Network Pharmacology Exploration Reveals Anti-Apoptosis as a Common Therapeutic Mechanism for Non-Alcoholic Fatty Liver Disease Treated with Blueberry Leaf Polyphenols

Abstract

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease characterized by excessive fat accumulation in the liver. The aim of this study is to elucidate the multi-target mechanism of polyphenols in blueberry leaves (PBL) on NAFLD by network pharmacology and to validate its results via biological experiments. Twenty constituents in PBL were preliminarily determined by liquid chromatography-tandem mass spectrometry. Subsequently, 141 predicted drug targets and 1226 targets associated with NAFLD were retrieved from public databases, respectively. The herb-compound-target network and the target protein-protein interaction network (PPI) were established through Cytoscape software, and four compounds and 53 corresponding targets were identified. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were performed to explore the biological processes of the predicted genes. The results of cell experiments demonstrated that PBL could significantly improve the viability of the NAFLD cell model, and the protein expressions of caspase-3 and Bcl-2 were consistent with the expected mechanism of action of PBL. Those results systematically revealed that the multi-target mechanism of PBL against NAFLD was related to the apoptosis pathway, which could bring deeper reflections into the hepatoprotective effect of PBL.

Keywords: NAFLD; apoptosis; network pharmacology; polyphenols in blueberry leaves.

Figure 1

The targets related to polyphenols in blueberry leaves (PBL) in the treatment of non-alcoholic fatty liver disease (NAFLD). (a) Distribution of PBL potential targets and NAFLD targets. (b) Protein–protein interaction (PPI) networks of PBL for the treatment of NAFLD. The importance of targets depends on the shade of purple and the grey line represents the interaction relationship.

Figure 2

Herb-compound-target network of PBL.

Figure 3

Gene Ontology (GO) enrichment analysis for potential targets of PBL.

Figure 4

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of PBL against NAFLD. Top 20 pathways enriched based on target genes (the abscissa is the gene ratio, the ordinate is pathway name, the size of the dot indicates the number of target genes, and the color represents the p value).

Figure 5

The effect of PBL on the viability of NAFLD-modeled cells. (a) Effects of palmitic acid (PA) on cell viability. HepG2 cells were incubated with different concentrations of PA for 48 h. (b) The result of PBL (0.78–100 µg/mL) on cell viability after 48 h of exposure. (c) Effect of PBL on PA-induced cell viability reduction. HepG2 cells were treated with PBL (0.78–100 µg/mL) and induced by PA (300 µM) for 48 h. (d) HepG2 cells evaluated by Oil Red O staining. The data represent as mean ± SEM for n = 3 studies. *** p < 0.001, **** p < 0.0001 compared with the control group.

Figure 6

PBL inhibits apoptosis in NAFLD-modeled cells. (a) Cells were analyzed by flow cytometry, the percentage of apoptotic cells. The suppression on apoptosis of PBL on NAFLD-modeled cells. The NAFLD-modeled cells were treated with PBL (6.25–25 µg/mL) for 48 h. Data are presented as mean ± SEM (n = 3). *** p < 0.001 compared with the control group. ^### p < 0.01 compared with the PA (300 µM) group. (b,c) Effects of PBL on Bcl-2, caspase-3 and protein expression in NAFLD-modeled cells. The data are represented as mean ± SEM (n = 3). * p < 0.05, ** p < 0.01 compared between the marked groups.