Mulberry extract upregulates cholesterol efflux and inhibits p38 MAPK-NLRP3-mediated inflammation in foam cells

Abstract

The accumulation of foam cells in arterial intima and the accompanied chronic inflammation are considered major causes of neoatherosclerosis and restenosis. However, both the underlying mechanism and effective treatment for the disease are yet to be uncovered. In this study, we combined transcriptome profiling of restenosis artery tissue and bioinformatic analysis to reveal that NLRP3 inflammasome is markedly upregulated in restenosis and that several restenosis-related DEGs are also targets of mulberry extract, a natural dietary supplement used in traditional Chinese medicine. We demonstrated that mulberry extract suppresses the formation of ox-LDL-induced foam cells, possibly by upregulating the cholesterol efflux genes ABCA1 and ABCG1 to inhibit intracellular lipid accumulation. In addition, mulberry extract dampens NLRP3 inflammasome activation by stressing the MAPK signaling pathway. These findings unveil the therapeutic value of mulberry extract in neoatherosclerosis and restenosis treatment by regulating lipid metabolism and inflammatory response of foam cells.

Keywords: MAPK signaling pathway; NLRP3 inflammasome; foam cells; mulberry; restenosis.

FIGURE 1

NLRP3 pathway is upregulated in neointima macrophages of restenosis patients. (a) Volcano plot (left) shows gene expression level in artery tissue of restenosis patients or healthy control with ‐log10 adjusted p‐value (y‐axis) versus log2 fold‐change (x‐axis). Up‐ and down‐regulated DEGs (with adjusted p‐value < .05 and log2 fold‐change > 2) are labeled with red and blue dots, respectively. Genes with no significant change are labeled with gray dots. (b) KEGG pathway enrichment analysis of the DEGs reveals the top enriched biological processes in artery tissue of restenosis patients compared to healthy control. (c) GO enrichment analysis of DEGs (BP, biological process; CC, cellular component; MF, molecular function). (d) Heat map shows differentially expressed genes (with adjusted p‐value < .05 and log2 fold‐change > 2) between restenosis patients or healthy control (n = 3). (e) The mRNA expression levels of NLRP3, caspase‐1, ASC and IL‐1β in artery tissue of restenosis patients or healthy control were validated by RT‐qPCR. Relative expressions were normalized to β‐Actin (n = 4 per group). (f) Protein expression of NLRP3, caspase‐1, ASC, and IL‐1β were determined by Western blotting. (g) Expression of NLRP3 and CD68 in normal and restenotic artery by immunofluorescence. Red, NLRP3; green, CD68; blue, DAPI nuclear staining. Compared with normal arterial tissue, **p < .01, ***p < .001. FC, Fold change; NC, Normal Control; Res, Restenosis.

FIGURE 2

Active compounds of mulberry extract target anti‐restenosis genes. (a) RAW 264.7 macrophages were treated with mulberry extract (0, 0.625, 1.25, 2.5, 5, and 10 mg/mL) for 24 h, and cell viability was measured by CCK‐8 method. (b) Predicted network of targets of mulberry‐derived compounds using Traditional Chinese Medicine Systems Pharmacology Database. Orange nodes represent the active compounds, purple nodes the predicted targets, and edges indicate the interactions between compounds and targets. The size of nodes is proportional to the degree of interaction. (c) Venn diagram shows overlapping genes of predicted targets of mulberry compounds and genes downregulated in restenosis tissue. (d) KEGG pathway enrichment analysis and (e) GO enrichment analysis of the overlapping genes in (c). Pathways involve in lipid metabolism and inflammation are highlighted with red box. (f) Protein–protein interaction (PPI) network analysis of the overlapping genes by Cytoscape. Node size is proportional to the degree of interaction.

FIGURE 3

Mulberry extract inhibits the expression of NLRP3 in foam cells via MAPK signaling pathway. Foam cells were induced by treating RAW264.7 macrophages with 50 μg/mL ox‐LDL for 24 hours. The cells were then given mulberry extract at low (5 mg/mL) or high concentration (10 mg/mL) as indicated. Following foam cell features were monitored. (a). Intracellular lipid level measured by Oil Red O staining. (b). Cholesterol ester content. (TC, total cholesterol; FC, free cholesterol; CE, cholesterol ester) qualified by cellular cholesterol assay of foam cells with or without treatment of mulberry extract as indicated. (c). Induced foam cells were treated with different concentrations of mulberry extract (0, 1, 5, 10 mg/mL) for 24 h and protein levels of NLRP3, caspase‐1, ASC, and IL‐1 β were determined by Western blotting. (d) The protein levels of NLRP3, ASC, p‐p38, p38 MAPK, and IL‐1β protein levels of cells undergoing indicated treatments were determined by Western blotting. Graph bars represent qualification of flow cytometry results (Data shown are mean ± SEM. Statistical significance was determined by two‐way ANOVA with correction for multiple comparisons. *p < .01, **p < .001, ***p < .0001, ##p < .01 and ###p < .001.

FIGURE 4

Mulberry extract treatment elevates cholesterol efflux and inhibits lipid accumulation and apoptosis in foam cells. (a) Pathways enrichment analysis of combined parameters of mulberry extract‐related metabolites and anti‐restenosis genes identified in this study using MetaboAnalyst. Cholesterol efflux pathway is highlighted by red box. (b) Metabolism pathways analysis using MetaboAnalyst. (c) The mRNA expression levels of ABCA1 and ABCG1 in induced foam cells with or without mulberry treatment as indicated were validated by RT‐qPCR. Relative expressions were normalized to β‐Actin gene. (d) Apoptosis of induced foam cells with or without mulberry treatment as indicated was detected using Annexin V flow cytometry (representative data are shown from three independent experiments). Statistical significance was determined by two‐way ANOVA with correction for multiple comparisons. ***p < .001, ****p < .0001, ##p < .01 and ###p < .001).