Abstract WP040: Nitroglycerin (GTN) Alleviates Ischemic Stroke Injury via GPR35-Dependent Regulation of Mitochondrial Dysfunction and Neuronal PANoptosis

Background: Mitochondrial dysfunction and subsequent neuronal death exacerbate injury after revascularization in acute ischemic stroke (AIS). PANoptosis, a coordinated cell death program integrating apoptosis, pyroptosis, and necroptosis, has emerged as a contributor to ischemia/reperfusion injury. GPR35 is a G protein–coupled receptor expressed in the gastrointestinal tract, immune cells, and central nervous system, where it modulates inflammation, mitochondrial function, and cell survival. GTN, a widely used vasodilator, has shown potential as a neuroprotective agent, but its role in modulating PANoptosis through mitochondrial regulation remains to be determined. Methods: Male Sprague-Dawley rats (n=138) were subjected to transient or permanent middle cerebral artery occlusion (tMCAO/pMCAO) with or without GTN (10 mg/kg) administered at reperfusion onset. To determine the causal role of GPR35, the selective GPR35 antagonist CID2745687 was given at ischemia onset. Infarct volumes and neurological deficits, ATP, ROS, COX I/II and nitric oxide (NO) levels were measured. Expression of GPR35, mitochondrial dynamics-related proteins (OPA1, MFN1/2, DRP1), and PANoptosis markers (apoptosis: caspase-3, Bax; pyroptosis: caspase-1, NLRP3, GSDMD; necroptosis: RIPK1, RIPK3, MLKL) were assessed via qRT-PCR, Western blotting, immunofluorescence and TUNEL staining. Results: GTN significantly reduced infarct volumes and neurological deficits in tMCAO but not in pMCAO. In tMCAO, GTN restored NO level and enhanced mitochondrial function (↑ATP, COX I/II; ↓ROS), upregulated GPR35, promoted mitochondrial fusion (↑OPA1, MFN1/2) and suppressed mitochondrial fission (↓DRP1). GTN also significantly reduced PANoptosis mediators across all three pathways. These protective effects were abolished by GPR35 activity inhibition, confirming GPR35 as a key mediator of GTN’s neuroprotective action. Conclusion: GTN confers neuroprotection in AIS with reperfusion by preserving mitochondrial dynamics, improving bioenergetics, and inhibiting PANoptosis through the NO-GPR35 axis. Pharmacological blockade experiments confirm GPR35 as an essential target in mediating these effects, highlighting GTN as a potential therapeutic adjunct in stroke.