**4. Discussion**

DIM, a dimer of indole-3-carbinol, is known to have some beneficial effects, such as antioxidant action [4], anti-cancer activity [5], hepatoprotection [6], and neuroprotection [7,8]. Recently, it was reported that DIM at 10 μM completely protected neuronal cells against hypoxia through reducing the expression of the aryl hydrocarbon receptor modulator and aryl hydrocarbon receptor induced by hypoxia [7]. In addition, the activation of the aryl hydrocarbon receptor by α-naphthoflavone promoted apoptosis through upregulating ROS generation in neuronal cells [26]. However, DIM could not protect neuronal cells against hypoxia in neuronal cells treated with siRNA targeted to the aryl hydrocarbon receptor modulator or hydrocarbon receptor [7]. Therefore, it remains unknown how DIM protects neuronal cells against oxidative stress-induced apoptosis.

Recently, we reported that *N*-acetyl serotonin protected neuronal cells against oxidative stress-induced apoptosis through activating both the TrkB/CREB/BDNF and Akt/Nrf2/antioxidant enzyme pathways [12], and *N*-palmitoyl serotonin protected neuronal cells against oxidative stress-induced apoptosis by stabilizing the activation of the BDNF autocrine loop, although it could not directly activate the phosphorylation of TrkB [11]. Based on these findings, we surmised that DIM may protect hippocampal neuronal cells against oxidative stress-induced apoptosis by maintaining the activation of both the TrkB/CREB/BDNF pathway and the Akt/Nrf2/ARE pathway. Therefore, we investigated the effect of DIM on oxidative stress-induced apoptosis in hippocampal neuronal cells using an in vitro study. From the results, we found that DIM protected hippocampal neuronal cells against oxidative stress-induced apoptosis by promoting the expression of both BDNF and antioxidant enzymes, such as HO-1, NQO-1, and GCLC. Moreover, in an in vivo study, DIM attenuated scopolamine-induced memory impairment by protecting hippocampal neuronal cells against oxidative damage in mice. Furthermore, such a neuroprotective effect of DIM was possibly associated with the stabilized activation of an antioxidant enzyme-generating system such as the Akt/Nrf2/ARE pathway in hippocampal neuronal cells. These results lead to the assumption that TrkB activation stabilized by DIM may be related to the activation of the Akt/Nrf2/ARE pathway in neuronal cells.

One possible mechanism for the neuroprotective action of DIM may be related to the upregulation of BDNF expression through activation of the TrkB/Akt/CREB/BDNF pathway [18]. BDNF, a ligand of TrkB with a stronger neuroprotective action than that of other neurotrophic factors, is mainly produced from astrocytes [13], microglia [14], and neuronal cells [15]. Here, the stimulation of TrkB by BDNF is known to promote proliferation and differentiation of neural stem cells [17]. In the signaling pathway, the activation of TrkB by BDNF induces the phosphorylation of Akt [27], and then the activated Akt is able to produce BDNF through the activation of CREB in neuronal cells [28]. Thus, maintaining the production of BDNF in neuronal cells may be an important strategy for the prevention or treatment of neurodegenerative diseases, such as Alzheimer's disease. Consistent with this, our present data indicates that DIM protects hippocampal neuronal cells against oxidative stress-induced apoptosis by elevating the phosphorylation of TrkB, Akt, and CREB. In support of this, the inclusion of K252a, an inhibitor of TrkB, neutralized the neuroprotective effect of DIM on oxidative stress-induced cell death of hippocampal neuronal cells. A similar result was observed previously when HT-22 cells were incubated with glutamate in the presence of *N*-acetyl serotonin [12]. Therefore, we suggest that DIM is able to protect neuronal cells by promoting the formation of BDNF through the activation of the TrkB/Akt/CREB/BDNF pathway in oxidative stress-exposed neuronal cells.

Another possible mechanism for the neuroprotective action of DIM is associated with the formation of antioxidant enzymes such as HO-1, NQO-1, and GCLC through the activation of the Akt/Nrf2/ARE pathway. In support of this, DIM augmented the expression of antioxidant enzymes in accordance with the Nrf2/ARE pathway [29]. Moreover, the action of DIM may be extended to activation of the TrkB/Akt/Nrf2/ARE pathway; DIM activates TrkB to produce p-TrkB as mentioned earlier. In turn, activated Akt liberates Nrf2 from the keap1-Nrf2 complex [12], and then Nrf2 is combined into the ARE region on DNA after it translocates into the nucleus [29]. As a result, Nrf2 promotes the formation of antioxidant enzymes, such as HO-1, NQO-1, and GCLC [29]. In support of this, our present study showed that DIM upregulated the expression of p-TrkB and p-Akt and also improved nuclear translocation of Nrf2. Consequently, DIM restored the level of antioxidant enzymes, such as HO-1, NQO-1, and GCLC, to the control level in oxidative stress-exposed hippocampal neuronal cells. In further support of the TrkB/Akt/Nrf2/ARE pathway, the inclusion of K252a, a TrkB inhibitor, or MK-2206, a selective Akt inhibitor, nullified the suppressive effect of DIM on both ROS generation and cell death in oxidative stress-exposed neuronal cells. Probably consistent with the in vitro results, DIM at low doses (10–20 mg/kg) improved scopolamine-induced memory impairment by protecting neuronal cells against oxidative damage in the hippocampal CA1 and CA3 regions of the brain in

mice. Additionally, the cholinolytic effect of scopolamine, mainly due to an increase of AChE activity and a decrease of ChAT activity, was remarkably abrogated by DIM. A similar result was observed previously with *N*-palmitoyl serotonin [19]. A reason for these results is that DIM is capable of crossing to the gastrointestinal tract as well as the blood–brain barrier [25]. In addition, the concentrations of DIM are equal to 0.8 to 1.6 mg/kg (48 to 96 mg/60 kg) in humans, based on body surface area [30].

Taking these results together, it is proposed that the activation of TrkB stabilized by DIM, followed by Akt activation, promotes the expression of both BDNF and antioxidant enzymes in neuronal cells. Therefore, the activation of the TrkB/Akt/Nrf2/ARE pathway, accompanied by stabilization of the TrkB/CREB/BDNF autocrine loop, may be important for the prevention and treatment of neurodegenerative diseases. In addition, the present study shows that DIM may be a candidate compound for the prevention and treatment of neurodegenerative diseases such as Alzheimer's disease.
