Caffeine Regulates The Phenotypic Polarization Of Microglia Through SIRT2 To Protect Hypoxia-ischemia Induced White Matter Damage And Its Mechanism

Objective:White matter damage(WMD)is one of the causes of cerebral palsy and cognitive impairment in preterm infants.The main pathological change is impaired myelination,and satisfactory treatment is still lacking.The pathogenesis of white matter damage in preterm infants is complex.At present,it mainly focuses on the theory of oxidative stress,amino acid toxicity and cytokine toxicity,and inflammatory reaction is often accompanied by that.Neuroinflammatory reaction plays an important role in the occurrence and development of hypoxic-ischemic brain damage.Some scholars have proposed the third stage of nerve injury after hypoxia-ischemia,which mainly involves the sustained release of cytokines and other harmful factors,as well as epigenetic changes,and may damage axon growth,synaptogenesis and neurogenesis.This stage mainly involves neuroinflammatory injury caused by microglia activation.Microglia are the main participants in neuroinflammation.Microglia activation can be divided into two main forms:M1 phenotype(pro-inflammatory)and M2 phenotype(anti-inflammatory),which play an important role in the development of white matter damage and are related to the severity of white matter damage.Therefore,microglia mediated inflammation is the main way to prevent the occurrence and development of brain white matter damage in preterm infants.Caffeine has been used to treat neonatal apnea in neonatal intensive care unit for more than 30 years.Recently,some studies suggest that caffeine can not only reduce the incidence of BPD,but also improve the long-term prognosis of nervous system in preterm infants.Clinical studies have shown that very low birth weight infants treated with caffeine in the early stage have improved short-term and long-term neurological outcomes.Experimental studies also show that caffeine can reduce apoptosis of developing brain neurons,enhance synaptic activity,improve myelination disorder and improve white matter damage(WMD)of preterm infants.The previous studies of our research group also showed that caffeine can promote the differentiation and maturation of oligodendrocytes in neonatal rats with ischemia hypoxia and improve long-term behavioral disorders.However,the direct protective effect of caffeine on white matter damage induced by hypoxia-ischemia and its mechanism are still unclear.Our previous pre-experiment found that the NLRP3 increased after hypoxia ischemia compared with the Control group,and improved after the application of caffeine.Therefore,the neuroprotective effect of caffeine may be related to the anti neuroinflammatory response.Sirtuin 2(SIRT2),is a NAD~+-dependent class III deacetylase,and also plays an important role in nervous system diseases.Neuroinflammation,synaptic dysfunction,metabolic abnormalities,oxidative stress and other pathological features will be affected by SIRT2.Firstly,it is one of the key factors regulating hippocampal function,necessary for normal axon formation,and plays a key role in synaptic plasticity and memory formation by regulating AMPA receptor acetylation.Importantly,SIRT2 can inhibit NF-κB activity and down regulate the expression of its target genes by reducing the acetylation and nuclear translocation of NF-κB p65 in microglia.Therefore,the involvement of SIRT2 in nervous system diseases may be related to the regulation of microglia activation.Therefore,based on the previous models,this study discussed:(1)whether the inflammatory response mediated by microglia activation is involved in the occurrence and development of brain white matter damage caused by hypoxia ischemia?(2)Does caffeine regulate the phenotypic polarization of microglia and protect brain white matter injury induced by hypoxia ischemia?(3)To explore the mechanism of SIRT2 involved in caffeine regulating the phenotypic polarization of microglia by proteomics.From the perspective of anti-neuroinflammation,we strive to clarify the mechanism of caffeine improving brain white matter injury,so as to provide a new theoretical basis for the clinical application of caffeine.Methods:1.3-day-old Sprague Dawley(SD)rats were randomly divided into sham operation(Sham)group and hypoxia-ischemia(HI)group(60 rats in each group).In HI group,WMD model was made after ligation of left common carotid artery at 3 days of age and exposure to hypoxia(8%oxygen)for 2.5 h.Samples were collected at 7 to 21days after model preparation.The pathological changes of brain tissue were observed by HE staining.Myelin protein MBP,synaptophysin(Syp),postsynaptic protein PSD-95and microglia marker Iba-1 were observed by immunohistochemistry,and microglia were analyzed by morphology;The NLRP3,CD86/Iba-1,CD206/Iba-1 of inflammatory corpuscles were observed by immunofluorescence staining;The expression of MBP,Syp,PSD-95,Iba-1,M1 phenotypic markers CD86 and i NOS,M2 phenotypic markers CD206and Arg-1,NLRP3 and its related proteins Caspase-1 and IL-1βwere detected by Western blot;The m RNA expression of inflammatory cytokines Il1b,Tnfa,Il10 and Tgfb was detected by RT-PCR;The concentrations of IL-1β,TNF-α;,IL-10 and TGF-βin brain homogenate were detected by ELISA.28 days after the model was made,Morris water maze was performed to detect the changes of learning,cognition and behavior of the two groups of rats.2.2-day-old rats were randomly divided into 5 groups(Sham group,HI group,Caffeine group,60 rats in each group;Caffeine+CGS21680 group,Caffeine+GW9662group,12 rats in each group):Sham group,HI group,Caffeine group,HI+Caffeine+CGS21680(Caffeine+CGS21680)group and HI+Caffeine+GW9662(Caffeine+CW9662)group.Caffeine group received continuous intraperitoneal injection of caffeine citrate(20mg/kg)for 5 days(once a day)from P2 to P6.Caffeine+CGS21680group received injection of caffeine citrate and A2AR agonist CGS21680(2mg/kg)for 5days(once a day).Caffeine+GW9662 group received injection of caffeine citrate and PPARγinhibitor GW9662(4mg/kg)for 5 days(once a day).The preparation of WMD model was still carried out at P3.Samples were collected at 7 to 21 days after model preparation.The weight changes of rats in each group were recorded.The pathological changes of brain tissue were observed by HE staining.MBP,Iba-1 and A2a R were observed by immunohistochemistry,and microglia were analyzed by morphology.NLRP3,Syp,PSD-95,CD86/Iba-1,CD206/Iba-1 and PPARγ/Iba-1 were observed by immunofluorescence;The expression of MBP,Syp,PSD-95,Iba-1,CD86,i NOS,CD206,Arg-1,NLRP3,caspase-1,IL-1β,A2a R,total PPARγ,p-PPARγ,nuclear PPARγand plasma PPARγwere detected by Western blot;The m RNA expression of inflammatory cytokines Il1b,Tnfa,Il10 and Tgfb was detected by RT-PCR;The concentrations of IL-1β,TNF-α,IL-10 and TGF-βin brain homogenate were detected by ELISA.28 days after the model was made,Morris water maze was performed to detect the changes of learning,cognition and behavior of the two groups of rats.3.2-day-old rats were randomly divided into three groups(27 rats in each group):Sham group,HI group and Caffeine group.The administration time and mode of caffeine were the same as before,and WMD model was still carried out at P3.At 14 days after hypoxia ischemia,3 rats in each group were randomly selected for proteomic identification and analysis of differential proteins,and GO and KEGG pathway enrichment analysis and PPI protein interaction analysis were carried out for these differential proteins.Differential proteins SIRT2,aquaporin 4(AQP4)and bradykinin-1(KNG-1)were detected by Western blot;SIRT2 was observed by immunohistochemistry.The protein of the second part model was used to detect SIRT2 at different time points.4.2-day-old rats were randomly divided into four groups(Sham group,HI group,Caffeine group,Caffeine+AK-7 group,30 rats in each group):Sham group,HI group,Caffeine group,HI+Caffeine+AK-7 group.The time and manner of caffeine administration were the same as before,and the preparation method of WMD model caused by hypoxia ischemia was the same as before.Caffeine+AK-7 group was given caffeine citrate and SIRT2 inhibitor AK-7(20mg/kg)for 5 days(once a day).Samples were collected at 14 days and 21days(only Syp and PSD-95 were detected)after model preparation.Immunohistochemistry was used to observe MBP;Syp,PSD-95,Iba-1,NLRP3,SIRT2,CD86/Iba-1,CD206/Iba-1 and PPARγ/Iba-1 were observed by fluorescence.The expression of SIRT2,PPARγ,NF-κB,p-PPARγ,phosphorylated NF-κB(p-NF-κB),nuclear PPARγ,nuclear NF-κB,MBP,Syp,PSD-95,Iba-1,CD86,i NOS,CD206,Arg-1,NLRP3,Caspase-1,IL-1βwere detected by Western blot;The m RNA expressions of Il1b,Tnfa,Il10 and Tgfb were detected by RT-PCR;The concentrations of IL-1β,TNF-α,IL-10 and TGF-βin brain homogenate were detected by ELISA.Using the second part of the model,SIRT2 was observed by fluorescence;SIRT2and total PPARγwere detected by Western blot;the m RNA expression of sirt2 was detected by RT-PCR.Results:1.(1)Pathological changes of paraventricular area in rats after hypoxia-ischemia:There were asymmetric enlargement of left ventricle,light staining of corpus callosum(CC)and subventricular zone(SVZ)in white matter,decreased number of cells,sparse structure and sieve-like changes at 7 to 21 days after hypoxia-ischemia.(2)Myelination and synaptic dysfunction in paraventricular region of rats after hypoxia-ischemia:Immunohistochemistry and Western blot showed that the expression of MBP in CC zone of HI group decreased at each time point compared with Sham group.The expression of Syp and PSD-95 decreased at 14 and 21 days after HI.(3)The learning and cognitive ability of rats decreased after hypoxia-ischemia:Morris water maze test showed that compared with Sham group,the escape incubation period of rats in HI group was prolonged in positioning test,and the number of crossing platforms and the frequency of target quadrant movement in space exploration test were significantly reduced in HI group.(4)Activation of NLRP3 inflammatory corpuscles in the paraventricular area of rats after hypoxia-ischemia injury:Immunohistochemistry and Western blot showed that the expression of NLRP3,Caspase-1 and IL-1βin the paraventricular area of rats in HI group was higher than that in Sham group at 7,14 and21 days after hypoxia-ischemia injury.(5)The activation of microglia in paraventricular area of rats after hypoxia-ischemia injury increased.Immunohistochemistry and Western blot showed that the expression of Iba-1 in CC and SVZ of microglia in HI group increased at 7,14 and 21 days after hypoxia-ischemia compared with Sham group.Morphological analysis showed that the morphology of microglia in HI group showed amoeba-like,rod shape and hypertrophic shape,the length of process length and the number of endpoint increased.The M1 phenotypic markers CD86/Iba-1 and M2phenotypic markers CD206/Iba-1 were increased,and the corresponding protein expressions of Iba-1,CD86,i NOS,CD206 and Arg-1 were also increased.(6)Changes of inflammatory factors in the paraventricular region of rats after hypoxic-ischemic injury:The results of RT-PCR and ELISA showed that the levels of transcription and secretion of M1 phenotype inflammatory factors IL-1βand TNF-αwere increased in HI group compared with Sham group.However,the secretion level of M2 phenotype inflammatory factors IL-10 and TGF-βincreased.2.(1)Caffeine intervention did not affect the changes of body weight in rats with hypoxic-ischemic brain damage:There was no significant difference in body weight among the three groups at 21 days after hypoxia ischemia.(2)Caffeine improved bilateral ventricular asymmetry and paraventricular pathological changes with hypoxic-ischemic injury:Compared with HI group,the increase of ventricular area and asymmetry of left and right ventricles were improved after caffeine treatment,and the number of cells in white matter increased and arranged neatly.(3)Caffeine improved the myelination of paraventricular area and synaptic dysfunction after hypoxia-ischemia in rats:Immunohistochemistry and Western blot showed that the expression of MBP in Caffeine group increased at all time points compared with HI group.Compared with HI group,the expression of Syp and PSD-95 increased at 21 days after hypoxia ischemia,but there was no significant difference between Caffeine group and HI group at 14 days after hypoxia ischemia.(2)Caffeine can alleviate the cognitive dysfunction of neonatal rats with hypoxia-ischemia induced white matter damage.In Morris water maze,the escape incubation period of rats after caffeine was shortened on the 4th and 5th day compared with HI group,and the exercise time of target quadrant and the times of crossing platform were improved compared with HI group.(3)Caffeine reduces the activation of paraventricular NLRP3 after hypoxic-ischemic injury:Immunofluorescence and Western blot showed that the expression of NLRP3,Caspase-1 and IL-1βin paraventricular decreased after caffeine administration.(4)Caffeine inhibited the expression of microglia in paraventricular region of rats after hypoxia-ischemia injury:Immunohistochemistry and Western blot showed that the number of Iba-1 in CC and SVZ decreased,the number of CD86/Iba-1 of M1 phenotype decreased,the number of CD206/Iba-1 of M2 phenotype increased,and the corresponding expressions of Iba-1,CD86 and i NOS protein decreased,and CD206 and Arg-1 protein increased.But the morphological analysis of microglia showed that caffeine treatment had no obvious improvement on microglia morphology.(5)Caffeine regulates the release of inflammatory cytokines in paraventricular region after hypoxic-ischemic injury:RT-PCR and ELISA showed that the transcription and secretion levels of M1 phenotype inflammatory cytokines IL-1βand TNF-αdecreased after caffeine treatment,while the transcription and secretion levels of M2 phenotype inflammatory cytokines IL-10 and TGF-βincreased.(6)Caffeine regulates PPARγpathway:Immunofluorescence and Western blot results showed that caffeine can increase the expression of PPARγtotal protein and nuclear protein,and also up-regulate the level of p-PPARγ,which were all down-regulate in HI group.(7)Caffeine regulates microglial phenotypic polarization and cytokine expression through PPARγ:Immunofluorescence and Western blot results showed that compared with Caffeine group,the number of CD86/Iba-1 increased and the number of CD206/Iba-1 decreased,and the corresponding expression of CD86 protein increased and CD206 protein decreased after administrating PPARγinhibitor GW9662.The results of RT-PCR showed that compared with Caffeine group,the levels of Il1b and Tnfa were higher,while the levels of Il10 and Tgfb were lower in Caffeine+GW9662group.(8)Caffeine mediates neuroinflammatory response and microglia phenotypic polarization by antagonizing A2a R:Western blot results showed that compared with Caffeine group,the expression of NLRP3,Caspase-1,IL-1βand CD86 increased,CD206decreased in Caffeine+CGS21680 group.In addition,RT-PCR results showed that Il1b and Tnfa increased,Il10 and Tgfb decreased in Caffeine+CGS21680 group compared with Caffeine group.3.(1)Screening of differentially expressed proteins in white matter damage induced by hypoxia ischemia in neonatal rats treated with caffeine:47 differentially expressed proteins were identified by proteomics.Compared with both Sham group and Caffeine group,27 proteins were up-regulated in HI group;20 proteins were down-regulated in HI group.The enrichment of GO and KEGG pathways showed that these pathways were related to myelin development and synapse formation.At the same time,NLRP3-related pathway and glial cell-related pathway were enriched.PPI protein interaction showed that SIRT2 and AQP4 were closely related to myelination protein,and SIRT2 could be enriched in NLRP3 component pathway.After verification,SIRT2 is a differential protein in caffeine improving brain white matter damage.(2)SIRT2 is an important differential protein for caffeine to improve white matter injury induced by hypoxia-ischemia in neonatal rats:Immunohistochemistry and Western blot showed that the expression of SIRT2 in HI group was lower than that in Sham group at each time point,and it was obviously improved after caffeine administration.4.(1)Caffeine improves myelination and long-term synaptic development through SIRT2:Immunofluorescence and Western blot results showed that the expression of MBP,Syp and PSD-95 was down-regulated after administration of SIRT2 inhibitor AK-7compared with Caffeine group.(2)Caffeine inhibited the activation of NLRP3 through SIRT2:Immunofluorescence and Western blot showed that the expression of NLRP3,Caspase-1 and IL-1βincreased after administration of SIRT2 inhibitor AK-7 compared with Caffeine group.(3)Caffeine inhibited the expression of Iba-1 through SIRT2:Immunofluorescence and Western blot showed that the expression of Iba-1 increased,the number of CD86/Iba-1 increased,the number of CD206/Iba-1 decreased,the corresponding expressions of Iba-1,CD86 and i NOS increased,and the expression of CD206 and Arg-1 decreased after administration of SIRT2 inhibitor AK-7 compared with Caffeine group.(4)Caffeine modulates the release of inflammatory cytokines through SIRT2:ELISA results showed that the concentrations of IL-1βand TNF-αwere higher,while the concentrations of IL-10 and TGF-βwere lower in Caffeine+AK-7 group than those in Caffeine group.(5)Caffeine increased the nuclear translocation and phosphorylation of PPARγthrough SIRT2:Immunofluorescence co-localization of PPARγ/Iba-1 and Western blot showed that the expression of PPARγtotal protein and nuclear protein decreased after administration of AK-7 compared with Caffeine group,and the p-PPARγ/total PPARγprotein also decreased.The expression of NF-κB nuclear protein also increased.(6)SIRT2 is an upstream protein of caffeine activated PPARγpathway after hypoxia ischemia:The results of immunofluorescence,Western blot and RT-PCR showed that SIRT2 expression was decreased in HI group,and caffeine could up regulate SIRT2 expression.After administration of PPARγinhibitor GW9662,SIRT2 did not change significantly at protein and transcription levels compared with Caffeine group.Conclusions:1.In hypoxic-ischemic white matter damage of neonatal rats,there are not only ventricular enlargement and myelination disorder of paraventricular white matter,but also abnormal synaptic development in paraventricular area and behavioral changes.2.Phenotypic polarization caused by continuous activation of microglia mediates neuroinflammatory response,which is involved in the occurrence and development of brain white matter damage caused by hypoxia ischemia.3.The brain protective effect of caffeine can not only reduce the disorder of myelination development,but also improve the long-term synaptic formation and cognitive impairment.4.Caffeine regulates the phenotypic polarization of microglia by activating PPARγthrough acting on the classical receptor A2a.5.Based on proteomic screening,SIRT2 was found and verified to be a key molecule for caffeine to promote myelination development and synapse formation and improve the pathological outcome of white matter damage.6.Caffeine promotes PPARγphosphorylation and nuclear translocation,regulates the phenotypic polarization of microglia,which may be related to the up-regulation of SIRT2,and inhibits neuroinflammatory response,which is one of the mechanisms of brain protection.