| Objective:To investigate the roles of electroacupuncture at "Baihui"(DU20)and "Shenting"(DU24)on learning and memory function and resting-state functional magnetic resonance imaging(rs-fMRI)in ischemic stroke rats.On the basis of neuroimaging,the plasticity changes of synaptic structure and function in brain regions were explored,and the molecular biological mechanism was further studied based on microRNA(miRNA)omics.Methods:1.The SPF male Sprague dawley(SD)rats were divided into sham operation group and ischemia model group according to the random number table method.Then,according to the preoperative neurological deficit score and behavioral test results,the rats were divided into three groups,the model group,the electroacupuncture group(EA)and the non-acupoint group.2.Rats underwent middle cerebral artery occlusion(MCAO)and received EA at DU20 and DU24 acupoints or non-acupoints treatment.The parameter of EA stimulation were dense disperse wave of 2/20 Hz,6V,30 minutes each time,once a day for 14 days.3.The learning-memory ability of all rats was test by the platform before treatment,which was measured by the platform and Morris Water Maze after treatment.4.The animal magnetic resonance imaging(MRI)include resting state-functional MRI and regional homogeneity(ReHo)was employed to analysis the variety of neuronal activity with all rats.5.The morphology of neuronal synapses was observed by Golgi staining,and long-term potentiation was detected by field potential electrophysiology in each group.6.The western blotting was used to measure the expression and phosphorylation of learning-memory related proteins.7.The miRNAs were used to detect the differential expression of miRNAs,as well as the prediction of target gene and the analysis of signal transduction pathways.Real-time quantitative PCR was used to verify the expression of miRNAs.Results:1.There was no significant difference in the platform test between the model group,the EA group and the non-acupoint group before treatment(P>0.05).After treatment,compared with the model group and the non-acupuncture group,the incubation period of the EA group was shortened in the platform test and Morris water maze(P<0.05).2.We found that there were infarctions in hippocampus,inner olfactory cortex,motor cortex,sensory cortex,dorsal thalamus and striatum and other brain areas of ischemic rats,and which were decreased by electro-acupuncture treatment at DU20 and DU24 acupoints(P<0.05).3.The rs-fMRI analysis of the ischemic stroke rats showed that the BOLD signal was decreased in bilateral amygdala,bilateral hippocampus,bilateral island cortex,olfactory cortex,bilateral pear cortex,bilateral sensory cortex,bilateral motor cortex,bilateral cingulate gyrus,bilateral dorsal thalamus,left auditory cortex and left striatum(P<0.005),which was increased in bilateral amygdala,bilateral hippocampus,right island cortex,right inner olfactory cortex,right piriform cortex and right sensory cortex after the treatment of the EA group(P<0.005),while increased in left amygdala,left piriform cortex and left striatum of the non-acupoint group(P<0.005).4.The synaptic plasticity was further analyzed in the hippocampal CA1 and entorhinal cortex.The number was decreased and shrinkage of dendritic spines in the model group at low power field.While the amount of branches,the number and density of dendritic spines were improved in the EA group at high power field(P<0.05).5.The synaptic functional plasticity test showed that the percentage of excitatory postsynaptic potential(fEPSP)was decreased in ischemic hippocampal CA3-CA1 and entorhinal cortex-hippocampal CA1 in ischemic stroke rats after HFS induction(P<0.05),and the expression and phosphorylation of the related learning and memory proteins were improved include the AMPA receptor and NMDA receptor in postsynaptic membrane(P<0.05).6.The expression of mRNAs in the ischemic hippocampus CA1 and entorhinal cortex showed that there were 48 miRNAs(P<0.05)that differential expression of miRNAs was more than 1.5 miRNAs in the ischemic hippocampus,in which 36 miRNAs were down-regulated and 12 miRNAs were up-regulated(P<0.05).While there were 90 miRNAs(P<0.05)that differential expression of miRNAs was more than 1.5 miRNAs in the entorhinal cortex,in which 70 miRNAs were down-regulated and 20 miRNAs were up-regulated(P<0.05).The expression of rno-miR-668 and rno-miR-3084b-5p were up-regulated in the ischemic hippocampus CA1 and entorhinal cortex,while they were down-regulated of rno-miR-28,rno-miR-10a,rno-miR-let-7b,rno-miR-425,rno-miR-7b,rno-miR-154,rno-miR-540,rno-miR-let-7f-2,rno-miR-186,rno-miR-219a,rno-miR-3072,rno-miR-344a,rno-miR-190b,rno-miR-100 and rno-miR-92a in the ischemic hippocampal CA1 and entorhinal cortex.7.The target gene prediction and signal transduction pathway were analyzed of miRNAs that the expression were up-regulated or down-regulated in ischemic hippocampal CA1 and entorhinal cortex,which showed that the cGMP-PKG signaling pathway(P<0.05)is related to learning-memory.It was confirmed that miR-219a down-regulated the key node PRKG2 target gene of cGMP-PKG signaling pathway by matics prediction and vitro test.8.It was found that miR-219a mimics could block the expression of miR-219a by electroacupuncture at DU20 and DU24 acupoints in vivo analysis,which also affect the phosphorylation of CREB in PRKG2 downstream,as well as to decrease the expression of the c-fos and c-jun protein that immediate early genes related learning-memory.Conclusion:1.Animals got EA treatment for 14 days showed a reduction in the infarct volumes,an increase of learning-memory ability and the BOLD signal Reho.These indicated that the improvement of learning-memory ability was associated with the function recovery of hippocampus and entorhinal cortex in stroke rats.2.Electroacupuncture at DU20 and DU24 acupoints could promoting the morphology recovery of hippocampus and entorhinal cortex in the ischemic stroke rats.Besides,the amplitude of LTP was increased in hippocampal CA3-CA1 and entorhinal cortex-hippocampal CA1,which promote the plasticity of the synaptic structure and function.3.The expression of miR-219a in stroke rats was inhibited by the treatment of electroacupuncture at DU20 and DU24 acupoints,and the target gene PRKG2 was up-regulated to improve the phosphorylation of CREB,which promoted the synthesis of learning-related proteins c-fos and c-jun. |
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