Fig. 4: Oxidative stress induces cPLA₂ activation and blockade of cytosolic phospholipase A₂ (cPLA₂) with AACOCF3 reduced cardiolipin (CL) loss, mitochondrial dysfunction, and apoptosis after spinal cord injury (SCI).

A Time course of mitochondrial cPLA₂ expression and activation after SCI. Data represent mean ± s.e.m., *P < 0.05, **P < 0.01 vs sham (Two-way ANOVA, Tukey’s multiple comparisons test, n = 6 rats/group). B An electron microscopic image shows phosphorylated cPLA₂ (p-cPLA₂) immunopositive peroxidase reaction products in a neuronal dendrite in the rat spinal ventral horn. p-cPLA₂ immunoreactivity (IR) was observed to localize in mitochondria (*) including both the outer (yellow arrow) and inner (red arrows) mitochondrial membranes, and on the cell membrane (green arrow) at 3 days after SCI. Bar = 0.25 μm. C cPLA₂ activation-induced CL loss was reversed by AACOCF₃, a cPLA₂ inhibitor. D SCI induced a decrease in the ratio of red/green in the MMP (Δψm) assay, which was reversed by AACOCF_3. (E–H) AACOCF₃, a cPLA₂ inhibitor, reversed SCI-induced cytochrome c release (E), Smac/DIABLO release (F), active caspase-3 expression (G), and cleaved PAPRP expression (H). Data represent mean ± s.e.m. *P < 0.05, **P < 0.01 (One-way ANOVA, Tukey’s multiple comparisons test, n = 6 rats/group). Cyt. c cytochrome c, Smac/D. Smac/DIABLO, A. casp-3 active caspase-3. I–M Oxidative stress induces cPLA₂ activation after SCI. I Immunohistochemistry shows a marked increase in p-cPLA₂-expression, a marker of cPLA₂ activation, at the injury epicenter and 3 mm rostral to it after a single injection of H₂O₂ into the T9 spinal cord. Right column images show that H₂O₂-induced p-cPLA₂ expression was substantially reversed by mepacrine (5 mg/kg), a PLA₂ inhibitor. The expression of p-cPLA₂ was found in neurons (arrows), swelling axons (open arrows), and glial cells (arrowheads). Bar, 100 μm. I–L Administration of XJB (10 mg/kg, i.p.) at 30 min post-injury significantly reduced SCI-induced mitochondrial cPLA₂ activation. J Representative images of phosphorylated cPLA₂ (p-cPLA₂), cPLA₂, and VDAC expression. K Compiled results in a bar graph for the ratio of p-cPLA₂/cPLA₂ expression. L Compiled results in a bar graph for the ratio of p-cPLA₂/VDAC expression. M Compiled results in a bar graph for the ratio of cPLA₂/VDAC expression. *P < 0.05, **P < 0.01 (One-way ANOVA, Tukey’s multiple comparisons test, n = 6). Data represent the mean ± s.e.m.