| bjective: To observe the effects of electro-acupuncture(Ea) stimulation combined with bone marrow mesenchymal stem cells(BMSCs) transplantation on the expression of myelin basic protein(MBP)、glutamate(Glu) and its receptors including N-methyl-D-aspartate receptor-2A(NMDAR2A) and glutamate receptor-2(Glu R2)in rats after intracerebral hemorrhage(ICH), and to explore the possible mechanism of combination therapy. Methods :(1) BMSCs culture: after the freeze BMSCs were recovered, passaged and induced, the cells concentration was adjusted to 2.5 x 107/m L for transplantation.(2) Build rat ICH model: ICH rats were replicated by injecting the collagenase and heparin into right caudate putamen, then evaluated the neurological severity degree three times by neurological severity score(NSS score) at 2h, 24 h, 48 h after operation, and the animal obtained nine points in any one or more as successful ICH model.(3) Animal grouping: the ICH rats were randomly divided into BMSCs group, normal saline(NS) group, Ea-BMSCs group and Ea group, and these groups were treated with 20 μl BMSCs, 20μl normal saline, 20 μl BMSCs combined electro-acupuncture(baihui acupoint and dazhui acupoint) and the simple electro-acupuncture therapy respectively. Every group was subdivided into 1,3,5,7 and 14 d five subgroups according to different treatment time. The NSS score was estimated and the blood serum was collected at the corresponding time point, then the rat was killed and its brain was kept.(4) MBP expression in serum was tested by ELISA.(5) Immunohistochemical staining was used to detected the expression of Glu and its receptors( Glu R2, NMDAR2A). Results:(1) The NSS scores of Ea-BMSCs group were lower than that of the NS group, Ea group, BMSCs group at 3d,5d,7d and 14d(P < 0.05). Compared with NS group at the same time point, the NSS scores of BMSCs group and Ea group decreased significantly at 3 d, 5 d, 7 d and 14 d(P < 0.05).(2) The expression of Glu in every group was increased at 1d and reached the peak at 3d, then decreased slowly. The expression of Glu in the Ea-BMSCs group was significantly lower than that of NS group, Ea group and BMSCs group at various time points(P <0.05). Compared with NS group, the expressions of Glu in the BMSCs group and Ea group reduced significantly at 3d, 5d, 7d(P<0.05).(3) The expression of NMDAR2 A in every group began to rise at 1d, and reached the peak at 3d, then decreased slowly. The expression of NMDAR2 A in Ea-BMSCs group declined obvious at 1-14d(P < 0.05) when compared with NS group, and declined significantly at 3d, 5d,7d(P < 0.05) when compared with Ea group, and reduced significantly at 3d, 5d(P < 0.05) when compared with BMSCs group. the expression of NMDAR2 A in Ea- BMSCs group declined obvious at 1-14 d. Compared with the expression of NMDAR2 A in NS group, BMSCs group declined at 1d- 7d and Ea group declined significant at 3d-7d(P<0.05).(4) The expression of Glu R2 in each group increased slightly at 1d, and showed weakly positive expression. But over time, the positive Glu R2 expression gradually increased and reached the peak at 7d. The Ea-BMSCs group, compared with the BMSCs group, Ea group and NS group, had higher expression of Glu R2 at each time point(P<0.05). Compared with the expression of Glu R2 in NS group, BMSCs group and Ea group increased at each time point(P<0.05).(5)The content of MBP in the BMSCs group was higher than that of the Ea-BMSCs group at 5, 7, 14d(P <0.05). The content of MBP in the Ea group was lower than that of the Ea-BMSCs group at 1, 3d, but higher than that of the Ea-BMSCs group at 14d(P <0.05). Compared with the content of MBP in NS group, the Ea-BMSCs group reduced at 5, 7, 14 d and BMSCs group reduced at 14d(P <0.05), and Ea group decreased at 5, 7, 14 d(P<0.05). Conclusion:(1)Electroacupuncture combined with BMSCs transplantation can reduce the expressions of Glu and NMDAR2 A surrounding bleeding area and alleviate brain injury mediated by excitotoxicity better than the simple electroacupuncture stimulation or BMSCs transplantation.(2)Compared with the simple electroacupuncture stimulation or BMSCs transplantation, electroacupuncture combined with BMSCs transplantation promote more expression of Glu R2, and reduce Ca2+ inflow and alleviate brain injury mediated by calcium overload.(3) Electroacupuncture combined with BMSCs transplantation can decrease the content of MBP in serum more than the electroacupuncture stimulation or BMSCs transplantation, and it is helpful to reduce the demyelination reaction. |
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