Exploring the Distinct Regulatory Actions and Molecular Pathways of Coptisine Through Network Pharmacology: Insights Into Its Influence on Platelet Activation and Thrombus Development

Figures

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  FIGURE 1. Network analysis of predicted targets of Coptisine in thrombosis. (A, B) Venn diagrams showing 90 targets, which are the intersection of the predicted targets of Coptisine and thrombosis‐related targets; (C) PPI network of the 90 common targets.

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  FIGURE 2. Enrichment analysis results of core targets of Coptisine against thrombosis. (A) Top 10 GO enrichment entries for the core targets of Coptisine against thrombosis (BP, biological process; CC, cellular component; MF, molecular function), (B) Top 20 KEGG pathway enrichment entries for the core targets of Coptisine against thrombosis, (C) Interaction network diagram of Coptisine‐thrombosis‐targets, (D) Protein–protein interaction network of the top five core targets, (E) KEGG pathway enrichment analysis of the five core targets (top 10 significant pathways), (F) Top 10 GO analysis of the 5 hub targets.

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  FIGURE 3. Molecular docking results and 2D docking diagrams of Coptisine (A, B) Docking results for NFKB1, (C, D) PTGS2, (E, F) STAT1, and (G, H) ACE.

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  FIGURE 4. Coptisine inhibits agonist‐induced platelet activation in vitro. (A) Chemical structure of Coptisine. (B) Effects of Coptisine on platelet viability. n = 5, ns = p > 0.05. (C, D, E) Effects of Coptisine on platelet aggregation induced by ADP, thrombin, or collagen. Data are expressed as mean ± SD. n = 4. *p < 0.05, **p < 0.01, ***p < 0.001 ns = p > 0.05. (F, G) Flow cytometric analysis of PE‐JON/A and FITC‐CD62P binding to platelets after stimulation with thrombin for 5 min at 37°C. Data are expressed as mean ± SD. n = 5. ###p < 0.001 versus the control, *p < 0.05, **p < 0.01, ***p < 0.001 versus the model group. ns = p > 0.05. (H) Effects of Coptisine on platelet ATP secretion. Data are expressed as mean ± SD. n = 5. ###p < 0.001 versus the control, *p < 0.05, **p < 0.01, ***p < 0.001 versus the model group. ns = p > 0.05.

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  FIGURE 5. Coptisine inhibits agonist‐induced platelet activation and adhesion (A) Scanning electron microscopy was used to observe the effects of Coptisine on platelet morphology, scale bar represents 5 μm, n = 3. (B) Effects of Coptisine on thrombin‐induced platelet adhesion; scale bar represents 10 μm. (C) Platelet numbers were measured using ImageJ software. Data are expressed as mean ± standard deviation. n = 4. ###p < 0.001, versus the control group, *p < 0.01, **p < 0.01, ***p < 0.001 versus the model group.

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  FIGURE 6. Effects of Coptisine on thrombin‐induced NF‐κB and MAPK signaling activation. (A) Platelets were pretreated with Coptisine at room temperature for 30 min, followed by stimulation with thrombin (0.025 U/mL). Representative Western blot images for phosphorylated P50 and total P50 (C) Representative Western blot images for phosphorylated P38 and total P38 (B, D). Histogram analysis of the immunoblots. Data are expressed as mean ± standard deviation. n = 4. ###p < 0.001 versus the control group, *p < 0.05, **p < 0.01, ***p < 0.001 versus the model group. (E, F, G) Levels of IL‐6, TNF‐α, and IL‐1β in mouse platelets were detected by enzyme‐linked immunosorbent assay (ELISA). All data are expressed as mean ± SD, n = 5, ###p < 0.001 versus the control group, ns = p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 versus the model group.

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  FIGURE 7. Coptisine inhibits thrombus formation but has no significant impact on the coagulation system. (A) In vivo experimental design. (B) Mice were orally administered coptisine (30 mg/kg) or vehicle control. The distal 2 mm of the tail was surgically excised and immediately immersed in pre‐warmed 0.9% NaCl solution, and the bleeding cessation time was recorded. n = 7, ns = p > 0.05. (C) H&E staining images of carotid artery thrombi (n = 3). (D–G) Effects of coptisine treatment on coagulation function indicators in rats: Prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and fibrinogen (FIB) levels. Results are expressed as mean ± SD. n = 5, ns = p > 0.05. Scale bar represents 100 μm.