| Objective:This paper aims to investigate the effect of dense and sparse wave forms of electro-nape-acupuncture on neuron apoptosis and autophagy related proteins in VD rats,and to optimize the treatment of VD with electro-nape-acupuncture,and to provide objective experimental evidence for clinical application.Methods:82 healthy male SD rats were randomly divided into 16 in sham operated group and 66 in model building group.Rats in the model building group were treated with modified Pulsinelli four-vessel occlusion method to prepare a rat VD model of global cerebral ischemia.The sham operated group only exposed four blood vessels and did not blot vessels.The 48 VD rats were randomly divided into 3 groups,the model group,the electro-nape-acupuncture dense wave group(dense wave group)and the electro-nape-acupuncture sparse wave group(sparse wave group),and added the sham operated group,it was just 4 groups,there are 16 rats in each group.The treatment was started with the eighth day after surgery.The dense wave group applied the dense wave(50Hz)electro-acupuncture at 'fengchi' and'gongxue' points,and the sparse wave group used sparse wave(2Hz)electro-acupuncture at 'fengchi' and 'gongxue' points.It should continue to electro-acupuncture stimulate points for 30 minutes,once a day,continue treatment for 14 days.The sham operated group and the model group were given the same conditions to catch,bind and fix without any perform treatment.After the treatment,a Y-maze test was performed to examine the behavioral changes in the rats.HE staining was used to observe the morphological changes of rat hippocampus.The TUNEL was used to detect the neuronal apoptosis in rat hippocampus.The transmission electron microscopy was used to detect neuronal autophagy in rat hippocampus.The Western blot was applied to detect Bcl-2,Bax,LC3,Beclin 1,p62,PI3K,p-AKT,p-mTOR proteins expressions in rat hippocampus.Results:1.The results of the Y-maze test:compared with the sham operated group,the spontaneous alternation rate of the model group was significantly decreased(P<0.01)and the stay time of the new arm in the model group was significantly reduced(P<0.01).Compared with the model group,the spontaneous alternation rate of the dense wave group and the sparse wave group was significantly increased(P<0.05,P<0.01)and the stay time of the new arm was significantly prolonged(P<0.01).Compared with the dense wave group,the spontaneous alternation rate of the sparse wave group was significantly increased(P<0.05),and there was no significant difference in the stay time of the new arms between the sparse wave group and the dense wave group(P>0.05).The time ratio of rats in each group compared with the random probability of 33.33%,the stay time of the new arm in the dense wave group was prolonged without significant difference(P>0.05),while the stay time of the new arm in the sparse wave was significantly prolonged(P<0.05).2.The HE staining results:the neuron cells in the rat hippocampus of the model group were poor in morphology,irregular in shape,arranged disorderly loose,interstitial edem,nuclear condensation,the gap between the neuron and the surrounding tissue was increased and loss of neurons obviously.The neurons in the hippocampus of the dense wave group and the sparse wave group were relatively intact and arranged in a uniform manner,the interstital edema was significantly reduced,the nucleus pyknosis was reduced,the degree of deep staining was reduced,the gap between the neuron and the surrounding tissue became smaller and loss of neurons.3.The results of TUNEL:compared with sham operated group,the number of apoptosis cells and the apoptotic index of neuron in the model group were increased significantly(P<0.01).Compared with the model group,the number of apoptosis cells and the apoptotic index of neuron in the dense wave group and the sparse group were decreased significantly(P<0.01).Compared with the dense wave group,the number of apoptosis cells and the apoptotic index of neuron in the sparse wave group were significantly decreased(P<0.05).4.The results of Western blot detection of apoptosis related proteins:compared with the sham operated group,the Bcl-2 protein expression was decreased(P<0.01),the Bax protein expression was increased(P<0.01)and the Bcl-2/Bax ratio was reduced(P<0.01)in the hippocampus of the model group rats.Compared with the model group,the Bcl-2 protein expression was increased(P<0.01),the Bax protein was expression decreased(P<0.01)and the Bcl-2/Bax ratio was increased(P<0.01)in the hippocampus of the dense wave group and the sparse group rats.Compared with the dense wave group,the Bcl-2 protein expression was increased(P<0.01),the Bax protein expression was decreased(P<0.01)and the Bcl-2/Bax ratio was increased(P<0.05)in the hippocampus of the sparse wave group rats.5.The transmission electron microscopy results:the hippocampal neurons of the model group rats exhibited many bilayer membrane structures,encapsulated cytoplasm,mitochondria and other oraganelles,forming autophagosomes.A small amount of autophagosomes were observed in the hippocampal neurons in the dense wave group and sparse wave group.6.The results of Western blot detection of autophagy associated proteins:compared with the sham operated group,the LC3-?/LC3-? ratio was increased,Beclin 1 protein expression was significantly increased and the p62 protein expression was decreased(P<0.01)in the hippocampus of the model group rats.Compared with the model group,the LC3-?/LC3-? ratio was decreased,Beclin 1 protein expression was significantly decreased and the p62 protein expression was increased(P<0.01)in the hippocampus of the dense wave group and sparse wave group rats.Compared with the dense group,the LC3-?/LC3-? ratio was decreased and the p62 protein expression was inceased(P<0.01)in the hippocampus of the sparse wave group rats.7.The results of Western blot detection of PI3K,p-AKT,p-mTOR protein:compared with the sham operated group,the PI3K,p-AKT and p-mTOR protein expressions were significantly increased in the hippocampus of the model group rats(P<0.01).Compared with the model group,the PI3K,p-AKT and p-mTOR protein expressions were significantly increased in the hippocampus of the dense wave group and sparse wave group rats(P<0.01).Compared with the dense wave group,the PI3K,p-AKT and p-mTOR protein expression were obviously significantly increased in the the hippocampus of the sparse wave group rats(P<0.01).Conclusion:1.Electro-Nape-Acupuncture can enhance spatial memory ability of VD model rats,and the sparse wave group is superior to the dense group.2.Electro-Nape-Acupuncture can reduce the damage of the hippocampal neurons in VD rats,play a neuroprotective role,and the sparse wave group is superior to the dense group.3.Electro-Nape-Acupuncture can increase the Bcl-2 protein expression,decrease the Bax protein expression,increase the Bcl-2/Bax ratio,reduce the nerve cell apoptosis index of the hippocampus tissue in VD rats,plays a role in inhibiting neuronal apoptosis,and the sparse wave group is superior to the dense group.4.Electro-Nape-Acupuncture can reduce LC3-?/LC3-? ratio,decrease Beclin 1 protein expression,increase p62 protein expression in hippocampus of VD model rats,plays a role in inhibiting hyperautophagy,and the sparse wave group is superior to the dense group.5.Electro-Nape-Acupuncture may increase the activity of PI3K/AKT/mTOR signaling pathway through up-regulating the related protein expression of PI3K,p-AKT and p-mTOR in VD model rats hippocampus,and thus adjust the apoptosis and autophagy of VD rat hippocampus neurons,reduce neuronal damage,improve the cognitive abiliy of VD rats,and the sparse wave group is superior to the dense group. |
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