Targeting the ACE/Ang II Axis with Captopril Attenuates Neuroinflammation and Cerebral Injury Following Fat Embolism

Figures

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  Figure 1. Temporal progression of cerebral injury, edema, and oxidative stress following cerebral fat embolism (CFE). (A) Representative images of Oil Red O staining of brain tissues in the Sham and CFE 2 h groups (magnification: 20×, scale bar: 100 μm and magnification: 40×, scale bar: 50 μm). Red arrows indicate the presence of lipid droplets (fat emboli) and subsequent microvascular occlusion. (B) Brain water content, evaluated by the dry/wet weight ratio (%), at 1, 2, and 4 h post-CFE. (C) Cortical malondialdehyde (MDA) levels at different time points following CFE. (D) Representative images of Hematoxylin and Eosin (H&E) staining showing neuronal morphology between sham (Sham) and 2 h post-CFE (CFE 2 h). White arrows indicate damaged, pyknotic, or necrotic neurons. Scale bar: 100 μm. Data are expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the Sham group.

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  Figure 2. Captopril alleviates CFE-induced brain edema, oxidative stress, and structural damage. (A) Western blot analysis and quantitative densitometry of ACE protein expression in the cortex at 1, 2, and 4 h post-CFE. (B) Dose-dependent effects of captopril (1, 5, 10, and 20 mg/kg) on brain edema (dry/wet ratio) at 2 h post-CFE. (C) Effect of 5 mg/kg captopril treatment on cortical MDA levels at 2 h post-CFE. (D) Representative images of hematoxylin and eosin (H&E)-stained brain sections from the sham (Sham), 2 h post-CFE (CFE 2 h), and 2 h post-CFE treated with 5 mg captopril (CFE 2 h + 5 mg Captopril) groups. White arrows indicate damaged or pyknotic neurons. Scale bar: 100 μm. Data are expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Sham group; # p < 0.05, ## p < 0.05 vs. CFE 2 h group.

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  Figure 3. Captopril modulates the cerebral renin-angiotensin system (RAS) following CFE. (A) Western blot analysis and relative optical density of ACE protein expression in the Sham, CFE 2 h, and CFE 2 h + captopril (5 mg/kg) groups. (B) Concentration of the pro-inflammatory mediator Angiotensin II (nmol/mL) assessed by ELISA. (C) Concentration of the neuroprotective peptide Angiotensin 1–7 (nmol/mL). (D) Concentration of the IL–1β (nmol/mL). Data are expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Sham group; # p < 0.05, ## p < 0.01 vs. CFE 2 h group.

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  Figure 4. Captopril suppresses CFE-induced iNOS expression. Western blot analysis and quantitative densitometry of inducible nitric oxide synthase (iNOS) protein levels in the cerebral cortex of the Sham, CFE 2 h, and CFE 2 h + captopril (5 mg/kg) groups. β-actin was used as an internal loading control. Data are expressed as mean ± SD. *** p < 0.001 vs. Sham group; ## p < 0.01 vs. CFE 2 h group.

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  Figure 5. CFE-induced ACE overexpression predominantly colocalizes with astrocytes and is attenuated by captopril. (A) Representative immunofluorescence images of the cerebral cortex in the Sham, CFE 2 h, and CFE 2 h + captopril (5 mg/kg) groups. Brain sections were co-stained for ACE (green) and the astrocyte marker GFAP (red). The merged images (ACE+/GFAP+, yellow) demonstrate the colocalization of ACE within reactive astrocytes (indicated by white arrows). Zoom Scale bar = 200 μm. (B) Quantification of ACE+/GFAP+ double-positive cells across the experimental groups. (C) Pearson’s correlation coefficient analysis, indicating the high degree of colocalization between ACE and GFAP following CFE, which was significantly disrupted by captopril treatment. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01 compared to the Sham group; # p < 0.05, compared to the CFE 2 h group.

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  Figure 6. Western blot analyses and quantification of relative optical densities for (A) VEGF, (B) HIF-1α, (C) phosphorylated ERK (p–ERK) relative to total ERK, and (D) phosphorylated Raf (p–Raf) relative to total Raf in the cerebral cortex of the Sham, CFE 2 h, and CFE 2 h + captopril (5 mg/kg) groups. β-actin was used as an internal loading control for VEGF and HIF–1α, while total ERK and total Raf served as controls for their respective phosphorylated forms. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the Sham group; # p < 0.05, ## p < 0.01, ### p < 0.001 compared to the CFE 2 h group.