Phoenixin-20 ameliorates brain infarction by promoting microglia M2 polarization in an ischemic stroke model

Abstract

Ischemic stroke is one of the most common causes of death worldwide. The transformation of microglia from the classic M1 to the alternative M2 state has been shown to have both deleterious and immunosuppressive roles in neuroinflammation. Microglial polarization toward the M2 phase is currently proposed to be a beneficial phenotype in brain ischemic injury. Phoenixin-20 is a newly identified pleiotropic neuropeptide expressed abundantly in different brain regions. In this study, we found that administration of Phoenixin-20 in ischemic stroke middle cerebral artery occlusion (MCAO) mice significantly reduced the brain infarction area but improved the neurological deficit score. Gene expression analysis showed Phoenixin-20 treatment inhibited pro-inflammatory M1 phase microglial markers: a cluster of differentiation molecule 11b (CD11b), cluster of differentiation molecule 86 (CD86), inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and increased anti-inflammatory M2 phase markers (found in Inflammatory Zone 1 (FIZZ1), Arginase 1 (Arg-1), Chitinase 3-like 3 (YM1), and interleukin-10 (IL-10)) in the infarcted brain. We further investigated the molecular mechanism of Phoenixin-20 in cultured microglia. We found that treatment with it induced signature genes expression in microglial M2 state, including Fizz1, Arg-1, YM1, and IL-10, indicating the promotion of microglial polarization toward the M2 state. Furthermore, we found that treatment with the M2 phase cytokine interleukin 4 (IL-4) induced the expression of microglial G Protein-Coupled Receptor (GPR173), which is the receptor of Phoenixin-20. Silencing of the microglial signal transducer and activator of transcription 6 (STAT6) partially blocked the effect of IL-4 on GPR173, suggesting that STAT6 is the upstream regulator of GPR173. Finally, we showed that the silencing of GPR173 completely abolished the effect of Phoenixin-20 in microglia, indicating the dependency of its regulatory role on GPR173. Collectively, our study demonstrates that Phoenixin-20 has a protective role in the acute stroke model. Our cell-based study demonstrates Phoenixin-20 promotes microglia toward M2 transformation, which could be the mechanism of its neuroprotection.

Keywords: GPR173; Microglia M2 polarization; Middle cerebral artery occlusion (MCAO); Neuroinflammation; Phoenixin-20; STAT6.