Figure 3.

Inhibition of NOX4 expression led to a remarkable decline in the levels of oxidative stress in the central nervous system after cerebral hemorrhage. (A) Representative western blot images demonstrate NOX4 expression changes in tissues surrounding the haematoma before and after NOX4 siRNA treatment. GAPDH blot used for densitometry. (B) Quantitative western blot analysis showing a significant decrease in NOX4 expression around the haematoma after NOX4 siRNA treatment. NOX4 band densities were normalized to the densities of GAPDH. Values are expressed as mean ± SEM. **p < 0.01 vs. Sham. ^##p < 0.01 si-NC vs. si-NOX4, ICH vs. si-NOX4. n = 5 rats/group. (C) NOX4 mRNA expression level before and after NOX4 siRNA administration. Relative mRNA levels (NOX4/GAPDH) were quantified. Values are expressed as mean ± SEM. **p < 0.01 vs. Sham; ^##p < 0.01 si-NC vs. si-NOX4, ICH vs. si-NOX4. n = 5 rats/group. (D) Representative immunofluorescence co-localization images showing that NOX4 (green) was knocked down in neurons (NEUN, red), astrocytes (GFAP, red), vascular endothelial cells (CD34, red), and microglial (Iba-1, red), Scale bar = 50 μm, n = 5 rats/group. (E) Representative immunohistochemical images illustrate changes in NOX4 protein expression before and after NOX4 siRNA treatment. Scale bar = 100 μm. (F) Representative immunohistochemical images showing the intensity of 3-nitrotyrosine expression around the haemorrhagic foci. Scale bar = 100 μm. (G) ROS in brain sections was examined by dihydroethidium (DHE) staining using a confocal microscope. (H–K) The levels of H₂O₂ (H), MDA (I), GPx (J), and SOD (K) in brain homogenate were measured, respectively. Values are expressed as mean ± SEM. **p < 0.01, ***p < 0.001 vs. Sham; ^##p < 0.01, ^###p < 0.001 si-NC vs. si-NOX4, ICH vs. si-NOX4. n = 5 rats/group.