The Mechanism of Montelukast Sodium to Improve Inflammation in Ischemic Stroke: Based on Astroglia-Microglia Interactions

Abstract

The present study aimed to elucidate the neuroprotective mechanism of montelukast sodium (MS) in ischemic stroke, with particular emphasis on astrocyte-microglia interactions. In vivo, adult male mice ( n = 10/group) were subjected to distal middle cerebral artery occlusion (dMCAO) and then intraperitoneally injected with MS (4 mg/kg) or an equivalent volume of saline for 7 consecutive days. In vitro, primary mouse astrocytes and microglia were isolated and co-cultured, then exposed to oxygen-glucose deprivation and reoxygenation (OGD/R) to mimic ischemic-reperfusion injury. Prior to MS intervention, astrocytes were transfected with HMGB1 overexpression plasmid (oeHMGB1) or short hairpin RNA against HMGB1 (shHMGB1) to overexpress or knock down HMGB1, or treated with an anti-HMGB1 antibody in the co-culture system. Primary outcomes included infarct volume (TTC staining), microglia activation (immunofluorescence), M1/M2-associated markers in microglia (RT-qPCR, immunofluorescence), S100 calcium-binding protein beta (S100β) levels, inflammatory mediators, and HMGB1/TLR4/NF-κB pathway activity (immunohistochemistry, RT-qPCR, Western Blot). MS administration reduced the cerebral infarction size in dMCAO mice, attenuated the upregulation of S100β and ROS levels, and suppressed HMGB1/TLR4/NF-κB signaling cascade in vivo. In the co-culture system, conditioned medium from MS-treated astrocytes promoted microglial polarization toward an M2-associated phenotype, as evidenced by increased Arg-1/CD206/IL-10 and decreased iNOS/TNF-α/CD16. The protective effects of MS were reversed by HMGB1 overexpression, but potentiated by HMGB1 neutralization. Crucially, pharmacological inhibition of TLR4 with TAK-242 mimicked the effects of MS, while the combination of MS and TAK-242 produced no additional benefits. Collectively, MS confers neuroprotection against cerebral ischemic injury by inhibiting HMGB1/TLR4/NF-κB signaling pathway and promoting microglial polarization.