β‐sitosterol Alleviates Pulmonary Arterial Hypertension in Rats With Involvement of the VEGF/PI3K/AKT Signaling Pathway

Figures

• 

  Figure 1. Network pharmacology analysis of SITO against PAH. (A) The chemical structure of SITO. (B) Venn diagram of overlapping targets between SITO active components and PAH. (C) PPI network of SITO active components and PAH targets. (D) Top 10 genes according to the Degree value.

• 

  Figure 2. Functional analysis of GO and KEGG. (A‐C) GO enrichment analysis of common genes, showing significant terms in biological processes (BP, A), cellular components (CC, B), and molecular functions (MF, C). (D) KEGG pathway enrichment analysis of key genes.

• 

  Figure 3. Molecular docking study of SITO binding to the three core target genes. (A). Binding conformation of SITO with EGFR protein (binding energy = −9.9 kcal/mol). (B). Binding conformation of SITO with IGF1 protein (binding energy = −6.8 kcal/mol) (C). Binding conformation of SITO with KDR protein (binding energy = −9.8 kcal/mol).

• 

  Figure 4. SITO alleviates MCT‐induced PAH hemodynamic abnormalities in rats. (A) Representative RVSP images. (B) M‐mode for quantification of tricuspid annular plane systolic excursion (TAPSE). (C) Bar charts showing mean RVSP measurements. (n = 6). (D) Bar charts showing TAPSE measurements (n = 6). All data were presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

• 

  Figure 5. SITO on MCT‐induced pulmonary artery remodeling. (A) Representative H&E images of the cross‐section of small pulmonary artery (PA), Scale bar, 20 μm. (B) Immunofluorescence staining of pulmonary arterioles: red, α‐SMA; green, PCNA; blue, DAPI (scale bar: 20 μm). (C) Pulmonary arteries fibrosis with Masson's trichrome staining, Scale bar, 20 μm. (D) Summarized data showing pulmonary arterial wall thickness. (E) Quantitative analysis of % media area (medial area/total vessel area × 100%,). (F) Summarized data showing averaged PA fibrosis ratio (the area of blue to the area of red). (G) Relative mRNA expression of TGF‐β1 in lung tissue. (H) Relative mRNA expression of Collagen I in lung tissue. All data were presented as the mean ± SEM. *p < 0.05, **p < 0.01 ***p < 0.001 and ****p < 0.0001.

• 

  Figure 6. Effects of SITO on right ventricular hypertrophy. (A) Representative H&E images of the RV cross‐sectional, Scale bar, 50μm. (B) Representative views of Masson's trichrome staining to detect RV fibrosis, Scale bar, 50μm. (C) the ratio of right ventricular (RV) to left ventricular (LV) wall plus septum (S) (RV/[LV + S]) (n = 6). (D) Summarized data showing the averaged area of RV cross‐­sectional cells (n = 6). (E) Summarized data showing the averaged RV interstitial fibrosis ratio (the area of blue to the area of red) (n = 6). (F) Relative TGF‐β1 mRNA expression in right ventricular tissue (n = 3). (G) Relative Collagen I mRNA expression in right ventricular tissue (n = 3). All data were presented as the mean ± SEM. *p < 0.05, **p < 0.01 ***p < 0.001 and ****p < 0.0001.

• 

  Figure 7. Effect of SITO on pulmonary inflammation. (A) mRNA expression levels of TNF‐α.(B) mRNA expression levels of IL‐6. (C) mRNA expression levels of IL‐1β. (D) mRNA expression levels of VEGF. (n = 3). All data were presented as the mean ± SEM. *p < 0.05, **p < 0.01 ***p < 0.001 and ****p < 0.0001.

• 

  Figure 8. Effect of SITO on the VEGF/PI3K/AKT signaling pathway and apoptosis/proliferation‐related protein expression in rats. (A–B) Protein levels of PCNA, Bcl‐2, and BAX were verified by Western blot. (C–D) Protein levels of VEGFR2, p‐VEGFR2, PI3K, p‐PI3K, AKT, and p‐AKT were verified by Western blot. Data are presented as the mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.