Ginkgolide B Blocks Vascular Remodeling after Vascular Injury via Regulating Tgfβ1/Smad Signaling Pathway

Figures

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  Figure 1. Ginkgolide B improves vascular remodeling and inflammation after vessel injury. (a) The schedule of animal experiment design. Collect blood from orbital venous plexus. (b) The detection of blood flow velocity by the Doppler echocardiography (n = 12 each group). The histological analysis performed using H&E staining (c) and the corresponding quantification (e) and Masson's staining (d) and the corresponding quantification (f) (scar bar: 50 μm; n = 4 each group). (g) The mRNA expression of Mcp1 (monocyte chemoattractant protein 1), Cd68 (cluster of differentiation 68), and α-Sma (α-smooth muscle actin) in tissues (n = 4 each group). All data are presented as mean ± SEM. ∗P < 0.05 versus RCA treatment with vehicle; #P < 0.05 versus LCA treatment with vehicle.

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  Figure 2. Ginkgolide B plays a protective role in vascular injury by the Tgfβ1/Smad signal pathway. (a) The protein level detection of Tgfβ1 and p-Smad2/3 and quantification in vivo. (b) The mRNA level detection of Pro-Tgfβ1 in vivo. The ELISA detection of Tgfβ1 (c), Mcp1 (d), and α-Sma (d) in serum. All data (n = 4 each group) are presented as mean ± SEM. ∗P < 0.05 versus RCA treatment with vehicle; #P < 0.05 versus LCA treatment with vehicle.

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  Figure 3. Ginkgolide B has no influence of Tgfβ1/Smad signal pathway in human umbilical vein endothelial cells (HUVECs). (a) The western blot analysis for Tgfβ1 and p-Smad2/3 in HUVECs stimulated with 3 ng/ml TGF-β1 for 15 min, 30 min, and 60 min (n = 4 each group). (b) The cell viability of HUVECs incubated with different concentrations of GB was detected by CCK8 (n = 8 each group). (c) The ELISA detection of Tgfβ1 in cell culture supernatant (n = 4 each group). (d) The western blot analysis for Tgfβ1 and p-Smad2/3 in HUVECs stimulated with 3 ng/ml TGF-β1 for 60 min and then treated with GB for 2 hours (n = 4 each group). All data are presented as mean ± SEM. ∗P < 0.05 versus control.

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  Figure 4. Ginkgolide B regulates the Tgfβ1/Smad signal pathway in human vascular smooth muscle cells (HVSMAs). (a) The western blot analysis for Tgfβ1 and p-Smad2/3 in HVSMAs stimulated with 3 ng/ml TGF-β1 for 15 min, 30 min, and 60 min (n = 4 each group). (b) The cell viability of HVSMAs incubated with different concentrations of GB was detected by CCK8 (n = 8 each group). (c) The ELISA detection of Tgfβ1 in cell culture supernatant (n = 4 each group). (d) The western blot analysis for Tgfβ1 and p-Smad2/3 in HVSMAs stimulated with 3 ng/ml TGF-β1 for 60 min and then treated with GB for 2 hours (n = 4 each group). All data are presented as mean ± SEM. ∗P < 0.05 versus control.

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  Figure 5. The protective role of Ginkgolide B is abolished after incubated with Tgfβ1 inhibitor in HVSMAs. (a) The western blot analysis for Tgfβ1 and p-Smad2/3 stimulated with different concentration of pirfenidone (PFD, Tgfβ1 inhibitor). (b) The western blot analysis for Tgfβ1 and p-Smad2/3 stimulated with 3 ng/ml TGF-β1 for 60 min and then treated with different concentration of pirfenidone. (c) The western blot analysis for Tgfβ1 and p-Smad2/3 stimulated with 3 ng/ml TGF-β1 for 60 min and then treated with GB or pirfenidone for 2 hours. (d) The ELISA detection of Tgfβ1 in cell culture supernatant. All data (n = 4 each group) are presented as mean ± SEM. ∗P < 0.05 versus control; #P < 0.05 versus treatment with TGF-β1; &P < 0.05 versus treatment with TGF-β1 and pirfenidone; ΦP < 0.05 versus treatment with TGF-β1 and GB.

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  Figure 6. The protective function of Ginkgolide B in vessel remodeling and inflammation caused by vascular injury is vanished in Tgfβ1 knockout mice. The mice were treated with pirfenidone capsules (presenting as PFD) and Yinxingneizhi Zhusheye (including Ginkgolide B) (presenting as GB). (a) The schedule of animal experiment design. Collect blood from orbital venous plexus. (b) The detection of blood flow velocity by the Doppler echocardiography and quantification (e) (n = 8 each group). The histological analysis performed using H&E staining (c) and the corresponding quantification (f) and Masson's staining (d) and the corresponding quantification (g) (scar bar: 50 μm; n = 4 each group). (h) The mRNA expression of Mcp1, Cd68, and α-Sma in tissues (n = 4 each group). All data are presented as mean ± SEM. ∗P < 0.05 versus RCA treatment with vehicle.

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  Figure 7. The molecular docking of Ginkgolide B and Tgfβ1. (a) 2D map. (b–d) The different form of 3D map.

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  Figure 8. Molecular mechanism involved in vascular remodeling. Vessel injury as in the case of PCI causes Tgfβ1, activates Smad2/3 phosphorylation, and contributes to the transcription of genes taken part in vascular remodeling. Ginkgolide B represses the production of Tgfβ1, further reduces Smad2/3 phosphorylation, and contributes to the decline transcription of genes taken part in vascular remodeling, which could decrease restenosis and thrombosis.