Effects Of Microglia-Expressed NAMPT On The Inflammation During The Chronic Phase Of Cerebral Ischemia And Its Mechanism

Background:Ischemic stroke is a high disability and high lethality of the disease.Cerebral ischemia-induced damage not only from the brain damage caused by acute ischemia,but also from the late secondary inflammatory response.At present,acute thrombolytic therapy for acute cerebral ischemia is effective.For the treatment of cerebral ischemia is very lack of means.Microglia is the main effector cell of inflammation and immunity in the brain,and the activation of the microglia mediates the inflammation of chronic ischemic stroke.There are two types of activated microglia called classical activation(M1 phenotype)and alternativeactivation(M2 phenotype),and it is generally believed that M1 microglia can release a variety of inflammatory factors on the recovery of brain function will have a harmful effect;and M2 microglia is considered to have anti-inflammatory effect.Therefore,the activation state M1/M2 phenotypes)of microglia can be used as a target event for the treatment of cerebral ischemic injury.Nicotinamide phosphate transferase(NAMPT),also known as visfatinand Pre-B cell colony-enhancing factor(PBEF),is a nicotinamide adenine dinucleoside plays an important role in the process of energy metabolism,senescence,inflammation,immunity of NAD and NAD-dependent enzymes(such as Sirtuins,PARPs)in cell metabolism.-NAMPT is widely distributed in the body,and can be released by peripheral inflammatory cells such as lymphocytes,macrophages,monocytes and so on.and further involved in the peripheral inflammatory process;NAMPT-specific inhibitor FK866 hasbeen reported a.significant anti-inflammatory effect.In the central nervous system,NAMPT is mainly expressed in neurons,and gliacells do not express NAMPT.Thus,the role of NAMPT in central inflammation,such as its role in cerebral ischemia inflammation,is rarely reported.Our laboratory found that NAMPT is abundantly expressed in the microglia in the brain of the aged mice,and chronic central inflammation is one of the main features of the aged brain.n addition,in the acute phase of cerebral ischemia,microglia cells did not express NAMPT;In the late stage of cerebral ischemia,microglial cells expressed NAMPT;NAMPT was not expressed in astrocytes at all time.We hypothesized that NAMPT is involved in microglia-mediated chronic inflammation in the microglial cells at the late stage of cerebral ischemia.High levels of NAMPT in the cerebrospinal fluid after blood flow may also originate from the release of microglia,in addition to injury from the injured neurons and peripheral NAMPT-infiltrated blood-brain barrier.These released NAMPTs are also involved in microglia-mediated Ischemic chronic inflammation.Therefore,this issue is concerned with the following questions:1)Whether microglia-induced expression of NAMPT is involved in the regulation of microglia-mediated inflammation in the chronic phase of cerebral ischemia,what mechanism involved?2)Does the microglia release NAMPT in ischemic and inflammatory conditions?What is the specific mechanism of NAMPT release from microglia?Objective:To investigate the regulation of NAMPT expression by microglial cells on the inflammatory response in chronic ischemic stroke and its mechanism in vivoand in vitro.To investigate the pathogenesis of NAMPT release from microglial cells and its influencing factors under ischemia and inflammation conditions.Method:Part ?The rats were treated with FK866 intracerebroventricular injection and the rats were treated with focal cerebral ischemia and reperfusion(MCAO 1h/R 14 d).The neurological function injury and recovery were observed by behavioral examination.The survival rate of neurons,the activation of astrocytes and microglia were observed by immunohistochemistry and immunofluorescence staining.Primary cultured microglia cells were treated with glucose-hypoxia/reoxygenation(OGD1 h/R24 h),and the expression of NAMPT in primary rat microglial cells was observed by fluorescence staining.After pretreatment with FK866,the morphological changes of microglial cells were observed after treated with OGD1 h/R24 h.After pretreatment with different concentrations of FK866,BV2 cells were treated with OGD1 h/R 24 h,the morphological changes of BV2 microglia were observed,and the release of inflammatory cytokines in microglial cells was detected by ELISA;RT-qPCR was used to detect the expression of M1 and M2 markers in BV2 cells after OGD1 h/R 0-24 h;BV2 cells were treated with FK866 pretreatment and OGD1 h/R for 24 h,then the expression of M1 and M2 markers in BV2 cells were detected by RT-qPCR;The expression of NF-?B in the cytoplasm and nucleus of BV2 cells was detected by OGD1 h/R 0-24 h;Then,the expression of NF-?B in the cytoplasm and nucleus was detected by FK866 pretreatment and OGD1 h/R24 h treatment;BV2 cells were treated with NF-?B inhibitorpretreatment and OGD1 h/R24 h,and the expression of TNF-?,IL-6 and NAMPT were detected by RT-qPCR.Part IIImmunofluorescence staining was used to observe the distribution and expression of NAMPT in primary cultured rat microglial cells.The release of NAMPT was detected by ELISA.The activity of LDH in supernatant was measured by LDH detection kit,and PI staining to identify cell necrosis;BV2 cells were treated with microglial-stimulating factor ATP alone or in combination with TNF-? under conditions of simulated ischemia in vitro(OGD/R).Immunofluorescent staining combined with specific labeling method was used to detect NAMPT whether co-localization with lysosome,autophagosome and endosome;The release of vesicles from BV2 cells was observed by transmission electron microscopy(TEM),and the vesicle diameter was measured as exosomes;Immunomicroscopic examination of BV2 showed that NAMPT was released by exosomes;The exosomes were collected by ultracentrifugation or commercial exosomes extraction kit.The amount of NAMPT in exosomes was detected by Western blotting.After pretreatment with phospholipase D inhibitor,BV2 cells were treated with OGD/R combined with ATP,exosomes were extracted from the supernatant of cells,and the amount of NAMPT released from exosomes was detected by Western blotting.Results:Part IIn animal experiments,pretreatment with FK866(10 nM)of NAMPT inhibitor could improve the neurological function and cerebral infarct volume induced by focal cerebral ischemia/reperfusion(MCAO 1 h/R 14 d),increase the survival rate of neurons,inhibit glial scar formation,inhibit microglia activation and proliferation;In primary cultured microglial cells,the activation of primary microglia and the up-regulation of NAMPT were induced by ischemia/hypoxia/reoxygenation(OGD1 h/R 24 h).FK866(1 nM)pretreatment inhibited OGD1 h/R24 h-induced primary microglia morphogenesis and activation;FK866(0.001-1 nM)inhibited OGD 1 h/R24 h-induced morphological changes of BV2 cells and the release of TNF-a,NAMPT,IL-6 in BV2 cells;FK866 reversed OGD1 h/R0-24 h induced the up-regulation of mRNA expression of TNF-a,IL-6 and the down-regulation of CD206 and Arg-1 mRNA expression of M1-type polarized BV2 cells;NF-?B nuclear translocation was induced by FK866(1 nM)in OGD1 h/R24 h;PDTC(50 ?M),an inhibitor of NF-?B,partially reversed OGD1 h/R24 h-induced microglia polarization.Part ?OGD/R treatment could induce primary microglia to release NAMPT;Immunofluoreseence staining showed that NAMPT had no co-localization with lysosome,autophagosome and endosome.NAMPT translocates into the cell after BV2 cells are treated with OGD/R or TNFa,and the combination of OGD/R or TNFa with ATP can "grow" a large number of NAMPT-positive vesicles bubble;The images of these vesicles were analyzed by transmission electron microscopy(TEM).The results showed that most of the vesicles were about 100 nm in diameter and corresponded to the size of exosomes.Immunoelectron microscopy confirmed the release of BV2 cells,many of these exosomes are NAMPT positive;These exosomes were collected by ultracentrifugation and detected by immunoblotting.It was found that NAMPT was expressed in exosomes,which indicated that BV2 cells released NAMPT through exosomes.We also found that phospholipase D inhibitor 1-butanol can inhibit the release of NAMPT through exosomes,indicating that phospholipase D is involved in the regulation of NAMPT release.Conclusions:1.NAMPT can be induced and released by microglial cells in the late stage of cerebral ischemia.The inhibition of NAMPT on the microglial cells could reverse the activation of NF-?B in the microglia cells induced by cerebral ischemia,While the reduction of microglia-mediated inflammation in the late stage of cerebral ischemia.2.Ischemia and inflammatory conditions induce microglia to release NAMPT through the exosomes pathway,and phospholipase D is involved in the regulation of NAMPT release.