N-3 PUFA Prevent Oxidative Stress in a Rat Model of Beta-Amyloid-Induced Toxicity

Abstract

Polyunsaturated fatty acids (PUFA) are involved in brain disorders associated to amyloid beta (Aβ) toxicity for which oxidative stress, neurochemical dysfunctions, and neuroinflammation are underlying mechanisms. Here, mechanisms through which lifelong exposure to n-3 PUFA-enriched or n-6/n-3 balanced diets could elicit a protective role in a rat model of Aβ-induced toxicity were investigated. To this aim, we quantified hippocampal reactive oxygen species (ROS) amount, 8-hydroxy-2'-deoxyguanosine and interleukin-10 levels, NADPH oxidase (NOX) 1, NOX2, superoxide dismutase 1, and glutathione contents, as well as plasmatic malondialdehyde. Moreover, in the same experimental groups, we assessed tryptophan, serotonin, and its turnover, kynurenine, and noradrenaline amounts. Results showed increased hippocampal ROS and NOX2 levels, serotonin turnover, kynurenine, and noradrenaline contents in Aβ-treated rats. Both n-6/n-3 balanced and n-3 PUFA enriched diets reduced ROS production, NOX1 and malondialdehyde levels, serotonin turnover, and kynurenine amount in Aβ-injected rats, while increasing NOX2, superoxide dismutase 1, and serotonin contents. No differences in plasmatic coenzyme Q10, reduced glutathione (GSH) and tryptophan levels were detected among different experimental groups, whereas GSH + oxidized glutathione (GSSG) levels were increased in sham animals fed with n-3 PUFA enriched diet and in Aβ-treated rats exposed to both n-6/n-3 balanced and n-3 enriched diets. In addition, Aβ-induced decrease of interleukin-10 levels was prevented by n-6/n-3 PUFA balanced diet. N-3 PUFA enriched diet further increased interleukin-10 and 8-hydroxy-2'-deoxyguanosine levels. In conclusion, our data highlight the possible neuroprotective role of n-3 PUFA in perturbation of oxidative equilibrium induced by Aβ-administration.

Keywords: beta-amyloid; inflammation; n-3 PUFA; oxidative stress.

Figure 1

Effects of standard, n-6/n-3 PUFA balanced and n-3 PUFA enriched diets on hippocampal ROS production, 8-OHdG and IL-10 levels, plasmatic MDA, and CoQ10 content in Aβ-treated rats. Measure of hippocampal ROS production (a, SHAM standard n = 3, Aβ standard n = 4, SHAM n-6/n-3 n = 4, Aβ n-6/n-3 n = 4, SHAM n-3 n = 4, Aβ n-3 n = 4 ), 8-OHdG (b, SHAM standard n = 4, Aβ standard n = 4, SHAM n-6/n-3 n = 4, Aβ n-6/n-3 n = 4, SHAM n-3 n = 4, Aβ n-3 n = 4), IL-10 (c, SHAM standard n = 4, Aβ standard n = 4, SHAM n-6/n-3 n = 4, Aβ n-6/n-3 n = 4, SHAM n-3 n = 3, Aβ n-3 n = 4 ), plasmatic MDA levels (d, SHAM standard n = 4, Aβ standard n = 4, SHAM n-6/n-3 n = 3, Aβ n-6/n-3 n = 4, SHAM n-3 n = 5, Aβ n-3 n = 4 ) in rats 7 days after Aβ icv injection (Aβ, 4 µM, black bar) or vehicle (SHAM, 5 µL vehicle, white bar) and plasmatic CoQ10 levels (e, SHAM standard n = 4, Aβ standard n = 4, SHAM n-6/n-3 n = 4, Aβ n-6/n-3 n = 4, SHAM n-3 n = 4, Aβ n-3 n = 4). Two-way ANOVA followed by Bonferroni multiple comparison test, ^§ p < 0.05, ^§§ p < 0.01, ^§§§ p < 0.001 Aβ-treated rats fed, either, with n-6/n-3 balanced and n-3 PUFA enriched diets versus SHAM rats; * p < 0.05, *** p < 0.001 SHAM rats fed with n-6/n-3 balanced and n-3 PUFA enriched diets versus SHAM standard diet; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 Aβ-treated rats fed with n-6/n-3 balanced and n-3 PUFA enriched diets versus Aβ, standard diet. Data were expressed as means ± mean standard error (SEM).

Figure 2

Effects of standard, n-6/n-3 PUFA balanced and n-3 PUFA enriched diets on NADPH oxidases (NOXs) expression in Aβ-treated rats. Effect of standard, n-6/n-3 PUFA balanced and n-3 PUFA enriched diets on hippocampal NOX1 (a, SHAM standard n = 5, Aβ standard n = 8, SHAM n-6/n-3 n= 4, Aβ n-6/n-3 n = 3, SHAM n-3 n = 3, Aβ n-3 n = 3) and NOX2 (b, SHAM standard n = 5, Aβ standard n = 8, SHAM n-6/n-3 n = 4, Aβ n-6/n-3 n = 4, SHAM n-3 n = 4, Aβ n-3 n = 4) expression in rats 7 days after Aβ icv injection (Aβ, 4 µM, black bar) or vehicle (SHAM, 5 µL, white bar). Two-way ANOVA followed by Bonferroni multiple comparison test, ^§ p < 0.05 Aβ-treated rats fed with standard diet versus SHAM rats; *** p < 0.001 SHAM rats fed with n-6/n-3 balanced and n-3 PUFA enriched diets versus SHAM standard diet; ^### p < 0.001, Aβ-treated rats fed with n-6/n-3 balanced and n-3 PUFA enriched diets versus Aβ standard diet. Data were expressed as means ± mean standard error (SEM).

Figure 3

Effects of standard, n-6/n-3 PUFA balanced and n-3 PUFA enriched diets on SOD1, GSH and GSH+GSSG levels in Aβ-treated rats. Representative image of Western blotting (a) and quantification of the optical density (b, SHAM standard n = 3, Aβ standard n = 4, SHAM n-6/n-3 n = 3, Aβ n-6/n-3 n = 4, SHAM n-3 n = 4, Aβ n-3 n = 4) of SOD1 band normalized to the actin protein value in the hippocampus of rats 7 days after icv injection of Aβ (Aβ, 4 µM, black bar) or vehicle (SHAM, 5 µL, white bar) receiving, from conception until 8-weeks-old, standard, n-6/n-3 balanced or n-3 PUFA enriched diets. GSH (c, SHAM standard n = 3, Aβ standard n = 4, SHAM n-6/n-3 n = 4, Aβ n-6/n-3 n = 4, SHAM n-3 n = 4, Aβ n-3 n = 4) levels in rats 7 days after icv injection of Aβ (Aβ, 4µM, black bar) or vehicle (SHAM, 5 µL, white bar) receiving from conception until 8-weeks-old standard, n-6/n-3 balanced or n-3 PUFA enriched diets. GSH + GSSG (d, SHAM standard n = 3, Aβ standard n = 4, SHAM n-6/n-3 n = 4, Aβ n-6/n-3 n = 4, SHAM n-3 n = 4, Aβ n-3 n = 4) levels in rats 7 days after icv injection of Aβ (Aβ, 4µM, black bar) or vehicle (SHAM, 5µL, white bar) receiving from conception until 8-weeks-old standard, n-6/n-3 balanced or n-3 PUFA enriched diets. Two-way ANOVA followed by Bonferroni multiple comparison test, ^§ p < 0.05, ^§§ p < 0.01, ^§§§ p < 0.001 Aβ-treated rats fed, either, with n-6/n-3 balanced and n-3 PUFA enriched diets versus SHAM rats * p < 0.05, *** p < 0.001 SHAM rats fed with n-6/n-3 balanced and n-3 PUFA enriched diets versus SHAM standard diet. Data were expressed as means ± mean standard error (SEM).

Figure 4

Effects of standard, n-6/n-3 PUFA balanced and n-3 PUFA enriched diets on TRP, 5-HT and 5-HT turnover levels in Aβ-treated rats. Measure of hippocampal TRP (a, SHAM standard n = 5, Aβ standard n = 5, SHAM n-6/n-3 n = 5, Aβ n-6/n-3 n = 4, SHAM n-3 n = 6, Aβ n-3 n = 4), 5-HT (b, SHAM standard n = 4, Aβ standard n = 4, >SHAM n-6/n-3 n = 6, Aβ n-6/n-3 n = 6, SHAM n-3 n = 6, Aβ n-3 n = 6) and 5-HT turnover (c, SHAM standard n = 3, Aβ standard n = 3, SHAM n-6/n-3 n = 5, Aβ n-6/n-3 n = 6, SHAM n-3 n = 6, Aβ n-3 n = 6) contents in rats 7 days after Aβ icv injection (Aβ, 4 µM, black bar) or vehicle (SHAM, 5µL, white bar) receiving, from conception until 8-weeks-old, standard, n-6/n-3 balanced or n-3 PUFA enriched diets. Two-way ANOVA followed by Bonferroni multiple comparison test, ^§§§ p < 0.001 Aβ-treated rats fed, either, with n-6/n-3 balanced and n-3 PUFA enriched diets versus SHAM> rats; * p < 0.05, **p < 0.01, *** p < 0.001 SHAM rats fed with n-6/n-3 balanced and n-3 PUFA enriched diets versus SHAM standard diet; ^# p < 0.05, ^### p < 0.01 Aβ-treated rats fed with n-6/n-3 balanced and n-3 PUFA enriched diets versus Aβ standard diet. Data were expressed as means ± mean standard error (SEM).

Figure 5

Effects of standard, n-6/n-3 PUFA balanced and n-3 PUFA enriched diets on KYN and NA levels in Aβ-treated rats. Effect of standard, n-6/n-3 PUFA balanced and n-3 PUFA enriched diets on KYN (a, SHAM standard n = 3, Aβ standard n = 4, SHAM n-6/n-3 n = 5, Aβ n-6/n-3 n = 3, SHAM n-3 n = 5, Aβ n-3 n = 3) and NA (b, SHAM standard n = 4, Aβ standard n = 4, SHAM n-6/n-3 n = 6, Aβ n-6/n-3 n = 6, SHAM n-3 n = 4, Aβ n-3 n = 6) content in the hippocampus of rats 7 days after Aβ icv injection (Aβ, 4 µM, black bar) or vehicle (sham, 5µL, white bar). Two-way ANOVA followed by Bonferroni multiple comparison test, ^§§ p < 0.01, ^§§§ p < 0.001 Aβ-treated rats fed, either, with n-6/n-3 balanced and n-3 PUFA enriched diets versus SHAM rats; ** p < 0.01, *** p < 0.001 SHAM rats fed with n-6/n-3 balanced and n-3 PUFA enriched diets versus SHAM standard diet; ^### p < 0.01 Aβ-treated rats fed with n-6/n-3 balanced and n-3 PUFA enriched diets versus Aβ standard diet. Data were expressed as means ± mean standard error (SEM).