Xinyin tablets affect mitophagy and cardiomyocyte apoptosis to alleviate chronic heart failure by regulating histone deacetylase 3(HDAC3)-mediated PTEN induced putative kinase 1(PINK1)/Parkin signaling pathway.

Abstract

Ethnopharmacological relevance: Xinyin, a Chinese patent medicine, is composed of Panax ginseng C.A.Mey. (Araliaceae), Ilex pubescens Hook. & Arn. (Aquifoliaceae), Leonurus japonicus Houtt. (Lamiaceae), Plantago asiatica L. (Plantaginaceae), Ophiopogon japonicus (Thunb.) Ker Gawl. (Asparagaceae), Astragalus membranaceus (Fisch.) Bunge, and Draba nemorosa L. (Brassicaceae). It has been used for the prevention and treatment of chronic heart failure (CHF) clinically. However, its underlying mechanism of action is far from completely understood.

Aim of the study: This study aimed to determine whether Xinyin alleviates CHF in SPF C57 mice and to explore the potential mechanism of action in H9c2 cells.

Materials and methods: Liquid chromatography tandem mass spectroscopy (LC-MS/MS) was performed to identify the chemical compounds in Xinyin. In vivo, 60 C57 mice were randomly divided into 6 groups: the sham group; model group; low-, medium-, and high-dose Xinyin groups; and perindopril group. Animals in the sham group underwent thoracotomy only. The others were subjected to coronary artery ligation. After 4 weeks of drug intervention, the cardiac function of the mice in each group was detected via echocardiography, the myocardial cells were evaluated via HE staining, and the degree of myocardial fibrosis was detected via Masson's trichrome staining. The expression of PINK1/Parkin signaling pathway-related genes (HDAC3, PINK1, Parkin, P62, LC3II/I, caspase-3, caspase-9, and Bax) was analyzed via RT‒qPCR and Western blotting. The effects of Xinyin on cardiomyocyte apoptosis and mitophagy mediated by the HDAC3 and PINK1/Parkin pathways in CHF model mice were evaluated. In vitro, H9c2 cardiomyocytes subjected to hypoxia were treated with different concentrations of Xinyin. The mRNA transcription levels of HDAC3, PINK1, Parkin, P62, LC3II/I, caspase-3, caspase-9, and Bax were measured via fluorescence quantitative PCR. Western blotting was used to detect the protein expression levels of PINK1, Parkin, P62, LC3 II/I, caspase-3, caspase-9, and Bax. TUNEL staining was used to detect the number of apoptotic bodies in the myocardium to evaluate the level of apoptosis. Transmission electron microscopy was used to observe changes in the number of mitophagosomes. Rapamycin (mitophagy agonist), Mdivi-1 (mitophagy inhibitor), ITSA-1 (HDAC3 agonist) and RGFP966 (HDAC3 inhibitor) were used to create intervention conditions. The effects of rapamycin or Mdivi-1 on PINK1/Parkin-mediated mitophagy were observed. Then, the effects of HDAC3 on the PINK1/Parkin signaling pathway, mitophagy and apoptosis in hypoxic cardiomyocytes were observed. Hypoxic cardiomyocytes were treated with Xinyin-containing serum or control serum to observe whether Xinyin could still play a protective role in cardiomyocytes when HDAC3 is activated or mitophagy is inhibited.

Results: 785 compounds were characterized from Xinyin, among which carbohydrates and glycosides, phenylpropanoids, terpenes were abundant, and a small number of amino acids, peptides and derivatives also existed in Xinyin. In vivo, Xinyin improved cardiac function (LVEF, LVFS, LVEDD, LVESD, and LVESV) and downregulated the expression of caspase-3, caspase-9, and Bax. The expression levels of PINK1 and Parkin subsequently increased. In vitro, the above findings were reinforced in H9c2 cardiomyocytes. Rapamycin and RGFP966 reduced the apoptosis of hypoxic H9C2 cardiomyocytes and increased mitophagy mediated by the HDAC3-mediated PINK1/Parkin signaling pathway.

Conclusions: Xinyin tablets have potential as an intervention for CHF by improving mitophagy and inhibiting cardiomyocyte apoptosis through the HDAC3-mediated PINK1/Parkin signaling pathway.