Mechanism Of Electroacupuncture On Myelin Repair By Regulating MiR-219 Following Ischemic Stroke

ObjectiveEstablished the Middle Cerebral Artery Occlusion（MCAO）model of rats to observing the effect of electroacupuncture（EA）on the expression of miR-219 and myelin repair,and to exploring the possible mechanism.Method1.Healthy male Sprague-Dawley（SD）rats were randomly divided into modeling group and sham group.The dead and unqualified rats were excluded according to the Zea Longa test-the scoring range is 0-4 points,1-3 points indicate successful modeling,0points indicate failure of modeling,and 4 points indicate excessive damage-after 24h following MCAO surgery.Then the rats with successful modeling were randomly divided into a model group（MCAO）and a treatment with EA group（MCAO+EA）.The rats in the MCAO+EA group were treated with EA for 30min,once a day for 9 days.The sham and MCAO groups did not receive any intervention,and the feeding conditions were consistent with the MCAO+EA group.The Rota-rod Test and Foot-fault Test were used to evaluated the recovery of neurological function in rats after EA treatment.Cerebral infarction volume was assessed by 2,3,5-triphenyltetrazolium chloride（TTC）staining.The morphology and thickness of myelin sheath and the number of myelinated axons in the corpus callosum on the ischemic side were observed by electron microscopy.quantitative real-time polymerase chain reaction（q RT-PCR）and Western Blot（WB）were used to detect the expression of miR-219 and myelin repair-related proteins in the ischemic penumbra of cerebral cortex.To clarify the effect of EA on the expression of miR-219 and myelin repair in MCAO rats.2.Healthy male SD rats were randomly divided into negative control group（MCAO+NC）,virus group（MCAO+AAV）and EA+virus group（MCAO+EA+AAV）.For MCAO+AAV group and MCAO+EA+AAV group,adenovirus AAV-miR-219 was injected into the lateral ventricle of the brain,and the target brain area（bregma was the zero point,1.5;-0.8;3.8）,the speed was 1μl/min,the dose was 5μl,and the virus titer was 1×10¹²GC.The virus titer of the MCAO+NC group was 0.5×10¹²GC,and the other conditions were consistent with the MCAO+AAV group.MCAO surgery was performed after 24h of completion of the lateral ventricular injection.The dead and unqualified rats were excluded by the Zea Longa test after 24h of MCAO surgery.The MCAO+EA+AAV group was treated with EA,once a day,30min each time,for 9 days.The Rota-rod Test and Foot-fault Test were used to evaluate the recovery of neurological function in rats.The morphology and thickness of myelin sheath and the number of myelinated axons were observed by electron microscopy.q RT-PCR and WB were used to detected the expression of miR-219and myelin repair-related proteins in the ischemic penumbra of cerebral cortex.To clarify the mechanism of EA.Result1.EA improved brain injury in MCAO rats.The results of neural behavioral tests and TTC staining showed that EA significantly improved the neurological deficit of MCAO rats,enhanced exercise endurance and motor coordination ability,and reduced the cerebral infarct volume.2.EA promoted myelin repair in MCAO rats.The results of electron microscopy showed that the myelin sheath in MCAO group was severely damaged,the thickness of myelin sheath was significantly reduced,the G value was increased,and the number of myelinated axons was decreased.However,EA increased the thickness of myelin sheath and the number of myelinated axons,and decreased the G value.3.EA up-regulated the expression of miR-219 and MBP,but inhibited the expression of hes5.The results of q RT-PCR showed that miR-219 was significantly decreased in the MCAO group,while EA up-regulated the expression of miR-219.The results of WB showed that hes5 was significantly increased and MBP was obviously decreased in MCAO rats,while EA inhibited the hes5 and promoted the MBP.4.miR-219 may be involved in EA promoting myelin repair in MCAO rats.OLN-93cells were used to detect the knockdown efficiency of AAV-miR-219.The results of q RT-PCR showed that the expression of miR-219 in OGD+AAV group was significantly lower than that in OGD+NC group.On this ground,AAV-miR-219 was injected into the lateral ventricle and EA was performed.The results of q RT-PCR showed that the expression of miR-219 in MCAO+AAV group was significantly lower than that in MCAO+NC group.Compared with MCAO+EA group,the expression of miR-219 in MCAO+EA+AAV group had a tendency to decrease,but the difference was not statistically significant（P=0.0582）.The results of WB showed that the expression of MBP in MCAO+EA+AAV group was significantly lower than that in MCAO+EA group,while the expression of hes5 tended to increase,but the difference was not statistically significant（P=0.1077）.Electron microscopy revealed a significant increase in G value and a significant decrease in the number of myelinated axons in the MCAO+EA+AAV group compared with the MCAO+EA group.The results of behavioral tests showed that compared with the MCAO+EA group,the MCAO+EA+AAV group had obvious neurological deficits.Conclusion1.EA improved the neurological deficit of MCAO rats,promoted the recovery of motor function,reduced the cerebral infarct volume,and played a neuroprotective role.2.EA up-regulated the expression of miR-219 and MBP,but down-regulateed the expression of hes5.EA also increased the myelin sheath thickness and the number of myelinated axons in MCAO rats,and promoted myelin sheath repair.3.Knockdown of miR-219 reversed the effects of EA in up-regulating MBP,increasing the number of myelinated axons,and improving neurological deficits.However,knockdown of miR-219 does not reverse the effect of EA on inhibiting hes5 and improving motor ability in MCAO rats.Therefore,upregulation of miR-219 may be the mechanism that EA promoting myelin repair in MCAO rats.