Ischemic stroke is one of the major causes of morbidity and mortality. The β‐1, 3‐galactosyltransferase 2 (B3galt2), a member of β‐1, 3‐galactosyltransferase family, is playing a vital role in the pathological process of cerebral ischemic injury, but its underlying mechanisms remain unclear. In the present study, we examined the involvement of oxidative stress and NLRP3 inflammasome activation in the neuroprotective effect of B3galt2. Cerebral ischemia/reperfusion (I/R) injury was simulated in a mouse middle cerebral artery occlusion (MCAO) model. Recombinant human B3galt2 (rh‐B3galt2) was administered intranasally 1 h post MCAO, and TGF‐β1‐siRNA was administered intracerebroventricularly 24 h before MCAO. Outcome measures included brain infarct volume, neurological function, blood‐brain barrier (BBB) permeability, neuronal apoptosis, oxidative stress, and the inflammatory response. First, we found that rh‐B3galt2 significantly alleviated brain infarct volume and BBB permeability, improved neurological function, and attenuated I/R‐induced neuron apoptosis and oxidative stress. Furthermore, rh‐B3galt2 attenuated pro‐inflammatory cytokines, NF‐κB, IL‐6, TNF‐α, and IL‐1β, and inhibited NLRP3 inflammasome activation. Finally, inhibition of TGF‐β1 by TGF‐β1‐siRNA abolished the anti‐oxidative and anti‐inflammatory effects of rh‐B3galt2 in mice after I/R. Collectively, our study demonstrated that rh‐B3galt2 exerts neuroprotective effects by regulating cerebral ischemia‐induced oxidative stress and NLRP3 inflammasome, which is mainly dependent on the heightening of the TGF‐β1 pathway. Thus, B3galt2 might be considered a new therapeutic target for ischemic stroke treatment.

The molecular mechanisms of blood-brain barrier (BBB) disruption in the early stage after ischemic stroke are poorly understood. In the present study, we investigated the potential role of nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) in ischemia-induced BBB damage using an animal middle cerebral artery occlusion (MCAO) model of ischemic stroke. Recombinant human NMNAT1 (rh-NMNAT1) was administered intranasally and Sirtuin 1 (SIRT1) siRNA was administered by intracerebroventricular injection. Our results indicate that rh-NMNAT1 reduced infarct volume, improved functional outcome and decreased BBB permeability in mice after ischemic stroke. Furthermore, rh-NMNAT1 prevented the loss of tight junction proteins (occludin and claudin-5) and reduced cell apoptosis in ischemic microvessels. NMNAT1-mediated BBB permeability was correlated with the elevation of nicotinamide adenine dinucleotide (NAD+)/NADH ratio and SIRT1 level in brain microvascular endothelial cells. In addition, rh-NMNAT1 treatment signi cantly decreased the levels of acetylated nuclear factor-κB, acetylated p53 and matrix metalloproteinase-9 in ischemic microvessels. Moreover, the protective effects of rh-NMNAT1 could be reversed by SIRT1 siRNA. In conclusion, these ndings indicate that rh-NMNAT1 protects BBB integrity after cerebral ischemia via the NAD+/SIRT1 signaling pathway in brain microvascular endothelial cells. NMNAT1 may be a novel potential therapeutic target for reducing BBB disruption after ischemic stroke.