Allicin, an Emerging Treatment for Pulmonary Arterial Hypertension: An Experimental Study

Figures

• 

  Figure 1. Validation of the establishment of PAH. (a) Survival percentage, (b) RV hypertrophy, (c) percentage of thickening of pulmonary arterial walls, and (d) representative images of the pulmonary arteries of the control (CON), with saline solution as the vehicle; PAH group (PAH) induced by 60 mg/kg/s.c. of MCT; and PAH with 16 mg/kg of allicin (PAH + A). Data are presented as mean ± standard error. Differences were tested by ordinary one-way ANOVA followed by the Bonferroni post hoc test for multiple comparisons, and significant differences between groups were set at p ≤ 0.05 n = 8, * p ≤ 0.05 vs. control; Δ p ≤ 0.05 vs. PAH.

• 

  Figure 2. Representative micrographs of immunohistochemistry detection of the effects of allicin on the alpha-smooth muscle actin (α-SMA) in lung tissue. α-SMA detection clearly shows the hyperplastic muscular layer of the PAH group. Immunoblotting assay of control group (CON), with saline solution as the vehicle; PAH group (PAH) induced by 60 mg/kg/s.c; and PAH with 16 mg/kg of allicin (PAH + A). All micrographs 40×.

• 

  Figure 3. Allicin effects on angiotensin concentration at the systemic and local level. Ang-II concentration in (a) lung tissue and (c) serum. Ang-(1-7) concentration in (b) lung tissue and (d) serum. Control group (CON), with saline solution as the vehicle; PAH group (PAH) induced by 60 mg/kg/s.c. of MCT; and PAH with 16 mg/kg of allicin (PAH + A). Data are presented as mean ± standard error. Differences were tested by ordinary one-way ANOVA followed by the Bonferroni post hoc test for multiple comparisons, and significant differences between groups were set at p ≤ 0.05. n = 8, * p < 0.05 vs. control; Δ p < 0.05 vs. PAH.

• 

  Figure 4. Effects of allicin on the protein expressions of ACE2, AT1R, and AT2R in lung tissue. Immunoblotting assay of (a) ACE2 and (b) representative micrographs of immunohistochemistry detection, and (c) protein expression of AT1R and (d) AT2R in lung tissue. Control group (CON), with saline solution as the vehicle; PAH group (PAH) induced by 60 mg/kg/s.c; and PAH with 16 mg/kg of allicin (PAH + A). For Western blotting, three randomly selected samples per group were analyzed. All micrographs 40×. Data are presented as mean ± standard error and were analyzed using the Kruskal–Wallis test. Significant differences between groups were set at p ≤ 0.05. n = 3, * p < 0.05 vs. CON; Δ p < 0.05 vs. PAH.

• 

  Figure 5. Allicin effects on endothelial function markers in lung and serum in PAH. Concentration of (a) NO, (b) BH4, and (c) BH2 in lung tissue. Concentrations of (d) NO, (e) BH4, and (f) BH2 in serum. Control group (CON), with saline solution as the vehicle; PAH group (PAH) induced by 60 mg/kg/s.c; and PAH with 16 mg/kg of allicin (PAH+A). Data are presented as mean ± standard error. Differences were tested by ordinary one-way ANOVA followed by the Bonferroni post hoc test for multiple comparisons, and significant differences between groups were set at p ≤ 0.05. n = 8, * p < 0.05 vs. control; Δ p < 0.05 vs. PAH.

• 

  Figure 6. Allicin effects on PDE5 and cGMP. (a) PDE5 protein expression in lung tissue and (b) representative micrographs of immunohistochemistry detection. Concentrations of cGMP in (c) lung and (d) serum. Control group (CON), with saline solution as the vehicle; PAH group (PAH) induced by 60 mg/kg/s.c; and PAH with 16 mg/kg of allicin (PAH + A). For Western blotting, three randomly selected samples per group were analyzed. All micrographs 40x. Data are presented as mean ± standard error and were analyzed using the Kruskal–Wallis test. Significant differences between groups were set at p ≤ 0.05. n = 3, * p < 0.05 vs. CON; Δ p < 0.05 vs. PAH. Data of GMP are presented as mean ± standard error. Differences were tested by ordinary one-way ANOVA followed by the Bonferroni post hoc test for multiple comparisons, and significant differences between groups were set at p ≤ 0.05. n = 8, * p < 0.05 vs. CON; Δ p < 0.05 vs. PAH.

• 

  Figure 7. Effects of allicin in total antioxidant capacity (TAC) and malondialdehyde (MDA) as oxidative stress markers. The concentration of (a) TAC and (b) MDA) in the lung. The concentration of (c) TAC and (d) MDA in serum. Control group (CON), with saline solution as the vehicle; PAH group (PAH) induced by 60 mg/kg/s.c; and PAH with 16 mg/kg of allicin (PAH + A). Data are presented as mean ± standard error. Differences were tested by ordinary one-way ANOVA followed by the Bonferroni post hoc test for multiple comparisons, and significant differences between groups were set at p ≤ 0.05. n = 8, * p < 0.05 vs. control; Δ p < 0.05 vs. PAH.

• 

  Figure 8. Effects of allicin on Nrf2 and Keap1 proteins on PAH. (a) Nrf2 by Western blot, and (b) representative micrograph of immunohistochemistry, where Nrf-2 is strongest in the arterial endothelium and muscle cells of PAH + A compared with the other groups. (c) Keap1 protein expression in lung tissue. Control group (CON), with saline solution as the vehicle; PAH group (PAH) induced by 60 mg/kg/s.c; and PAH with 16 mg/kg of allicin (PAH + A). For Western blotting, three randomly selected samples per group were analyzed. All micrographs 40×. Data are presented as mean ± standard error and were analyzed using the Kruskal–Wallis test. Significant differences between groups were set at p ≤ 0.05. n = 3, * p < 0.05 vs. control; Δ p < 0.05 vs. PAH.

• 

  Figure 9. Allicin effects on markers of hypoxia in PAH. Protein expression of (a) hypoxia-inducible factor 1-alpha (HIF-1α) by Western blot, (b) representative micrograph of immunohistochemistry, where nuclear staining of HIF 1α is shown (black arrows) in PAH and was not observed in PAH + A group, and (c) protein expression of vascular endothelial growth factor (VEGF). Control group (CON), with saline solution as the vehicle; PAH group (PAH) induced by 60 mg/kg/i.p; and PAH with 16 mg/kg of allicin (PAH + A). For Western blotting, three randomly selected samples per group were analyzed. All micrographs 40×, except HIF-1 in PAH that is 100×. Data are presented as mean ± standard error and were analyzed using the Kruskal–Wallis test. Significant differences between groups were set at p ≤ 0.05. n = 3, * p < 0.05 vs. control; Δ p < 0.05 vs. PAH.

• 

  Figure 10. Experimental design.