Introduction
We have previously reported that a peroxisome proliferator-activated receptor (PPAR-Y) agonist protects against cerebral injury by anti-oxidant mechanisms 1 . The neuroprotective effect of a PPAR-Y agonist, pioglitazone induced SOD-1, suggesting that PPAR-Y activation was involved as a mechanism of the protection against cerebral injury. PPAR-Y agonists also have the ability to decrease reactive oxygen species generation. Here, we investigate the effects of pioglitazone on the expression of the superoxide generating enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in cerebral tissue.
Methods
Twelve male Sprague-Dawley rats were treated with pioglitazone (20 mg/kg per day, n=6) or vehicle (n=6) for 4 days at which time they, underwent 90-minutes of MCAO. The brain was removed 2 hours into reperfusion, and tissue was obtained from the cortex of the ischemic hemisphere and examined for CuZn-SOD (SOD 1) and 22-kDa, 47-kDa (NADPH subunits) and nNOS levels by Western blot. Briefly, small brain tissue samples (40 mg) were added to PBS buffer and homogenized. After centrifugation at 500 g for 3 minutes, the supernatant was removed. The amount of protein, which was loaded for Western blotting, was 15 μg. To correct for loading, we used the expression of GAPDH as the housekeeping protein. Antibodies against SOD 1 and 22-kDa, 47-kDa and nNOS were used for Western blotting. After protein blotting, membranes were incubated with horseradish peroxidase-conjugated anti-goat monoclonal antibody. Antigen detection was performed with a chemiluminescence detection system. Results were obtained by calculating a ratio of the SOD 1 protein levels to 22-kDa, 47-kDa and nNOS protein levels and reported as relative optical density. Significant differences between groups were determined with the Mann–Whitney test.
Results
The level of CuZn-SOD was increased in the cortex in treated animals (42.7 ± 22.2 vs. 12.5 ± 10.4; treated group vs. vehicle group, mean ± SD) (P<0.05). The ratio of the SOD 1 protein level to the 47-kDa subunit protein level of NADPH oxidase in the ischemic cortex 2 hours after transient MCAO was significantly increased by treatment with pioglitazone (0.74 ± 0.41 vs. 0.22 ± 0.15) (P<0.05) (figure 1.). Additionally, the ratio of the SOD 1 protein level to the nNOS in the ischemic cortex, also significantly increased by treatment with pioglitazone (2.87 ± 1.59 vs. 0.51 ± 0.44) (P<0.05).
SOD-1/p47kDa in ischemic cortex
Conclusions
These data, which show that a PPAR-Y agonist increased the ratio of the SOD 1 (as a free radical scavenger) protein levels to the 47-kDa subunit protein level of NADPH oxidase (as a production of free radical) suggest that the role of PPAR- is specific to events occurring during reperfusion. Our data point to free radical scavenging as the mediator of this neuroprotection.