Anthraquinone Emodin Inhibits Tumor Necrosis Factor Alpha-Induced Calcification of Human Aortic Valve Interstitial Cells via the NF-κB Pathway

FIGURE 1. Viability and cell cycle of hVICs with treatments at the concentration of EMD ranging from 1 to 20 μM. (A) Morphologies of hVICs with different treatments for 3 days. (B) Cell proliferation curve from cck8 assay, n = 5. (C,D) Cell cycle analysis, (S-phase) were counted and made statistical comparison. ∗p < 0.05 (vs. control) and #p < 0.05 (vs. TNF-α) were accepted as significant difference, n = 3.

FIGURE 2. Emodin inhibits TNF-α-induced calcification of hVICs for 12 and 18 days (d). (A) Alizarin Red S positive staining. (B,C) Absorbance value (OD) for 12 days (B) and 18 days (C) conditioned culuring, ∗ and #p < 0.05 were accepted as significant difference when, respectively compared to control (absence of both TNF-α and EMD), and to TNF-α group, n = 3.

FIGURE 3. Global gene expression profiles under the TNF-α conditioned culture with or without EMD. (A) Heatmap for global gene expression, # indicates number, experiment repeated three times, n = 3; (B) Volcano map of TNF-α and TNF-α plus EMD expression genes, FC (fold change) > 1 was accepted as positive differentially expressed genes, up for 1131; down for 743. (C) GO enrichment of those selected DEGs including biological process, cellular component, molecular function.

FIGURE 4. KEGG pathway enrichment analysis. (A) pathway enrichment bubble map, a larger P-value (-Log10) indicates a higher degree of enrichment. (B) Venn interaction of DEGs in the TNF, PI3K-Akt, MAPK, NF-kappa B, and Ras signaling pathways. RELA is the consensus differentially expressed gene.

FIGURE 5. Emodin involves in NF-κB signal pathway. (A) Heatmap for 34 typical functional DEGs; (B) up-down comparison of the selected gene expression (expression level represents average value, n = 3); (C) qRT-PCR for detecting BMP2, RELA, RUNX2, TNF, TRAF1 gene expression of hVIC with conditioned culturing, ∗ and #p < 0.05 were accepted as significant difference when, respectively compared to control (absence of both TNF-α and EMD), and to TNF-α group, n = 3; (D) Western blotting bands for TRAF1 and RELA protein synthesis, (TNF-α: 30 ng/ml, EMD: 10 μM). (E) Graphical indication of emodin involving in NF-κB pathway.