| Objective:To explore whether electroacupuncture at "GV20" and "GV24" acupoints can promote the transcriptional expression of memory engram genes and reduce synaptic morphological damage by regulating hippocampal histone acetylation,so as to improve the acquisition and maintenance of spatial memory in rats with vascular cognitive impairment(VCI).Methods:Seventy Sprague Dawley(SD)rats were selected as the experimental subjects,which were randomly divided into sham operation group(n=10)and real operation group(n=60).The VCI model was made by bilateral common carotid artery occlusion(Two Vessel Occlusion,2VO)in the real operation group.In the sham operation group,only bilateral common carotid arteries were exposed without occlusion.The qualified rats of real operation group were randomly divided into model group,electroacupuncture group,control virus group,control virus electroacupuncture group,inhibition virus group and inhibition virus electroacupuncture group,with 10 rats in each group.Viruses were injected into the hippocampal CA1 region of the latter 4 groups,sh RNA(scramble/scramble)virus was injected into the control virus group and the control virus electroacupuncture group,and sh RNA(CBP/P300)virus was injected into the inhibition virus group and the inhibition virus electroacupuncture group.Intervention methods: The electroacupuncture group,control virus electroacupuncture group and inhibition virus electroacupuncture group were treated with electroacupuncture at "GV20" and "GV24" acupoints,the parameters were dense wave,frequency 1-20 Hz,stimulation intensity 1m A,30 min each time,2 days off every 5 days for a total of 4 weeks.The sham operation group,model group,control virus group and inhibition virus group were caged and reared without any treatment after grabbing 30 min under the same conditions.All rats were performed Barnes maze before intervention and Morris water maze after intervention to detect the acquisition and maintenance of spatial memory.Golgi staining was used to detect the density of dendritic spines in hippocampal CA1 region.Real-time fluorescence quantitative PCR was used to detect the transcription of hippocampal CBP(CREB Binding Protein),P300(E1A Binding Protein P300),c-fos,Egr-1(Early growth response gene 1)and Arc(Activity-regulated cytoskeletal).Western blotting was used to detect the acetylation of histone H3 and H4 and the expression of CBP,P300,c-fos,Egr-1and Arc in hippocampus.Results:(1)Effects of electroacupuncture on transcription and expression of histone acetyltransferase CBP and P300 in the hippocampus of VCI rats: The m RNA and protein levels of CBP and P300 in the model group decreased compared with the sham operation group(P<0.05),and increased in the electroacupuncture group compared with the model group(P<0.05).Compared with the control virus group,the m RNA and protein levels of CBP and P300 in the inhibition virus group were decreased(P<0.05).The m RNA and protein levels of CBP and P300 in the inhibition virus electroacupuncture group were lower than those in the control virus electroacupuncture group(P<0.05).There was no difference between the model group and the control virus group,and between the electroacupuncture group and the control virus electroacupuncture group(P>0.05).(2)Effects of electroacupuncture on the acetylation modification of histone H3 and H4 in the hippocampus of VCI rats: The acetylation levels of histone H3 and H4 in the model group decreased compared with the sham operation group(P<0.05),and increased in the electroacupuncture group compared with the model group(P<0.05).Compared with the control virus group,the acetylation levels of histone H3 and H4 in the inhibition virus group were decreased(P<0.05).The acetylation levels of histone H3 and H4 in the inhibition virus electroacupuncture group were lower than those in the control virus electroacupuncture group(P<0.05).There was no difference between the model group and the control virus group,and between the electroacupuncture group and the control virus electroacupuncture group(P>0.05).(3)Effect of electroacupuncture regulating histone acetylation modification on acquisition and maintenance of spatial memory in VCI rats:Barnes maze test before intervention showed that compared with the sham operation group,the escape latency and the number of entering the wrong hole in the other groups were increased(P<0.05),while the percentage of time in the target quadrant and the number of contact with the target hole were decreased(P<0.05).There was no difference among the groups except the sham operation group(P>0.05).After intervention,Morris water maze test showed that compared with the sham operation group,the escape latency in the model group was prolonged(P < 0.05),the percentage of time in the target quadrant and the number of crossing the platform were decreased(P<0.05).Compared with the model group,the escape latency in the electroacupuncture group was shortened(P<0.05),and the percentage of time in the target quadrant and the number of crossing the platform were increased(P<0.05).Compared with the control virus group,the escape latency in the inhibition virus group was further prolonged(P<0.05),the percentage of time in the target quadrant and the number of crossing the platform were further decreased(P<0.05).Compared with the control virus electroacupuncture group,the escape latency was prolonged(P<0.05),and the percentage of time in the target quadrant and the number of crossing the platform were decreased(P<0.05)in the inhibition virus electroacupuncture group.There was no difference between the model group and the control virus group,and between the electroacupuncture group and the control virus electroacupuncture group(P>0.05).(4)Effect of electroacupuncture regulating histone acetylation modification on synaptic morphology in hippocampus CA1 region of VCI rats: Golgi staining experiment showed that compared with the sham operation group,the density of dendritic spines in the model group decreased(P<0.05).Compared with the model group,the density of dendritic spines in the electroacupuncture group was increased(P<0.05).Compared with the control virus group,the dendritic spine density decreased in the inhibition virus group(P<0.05).Compared with the control virus electroacupuncture group,the dendritic spine density decreased in the inhibition virus electroacupuncture group(P<0.05).There was no difference between the model group and the control virus group,and between the electroacupuncture group and the control virus electroacupuncture group(P>0.05).(5)Effect of electroacupuncture regulating histone acetylation modification on transcription and expression of c-fos and Egr-1 in hippocampus of VCI rats: The m RNA and protein levels of c-fos and Egr-1 in the model group decreased compared with the sham operation group(P<0.05),while the m RNA and protein levels of c-fos and Egr-1 in the electroacupuncture group increased compared with the model group(P<0.05).Compared with the control virus group,the m RNA and protein levels of c-fos in the inhibition virus group decreased(P<0.05),but the m RNA and protein levels of Egr-1 had no difference(P>0.05).Compared with the control virus electroacupuncture group,the m RNA and protein levels of c-fos in the inhibition virus electroacupuncture group decreased(P<0.05),but the m RNA and protein levels of Egr-1 had no difference(P>0.05).There was no difference in the m RNA and protein levels of c-fos and Egr-1 between the model group and the control virus group,and between the electroacupuncture group and the control virus electroacupuncture group(P>0.05).Conclusion:Electroacupuncture at "GV20" and "GV24" acupoints can up-regulate the acetylation level of histone H3 and H4 in hippocampus,promote the transcription and expression of memory engram gene c-fos,reduce synaptic morphological damage,which is one of the mechanisms to improve the acquisition and maintenance of spatial memory in VCI rats. |
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