The nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome plays a crucial role in the prognosis of subarachnoid hemorrhage (SAH). WNK1 kinase negatively regulates NLRP3 in various inflammatory conditions, but its role in early brain injury (EBI) after SAH remains unclear. In this study, we used an in vivo SAH model in rats/mice and AAV-WNK1 intraventricular injection to investigate its neuroprotective mechanisms. WNK1 expression was significantly reduced in SAH patient blood and SAH model brain tissue, correlating negatively with microglial activation. AAV-WNK1 alleviated brain edema, neuronal necrosis, behavioral deficits, and inflammation by inhibiting NLRP3 inflammasome activation. In hemin-stimulated BV-2 cells, WNK1 overexpression reduced NLRP3 activation and inflammatory cytokines. Chloride counteracted WNK1's inhibitory effects, and WNK1 suppressed P2X7R-induced NLRP3 activation. Mechanistically, WNK1 functioned via the OXSR1/STK39 pathway. These findings highlight WNK1 as a key regulator of intracellular chloride balance and neuroinflammation, presenting a potential therapeutic target for SAH treatment.
Animals ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Subarachnoid Hemorrhage/complications* ; Inflammasomes/metabolism* ; Rats ; Mice ; Neuroinflammatory Diseases/metabolism* ; WNK Lysine-Deficient Protein Kinase 1/genetics* ; Male ; Humans ; Chlorides/metabolism* ; Mice, Inbred C57BL ; Rats, Sprague-Dawley ; Brain Injuries/metabolism* ; Microglia/metabolism* ; Protein Serine-Threonine Kinases

OBJECTIVETo investigate the association between single nucleotide polymorphisms (SNPs) in Wnk1 gene and ischemic stroke in Chinese Han population.

METHODSA hospital-based case-control study was carried out. The ischemic stroke group included 294 Chinese Han subjects, who were admitted with non-fatal ischemic stroke in departments of neurology of 5 hospitals in Xinjiang during January 2008 through December 2009. Control group included 314 age and sex-matched Han subjects without an inquired history of stroke, hospitalized in departments of surgery of these 5 hospitals. Ten tagging SNPs (tSNPs) of the Wnk1 gene were genotyped, and the association between these tSNPs and ischemic stroke were evaluated. The tSNPs (rs3858703, rs11611246, rs7305065, rs1990021, rs34408667, rs12309274, rs1012729, rs956868, rs12828016 and rs953361) were determined by the Multiplex SNaPshot platform. The data were analyzed by using t-test, Ξ2-test and logistic regression. Linkage disequilibrium and haplotype were analyzed by Haploview software.

RESULTSThe rates of alcohol drinking, hypertension ,diabetes and hyperlipidemia in ischemic stroke group were higher than those in control group (37.1% vs 21.0%, 62.9% vs 36.6%, 18.0% vs 6.1% and 36.4% vs 17.5%, respectively, all P<0.01). No significant difference in smoking rate was found between two groups. The genotyping loss rates of all sites were less than 1%. All the tSNPs were examined by Hardy-Weinberg equilibrium test except rs34408667. tSNP rs11611246 in the 4th intron of the Wnk1 gene was significantly associated with ischemic stroke. The distribution frequency of T allele in cases was significantly lower than that in male controls (30.3% vs 35.7%, P =0.046). When the samples were further stratified according to gender, rs11611246 was found to be associated with a reduced risk of ischemic stroke in male cases than in controls. GT and TT genotype frequencies were 43.3% and 7.2% in male cases, 43.1% and 15.2% in male controls, respectively (P=0.038). The T allele was associated with a reduced risk of ischemic stroke, with a per-allele OR of 0.702(95%CI:0.517-0.953, P=0.023) in male cases than in male controls. The significance remained after adjusting the covariates of age (P=0.022), or the covariates of age, BMI, cigarette smoking, alcohol drinking, hypertension, diabetes and hyperlipidemia (P=0.008). No association between other 9 tSNPs and ischemic stroke was noted in Chinese Han subjects.

CONCLUSIONThe polymorphism of rs11611246 on the 4th intron of Wnk1 gene is associated with a reduced risk of ischemic stroke in Chinese Han population and the T allele might be a protective factor for ischemic stroke in male Chinese Hans.

Aged ; Asian Continental Ancestry Group ; genetics ; Brain Ischemia ; genetics ; Female ; Genotype ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; Male ; Middle Aged ; Minor Histocompatibility Antigens ; Polymorphism, Single Nucleotide ; Protein-Serine-Threonine Kinases ; genetics ; Stroke ; genetics ; WNK Lysine-Deficient Protein Kinase 1