Assessing competence of broccoli consumption on inflammatory and antioxidant pathways in restraint-induced models: estimation in rat hippocampus and prefrontal cortex

Abstract

A growing body of evidence advocated the protective and therapeutic potential of natural compounds and phytochemicals used in diets against pathological conditions. Herein, the outcome of dietary whole broccoli consumption prior to restraint stress has been investigated in the hippocampus and prefrontal cortex of male rats, two important regions involved in the processing of responses to stressful events. Interestingly, a region-specific effect was detected regarding some of antioxidant defense system factors: nuclear factor erythroid-derived 2-related factor 2 (Nrf-2) antioxidant pathway, mitochondrial prosurvival proteins involved in mitochondrial biogenesis, and apoptotic cell death proteins. Dietary broccoli supplementation modulated the restraint-induced changes towards a consistent overall protection in the hippocampus. In the prefrontal cortex, however, despite activation of most of the protective factors, presumably as an attempt to save the system against the stress insult, some detrimental outcomes such as induced malate dehydrogenase (MDA) level and cleaved form of caspase-3 were detectable. Such diversity may be attributed in one hand to the different basic levels and/or availability of defensive mechanisms within the two studied cerebral regions, and on the other hand to the probable dose-dependent and hormetic effects of whole broccoli. More experiments are essential to demonstrate these assumptions.

Figure 1

Biochemical assessment of antioxidant defense system in the hippocampus (a) and prefrontal cortex (b) tissues derived after 6 hours of restraint-induced stress in experimental groups (n = 6), SOD activity, and GSH and MDA levels; bars indicate the mean ± SEM; one-way ANOVA; **P < 0.01, ***P < 0.01  compared with the control; ^# P < 0.05  ^## P < 0.01, ^### P < 0.001 compared with the stress.

Figure 2

Western blot analysis to measure the expression of major mitochondrial prosurvival proteins involved in the mitochondrial biogenesis, PGC-1α, NRF-1, and TFAM, in the hippocampus and prefrontal cortex tissues derived after 6 hours of restraint-induced stress in experimental groups. (a) Immunoblot bands of PGC-1α, NRF-1, and TFAM, as well as their relevant β-actin bands. ((b)–(d)) The densities of corresponding bands were measured and the ratios to β-actin were calculated and represented as arbitrary units on the graphs for each experimental group (n = 6). Bars indicate the mean ± SEM; one-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

Western blot analysis to measure the expression of the major proteins of the antioxidant Nrf-2 pathway, nuclear Nrf-2, HO-1, and NQO-1 in the hippocampus and prefrontal cortex tissues derived after 6 hours of restraint-induced stress in experimental groups. (a) Immunoblot bands of NQO-1, HO-1, and nuclear Nrf-2, as well as their relevant β-actin and lamin B2. ((b)–(d)) The densities of corresponding bands were measured and their respective ratios to β-actin and lamin B2 and were calculated and represented as arbitrary units on the graphs for each experimental group (n = 6). Bars indicate the mean ± SEM; one-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

Western blot analysis to measure the expression of inflammatory proteins, nuclear NF-κB p65, and TNF-α in the hippocampus and prefrontal cortex tissues derived after 6 hours of restraint-induced stress in experimental groups. (a) Immunoblot bands of nuclear NF-κB p65 and TNF-α, as well as their relevant lamin B2 and β-actin bands. ((b) and (c)) The densities of corresponding bands were measured and the ratios to lamin B2 and β-actin were calculated and represented as arbitrary units on the graphs for each experimental group (n = 6). Bars indicate the mean ± SEM; one-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5

Western blot analysis to measure the expression of the important proteins of the caspase-dependent and caspase-independent apoptotic cell death, cleaved caspase-3 and AIF, respectively, in the hippocampus and prefrontal cortex tissues derived after 6 hours of restraint-induced stress in experimental groups. (a) Immunoblot bands of cleaved caspase-3 and nuclear AIF, as well as their relevant β-actin and lamin B2 bands. Changes in the expression of 17 kDa fragment of cleaved caspase-3 are considered. ((b) and (c)) The densities of corresponding bands were measured and the ratios to β-actin and lamin B2 were calculated and represented as arbitrary units on the graph for each experimental group (n = 6). Bars indicate the mean ± SEM; one-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001.