Novel Pyrazolo[3,4-b] Pyridine Derivative (HLQ2g) Attenuates Hypoxic Pulmonary Hypertension via Restoring cGKI Expression and BMP Signaling Pathway

Figures

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  FIGURE 1. HLQ2g can stimulate sGC and upregulate cGMP. (A): The structure of Riociguat. (B): The structure of HLQ2g. (C): Dose-dependent curve of HLQ2g to elevate the cGMP levels in the absence or presence of 10 μM ODQ in HPASMCs without IBMX, values are the average of three independent experiments. (D): Dose-dependent curve of HLQ2g to elevate the cGMP levels in the absence or presence of 10 μM ODQ in HPASMCs under condition of IBMX (10 μM), values are the average of three independent experiments.

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  FIGURE 2. Hemodynamics, right heart remodeling, pulmonary vascular remodeling in Hypoxia-induced PH rats. (A): RVSP waveform; (B): RVSP summary graph; (C): RV/LV + S (right ventricle/left ventricle + interventricular septum) statistics; (D): RV/Tibial length (right ventricle/tibial length) statistics; (E): Statistical chart of HE staining (100 μm); (WT%) = (outside diameter-inside diameter)/(outside diameter) × 100. (F): Representative image of HE staining. Control: the control rats; Hypoxia: Hypoxia-induced PH rats. Data are expressed as mean ± SD. n = 3–5. Vs. the control, ***p < 0.001; vs. the Hypoxia group, #p < 0.05, ##p < 0.01, ###p < 0.001.

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  FIGURE 3. In hypoxia-induced PH rat model, HLQ2g can regulate cGKI protein and BMPR2 pathway. (A): The expression of cGKI, BMPR2, p-Smad1/5/8, and Id1 in pulmonary artery of rats; (B–E): The quantitative analysis results of A. Control: the control rats; Hypoxia: Hypoxia-induced PH rats. Data are expressed as mean ± SD. n = 3. Vs. the control, *p < 0.05, **p < 0.01; vs. the Hypoxia group, #p < 0.05, ##p < 0.01.

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  FIGURE 4. HLQ2g can inhibit the proliferation of HPASMCs. (A, B): CCK8 detected cell proliferation. (C): Quantitative analysis of I. (D): The expression of PCNA protein in HPASMCs stimulated by hypoxia. (E): The expression of PCNA protein in HPASMCs induced by PDGF. (F): Quantitative analysis of D. (G): Quantitative analysis of E. H: Quantitative analysis of J. (I, J): EdU detected cell proliferation. Riociguat (1 μM); HLQ2g (1 μM); Vehicle: 1% DMSO; Control: normal control group; PDGF: platelet-derived growth factor BB treatment group (20 ng/ml; 24 h). Data are expressed as mean ± SD, n = 3–5. Vs. the control, **p < 0.01, ***p < 0.001; vs. the PDGF/Hypoxia group, #p < 0.05, ##p < 0.01, ###p < 0.001.

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  FIGURE 5. HLQ2g can inhibit the migration of HPASMCs. (A, B): Scratch test results (100 μm); (C): Statistical analysis of A; (D): Statistical analysis of B. Riociguat (1 μM); HLQ2g (1 μM). Data are expressed as mean ± SD, n = 3. Vs. the control, ***p < 0.001; vs. the PDGF/Hypoxia group, ###p < 0.001.

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  FIGURE 6. HLQ2g can upregulate cGKI expression and BMP signaling pathway in Hypoxia-induced HPASMCs. (A): The protein expression of cGKI, BMPR2, p-Smad1/5/8, and Id1 in HPASMCs determined by Western blot. Riociguat (1 μM). (B–E): Quantitative analysis results of graph A. Data expressed as mean ± SD, n = 3. Vs. the control, **p < 0.01, ***p < 0.001; vs. the Hypoxia group, #p < 0.05, ##p < 0.01, ###p < 0.001.

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  FIGURE 7. Rp-8-Br-PET-cGMPS can block the effect of HLQ2g. (A): CCK8 detected cell proliferation. (B, G): Transwell test results (200 μm). (C): The protein expression of BMPR2, p-Smad1/5/8, and Id1 in HPASMCs determined by Western blot. (D–F): Quantitative analysis results of graph C. HLQ2g (1 μM); Rp-8: Rp-8-Br-PET-cGMPS (30 μM). Data expressed as mean ± SD, n = 3–5. Vs. the control, *p < 0.05, **p < 0.01, ***p < 0.001; vs. the Hypoxia group, #p < 0.05, ##p < 0.01, ###p < 0.001; vs. the Hypoxia + HLQ2g group, ^p < 0.05, ^^p < 0.01, ^^^p < 0.001.