3.1 Active Compounds and Target Genes Detection
Details on revealing the pharmacological mechanisms of NRR against arrhythmia are shown in Figure 1. There were 185, 235 and 58 components obtained from TCMSP, SymMap and ETCM database, respectively. After screening by pharmacokinetic properties and removaling duplicate, 21 active components were included, dividing into 5 categories: 15 coumarin (e.g., Cnidilin, Ammidin, Phellopterin, etc.), 1 flavonoid (Diosmetin), 1 nitrogen-containing compound (Isoindigo), 2 triterpenoids (beta-sitosterol and Sitosterol) and 2 others (Figure 2). The details of 21 compounds see in table S1. Then, the targets were predicted by UniProt database, and a total of 159 putative targets were identified, including 105 in coumarin, 10 in flavonoid, 3 in nitrogen-containing compound, 40 in triterpenoids and 1 in others. A compound-target network was constructed as shown in Figure 3. Eventrally, 57 targets of NRR against arrhythmia were obtained after the removal of repetitions.
3.2 Putative NRR Target Proteins in Arrhythmia
There were 96342 and 5584 arrhythmia-associated gene acquired from CTD and GeneCards databased, respectively. With prioritized inference and relevance scores, 452 and 97 genes were identified (Table S3&S4). By integrating the data from the two databases, 265 targets were included and considered as the key putative arrhythmia-associated proteins. As shown in the Venn diagram, a total of 19 overlapping genes of NRR and arrhythmia were obtained (Figure 4A), including CDK2, BAX, CASP3, etc., which were taken as the key targets for the treatment of arrhythmia with NRR.
3.3 PPI Construction and Hub Genes Identification
A function-associated protein network of the interaction between NRR and arrhythmia was constructed, which included 19 nodes and 40 edges (Figure 4B). The topological parameters were analyzed based on Analyze Network (Table S5). Only one module was divided through MCODE, which considered as the core module (Figure 4C). Futhermore the core proteins were identified via CytoHubba, including CASP3 (caspase 3), CASP8 (caspase 8), CASP9 (caspase 9), CDK2 (Cell division protein kinase 2) and BAX (Apoptosis regulator BAX); and these core targets all belonged to the core module (Figure 4D).
3.4 Biological Function of NRR Targeting on Arrhythmia
To perform further insight into the mechanisms underlying NRR effects on arrhythmia at the systematic level, GO and KEGG pathway analyses were carried out. There were 141 GO terms and 31 KEGG pathways enriched from the 19 overlapping targets (Tables S6&S8), and 33 GO terms and 16 KEGG pathways enriched from 5 core targets (Tables S7&S9). The GO-biological process mainly related to response to drug, response to lipopolysaccharide, response to estradiol, extrinsic apoptotic signaling pathway in absence of ligand, apoptotic process etc. Among the pathways enriched by overlapping targets and core targets, 15 are consistent. The top 3 pathways enriched by 5 core targets were p53 signaling pathway, Hepatitis B and Apoptosis. A drug-compound-targets-pathways network was builded base on the KEGG enrichment analysis (Figure 5).
3.5 Molecular Docking
The affinity between the two counterparts can be predicted by calculating the binding energy. Ten active NRR componds with good pharmacokinetic properties were molecularly docked with 5 core receptors, including CASP3 (PDBID: 1NME), CASP8 (PDBID: 2K7Z), CASP9 (PDBID: 3V3K), BAX (PDBID: 2LR1), CDK2 (PDBID: 2R3J). The binding energy of less than 0 meaned that two molecules combine spontaneously. The lower the binding energy, the stronger the affinity of the compounds to the targets. A total of 50 ligand-receptor combinations were computed. The majority of NRR components bind well with the key receptors, in which 44 combinations had affinities of < -7kcal/mol, account for 88%. CDK2 and C9 had the strongest binding energy, at -9.7 kcal/mol. The molecular binding findings are shown in Fig. 6.
3.6 Experimental Validation on the Targets of NRR in Treating Arrhythmia
The ECG of sham, vehicle and NRR-treated group were showed in Fig. 8. CASP3, CASP8, CASP9, BAX and CDK2 mRNA expression were consistently altered in individual samples from the sham, vehicle and NRR groups. The expression of CASP3, CASP8, CASP9, BAX and CDK2 were upregulated after arrhythmia, while the CASP3, CASP8, CASP9, BAX and CDK2 expression were downregulated after treated with NRR for 14 days.