Probucol Increases Striatal Glutathione Peroxidase Activity and Protects against 3-Nitropropionic Acid-Induced Pro-Oxidative Damage in Rats

<div><p>Huntington’s disease (HD) is an autosomal dominantly inherited neurodegenerative disease characterized by symptoms attributable to the death of striatal and cortical neurons. The molecular mechanisms mediating neuronal death in HD involve oxidative stress and mitochondrial dysfunction. Administration of 3-nitropropionic acid (3-NP), an irreversible inhibitor of the mitochondrial enzyme succinate dehydrogenase, in rodents has been proposed as a useful experimental model of HD. This study evaluated the effects of probucol, a lipid-lowering agent with anti-inflammatory and antioxidant properties, on the biochemical parameters related to oxidative stress, as well as on the behavioral parameters related to motor function in an <i>in vivo</i> HD model based on 3-NP intoxication in rats. Animals were treated with 3.5 mg/kg of probucol in drinking water daily for 2 months and, subsequently, received 3-NP (25 mg/kg i.p.) once a day for 6 days. At the end of the treatments, 3-NP-treated animals showed a significant decrease in body weight, which corresponded with impairment on motor ability, inhibition of mitochondrial complex II activity and oxidative stress in the striatum. Probucol, which did not rescue complex II inhibition, protected against behavioral and striatal biochemical changes induced by 3-NP, attenuating 3-NP-induced motor impairments and striatal oxidative stress. Importantly, probucol was able to increase activity of glutathione peroxidase (GPx), an enzyme important in mediating the detoxification of peroxides in the central nervous system. The major finding of this study was that probucol protected against 3-NP-induced behavioral and striatal biochemical changes without affecting 3-NP-induced mitochondrial complex II inhibition, indicating that long-term probucol treatment resulted in an increased resistance against neurotoxic events (i.e., increased oxidative damage) secondary to mitochondrial dysfunction. These data appeared to be of great relevance when extrapolated to human neurodegenerative processes involving mitochondrial dysfunction and indicates that GPx is an important molecular target involved in the beneficial effects of probucol.</p> </div

3-NP treatment inhibits complex II activity.

<p>Treatments were conducted as previously mentioned (see Methods Section). Complex II activity in striatum is expressed as nmol.min⁻¹.mg protein⁻¹ and presented as the mean ± S.E.M. (n = 6 rats/group). ** p < 0.01 and *** p < 0.001 compared with the control group using two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.</p

Probucol attenuates motor impairment induced by 3-NP in rats.

<p>Treatments were conducted as previously mentioned (see Methods Section). Locomotor (A) and exploratory (B) activities in the open field test as well as the latency for the first fall (C) and the number of falls in the rotarod (D) were evaluated 24 h after the last 3-NP administration. These results are expressed as the total number of crossings (A), total number of rearings (B), the latency for the first fall(s) (C) and the total number of falls. The data are presented as the mean ± S.E.M. (n =10 rats/group). *p < 0.05 and *** p < 0.001 compared with the control group and # p < 0.05, # # p < 0.01 and # # # p < 0.001 compared with the 3-NP group using two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.</p

Probucol reduces 3-NP-induced lipid peroxidation in rats.

<p>Treatments were conducted as previously mentioned (see Methods Section). Striatal thiobarbituric acid reactive substance (TBARS) levels are expressed as nmol of MDA/mg protein. The data are presented as the mean ± S.E.M. (n = 6 rats/group). *** p< 0.001 compared with the control group and # # p < 0.01 and # # # p < 0.001 compared with the 3-NP group using two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.</p

Figure 7

<p><b>Effects of 3-NP and/or probucol on iNOS, GFAP</b> and <b>caspase 3/cleaved caspase 3 expression.</b></p

Effects of 3-NP and/or probucol on striatal GSH levels, glutathione peroxidase and glutathione reductase activities.

<p>Treatments were conducted as previously mentioned (see Methods Section). The GSH levels (A) are expressed as µmol GSH·mg protein^-1. GR activity (B) and GPx activity (C) are expressed as the nmol of NADPH oxidized/min/mg protein. The data are presented as the mean ± S.E.M. (n= 6 rats/group). †† p < 0.01 and † † † p < 0.001 main effect of probucol, ** p < 0.01 and *** p < 0.001 compared with the control group and # p < 0.05 compared with the 3-NP group using two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.</p

Probucol attenuates the increase in superoxide dismutase (SOD) and catalase activities in the rat striatum.

<p>Treatments were conducted as previously mentioned (see Methods Section). SOD activity (A) is expressed as SOD units/mg of protein. Catalase activity (B) is expressed as µmol of H₂O₂/min/mg protein. The data are presented as the mean ± S.E.M. (n = 6 rats/group). * p < 0.05 and ** p < 0.01 compared with the control group, and # p < 0.05 and # # p < 0.01 compared with the 3-NP group using two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.</p

Probucol prevents 3-NP-induced decreases in body weight.

<p>The animals were pretreated with probucol (3.5 mg/kg/day) or vehicle (1% of DMSO) in drinking water daily for 2 months and administered intraperitoneally with 3-NP (25 mg/kg) or vehicle, once a day for 6 consecutive days. The body weight values are expressed as the percentage of change in body weight after 3-NP or vehicle administration and presented as the mean ± S.E.M. (n = 10 rats/group). ***p < 0.001 compared with the control group and ## p < 0.01 and ### p < 0.001 compared with the 3-NP group using two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.</p