Fisetin alleviates pulmonary arterial hypertension by inhibiting the TGF-β1/Smad 2/3 signaling pathway

Figures

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  Fig. 1. Fisetin ameliorates MCT-induced pulmonary vascular remodeling and inhibits PASMC proliferation in rats. (a) Experimental approach. Rats were randomly divided into four groups (n = 6) and treated for four weeks as follows: Control: daily saline; MCT: a single intraperitoneal administration of MCT (55 mg/kg); MCT + Fisetin: a single MCT injection, followed by daily intragastric Fisetin (50 mg/kg/day); MCT + Sildenafil: a single MCT injection, followed by daily intragastric sildenafil (20 mg/kg/day). (b) Statistical analysis of RVSP (n = 6). (c) Statistical analysis of RVHI (n = 6). (d) Representative images of pulmonary arteries (H&E, Masson's trichrome, and α-SMA staining; 20 ×, scale bar = 100 μm n = 3–6). (e, f) Quantitative analysis of WA% and WT% (n = 6). (g, h) Representative Western blot and corresponding quantification of PCNA (n=3). Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. MCT group

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  Fig. 2. Fisetin inhibits inflammation in MCT-induced PAH rats, and its mechanism was investigated via protein–protein docking. (a-c) The concentrations of IL-1β, IL-6, and TNF-α in lung tissues homogenates were determined by ELISA (n = 3). (d-f) Molecular docking of IL-1β, IL-6, and TNF-α with TGF-β1. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. MCT group

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  Fig. 3. Fisetin inhibits the TGF-β1/Smad2/3 signaling pathway in MCT-induced PAH rats and molecular docking. (a) The results of the molecular docking between Fisetin and TGF-β1. (b-e) Representative Wertern blot and corresponding quantification of TGF-β1, Smad2/3, and p-Smad2/3 (n = 3). Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

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  Fig. 4. Fisetin inhibits the migration and proliferation of PDGF-PASMCs. (a-b) Viability of PASMCs treated with Fisetin, with or without PDGF-induced, was assessed by CCK-8 assay (n = 3–4). (c-d) Representative IF images of MMP9 (red) and DAPI (blue) (c) and MMP9 fluorescence intensity analysis (d) (n = 3, Scale bar = 20 μm. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

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  Fig. 5. Fisetin inhibits autophagy in PDGF-induced PASMCs. (a-d) Representative IF images of LC3B, Beclin1, and p62 (red) and DAPI (blue) (a) and LC3B, Beclin1, and p62 fluorescence intensity analysis (b-d) (n = 3, Scale bar = 20 μm). (e) TEM images showing autophagosomes(indicated by red arrows) and autolysosomes(indicated by black arrows) in PASMCs (indicated by black arrows), (n = 3, Scale bar = 1.0 μm). (f-i) Representative Western blot and corresponding quantification of LC3BⅡ, Beclin1, and p62 (n = 3). (j-l) Protein–protein docking of TGF-β1 with LC3B (j), Beclin1 (k), and p62 (l). Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

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  Fig. 6. Fisetin inhibits the TGF-β1/Smad2/3 signaling pathway in PDGF-BB-induced PASMCs. (a-c) Representative IF images of TGF-β1 and Smad2/3 (red) and DAPI (blue) (a) and TGF-β1 and Smad2/3 fluorescence intensity analysis (b,c) (n = 3, Scale bar = 20 μm). (d-f) Representative Western blot and corresponding quantification of Smad2/3, and p-Smad2/3 (n = 3). Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001