Myocardial protective effect and transcriptome profiling of Naoxintong on cardiomyopathy in zebrafish.

BACKGROUND: Cardiomyopathy is a kind of cardiovascular diseases, which makes it more difficult for the heart to pump blood to other parts of the body, eventually leading to heart failure. Naoxintong (NXT), as a traditional Chinese Medicine (TCM) preparation, is widely used in the treatment of cardiovascular diseases, including cardiomyopathy, while its underlying mechanism has not been fully elucidated. The purpose of this study is to investigate the therapeutic effect of NXT on cardiomyopathy and its molecular mechanism in zebrafish model. METHODS: The zebrafish cardiomyopathy model was established using terfenadine (TFD) and treated with NXT. The therapeutic effect of NXT on cardiomyopathy was evaluated by measuring the heart rate, the distance between the sinus venosus and bulbus arteriosus (SV-BA), the pericardial area, and the blood flow velocity of zebrafish. Then, the zebrafish hearts were isolated and collected; transcriptome analysis of NXT on cardiomyopathy was investigated. Moreover, the heg1 mutant of zebrafish congenital cardiomyopathy model was used to further validate the therapeutic effect of NXT on cardiomyopathy. Additionally, UPLC analysis combined with the zebrafish model investigation was performed to identify the bioactive components of NXT. RESULTS: In the TFD-induced zebrafish cardiomyopathy model, NXT treatment could significantly restore the cardiovascular malformations caused by cardiac dysfunction. Transcriptome and bioinformatics analyses of the TFD and TFD + NXT treated zebrafish developing hearts revealed that the differentially expressed genes were highly enriched in biological processes such as cardiac muscle contraction and heart development. As a cardiac development protein associated with cardiomyopathy, HEG1 had been identified as one of the important targets of NXT in the treatment of cardiomyopathy. The cardiovascular abnormalities of zebrafish heg1 mutant could be recovered significantly from NXT treatment, including the expanded atrial cavity and blood stagnation. qRT-PCR analysis further showed that NXT could restore cardiomyopathy phenotype in zebrafish through HEG1-CCM signaling. Among the seven components identified in NXT, paeoniflorin (PF) and salvianolic acid B (Sal B) were considered to be the main bioactive ones with myocardial protection. CONCLUSION: NXT presented myocardial protective effect and could restore myocardial injury and cardiac dysfunction in zebrafish; the action mechanism was involved in HEG1-CCM signaling.

Naoxintong restores ischemia injury and inhibits thrombosis via COX2-VEGF/ NFκB signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Naoxintong (NXT) is a traditional Chinese medicine preparation that is often used in combination with aspirin in the treatment of cardiovascular diseases (CVD). One of the main symptoms of CVD is hypoxic-ischemia (HI). The purpose of this study is to find out the molecular nodes targeted by NXT and its related molecular pathways in vascular repair. MATERIALS AND METHODS: First, human vein umbilical endothelial cells (EA.hy926) were utilized to set up the Oxygen-Glucose Deprivation-Reoxygenation (OGD/R) model and treated with NXT. Cell proliferation, damage and apoptosis were detected by MTT, LDH, and flow cytometry assays. Second, transcriptional responses of OGD/R cells to NXT treatment were investigated. qRT-PCR, western blotting and inhibitor assays were performed. Third, the anti-thrombotic effect of NXT was evaluated by the zebrafish thrombosis model. Morphological observation, histological staining and qRT-PCR assays were implemented on zebrafish model to further observe in vivo the therapeutic effects of NXT on ischemia and thrombosis. RESULTS: In OGD/R EA.hy926 cells, NXT treatment could reduce ischemic vascular injury, increase cell viability and decrease the proportion of apoptosis. Through RNA-seq analysis, 183 differentially expressed genes (DEGs) were screened with 110 up-regulated genes and 73 down-regulated genes between OGD/R and OGD/R + NXT treated EA.hy926 cells. VEGF and NFκB pathways were enriched. Among these genes, COX2 was identified as one of important targets via which NXT could restore vascular injury. COX2 inhibitor (NS-398), and aspirin, a drug that prevents the development of CVD by targeting COX2, exhibited similar effects to NXT in the treatment of OGD/R EA.hy926 cells. In zebrafish thrombosis model, NXT could attenuate tail venous thrombus and recover the quantity of heart red blood cells. Furthermore, NXT could prevent the formulation of thrombosis and eliminate inflammation in zebrafish by COX2-VEGF/NFκB signaling. CONCLUSION: Our studies implicated that NXT could restore HI injury and inhibit thrombosis through COX2-VEGF/NFκB signaling, which is consistent with the molecular target of aspirin. This finding might explain the principle of NXT combined with aspirin in the treatment of cardiovascular diseases.

Generation and Application of the Zebrafish heg1 Mutant as a Cardiovascular Disease Model.

Cardiovascular disease (CVD) is the leading cause of global mortality, which has caused a huge burden on the quality of human life. Therefore, experimental animal models of CVD have become essential tools for analyzing the pathogenesis, developing drug screening, and testing potential therapeutic strategies. In recent decades, zebrafish has entered the field of CVD as an important model organism. HEG1, a heart development protein with EGF like domains 1, plays important roles in the development of vertebrate cardiovascular system. Loss of HEG1 will affect the stabilization of vascular endothelial cell connection and eventually lead to dilated cardiomyopathy (DCM). Here, we generated a heg1-specific knockout zebrafish line using CRISPR/Cas9 technology. Zebrafish heg1 mutant demonstrated severe cardiovascular malformations, including atrial ventricular enlargement, heart rate slowing, venous thrombosis and slow blood flow, which were similar to human heart failure and thrombosis phenotype. In addition, the expression of zebrafish cardiac and vascular markers was abnormal in heg1 mutants. In order to apply zebrafish heg1 mutant in cardiovascular drug screening, four Traditional Chinese Medicine (TCM) herbs and three Chinese herbal monomers were used to treat heg1 mutant. The pericardial area, the distance between sinus venosus and bulbus arteriosus (SV-BA), heart rate, red blood cells (RBCs) accumulation in posterior cardinal vein (PCV), and blood circulation in the tail vein were measured to evaluate the therapeutic effects of those drugs on DCM and thrombosis. Here, a new zebrafish model of DCM and thrombosis was established, which was verified to be suitable for drug screening of cardiovascular diseases. It provided an alternative method for traditional in vitro screening, and produced potential clinical related drugs in a rapid and cost-effective way.