Chemical structure of LEO.

The procedure of LEO antithrombotic experimental assay.

Effect of LEO on AH-induced thrombosis in zebrafish larvae. (A) Images of erythrocytes staining in the heart of zebrafish, red circle indicates the staining of erythrocytes in the heart. (B) The RBCs intensity (% of control). Data are represented as mean ± SD (n = 20). #p < 0.05, ##p < 0.01, ###p < 0.001 vs Control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs AH group.

Effect of LEO on AH-induced thrombosis in zebrafish larvae. (A) Images of venous thrombosis in the tail of zebrafish, red circle indicates the thrombus in the caudal vein. (B) The effect of LEO on thrombus area of zebrafish. (C) The vein thrombosis inhibition rate (% of control). Data are represented as mean ± SD (n = 20). #p < 0.05, ##p < 0.01, ###p < 0.001 vs Control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs AH group.

LEO inhibit oxidative damage. (A) Images of ROS in zebrafish, (B) ROS level (n = 15) (C) MDA level (n = 3) (D) LDH level (n = 3) (E) SOD level (n = 3) (F) GSH level (n = 3). Values are presented as means ± SD. #p < 0.05, ##p < 0.01, ###p < 0.001 vs Control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs AH group.

Effect of LEO on NO and ET-1 levels in zebrafish treated with AH. (A) NO level, (B) ET-1 level. Values are presented as means ± SD (n = 3). #p < 0.05, ##p < 0.01, ###p < 0.001 vs Control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs AH group.

Effect of LEO on the H&E staining of tail vein in zebrafish larval (×200 and ×400). The blue dots represent erythrocyte.

The related mRNA expression of platelet activity and coagulation cascade in control group, AH group and AH + LEO (2.5, 5, 10 μM) groups. (A) The mRNA expressions of pkcα. (B) The mRNA expressions of pkcβ. (C) The mRNA expressions of vwf. (D) The mRNA expressions of fga. (E) The mRNA expressions of fgb. (F) The mRNA expressions of fgg. (G) The mRNA expressions of f2. Data are expressed as mean ± SD (n = 3). #p < 0.05, ##p < 0.01, ###p < 0.001 vs Control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs AH group.

The expression of related protein in control group, AH group and AH + LEO (2.5, 5, 10 μM) groups. (A) The expression of PI3K and phospho-PI3K protein. (B) The expression of Akt and phospho-Akt protein. (C) The expression of ERK and phospho-ERK protein. (D) The expression of FIB protein. Data are represented by mean ± SD (n = 3). #p < 0.05, ##p < 0.01, ###p < 0.001 vs Control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs AH group.

Metabolomic analysis of zebrafish larvae samples from control, AH, and LEO groups. (A) Representative base peak chromatogram (BPC) of the control, AH, and LEO groups in the positive ion mode. (B) Representative base peak chromatogram (BPC) of the control, AH, and LEO groups in the negative ion mode. (C) PCA score plot of metabolites in the positive ion mode. (D) OPLS-DA score plot of metabolites in the positive ion mode.

The normalized intensity of differential metabolites in control, AH and LEO groups. (A) and (B) Relative intensities of the 23 important differential metabolites. (C) Venn diagram of primary metabolic differentials between different groups. a p < 0.01, b p < 0.001, vs control group; c p < 0.01, d p < 0.001, vs AH group.

Disturbed pathway in response to AH-induced thrombosis and LEO treatment. (A) Summary of pathway analysis with Metabo-Analyst. (B) Metabolic pathways (purple bold) participating in the anti-thrombosis process of LEO against AH-induced thrombosis in zebrafish. The metabolites (red) were the identified biomarkers in the present study. Compared with the control group, the up-regulation is represented by ↑, and the down-regulation is represented by ↓.

The potential mechanism linking dysregulated oxidative stress, platelets activation, and coagulation cascade with thrombosis.