Huangqi Chifeng decoction alleviates cerebral ischemia and reperfusion injury by targeting NFKBIA, NF-κB1 p105 and PIAS1 to multi-dimensionally inhibit NF-κB/NLRP3 signaling: A multi-omics analysis.

BACKGROUND: Cerebral ischemia-reperfusion (I/R) injury remains a critical challenge in ischemic stroke management, with limited therapeutic options. Huangqi Chifeng Decoction (HQCF), a classical traditional Chinese medicine (TCM) formula, exhibits clinical efficacy in stroke treatment, yet its protective mechanisms remain poorly characterized.

PURPOSE: This study aims to assess the protective effects of HQCF treatment on cerebral I/R injury in mice, and to uncover the underlying molecular mechanism.

METHODS: The therapeutic effects of HQCF on cerebral I/R injury were evaluated by neurobehavioral tests, TTC staining, H&E staining and Nissl staining using the mouse model of middle cerebral artery occlusion-reperfusion (MCAO/R). Then, the compositional analysis was conducted using Q-Orbitrap LC-MS/MS. Subsequently, multi-omics, including proteomics, transcriptomics, metabolomics were performed to elucidate HQCF's therapeutic mechanisms. Key pathways and targets were validated through western blot, molecular docking, cellular thermal shift assay (CETSA), and immunofluorescence. Pathway specificity was further validated using the NF-κB pathway agonist, NF-κΒ activator 2.

RESULTS: HQCF intervention markedly alleviated neurological deficits and reduced infarct volumes in MCAO/R mice, with concomitant improvement in histopathological features, as demonstrated by comparison with the positive control drug edaravone. Integrative multi-omics profiling revealed that HQCF mitigates I/R injury partially through inhibiting NF-κB/NLRP3 signaling, with NF-κB-inhibitor alpha (NFKBIA), PIAS1 and NF-κB1 p105, the critical inhibitors of NF-κB activation, as the key potential targets of HQCF. LC-MS/MS, molecular docking and CETSA studies confirmed NFKBIA, PIAS1 and NF-κB1 p105 represent novel pharmacological targets of Oleanolic acid, Astragaloside A and Formononetin, respectively, highlighting their critical roles in mediating HQCF's neuroprotective effects. Mechanistically, HQCF promoted NFKBIA, PIAS1 and NF-κB1 p105-mediated inhibition of NF-κB activation, thereby suppressing NLRP3/GSDMD-driven pyroptosis, eventually attenuating neuronal injury in peri-infarct regions. Additionally, in vivo experiments revealed that NF-κΒ activator 2 reversed the protective effects of HQCF on cerebral I/R injury.

CONCLUSION: This study systematically demonstrates that HQCF mitigates cerebral I/R injury via multi-dimensionally inhibiting NF-κB/NLRP3 signaling-mediated neuronal pyroptosis, including targeting the precursor NF-κB1 p105, binding the NFKBIA in the cytoplasm, and targeting PIAS1 in the nuclear. These findings provide novel mechanistic insights into HQCF' therapy on cerebral I/R injury, further confirm the advantage of TCM in treating cerebrovascular disease via multitarget strategy and advance translational investigations of HQCF.