Identification of Hub Genes in Protective Effect of Astragaloside IV on Aconitine-Induced Cardiac Damage in Zebrafish Based on Bioinformatics Analysis

Abstract

Accumulating evidence suggests that Astragaloside IV (AS-IV) improves cardiac function and protects the cardiovascular system. However, the molecular targets involved remain ambiguous. In this work, we report research suggesting that AS-IV can antagonize arrhythmias and reduce the cardiac damage induced by aconitine in zebrafish. Zebrafish have certain benefits with respect to studying the effect of drugs on cardiovascular disease. The possible mechanisms involved are analyzed, and hub gene targets are predicted. First, a model of cardiac damage induced by aconitine was created, and then a safe drug concentration of AS-IV was screened, and the appropriate drug dose gradient was selected within a safe drug concentration range. Second, we confirmed the protective effect of AS-IV in the cardiovascular system by observing changes in zebrafish heart rates and the cardiac and vascular structure. Third, we aimed to demonstrate the antagonistic mechanism of AS-IV on heart rate and cardiac damage induced by aconitine in zebrafish, with differentially expressed genes (DEGs) detected by RNA sequencing. The DEGs were then further analyzed by bioinformatic techniques, such as function enrichment analysis, protein-protein interaction network, and DNA-microRNA networks, for example. Next, we predicted the hub genes of the cardiac protective effects of AS-IV. Finally, we validated these genes in different transcriptome sequence datasets of cardiac damage. Thus, we conclude that miR-26b-5p/ATF3/JUN are key targets of AS-IV and play an important role in maintaining cardiac homeostasis and regulating cardiac remodeling.

Keywords: RNA sequencing; aconitine; astragaloside IV; bioinformatics analysis; cardiac damage; zebrafish.

Figure 1

Workflow for AS-IV intervention in aconitine-induced cardiac damage in zebrafish.

Figure 2

(A) Heart rates of zebrafish in aconitine group at 72 hpf (n = 10). (B) SV-BA distance, comparing control group with aconitine group (n = 10). (C) Effect of aconitine on the morphology of zebrafish heart.

Figure 3

(A) Effects of different doses of AS-IV on mortality, and development of the heart and other parts of the zebrafish. (B) At 50 mg/L AS-IV, abnormal tail vascular development and tail malformation starts to become apparent. (C) Comparison of heart rates of zebrafish in each group (histogram). (D) Comparison of distance SV-BA in each group (trend graph). (E) Changes in heart morphology of zebrafish in each group. (a) Control group, (b) Aconitine group, (c) AS-IV 10 mg/L group, (d) AS-IV 25 mg/L group, (e) AS-IV 40 mg/L group.

Figure 4

(A) Statistical chart of the number of DEGs. The horizontal axis represents comparison of different samples, the vertical axis represents the number of DEGs, red represents up-regulation, blue represents down-regulation. (B) Volcano Plot of DEGs. Each dot in the volcano plot of DEGs represents a gene. The red dots in the plot represent up-regulated DEGs, the blue dots represent down-regulated DEGs, and the black dots represent non-differentially expressed genes. The dotted line means the threshold lines of FDR and fold change (C) Biological Process diagram of DEGs. X axis represents -log10 (p-value). Y axis represents enrichment names. (D) Cellular component diagram of DEGs. (E) Molecular function diagram of DEGs. (F) Diagram of KEGG pathway.

Figure 5

(A) Hub genes identified by Cytoscape network analysis. The shade of the color indicates the degree of the gene. The list on the right is the result of the top 20 genes. (B) Protein-protein interaction data network of ATF3. Red, up-regulation; blue, down-regulation. Color depth and size of the dots were proportional to the fold change and correlated degree, respectively; thickness of the line between the two nodes indicated the connection strength positively. (C) DNA-microRNA network of ATF3. The shade of the color indicates the degree of the genes. The list on the right is the result of the top 5 genes.