The integration strategy of Didang decoction against cerebral ischemia-reperfusion injury: Regulation of apoptosis-autophagy-inflammation network and validation of PI3K/Akt pathway

ETHNOPHARMACOLOGY RELEVANCE: Didang decoction (DDD) is a classic prescription for blood-breaking, blood-stasis-removing and collateral-dredging in Treatise on Febrile Diseases and Synopsis of the Golden Chamber. DDD is mainly used to treat "blood stasis syndrome" and is associated with diseases such as thrombosis, ischemic stroke, and microcirculatory disorders in modern medicine. Its core mechanism is believed to improve tissue ischemia and inflammatory response by blood-breaking and blood-stasis-removing (activating blood circulation and removing stasis). However, the effects of its components and their multi-target regulatory mechanisms on cerebral ischemia-reperfusion injury (CIRI) have not been elucidated. AIM OF THE STUDY: To explore the metabolic mechanism of DDD on CIRI and its potential therapeutic effect. MATERIALS AND METHODS: This study developed a rat model of middle cerebral artery occlusion-reperfusion. The efficacy and optimal dose of DDD were first detected by mNss, TTC, HE, and Nissl. Secondly, network pharmacology and non-targeted metabolomics were used to predict the treatment targets and components of Didang decoction. The specific mechanism of action of Didang decoction was finally confirmed through inflammatory factor detection, co-expression immunofluorescence, TEM, and WB. RESULTS: The DDD H dose group significantly reduced the neurological deficit score, reduced the volume of cerebral infarction, and restored brain tissue damage. In total, 77 components of Didang decoction were identified based on UHPLC-MS/MS technology. Among these, Kaempferol, Rhein, Alizarin, and other components were associated with neuroprotection. Metabolomics screening revealed that DDD upregulated 42 metabolites and downregulated 78 metabolites. Network pharmacology predicted that core targets including AKT1 and IL6 coordinated the apoptosis-autophagy-inflammation network by modulating the PI3K/Akt signaling pathway. Animal experiments further confirmed that DDD significantly suppressed the expression of Bax, Cleaved-Caspase-3, Cytochrome C, LC3-II, Becline1, ULK1, NLRP3, and P-NF-кBp65 proteins; DDD also downregulated the levels of proinflammatory factors and activated the key nodes of the PI3K/Akt/mTOR pathway. CONCLUSION: For the first time, this study systematically revealed the molecular mechanism by which Didang Decoction synergistically modulates metabolic networks and alleviates cerebral ischemia-reperfusion injury via multiple components.