Network and Experimental Pharmacology on Mechanism of Yixintai Regulates the TMAO/PKC/NF-κB Signaling Pathway in Treating Heart Failure

Abstract

Objective: This study aims to explore the mechanism of action of Yixintai in treating chronic ischemic heart failure by combining bioinformatics and experimental validation.

Materials and methods: Five potential drugs for treating heart failure were obtained from Yixintai (YXT) through early mass spectrometry detection. The targets of YXT for treating heart failure were obtained by a search of online databases. Gene ontology (GO) functional enrichment analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses were conducted on the common targets using the DAVID database. A rat heart failure model was established by ligating the anterior descending branch of the left coronary artery. A small animal color Doppler ultrasound imaging system detected cardiac function indicators. Hematoxylin-eosin (HE), Masson's, and electron microscopy were used to observe the pathological morphology of the myocardium in rats with heart failure. The network pharmacology analysis results were validated by ELISA, qPCR, and Western blotting.

Results: A total of 107 effective targets were obtained by combining compound targets and eliminating duplicate values. PPI analysis showed that inflammation-related proteins (TNF and IL1B) were key targets for treating heart failure, and KEGG enrichment suggested that NF-κB signaling pathway was a key pathway for YXT treatment of heart failure. Animal model validation results indicated the following: YXT can significantly reduce the content of intestinal microbiota metabolites such as trimethylamine oxide (TMAO) and improve heart failure by improving the EF and FS values of heart ultrasound in rats and reducing the levels of serum NT-proBNP, ANP, and BNP to improve heart failure. Together, YXT can inhibit cardiac muscle hypertrophy and fibrosis in rats and improve myocardial ultrastructure and serum IL-1β, IL-6, and TNF-α levels. These effects are achieved by inhibiting the expressions of NF-κB and PKC.

Conclusion: YXT regulates the TMAO/PKC/NF-κB signaling pathway in heart failure.

Keywords: PKC/NF-κB pathway; TMAO; heart failure; network pharmacology; traditional Chinese medicine; yixintai.

Figure 1

HPLC of YXT Granules. (A) HPLC of YXT Granules test sample; (B) HPLC of YXT Granules mixed reference substance; (C) Ten batches of YXT fingerprint overlay (S1-S10) and control fingerprint (R).

Figure 2

Differential analysis of heart failure. The DEGs are represented as a volcano figure (A), heatmap (B), Box plot (C) and PCA score plot (D) comparing the control and HF groups.

Figure 3

YXT intervenes in “potential DEGs” of heart failure.

Figure 4

Core DEGs PPI topology analysis.A:The PPI network used Cityscape 3.7.1 software for visual analysis. (B) PPI network of more significant proteins extracted from (A) by filtering 2 parameters: CASP1, CNR2.The redder the target color, the larger the degree, and the yellower the color, the smaller the degree.

Figure 5

“Core DEGs” Difference Group Comparison Chart. (A) IL-1β; (B) TNF-α; (C) MMP3; (D) MMP9; € MMP13.

Figure 6

Potential gene GO and KEGG analysis; (A) GO analysis; (B) KEGG analysis; (C) KEGG target pathway diagram.

Figure 7

Visualization of the interface between Core DEGs and Active Ingredients: (A) Result of docking IL1B with Calycosin; (B) Result of docking IL1B with Formononetin. (C) Result of docking IL1B with Calycosin-7-O-β-D-glucoside; (D) Result of docking IL1B with hydroxysafflor yellow A; (E) Result of docking IL1B with Salvianolic Acid B; (F) Results of docking between TNF and Calycosin; (G) Results of the docking between TNF and Formononetin; (H) The docking result of TNF and Calycosin-7-O-β-D-glucoside; (I) The docking result of TNF and hydroxysafflor yellow A; (J) The docking result of TNF and Salvianolic Acid B.

Figure 8

Cardiac ultrasound results of rats in each group. (A) Cardiac global function was quantitatively assessed from two-dimensional echocardiographic short axis images among the different groups; (B) Cardiac systolic function was determined by measuring ejection fraction (EF) and fraction shortening (FS).n=6. ^##P<0.01 vs sham group; **P<0.01 vs model group; All values are depicted as mean ± SD.

Figure 9

HE and Masson staining images of rat hearts in each group (× 200). (A) HE staining images; (B) Masson staining images.

Figure 10

Morphological structure diagram of mitochondria in rat cardiomyocytes (× 3000).

Figure 11

YXT’s effect on ANP, BNP, NT-pro BNP, IL-1β, IL-6, and TNF-α levels in HF rats of each group. (A) serum ANP content. (B) serum BNP content. (C) serum NT-pro BNP content. (D) serum IL-1β content. (E) serum IL-6 content. (F) serum TNF-α content.n=6 ^##P<0.01 vs Sham group. **P<0.01 vs model group; *P<0.05 vs model group. All values are indicated as mean ± SD.

Figure 12

Effect of YXT on TMAO levels in HF rats. ^##P<0.01 vs sham group; **P<0.01 vs model group. model group. All values are indicated as mean ± SD.

Figure 13

Effects of YXT on NF-κB/PKC signaling pathway in HF rates; (A) PKC mRNA expression. (B) NF-κB p65 mRNA expression. (C) Relative protein expression of PKC. (D) Relative protein expression of NF-κB. (E) Protein analysis of six groups detected the protein expression of PKC and NF-κB. n=5. ^##P<0.01 vs sham group; **P<0.01 vs model group. All values are indicated as mean ± SD.