Bellidifolin Improves Pulmonary Artery Smooth Muscle Cells Proliferation by Targeting the IGFBP5-Mediated PI3K-AKT-mTOR Pathway and Dilates the Pulmonary Artery

Figures

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  Figure 1. (a,b) Effect of BEL (6–96 μmol/L) on NE-precontracted pulmonary artery rings of Endo+ and Endo− (n = 6). (c) Effect of different concentrations of BEL on KCl-precontracted pulmonary artery rings (Endo+) (n = 6). (d) Effect of high BEL (90 μmol/L) concentration on internal calcium release and external calcium inflow (n = 3). The results are expressed as mean ± SD (** p < 0.01 and *** p < 0.001).

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  Figure 2. (a,b): Intracellular Ca2+ concentration in rat PASMCs measured by flow cytometry. (c,d) Intracellular Ca2+ concentration in rat PASMCs measured by immunofluorescence. The results are expressed as mean ± SD (n = 3). (* p < 0.05, ** p < 0.01 and *** p < 0.001).

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  Figure 3. (a–c): Effect of BEL (10–160 μmol/L) on the proliferation of PASMCs at 24 (a), 48 (b), and 72 h (c). (d) Effect of BEL (10–160 μmol/L) on the proliferation of PASMCs induced by CoCl2 24 h. (e) Effect of BEL (10–160 μmol/L) on the proliferation of PASMCs induced by CoCl2 48 and 72 h. The results are expressed as mean ± SD (n = 3).

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  Figure 4. BEL treatment arrested the cell cycle at the G2/M phase. (a) PASMC circulation in each group was detected. The black curve in a indicates the overall model fit. (b) Quantification of the S, G2/M and G1 phases of each group of cells. The results are expressed as mean ± SD (n = 3). (ns, not significant (p > 0.05), * p < 0.05, ** p < 0.01 and *** p < 0.001).

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  Figure 5. Effect of BEL on PASMC migration. (a) Effect of BEL (20, 50, and 80 μmol/L) on the migration of PASMCs induced by CoCl2. (b) Quantification of wound closure. The results are expressed as mean ± SD (n = 3) (*** p < 0.001).

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  Figure 6. Effect of BEL on the expression of Acta2 (a), Tagln (b), Pcna (c), Spp1 (d), and Vim (e) mRNA in PASMCs treated with CoCl2. Effect of BEL on the expression of α-SMA (f), SM22α (g), PCNA (h), OPN (i), and vimentin (j) proteins in PASMCs induced by CoCl2. The results are expressed as mean ± SD (n = 3). (* p < 0.05, ** p < 0.01 and *** p < 0.001). Original western blot images see Supplementary Material File S1.

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  Figure 7. Transcriptome-quantitative proteomics-combined analysis. (a) Venn diagram showing the intersection analysis of DEGs and DEPs between the CoCl2 group and control groups. (b) Venn diagram showing the intersection analysis of DEGs and DEPs between the CoCl2 (10 μmol/L) group and the CoCl2 (10 μmol/L) + BEL (50 μmol/L) groups. (c) Heat map showing the expression profiles of DEGs and DEPs in the CoCl2 (10 μmol/L) + BEL (50 μmol/L) group vs. the CoCl2 (10 μmol/L) group. (d,e) The volcano plot displays the annotation information for genes and proteins that show differences in omics expression. (f) KEGG analysis (the CoCl2 (10 μmol/L) + BEL (50 μmol/L) group vs. the CoCl2 (10 μmol/L) group). (g) GO analysis (the CoCl2 (10 μmol/L) + BEL (50 μmol/L) group vs. the CoCl2 (10 μmol/L) group). The red boxes indicate the key genes and proteins /pathways identified through screening in the (c–f). In the four quadrants of (d,e), gray dots represent features with no significant differences in either omics dataset; light-colored dots indicate features that are significant in only one of the single-omics analyses; dark-colored dots denote features that show significant differences in both…

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  Figure 8. RT-qPCR, immunocytochemistry and Western blot validation of differentially expressed genes and proteins. (a,b) Representative results of Aldh1A1, Mgp, and Igfbp5 immunocytochemistry staining. (c) Representative results of Col4a6 Western blot analysis. (d–f) Representative results of the relative expression levels of Aldh1A1, Igfbp5, and Mgp mRNA. The results are expressed as mean ± SD (n = 3) (* p < 0.05, ** p < 0.01, and *** p < 0.001). Original western blot images see Supplementary Material File S1.

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  Figure 9. The molecular docking image of the key proteins and BEL. (a,b) The representative image of Igfbp5’s CETSA, (n = 3). Schematic diagrams of molecular docking: (c) Igfbp5 with BEL, (d) Aldh1a1 with BEL, (e) Igfbp5 with NBI31772, and (f) Aldh1a1 with CM037. Original western blot images see Supplementary Material File S1.

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  Figure 10. (a–d) The protein expression of p-Akt and p-mTOR was detected by Western blot in the PASMCs. The results are expressed as mean ± SD (n = 3) (* p < 0.05 and ** p < 0.01). Original western blot images see Supplementary Material File S1.

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  Figure 11. BEL suppresses abnormal PASMC proliferation through the Igfbp5-mediated PI3K-Akt-mTOR signaling axis. (a–d) Protein expression levels of p-PI3K, p-Akt, and p-mTOR in PASMCs detected by Western blot. BEL suppresses abnormal PASMC proliferation through the Igfbp5-mediated PI3K-Akt-mTOR signaling axis. (e,f) Effects of BEL on PASMC proliferation through Igfbp5. The red scale bar in the lower right corner of e represents 50 μm. Data are expressed as mean ± SD (n = 3) (* p < 0.05, ** p < 0.01 and *** p < 0.001). Original western blot images see Supplementary Material File S1.