| Neuroimmunity is one of the important mechanisms of cerebral ischemia reperfusion injury.Studies shown that microglia cells are activated rapidly after ischemia.Under different microenvironment and conditions,microglia have the dual properties of aggravating brain injury and protecting neurons.Therefore,how to regulate the secretion phenotype of microglia is particularly important for the treatment of ischemic stroke.Electroacupuncture(EA)has been used for treatment of this disease for thousands of years.EA is a combination method of traditional medicine and modern medicine in China.EA can significantly improve the symptoms of neurological deficit caused by cerebral ischemia,promote the rehabilitation of stroke,and is widely used in clinical treatment of stroke and pain related diseases.In this study,EA was adopted as the treatment method.ANXA1,a medium expressed in microglial cells,was selectedas the intervention target to study the mechanism of the protective effect of microglial cell phenotype conversion induced by EA on cerebral ischemia.Methods:Methods: Healthy adult C57 mice were selected and MCAO/R model was established.Lateral ventricle injection of Ad-shANXA1 inhibited the expression of ANXA1 gene,and lateral ventricle injection of Boc-2 antagonized the ANXA1 receptor FPR.TTC staining,infarction volume after ischemia,neurological function score and neuronal protein expression were used to determine the effect of EA on brain protection.Water maze and open field experiments were used to study the effects of EA on learning and memory in ischemic injury mice.The protein and gene expressions of ANXA1,FPR,Arg-1,BDNF,IL-1β,TNF-α and i NOS in each group were detected by Western Blot and RT-PCR.The number,activity and phenotype of neurons and microglia were observed by staining NEUN and IBA-1 with double immunofluorescence staining.Results1.EA upregulated the expression of ANXA1 and ANXA1 receptor FPR were in MCAO/R mice.The mice were divided into control group(Sham),model group(MCAO/R)and model + EA group(MCAO/R+EA).The expression of ANXA1 and its receptor FPR were detected by Western Blot and RT-PCR.The results showed that compared with the Sham group,the expression of ANXA1 and FPR were both up-regulated by ischemia injury in the MCAO/R group,while the expression of ANXA1 and FPR were further enhanced after EA intervention,which was significantly different compared with the MCAO/R group.The results suggest that the ANXA1-FPR pathway may be involved in the neuroprotective effect of EA against cerebral ischemia.2.EA exerted neuroprotective effects on MCAO/R mice via ANXA1(1)The transfection and inhibition efficiency of Ad-shANXA1 following by intracerebroventricular injections experimentsMice were divided into Control group,SCR and shANXA1 group.IBA-1 was used to labele microglia activity.Immunofluorescence results showed that the activity of IBA-1 was not significantly changed compared with that of SCR,suggesting that microglia activity was not activated by lateral ventricular injection procedure.Further,we observed the transfection and inhibition efficiency of Ad-shANXA1 by immunofluorescence and Western Blot.The results of IFC showed that there was no significant difference between Control and SCR,and the fluorescence intensity of shANXA1 group was significantly lower than that of SCR.The results above suggested that the expression of ANXA1 was significantly inhibited after lateral ventricular injection of Ad-shANXA1.(2).The neurological deficit symptoms and the volume of cerebral infarction caused by MACO/R were improved by EA treatment via ANXA1The mice were divided into Sham,MCAO/R,MCAO/R+EA,MCAO/R+EA+Boc-2 and MCAO/R+EA+shANXA1 group.The results showed that no neurological deficiency was found in Sham group.Compared with Sham group,the signs of neurological deficiencywere obvious in MCAO/R group.Compared with the MCAO/R group,EA significantly improved the signs of neural function defects caused by ischemia reperfusion.Boc-2 and Ad-shANXA1 respectively antagonized the ANXA1 receptor FPR and inhibited the expression of ANXA1.The results showed that the neurological deficiency induced by ischemia-reperfusion was significantly reversed by Boc-2 and shANXA1.TTC staining showed that the cerebral area of the embolization side of MCAO/R mice had obvious infarction,accounting for about 30% of the whole cerebral slice.Compared with the MCAO/R group,the infarct area was significantly reduced after EA treatment,and the infarct area was reduced by about 50% compared with the MCAO/R group.Compared with MCAO/R+EA group,the infarct volume of MCAO/R+EA+Boc-2 or MCAO/R+EA+shANXA1 group was significantly increased.The results showed that EA reduced the size of infarct after cerebral ischemia via ANXXA1.(3).The ability of learning and memory caused by MACO/R was enhanced by EA treatment via ANXA1.In the platform hiding period,the latency of escape and the distance to find the platform were decreased in each group,but the mice in the EA intervention group showed stronger learning and memory ability.The effect of EA was dependent on ANXA1,and blocking ANXA1 could reverse the effect of EA.During the Probe test period,the distance and time the mice swam in the target quadrant,the number of times they crossed the platform,and the incubation period for finding the platform were observed.The results showed that compared with the Sham group,the incubation period of searching for the platform was significantly prolonged,while the distance and time of swimming in the target quadrant and The Times of crossing the platform were significantly decreased in the MCAO/R group.Compared with MCAO/R group,the incubation period of the EA group was shortened,and the distance and time of swimming in the target quadrant and the times of crossing the platform were lengthened to different extent.Compared with MCAO/R+EA group,antagonism to ANXA1 in MCAO/R+EA+Boc-2 and MCAO/R+EA+shANXA1 group could weaken the effect of EA,prolong the latency time of finding the platform,and reduce the distance and time of swimming in the target quadrant and the number of crossing the platform.The results suggest that ANXA1 mediated EA can improve the learning and memory ability of ischemic injury mice.(4).EA alleviated the depression and enhanced exploration desire in MCAO/R mice via ANXA1.Mice were divided into Sham,MCAO/R,MCAO/R+EA,MCAO/R+EA+ Boc-2and MCAO/R+EA+ shANXA1 groups.Open field test was used to observe the increase of total movement distance,total resting time,time and times of entering the central region within 10 minutes.The results are as follows: compared with control group,MCAO/R group total rest time increased,the total movement distance and the time duration of the central region and into the central area of less,and MCAO/R group,electric acupuncture intervention group always stationary time reduced,the time duration of the total movement distance,into the central area and into the center area of the number of significant increased,lateral ventricle injection Boc-2 or Ad-shANXA1 mice treated with electric acupuncture treatment,the curative effect is not obvious.These results suggested that after ischemic injury,mice were in a state of depression and decreased desire for exploration,and EA therapy could alleviate the state of depression and enhance the desire for exploration in mice,and the effect of EA was dependent on ANXA1,and blocking ANXA1 could reverse the protective effect of EA.(5).The loss of neurons induced by MACO/R was reduced by EA treatment via ANXA1.The mice were divided into Sham,MCAO/R,MCAO/R+EA,MCAO/R+EA+Boc-2 and MCAO/R+EA+shANXA1 groups.The results showed that compared with Sham group,the number of NeuN positive neurons and protein level in MCAO/R group were significantly reduced.Compared with MCAO/R+EA group,EA significantly increased NeuN positive neurons and NeuN protein level.Compared with the MCAO/R+EA group,as the ANXA1 sh RNA or the ANXA1 receptor antagonist Boc-2 adapted,the effect of EA was reversed,that the number of neun-positive neurons and NeuN protein levels in the brain of the mice was down-regulated.The results suggested that ANXA1 mediated the neuronal protective effect after EA targeting of MCAO/R in mice.3.EA induced the transformation of microglia phenotype from M1 type to M2 type via ANXA1(1)The activity of microglia induced by MACO/R was changed by EA via ANXA1.The mice were divided into Sham,MCAO/R,MCAO/R+EA,MCAO/R+EA+Boc-2 and MCAO/R+EA+shANXA1 groups.According to the count of active microglia,the number of activated microglia in the MCAO/R group was significantly higher than that in the Sham group in the cortical and hippocampal regions.Compared with MCAO/R+EA group,EA reduced the number of activated microglia cells.Compared with MCAO/R+EA,microglial cell activity remained high in mice treated with EA after lateral ventricle injection of Boc-2 and Ad-shANXA1.Microglia morphology observation showed: Sham group of microglia is high branching resting state,MCAO/R of microglia activation conditions increase the cell body,shorter or disappear,cell morphology in ameboid,cupping treatment could reduce the number of sample amoebic microglia,the curative effect by ANXA1 blockers can also be suppressed.It suggested that the EA could induce changes in microglial cell activity through ANXA1 and improve the tolerance of neurons to ischemia and hypoxia.(2).EA treatment promoting microglia phenotype changed from M1 to M2 mediated by ANAX1.Mice were divided into Sham,MCAO/R,MCAO/R+EA,MCAO/R+EA+Boc-2and MCAO/R+EA+shANXA1 groups.MCAO/R+EA+shANXA1 groups were detected by Western Blot and RT-PCR to detect the expression of M1 phenotype representative molecules(IL-1β、TNF-α and iNos)and M1 phenotype representative molecules(Arg1 and BDNF)and mRNA.The results showed that compared with Sham group,the expression levels of IL-1β、TNF-α and iNos proteins and mRNA in the cerebral infarction area of MCAO/R group were significantly increased.The expression levels of IL-1β、TNF-α and iNos protein and mRNA were significantly reduced by EA.MCAO/R+EA+Boc-2 and MCAO/R+EA+shANXA1 groups showed significantly higher expression of il-1β expression and mRNA expression in TNF-αpositive protein and mRNA than MCAO/R+EA group,The expression of iNos gene in the EA group was significantly lower than that in the MCAO/R group,while the expression of iNos mRNA in the MCAO/R+EA+shANXA1 group was significantly higher than that in the MCAO/R+EA group.Suggesting that the inhibition of M1 phenotype molecule expression in microglial cells was reversed by Boc-2 and Ad-shANXA1.Compared with Sham group,Arg1 and BDNF protein and mRNA expression in the cerebral infarction area of mice in MCAO/R group were significantly up-regulated.Compared with MCAO/R group,Arg1 and BDNF levels were significantly increased in MCAO/R+EA group after EA treatment.EA significantly upregulated Arg1 and BDNF protein and mRNA expression levels.The expression of Arg1 and BDNF protein and mRNA in the MCAO/R+EA+s Boc-2 or MCAO/R+EA+shANXA1 group was significantly lower than that in the MCAO/R+EA group,suggesting that the promotion of M2 phenotype molecule expression in microglial cells was reversed by Boc-2 and Ad-shANXA1.The results indicated that ANXA1 mediated the switch of M1 to M2 phenotype microglia by EA.Mice were divided into Control group and EA group.The protein expressions of BDNF,Arg1,TNF-α and IL-1β were detected by Western Blot.The protein expression results showed that the protein expressions of IL-1β and TNF-α were significantly down-regulated but Arg-1 and BDNF were significantly up-regulated after EA.These results suggest that EA can regulate the expression of ANXA1 and neuroinflammation in the brain tissues of mice under normal conditions.4.EA showed no effect on the expression of ANXA1 in peripheral blood cells in MCAO/R miceMice were divided into Control,MCAO/R,MCAO/R+EA and MCAO/R+EA+shANXA1 groups.Western Blot was used to detect the expression of ANXA1 in peripheral blood cells.The results showed that there was no significant difference in the expression of ANXA1 in peripheral blood cells between the Control group and the MCAO/R group.Compared with MCAO/R group and MCAO/R+EA group,the expression of ANXA1 protein showed no significant statistical difference.Compared with MCAO/R+EA group and MCAO/R+EA group,the expression of ANXA1 protein did not get increased or inhibited.These results suggest that ANXA1 in peripheral blood cells is not involved in the regulation of ANXA1 in brain by EA.Conclusion:EA can change the morphology and phenotype of microglia by ANXA1 and FPR in the brain,promote the release of anti-inflammatory cytokines,and enhance the tolerance of neurons to ischemia and hypoxia,thereby protecting damaged neurons. |
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