Abstract
Background/Aim Danhong injection (DHI) is a Chinese patent drug used for relieving cardiovascular diseases. Recent studies have suggested that DNA methylation plays a pivotal role in the maintenance of cardiac fibrosis (CF) in cardiovascular diseases. This study was aimed at identifying the effect and the underlying mechanism of DHI on CF, especially the DNA methylation. Methods A CF murine model was established by thoracic aortic constriction (TAC). A 28-day daily treatment with or without DHI via intraperitoneal injection was carried out immediately following TAC surgery. The changes in cardiac function, pathology, and fibrosis following TAC were measured by echocardiography and immunostaining. We used methyl-seq analysis to assess the DNA methylation changes in whole genes and identified the methylation changes of two Ras signaling-related genes in TAC mice, including Ras protein activator like-1 (Rasal1) and Ras-association domain family 1 (Rassf1). Next, the methylation status and expression levels of Rasal1 and Rassf1 genes were consolidated by bisulfite sequencing, quantitative reverse transcription polymerase chain reaction (RT-qPCR), and Western blotting, respectively. To determine the underlying molecular mechanism, the expressions of DNA methyltransferases (DNMTs), Tet methylcytosine dioxygenase 3 (TET3), fibrosis-related genes, and the activity of Ras/ERK were measured by RT-qPCR and Western blotting. Results DHI treatment alleviated CF and significantly improved cardiac function on day 28 of TAC. The methyl-seq analysis identified 42,606 differential methylated sites (DMSs), including 19,618 hypermethylated DMSs and 22,988 hypomethylated DMSs between TAC and sham-operated mice. The enrichment analysis of these DMSs suggested that the methylated regulation of Ras signal transduction and focal adhesion-related genes would be involved in the TAC-induced CF development. The results of bisulfite sequencing revealed that the TAC-induced methylation affected the CpG site in both of Rasal1 and Rassf1 genes, and DHI treatment remarkably downregulated the promoter methylation of Rasal1 and Rassf1 in CF hearts. Furthermore, DHI treatment upregulated the expressions of Rasal1 and Rassf1, inhibited the hyperactivity of Ras/ERK, and decreased the expressions of fibrosis-related genes. Notably, we found that DHI treatment markedly downregulated the expression of DNMT3B in CF hearts, while it did not affect the expressions of DNMT1, DNMT3A, and TET3. Conclusion Aberrant DNA methylation of Rasal1 and Rassf1 genes was involved in the CF development. DHI treatment alleviated CF, prevented the hypermethylation of Rasal1 and Rassf1, and downregulated DNMT3B expression in CF hearts.
Highlights
Cardiac fibrosis (CF) is an integral constituent of every form of chronic heart disease, and its relevance for disease progression is increasingly being recognized [1]
We found that the Ea in the H-Danhong injection (DHI) group was improved compared with that in the thoracic aortic constriction (TAC) group (P < 0:05), whereas there was no statistically significant difference in the Ea/annular velocity (Aa) (P > 0:05)
To investigate the mechanisms underlying the methylation of the Ras signaling pathway induced by TAC and the antifibrosis effect of DHI, we focused our investigation on the proteins and genes that mediated DNA methylation
Summary
Cardiac fibrosis (CF) is an integral constituent of every form of chronic heart disease, and its relevance for disease progression is increasingly being recognized [1]. Ras protein activator like-1 (RASAL1) and Ras-association domain family 1 (RASSF1) function as inhibitors of Ras signaling, and the aberrant promoter methylation of RASAL1 and RASSF1 contributes to pressure overload-induced CF [3, 4]. Oxidative Medicine and Cellular Longevity increased fibrosis was associated with significantly elevated promoter methylation of RASAL1, decreased RASAL1 expression, and increased Ras-GTP activity [3]. Tao et al reported that upregulation of p-ERK1/2 was detected in activated cardiac fibroblasts with decreased RASSF1A expression, which is important for pathogenesis of CF and activation of fibroblasts [4]. Administration of the DNA methylation inhibitor could relieve the CF and hypertrophy and improve the cardiac function [5]. A highly efficient, specific, and safe compound targeting the aberrant DNA methylation in CF patients has not been identified yet [6]
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