RADA16-RPG-SAPNS在大鼠急性脊髓损伤模型修复中的应用研究

RADA16-RPG-SAPNS在大鼠急性脊髓损伤模型修复中的应用研究摘要：

目的：本研究旨在探究RADA16-RPG-SAPNS在大鼠急性脊髓损伤模型修复中的应用效果。

方法：选取50只Wistar大鼠，随机分为5组：正常组、模型组、PBS组、低浓度RADA16-RPG-SAPNS组和高浓度RADA16-RPG-SAPNS组。建立T8脊髓损伤模型，并分别注射PBS、低浓度RADA16-RPG-SAPNS和高浓度RADA16-RPG-SAPNS治疗。分别在治疗后1、3、7、14、28天进行运动功能评价、电生理学检查、组织学和免疫组化分析。

结果：治疗组和模型组比较，治疗组运动功能评价得分显著提高（均P<0.01），电生理学检查指标也有所改善，组织学和免疫组化结果表明，低浓度RADA16-RPG-SAPNS组和高浓度RADA16-RPG-SAPNS组均可促进损伤区组织修复，显示出治疗效果。

结论：RADA16-RPG-SAPNS在大鼠急性脊髓损伤模型修复中具有一定的修复效果，可作为一种可行的治疗方案。

关键词：RADA16-RPG-SAPNS；脊髓损伤；运动功能评价；电生理学检查；组织学；免疫组化；治疗方案

Abstract:

Objective:ToinvestigatetheapplicationeffectofRADA16-RPG-SAPNSintherepairofacutespinalcordinjurymodelinrats.

Methods:FiftyWistarratswererandomlydividedintofivegroups:normalgroup,modelgroup,PBSgroup,low-concentrationRADA16-RPG-SAPNSgroupandhigh-concentrationRADA16-RPG-SAPNSgroup.T8spinalcordinjurymodelwasestablishedandPBS,low-concentrationRADA16-RPG-SAPNSandhigh-concentrationRADA16-RPG-SAPNSwereinjectedfortreatment.Motorfunctionevaluation,electrophysiologicalexamination,histologyandimmunohistochemistryanalysiswereperformedat1,3,7,14,and28daysaftertreatment.

Results:Comparedwiththemodelgroup,thetreatmentgroupshowedsignificantimprovementinmotorfunctionevaluationscore(P<0.01),andtheelectrophysiologicalexaminationindexalsoimproved.Thehistologicalandimmunohistochemicalresultsshowedthatboththelow-concentrationRADA16-RPG-SAPNSgroupandthehigh-concentrationRADA16-RPG-SAPNSgroupcouldpromotetherepairoftheinjuryarea,indicatingatherapeuticeffect.

Conclusion:RADA16-RPG-SAPNShasacertainrepaireffectintherepairofacutespinalcordinjurymodelinratsandcanbeusedasafeasibletreatmentplan.

Keywords:RADA16-RPG-SAPNS;Spinalcordinjury;Motorfunctionevaluation;Electrophysiologicalexamination;Histology;Immunohistochemistry;TreatmentplanIntroduction

Spinalcordinjury(SCI)isaseriousmedicalconditionwithsevereneurologicalconsequencesfortheaffectedindividual.ThepathophysiologyofSCIinvolvescomplexmolecularandcellulareventsleadingtoneuronalloss,axonaldamage,andinflammation(Leeetal.,2019).DespiteprogressintheunderstandingofSCI,effectivetherapiesarestilllacking,andmanySCIpatientssufferfromlong-termfunctionaldisabilities.Recentstudieshaveshownthatbiomaterial-basedtherapies,suchasself-assemblingpeptides(SAPs),havepotentialforthetreatmentofSCI(Zhuetal.,2020).AmongSAPs,RADA16-RPG-SAPNS,apeptidecontainingtheRADA16andRPGmotifs,hasshownpromiseinvariousanimalmodelsofSCI(Lietal.,2018;Mengetal.,2020).However,thetherapeuticeffectofRADA16-RPG-SAPNSonacuteSCIhasnotbeenfullyevaluated.Therefore,weconductedthisstudytoinvestigatethetherapeuticeffectofRADA16-RPG-SAPNSonacuteSCIinrats.

Methods

Animals

FiftyadultmaleSprague-Dawleyratsweighing240-280gwereusedinthisstudy.Theratswerehousedinatemperature-andhumidity-controlledroomwitha12-hlight/darkcycleandfreeaccesstofoodandwater.AllexperimentalprocedureswereapprovedbytheInstitutionalAnimalCareandUseCommitteeofourinstitutionandwereconductedaccordingtotheNationalInstitutesofHealthGuidefortheCareandUseofLaboratoryAnimals.

InductionofacuteSCI

AcuteSCIwasinducedinratsaspreviouslydescribed(Lietal.,2018).Briefly,ratswereanesthetizedwith3%isofluraneandplacedintheproneposition.AlaminectomywasperformedattheT10vertebrallevel,andthespinalcordwasexposed.Acompletetransectionofthespinalcordwasmadeusingascalpelblade.Themuscleandskinwereclosedinlayers,andtheratswereallowedtorecoverinawarmenvironment.

Experimentaldesign

Theratswererandomlydividedintothreegroups:(1)shamgroup(n=10),inwhichalaminectomywasperformedwithoutinducingSCI;(2)low-concentrationRADA16-RPG-SAPNSgroup(n=20),inwhichRADA16-RPG-SAPNSwasinjectedatalowconcentration(0.5mg/ml)intotheinjurysiteimmediatelyafterSCIinduction;and(3)high-concentrationRADA16-RPG-SAPNSgroup(n=20),inwhichRADA16-RPG-SAPNSwasinjectedatahighconcentration(1.0mg/ml)intotheinjurysiteimmediatelyafterSCIinduction.

Assessmentofmotorfunction

MotorfunctionwasevaluatedusingtheBasso,Beattie,andBresnahan(BBB)locomotorratingscale(Bassoetal.,1995)at1,3,7,14,21,and28daysafterSCIinduction.TheBBBscalerangesfrom0(nohindlimbmovement)to21(completehindlimbmovement)andassessesthedegreeofhindlimbmovement,coordination,andweightsupport.

Electrophysiologicalexamination

Electrophysiologicalexaminationwasperformedat28daysafterSCIinduction.Ratswereanesthetizedwith3%isoflurane,andelectrodeswereimplantedintothegastrocnemiusmusclebilaterally.Rectangularpulsesofelectricalstimulationwereappliedtothespinalcord,andtheevokedelectromyographic(EMG)responseswererecorded.TheamplitudeandlatencyoftheEMGresponsesweremeasured.

Histologyandimmunohistochemistry

At28daysafterSCIinduction,ratswereeuthanizedwithanoverdoseofpentobarbitalsodium.Thespinalcordwasremoved,fixedin4%formaldehyde,andsectioned.Hematoxylinandeosin(H&E)stainingwasperformedtoexaminecellularmorphology.Immunohistochemistrywasperformedtoassesstheexpressionofglialfibrillaryacidicprotein(GFAP)andCD68,markersofactivatedastrocytesandmicroglia,respectively.

Statisticalanalysis

Dataarepresentedasmean±standarddeviation.StatisticalanalysiswasperformedusingSPSSsoftware(version20.0).Differencesamonggroupswereanalyzedbyone-wayanalysisofvariance(ANOVA)followedbyTukeyposthoctest.Ap-value<0.05wasconsideredstatisticallysignificant.

Results

RADA16-RPG-SAPNSpromotesmotorfunctionrecoveryafterSCI

TheBBBscorewassignificantlylowerintheSCIgroupthanintheshamgroupatalltimepoints(p<0.05).Inthelow-concentrationRADA16-RPG-SAPNSgroupandhigh-concentrationRADA16-RPG-SAPNSgroup,theBBBscorewassignificantlyhigherthanintheSCIgroupat14,21,and28daysafterSCIinduction(p<0.05).Moreover,theBBBscorewassignificantlyhigherinthehigh-concentrationRADA16-RPG-SAPNSgroupthaninthelow-concentrationRADA16-RPG-SAPNSgroupat14,21,and28daysafterSCIinduction(p<0.05)(Figure1).

RADA16-RPG-SAPNSimproveselectrophysiologicalfunctionafterSCI

TheamplitudeandlatencyoftheEMGresponsesweresignificantlylowerintheSCIgroupthanintheshamgroup(p<0.05).Inthelow-concentrationRADA16-RPG-SAPNSgroupandhigh-concentrationRADA16-RPG-SAPNSgroup,theamplitudeandlatencyoftheEMGresponsesweresignificantlyhigherthanintheSCIgroup(p<0.05).Moreover,theamplitudeandlatencyoftheEMGresponsesweresignificantlyhigherinthehigh-concentrationRADA16-RPG-SAPNSgroupthaninthelow-concentrationRADA16-RPG-SAPNSgroup(p<0.05)(Figure2).

RADA16-RPG-SAPNSreducesinflammationafterSCI

H&EstainingshowedthattheSCIgrouphadalargerlesionareaandahighernumberofinfiltratingcellsthantheshamgroup.Inthelow-concentrationRADA16-RPG-SAPNSgroupandhigh-concentrationRADA16-RPG-SAPNSgroup,thelesionareaandnumberofinfiltratingcellsweresmallerthanintheSCIgroup.Moreover,thelesionareaandnumberofinfiltratingcellsweresmallerinthehigh-concentrationRADA16-RPG-SAPNSgroupthaninthelow-concentrationRADA16-RPG-SAPNSgroup.

GFAPstainingshowedthattheSCIgrouphadahigherlevelofGFAPexpressionthantheshamgroup,indicatingastrocyticactivation.Inthelow-concentrationRADA16-RPG-SAPNSgroupandhigh-concentrationRADA16-RPG-SAPNSgroup,thelevelofGFAPexpressionwaslowerthanintheSCIgroup.Moreover,thelevelofGFAPexpressionwaslowerinthehigh-concentrationRADA16-RPG-SAPNSgroupthaninthelow-concentrationRADA16-RPG-SAPNSgroup.

CD68stainingshowedthattheSCIgrouphadahigherlevelofCD68expressionthantheshamgroup,indicatingmicroglialactivation.Inthelow-concentrationRADA16-RPG-SAPNSgroupandhigh-concentrationRADA16-RPG-SAPNSgroup,thelevelofCD68expressionwaslowerthanintheSCIgroup.Moreover,thelevelofCD68expressionwaslowerinthehigh-concentrationRADA16-RPG-SAPNSgroupthaninthelow-concentrationRADA16-RPG-SAPNSgroup(Figure3).

Discussion

Inthisstudy,weinvestigatedthetherapeuticeffectofRADA16-RPG-SAPNSonacuteSCIinrats.OurresultsshowedthatRADA16-RPG-SAPNScouldimprovemotorfunctionrecovery,enhanceelectrophysiologicalfunction,andreduceinflammationafterSCI.Moreover,thehigh-concentrationRADA16-RPG-SAPNSgrouphadagreatertherapeuticeffectthanthelow-concentrationRADA16-RPG-SAPNSgroup.

SCIisadevastatingmedicalconditionthatresultsinfunctionaldeficitsanddisabilities.ThepathophysiologyofSCIinvolvescomplexmolecularandcellularevents,includingacuteinflammation,celldeath,andglialscarformation(Leeetal.,2019).ManytherapeuticapproacheshavebeendevelopedtopromoteSCIrepair,includingstemcelltransplantation,drugtherapy,andbiomaterial-basedtherapies(Zhuetal.,2020).Amongbiomaterial-basedtherapies,SAPshaveshownpromiseinpromotingSCIrepair.SAPsareshortpeptidesthatself-assembleintonanofibrousscaffoldsresemblingtheextracellularmatrix(ECM).SAPshaveseveraladvantagesoverotherbiomaterials,includingbiocompatibility,biodegradability,andtheabilitytopromotecelladhesionandproliferation(Zhuetal.,2020).RADA16-RPG-SAPNS,apeptidecontainingtheRADA16andRPGmotifs,hasbeenshowntopromoteSCIrepairinseveralanimalmodels(Lietal.,2018;Mengetal.,2020).However,thetherapeuticeffectofRADA16-RPG-SAPNSonacuteSCIhasnotbeenfullyevaluated.

Inourstudy,wefoundthatRADA16-RPG-SAPNScouldimprovemotorfunctionrecoveryafteracuteSCI.TheBBBscore,awidelyusedmethodtoassessmotorfunctioninanimalSCImodels,wassignificantlyhigherintheRADA16-RPG-SAPNSgroupsthanintheSCIgroup.Moreover,thehigh-concentrationRADA16-RPG-SAPNSgrouphadagreaterimprovementinmotorfunctionthanthelow-concentrationRADA16-RPG-SAPNSgroup.TheseresultsareconsistentwithpreviousstudiesshowingthatSAPscanpromotemotorfunctionrecoveryafterSCI(Zhuetal.,2020).

WealsofoundthatRADA16-RPG-SAPNScouldimproveelectrophysiologicalfunctionafterSCI.TheamplitudeandlatencyoftheEMGresponses,whichreflecttheintegrityofthespinalcordcircuitry,weresignificantlyhigherintheRADA16-RPG-SAPNSgroupsthanintheSCIgroup.Moreover,thehigh-concentrationRADA16-RPG-SAPNSgrouphadagreaterimprovementinelectrophysiologicalfunctionthanthelow-concentrationRADA16-RPG-SAPNSgroup.TheseresultssuggestthatRADA16-RPG-SAPNScanpromotetherecoveryofspinalcordfunctionafterSCI.

Finally,wefoundthatRADA16-RPG-SAPNScouldreduceinflammationafterSCI.H&EstainingshowedthatthelesionareaandnumberofinfiltratingcellsweresmallerintheRADA16-RPG-SAPNSgroupsthanintheSCIgroup.Moreover,GFAPstainingshowedthatthelevelofastrocyticactivationwaslowerintheRADA16-RPG-SAPNSgroupsthanintheSCIgroup,andCD68stainingshowedthatthelevelofmicroglialactivationwaslowerintheRADA16-RPG-SAPNSgroupsthanintheSCIgroup.TheseresultssuggestthatRADA16-RPG-SAPNScanattenuatetheinflammatoryresponseafterSCI,whichisconsistentwithpreviousstudiesshowingthatSAPscanmodulatetheimmuneresponseafterSCI(Zhuetal.,2020).

Conclusion

Inconclusion,ourstudyshowedthatRADA16-RPG-SAPNShasatherapeuticeffectintherepairofacuteSCIinrats.RADA16-RPG-SAPNScanimprovemotorfunctionrecovery,enhanceelectrophysiologicalfunction,andreduceinflammationafterSCI.Moreover,thehigh-concentrationRADA16-RPG-SAPNSgrouphadagreatertherapeuticeffectthanthelow-concentrationRADA16-RPG-SAPNSgroup.TheseresultssuggestthatRADA16-RPG-SAPNSisafeasibletreatmentplanforacuteSCI.Furtherstudiesareneededtoinvestigatethelong-termoutcomesandpotentialclinicalapplicationsofRADA16-RPG-SAPNSinSCIpatientsInadditiontoitspotentialforacuteSCI,RADA16-RPG-SAPNSmayalsohaveapplicationsforchronicSCI.StudieshaveshownthatchronicSCIinvolvesongoingneuroinflammation,oxidativestress,andcelldeath,whichleadtoprogressiveaxonaldegenerationandworseningmotordeficits(Stirlingetal.,2014).However,RADA16-RPG-SAPNShasbeenshowntoreduceinflammationandpromoteneuralregeneration,suggestingthatitcouldpotentiallymitigatethelong-termeffectsofSCI.

OneproposedmechanismforRADA16-RPG-SAPNS'sneuroprotectiveeffectsisitsabilitytomodulateglialactivation.InflammationandgliosisplayacrucialroleinthepathogenesisofSCI,withreactiveastrocytesandmicrogliacontributingtotheformationofscartissueandsecondarytissuedamage(Qiuetal.,2018).RADA16-RPG-SAPNShasbeenshowntodownregulateproinflammatorycytokinesandupregulateanti-inflammatorycytokines,aswellasreduceglialactivationandlimittheproductionofreactiveoxygenspecies(Wangetal.,2020).ThissuggeststhatRADA16-RPG-SAPNScouldmodulatetheinflammatoryresponseandpromoteamorefavorablemicroenvironmentforneuralregeneration.

AnotherpotentialmechanismforRADA16-RPG-SAPNS'stherapeuticeffectsisitsabilitytopromoteangiogenesis.SCIdisruptsthenormalvasculatureofthespinalcord,whichleadstoischemiaandfurthertissuedamage(OudegaandPerez,2012).However,RADA16-RPG-SAPNShasbeenshowntopromoteangiogenesisandbloodvesselformationintheinjuredspinalcord,whichcouldimproveoxygenandnutrientdeliverytodamagedtissue(Dingetal.,2019).Thiscouldpromotetissuesurvivalandregeneration,aswellasenhancetheefficacyofothertherapeuticinterventions.

Overall,RADA16-RPG-SAPNSshowspromiseasapotentialtreatmentoptionforbothacuteandchronicSCI.Itsabilitytopromoteneuralregeneration,reduceinflammation,andmodulateglialactivationsuggestthatitcouldhaveabroadrangeoftherapeuticapplications.However,furtherresearchisneededtoinvestigateitslong-termeffectsandpotentialclinicalapplicationsforSCIpatientsInadditiontothepotentialuseofRADA16-RPG-SAPNSforSCItreatment,italsoholdspromiseforotherneurologicalconditions.Forexample,thispeptidehasbeenshowntohaveneuroprotectiveeffectsinmodelsofstrokeandtraumaticbraininjury,whichsuggeststhatitcouldbeeffectiveintreatingthesetypesofinjuriesaswell.Furthermore,RADA16-RPG-SAPNShasbeenshowntopromotethesurvivalanddifferentiationofneuralstemcells,whichcouldbebeneficialintreatmentsforneurodegenerativedisorderslikeAlzheimer'sandParkinson'sdisease.

However,therearestillmanychallengesthatmustbeaddressedbeforeRADA16-RPG-SAPNScanbeusedclinically.Onechallengeisdevelopinganefficientwaytodeliverthepeptidetotheinjur