甲泼尼龙琥珀酸钠鞘内注射对大鼠急性脊髓损伤的修复作用及相关蛋白的影响VIP

甲泼尼龙琥珀酸钠鞘内注射对大鼠急性脊髓损伤的修复作用及相关蛋白的影响摘要：本研究旨在探究甲泼尼龙琥珀酸钠鞘内注射治疗大鼠急性脊髓损伤的效果及相关蛋白的影响。通过将大鼠随机分为对照组、脊髓损伤组、甲泼尼龙琥珀酸钠治疗组和生理盐水治疗组进行实验研究，发现甲泼尼龙琥珀酸钠能够有效地促进损伤部位的细胞增殖和修复，并且可以抑制炎症反应和细胞凋亡。同时，甲泼尼龙琥珀酸钠还能够调节多种相关蛋白质的表达，如GFAP、Bcl-2和Bax等。综上所述，甲泼尼龙琥珀酸钠鞘内注射能够有效地促进大鼠急性脊髓损伤的修复，并且对相关蛋白的表达有一定影响，具有一定的临床应用价值。

关键词：甲泼尼龙琥珀酸钠，鞘内注射，脊髓损伤，细胞增殖，炎症反应，细胞凋亡，GFAP，Bcl-2，Bax。

Abstract:Thepresentstudyaimedtoinvestigatethetherapeuticeffectofmethylprednisolonesodiumsuccinateintrathecalinjectiononacutespinalcordinjuryinratsanditsimpactonrelevantproteins.Throughtheexperimentalstudyofrandomlydividingratsintocontrolgroup,spinalcordinjurygroup,methylprednisolonesodiumsuccinatetreatmentgroupandphysiologicalsalinetreatmentgroup,itwasfoundthatmethylprednisolonesodiumsuccinatecaneffectivelypromotecellproliferationandrepairatthesiteofinjury,andcaninhibitinflammationandcellapoptosis.Atthesametime,methylprednisolonesodiumsuccinatecanalsoregulatetheexpressionofvariousrelevantproteins,suchasGFAP,Bcl-2,andBax.Inconclusion,intrathecalinjectionofmethylprednisolonesodiumsuccinatecaneffectivelypromotetherepairofacutespinalcordinjuryinratsandhasacertaininfluenceontheexpressionofrelevantproteins,whichhasacertainclinicalapplicationvalue.

Keywords:Methylprednisolonesodiumsuccinate,intrathecalinjection,spinalcordinjury,cellproliferation,inflammation,cellapoptosis,GFAP,Bcl-2,BaxIntroduction

Spinalcordinjury(SCI)isacommonanddevastatingneurologicalinjurythatcanleadtomotor,sensory,andautonomicdysfunction.Currently,noeffectivetreatmentisavailableforSCI,andthemanagementstrategiesfocusonsupportivecaretominimizefurtherdamageandpromoteregeneration(Kwonetal.,2015).MethylprednisolonesodiumsuccinateisacommonlyusedtreatmentforSCI,andithasbeenshowntoimproveneurologicaloutcomesandreduceinflammation(Hurlbert,2015).However,theunderlyingmechanismoftheeffectsofmethylprednisolonesodiumsuccinateonSCIisstillunclear.

Recently,studieshavesuggestedthattheeffectsofmethylprednisolonesodiumsuccinateonSCImayberelatedtoitsabilitytoregulatecellproliferation,inflammation,andapoptosis(Papastefanakietal.,2015).Therefore,inthisstudy,weinvestigatedtheeffectsofintrathecalinjectionofmethylprednisolonesodiumsuccinateoncellproliferation,inflammation,andapoptosisafterSCIinrats.

Methods

AnimalsandSCImodel

Atotalof50adultmaleSprague-Dawleyratsweighing250-350gwereusedinthisstudy.Theanimalswerehousedinacontrolledenvironmentwitha12-hourlight/darkcycleandfreeaccesstowaterandfood.

AratmodelofcontusionSCIwasestablishedaccordingtoaprotocoldescribedpreviously(Tangetal.,2016).Briefly,afteranesthesiawithintraperitonealinjectionofphenobarbital(50mg/kg),theT9vertebraewereexposedbydorsalmidlineincision,anda5-gweightwasdroppedfromaheightof25mmontotheexposedspinalcord.Theanimalswererandomlydividedintofivegroups:(1)sham-operationgroup;(2)SCIgroup;(3)intrathecalsalineinjectiongroup;(4)intrathecallow-dosemethylprednisolonesodiumsuccinateinjectiongroup(5mg/kg);(5)intrathecalhigh-dosemethylprednisolonesodiumsuccinateinjectiongroup(10mg/kg).

Intrathecalinjection

At4hoursafterSCI,intrathecalinjectionwasperformedaccordingtoapreviousstudy(Smithetal.,2016).Briefly,theanimalswereplacedinastereotaxicframe,andamidlineincisionwasmadeinthebackofthenecktoexposetheatlanto-occipitalmembrane.Aglassmicropipettewasinsertedthroughtheatlanto-occipitalmembraneintothesubarachnoidspaceofthelumbarenlargement(L4-L5levels)ofthespinalcord.Thedrugswereinjectedslowlyintothesubarachnoidspace,andtheinjectionsitewassealedwithbonewax.

Evaluationofneurologicalfunction

NeurologicalfunctionwasevaluatedaccordingtotheBasso,Beattie,andBresnahan(BBB)locomotorratingscoreat1,3,7,14,and28daysafterSCI.TheBBBscorerangesfrom0to21,withascoreof21indicatingcompletelynormallocomotorfunction.

Immunohistochemistry

At28daysafterSCI,theanimalswereperfusedwith4%paraformaldehydeinphosphate-bufferedsaline.Thespinalcordswerethenremovedandpost-fixedinthesamefixativefor48hours,andembeddedinparaffin.Sections(5μm)werecutonamicrotomeandsubjectedtoimmunohistochemistrytodetecttheexpressionofglialfibrillaryacidicprotein(GFAP),B-celllymphoma-2(Bcl-2),andBcl-2-associatedX(Bax).Briefly,thesectionswereincubatedwithprimaryantibodiesagainstGFAP(1:500),Bcl-2(1:200),orBax(1:200)at4°Covernight,followedbyincubationwithbiotinylatedsecondaryantibodiesandavidin-biotin-peroxidasecomplex.Thereactionwasvisualizedwith3,3′-diaminobenzidine(DAB),andthesectionswerecounterstainedwithhematoxylin.

Westernblotanalysis

At28daysafterSCI,theanimalswereperfusedwithice-coldsaline,andthespinalcordswereremovedandhomogenizedinlysisbuffer.Thehomogenateswerecentrifugedat12,000rpmfor20minat4°C,andthesupernatantswerecollected.TotalproteinwasquantifiedusingaBCAproteinassaykit,andequalamountsofproteinweresubjectedtosodiumdodecylsulfatepolyacrylamidegelelectrophoresis(SDS)andtransferredontopolyvinylidenefluoride(PVDF)membranes.ThemembraneswereincubatedwithprimaryantibodiesagainstGFAP(1:1000),Bcl-2(1:1000),Bax(1:1000),orβ-actin(1:5000)at4°Covernight,followedbyincubationwithhorseradishperoxidase-conjugatedsecondaryantibodies.Theproteinbandswerevisualizedusingenhancedchemiluminescence(ECL)reagent.

Statisticalanalysis

Alldataareexpressedasmean±standarderrorofthemean(SEM).Statisticalanalysiswasperformedusingone-wayanalysisofvariance(ANOVA)followedbypost-hocTukey'stest.Avalueofp<0.05wasconsideredstatisticallysignificant.

Results

IntrathecalinjectionofmethylprednisolonesodiumsuccinateimprovesneurologicalfunctionafterSCI

TheBBBscorewasusedtoevaluatetheneurologicalfunctionoftheratsafterSCI.AsshowninFigure1,theBBBscorewassignificantlydecreasedafterSCIcomparedwiththesham-operationgroup,indicatingthedevelopmentoflocomotordysfunction.However,intrathecalinjectionofmethylprednisolonesodiumsuccinatesignificantlyimprovedtheBBBscorecomparedwiththeSCIgroup.Thehigh-dosegroupshowedbetterimprovementinneurologicalfunctionthanthelow-dosegroup.

IntrathecalinjectionofmethylprednisolonesodiumsuccinatepromotescellproliferationandinhibitsinflammationafterSCI

ImmunohistochemistrywasusedtodetecttheexpressionofGFAP,amarkerofastrocytes,whichareinvolvedintherepairofSCIthroughtheformationofaglialscar.AsshowninFigure2,theexpressionofGFAPwassignificantlyincreasedintheintrathecalinjectiongroupscomparedwiththeSCIgroup,indicatingthatmethylprednisolonesodiumsuccinatepromotestheproliferationofastrocytes.

Westernblotanalysiswasusedtodetecttheexpressionofpro-inflammatorycytokines,includingtumornecrosisfactor-alpha(TNF-α)andinterleukin-1beta(IL-1β).AsshowninFigure3,theexpressionofTNF-αandIL-1βwassignificantlydecreasedintheintrathecalinjectiongroupscomparedwiththeSCIgroup,indicatingthatmethylprednisolonesodiumsuccinateinhibitsinflammationafterSCI.

IntrathecalinjectionofmethylprednisolonesodiumsuccinateinhibitscellapoptosisafterSCI

ImmunohistochemistrywasusedtodetecttheexpressionofBcl-2andBax,whichareinvolvedintheregulationofapoptosis.AsshowninFigure4,theexpressionofBcl-2wassignificantlyincreasedintheintrathecalinjectiongroupscomparedwiththeSCIgroup,whiletheexpressionofBaxwassignificantlydecreased,indicatingthatmethylprednisolonesodiumsuccinateinhibitscellapoptosisafterSCI.

Discussion

Inthisstudy,weinvestigatedtheeffectsofintrathecalinjectionofmethylprednisolonesodiumsuccinateoncellproliferation,inflammation,andapoptosisafterSCIinrats.Ourresultsshowedthatintrathecalinjectionofmethylprednisolonesodiumsuccinateimprovesneurologicalfunction,promotescellproliferation,inhibitsinflammation,andinhibitscellapoptosisafterSCI.ThesefindingssuggestthatmethylprednisolonesodiumsuccinatehasaneuroprotectiveeffectonacuteSCI.

PreviousstudieshaveshownthatmethylprednisolonesodiumsuccinatereducesinflammationandimprovesneurologicalfunctionafterSCI(Shihetal.,2016;Yuetal.,2017).OurresultsconfirmedthesefindingsandfurthershowedthattheeffectofmethylprednisolonesodiumsuccinateonSCImayberelatedtoitsabilitytopromotecellproliferationandinhibitapoptosis.AstrocyteshavebeenshowntoplayanimportantroleintherepairofSCIthroughtheformationofaglialscar,whichhelpstopreventfurtherdamagetothespinalcord(FawcettandAsher,1999).Ourresultsshowedthatintrathecalinjectionofmethylprednisolonesodiumsuccinatepromotestheproliferationofastrocytes,suggestingthattheglialscarmaycontributetotheneuroprotectiveeffectofmethylprednisolonesodiumsuccinateonSCI.Inaddition,pro-inflammatorycytokinessuchasTNF-αandIL-1βareinvolvedinthesecondaryinjuryafterSCI(Tianetal.,2016).OurresultsshowedthatintrathecalinjectionofmethylprednisolonesodiumsuccinateinhibitstheexpressionofTNF-αandIL-1β,suggestingthattheanti-inflammatoryeffectofmethylprednisolonesodiumsuccinatemaycontributetotheimprovementofneurologicalfunctionafterSCI.

Ourstudyhassomelimitations.First,weonlyinvestigatedtheeffectsofmethylprednisolonesodiumsuccinateonacuteSCI.FurtherstudiesareneededtoinvestigatewhethertheeffectsofmethylprednisolonesodiumsuccinateonchronicSCIarethesame.Second,weonlyinvestigatedtheunderlyingmechanismoftheeffectsofmethylprednisolonesodiumsuccinateoncellproliferation,inflammation,andapoptosis.Additionalstudiesareneededtoinvestigatetheregulationofothermechanisms,suchasoxidativestressandmitochondrialdysfunction,whichareinvolvedinthepathophysiologyofSCI.

Inconclusion,ourstudyshowedthatintrathecalinjectionofmethylprednisolonesodiumsuccinatecaneffectivelypromotetherepairofacutespinalcordinjuryinratsandhasacertaininfluenceontheexpressionofrelevantproteins.ThesefindingshaveclinicalapplicationvalueforthetreatmentofSCIFurthermore,ourstudysuggeststhattheoptimaltiminganddoseofmethylprednisoloneadministrationshouldbecarefullyconsideredinclinicalpractice,aslargerdosesordelayedadministrationmayexacerbatesecondaryinjuryprocesses.Inaddition,futureresearchshouldexplorealternativetherapeuticstrategies,suchasstemcelltherapyandgenetherapy,whichhavethepotentialtoimproveoutcomesinpatientswithSCI.

Despitethelimitationsofourstudy,suchasthesmallsamplesizeandthefocusononlyafewrelevantproteins,thesefindingscontributetoourunderstandingofthemechanismsunderlyingtheeffectsofmethylprednisoloneonSCI.Ultimately,acomprehensiveapproach,whichaccountsforthecomplexpathophysiologyofSCI,willberequiredtodevelopmoreeffectivetreatmentsforthisdevastatingcondition.Bybuildingonthesefindings,wemaybeabletodevelopnewtherapiesthatcanrestorefunctionandimprovethequalityoflifeforindividualswithSCISpinalcordinjury(SCI)isadevastatingconditionthatcanresultinlong-termdisability,lossofqualityoflifeandreducedlifeexpectancy.DespiteadvancesinourunderstandingofthepathophysiologyofSCI,thereiscurrentlynocureforthiscondition.Methylprednisolone(MP),asyntheticglucocorticoid,hasbeenusedasatreatmentoptionforacuteSCIforovertwodecades.However,themechanismsunderlyingthetherapeuticeffectsofMPonSCIarenotwellunderstood.

RecentresearchhasshedlightonsomeofthemolecularmechanismsthatunderliethebeneficialeffectsofMPonSCI.Forinstance,studieshavesuggestedthatMPmayexertitsprotectiveeffectsbyinhibitingtheactivationofseveralpro-inflammatorycytokines,includinginterleukin-1β(IL-1β),tumornecrosisfactor-α(TNF-α)andinterleukin-6(IL-6)(Qiaoetal.,2018).Thereductioninpro-inflammatorycytokinesisthoughttodecreasetheinfiltrationofimmunecellsintotheinjuredtissue,whichcancausefurtherdamagetothespinalcord.

MPhasalsobeenshowntoreduceoxidativestressintheinjuredspinalcord.OxidativestressisknowntoplayakeyroleinthepathologyofSCIandcontributestoneuronaldamageandcelldeath(Heetal.,2015).MPmayexertitsantioxidanteffectsbyincreasingtheexpressionofantioxidantenzymes,suchassuperoxidedismutase(SOD)andglutathioneperoxidase(GPx)(SharmaandSharma,2020).

Furthermore,MPhasbeenshowntoaffecttheexpressionofcertainproteinsinvolvedintheregulationofneuronalsurvivalandaxonalregeneration.Forinstance,MPhasbeenshowntoupregulatetheexpressionofbrain-derivedneurotrophicfactor(BDNF),aproteinthatpromotesneuronalsurvivalandaxonalregeneration(Xuetal.,2017).Additionally,MPhasbeenshowntoincreasetheexpressionoftropomyosinreceptorkinaseB(TrkB),areceptorforBDNF,whichmayfurtherenhancetheneuroprotectiveeffectsofMP(Liuetal.,2018).

Despitethesefindings,themechanismsunderlyingtheeffectsofMPonSCIarecomplexandmultifaceted,andmoreresearchisneededtofullyunderstandhowMPexertsitstherapeuticeffects.Moreov