Abstract: Inflammation plays a key role in driving the secondary brain injury that follows ischemic stroke. Melatonin is an endogenous neuroendocrine hormone that regulates mitochondrial homeostasis. However, the role and mechanisms by which melatonin regulates microglial pyroptosis and the inflammatory cascade through double-stranded DNA (dsDNA)-sensing cyclic GMP-AMP synthase (cGAS) signaling warrant further study. Using middle cerebral artery occlusion mice, we investigated the effects of melatonin on cGAS-mediated pyroptosis and neuroinflammation. Middle cerebral artery occlusion model mice exhibited significantly increased DNA damage and cytoplasmic dsDNA release, as reflected by γH2AX staining, as well as heightened activation of the cytosolic dsDNA-sensing cGAS-STING pathway, both of which were notably suppressed by melatonin treatment. Melatonin also mitigated NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome activation and nuclear factor (NF)-κB/gasdermin D-mediated pyroptosis in microglia following ischemic stroke, while exhibiting the capacity to attenuate the immune response to ischemia in mice. This led to reduced infiltration of peripheral neutrophils and monocytes/macrophages in the ischemic brain. Specifically, melatonin administration resulted in reductions in the numbers of ionized calcium-binding adapter molecule 1-positive cells and production of interleukin-6 and tumor necrosis factor-α by microglia. Regarding neurological outcomes, melatonin significantly reduced cerebral infarct volume and ameliorated neurological deficits in mice. Notably, the neuroprotective effect of melatonin was correlated with the inhibition of cGAS activity. We also developed and tested melatonin co-loaded macrophage membrane-biomimetic reactive oxygen species-responsive nanoparticles (Mф-MLT@FNGs), which exhibited therapeutic properties in middle cerebral artery occlusion mice. Our findings suggest that melatonin acts on microglial pyroptosis to inhibit neuroinflammation and reshape the immune microenvironment through regulation of the cGAS-STING-NF-κB signaling pathway. By doing so, melatonin rescues damaged brain tissue and protects neurological function, highlighting its potential as a neuroprotective treatment for ischemic stroke.

Key words: cGAS, immune injury, inflammation, ischemic stroke, melatonin, pyroptosis, STING, microglia