An integrated approach elucidates Xiongzhi Tongluo formula against ischemic stroke.

Abstract

BACKGROUND: Ischemic stroke (IS) remains a critical challenge in stroke management. Xiongzhi Tongluo formula (XZTLF), a traditional Chinese herbal prescription, has shown potential in treating ischemic stroke, yet its mechanisms remain elusive. METHODS: This integrated study combined clinical and in vivo approaches to investigate XZTLF. We first employed network node similarity algorithms, ultra-high performance liquid chromatography quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MS)-based phytochemical profiling, and network pharmacology to identify potential molecular targets for ischemic stroke (IS) intervention. The predicted hub genes were then validated through molecular docking and a multicenter randomized controlled trial in patients with acute IS. For in vivo validation, a middle cerebral artery occlusion (MCAO) rat model was used. Rats were treated with XZTLF via oral gavage at doses of 2.97 or 11.88 g/kg/day for 7 days, using edaravone dexborneol (10 mg/kg, i.p.) as a positive control. Key targets and mechanisms of action were further assessed through biochemical and histological analyses of rat brain tissue. RESULTS: XZTLF demonstrated significantly closer network-based proximity to IS targets compared to control prescriptions. Chemical systematic pharmacology analysis identified 173 bioactive constituents in XZTLF. Network pharmacology screening revealed eight core targets (NFKB1, BCL2L1, MAPK1, STAT1, CDKN2A, AR, RAF1, and MMP2), which were subsequently validated in IS patients through ELISA quantification. In MCAO rat models, XZTLF administration significantly reduced cerebral infarct volume and improved neurological function. Mechanistically, XZTLF attenuated neuroinflammation by downregulating pro-inflammatory mediators (including IL-6, TNF-α, and NF-κB), mitigated oxidative damage (MDA, SOD, CAT), and suppressed hyperactivation of the PI3K/AKT signaling pathway. CONCLUSION: XZTLF shows potential for treating IS through multimodal regulation of neuroinflammatory cascades, redox homeostasis, and autophagy pathways, based on integrated preclinical and preliminary clinical findings.