Dexmedetomidine Reduces Chronic Stress–Related Thrombosis in a Mouse FeCl 3 Model

Figures

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  FIGURE 1. Analysis of targets associated with dexmedetomidine (Dex) and thrombosis. (A) Venn diagram illustrating the targets related to Dex and thrombosis. It includes 166 Dex‐related targets (right), 572 thrombosis‐related targets (left), and 42 targets associated with both Dex and thrombosis (center). (B) OA anti‐thrombosis PPI network. A larger area indicates larger nodes; blue color indicates a higher association, while lighter colors indicate less association. The core target is represented in the inner circle. (C) KEGG pathway enrichment analysis (DAVID). The Y‐axis represents pathways, the X‐axis represents FDR, and p values are indicated by color changes. Bubble size reflects the number of genes enriched in each pathway.

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  FIGURE 2. Dexmedetomidine alleviates FeCl3‐induced thrombus formation in stressed mice. (A) Schematic representation of the chronic stress model protocol and the arterial thrombosis induction process. (B and C) Representative photos and quantitative analysis of thrombus area, weight, and length across four experimental groups (n = 10/group for weight and length; n = 6/group for area). (D) Representative hematoxylin and eosin (H&E) staining images of thrombus, showing corresponding histological features. (E) Representative images (immunofluorescence: Left panels; immunostaining: Right panels) and quantitative data showing the number of CD31+ endothelial cells in the thrombi of two experimental groups (n = 6/group). Data are expressed as mean ± SEM, with statistical differences analyzed using one‐way ANOVA and Tukey's post hoc test for panel C, and unpaired Student's t‐tests for panel D. Scale bars: 1 mm and 500 μm.

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  FIGURE 3. Dexmedetomidine reduces pathological protein expression and plasma hypercoagulability induced by chronic stress. Mice were intraperitoneally injected with NaCl or Dex and subjected to 4 h of daily chronic stress for 14 consecutive days. Carotid arteries were collected for protein analysis. Western blot analysis was performed on total proteins extracted from thrombosed carotid arteries. (A and B) Representative Western blots and quantitative data show the expression levels of gp91phox, SOD‐2, Bcl‐2, PAR‐2, PAI‐1, C‐cas‐3, cytochrome c, and p‐Akt across four experimental groups (n = 4/group). (C and D) Plasma concentrations of ADAMTS13 and vWF were assessed using ELISA after 14 days of chronic stress in four groups (n = 8/group). Data are expressed as mean ± SEM, with statistical significance determined by one‐way ANOVA and Tukey's post hoc test for panel B, and unpaired Student's t‐tests for panel C.

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  FIGURE 4. Dexmedetomidine reduces the expression of inflammation‐ and proteolysis‐related target genes in the carotid arteries of stressed mice. (A–F) QPCR analysis shows the levels of MMP‐2, TLR4, MCP‐1, VCAM‐1, ICAM‐1, and TNF‐α in four experimental groups (n = 5). Data are presented as mean ± SEM, and statistical significance was assessed using one‐way ANOVA and Tukey's post hoc test for panels A–F.

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  FIGURE 5. The α2‐adrenergic receptor antagonist yohimbine abrogates the protective effect of Dex on chronic stress‐induced endothelial damage and thrombus formation. Yohimbine was administered alongside Dex. After 14 days of stress, thrombosis was induced in the carotid artery using FeCl3 for 15 min, followed by experimental analysis. (A and B) Representative photos and quantitative data for the levels of wet thrombus length and weight in two experimental groups (n = 6). (C and D) Representative images (H&E staining, CD31+ immunofluorescence, and immunohistochemistry) and quantification of thrombus area and endothelial cell counts in the two experimental groups (n = 6). Data are presented as mean ± SEM and analyzed using unpaired Student's t‐tests. Scale bars: 1 mm, 500 μm, 100 μm.

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  FIGURE 6. Yohimbine abrogates Dex‐mediated beneficial alterations in targeted protein levels in FeCl3‐treated arteries after 14 days of the stress protocol. (A and B) Representative images and quantitative data show the levels of Bcl‐2, cytochrome c, PAI‐1, PAR‐2, and P‐Akt in the thrombotic arterial tissues of two experimental groups (n = 6). (C and D) Plasma levels of ADAMTS13 and vWF were measured using ELISA in two groups (n = 8). Data are expressed as mean ± SEM and analyzed using unpaired Student's t‐tests for panels B and C.

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  FIGURE 7. Yohimbine increases the expression of inflammation‐ and proteolysis‐related target genes in the carotid arteries of stressed mice treated with Dex. (A–F) QPCR analysis shows the levels of MMP‐2, TLR4, MCP‐1, VCAM‐1, ICAM‐1, and TNF‐α in four experimental groups (n = 5). Data are presented as mean ± SEM, and statistical significance was assessed using one‐way ANOVA and Tukey's post hoc test for panels A–F.

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  FIGURE 8. Dex treatment reduces stress serum–induced ROS production and apoptosis; these effects were diminished by yohimbine treatment. Following pretreatment with Dex, with or without yohimbine, for 30 min, the HUVECs were cultured in EBM‐2 containing 5% non‐stress serum (NS‐serum) and 5% stress serum (S‐serum), respectively, for 24 h. The cells were then subjected to evaluations of ROS production (immunofluorescence) and apoptosis (TUNEL) assays (n = 4 for each of the four groups). (A, B) Representative western images and quantitative data of ROS production and apoptosis in 4 experimental groups (n = 5). (C, D) Representative Western blot images and quantitative data for the levels of BCL‐2, PAR‐2, and cytochrome c in four groups (n = 4). Data are presented as mean ± SEM and were analyzed using one‐way ANOVA and Tukey's post hoc test for panels B and C. Scale bars: 100 μm.