Enhancing Annexin-1and NFPRs Interaction Regulates Microglial Activation To Protect Neuron From Ischemia Like Injury

Objective:To explore whether annexin-1-nFPRs signaling affects microglial phenotype and migration in primary microglia or BV-2cell lines, to determine annexin-1-nFPRs signaling mediated neuroprotective effect and mechanism in the ischemic microenvironment.Methods:RT-PCR and Western blot were performed to determine which cell types express annexin-1and nFPRs in the central nervous system, intracellular [Ca2+] change was measured by calcium image analysis, the expression of Annexin-1, Iba-1, nFPRs, arginase-1, HA proteins was detected by Western blot, and neuronal viability was determined by MTT assay, these cytokines release in microglia-neuron coculture was measured by ELISA kits, in vitro migration assays was performed to determine microglial migration numbers by transwell chamber.Results:1. Annexin-1is sparingly expressed in microglia, oxygen and glucose deprivation (OGD) treatment and neuron-conditioned media (NCM) treatment could significantly upregulate annexin-1expression, Interestingly, Iba-1, a common marker of microglial activation, was upregulated after OGD and NCM treatment, and shown similar tendency with annexin-1upregulation.2. Similarly, annexin-1related receptors nFPRs are also sparingly expressed in microglia, annexin-1mimetic peptide Ac2-26could activate nFPRs, evoked [Ca2+]i increased in microglia and upregualted these receptors, moreover, nFPRs were upregulated by NCM stimulation.3. Ac2-26protects neuron from ischemia like injury via activating nFPRs on microglia.4. Enhancing annexin-1and nFPRs interaction alters microglia into anti-inflammatory phenotype (M2phenotype) and promotes microglial migration.5. Glutamate and ATP could significantly upregulate annexin-1expression, conversely, treated microglia with NMDA receptors antagonist-MK-801and ATP degrating enzyme-Apyrase in the NCM could block NCM induced annexin-1upregulation, suggested that neurotransmitter glutamate and ATP are involved in annexin-1mediated neuroprotective effect.Conclusion:Taken together, we could propose that injured neurons release glutamate and ATP to elicit microglial response, and upregulated or secreted annexin-1interaction with nFPRs through an autocrine and/or paracrine fashion to favor microglial M2polarization and promote migraton, which represents a positive feedback process.