Exploring the mechanisms of Gualou Xiebai Banxia decoction against myocardial ischemia by integrating network pharmacology, metabolomics, lipidomics, and experimental validation

Ethnopharmacological relevance: Gualou Xiebai Banxia decoction (GXB) is a classic formula originated from "Synopsis of the Golden Chamber", with efficacy in activating cardiac "yang", resolving phlegm, and promoting blood circulation. Nearly 2000 years of clinical practice have demonstrated the efficacy of GXB in managing myocardial ischemia (MI). Nevertheless, its regulatory effects on metabolic disturbances associated with MI and the underlying mechanisms remain obscure.

Purpose: This study aims to evaluate the therapeutic effects of GXB against MI, characterize the metabolomic and lipidomic alterations related to GXB treatment, and investigate the potential mechanisms of action.

Methods: UHPLC-Q-Orbitrap HRMS combined with network pharmacology were implemented to explore the active ingredients and potential targets of GXB in the treatment of MI. Subsequently, an isoprenaline (ISO)-induced rat model of MI was established to assess the cardioprotective effects of GXB using echocardiography, biochemical indicators, and histopathological analysis. Comprehensive metabolomics and lipidomics were conducted to characterize endogenous metabolic alterations following GXB intervention. A components-targets-differential metabolites network was constructed through integrated analysis. Further Molecular Biology experiment verification was performed in ISO-induced rat model and H9c2 cell injury model. Notably, inhibitors were used in H9c2 cells to validate the role of PLA2 and PKC in GXB's protective effects. The levels of key proteins, oxidative stress markers and inflammatory cytokines were detected.

Results: A total of 123 constituents were identified in GXB, with 18 detected in plasma. The "constituents-targets-disease" interaction network was constructed based on absorbed components. GXB improved cardiac function, alleviated cardiac injury, reduced oxidative stress and inflammation in MI rats. Metabolomic and lipidomic analyses revealed 19 differential metabolites and 122 lipids associated with GXB treatment, primarily involved in lipid metabolism, amino acid metabolism, and energy metabolism. Conjoint analysis identified four key targets, including PLA2G2A, PLA2G4A, PRKCA, and PRKCE, which were mainly linked to lipid metabolism. GXB treatment decreased the expressions of these four proteins, reduced oxidative stress and inflammation. In vitro functional experiments confirmed that inhibition of PLA2 and PKC proteins mimicked the protective effects of GXB.

Conclusion: This study demonstrates that GXB protects against MI by regulating lipid metabolism and attenuating inflammation via inhibiting the PLA2/PKC-NOX signaling axis, providing evidence for its clinical application.