Effect And Mechanism Of DJ-1 On Alleviating Cerebral Ischemia-reperfusion Inflammatory Injury By Affecting Microglia Polarization Through Sirt1

BackgroundAfter cerebral ischemia-reperfusion injury,activated microglia can be polarized into different phenotypes(M1 type that promotes inflammation or M2 type that resists inflammation)to regulate the inflammatory response process of nerve tissue.As a member of the deacetylase family,Sirt1 plays an important role in microglia/macrophage polarization and autophagy.DJ-1 is a multifunctional protein,which plays an important role in cell apoptosis and transcription.Our previous studies showed that DJ-1 has an anti-inflammatory effect on cerebral ischemia-reperfusion injury.Whether the neuroprotective effect of DJ-1 is related to the polarization of microglia is unclear.PurposeIn order to study in the middle cerebral artery occlusion/reperfusion(MCAO/R)model,DJ-1 affects the polarization of microglia and specific molecular mechanisms.Methods(1)We use the middle cerebral artery occlusion/reperfusion(MCAO/R)model to simulate ischemia-reperfusion injury.The middle cerebral artery occlusion is 1 hour,reperfusion is 12 hours,24 hours,and48 hours.Western blot was used to detect the expression of microglia markers(CD86,Arg1,IL-10)at the three reperfusion time points.(2)The MCAO/R model was established.DJ-1si RNA was injected24 h before the MCAO/R model was established to interfere with the expression of DJ-1.Injection site rat lateral ventricle.Observe the influence of DJ-1 interference on neurological function,cerebral infarction volume and microglia morphology after brain I/R injury.Western blot was used to detect the influence of DJ-1 interference on the polarization of microglia.ND-13(a peptide synthesized based on the amino acid sequence of DJ-1)was given while interfering with DJ-1,and the effects on rat neurological function,cerebral infarction volume,and microglia polarization were observed.(3)The MCAO/R model was established.DJ-1 si RNA was injected into the lateral ventricle of the rat 24 hours before the establishment of the MCAO/R model,and EX-527,a specific inhibitor of Sirt1,was injected intraperitoneally when the rat recovered after the model was established.Firstly,the interaction between DJ-1 and Sirt1 was detected by co-immunoprecipitation(Co-IP).Western blot was used to detect the influence of inhibiting Sirt1 on the polarization of microglia while DJ-1interfered.Secondly,the expression of Beclin1,LC3II/LC3 I,and p62 was detected by Western blot,and the ultrastructure of autophagosomes was observed by transmission electron microscope(TEM).Finally,immunofluorescence and Western blot were used to observe the expression of Atg5,Atg12 and Atg16L1,and Co-IP was used to detect the interaction between the autophagy-related complex Atg5-Atg12-Atg16L1.Results(1)After 48 hours of reperfusion,the expression of M1 type microglia marker CD86 reached a peak.The M2 type microglia markers(Arg1,IL-10)reached the peak at 24 h after reperfusion.(2)Compared with the MCAO/R group,DJ-1 interfered with the rat’s neurological function and significantly increased the cerebral infarction volume.The number of Iba1-positive microglia did not change significantly,but the morphology was changed.The expression of M1 type cytokines(CD86,IL-6)increased,and the expression of M2 type cytokines(Arg1,IL-10)decreased.ND-13 can reverse the effects of DJ-1interference on nerve function damage,increase in cerebral infarction volume and microglia polarization.(3)In the MCAO/R model,DJ-1 and Sirt1 are combined with each other.After DJ-1 interference,the expression of Beclin1,LC3II/LC3 I,p62,Atg5,Atg12,Atg16L1 decreased,and the number of autophagosomes under TEM decreased.Compared with the DJ-1 si RNA group,inhibiting Sirt1 while DJ-1 interfered further increased the release of CD86 and IL-6,and decreased the expression of Arg1 and IL-10 more significantly.The expression of Beclin1,LC3II/LC3 I,p62,Atg5,Atg12,Atg16L1 was further reduced,and the number of autophagosomes under transmission electron microscope was very small.ConclusionDJ-1 may reduce cerebral I/R inflammation damage by affecting the polarization of microglia.The mechanism by which DJ-1 affects the polarization of microglia may be that DJ-1 activates the autophagy-related complex Atg5-Atg12-Atg16L1 through Sirt1 to promote the autophagy process,thereby regulating the microglia during cerebral I/R injury polarization.DJ-1 may be a drug target for the treatment of cerebral I/R injury.