Paeoniflorin Ameliorates Chronic Hypoxia/SU5416-Induced Pulmonary Arterial Hypertension by Inhibiting Endothelial-to-Mesenchymal Transition

Figures

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  Figure 1. Male SD rats received a single subcutaneous injection of SU5416 (20 mg/kg) or vehicle and were placed in hypoxia for 3 weeks (10% O2). These rats received daily doses of 300 mg/kg of paeoniflorin (PF) or saline by gavage once daily for 3 weeks from the first day. Rats were sacrificed at 21 days after the SU5416 administration to evaluate the preventive efficacy of PF in PAH.

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  Figure 2. Effects of PF on hemodynamic parameter and pulmonary vascular remodeling in the SU5416/hypoxia (SuHx) rat model of pulmonary arterial hypertension (PAH). (A) Assessment of the right ventricular systolic pressure (RVSP) in rats (n = 6). Lung sections were stained with hematoxylin-eosin (H&E) solution for histopathological evaluation or with Masson’s trichrome for collagen assessment. (B) The degree of pulmonary vascular remodeling was evaluated by the pulmonary arterial medial wall thickness (n = 6). (C) The degree of pulmonary vascular adventitial fibrosis was evaluated by the percentage of the collagen-positive area (n = 6). (D) Representative images of H&E staining in lung sections. (E) Representative images of Masson’s trichrome staining in lung sections. Data were indicated as the mean ±SEM. *P < 0.05 vs untreated control; #P < 0.05 vs untreated SuHx group.

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  Figure 3. Effect of PF on right ventricular hypertrophy and fibrosis in the SuHx rat model of PAH. (A) Assessment of right ventricular hypertrophy (the ratio of right ventricular (RV) weight to left ventricular plus septal weight in rats (n = 6). RV remodeling was demonstrated by H&E staining, and RV fibrosis was demonstrated by picrosirius red staining. (B) RV remodeling was evaluated by cross-sectional area (CSA) of RV cardiomyocytes (n = 6). (C) Interstitial RV fibrosis was evaluated by the percentage of the collagen-positive area (n = 6). (D) Representative images of H&E staining in RV sections. (E) Representative images of picrosirius red staining in RV sections. Data were indicated as the mean ±SEM. *P < 0.05 vs untreated control; #P < 0.05 vs untreated SuHx group.

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  Figure 4. Effect of PF on endothelial-to-mesenchymal transition (EndMT) in the SuHx rat model of PAH. (A) Representative Western blots of VE-cadherin, fibronectin, and vimentin were shown. (B–D) Quantitative analysis of VE-cadherin, fibronectin, and vimentin was shown. Data were indicated as the mean ±SEM. n = 3–4 independent experiments, respectively. *P < 0.05 vs untreated control; #P < 0.05 vs untreated SuHx group.

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  Figure 5. Hypoxia induce EndMT in HPAECs. HPAECs were exposed to hypoxia for 0 h, 6 h, 12 h, 24 h, 48 h, and 72 h, respectively. The expression of EndMT markers was evaluated by Western blotting and immunofluorescence staining. (A) Representative Western blots and quantitative analysis of VE-cadherin, α-SMA, vimentin, fibronectin, snail and twist were shown. n = 3, 4, 3, 3, 5, 3 independent experiments, respectively. (B) Double immunofluorescence staining of HPAECs for VE-cadherin (red) and vimentin (green) was performed (n = 3). *P < 0.05 vs normoxia.

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  Figure 6. Effect of PF on hypoxia-induced EndMT in HPAECs. HPAECs were cultured under normoxia or hypoxia with or without paeoniflorin (10 μΜ) treatment for 48 h. The expression of EndMT markers was evaluated by Western blotting and immunofluorescence staining. (A) Representative Western blots and quantitative analysis of VE-cadherin, α-SMA, vimentin, fibronectin, snail and twist were shown. n = 3, 4, 5, 3, 5, 3 independent experiments, respectively. (B) Immunofluorescence staining for VE-cadherin and vimentin was detected (n = 3). *P < 0.05 vs normoxia; #P < 0.05 vs hypoxia.

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  Figure 7. BMPR2 is critical for the anti-EndMT of PF in HPAECs. (A) HPAECs were exposed to hypoxia (1% O2) with or without PF treatment, and representative Western blots and quantitative analysis of BMPR2 were shown (n = 3). (B) Representative Western blots and quantitative analysis of p-SMAD1/5/SMAD1/5 were shown (n = 4). (C) HPAECs were transfected by negative control (NC) siRNA or BMPR2 siRNA and representative Western blots and quantitative analysis of BMPR2 were shown (n = 3). (D) HPAECs transfected with BMPR2 siRNA or NC siRNA for 24 h were cultured under normoxia or hypoxia with or without paeoniflorin for 48 h. Representative Western blots and quantitative analysis of BMPR2, VE-cadherin, fibronectin, and vimentin were shown. n = 3, 4, 3 independent experiments, respectively. Data were indicated as the mean ±SEM. *P < 0.05 vs normoxia; #P < 0.05 vs hypoxia; &P < 0.05 vs hypoxia + NC siRNA + PF.