| BackgroundWith the aging of the population,the incidence of cerebrovascular diseases,especially ischemic encephalopathy.For ischemic encephalopathy,restoring blood and oxygen supply to brain tissue as soon as possible is one of the effective means to save its life and reduce the rate of disability.However,due to the recovery of blood oxygen supply to the brain tissue,brain dysfunction is more serious,named cerebral ischemia reperfusion injury(IRI).Inflammatory response plays a key role in cerebral ischemia reperfusion injury.As the main source of inflammatory cytokines in neuroinflammation,the exploration of the mechanism of microglia/macrophage polarization in cerebral ischemia reperfusion has become a research hotspot.The polarization of microglia/macrophage,the resident immune cells in the brain,plays an important role in the injury and repair associated with ischemia reperfusion(I/R).Microglia in resting state are activated after ischemic hypoxia injury in brain tissue and release inflammatory cytokines.Microglia are activated through structural,phenotypic and functional changes,and play a dual role in neuron injury and repair,i.e.,the transformation from M1 phenotype to M2 phenotype.M1 phenotype "pro-inflammatory cells"(mainly marked by i NOS and CD86)produce inflammatory mediators such as IFN-β and IL-1β,which tend to induce neuron injury and death.M2 phenotype(mainly marked by Arg1 and CD206),also known as "repair cells",is conducive to tissue repair and secretion of anti-inflammatory factors such as IL-10、IL-4、 TGF-β,etc.The polarization process of microglia from M1 to M2 is time-sensitive.Microglia/macrophages express M2 signal during acute ischemia and hypoxia injury.In the late stage of injury,M1 phenotype plays a dominant role.The transformation from M2 to M1 was not conducive to the repair of brain tissue.Therefore,in the early stage of ischemia and hypoxia,microglia/macrophages were mainly induced by M2 phenotype to play anti-inflammatory and neuroprotective effects,which has become the focus of our research.DJ-1 is the third gene that has been found to be associated with familial Parkinson’s disease.It is a mature ROS scavenger with a variety of other functions,including protein molecular chaperone,protease,autophagy regulation,etc.DJ-1 is subcellular located in the mitochondrial matrix and intima,participating in mitochondrial physiological functions and protecting mitochondria against oxidative stress.DJ-1 was diffusely overexpressed in the cytoplasm of neurons of the adult rat brain nervous system and all glial cells,and was not affected by function or anatomical location.A large number of studies have confirmed the neuroprotective effect of DJ-1.We reported that interfering with DJ-1 can aggravate cerebral ischemia reperfusion injury in rats,increase the infarct volume and neurological function damage.As an effective therapeutic target for stroke,DJ-1 has been highly regarded in terms of anti-inflammatory effects.Current studies have shown that DJ-1 can regulate the polarization of microglia/macrophages.However,whether DJ-1 participates in microglia/macrophages polarization and plays a neuroprotective role in cerebral ischemia/reperfusion injury,the specific mechanism remains unclear.ObjectiveTo investigate the regulation of microglial/macrophage polarization in cerebral ischemia/reperfusion injury by DJ-1 and its potential mechanism.MethodsWe established the Middle Cerebral Artery Occlusion/Reperfusion(MCAO/R)in adult male SD rats and Oxygen/Glucose Deprivation/Reoxygenation(OGD/R)model in the highly aggressively proliferating immortalized(HAPI)microglial cell line,to simulate the in vivo and in vitro cerebral ischemia-reperfusion injury.Part1(1)Laser Doppler cerebral blood flow meter was used to measure the change of cerebral blood flow in the middle cerebral artery of rats during MCAO/R.(2)By establishing MCAO/R model at different time points,the middle cerebral artery of rats was embolized for 10 min,30min,1h,and reperfusion for24 h respectively.TTC staining was used to observe the cerebral infarction volume of rats.(3)24h before MCAO/R model,three different sequences of DJ-1 siRNA and different concentrations of DJ-1 based polypeptide ND13 were given by intracerebroventricular injection.The interference efficiency of DJ-1 and the expression levels of microglia/macrophage markers Arg1 and i NOS were observed by Western blot.(4)By establishing OGD/R model at different time points,HAPI cells were hypoxic for 1h,2h,4h and 6h respectively,and then reoxygenated for 24 h.CCK-8 assay was used to detect microglia/macrophage proliferation index.24 h before the establishment of OGD/R model,HAPI cells were co-cultured with different concentrations of ND13(0u M,5u M,10 u M,15 u M,20 u M).Microglia/macrophage proliferation index was detected by CCK-8 assay.Part2(1)Established MCAO/R model treated with DJ-1siRNA or ND13.(2)Western blot assay was used to detect the effects of DJ-1siRNA or ND13 on the expression levels of microglia/macrophage markers Arg1,CD163,i NOS and CD86.(3)Western blot assay was used to detect the effects of DJ-1siRNA or ND13 on the expression levels of inflammatory cytokines IL-10,IL-4,TNF-α and IL-1β.Part3(1)Western blot assay was used to detect the effects of DJ-1siRNA or ND13 on the expression levels of pathway proteins P62 and immunoprecipitation(Co-IP)assay was to detect the interaction between P62 and TRAF6.(2)Western blot assay was used to detect the effects of DJ-1siRNA or ND13 on the expression levels of pathway proteins TRAF6,IRF5 and IKKαβ.(3)MCAO/R model was established.24 hours before the model,ND13 and XRK3F2(a specific inhibitor of P62)was injected simultaneously through the lateral ventricle.(4)Observe the effects of ND13 and XRK3F2 on the neurological function injury.TTC was detected cerebral infarction volume,HE and NISSL was for brain tissue cell morphology of rats with MCAO/R.Part 4(1)The effects of ND13 and XRK3F2 on the expression levels of microglia/macrophage markers(Arg1,CD163,i NOS and CD86)and the levels of inflammatory cytokines(IL-10,IL-4,TNF-α and IL-1β)in MCAO/R and OGD/R models were detected by Western blot assay.(2)In order to detect the effect ND13 and XRK3F2 in MCAO/R and OGD/R models,co-immunoprecipitation(Co-IP)assay was used to detect the interaction between P62 and TRAF6,Western blot was used to detect the expression levels of pathway proteins P62,TRAF6,IRF5 and IKKαβ.(3)Double-label immunofluorescence assay was used to observe IRF5 localization and nucleation after ND13 and XKR3F2 co-treated.Results Part1(1)The MCAO model was established successfully if the blood flow decline rate of middle cerebral artery in rats was greater than 70%.(2)The neurological deficit was obvious after 1h of middle cerebral artery embolization,and the infarct volume was significantly higher than that in the10 min and 30 min groups.(3)The interference efficiency of DJ-1siRNA(Sense 5’-3’CCCAUUGGCUAAGGACAAATT,Antisense5’-3’UUUGUCCUUAGCCAAU GGGTT)was the most obvious in MCAO/R model.The expression level of Arg1 was significantly upregulated when ND13 concentration was 0.2μg/μl.(4)CCK-8 index of HAPI cells at OGD4h/R24 h was about 48.3%.(5)In the OGD/R model of HAPI cells,the proliferation ability of HAPI cells was significantly higher when the concentration of ND13 was 15μM than that of other concentration groups.Part2(1)The MCAO/R model treated with siRNA interference or ND13 was successfully established.(2)After DJ-1siRNA interfered with the expression of DJ-1,the level of M1 marker proteins(i NOS and CD86)of microglia/macrophages were increased.After ND13 treatment,the level of microglia/macrophage M2 marker proteins(Arg1 and CD163)were increased.(3)After DJ-1siRNA interfered with the expression of DJ-1,the expression of pro-inflammatory inflammatory factors(TNF-α and IL-1β)were increased.After ND13 treatment,the expression of anti-inflammatory inflammatory factors(IL-10 and IL-4)were increased.Part3(1)After DJ-1siRNA interfered with the expression of DJ-1,the expression level of P62 was increased.After ND13 treatment,the expression level of P62 was decreased.However,IKKαβ level was decreased after DJ-1 siRNA interference and was upregulated after ND13 treatment.Co-immunoprecipitation(Co-IP)assay confirmed that the interaction between P62 and TRAF6 was weakened after ND13 treatment(2)After DJ-1siRNA interfered with the expression of DJ-1,the expression levels of TRAF6 and IRF5 were increased.After ND13 treatment,the expression levels of TRAF6 and IRF5 were decreased.However,IKKαβ level was decreased after DJ-1 siRNA interference and was upregulated after ND13 treatment.(3)The MCAO/R model treated with ND13 and XRK3F2 was successfully established.(4)Compared with the treatment of ND13 alone,the neurological score and cerebral infarction volume was reduced;the ischemic injury of neuron was alleviated after treated with ND13 and XRK3F2 together.Part4(1)Both in MCAO/R and OGD/R model,after the combination of ND13 and XRK3F2,the level of microglia/macrophage M2 marker proteins(Arg1 and CD163)was further increased,and the expression of anti-inflammatory inflammatory cytokines(IL-10 and IL-4)was further increased compared with the treatment of ND13 alone.(2)Both in MCAO/R and OGD/R model,co-immunoprecipitation(Co-IP)assay confirmed that the interaction between P62 and TRAF6 was further weakened after ND13 and XRK3F2 co-treated.At the same time,the expression levels of P62,TRAF6 and IRF5 were further decreased.However,the expression of IKKαβ in ND13 and XRK3F2 group was significantly lower than that in ND13 group.(3)IRF5 is expressed in cytoplasm,and a small amount is expressed in mitochondria.After OGD/R,the signal of IRF5 co-localization with the nucleus was enhanced.ND13 enhanced the activity of DJ-1 and decreased the expression of IRF5.After treatment with ND13 and XRK3F2,the number of IRF5 positive cells and positive signal intensity decreased compared with that in ND13 group.Conclusion:DJ-1 regulates microglia/macrophages polarization and inflammatory factors,alleviates the inflammatory injury caused by cerebral ischemia/reperfusion.DJ-1 inhibits the expression of P62 and affects the interaction of P62-TRAF6.DJ-1 regulates the TRAF6/IRF5 signaling pathway through P62. |
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