Melatonin Ameliorates Ischemic Stroke by Inhibiting M1 Polarization of Microglia through Suppression of the MAPK/ERK Pathway

Abstract

Background: Melatonin (MT) has been recognized for its neuroprotective effects. Studies have proved that polarization of microglia from a classic proinflammatory state (M1) to an anti-inflammatory state (M2) is a therapeutic approach for treating ischemic stroke (IS). Therefore, this study aims to investigate the potential mechanism of MT on microglia polarization after IS. Methods: The rat model of middle cerebral artery occlusion (MCAO) was established according to the Longa wire embolization method. BV2 microglial IS model was induced by oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro. After MT intervention, Quantitative Real-Time PCR (qRT-PCR) and western blot were used to analyze mitogen-activated protein kinase (MAPK)/extracellular-signal regulated kinase (ERK) pathway-related proteins and polarization markers expression, respectively. Enzyme-linked immunosorbent assay (ELISA) was used to measure the inflammatory factors. Hematoxylin-eosin (HE) and 2,3,5-triphenyl tetrazolium chloride (TTC) staining were used to observe the pathological changes and the volume of cerebral obstruction in rat brains, respectively. The markers of microglial polarization were evaluated by immunofluorescence staining. Cell Counting Kit-8 (CCK-8) and flow cytometry were used for the detection of viability and apoptosis. Results: In vivo studies confirmed that the expression of MAPK/ERK pathway proteins (p-ERK1/ERK1 and p-ERK2/ERK2) and proinflammatory factors inducible nitric oxide synthase (iNOS), interleukin-1beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) upregulated, and the levels of anti-inflammatory factors macrophage mannose receptor (CD206) and interleukin-10 (IL-10) downregulated in MCAO rats. MT treatment significantly reduced the volume of cerebral infarction in MCAO rats, inhibited the increase of proinflammatory markers level, and increased anti-inflammatory markers level. In vitro studies confirmed that M1 polarization was reduced, but M2 polarization was increased in MT-treated BV2 microglia. In addition, MT reduced the expression of p-ERK1/ERK1 and p-ERK2/ERK2, increased cell proliferation activity, and restrained apoptosis. Meanwhile, the addition of MAPK/ERK pathway inhibitors produced similar results to MT treatment. Activation of the MAPK/ERK pathway weakened the effect of MT on M2 polarization of microglia and promoted IS brain injury. Conclusions: MT alleviated IS-induced inflammation response, apoptosis, and M1 polarization and increased cell viability by inhibiting the activation of the MAPK/ERK pathway, thereby indicating that MT is a potential and novel therapeutic drug for IS.