Study On The Effect Of Electroacupuncture On Anti Oxidative Stress In Mice With Cerebral Ischemia Reperfusion Injury Based On Nrf2 Pathway

Objective:Through the observation of electroacupuncture on focal cerebral ischemia-reperfusion injury in mouse cerebral cortex after γ-GCS effect of protein and its mRNA expression,explores oxidative stress factor Nrf2 in electroacupuncture on cerebral ischemia-reperfusion injury effect and mechanism for clinical electro-acupuncture on cerebral ischemia-reperfusion injury of the mechanism to provide a new theoretical basis.Method:Using wild-type mice(WT)and the Nrf2 gene knockout mice(KO),group by random number table,divided into WT Sham group,WT model groups,WT electro-acupuncture group,KO Sham groups,KO model groups,KO electro-acupuncture group.Sham-operated group only to separate blood vessels in mice,not plug thread,remaining the same as model groups.Model group prepared by modified Longa thread right Middle cerebral artery occlusion in mice model of ischemic wire bolt after 2 h.Electric needle group after wire bolt for electric needle "baihui",and "dazhuil",which "baihui" points forward right,"dazhui"points about 30° oblique thorn,depth about 3-5mm,access electric needle,"baihuil" points received negative,"dazhui" points received cathode,added density wave,frequency 2/15Hz,to needle handle minor fibrillation,and points district local minor jitter for degrees,continuous treatment 15min.After cerebral ischemia-reperfusion 24h according to Julio’s behavioral neurological assessment evaluating the functional activity of mice;mouse brain weighed wet and dry weight,calculation of water content and TTC staining and determination of cerebral infarct volume.Eye blood sampling,separate supernatant,enzyme-linked immunosorbent assay(ELISA)in serum contents of GSH and GSSG ratio and GSH/GSSG changes.From ischemia cortex observed using immunohistochemistry,RT-PCR,observation of γ-GCS protein and mRNA expression.All data were calculated by SPSS 23.0.Results:1.Nerve function of cerebral ischemia reperfusion mice behavior observationWith the corresponding sham operation group,WT model group,KO model group,KO EA group of nerve function were significantly reduced(P<0.01);WT group neural function score was(13.20+1.30)WT,and the sham operation group decreased significantly(P<0.05).The difference between WT electroacupuncture group and WT model group was significant(P<0.05).The WT model group was compared with the KO model group,and the neurobehavioral performance was statistically significant(P<0.05).The difference was statistically significant in the WT electroacupuncture group compared with the KO EA group(P<0.01).2.water content in brain of cerebral ischemia-reperfusion in miceCompared with the corresponding sham operated group,the model group and the EA group were significantly different(P<0.01).After electroacupuncture,the cerebral edema in WT group was relieved,and the water content was significantly lower than that in the WT model group(P<0.01).Compared with WT mice,the brain water content of KO mice increased significantly compared with that of mice(P<0.01).The water content of WT EA group was significantly lower than that of KO electroacupuncture group(P<0.01).3.Cerebral infarction ischemia-reperfusion brain tissue in mice(TTC staining)After reperfusion with 24h in mice,there was no white infarct in TTC staining in the sham operation group,and even red staining occurred.Model group and electric acupuncture group showed different size of white infarct area,but the white area of WT electroacupuncture group was smaller than that of WT model group,KO model group and KO electroacupuncture group.The volume of infarction in WT electroacupuncture group was the smallest(22.97+2.44%),compared with the model group,the difference was significant(P<0.01).The cerebral infarction volume of the WT model group was smaller than that of the KO model group,with statistical significance(P<0.05).The white area of brain tissue of WT electroacupuncture group was lower than that of KO electroacupuncture group(P<0.01).4.The brain tissue of mice with cerebral ischemia-reperfusion and pathological morphologyUnder 40x10 visible microscope:nerve cell cytoplasm is pale red,nuclear is bluish black.Sham brain Morphosis and Ultrastructure of normal,visible nerve pyramidal cell number,abundant cytoplasm,large nucleus,rounded,no pathological damage.WT mice brain tissue of ischemic neuronal cell bodies reduce the deformation,hyperchromatic nuclei condensation,visible part of the cell nucleus and cytoplasm of ill-defined,nuclear pyknosis triangular or irregularly shaped.Nerve tissue interstitial edema,dyeing and reticular structure is loose and light,even parts only necrotic tissue and no significant cell structure of red dye.WT EA group was significantly decreased infarct model group,microscopic pathology is not obvious,cell morphology,normal size,occasionally with different degree of degeneration of nerve cells,brain edema reduction.KO model and EA group cell necrosis was around vacuoles,edema,and nucleus dissolved serious5.The cerebral ischemia-reperfusion in mice serum content of GSH and GSSG and GSH/GSSGWT compared with the sham operation group,WT model group,the content of serum GSH decreased significantly(P<0.05),the content of GSSG increased significantly(P<0.01),the ratio of GSH/GSSG decreased significantly(P<0.01);the content of GSH of WT group increased significantly(P<0.05),the content of GSSG was decreased(P<0.05)the ratio of GSH/GSSG,increased significantly(P<0.01);and WT group GSH content and GSH/GSSG ratio were significantly higher than that of WT model group(P<0.01),the content of GSSG decreased significantly(P<0.01).KO compared with the sham operation group,KO model group,KO EA group the serum GSH content and GSH/GSSG ratio decreased significantly(P<0.01),the contents of GSSG increased significantly(P<0.05);and there were no significant difference between the KO model group,KO EA group between group GSH,GSSG content,GSH/GSSG ratio(P>0.05).The serum GSH content,GSSG content and GSH/GSSG ratio in the KO model group were significantly lower than that in the WT model group(P<0.01),and the serum GSH content and GSH/GSSG ratio in the KO electroacupuncture group were significantly lower than those in the WT electroacupuncture group(P<0.01).6.Cerebral ischemia-reperfusion in mice brain γ--GCS protein expressionFrom optical microscope observation of defect of cerebral ischemia-reperfusion brain positive cell of γ-GCS,γ-GCS protein appears brown yellow,widespread in mouse cerebral cortex,spinal cells express the obvious,Sham-operated group Brown-positive cells,electro-acupuncture positive cell staining was significantly better after.But KO Mice occasional expressions of positive cells,lighter in coloring,and expression of cytoplasmic and nuclear are not obvious.Positive cells were detected in all sham operated groups.WT compared with the sham operation group,model group,positive cells were more in number two,with significant difference(P<0.01);WT group positive cells were the most(33+3.39),significantly higher than that of WT model group(P<0.01).Compared with the KO sham operated group,the positive cells in the KO model group and the KO EA group increased significantly(P<0.01).The number of positive cells in the WT model group was significantly higher than that in the KO model group(P<0.01).The number of positive cells in WT EA group was significantly higher than that in KO electroacupuncture group(P<0.01).The WT model group and the KO model group were compared with the corresponding sham operation,with statistical significance(P<0.05).Two groups of numerical acupuncture group were compared with the corresponding sham operation group,the difference was significant(P<0.01),there was statistical significance.The difference between the WT model group and the WT electro acupuncture group was statistically significant(P<0.01).WT model group and KO model group,WT EA group and KO EA group average gray level and average optical density value were statistically significant difference(P<0.01).7.Defect of cerebral ischemia-reperfusion in mice brain γ--GCS mRNA expressionFrom the detection of RT-PCR γ--GCS expression level of mRNA showed that the model group and EA group than in sham operation group and the corresponding WT,the difference was significant(P<0.01);and the brain tissue of WT group in-GCS gamma mRNA expression was also higher than that of WT model group(P<0.05).Compared with the KO sham operation group,the expression of,-GCS and mRNA in the KO model group and KO electroacupuncture group were significantly higher(P<0.05).The expression of gamma-GCS mRNA in wild mice was significantly higher than that of corresponding Nrf2 knockout mice,regardless of sham operation,model or electroacupuncture.Conclusion:1.Electro-acupuncture can improve the WT mice after cerebral ischemia-reperfusion injury in neurological damage and brain tissue morphology,prompts electro-acupuncture on ischemic cerebrovascular disease damages brain tissue have a protective effect.2.EA can promote γ-GCS protein expression,GSH content increased,electroacupuncture can promote the expression of antioxidant proteins,increase the body’s antioxidant capacity.3.Electroacupuncture the Nrf2 gene knockout mice,γ--GCS protein expression with no apparent improvement,suggesting that regulation of electroacupuncture on cerebral ischemia-reperfusion injury of γ--GCS protein expression,closely associated with Nrf2.In conculusion,this subject first through Nrf2 gene knock except mice preparation brain deficiency blood again perfusion model,observation Nrf2 signal pathway on electric needle intervention role of effect,and thought electric needle can through Nrf2 signal pathway regulation downstream of gamma-GCS gene and protein of expression,upgrade body of anti-oxidation capacity,play on deficiency bloody brain vascular disease of protection role,this for clinical treatment brain vascular disease provides new thought.