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Huoxin Pill Reduces Myocardial Ischemia Reperfusion Injury in Rats via TLR4/NFκB/NLRP3 Signaling Pathway

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Abstract

Objective

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

Methods

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

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

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 TLR4NFκB/NLRP3 signaling pathway.

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Authors and Affiliations

  1. Institute of Basic Medical Sciences of Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing Key Laboratory of Chinese Materia Pharmacology, National Clinical Research Center of Traditional Chinese Medicine for Cardiovascular Diseases, Beijing, 100091, China

    Ce Cao, Yu-tong Qi, Ao-ao Wang, Zi-yan Wang, Zi-xin Liu, Hong-xu Meng, Lei Li &  Jian-xun Liu

  2. Institute of Chinese Medicine Sciences, Guangdong Pharmaceutical University, Guangzhou, 510006, China

    Ce Cao &  Jian-xun Liu

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  1. Ce Cao
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Contributions

Liu JX and Li L conceived and designed the experiments. Cao C wrote the article. Qi YT, Meng HX and Wang AA performed the experiments. Wang ZY and Liu ZX were in charge of the collection of references. All authors agreed to be responsible for the content of the work. All authors agreed to the publication of this article.

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Correspondence to Jian-xun Liu.

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Additional information

Supported by the National Natural Science Foundation of China (No. 82174015 and No. 82030124), Scientific and Technological Innovation Project of China Academy of Chinese Medical Sciences (No. CI2021A04609)

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Cao, C., Qi, Yt., Wang, Aa. et al. Huoxin Pill Reduces Myocardial Ischemia Reperfusion Injury in Rats via TLR4/NFκB/NLRP3 Signaling Pathway. Chin. J. Integr. Med. 29, 1066–1076 (2023). https://doi.org/10.1007/s11655-023-3640-1

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