Qishen Yiqi Dripping Pills Activate HDAC3-Mediated Nrf2-PITX2 Signaling Pathway to Reduce Oxidative Stress and Alleviate Myocardial Infarction Injury in Mice.

objectiveTo clarify the mechanism by which Qishen Yiqi Dripping Pill (QSYQ) inhibits oxidative stress injury in myocardial infarction (MI), with a focus on verifying whether its effect is mediated through the histone deacetylase 3 (HDAC3)/nuclear factor erythroid 2-related factor 2 (Nrf2)/paired-like homeodomain transcription factor 2 (PITX2) pathway and ultimately determining its active ingredient.

methodsThe mice were divided into 6 groups using simple randomization, with 10 mice in each group: the sham group, the model group, the QSYQ-L (19.5 mg/kg), QSYQ-M (39 mg/kg), and QSYQ-H (78 mg/kg) groups, and the metoprolol (19.5 mg/kg) group. A mouse model of MI was established by permanent ligation of the left anterior descending coronary artery. The experimental groups (QSYQ and metoprolol) received daily gavage of the corresponding agents for 7 days following surgery. Cardiac function was assessed longitudinally using echocardiography, electrocardiogram, and laser speckle flowmetry. Myocardial injury was evaluated through hematoxylin and eosin staining, infarct size and area-at-risk were determined using triphenyl tetrazolium chloride and evans blue dual staining, and cardiac troponin I (cTnI), creatine kinase myocardial band (CK-MB), and lipid peroxidation (LPO) were analyzed via enzyme-linked immunosorbent assay. The chemical constituents of QSYQ and their metabolites were profiled using high-performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry, and their potential targets were predicted via network pharmacology. Key protein interactions and localizations were confirmed by co-immunoprecipitation and immunofluorescence, respectively. Target engagement was further validated by molecular docking and in vitro oxygen-glucose deprivation/reoxygenation models in H9c2 cells, using cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) assays.

resultsQSYQ administration demonstrated significant cardioprotective effects, markedly reducing myocardial infarct size and attenuating pathological tissue damage. Concurrently, it improved cardiac functional parameters, notably ejection fraction and fractional shortening, while enhancing myocardial perfusion efficiency (P<0.01). Network pharmacology analysis suggests that oxidative stress modulation is a primary therapeutic mechanism. At the molecular level, QSYQ effectively suppressed HDAC3 expression while activating the Nrf2/PITX2 antioxidant pathway. Protein interaction validation through co-immunoprecipitation and structural analysis confirmed the formation of an HDAC3-Nrf2-PITX2 regulatory complex. Crucially, cryptotanshinone, a core bioactive constituent of QSYQ, exhibited high-affinity binding to HDAC3 (P<0.01), which was validated by CETSA and DARTS experiments.

conclusionsQSYQ effectively ameliorates MI injury, at least in part, by inhibiting HDAC3, which consequently activates the Nrf2/PITX2 signaling pathway and suppresses oxidative stress damage. Mechanistically, cryptotanshinone is identified as the critical molecular entity directly targeting HDAC3 to mediate this cardioprotective effect.