甲潑尼龍琥珀酸鈉鞘內(nèi)注射對大鼠急性脊髓損傷的修復作用及相關(guān)蛋白的影響

甲潑尼龍琥珀酸鈉鞘內(nèi)注射對大鼠急性脊髓損傷的修復作用及相關(guān)蛋白的影響摘要：本研究旨在探究甲潑尼龍琥珀酸鈉鞘內(nèi)注射治療大鼠急性脊髓損傷的效果及相關(guān)蛋白的影響。通過將大鼠隨機分為對照組、脊髓損傷組、甲潑尼龍琥珀酸鈉治療組和生理鹽水治療組進行實驗研究，發(fā)現(xiàn)甲潑尼龍琥珀酸鈉能夠有效地促進損傷部位的細胞增殖和修復，并且可以抑制炎癥反應(yīng)和細胞凋亡。同時，甲潑尼龍琥珀酸鈉還能夠調(diào)節(jié)多種相關(guān)蛋白質(zhì)的表達，如GFAP、Bcl-2和Bax等。綜上所述，甲潑尼龍琥珀酸鈉鞘內(nèi)注射能夠有效地促進大鼠急性脊髓損傷的修復，并且對相關(guān)蛋白的表達有一定影響，具有一定的臨床應(yīng)用價值。

關(guān)鍵詞：甲潑尼龍琥珀酸鈉，鞘內(nèi)注射，脊髓損傷，細胞增殖，炎癥反應(yīng)，細胞凋亡，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