Network Pharmacology-Based Strategy for Predicting Therapy Targets of Citri Reticulatae Pericarpium on Myocardial Hypertrophy

Abstract

Objective: Through a network pharmacology method, we screened the main active compounds of Citri Reticulatae Pericarpium (CRP), constructed a drug-ingredient-disease-target network, explored the molecular mechanism of its treatment of myocardial hypertrophy, and validated it by using molecular biology approach.

Methods: Traditional Chinese Medicine Systems Pharmacology (TCMSP) and GeneCards were utilised to collect the effective component in CRP and the targets of CRP and myocardial hypertrophy. The STRING database constructed the protein interaction network. The drug-ingredient-disease-target network was outlined by the Cytoscape 3.9.0 software. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using the Metascape database. Real-time PCR (RT-PCR) and Western blotting were utilised to determine the mRNA and protein level of the critical targets of CRP therapy for myocardial hypertrophy.

Results: We found that five practical components of CRP exerted therapeutic effects on myocardial hypertrophy by modulating 41 targets. Further analysis revealed that naringenin was the essential active compound in CRP that regulated myocardial hypertrophy. In addition, we showed that the active compounds of CRP might exert antihypertrophy effects via regulating essential target proteins such as AKT1-, MAPK3-, PPARA-, PPARG-, and ESR1-mediated signaling pathways such as cell proliferation, nuclear receptor activation, and oxidative stress. The molecular biology experiments demonstrated that naringenin inhibited the mRNA level of NPPA and NPPB induced by Ang II and regulated related targets such as AKT1, MAPK3, PPARA, PPARG, and ESR1.

Conclusion: CRP could inhibit myocardial hypertrophy through multitarget and multiapproach.

Figure 1

The workflow of key target gene prediction and validation of CRP therapy for myocardial hypertrophy.

Figure 2

Potential target genes and PPI network map of CRP therapy for myocardial hypertrophy. (a) The Venny results of potential target genes of CRP therapy for myocardial hypertrophy. (b) The PPI network map of 41 target genes. (c) Count and list the top 30 genes of the PPI network map.

Figure 3

The CRP-myocardial hypertrophy-potential target gene network.

Figure 4

GO and KEGG analyses of potential target genes of CRP in myocardial hypertrophy. The GO analysis for biological process (a), molecular function (b), and cellular components (c) of potential target genes of CRP in myocardial hypertrophy. (d) The top 9 remarkably enriched KEGG analysis for the signaling pathway of potential target genes of CRP in myocardial hypertrophy.

Figure 5

Effect of naringenin on the mRNA expression of NPPA and NPPB. H9C2 cells were treated with 20 μM naringenin for 1 h followed by stimulation with Ang II for 24 h. The mRNA expressions of NPPA (a) and NPPB (b) were detected by real-time PCR. ^∗P < 0.05 vs. the group without treatment, ^#P < 0.05 vs. the group treated with Ang II, n = 5.

Figure 6

Effect of naringenin on the expression of essential target genes (AKT, PPARA, PPARG, ESR1, and ERK1/2). H9C2 cells were treated with 20 μM naringenin for 1 h followed by stimulation with Ang II for 24 h. The mRNA expression of PPARA (a), PPARG (b), and ESR1 (c) were detected by real-time PCR. (d–f) The protein expression of ERK and AKT were checked by Western blotting. ^∗P < 0.05 vs. the group without treatment, ^#P < 0.05 vs. the group treated with Ang II, n = 5.