Atractylenolide-1 Targets FLT3 to Regulate PI3K/AKT/HIF1-α Pathway to Inhibit Osteogenic Differentiation of Human Valve Interstitial Cells

FIGURE 1. Viability and calcification of VICs treated with AT-1. (A) Chemical structure of atractylenolide-1. (B) The CCK8 method was used to analyze the effect of treatment with different atractylenolide-1 concentrations on VICs. (C) IC50 of atractylenolide-1 in VIC cells. (D) Effect of no AT-1 and 20 μM AT-1 on the proliferation of OM-induced VIC cells in 5 days. (E) Cells with different treatments (control (normal culture medium), OM (osteogenic medium), OM+10 μM AT-1 (osteoblastic medium plus10 μM AT-1 treatment), OM+20 μM AT-1 (osteoblastic medium plus 20 μM AT-1 treatment), and OM + AGP (osteoblastic medium plus 10 μM andrographolide) were stained with Alizarin Red S. (F) Alizarin Red staining statistics. *p < 0.05, **p < 0.01 indicated a significant difference compared to the control group and #p < 0.05, ##p < 0.01 indicated a significant difference compared to the OM group, n = 3.

FIGURE 2. Effect of AT-1 on OM-induced calcific-related gene and protein expression in VICs. (A) VICs were stimulated with OM and then treated or not with AT-1 for 48 h. The mRNA RUNX2 and ALP expression levels were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). (B, C) VICs were stimulated with OM and then treated or not with AT-1 and AGP for 48 h and the expression levels of RUNX2 and ALP protein were detected by Western blotting. (D) Statistical analysis of Runx2 and ALP protein expression. (E) The ALP activity of the VICs was measured using the ALP biochemical kit. (F) The expression of RUNX2 in VICs was evaluated at 48 h by immunofluorescence. *p < 0.05, **p < 0.01 indicated a significant difference compared to the control group and #p < 0.05, ##p < 0.01 indicated a significant difference compared to the OM group, n = 3.

FIGURE 3. Changes in gene expression under different treatments analyzed by mRNA-seq. (A) Schematic diagram of the mRNA transcriptome sequencing process. (B) Venn interaction of DEGs of the Control versus the OM group, and the OM group versus the OM + AT-1 group. (C) GO enrichment of the commonly detected DEGs, which included molecular function, cellular components, and biological processes. (D) KEGG pathway enrichment bubble map, where a larger p-value (-log10) indicates a higher degree of enrichment.

FIGURE 4. The effect of AT-1 on the PI3K/AKT/HIF1-α pathway and investigations into its upstream targets. (A) Effect of different AT-1 and AGP concentrations on the expression of PI3K, AKT, and P-AKT proteins (B) Statistical analysis of PI3K/β-actin and P-AKT/AKT. (C) Effect of different AT-1 and AGP concentrations on the expression of HIF1-α protein. (D) Statistical analysis of HIF1-α/β-actin. (E) The predicted AT-1 targets and PI3K pathway proteins were subjected to PPI network association analysis to identify the upstream targets of AT-1 regulating the PI3K pathway. *p < 0.05, **p < 0.01 indicated a significant difference compared to the control group and #p < 0.05, ##p < 0.01 indicated a significant difference compared to the OM group, n = 3.

FIGURE 5. AT-1 inhibits the phosphorylation of FLT3 and the activation of PI3K/AKT/HIF1-α, thereby inhibiting the osteogenic differentiation of VICs. (A) Molecular docking technology was used to detect the binding ability of AT-1 to FLT3 protein. (B) Effect of different AT-1 concentrations on the level of phosphorylated FLT3 protein in OM-induced VIC cells. (C) Statistical analysis of P-FLT3/FLT3. (D) Effect of AT-1 treatment with or without FLT3 inhibitor on the regulation of PI3K/AKT/HIF1-α in OM-induced VICs. (E) Statistical analysis of PI3K/β-actin, P-AKT/AKT, and HIF1-α/β-actin. (F) Cells with different treatments (control, OM, OM+20 μM AT-1, and OM+20 μM AT-1+10 nM SKLB4771) were stained with Alizarin Red S. (G) Alizarin Red staining statistics. *p < 0.05, **p < 0.01 indicated a significant difference compared to the control group and #p < 0.05, ##p < 0.01 indicated a significant difference compared to the OM group, n = 3.