Study of NOX4/ROS/NF-κB signaling pathway in anti-ischemia-reperfusion injury effect and mechanism of STQJD

This study aimed to investigate the effects of Shu-Tiao Qi-Ji Decoction (STQJD) on a rat model of cerebral ischemia-reperfusion injury and to elucidate its mechanism of action. The therapeutic effects of STQJD on ischemic stroke (IS) were assessed using neurobehavioral scores, 2,3,5-Triphenyltetrazolium chloride (TTC), Nissl and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Network pharmacology was used to investigate the potential mechanisms of STQJD. Reactive Oxygen Species (ROS) content and expression levels of inflammatory markers in rat serum were detected. The mRNA and protein expression levels of Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), NADPH oxidase 4 (NOX4), Tumor necrosis factor-alpha (TNF-α), NOD-like receptor family pyrin domain containing 3 (NLRP3), Apoptosis-associated speck-like protein containing a CARD (ASC), Toll-like receptor 4 (TLR4), and Cysteine-dependent aspartate-specific protease-1 (Caspase-1) in the brain tissue of rats were detected using RT-qPCR and Western blotting. Immunofluorescence was used to detect NF-κB and TNF-α protein expression. STQJD markedly improved neurological function, reduced cerebral infarct volume, and mitigated neuronal damage and apoptosis in rats subjected to Middle Cerebral Artery Occlusion (MCAO). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that STQJD's anti-IS effects are linked to NOX4/TLR4/NF-κB/TNF-α signaling pathways. STQJD reduced serum levels of inflammatory markers, including TNF-α, Interleukin-1 beta (IL-1β), and Interleukin-18 (IL-18), as well as mRNA and protein expression of TLR4, NOX4, NLRP3, NF-κB, ASC, TNF-α, and Caspase-1 in the brain tissues of MCAO rat levels. STQJD effectively ameliorates IS in MCAO/R rats by modulating the NOX4/TLR4/NF-κB/TNF-α signaling pathway, reducing inflammation and oxidative damage, and exerting neuroprotective effects.