| BackgroupAcute ischemic cerebrovascular disease is one of the three major diseases leading to human death,which is also the main cause of disability.The penumbra area around the cerebral infarction core due to ischemia / reperfusion injury mechanism lead to a large number of neurons death and neurological function continued to deteriorate,so iniating active researchs on cerebral ischemia / reperfusion injury mechanism and takeing effective measures to rescue the dying neurons are the key stratege to improve neurological function and reduce disability rate at early stage.A large number of studies have shown that excessive inflammatory response is the initial step and the pivot in the process of cerebral ischemia / reperfusion injury,which is the main factor that causes the early death and neurological deterioration after stroke.Nuclear factor-kappa appa B(NF-kB)signaling pathway controls the central link in the inflammatory reaction,and the regulation mechanism of NF-kB signaling pathway is the footstone for early anti-inflammatory treatment after cerebral ischemia /reperfusion.Zinc finger protein A20 is a decisive negative feedback suppressor in the NF-kB signaling pathway,and thereby a potential therapeutic target for inhibiting the excessive activation of NF-kB signaling pathway.However,until now,there has been no report about the temporal and spatial expression of zinc finger protein A20 after cerebral ischemia / reperfusion insult and its protective effects on brain inflammatory injury.Acupuncture is a treasure of traditional Chinese medicine,known as a regulation of external treatment for internal diseases.Electroacupuncture is the combination of the traditional acupuncture and the electro-therapy which is becoming the main form of acupuncture treatment.Many domestic and foreign studies have shown thatelectro-acupuncture has a good anti-inflammatory effect,as well as a long history for the treatment of ischemic cerebrovascular disease.Although the electro acupuncture treatment of ischemic stroke and inflammatory reaction through regulation of NF-kB signaling pathway has been reported widely,the lack of electroacupuncture regulation of zinc finger protein A20 after cerebral ischemia / reperfusion means missing the core mechanism of electroacupuncture how to inhibit NF-kB signaling pathway.Therefore,this study consists of three parts:(1)Clearing A20 expression characteristics in focal ischemia / reperfusion brain tissue and its role on cerebral ischemic injury.(2)Observeing the regulational effect of electroacupuncture on the expression of zinc finger protein A20 and elucidating the mechanism of A20 involved in the electroacupuncture’s inhibition of NF-k B signaling pathway activity and brain inflammatory injury.(3)Investigateing the relationship between neurons exprssiong A20 and the regulation of electroacupuncture on the expression of A20 in the inhibition of excessive inflammatory injury after focal cerebral ischemia / reperfusion,and exploreing the possible pave for electroacupuncture putting its neuroprotective effect on neurons.Part 1:Expression characteristics of zinc finger protein A20 in focal ischemia /reperfusion brain tissue of SD rats and its effect on cerebral ischemic injuryObjectives: To observe the expression characteristics of zinc finger protein A20 in focal ischemia / reperfusion brain tissue of SD rats and its effect on cerebral ischemic injury.Methods: Male SD rats were used to establish the focal cerebral ischemia /reperfusion model through using a suture transiently obstructed the right middle cerebral artery(MCAO).The rats were divided into 5 groups: sham operation(sham)group,ischemia / reperfusion(MCAO)group,A20 overexpression(MCAO + LV-A20)group,A20 silencing(MCAO + LV-sh A20)group and empty virus vector(MCAO+Vehicle)group.The temporal and spatial expression of zinc finger protein A20 in brain tissue were detected by real time fluorescence quantitative polymerase chain reaction(RT-q PCR),by Western blot and immunofluorescence(IF)respectively.Byestablishment of a lentivirus vector carrying A20 gene overexpression and silencing system,which transfected SD rat brain tissue with intracerebroventricular injection,effects of zinc finger protein A20 on the neurological function and volume of cerebral infarction of rat after focal cerebral ischemia / reperfusion injury were evaluated by a18-point assessing system and 2,3,5-triphenyltetrazolium chloride staining(TTC)respectively at indicated time-points.Results:(1)Temporal expression characteristics:The expression of A20 m RNA and protein in rat ischemia/reperfusion brain tissue was quantified using RT-q PCR and western blotting,respectively,at the indicated time points(6,12,24,48,and 72 h after reperfusion).The m RNA and protein expression of A20 in brain tissue of sham group was very small.Beginning at 6 h after reperfusion the simple ischemia / reperfusion insult induced higher A20 m RNA transcription than that in the Sham group(except at 72 h,all remaining time points P<0.001),the peak occurred at 24 h after reperfusion and levels then dropped rapidly to normal comparable with the Sham group at 72 h after reperfusion(P>0.05).Similarly,the amount of A20 protein in the ischemic area were only increased after 12,24,and 48 h of reperfusion in the MCAO group compared with the Sham group(P<0.05,P<0.001,P<0.05,respectively).(2)Spatial expression characteristics: The spatial distribution of A20 in the brain tissues of rats was identified using immunofluorescence staining.There was little A20 expression in the brains of rats in the Sham group.Twenty-four hours after reperfusion the number of A20 immunopositive cells was increased significantly in the brains of rats in the MCAO group compared with the Sham group(P<0.001).The positive cells were located mainly in the focal cerebral ischemia / reperfusion cortex.To further determine the main cellular localization of A20 in the rat brain double-immunofluorescence staining was performed to assess the localization of A20 in neurons,astrocytes,and microglia by co-staining for Neu N,GFAP,and Iba-1,respectively.Following 2 h of ischemia and 24 h of reperfusion increased levels of A20 protein were found mainly in the neurons,whereas very little was observed in microglia and astrocytes.(3)The role of zinc finger protein A20 in focal cerebral ischemia /reperfusion injury: To investigate the effects of A20 on cerebral ischemia/reperfusioninjury neurobehavioral evaluations were performed and the infarct volume was assessed using an 18-point neurologic deficit score and 2,3,5-triphenyltetrazoliumchloride(TTC)staining,respectively.Beginning after 6 h of reperfusion following 2 h of ischemia there was no clear difference in neurological evaluation among groups.However,72 h after reperfusion rats in the MCAO + LV-A20 group exhibited far more notable mitigation of their neurologic dysfunction compared with those in the MCAO group(P < 0.05).In contrast,that was very little improvement in rats in the MCAO + LV-sh A20 group compared with the MCAO group(P < 0.05).There was also no significant difference in neurologic score between the MCAO and MCAO + Vehicle groups(P ? 0.05).Consistent with this,rats in the MCAO+LV-A20 group exhibited smaller brain infarct volumes compared with those in the MCAO group 72 h after reperfusion(P < 0.001).In addition,the infarct volume was larger in the MCAO+LV-sh A20 group than the MCAO group(P < 0.001).The mean infarct volume was similar in the MCAO + vehicle and MCAO groups(P > 0.05).Therefore,these data suggest that the stroke outcome could be improved by the overexpression of A20 but exacerbated by A20 deficiency.Conclusion: The focal cerebral ischemia / reperfusion can stimulate the expression of zinc finger protein A20,upregulated zinc finger protein A20 mainly express in focal cerebral ischemia / reperfusion cortex neurons;zinc finger protein A20 has a protective effect against focal cerebral ischemia / reperfusion injury.Part 2:Effects of Electroacupuncture on the expression of A20 and inflammatory injury after focal cerebral ischemia / reperfusion in SD rats and its mechanism Objectives: To observe the effect of electroacupuncture on regulation of A20 expression in SD rats brain tissue with focal cerebral ischemia / reperfusion,and clarify whether A20 was required for electroacupuncture inhibiting NF-kB signaling pathway and mitigating ischemic inflammatory brain inflammatory damage.Methods: Male SD rats were divided into 5 groups: sham operation(sham)group,ischemia / reperfusion(MCAO)group,electroacupuncture(MCAO + EA)group,A20 silencing electroacupuncture(MCAO + EA+ LV-sh A20)group and empty virus vector(MCAO+Vehicle)group.After establishing MCAO model,the rats received electroacupuncture treatment at the acupoints of the “Baihui(GV 20)”,“Hegu(L14)” and “Taichong(Liv3)” with a certain frequency and intensity.The effects of electroacupuncture on the expression of zinc finger protein A20 in brain tissue were detected by RT-q PCR,Western-blot and immunofluorescence;by A20 gene silencing,the role of zinc finger protein A20 in the effects of electro-acupuncture treatment on neurobehavioral scores,infarction volumes,cytokine levels,glial cell activation,and the NF-κB signaling pathway were assessed at the indicated time points.Results:(1)electroacupuncture upregulates A20 expression following focal cerebral ischemia-reperfusion: The expression of A20 m RNA and protein in rat ischemia / reperfusion brain tissue was quantified using RT-q PCR and western blotting,respectively,at the indicated time points(6,12,24,48,and 72 h after reperfusion),A20 m RNA levels were increased significantly further in the MCAO+EA group compared with the MCAO group(each time point P<0.001),demonstrating earlier peak expression at 12 h after reperfusion and m RNA levels still remained high at 72 h after reperfusion.Similarly,the amount of A20 protein in the ischemic area was increased significantly in the MCAO +EA group compared with the MCAO group at each time point(at 12 h P<0.001,at 24 h P<0.001,at 48 h P<0.01,at72 h P<0.01,respectively).The spatial distribution of A20 in the brain tissues of rats was identified using immuno-fluorescence staining,the A20-positive cell count in the MCAO + EA group was increased significantly compared with the MCAO group(P<0.001).(2)A20 was required for electroacupuncture exerting neuroprotective effects:The effects of A20 silencing on electroacupuncture induced neuroprotection were assessed by performing neurobehavioral evaluations and measuring infarct volume 72 h after reperfusion,Compared with the MCAO group,the neurologic function of rats in the MCAO + EA group was improved(P < 0.05)and the infarct volume was significantly smaller(P < 0.001).In contrast,the conditions were worsened in rats in the MCAO + EA + LV-sh A20 group,as evidenced by a poor neurologic deficit and larger infarction volume compared with the MCAO + EA group(P < 0.05,P < 0.001).Finally,the neurologic scores and infarct volume were similar in the MCAO + EA + Vehicleand MCAO + EA groups(P ?0.05).(3)A20 was also required for the anti-inflammatory effects of electroacupuncture: the levels of TNF-α and IL-1β were measured using ELISA to determine the scale of the inflammatory response in rat brain tissue 24 h after cerebral,both cytokines in focal ischemia/reperfusion brain tissue were increased significantly in the MCAO group compared with the Sham group(P < 0.001,P < 0.001,respectively),but were decreased significantly in the MCAO + EA group compared with the MCAO group(P<0.05,P<0.001,respectively).Moreover,an antiinflammatory effects of EA were reduced significantly by silencing A20 m RNA in the MCAO+EA+LV-sh A20 group,since the brains exhibited higher TNF-α and IL-1βlevels than those in the MCAO + EA group(P<0.001,P<0.001,respectively).There was no difference in TNF-α and IL-1β levels between the MCAO + EA and MCAO +EA + Vehicle groups(P ? 0.05,P ? 0.05,respectively).The activation of innate immunocytes in brain was detected by using RT-q PCR to measure the m RNA levels of A1 and GFAP,which are markers of microglia and astrocyte activation,respectively.Twenty-four hours after reperfusion the relative levels of Α1 and GFAP were clearly elevated in the MCAO group compared with the Sham group(P < 0.001,P < 0.001,respectively).However,levels were significantly lower in the MCAO + EA group compared with the MCAO group(P<0.001,P<0.05,respectively).Furthermore,compared with the MCAO + EA group the relative levels of Α1 and GFAP were significantly higher in the MCAO + EA + LV-sh A20 group(P<0.001,P<0.001,respectively).There was no difference between the MCAO + EA and MCAO + EA +Vehicle groups(P ? 0.05,P ? 0.05,respectively).The activation state of glial cells was investigated further by using immunofluorescence staining to assess morphological changes.Glial cell activation patterns appeared in the ischemia/reperfusion cortex of rats.Activated astrocytes show increased GFAP immunoreactivity and thickening of the cytoplasmic processes,and activated microglia present with enlarged and brightened cell bodies together with shortened and thickened processes.Glial cells in the brains of rats in the sham group were in the resting state,with a ramified morphology and delicate processes.Glial cell activation was the most extensive and evident in thecerebral ischemia/reperfusion area of the MCAO + EA + LV-sh A20 group;however,it was inhibited by EA in the brains of rats in the MCAO + EA group.An intermediate phenotype was observed in the MCAO group.The morphological changes in the glial cells were consistent with the expression of GFAP and A1 m RNA observed in the same groups.(4)The upregulation of A20 was required for electroacupuncture to inhibit NF-κB signaling pathway: we determined whether the increased neuronal A20 expression was essential for the therapeutic effects of electroacupuncture on the post-ischemia inflammatory injury by inhibiting the NF-κB signal pathway,Western blotting was used to measure A20,p-IKKβ,IKKβ,p-IκBα,IκBα,nuclear p65,and cytoplasmic p65 proteins in the focal ischemia/ reperfusion area 24 h after reperfusion.Twenty-four hours after reperfusion there were increased A20 protein levels,a reduced phosphorylation ratio of IKKβ and IκBα,and suppressed nuclear translocation of NF-κB p65 in the MCAO+EA group compared with the MCAO group(P<0.05,P<0.05,P<0.001,P<0.01,respectivel).In contrast,little A20 protein expression was detected in the MCAO+EA+LV-sh A20 group,which also exhibited significantly elevated phosphorylation ratios of IKKβ and IκBα and the increased nuclear translocation of NF-κB p65 compared with the MCAO+EA group(P<0.001,P<0.001,P<0.001,P<0.001,respectively).These results suggest that A20 expression is an essential for the ability of EA to inhibit NF-κB signaling.Conclusion: Electroacupuncture increased the expression of A20 in focal cerebral ischemia / reperfusion cortical neurons,and upregulation of A20 is the key target and important mechanism for electroacupuncture inhibiting NF-κB signaling pathway mediated ischemic inflammatory brain injury.Part 3:The relationship between cortical neurons insulted by focal cerebral ischemia / reperfusion and the regulation of A20 by electroacupuncture in the inhibition of excessive inflammatory injury Objectives: To investigate the mutual effects of cortical neurons and electroacupuncture on the regulation of A20 in focal cerebral ischemia / reperfusion induced excessive inflammatory injury.Methods: Male SD rats were divided into 4 groups:ischemia / reperfusion(MCAO)group,electroacupuncture(MCAO + EA)group,A20 silencing electroacupuncture(MCAO + EA+ LV-sh A20)group and empty virus vector(MCAO+ Vehicle)group,and 24 h after reperfusion was chosen as check time-point.Double staining immunofluorescence was used to demonstrate NF-κB p65 express in neuron and corresponding morphological changes of neurons in focal cerebral ischemia/ reperfusion cortex,and correlations between A20-positive cell counts and neurologic scores,infarct volume,TNF-α and IL-1β content,and the relative m RNA levels of A1 and GFAP were investigated using Pearson’s correlation coefficients.Results:(1)Double immunofluorescence staining demonstrated only a small amount of NF-κB p65 were detected in the cytoplasm of individual neuron in the sham rat brains.Whereas,at reperfusion 24 h,sorts of brain cells had expressed NF-κB p65 in the focal ischemia/reperfusion cortex of MCAO rats.Especially,a large number of neurons not only had significant NF-κB p65 immunofluorescence in the cytoplasm,but also shown the positive staining in the nucleus,suggesting nuclear translocation.Moreover,these neurons arranged loosely and disconnectedly,with partial cell body representing swollen.Contrarily,EA group has shown neuron arrangement basically kept closely and orderly in the ischemia/ reperfusion cortex,and despite neuronal NF-κB p65 expression was also abundant,most of them were confined to the cytoplasm.However,A20 silencing seriously damaged EA blocking NF-κB p65 nuclear translocation.The dense NF-κB p65 immunofluorescence staining was detected covering throughout cytoplasm and nucleus of nearly all neurons in the ischemia/reperfusion cortex of MCAO + EA + LV-sh A20 rats.Accordingly,cell number in this area was decreased significantly,and the residual neurons arranged sparsely,with shrank body and broken nucleus.Thus these results precisely indicated that A20 was involved in EA regulating neuronal NF-κB signaling and also effectively influenced the neuronal morphological characteristics,which could affect the neuron survive.(2)There was a clear positive correlation between neuronal A20-positive cell counts and neurologic scores(r = 0.8140,P < 0.0001),and a significant negative correlation between neuronal A20-positive cell counts and infarct volume(r = –0.7591,P < 0.0001),TNF-α content(r = –0.8504,P < 0.0001),IL-1β content(r = –0.8362,P < 0.0001),relative A1 m RNA levels(r = –0.8704,P < 0.0001),and relative GFAP m RNA levels(r = –0.8209,P <0.0001).Conclusion: Focal cerebral ischemia / reperfusion cortical neurons is the main body and platform for electroacupuncture regulating A20 expression to inhibit NF-κBp65 nuclear translocation and attenuate brain inflammatory injury,on the other hand through upregulation of neuronal A20 and inhibition of neuronal NF-κBp65nuclear translocation,electroacupuncture can effectively prevent focal cerebral ischemia/ reperfusion cortical neurons morphological damage. |
