Objective:

2

To 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.

Methods:

2

The mice were divided into 6 groups using simple randomization

with 10 mice in each grou

p: 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.

Results:

2

QSYQ 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.

Conclusions:

2

QSYQ 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.