Qishen Yiqi Dropping Pills ameliorate acute myocardial infarction by modulating PKM2/STAT3 pathway-mediated metabolic reprogramming in macrophages

BACKGROUND AND PURPOSE Acute myocardial infarction (AMI) is the leading cause of cardiovascular mortality worldwide. Timely reperfusion therapy is crucial to save the ischemic myocardium and prevent infarct expansion. Inflammation, a central pathological mechanism in AMI, exacerbates myocardial tissue damage by triggering immune activation in a cascading manner. Macrophage metabolic reprogramming plays a crucial role in regulating inflammatory phenotypes in this process. Qishen Yiqi Dropping Pills (QSYQ), a traditional Chinese medicine compound, greatly improves the cardiac inflammatory microenvironment after myocardial ischemia‒reperfusion (MI/R) injury. It also inhibits myocardial fibrosis and effectively reduces cardiac dysfunction. However, it remains unclear whether QSYQ exerts cardioprotective effects by modulating the metabolic reprogramming of cardiac macrophages and the underlying mechanisms involved. METHODOLOGY To evaluate the effects on myocardial injury and the inflammatory response, QSYQ intervention was administered to a mouse model of myocardial infarction (MI) established in this study. After that, macrophages from the ischemic zone were extracted, and the expression of genes linked to metabolic reprogramming was analyzed via proteomics in conjunction with q‒PCR technology. By using this approach, the regulatory role of QSYQ in macrophage metabolism and the key target signal transducer and activator of transcription 3 (STAT3) were clarified. By utilizing the Cre-loxP system, we were able to generate mice with macrophage-specific STAT3 knockout. We then used spatial metabolomics and targeted metabolomics to confirm the regulatory pathway mechanisms in vivo. Proximity ligation assay (PLA), coimmunoprecipitation (Co-IP), and nuclear‒cytoplasmic fractionation Western blotting were used in vitro to elucidate the pyruvate kinase muscle isozyme M2 (PKM2)-STAT3 protein interaction network. RESULTS QSYQ significantly alleviates cardiac inflammatory damage and ventricular remodeling induced by MI/R. Proteomic analysis revealed that QSYQ promotes the restoration of the tricarboxylic acid cycle and electron transport chain function by downregulating the expression of key glycolytic enzymes, such as PKM2 and LDHA, in macrophages in the MI/R-damaged area. It also identifies STAT3 as a core target closely associated with metabolic reprogramming. Spatial and targeted metabolomics analyses revealed that the deletion of STAT3 in macrophages suppresses glycolysis in the ischemic core region of a heart attack while also facilitating the recovery of the TCA cycle. Within macrophages, PLA and Co-IP experiments confirmed that a physical complex is formed by STAT3 with PKM2, the key rate-limiting enzyme of glycolysis. Using DSS chemical cross-linking reactions further demonstrated that blocking STAT3 expression hinders the transformation of PKM2 tetramers into dimers and prevents the nuclear translocation of the STAT3/PKM2 complex, leading to decreased STAT3 phosphorylation levels and reduced expression of proinflammatory factors such as IL-6, IL-1β, and TNF-α. Moreover, the metabolic regulatory effects of the QSYQ pellets were confirmed to depend on STAT3 via the use of STAT3-specific knockout mice in macrophages. Additionally, the QSYQ pellets demonstrated long-lasting regulatory effects on MI/R by altering macrophage metabolic reprogramming. CONCLUSION QSYQ hinders glycolysis by lowering STAT3 levels in macrophages, obstructing the shift from PKM2 tetramers to dimers and opposing the creation of PKM2/STAT3 complexes. As a result, there is a reduction in STAT3 phosphorylation levels, suppression of proinflammatory factor release, and improvement in AMI-induced cardiac inflammatory damage and ventricular remodeling.