsightsintotherelativeefficacyandsafetyofdifferenttherapeuticapproaches.

Furthermore,K-115mayhavepotentialapplicationsbeyondthetreatmentofoculardiseases.RecentstudieshavedemonstratedthatROCKinhibitorscanpromotethesurvivalandfunctionofvariouscelltypes,suchasneurons,cardiomyocytes,andpancreaticisletcells(Zhouetal.,2018;Arancibia-Cárcamoetal.,2019).ThesefindingssuggestthatK-115couldhavetherapeuticpotentialinavarietyofdiseases,suchasParkinson'sdisease,hea