Ginsenoside CK targets PHD2 to prevent platelet adhesion and enhance blood circulation by modifying the three-dimensional arrangement of collagen

Figures

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  Figure 1. CK ameliorates acute soft tissue injuries in rats. (A) Schematic representation of the construction process for rat soft tissue injury model. (B) Measurement of swelling at the injury site. (C) Soft tissue injury score statistics and H&E (D) and Masson (E) staining analysis of soft tissue. (F–I) Determination of whole blood viscosity under low (1 and 3 s), medium (30 s), and high (200 s) shear conditions. RBC (J), WBC (K), LYMPH (L), and NEUT (M) counts in rats with soft tissue injuries. ###P < 0.001 vs. con group; ∗∗P < 0.01, ∗∗∗P < 0.001 vs. mod group, (n = 6).

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  Figure 2. Untargeted plasma metabolomics analysis and evaluation on rat soft tissue injury. (A) Cluster analysis of significantly differential metabolites affected by CK. (B) Enrichment analysis of metabolic pathways based on significantly differential metabolites. (C) The impact of CK on the key metabolites 2-OG and Vc in HIF-1 signaling pathway. (D) Schematic diagram of the role of PHD2 and its associated metabolites in the HIF-1α signaling pathway. (E) Western blot detection of the effect of CK on the expression of PHD2, HIF-1α and VEGF. ∗P < 0.05, ∗∗P < 0.01 vs. mod group, (n = 6).

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  Figure 3. PHD2 protein is identified as a target of CK. (A) DARTS assay of CK's effect on damaged soft tissue proteins on SDS-PAGE. (B) Identification of potential target proteins of CK by HPLC–MS/MS analysis. (C) Western blot of PHD2 target proteins on DARTS samples. ∗P < 0.05 vs. mod group (n = 3). (D) Molecular docking of CK molecules and PHD2 protein. (E) FTSA analysis of PHD2 protein treated with or without 50 μmol/L CK. (F) SPR analysis of CK and PHD2 (KD = 18.0 μmol/L). (G) MST analysis of CK and PHD2 (KD = 17.6 μmol/L).

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  Figure 4. CK affects collagen structure by inhibiting prolyl hydroxylase. (A) Schematic diagram of hydroxylation modification of collagen structure by PHD2. (B) MST analysis of CK on PHD2–collage interaction. (C) Effect of CK on HYP content in collagen. #P < 0.05 vs. collagen group; ∗∗∗P < 0.001 vs. PHD2 group (n = 5). (D) Protein profile identification flow chart of collagen α1 chain. The effect of CK on the hydroxylation ratio (E) or hydroxylation number (F) of representative collagen α1 peptides. (G) SEM imaging analysis of architecture for collagen with or without PHD2 or CK administration.

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  Figure 5. CK reduces platelet adhesion and disrupts the binding between VWF and collagen. (A) Effect of CK on fluorescence imaging of platelet adhesion with collagen protein which coated on microporous plates. ###P < 0.001 vs. con group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. mod group (n = 3). (B) VWF-collagen interaction diagram. CK affects PHD2 mediated (C) collagen–platelet adhesion (D) collagen–VWF binding, and (E) the adhesion of collagen–VWF-platelet. ###P < 0.001 vs. con group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. mod group (n = 6). (F) MST analysis of VWF and collagen. (G) The impact of PHD2 on VWF–collagen interaction. (H) The effect of CK on the interaction between VWF and collagen.

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  Figure 6. CK improves blood circulation by changing collagen structure in soft tissue injury rats. (A) Van Gieson staining for collagen on damaged soft tissues. (B) Fluorescence imaging of exposed 3D structure of collagen on soft tissue slices using antibody fluorescence detection. (C) The impact of CK on collagen HYP content. (D) Extraction and SDS-PAGE separation of collagen protein. (E) Sequence alignment of representative peptide segments of collagen α1 chain among rat, mouse and human. (F) Protein profile assay for the impact of CK on the total hydroxylation modification number in rat soft tissues. (G) Identification of representative peptide MS/MS spectra for prolyl hydroxylation modification. (H) Immunohistochemical analysis of VWF-collagen on injured soft tissues. The effect of CK on blood parameters in soft tissue injury rats, including RBC aggregation index (I), HCT (J), PLT (K) and MPV (L). #P < 0.05, ##P < 0.01, ###P < 0.001 vs. con group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. mod group (n = 6).

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  Figure 7. CK inhibits pulmonary thrombus in mouse DIC model. (A) Hemorrhage illustration in the mouse tail in the DIC model. (B, C) Measurement of bleeding time and volume. (D) Masson staining and (E) H&E staining for pathological analysis of lung tissue. (F, G) Immunofluorescence analysis of thrombin and fibrinogen. (H) Fluorescence co-localization analysis of VWF and collagen protein in the lungs. ##P < 0.01, ###P < 0.001 vs. con group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. mod group (n = 6).

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  Figure 8. Construction of sh-Phd2 mice confirms that PHD2 is a potential target for inhibiting platelet adhesion and thrombus. (A) Molecular docking of PHD2 inhibitors Rox, Vad, Dap and IOX2 with PHD2 protein, and dashed lines indicate catalytic pocket. (B) Effect of PHD2 inhibitors on platelet adhesion; ns, multiple comparisons test no significant (n = 3). (C) Determination and analysis of the enzymatic activity of PHD2 in the presence of PHD2 inhibitors. ##P < 0.01, ###P < 0.001 vs. con group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, vs. mod group; ΔP < 0.05, Dap vs. Rox group (n = 3). (D) Construction scheme of sh-Phd2 knockdown mice. (E) Western blot analysis for assessing the knockdown efficiency of PHD2 in the sh-Phd2 group. ∗∗P < 0.01, vs. sh-con, (n = 3). (F, G) Measurement of tail bleeding time and volume of DIC model mice. (H) Masson staining for thrombus analysis of lung tissue. (I, J) Fibrinogen and thrombin immunofluorescence analysis and statistics. (K) Fluorescence co-localization analysis of VWF and collagen in lung tissue. ##P < 0.01, ###P < 0.001, vs. con group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. mod group; ΔP < 0.05, ΔΔP < 0.01, ΔΔΔP < 0.001, vs. mod group in sh-Phd2…

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  Figure 9. Effect of PHD2 inhibitor combined with ASP on FeCl3-induced arterial thrombosis. (A) Occlusive carotid artery thrombus images. (B) Effect of PHD2 inhibitors in combination with ASP on thrombus wet weight and (C) thrombus length. (D–G) Analysis of key coagulation parameters in plasma including PT, APTT, TT, and FIB. (H) Analysis of arterial thrombus histopathology by Masson staining. ###P < 0.001, vs. ctrl group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. mod group; ΔP < 0.05, ΔΔP < 0.01, ΔΔΔP < 0.001, vs. respective monotherapy group (n = 6).

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  Figure 10. The schematic diagram for antiplatelet adhesion by regulating the three-dimensional structure of collagen. (A) Targets for antiplatelet thrombosis with current research. Light red shading indicates clinically approval drug targets, dark red highlighting denotes newly emerging tactics, and blue representing the collagen target illuminated in this paper. (B) The mechanism diagram targets the PHD2 protein to modulate the three-dimensional structure of collagen for inhibiting platelet adhesion.