Protectiveeffectoftrans-resveratrol
onhippocampalneuronsofratswithAp25.35一inducedAlzheimer'sdisease
Objective:Toinvestigatetheprotectiveeffectoftrans.resveratrol
onhippocampalneuronsofratswithA1325.35一inducedAlzheimer’sdisease,andexploreitspossiblemechanisms.
Methods:Ratsare
randomlydividedintoshamgroup,positivecontrolgmup(donepezil),modelgroup(AH25.35),low-andhigh-doseofTRgroups,respectiveIy(n:9).
AfterinjectionofA]825.35intohippocampus,ratswereadministratedwith
1.0mg.kg-1
donepezilandl0and40mg‘kg~TRoncedailybygavageforconsecutive15
daysrespectively,whileshamandmodelgroupswereadministrated谢thv01ume.matched
normalsalinebygavage.The
learningandmemoryabilityofratsweredetectedbvMorris
wat钉mazetest?neuronalinjuryinhippocampusWasobserved
byhematoty.eosinstaining’
andthemRNAexpressionlevelsof
caspasel2weredetectedbyrealtimePCRandthe
Protemexpresslonsofcaspase8andcaspase12wereexaminedby
immunohistOChemistryandWesternblotintherathippocampus.
Results:IntheratshippocampusafterinjectionofA]325.35,the
escapelatencies(s)of
sh锄group,positivecontrolgroup(donepezil),modelgroup(A陈35),low.and
high
-doseofTRgroups
onthefirstdayinthenavigationtestbyMorriswatermazetestwere
59?0士1
8?2,65?2士27.8,98.8士19.9,77.7士20.4,66.6士25.9respectively;theescape
latencies(s)ontheseconddaywere
25.0+14.5,32.4士21.2,72.8士25.0,52.4+14.8,
33?3士15?1respectively;theescapelatencies(s)onthethirddaywere20.3士12.0,
25?1士12?9,67?2士17.8,44.8士23.2,26.2士19.7respectively;theescapelatencies(s)onthe
founhdaywere
10.3士4.8,12.0士5.8,62.6士22.1,30.3士11.5,14.2士4.7;Theadjusted
escapelatenciesonthefifthdayinspatialprobetestwere32.8士8.4,25.3士5.5,
一
6
15.6+1.2,21.7+4.8,28.0+4.9respectively.ThemRNAexpressionsofcaspasel2of
shamgroup,positivecontrolgroup(donepezil),modelgroup(A1325.3s),low-andhigh
-doseofTRgroupsbyrealtimePCRwere55.70士10.12,91.11+7.06,181.49士38.68,
138.43.26士15.28,98.06士23.05respectively;Theprotein
expressionsofcaspase8by
westernblotwere0.18士0.03,0.29士0.02,0.64士0.04,0.57士0.05,0.31士0.03
respectively;Theproteinexpressionsofcaspase12byWesternblotwere0.19士0.05,0.29士0.03,0.68士0.03,0.60士0.05,0.32士0.04respectively.Comparedtothe
sham—treatedrats,A1325.35injectionsignificantly
meanescapelmencyinthe
prolongedthe
navigationtestandshortenedtheadjustedescapelatencyinspatialprobetest(P<0.o1),
NeumalinjuryinhippocampusandthemRNAexpressionofcaspasel2,aSwellasthe
proteinexpressionsofcaspase8andcaspase12inrathippocampuswere
increased(P<0.01).Comparedwiththemodelrats,Trans?resveratrol-treated
groups
significantlyshortenedthemeanescapelatencyinthenavigationtestandprolongedthe
adjustedescapelatencyinspatialprobetest∽<0.05),andattenuatedneuronalinjuryinrathippocampus,andsignificantlydecreasedthemRNAexpressionsofCaspasel2,aSwellaStheproteinexpressionsfcaspase8andCaspasel2inrathippocampus(P<O.05).
Conclusion:Thereisprotectiveeffectoftrans?resveratrolonhippocampalneuronsof
ratswithAp25.35‘inducedAlzheimer’Sdisease,itsmechanismsmaybeatleastpartlyduetodecreasingthemRNAandproteinsexpressionofcaspase8andcaspase12inrat
hippocampus.
KeyWords:trans-resveratrol;蝤ppoc举;caspase8;caspasel2;
7
既往研究TR对AB。¨j诱导AD大鼠有保护作用,其机理可能部分与抑SwJiNOS、caspase8、caspase9mRNA表达有关n0’川。目前关于TR对AD的保护作用研究,通过内质网应激凋亡通路caspasel2表达的影响,国内外研究较少。本实验以相关文献报道的白藜芦醇抗AD作用为依据,通过观察TR对AD大鼠海马神经元细胞形态影响,探讨TR对AD的保护作用及潜在的机制。进一步证实了TR对AD大鼠海马神经元的保护作用,可能与抑制caspasel2mRNA,caspasel2和caspase8蛋白表达有关,为TR临床应用打下基础,同时提供TR治疗AD的药理学依据及可能的发病机制。
反应体系的组成如下表:
试剂使用量SYBRsuperMix荧光混合物7.5ul
模板混合液(上游、下游引物)0.5ul
DEPC水
稀释6.5倍cDNA反应总体积4u13.0ul15ul
反应条件如下:
Stage1:预变性
95℃10min1Cycle
Stage
2:PCR反应40Cycles
95℃10s:57.9℃1min
Stage3:融解曲线分析
80Cycles95℃1min;55℃lmin;55℃10S。
③引物:
引物合成TaKaRa生物工程公司,见表1。
表1引物序列
Table1Thesequenceofprimers
n
Forwardprimer(5'-3’)ene
Lj7Reverseprimer(5'-3’)
Caspase12CAATTCCGACAAACAGCTGAGTTTACATGGGCCACTCCAACATTTAC3-actinGGAGATTACTGCCCTGGCTCCTAGACTCATCGTACTCCTGCTTGCTG
5)结果分析采用相对定量法
以Ct值为统计参数依次计算出如下数据:
a.Ctaverage=(Ctl+Ct2)/2(重复管)
b.dCt=Ctaverage-中间值
C.基因的表达=2^(-dCt)
d.相对定量=目的基因的表达/内参基因的表达