| Objective: To observe the expression changes of the cerebral edema associatedAQP-4，AQP-9and MMP-9proteins in surrounding the hemorrhage area ofintracerebral hemorrhage (ICH) rats subjected to electro-acupuncturestimulation combined with BMSCs transplantation, so as to explore whether thecombination therapy can promote the water transport in surrounding thehemorrhage focous，reduce brain edema and improve neurological symptoms.Methods:1. Establishment of the ICH rat models. The ICH models werebuilded by injecting the collagenase and heparin into right caudate nucleus ofthe SD rats via brain stereotaxic instrument. Neurological severity score (NSS)was used to evaluate models atfer2hours,24hours and48hours, and the ratswere selected with scores exceeded9for further experiments.2. Cultivation ofthe BMSCs. Atfer routinely recovered and passaged, GFP-labeled BMSCs wereinductively cultured for24hours when the growth to cell alignment is about80%, then the BMSCs were induced by the serum-free inductive liquid beforetransplantation6hours，collected and reserved with the concentration of2.5x107/ml.3. The experimental rats were divided into four groups at3days atfermodel-build. The saline group (control group), the animals were injected with20^1physiological saline into the right caudate nucleus. The BMSCs transplantation group (BMSCs group), the animals were injected with20[ilBMSCs into the right caudate nucleus. The electro-acupuncture therapy group(Ea group), the ICH rats were electro-acupunctured the Baihui and Dazhui withthe3hz and lv continuous wave, for10minutes every day. Theelectro-acupuncture combined with BMSCs transplantation group (Ea-BMSCsgroup), the animals were injected with20(il BMSCs into the right caudatenucleus, and electro-acupuncturing the Baihui and Dazhui Similar to theEa-BMSCs group atfer injection2hours.4. According to the treatment time，each group is redivided into ifve subgroups namely1-day,3-day,5-day,7-dayand14-day group. Then, collected serum, the rats were sacrificed and removedthe brain at the above-mentioned time points.5. The distributions ofaquaporins-4(AQP-4), aquapoirns-9(AQP-9) and matrix metalloproteinase-9(MMP-9) were observed in brain tissue by immunohistochemical method, andAQP-4expression was detected in surrounding the focal hemorrhage bywestern blotting. The water content surrounding the bleeding stove wasdetected by dry and wet method; and interleukin-2(IL-2) in serum was detectedby radioimmunoassay (RIA), Results:1. The water content in surrounding thehemorrhage. The brain water content of each group began to rise at the3rd day,and reached to the peak at the5th day. The brain water content of Ea group waslower than the others groups at each time point.2. The expression change ofthe AQP-4in surrounding the hemorrhage.①The AQP-4protein began toirse at the3rd day in the control group, the BMSCs group and the Ea-BMSCs hgroup, then, reached to the peak at the5t day. In addition，the AQP-4proteinbegan to rise again at the7th day in the BMSCs group and the Ea-BMSCs group,②The AQP-4protein of the BMSCs group and the Ha-BMSCs group werelower than the control group at the5lh day (p<0.05)，but higher than the controltgroup at the14h day (p<0.05).Moreover, the AQP-4protein of the Ea-BMSCsgroup was lower than the BMSCs group at the5th day (p<0.05), and there wasno statistical difference between the BMSCs group and the Ea-BMSCs groupat the14th day.3. The expression change of the AQP-9in surrounding thehemorrhage.①The AQP-9protein began to rise at the lsl day in the controlgroup，the BMSCs group and the Ea-BMSCs group, and twice reached to thepeak at the3rd day and ththe7day，but there was only one peak at the7th day inthe Ea group.(2)The AQP-9protein of the Ea-BMSCs group was higher thanthe control group, the BMSCs group and the Ea group at the3rd day(p<0.05),but there were no statistical differences between the BMSCs group，controlgroup and the Ea group.③At the7th day，the AQP-9protein of each group washigher than the control group(p<0.05), just the Ea-BMSCs group was higherthan the Ea group (/?<0.05)，but there was no statistical differences betweenthe Ea-BMSCs group and the BMSCs group.4. The expression change of theMMP-9in surrounding the hemorrhage.?The MMP-9protien of theEa-BMSCs group and the BMSCs group began to rise at the1st day，andreached to the peak at the3rd day, then falling slowly，but the upwardtendency was shown in the Ea-BMSCs group at the7th day. The slowly declining trendcy was shown in the control group and the Ea group.②Atthe1st day, the MMP-9protien of the Ea-BMSCs group and the Ea group werelower than the BMSCs group and the control group(p<0.05).③At the3rd day,the MMP-9protien of the Ea-BMSCs group and the Ea group were lower thanthe control group(/?<0.05)，but the BMSCs group was higher than the controlthgroup(p<0.05).?At the14day，the MMP-9protien of the Ea-BMSCsgroup and the BMSCs group were higher than the control group(p<0,05), andthe Ea group was lower than the control group(p<0.05), but there wasno statistical difference between the BMSCs group and the Ea-BMSCs group,5, The level of IL-2in serum,①There was no difference in the all groups atthse1t day.②The IL-2of the BMSCs group was higher than the other groupsrat the3d day (P<0.05), then quickly reduce. It closed to the control group atthte5h day，and then began to rise?③The IL-2of the Ea-BMSCs group wassignificantly higher than the control group and the BMSCs group at the5th day(P<0，05)，and then the IL-2has been slow down at a higher level?④The IL-2of the Ea group was significantly lower than thde other groups at the3r day (P<0.05)，but it was higher than the BMSCs group and the control group at the5thdhay (P<0.05). At the14t day, it was significantly higher than the other groups(P <0.05). Conclusion：1, Electroacupuncture combined with BMSCstransplantation can inhibit AQP-4expression and reduce the brain edema, and itcan create better environment for the BMSCs and promote survival andmigration of BMSCs.2. Electroacupuncture combined with BMSCs transplantation can increase the expression of AQP-9, and it can accelerate thedissipation of brain edema and promote the recovery of brain energymetabolism.3. Electroacupuncture combined with BMSCs transplantation caninhibit the MMP-9expression in the early transplantation to reduce brainedema，and it can promote the speed of the repair in the recovery peirod.4.BMSCs transplantation can cause a significant irse in IL-2, and it hint that theBMSCs transplantation may induce gratf versus host reaction.Electroacupuncture combined with BMSCs transplantation can reduce the IL-2to ease of transplant rejection. |
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