Objective:

2

To explore the protective effect of Huoxin Pill (HXP) on acute myocardial ischemia-reperfusion (MIRI) injury in rats.

Methods:

2

Seventy-five adult SD rats were divided into the sham-operated group

model group

positive drug group (diltiazem hydrochloride

DH)

high dose group (24 mg/kg

HXP-H) and low dose group (12 mg/kg

HXP-L) of Huoxin Pill (

n

=15 for every group) according to the complete randomization method. After 1 week of intragastric administration

the left anterior descending coronary artery of the rat's heart was ligated for 45 min and reperfused for 3 h. Serum was separated and the levels of creatine kinase (CK)

creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH)

superoxide dismutase (SOD)

and malondialdehyde (MDA)

hypersensitive C-reactive protein (hs-CRP) and interleukin-1β (IL-1β) were measured. Myocardial ischemia rate

myocardial infarction rate and myocardial no-reflow rate were determined by staining with Evans blue and 2

3

5-triphenyltetrazolium chloride (TTC). Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine (BATMAN) databases were used to screen for possible active compounds of HXP and their potential therapeutic targets; the results of anti-inflammatory genes associated with MIRI were obtained from GeneCards

Drugbank

Online Mendelian Inheritance in Man (OMIM)

and Therapeutic Target Datebase (TTD) databases was performed; Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were used to analyze the intersected targets; molecular docking was performed using AutoDock Tools. Western blot was used to detect the protein expression of Toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NFκB)/NOD-like receptor protein 3 (NLRP3).

Results:

2

Compared with the model group

all doses of HXP significantly reduced the levels of LDH

CK and CK-MB (

P

<

0.05

P

<

0.01); HXP significantly increased serum activity of SOD (

P

<

0.05

P

<

0.01); all doses of HXP significantly reduced the levels of hs-CRP and IL-1β (

P

<

0.05

P

<

0.01) and the myocardial infarction rate and myocardial no-reflow rate (

P

<

0.01). GO enrichment analysis mainly involved positive regulation of gene expression

extracellular space and identical protein binding

KEGG pathway enrichment mainly involved PI3K-Akt signaling pathway and lipid and atherosclerosis. Molecular docking results showed that kaempferol and luteolin had a better affinity with TLR4

NFκB and NLRP3 molecules. The protein expressions of TLR4

NFκB and NLRP3 were reduced in the HXP group (

P

<

0.01).

Conclusions:

2

HXP has a significant protective effect on myocardial ischemia-reperfusion injury in rats

and its effect may be related to the inhibition of redox response and reduction of the inflammatory response by inhibiting the TLR4/NFκB/NLRP3 signaling pathway.