3,3'-Diindolylmethane Promotes BDNF and Antioxidant Enzyme Formation via TrkB/Akt Pathway Activation for Neuroprotection against Oxidative Stress-Induced Apoptosis in Hippocampal Neuronal Cells

Abstract

3,3'-Diindolylmethane (DIM), a metabolite of indole-3-carbinol present in Brassicaceae vegetables, possesses various health-promoting effects. Nonetheless, the effect of DIM on neurodegenerative diseases has not been elucidated clearly. In this study, we hypothesized DIM may protect neuronal cells against oxidative stress-induced apoptosis by promoting the formation of brain-derived neurotrophic factor (BDNF) and antioxidant enzymes through stabilizing the activation of the tropomyosin-related kinase receptor B (TrkB) cascade and we investigated the effect of DIM on oxidative stress-mediated neurodegenerative models. DIM protected neuronal cells against oxidative stress-induced apoptosis by regulating the expression of apoptosis-related proteins in glutamate-treated HT-22 cells. Additionally, DIM improved the expression of BDNF and antioxidant enzymes, such as heme oxygenase-1, glutamate-cysteine ligase catalytic subunit, and NAD(P)H quinine oxidoreductase-1, by promoting the activation of the TrkB/protein kinase B (Akt) pathway in the cells. Consistent with in vitro studies, DIM attenuated memory impairment by protecting hippocampal neuronal cells against oxidative damage in scopolamine-treated mice. Conclusionally, DIM exerted neuroprotective and antioxidant actions through the activation of both BDNF production and antioxidant enzyme formation in accordance with the TrkB/Akt pathway in neuronal cells. Such an effect of DIM may provide information for the application of DIM in the prevention of and therapy for neurodegenerative diseases.

Keywords: 3,3’-diindolylmethane; antioxidant enzymes; brain-derived neurotrophic factor; hippocampal neuronal cells; neurodegenerative disease.

Figure 1

Effect of 3,3′-diindolylmethane (DIM) on glutamate-induced cytotoxicity, and reduction of ROS and glutathione levels in HT-22 cells. HT-22 cells, seeded on a 96 well-plates and incubated for 24 h, were incubated with or without DIM (0–80 μM) for 30 min before glutamate challenge (5 mM). After 12 h, cell viability, ROS level, and GSH level were measured as described in Materials and Methods. (A) Cell viability, (B) ROS level, and (C) GSH level. Data are the mean ± SD values of triple determinations. ** p < 0.01 versus glutamate-treated group. – is absence, + is presence.

Figure 2

Suppressive effect of 3,3′-diindolylmethane on glutamate-induced apoptosis in HT-22 cells. HT-22 cells were seeded on a 60 mm dish, and then incubated for 24 h. The cells were challenged with glutamate after preincubation with or without DIM (0–40 μM) for 30 min. After 12 h, the expression of Bcl-2, Bax, cytochrome c, cleaved caspase-3, AIF, or β-actin was examined as described in Materials and Methods. The data were contained from three independent experiments. ** p < 0.01 versus glutamate-treated group. – is absence, + is presence.

Figure 3

Effect of 3,3′-diindolylmethane on phosphorylation of TrkB, Akt, or CREB, and expression of BDNF, Nrf2, or antioxidant enzymes. The experiments were performed as described in the Figure 2 legend. The data were obtained from three independent experiments. (A) p-TrkB, p-Akt, p-CREB, and BDNF and (B) nuclear and cytosolic Nrf2, HO-1, GCLC, and NQO-1. ** p < 0.01 versus glutamate-treated group. − is absence, + is presence.

Figure 4

Inhibitory effect of K252a or MK-2206 on neuroprotective action of DIM. HT-22 cells were preincubated with or without DIM in combination with K252a or MK-2206 for 30 min before glutamate challenge. After 12 h, cell viability and ROS level were measured as described in Materials and Methods. (A), (B) Cell viability and (C), (D) ROS level. Data are the mean ± SD values of quintuple determinations. ** p < 0.01 versus glutamate-treated group; ^# p < 0.05 and ^## p < 0.01 versus DIM with glutamate-treated group.

Figure 5

Inhibitory effect of 3,3′-diindolylmethane on memory impairment induced by scopolamine in mice. Mice were orally administrated with DIM (10 or 20 mg/kg) before scopolamine treatment (2 mg/kg, i.p.). After 1 h, the mice were tested for Morris water maze or passive avoidance (Acquisition trial). (A) Escape latency time, (B) number of platform area crossings, and (C) latency time. Data are the mean ± SEM values of sextuple determinations. *P< 0.05 and **P< 0.01 versus scopolamine-treated group.

Figure 6

Effect of 3,3′-diindolylmethane on antioxidant biomarkers and cholinergic enzymes in brain tissue of mice treated with scopolamine. Experiments were performed as described in the Figure 5 legend. Lipid peroxidation, GSH, and the activities of GPx, GR, AChE, or ChAT in brain tissues were determined as described in Materials and Methods. (A) Lipid peroxidation, (B) GSH, (C) GPx activity, (D) GR activity, (E) AChE activity, and (F) ChAT activity. Data are the mean ± SEM values of sextuple determinations. * p < 0.05 and ** p < 0.01 versus scopolamine-treated group.

Figure 7

Neuroprotective effect of 3,3′-diindolylmethane on hippocampal CA1 and CA3 regions of mice treated with scopolamine. Experiments were performed as described in the Figure 5 legend. Neuronal cell staining of brain tissues was carried out as described in Materials and Methods. The results were obtained from three independent experiments. (A) CA1 region and (B) CA3 region. ** p < 0.01 versus scopolamine-treated group.