Intermittent hypoxic training regulates the microglia phenotype by increasing melatonin receptor expression.

Abstract

Microglia is a resident immune cell in the brain. Microglia could be polarized into two phenotypes: pro‐inflammatory (M1) and anti‐inflammatory (M2). The pro‐inflammatory phenotype of microglia is known to harmful in ischemic stroke disease processes. An intervention, therefore, that could shift the phenotype of microglia toward the anti‐inflammatory phenotype could be beneficial in cerebral protection against ischemic stroke. Intermittent hypoxic training (IHT) protects the microglia against hypoxia and reoxygenation stress and increases the M2/M1 ratio. The involvement of the melatonin, an antioxidant, was proposed from the many laboratories. We studied the function of melatonin in the polarization of microglia in the IHT.MethodsEOC20, mouse microglia, have been assigned to the control, oxygen‐glucose deprivation (OGD), IHT, OGD + IHT. The cell viability of each group was assayed by using Calcein‐AM assay. The phenotype of EOC20 cells was determined with immunocytochemistry staining CD 68 (M1) and CD 206 (M2) and quantified by flow cytometry. The involvement of melatonin (30 μM) and melatonin receptor was determined by using melatonin and melatonin receptor antagonist, Luzindole (50 μM). Protein contents of melatonin receptor 1B, Akt, Erk1/2 were measured by immunoblot. Two factors (treatment and protocol) ANOVA and Tukey post hoc test was used to find statistical significance. P<0.05 was considered a significant value.ResultCell viability in the OGD group was significantly decreased comparing to the control, IHT, OGD+ IHT groups. Meaningful elevation in the melatonin 1B receptor and phosphorylation of Akt and Erk1/2 were detected, but those protein contents were blunted with Luzindole treatment. M2/M1 ratio was altered with melatonin treatment, control group 0.32 and melatonin group 2.7.ConclusionMicroglia was activated and polarized to the pro‐inflammatory phenotype after OGD and reoxygenation. However, IHT was able to mitigate cell death and shift the phenotype to the anti‐inflammatory phenotype. IHT‐induced favorable effects including phenotype of microglia were diminished by inhibiting melatonin 1B receptor with Luzindole. Results in the current study proposed that polarization of microglia influenced by the activation of the melatonin pathway.