Danhong Injection Alleviates Cardiac Fibrosis via Preventing the Hypermethylation of Rasal1 and Rassf1 in TAC Mice.
Danhong Injection Alleviates Cardiac Fibrosis via Preventing the Hypermethylation of Rasal1 and Rassf1 in TAC Mice.
复制标题
丹红注射液通过预防TAC小鼠Rasal1和Rassf1高甲基化减轻心脏纤维化
DOI：
10.1155/2020/3158108
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发表时间：
2020
影响因子：
--
通讯作者：
Liu H
中科院分区：
文献类型：
--
作者：
Li S;Li P;Liu W;Shang J;Qiu S;Li X;Liu W;Shi H;Zhou M;Liu H
丹红注射液是治疗心血管疾病的中成药。最近的研究表明，DNA甲基化在心血管疾病中心脏纤维化（CF）的维持中起着关键作用。本研究旨在探讨DHI对CF的影响及其机制，尤其是DNA甲基化的影响。 采用胸主动脉缩窄术（TAC）建立CF小鼠模型。在TAC手术后立即进行28天的每日治疗，通过腹膜内注射使用或不使用DHI。通过超声心动图和免疫组化检测心脏功能、病理和纤维化的变化。我们使用甲基序列分析来评估整个基因的DNA甲基化变化，并确定了TAC小鼠中两个Ras信号相关基因的甲基化变化，包括Ras蛋白激活物样1（Rasal 1）和Ras相关结构域家族1（Rassf 1）。接下来，分别通过亚硫酸氢盐测序、定量逆转录聚合酶链反应（RT-qPCR）和蛋白质印迹法巩固Rasal 1和Rassf 1基因的甲基化状态和表达水平。通过RT-qPCR和Western blotting检测DNA甲基转移酶（DNMTs）、泰特甲基胞嘧啶双加氧酶3（TET 3）、纤维化相关基因的表达以及Ras/ERK活性，以确定潜在的分子机制。 DHI治疗减轻了TAC第28天的CF并显著改善了心功能。甲基序列分析确定了42，606个差异甲基化位点（DMS），包括TAC和假手术小鼠之间的19，618个高甲基化DMS和22，988个低甲基化DMS。这些DMS的富集分析表明，Ras信号转导和粘着斑相关基因的甲基化调控可能参与TAC诱导的CF的发展。亚硫酸氢盐测序结果显示，TAC诱导的Rasal 1和Rassf 1基因启动子区CpG位点甲基化均受到影响，DHI处理显著下调CF心肌Rasal 1和Rassf 1基因启动子区甲基化。DHI可上调Rasal 1和Rassf 1的表达，抑制Ras/ERK的过度活化，降低纤维化相关基因的表达。值得注意的是，我们发现DHI处理显著下调CF心脏中DNMT 3B的表达，而不影响DNMT 1、DNMT 3A和TET 3的表达。 Rasal 1和Rassf 1基因的异常甲基化参与了CF的发生。DHI治疗减轻CF，防止Rasal 1和Rassf 1的高甲基化，并下调CF心脏中DNMT 3B的表达。
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. 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. 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. 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.
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影响因子：
4.8
作者：
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通讯作者：
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影响因子：
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作者：
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影响因子：
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作者：
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