| Objective: In this study,we use a mouse model of bilateral common carotid artery occlusion(BCCAO)to mimic prosencephalic ischemia/reperfusion injury.Before cerebral ischemia they were subjected to repetitive electroacupuncture(EA)stimulation.And then to dectect the expression of Rheb 、 mTOR 、 p-mTOR 、LC3-Ⅱand caspase-3 in the cerebral hippocampus and to evaluate the Neurological behavior scores(N BS)and the number of apoptotic neurons at 72 h after reperfusion.Our study was to explore the role of mTOR signaling pathway in cerebral ischemia/reperfusion and the mechanism of EA preconditioning-induced neuroprotection.It can provide a new theoretical basis for clinical prevention and treatment of ischemic cerebral disease.Methods: Totally 36 three-month male C57/BL6 mice(weighing 22-25g)were randomly divided into three groups(n=12):sham(S)group,ischemia/reperfusion(I/R)group and electroacupuncture preconditioning(EA)group.In this study,we use a mouse model of 15 minutes bilateral common carotid artery occlusion(BCCAO)and then reperfusion 72 h to mimic cerebral ischemia/reperfusion injury.In the sham group,both common carotid arteries of mice were only exposed but not clipped;In the I/R group,mice were subjected to 15 minutes bilateral common carotid artery occlusion(BCCAO)and then reperfusion 72h;In the EA group,before being subjected to BCCAO,the acupoint“Baihui”of mice was stimulated at an intensity of 1 mA and a frequency of 2/15 Hz for 30 minutes for 5 consecutive days by the electroacupuncture.The Neurological behavior scores(NBS)of all the mice were evalua ted at the 72 h after reperfusion.Neuronal morphology and apoptosis in the hippocampus CA1 were assessed by hematoxylin-eosin(HE)staining and terminal deoxynucleotide transferase-mediated d UTP nick-end labeling(TUNEL)staining,and the expression of Rheb、mTOR、p-mTOR(ser2448)、LC3-Ⅱand caspase-3 were analyzed by Western blot at 72 hours after reperfusion.Results: 1.At 72 hours after cerebral reperfusion,the Neurological behavior scores(NBS)in the group I/R was much higher in comparison with the sham group(P<0.05).Compared with the I/R group,the Neurological behavior scores(N BS)in the group EA was decreased(P<0.05).2.In this study,hematoxylin-eosin staining(HE)was used to observe hippocampal CA1 neuronal morphology and we use Tunel staining(TUNEL)to count the number of apoptotic cells.In the sham group,the hippocampal CA1 neuronal morphology were almost normal,and only a small amount of degenerate neuron was observed.The neuronal morphology in the I/R group was much worse than the sham group.However,in comparison with the group I/R,the neuronal degeneration was less severe and the placement was less disordered in the EA group.In the sham group,barely any apoptotic cells(TUN EL-positive cells)were detected in hippocampus CA1 region,but lots of apoptotic cells were seen in the I/R group.Compared with the sham group,the number of apoptotic cells in the I/R group was remarkably higher(P<0.05).But in comparison with the group I/R,the number of apoptotic cells in the EA group decreased(P<0.05).3.Western blot was used to detected the expression of Rheb、mTOR、p-mTOR、LC3-Ⅱand caspase-3.Compared with sham group,the level of Rheb、mTOR、p-mTO R、LC3-Ⅱand caspase-3 expression were upregulated significantly in the I/R group(P<0.05).And In comparison with the group I/R,the expression of Rheb、mTOR and p-mTOR were increased but the expression of LC3-Ⅱand caspase-3 were decreased in the EA group(P<0.05).Conclusion: 1.The level of Rheb、 mTOR、p-mTO R、 LC3-Ⅱexpression in the cerebral hippocampus increased after cerebral ischemia/reperfusion.It suggested that mTOR and autophagy may play an important role in cerebral ischemia / reperfusion.2.EA preconditioning can upregulate the expression of Rheb、mTOR、p-mTO R and downregulate the expression of LC3-Ⅱ、caspase-3 in the cerebral hippocampus,which can also reduce the number of apoptotic neurons.It demonstrated that EA preconditioning-induced neuroprotection may be caused by inhibiting autophagy via activating mTOR. |
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