NAD+ Attenuates Neuroinflammation in Ischemic Stroke by Suppressing the cGAS- STING Pathway via Mitochondrial Homeostasis Restoration

Stroke remains one of the leading causes of death and disability worldwide, with neuroinflammation playing a critical role in its pathogenesis. The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is a major driver of innate immune responses activated by endogenous DNA, including mitochondrial DNA (mtDNA), and contributes significantly to post-ischemic neuroinflammation. Nicotinamide adenine dinucleotide (NAD⁺), a vital metabolic coenzyme, has demonstrated anti-inflammatory potential in various diseases, including stroke; however, its specific role in regulation of neuroinflammation following stroke remains unclear. In this study, we examined the protective effects of NAD⁺ against ischemic stroke and explored its role in modulating cGAS-STING-mediated neuroinflammatory responses. Utilizing a transient middle cerebral artery occlusion/reperfusion mouse model and an oxygen-glucose deprivation/reoxygenation BV2 microglia model, we observed that NAD⁺ treatment significantly reduced infarct volume and brain edema, and improved neurological outcomes. NAD⁺ reduced the levels of pro-inflammatory cytokines and facilitated the polarization of microglia from pro-inflammatory M1 to the anti-inflammatory M2 phenotype. This effect was associated with the inhibition of cGAS-STING-TBK1 signaling pathway activation. Pharmacological inhibition or genetic knockdown of STING largely abrogated the anti-inflammatory effects of NAD⁺ in BV2 microglia, indicating a STING-dependent mechanism. Furthermore, NAD⁺ restored mitochondrial respiratory function and reduced cytoplasmic mtDNA leakage, suggesting that the improvement of mitochondrial homeostasis may underlie the suppression of cGAS-STING-TBK1 signaling pathway by NAD⁺. Our findings indicate that NAD⁺ alleviates neuroinflammation following brain ischemia/reperfusion injury by regulating mitochondrial homeostasis and attenuates the cGAS-STING-TBK1 signaling pathway activation in microglia, thereby providing new therapeutic insights for ischemic stroke.