Electroacupuncture Promotes Axon Regrowth By Attenuating MAIs-induced Rho A/ROCK Pathway In Cerebral Ischemia-reperfusion Rats

ObjectiveCerebral ischemia reperfusion injury(CIRI)has complicated pathogenesis.Central inhibition of microenvironment in rats with cerebral ischemia-reperfusion injury,Rho A/rock-mediated inhibition of nerve regeneration pathway and neuroplasticity protein are closely related,which is one of the research hotspot.In this paper,the effects of electroacupuncture on central inhibitory microenvironment,Rho A/ROCK nerve regeneration pathway and neuroplasticity protein in rats with CIRI were investigated to provide experimental basis for clinical treatment of ischemic stroke.MethodsRats were randomly divided into following four groups:Ischemia/reperfusion group(I/R),sham operation group(Sham),electroacupuncture group(EA),and ROCK inhibitor Fasudil group(Fasd).In this experiment,the modeling was carried out several times.75 experimental rats were shared,64 rats were successfully modeled,16 rats per group.Rats in I/R group received MCAO for 2 h,followed by reperfusion for 7d.Rats in Sham group received the same surgical procedures as those in I/R group,but no suture was inserted into the ICA.In EA group,rats received electroacupuncture(EA)therapy at GV20 and left ST36 with disposable,sterile acupuncture needles,then fixed these needles with two electrodes,with dilatational wave stimulation at a frequency of 2/100 Hz(intensity,1 mA)for 20 min using an EA device.Rats in Fasd group received Fasudil HCl by intraperitoneal injection once daily at a dose of 15 mg/kg.Rats in EA group,and Fasd group received the first treatment at the onset of reperfusion,and they were treated once a day for a total of 7 days.No other treatments were given in Sham group and I/R group in addition to grabbing stimuli once a day.TTC staining was used to calculate the volume of cerebral infarction,Garcia JH's neurological function test was carried out in the 7th day after the surgery to evaluate the neurological function,and the morphological changes of hippocampal neurons were observed by transmission electron microscopy(TEM).Myelin-associated inhibitors((MAIs)(NogoA,MAG,OMgp,and NgR),Rho A/ROCK pathway-related molecules(Rho A,ROCK2,MLC1,MYPT1)and neuroplasticity related proteins(GAP-43,BDNF,Synaptophysin)in the central microenvironment were detected by Western blot,immunofluorescence,and real-time fluorescence quantitative PCR.To verify the scientific hypothesis that electroacupuncture can negatively regulate the Rho A/ROCK neural regeneration inhibitory pathway induced by myelin related inhibitors,promote the expression of neuroplasticity related proteins,and attenuate cerebral ischemia reperfusion injury.Results1.Electroacupuncture can improve the cerebral infarct size and neurobehavioral in model of MCAOTTC staining indicated that the infarct size in I/R group was significantly larger than Sham group(P<0.001),and EA or Fasudil treatment could obviously reduce the infarct size after cerebral I/R(P<0.01).Moreover,Garcia JH's neurological scoring indicated that compared with the Sham group,the scores of I/R group declined significantly(P<0.001),while EA or Fasudil treatment could increase the scores obviously(P<0.001).No statistical significance was found between EA group and Fasd group on the infarct size and Garcia JH's scoring.The above results indicated that EA could reduce the volume of cerebral infarction and improve the nerve function.2.Electroacupuncturecan ameliorate the ultrastructure of the hippocampus in cerebral I/RTEM of brain hippocampus neurons showed that in the sham group,the cell membrane was clear and complete,and the nucleus(N)was large and round,dominated by euchromatin;Mitochondria distributed evenly and the membranes were clear.Rough endoplasmic reticulum(RER)was slightly dilated and ribosomes were attached to the surface.However,in the I/R group,the nucleus was irregular,with serrate edges and a small amount of heterochromatin clumped and scattered.Mitochondria were abundant with severe swelling,matrix electron density decreased,cristae expansion and shortening,and some outer membrane disintegration.RER was moderately dilated and the ribosomes on the surface were partially detached.While these changes were obviously improved by electroacupuncture treatment or intraperitoneal injection of Fasudil.In the EA group,the cell membrane was clear and complete without obvious damage,and the nucleus was large,round,and clear.Mitochondria were severely swollen and cristae expanded.RER expanded slightly and some ribosomes fell off.And in the Fasd group,the cell membrane was clear and complete;The nucleus was large and round;Mitochondria were moderately swollen,and nearly half of mitochondrial membranes were damaged.RER expanded slightly and ribosomes were attached on the surface.Golgi apparatus(Go)expanded slightly.These results indicated that EA treatment can reduce cerebral I/R injury by improving the ultrastructure of ischemic neurons.3.Electroacupuncture can improve the unfavorable milieu by attenuating the expression of MAIs in cerebral I/RNogoA,MAG,and OMgp are the main MAIs proteins existing in CNS.The results of western blot showed that the expression of NogoA in the I/R group was significantly higher thanthe sham group(P<0.001),OMgp,MAG,and NgR expression showed an obvious upward trend(P>0.05).Additionally,EA treatment significantly decreased the levels of OMgp,NogoA,and NgR(P<0.05),but increased MAG slightly(P>0.05).Immunofluorescence results showed that the expression of NogoA,and MAG in the I/R group was significantly higher than in the Sham group(P<0.01).Additionally,EA significantly decreased the expression of NogoA after cerebral I/R(P<0.05),and the level of MAG showed an upward trend after EA treatment(P>0.05).Real-time PCR showed that in contrast to the sham group,in the I/R group,and the mRNA level of MAG,OMgp and NgR showed an upward trend(P>0.05),and EA treatment significantly reduced the mRNA level of NgR(P<0.05),and partially decreased the mRNA level of MAG,OMgp and NogoA(P>0.05).Overall,the above results showed that EA abrogated the unfavorable milieu by attenuating the expression of MAIs in cerebral I/R.4.Electroacupuncture attenuated RhoA/ROCK-mediated neural regeneration inhibition pathway in cerebral I/RThe Rho A/ROCK signaling pathway mediates inhibitory signaling and is an important pathway to block central nerve regeneration.For the western blot,we found that compared with the Sham group,in the I/R group,the protein levels of ROCK2 and myosin phosphatase target subunit-1(MYPT1)increased significantly(P<0.05,P<0.01),Rho A and MLC1 showed an upward trend(P>0.05),while EA and Fasudil treatment significantly lowered ROCK2,MYPT1,and MLC1 expression(P<0.05),there was no significant difference between EA and Fasd groups.In addition,Immunofluorescence also revealed that compare with the Sham group,the expressions of Rho A and ROCK2 increased obviously in I/R group(P<0.01),this upward trend can be attenuated significantly through EA or Fasudil(P<0.05).Moreover,the mRNA expression of Rho A,ROCK2,MLC1,and MYPT1 was not significantly changed after cerebral I/R injury(P>0.05),while EA treatment can significantly inhibit the mRNA expression of ROCK and MLCl(P<0.05),and in contrast to the I/R group,the mRNA level of MYPT1 in EA group showed an obvious decline trend(P>0.05).5.Expression of neuroplasticity related proteins(GAP43,BDNF,Synap)in CIRI rat brain tissues by electroacupunctureNeuroplasticity is an important basis for brain function rehabilitation.The western blot results showed that compared to the sham group,in the I/R group,the level of GAP43,and BDNF was significantly lower(P<0.01),and Synap expression was not significantly changed.Additionally,EA treatment significantly increased the levels of GAP43 and BDNF(P<0.01,P<0.05),and the level of Synap did not significantly change after EA treatment(P>0.05).Immunofluorescence results also showed that EA increased the expression of BDNF in the region of ischemic penumbra cortex after cerebral I/R(P<0.05).Moreover,we also determined the mRNA levels of GAP43,BDNF,and Synap.In contrast to the sham group,in the I/R group,the mRNA level of GAP43 and Synap was lower(P<0.05),and BDNF expression showed a downward trend(P>0.05).EA or Fasudil could partially increase the mRNA level of GAP43 and BDNF(P>0.05),and there was no significant difference between EA group and Fasd group.Overall,the above results showed that EA and Fasudil promoted neural regeneration by upregulating the expression of GAP43,and BDNF in cerebral I/R.Conclusion:1.After 7 days of CIRI,infarct lesions were observed in the blood supply area of the right MCA of rats in the model group,and the ultrastructure of neurons in the hippocampus on the injured side was seriously damaged,resulting in low neurological function score.Electroacupuncture at GV20 and ST36 can educe the cerebral infarct size,improve neurobehavioral,and reduce neuron injury in model of MCAO.2.After 7 days of CIRI,the expression of inhibitory factors NogoA and MAG in the central microenvironment of brain tissues of rats in the model group was significantly increased.Electroacupuncture at GV20 and ST36 can improve the unfavorable milieuby attenuating the expression of MAIs(NogoA,OMgp,NgR)in cerebral I/R rats.3.After 7 days CIRI,the expression of RhoA/ROCK path-related proteins(RhoA,ROCK2 and MYPT1),an inhibitory signal of nerve regeneration,was significantly increased in brain tissue.Electroacupuncture at GV20 and ST36 can negatively regulate RhoA/ROCK-mediated neural regeneration inhibition pathway in cerebral I/R rats.4.After 7 days CIRI,the expression of neuroplasticity related proteins(GAP43,BDNF and Synap)in brain tissue was low.Electroacupuncture at GV20 and ST36 can promote neural regeneration by upregulating the expression of GAP43,and BDNF in cerebral I/R rats.Innovation:Based on the previous work,this study investigated the effect of EA intervention on axonal regeneration signals after cerebral I/R injury,mainly from three aspects:the inhibitory factors in the central injury microenvironment,the inhibitory signal RhoA/ROCK pathway of axonal regeneration and the expression of neuroplasticity related proteins.Moreover,the functional differences between electroacupuncture at GV20 and ST36 acupoints combination and the positive control drug fasudil hydrochloride were compared,which was innovative in scientific thinking and research content.This study focused on the MAIs-mediated Rho A/ROCK nerve regeneration inhibition pathway in CIRI rats and the intervention of electroacupuncture at GV20 and ST36.Rho A/ROCK neural regeneration inhibition pathway mediated by MAIs was selected.CIRI rat model was established by suture method,and Garcia JH's neurological scoring were used for neurobehavioral evaluation.The pathomorphological changes of brain tissue were observed by TTC staining and TEM.Expressions of MAIs and its receptor(NogoA,MAG,OMgp,NgR),Rho A/ROCK pathway-related proteins(Rho A,ROCK2,MLC1,MYPT1)and neuroplasticity related proteins(GAP43,BDNF,Synap)in brain tissues were detected by Western Blot,immunofluorescence,and quantitative real-time PCR.To verify that the scientific hypothesis that electroacupuncture at GV20 and ST36 can improve the central injury microenvironment,inhibit Rho A/ROCK-mediated neural regeneration inhibition pathway,and promote axonal regeneration,so as to alleviate of CIRI.It provided new ideas for the prevention and treatment of cerebral I/R by electroacupuncture.