| Ischemic cerebrovascular disease has high mobidity, mortality anddisability rate. The structure and function reconstruction of the cerebralcirculation are the basis and guarantee of the neural network reconstructionafter cerebral ischemia. Therefore, there is a very important significance tolucubrate the angiogenesis in ischemic area after cerebral ischemia.The revascularization after cerebral ischemia depending on theproliferation and reparation by endothelial cells(ECs) is not enough, whilethe angiogenesis process through proliferation, migration anddifferentiation by endothelial progenitor cells(EPCs) is a new strategy inangiogenesis research. In the past, cell transplantation is a more appliedapproach in the study of EPCs than the way only on body’s overall level.The endogenous EPCs can effectively promote angiogenesis after cerebralischemia, while there in deed exist many unresolved questions in exogenous EPCs. So it is necessary to investigate the angiogenesis effect ofendogenous EPCs. Phosphatidylinositol-3-kinase/protein kinase B(PI3K/AKT) signal pathway and SDF-1α/CXCR4axis were closely relatedto the mobilization of EPCs in bone marrow, but both of them lack of theresearch on overall level. Acupuncture, a traditional Chinese non-medicinemethod, treated cerebral ischemia more than one thousand of year, canpromote angiogenesis in cerebral ischemic area. This effect may be relatedwith that electroacupuncture can mobilize EPCs homing to peripheralblood and ischemic area to promote angiogenesis, but its mechanism is stillunclear. Our research aim to investigate the change rule of theCD34+VEGFR2+EPCs number, CD34+EPCs number and the express ofSDF-1α and pAKT in bone marrow, peripheral blood, cerebral ischemiccortex after acupuncture intervene. To explore the mechanism ofelectroacupuncture in promoting angigenesis after cerebral ischemia, anddetermine whether it is related with electroacupuncture upregulating theexpression of SDF-1α and pAKT then inducing bone marrow EPCsmobilizating and migrating to the ischemic brain.Objective:1. To investigate the effect of electroacupuncture on the neurologicalrecovery after focal cerebral ischemia/reperfusion；2. To investigate the effect of electroacupuncture on the number of CD34+VEGFR2+EPCs and CD34+EPC in rat bone marrow andperipheral blood after focal cerebral ischemia/reperfusion;3. To investigate the effect of electroacupuncture on the level of SDF-1αin rat bone marrow, peripheral blood and ischemic cortex after focalcerebral ischemia/reperfusion;4. To investigate the effect of electroacupuncture on the proteinphosphorylationin of AKT in rat bone marrow after focal cerebralischemia/reperfusion;5. To investigate the effect of electroacupuncture on the expression ofEPCs derived vessels in ischemic cortex after focal cerebralischemia/reperfusion;6. To investigate and certify bone marrow derived EPCs could mobilize toperipheral blood and home to ischemic cortex to promote angiogenesisby search for the concentration gradient of SDF-1α among bone marrow,peripheral blood and ischemic brain after focal cerebralischemia/reperfusion;7. To in-depth explore the mechanism that electroacupuncture mobilizeendogenous EPCs promoting angiogenesis after focal cerebralischemia/reperfusion.Method:A total of255SD rats received filament occlusion of the right middle cerebral artery for2h followed by reperfusion for12h,1d,2d,3d,7drespectively. Rats were randomly divided into sham group, modelgroup(I/R group) and electroacupuncture group(I/RE group).Electroacupuncture was given at the Baihui (GV20)/Siguan (Hegu(LI4)/Taichong (LR3)) acupoints in the I/RE group,30min/d, once aday. The modified neurological severity score(mNSS) were used toevaluate the neural functional recovery after focal cerebralischemia/reperfusion. The number of CD34+VEGFR2+EPCs andCD34+EPCs were quantified by fluorescence-activated cell sortinganalysis(FACS). The level of SDF-1α in bone marrow and peripheral blood,level of p-AKT in bone marrow, expression of CD34+VEGFR2+vessels andSDF-1α in the ischemic brain were detected by real-time PCR, western blotand double immunofluorescence labeling approach respectively.Result:1. The modified neurological severity score(mNSS)After focal MCAO/R, rats in I/R group and I/RE group showedneurological deficit behavior more or less at12h、1d、2d、3d、7d.Electroacupuncture significantly improved the functional outcomecompared with model group at3and7days(P<0.01), while there were nosignificant functional improvement at12h,1d,2d compared with I/Rgroup(P>0.05). As time went on, the mNSS score all slightly decreased gradually.2. The effect of electroacupuncture on the number of CD34+VEGFR2+EPCs in rat peripheral blood after focal cerebral ischemia/reperfusionEPCs number in I/R group began to increase at12h afterreperfusion(P<0.05), and peaked at1d(P<0.01) then gradually decreased,but it was still higher than sham group at2d and3d(P<0.05), while therewas no significant difference at7d compared with sham group(P>0.05).Compared with I/R group, there was no statistical significance at12h inI/RE group(P>0.05), but it obviously increased at1d to3d(P<0.05) thendecreased at7d(P>0.05).3. The effect of electroacupuncture on the number of CD34+EPCs inrat peripheral blood after focal cerebral ischemia/reperfusionCompared with sham group, the CD34+EPCs number in peripheralblood were higher at12h,24h,48h after reperfusion in I/R group(P<0.01),and peaked at24h(P<0.01), in which the increase were more obvious inI/RE group at each time point(P<0.01). The CD34+EPCs number at12h,24h,48h after reperfusion in I/RE group were increased gradually andobviously higher than I/R group.4. The effect of electroacupuncture on the number of CD34+VEGFR2+EPCs in rat bone marrow after focal cerebral ischemia/reperfusionCompared with sham group, EPCs number were significantupregulated at12h,1d,2d after reperfusion and peaked at12h(P<0.05) then gradually reduced. In I/RE group, EPCs number obviously increased at1d,2d(P<0.01) compared with I/R group and peaked at1d(P<0.01), while therewas no significant difference at3d compared with I/R group(P>0.05) but itwas still higher than sham group(P<0.05).5. The effect of electroacupuncture on the number of CD34+EPCs inrat bone marrow after focal cerebral ischemia/reperfusionThe CD34+EPCs number in bone marrow at12h in I/R group was thehighest than24h and48h after reperfusion, but the number in I/R groupalways higher than sham group(P<0.05), while the increase were moreobvious in I/RE group at each time point(P<0.01). Compared with I/Rgroup, the number were obviously increased at12h and24h in I/RE group,but there were no significant difference at48h.6. The effect of electroacupuncture on the level of p-AKT protein in ratbone marrow after focal cerebral ischemia/reperfusionThere were few expression of p-AKT in the bone marrow of the shamgroup. The p-AKT level increased gradually at12h,24h,48h afterMCAO/R in I/R group, and peaked at7d(P<0.05), while the increase weremore obvious in I/RE group at each time point(P<0.01). Compared with I/Rgroup, the p-AKT level were obviously increased at each time point in I/REgroup(P<0.01).7. The effect of electroacupuncture on the mRNA level of SDF-1αin rat bone marrow, and peripheral blood after focal cerebral ischemia/reperfusionIn the peripheral blood, compared with I/R group, the SDF-1αmRNA expression were highly increased at1d,2d,3d in I/REgroup(P<0.05), but there was no significant difference at12h(P>0.05). Inthe bone marrow, SDF-1α mRNA expression in both I/R and I/RE grouppeaked at12h after reperfusion(P<0.05), but there were no significantdifference between the two groups(P>0.05). Then SDF-1α mRNAexpression began to reduce step by step, but rose again at7d, in which itwas obviously higher at1d in I/RE group than I/R group(P<0.05).8. The effect of electroacupuncture on the mRNA and protein level ofSDF-1α in rat cerebral ischemic cortex after focal cerebralischemia/reperfusionCompared with sham group, the mRNA and protein expression ofSDF-1α were significantly increased in I/R and I/RE group(P<0.05), inwhich the increase in I/RE group was more obvious(P<0.05). The mRNAand protein level of SDF-1α at1d,3d,7d in I/RE group were higher thanI/R group and peaked at3d after reperfusion(P<0.05).9. The effect of electroacupuncture on the expression of CD34+VEGFR2+vessels and mRNA level of CD34and VEGFR2in cerebral ischemic cortexafter focal cerebral ischemia/reperfusionThe number of CD34+VEGFR2+vessels gradually increased afterMCAO/R in both I/R and I/RE group, and peaked at7d(P<0.05). Compared with I/R group, there was no significant difference at1d in I/REgroup (P>0.05), but it was obviously increased at3d and7d(P<0.05). ThemRNA expression of CD34and VEGFR2had no significant difference at1d in I/RE group compared with I/R group(P>0.05). however, they bothobivious increased at3d and7d in I/RE group than I/R group(P<0.05).Conclusion:1. Electroacupuncture improves neurological function recovery after focalcerebral ischemia/reperfusion;2. Electroacupuncture promote the angigenesis in ischemic brain byactivating PI3K/AKT pathway, accelerating the phosphorylationin ofAKT, decreasing the SDF-1α level in bone marrow, promotingCD34+VEGFR2+EPCs and CD34+EPCs mobilizing into peripheralblood from bone marrow, increasing the number ofCD34+VEGFR2+EPCs and CD34+EPCs, and thus upregulating theexpression of CD34, VEGFR2and CD34+VEGFR2+vessels;3. Electroacupuncture can increase and accelerate the forming of theSDF-1α concentration gradient among bone marrow, peripheral bloodand ischemic brain, promoting bone marrow derived CD34+EPCs,CD34+VEGFR2+EPCs mobilizing to peripheral blood and homing intoischemic cortex to promote angiogenesis by search for theconcentration gradient after focal cerebral ischemia/reperfusion. |
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