Oxygen-glucose Deprivation-induced PBEF Secretion In BV2Microglial Cells

Background:Pre-B cell colony enhancing factor (PBEF), is also called visfatin and nicotinamide phosphoribosyltransferase (Nampt). It has been found to have many functions as a cytokine, the rate-limiting enzyme in NAD biosynthesis and an adipokine. Interest in PBEF has increased considerably for its potential involvement in a wide range of pathophysiological processes.PBEF is expressed in peripheral and in the central nervous system. In peripheral, PBEF regarding as a novel mediator of inflammation, is secreted to the extracellular environment by cells including lymphocytes, monocytes, macrophages, and its secretion is cell-dependent. In the central nervous system(CNS), PBEF is mostly expressed in neuron and vascular endothelial cell. After cerebral ischemia, PBEF is mainly expressed in neurons and some of microglia, but not expressed in astrocytes. Microglial cells are the immune and inflammation-related cells in the brain and the surveilant in the CNS. Microglial cells are sharing the same origin as monocytes and macrophages. Therefore, we suppose that microglial cells express and secret PBEF under inflammatory conditions, for instance, during brain ischemia. The PBEF released from microglia maybe function as a novel inflammatory factor to promote or maintain inflammation in the CNS.However, the mechanism of PBEF secretion has not to be clarified yet up to now. PBEF does not contain a consensus secretory signal sequence. Therefore PBEF seems to be secreted through a non-endoplasmic reticulum(ER)/Golgi-dependent protein secretion pathway. Therefore, in the present study we try to answer these questions:1) Do microglial cells express and release PBEF during brain ischemia?2) Which pathway is involved in PBEF secretion?3) What is the main factor that affacts PBEF secretion in microglial cells?Objectives:To observe the effects of oxygen-glucose deprivation/recovery (OGD/R) induced PBEF secretion from BV2cells, and to explore the pathways and the factors which are involved in PBEF secretion.Method:The intracellular PBEF content in BV2cells and the amount of PBEF in the culture medium were determined by Western Blotting and ELISA, respectively after the treatment of one hour-OGD and recovery for different time in BV2cells. RT-PCR was used to determine PBEF mRNA expression. The ER/Golgi secretory pathway inhibitors, protein synthesis inhibitor and nonclassical secretion inhibitors were used to determine the basic pathway that involed in PBEF secrtion. The subcelluar localization of PBEF was detected by immunostaining, and the same method was used to observe the co-localized of PBEF and cellular organs before and after OGD treatment when BV2cells were transfected with MitRed or LAMP1-RFP or LC3-GFP fusion protein expression vector. ATP and Calcium-related compounds were used to determine the role of these factors in PBEF secretion.Results:Part I Oxygen-glucose deprivation-induced PBEF secretion in BV2cellsThe intracellular PBEF content decreased after the treatment of one hour-OGD and6h-, 12h-and24h-recovery (OGD1h/R6,12,24h) in BV2cells. Meanwhile, OGD1h/R6,12,24h increased the amount of PBEF in the culture medium significantly. And BV2cells upregulated PBEF mRNA expression after exposure to OGD1h/R6,12,24h. OGD1h/R12h-induced PBEF secretion was not inhibited by brefeldin A (an ER/Golgi secretory pathway inhibitor) and cycloheximide (a protein synthesis inhibitor). While Glyburide (an ABC family transporters inhibitor) or DIDS (an ABCA1inhibitor) inhibited PBEF secretion, and NH4CI affected the secretion of PBEF. Immunofluorescence staining indicated PBEF was mainly localized in cytoplasm and fewer in nucleus, and PBEF was co-localized with mitochondria and lysosome, but not autophagosome. OGD1h/R12h caused intracellular PBEF redistribute and accumulate to the cell periphery. PBEF secretion was enhanced by exogenous ATP and Ca2+, and the secretion was inhibited by apyrase, P2receptor blocker PPADS and P2X7receptor blocker BBG.Part II Oxygen-glucose deprivation-induced PBEF and other inflammatory cytokine secretionOxygen-glucose deprivation and recovery caused BV2cells secret PBEF and other inflammatory cytokines including TNF-a and IL-6. It suggested that there was the pre-condition for the interaction between the released PBEF and other inflammatory cytokine.Part Ⅲ The role of the secreted PBEF in OGD-induced BV2cell injuryExogenous PBEF had no effect on BV2cell viability in normal condition, while OGD/R induced BV2cell injury was reversed by exogenous PBEF in a concentration-dependent manner.Conclusions:1. PBEF expression was upregulated and PBEF secretion was induced after oxygen-glucose deprivation/recovery-treatment in BV2cells; PBEF was secreted via a non-classical protein-secretion pathway, ABCA1transporter and secretory lysosome were involved in PBEF secretion; PBEF secretion was enhanced by the increment of intracellular calcium ion concentration and exogenous ATP and P2X7receptor participated in regulation of PBEF secretion from BV2cells.2. Oxygen-glucose deprivation induced PBEF secretion, together with TNF-a and IL-6release.3. Extracellular PBEF reversed oxygen-glucose deprivation-induced injury in BV2cells.