Sulforaphane Reverses the Amyloid-β Oligomers Induced Depressive-Like Behavior

Abstract

Background:

Depression is one of the most common behavioral and psychological symptoms in people with Alzheimer’s disease (AD). To date, however, the molecular mechanisms underlying the clinical association between depression and AD remained elusive.

Objective:

Here, we study the relationship between memory impairment and depressive-like behavior in AD animal model, and investigate the potential mechanisms.

Methods:

Male SD rats were administered amyloid-β oligomers (AβOs) by intracerebroventricular injection, and then the depressive-like behavior, neuroinflammation, oxidative stress, and the serotonergic system were measured in the brain. Sulforaphane (SF), a compound with dual capacities of anti-inflammation and anti-oxidative stress, was injected intraperitoneally to evaluate the therapeutic effect.

Results:

The results showed that AβOs induced both memory impairment and depressive-like behavior in rats, through the mechanisms of inducing neuroinflammation and oxidative stress, and impairing the serotonergic axis. SF could reduce both inflammatory factors and oxidative stress parameters to protect the serotonergic system and alleviate memory impairment and depressive-like behavior in rats.

Conclusion:

These results provided insights into the biological mechanisms underlying the clinical link between depressive disorder and AD, and offered new drug options for the treatment of depressive symptoms in dementia.

Keywords

Alzheimer’s disease amyloid-β oligomers depression neuroinflammation oxidative stress sulforaphane

Alzheimer’s disease (AD) is a neurodegenerative disorder that leads to severe memory impairment and cognitive decline [1]. Recent studies have shown that over 90% of AD patients are accompanied by significant behavioral and psychological symptoms, and affective-related behavior increases the severity of cognitive decline in AD patients [2, 3]. The high degree of co-presentation of behavioral, psychological, and cognitive symptoms in AD suggests a possible common perturbation of signaling pathways or neural circuitry specific to each symptom domain. However, the molecular mechanisms underlying the pathophysiologic association between depression and AD remain elusive. Given the complex nature of AD, a deeper understanding of the mechanisms underlying major symptom domains, including memory impairment and depression, may contribute to the discovery of novel and successful therapeutic targets.

More and more studies suggest that early memory impairments of AD might be explained by the presence of soluble forms of amyloid-β oligomers (AβOs), rather than the insoluble forms of amyloid-β (Aβ) peptides and senile plaques [4, 5]. Neuroinflammation and oxidative stress are thought to be the brain’s response to AβOs induced injury, which play a key role in the pathogenesis of AD and depression [6 –8]. Therefore, we hypothesized that AβOs might be linked to depressive symptoms of AD by inducing the neuroinflammation and oxidative stress. Thus, drugs that enhance endogenous anti-inflammatory and anti-oxidative stress pathways would be beneficial to both memory impairment and depressive-like behavior. This may be achieved by using sulforaphane (SF), which dissociates Nrf2 from keap-1, promotes its translocation to the nucleus, and induces anti-oxidant and anti-inflammatory responses [8, 9]. In order to test the above hypothesis, this study was primarily aimed at examining the effects of AβOs on behavioral and psychological abnormalities in rats, to explore the inflammatory and oxidative stress response in the brain of rats. The second aim of this work was to assess the treatment effect of SF on the behavioral and psychological abnormalities inducing by AβOs.

MATERIAL AND METHODS

Ethics statement

All the experimental procedures followed the rules in the “Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research”. The study protocol was approved by the Ethics Committee of Capital Medical University, and every effort was made to minimize the number and suffering of animals.

Animals

Adult male Sprague-Dawley rats (250–300 g, 12 weeks) were hosted in pairs in polycarbonate cages (460*300*180 mm, four per cage), in a sound-attenuated room with controlled temperature (23±1°C) and 12:12 h light-dark cycles (lights on at 8:00 AM). Food and water were available at free-demand. One week before the experiments, the rats were transferred to individual cages (295*190*150) under similar housing conditions (sterile bedding was changed every 2 days or before if required). In order to decrease the stress induced by human manipulation, animals were handled daily during the week before behavioral testing. One hour before the assays, the rats were moved to the experimental room, which was a separated sound-attenuated space with adequate illumination. For our research, we tried to use the minimum necessary number of animals and avoid their suffering.

AβOs preparation

AβOs were generated as described previously [10]. Briefly, 1 mg Aβ₄₂ lyophilized powder (abcam) was suspended in hexafluoroisopropanol to a concentration of 1 mM, and the solvent was evaporated to produce dried films, which were subsequently dissolved in dimethylsulfoxide to make a 5 mM solution. Then, the DMSO-Aβ₄₂ solution was diluted with cold phosphate-buffered saline (PBS) containing 0.05% sodium dodecyl sulfate (SDS) to 200μM, followed by a 24 h incubation at 4°C. Oligomer preparations were centrifuged at 13,000 rpm for 10 min at 4°C to remove insoluble aggregates (protofibrils and fibrils), and the supernatants containing soluble AβOs were stored at 4°C. Protein concentration was determined using the BCA assay (Thermo Scientific Pierce).

Intracerebroventricular (i.c.v.) injection of AβOs

Sprague-Dawley male rats were used to test the effects of AβOs infusions on memory and depressive-like behavior. The animals were anesthetized with 2.5% isoflurane inhalation and were gently restrained during the i.c.v. procedure. After disinfecting the skin with alcohol, the scalp was shaved to expose the skull, microinjections were performed using a 10μL Hamilton syringe fitted with a 26-gauge needle (according to Paxinos and Watson atlas coordinates: from bregma: ML = 1.5 mm; AP = 1 mm; DV = 3.5 mm) [11]. Rats received a single, i.c.v injection of 10μL AβOs (500 pmol), and control rats received injections of an equal volume of PBS and 2% DMSO, and an injection rate of 1μL/min over a period of 10 min. At the end of infusion, the needle was left in place for an additional 3 to 5 min before being slowly withdrawn, to allow diffusion from the tip and prevent reflux of the solution.

Treatment with sulforaphane

Rats were treated daily with SF (Abcam) or corn oil. SF in corn oil was injected intraperitoneally at 5 mg/kg per day for 7 days. The dose was chosen based on previous studies in the literature [12].

Behavioral tests

The behavioral tests were done in the following sequence: open field test (OFT), sucrose preference test (SPT) and forced swimming test (FST) [13]. The timeline is in the Fig. 1A.

Fig. 1

SF improved depressive-like behavior inducing by AβOs. A) Schematic representation of experimental protocol. Behavioral analysis was monitored by using open field test (OFT), sucrose preference test (SPT), forced swim test (FST), and Morris water maze (MWM). B) The number of horizontal activity. C) The number of vertical activity. D) Locomotion ration in OFT. E) The time in DF center. F) Sucrose preference in SPT. G) Overall fluid consumption in SPT. H) Immobility time in FST. I) The number of jumps in FST. SF, sulforaphane; AβOs, amyloid-β oligomers; i.c.v., intracerebroventricular; S.C, sample collection. Values are expressed as mean±SEM. (n = 9/group), ^**p < 0.01.

The OFT was performed to determine the number of locomotor crossing and rearing, and evaluate the movement and exploratory behavior of rats. The test device was a self-made uncapped box (100 cm×100 cm×40 cm). The bottom of the box was divided into 25 equal grids by black line. The OFT was carried on 8:00–12:00 am without interference. Each rat was gently placed in a central square to observe the behavior for 5 min. A digital video camera was installed above the apparatus. To neutralize odors, the arena was cleaned with 70% ethanol before each subject was tested.

The anhedonia state as the core symptom of depressive-like behavior was investigated by SPT. At the beginning of SPT, all rats were fed alone in cages with two bottle of sucrose solution (1%, w/v) on each side of the cage for 24 h habituation period. And the rats were deprived of water for 16 h. Then, the rats were provided with two pre-weighed bottles for 1 h, one containing 1% sugar water and the other was pure water, and placed randomly to avoid the effect of space preference on the experimental results. The reduced sucrose preference, an index of anhedonia, was calculated according to the following formula: sucrose consumption / (water consumption + sucrose consumption)×100%.

The FST usually used to evaluate the effects of anti-depressants and depressive-like behaviors in rodents. The rats were arranged to pre-swim into a cylindrical tank (25 cm diameter, 45 cm height) containing clean water at 24±1°C (35 cm deep) for 15 min, and then put back into cage after drying. Twenty-four hours later, the rats were forced to swim individually for 5 min as formal test, and the immobility time were record during swimming period. All experiments were done by trained recorders blinded to the treatment.

Morris water maze

Spatial memory and learning were evaluated using the hippocampus-dependent Morris water maze (MWM) test [14]. The water maze consisted of a circular water tank that was partially filled with water (24±1°C). The pool was divided virtually into four equal quadrants labeled N–S–E–W. A colorless escape plat (10 cm in diameter) was hidden 1.5 cm below the surface of the water in a fixed location. The maze was located in a quiet test room, surrounded by many visual cues outside of the maze which was visible from within the pool and could be used by the rats for spatial orientation.

The experiments were conducted two sessions per day for 5 days, each session comprising four trials, with an intertribal interval of 60 s and an intersession interval of 2 h. In each trial, the animals were gently placed in the middle of the circular edge in a randomly selected quadrant, with the nose pointing toward the wall. If animals failed to find the escape platform within 120 s by themselves, they were placed on the platform for 30 s by the experimenter and their escape latency was accepted as 120 s. After climbing onto the platform, the animal remained there for 30 s before the commencement of the next trial. On the sixth day, the platform was removed, and the same water inlet point was selected. The 120 s swimming trajectory of the rats was photographed by a camera, the frequency of crossing the “platform position” and the time spent in the target quadrant were recorded. The test was performed from 10:00 AM to 17:00 PM to exclude variations in performance resulting from circadian rhythmicity.

Sample collection for biochemical measurements

The rats’ brains were immediately removed and were dissected on ice. The dorsal raphe nucleus and striatum tissue were isolated from the brain and kept at –80°C.

Protein extractions

Ice cold Tris-buffered saline (TBS) consisting of 20 mM Tris-HCl, 150 mM NaCl, pH 7.4, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitor cocktail (Roche) were added to the brain tissue and homogenized at a setting of mechanical Dounce homogenizer, following centrifugation for 30 min at 13,000 g. The protein content of the brain homogenates was determined using a Pierce BCA protein assay kit (Thermo Scientific).

Examination for malondialdehyde (MDA) and glutathione (GSH)

The striatum tissue samples were homogenized with 10 times (w/v) ice-cold 0.1 M phosphatebuffer (PB), pH 7.4. The homogenate was centrifuged at 3000 rpm for 15 min, and the supernatant was used for the estimation of MDA and GSH using detection kit (Jiancheng, China). Detection was carried out according to the manufacturer’s instructions, and each sample was tested in triplicate.

Western blot

The levels of tryptophan hydroxylase (TPH) in the dorsal raphe nucleus (DRN), serotonin transporter (SERT) in the striatum and IL-1β, TNFα in the cortex were measured using western blot. According to the rat brain atlas [11], the DRN and striatum region were identified under a binocular microscope and was punched out from brain slices. The protein concentrations of samples were determined using a BCA protein assay kit (Thermo Scientific). Samples were loaded on SDS/PAGE gels, and separated proteins were transferred to nitrocellulose membranes. The membranes were blocked in a solution of 5% fat-free milk for 30 min at 20°C and incubated overnight at 4°C with one of the following rabbit primary antibodies: anti-TPH2 (abcam); anti-SERT (sigma); anti-IL-1β (abcam); anti-TNFα (abcam); anti-β-actin (Santa Cruz). Primary antibody incubation was followed by incubation at 25°C for 1 h with the goat HRP-labeled secondary antibody and finally by visualization using enhanced chemiluminescence reagents (Beyotime Institute of Biotechnology). The membranes were scanned, and optical densities were determined using ImageJ software. The band density was all normalized to β-actin when analyzing.

Immunohistochemistry and densitometric analysis

The Sprague Dawley rats were anesthetized with choral hydrate (300 mg/kg) pentobarbital, perfused transcardially with saline and then with 500 ml of 4% paraformaldehyde in phosphate buffer (PB; 0.1 M, pH 7.4). Brains were dissected, postfixed in the same fixative solution for 2 h at room temperature, equilibrated with 30% sucrose in PB at 4°C. Brains were sliced into 40μm thick sections using a cryostat. Sections were processed for immunohistochemistry as follows. Floating sections were incubated with 1% hydrogen peroxide in PBS for 10 min at room temperature to inhibit endogenous peroxidase, washed three times with PBS, incubated for 1 h in 3% normal goat serum in TBS, pH 7.4 at 22°C, incubated in rabbit TPH2 (abcam) and SERT (sigma) antibody, diluted 1:300 in TBS containing 3% normal goat serum for 24 h at 4°C, rinsed, incubated for 1 h at 25°C in 1:200 dilution of biotinylated goat anti-rabbit secondary antibody (Santa Cruz) for 1 h, rinsed, incubated with avidin-biotin peroxidase complex (ABC) reagent for 1 h (ZSGB-BIO), rinsed, and then incubated in a solution containing 22μg/ml diaminobenzidine (DAB) and 0.003% hydrogen peroxide (H₂O₂) for color deposition. Sections were mounted on coated slides, dehydrated, cover slipped, viewed and photographed using Nikon microscope and digital camera.

Statistics

The statistical analyses were performed using one-way ANOVA followed by Bonferroni’s post hoc comparison. To identify a learning deficit, we used two-way repeated measures ANOVA followed by Bonferroni’s post hoc comparison to analyze a ‘day by treatment’ interaction for escape latency. The probe trial was assessed using single sample t-test to check if treatment groups had preference for the target zone. p < 0.05 was considered significant.

RESULTS

AβOs induces depression-like behavior, and SF ameliorates it

Figure 1B–E shows the number of horizontal activity, vertical activity, locomotion ratio, and time in OF center obtained from OFT, respectively. One-way ANOVA showed that there were overall group differences for the number of horizontal activity (F_3,32 = 16.05, p < 0.0001), vertical activity (F_3,32 = 7.552, p = 0.0006), locomotion ratio (F_3,32 = 52.72, p < 0.0001), and time in OF center (F_3,32 = 5.643, p = 0.0032). Post-hoc Bonferroni comparison showed that the AβOs group had fewer number of horizontal activity (p < 0.0001), vertical activity (p = 0.0019), locomotion ratio (p = 0.0059), and time in OF center (p = 0.0085) than the control group. On the other hand, as compared with SF group, AβOs + SF group did not show any significant difference for the horizontal activity (p = 0.2709), vertical activity (p = 0.6382), locomotion ratio in OFT (p = 0.0657), and time in OF center (p = 0.9528). In addition, SF per se had no significant effect on the horizontal activity, vertical activity, locomotion ratio or time in OF center.

As shown in Fig. 1F and G, one-way ANOVA showed that there was overall group difference for sucrose preference (F_3,32 = 28.12, p < 0.0001) while not for total fluid consumption (F_3,32 = 0.1495, p = 0.9292). Post-hoc Bonferroni comparison showed that, compared with the control group, the AβOs group demonstrated significant decrease in sucrose preference (p < 0.0001). On the other hand, as compared with SF group, AβOs + SF group did not show any significant difference for sucrose preference (p = 0.1476). In addition, SF per se had no significant effect on sucrose preference.

As shown in Fig. 1H and I, one-way ANOVA showed that there was overall group difference for the performance of rats in FST, including immobility time (F_3,32 = 54.8, p < 0.0001) and the jump numbers (F_3,32 = 10.27, p < 0.0001). Post-hoc Bonferroni comparison showed that, the AβOs group significantly increased the immobility time (p < 0.0001) and decreased the jump numbers (p = 0.0005) of rats compared with the control group. On the other hand, as compared with the SF group, AβOs + SF group didn’t show any significant difference for the immobility time (p = 0.4145) and the jump numbers of rats (p = 0.2550). Moreover, SF per se had no significant effect on the immobility time or the jump numbers.

AβOs impair spatial learning in the Morris water maze, and SF restores it

As shown in Fig. 2A, in the MWM, all animals showed progressive decline in the escape latency with training. Two-way ANOVA showed that there was overall group difference for sucrose preference (F_4,160 = 164.7, p < 0.0001). A significant increase in escape latency was observed in the AβOs group after the third training day compared to the control group (day 3, p < 0.0001; day 4, p = 0.0010; day 5, p = 0.0001). On the other hand, as compared with the SF group, AβOs + SF group did not show any significant difference for escape latency with training (day 3, p > 0.9999, day 4, p = 0.8511, day 5, p > 0.9999). As shown in Fig. 2B and C, one-way ANOVA showed that there was overall group difference for the probe trial (F_3,32 = 8.276, p = 0.003) and the number of platform crossing (F_2.53,20.24 = 6, p = 0.0059). Post-hoc Bonferroni comparison showed that the AβOs group significantly decreased the probe trial (p = 0.0004) and the number of platform crossing (p = 0.0049) compared with the control group. On the other hand, as compared with the SF group, AβOs + SF group did not show any significant difference for the probe trial (p = 0.2805) and the number of platform crossing (p > 0.9999). In addition, we did not find any difference in swimming speed among four groups on the first training day (F_3,32 = 0.2048, p = 0.8923), which excludes any potential influence of motor disabilities on escape latency. SF per se had no significant effect on the rat performance. In the probe trial, the AβOs + SF groups had more preferred for the target zone, and the exploration rates was 30.14% in the target zone. We did the pre-testing of floating behavior before the MWM, and two rats were eliminated because showing floating or thigmotaxis behaviors.

Fig. 2

SF improved the cognitive deficits of the AβOs treatment rats in Morris water maze. A) Latency to reach escape platform. B) Time spent in target quadrant. C) The number of crossing the platform. D) Swimming speed on the first training day. SF, sulforaphane; AβOs, amyloid-β oligomers. Values are expressed as mean±SEM. (n = 9/group), ^**p < 0.01.

AβOs damage the serotonergic system of the brain, and sulforaphane reverses it

Brain sections from rats, including the DNR and the striatum, were stained with antibody to TPH2 or SERT, which is a marker of the serotonergic neuron or neurite. As shown in Fig. 3B and C, western blot investigated the protein level of TPH2 in the DNR. One-way ANOVA showed that there was overall group difference for the expression of TPH2 (F_3,20 = 19.35, p < 0.0001). Post-hoc Bonferroni comparison showed that the AβOs group significantly decreased the expression of TPH2 (p = 0.0004) compared with the control group. On the other hand, as compared with the SF group, AβOs + SF group did not show any significant difference for the expression of TPH2 (p = 0.1409). As shown in Fig. 3D and E, immunohistochemical staining observed the serotonergic neuron in the DNR. One-way ANOVA showed that there was overall group difference for the number of TPH2 immunopositive neurons in the DRN of rats (F_3,20 = 8.716, p = 0.0007). Post-hoc Bonferroni comparison showed that the AβOs group significantly decreased the number of TPH2 immunopositive neurons (p = 0.0012) compared with the control group. On the other hand, as compared with the SF group, AβOs + SF group did not show any significant difference for the number of TPH2 immunopositive neurons (p = 0.9964).

Fig. 3

SF reduced the AβOs induced the damage of serotoninergic neuron. A) An atlas view of coronal section showing the DRN region. For western blotting analysis, the DRN tissues were punched out from brain slices as indicated by a red circle. B, C) Western blot analysis for TPH2 protein levels in the DRN region. Bar graphs illustrate the protein expression of TPH2 (n = 6/group). D, E) Immunohistochemical study of TPH2 expression in the DRN region (n = 3/group). F) The correlation analysis between the immobility time and expression of TPH2 (p = 0.0002). TPH, tryptophan hydroxylase; DRN, dorsal raphe nucleus; SF, sulforaphane; AβOs, amyloid-β oligomers. Values are expressed as mean±SEM. ^**p < 0.01.

SERT is primarily located on serotonergic nerve terminals and axons in the brain. We also investigate the SERT density in the striatum. As shown in Fig. 4, one-way ANOVA showed that there was overall group difference for the expression of SERT, including western blot (F_3,20 = 16.08, p < 0.0001) and immunohistochemistry (F_3,20 = 10.85, p = 0.0002). Post-hoc Bonferroni comparison showed that, the AβOs group significantly increased the expression of SERT, including western blot (p = 0.0003) and immunohistochemistry (p = 0.0010) compared with the control group. On the other hand, as compared with the SF group, AβOs + SF group did not show any significant difference for the expression of SERT in western blot (p = 0.5086) and immunohistochemistry (p = 0.9990). In addition, SF per se had no significant effect on the expressions of TPH2 or SERT. We did the correlation analysis between the expression of serotonergic system in the brain with behavioral findings, the immobility time and expression of TPH2 are negatively correlated (p = 0.0002), the immobility time and expression of SERT are not correlated (p = 0. 0702).

Fig. 4

SF reduced the AβOs inducing the damage of SERT. A) An atlas view of coronal section showing the striatum region. For western blotting analysis, the striatum tissues were punched out from brain slices as indicated by a red circle. B, C) Western blot analysis for SERT protein levels in the striatum region. Bar graphs illustrate the protein expression of SERT (n = 6/group), D, E) Immunohistochemical study of SERT expression in the striatum (n = 3/group). F) The correlation analysis between the immobility time and expression of SERT (p = 0.0702). SERT, serotonin transporter; SF, sulforaphane; AβOs, amyloid-β oligomers. Values are expressed as mean±SEM. ^**p < 0.01.

AβOs induce the oxidative stress and neuroinflammation in the brain of rats, and sulforaphane reverses the phenomenon

As shown in Fig. 5, one-way ANOVA showed that there was overall group difference for the level of MDA (F_3,20 = 36.59, p < 0.0001) and the level of GSH (F_3,20 = 5.716, p = 0.0054). Post-hoc Bonferroni comparison showed that, the AβOs group significantly increased the level of MDA (p < 0.0001) and decreased the level of GSH (p = 0.0076) compared with the control group. On the other hand, as compared with the SF group, AβOs + SF group did not show any significant difference for the levels of MDA (p = 0.1486) and GSH (p = 0.9732). In addition, SF per se had no significant effect on the expressions of MDA and GSH.

Fig. 5

SF inhibits oxidative stress induced by AβOs. Measurement expression of (A) GSH and (B) MDA in brain tissue. SF, sulforaphane; AβOs, amyloid-β oligomers. Values are expressed as mean±SEM, (n = 6/group) ^**p < 0.01.

IL-1β and TNF-α are biomarkers of neuroinflammation. As shown in Fig. 6, one-way ANOVA showed that there was overall group difference for the levels of IL-1β (F_3,20 = 44.06, p < 0.0001) and TNF-α (F_3,20 = 25.97, p < 0.0001). Post-hoc Bonferroni comparison showed that, the AβOs group significantly increased the level of IL-1β (p < 0.0001) and TNF-α (p < 0.0001) compared with the control group. On the other hand, as compared with the SF group, AβOs + SF group did not show any significant difference for the levels of IL-1β (p = 0.9325) and TNF-α (p = 0.8915). In addition, SF per se had no significant effect on the expressions of IL-1β or TNF-α.

Fig. 6

SF inhibits the neuroinflammation induced by AβOs. Western blot analysis for the expression of IL-1β and TNF-α in the brain tissue (A). Bar graphs illustrate the quantification of (B) IL-1β and (C) TNF-α. SF, sulforaphane; AβOs, amyloid-β oligomers. Values are expressed as mean±SEM, (n = 6/group) ^**p < 0.01.

DISCUSSION

Although clinical and epidemiological studies have revealed a strong connection between AD and depression, the mechanisms connecting these two disorders at the molecular and cellular levels remain to be elucidated. The findings of the present study indicated that AβOs was able to produce depressive-like behavior as assessed through FST, OFT, and SPT. Moreover, declined levels of TPH2 and SERT in DRN and striatum further confirmed the depressive-like behavior. At the same time, we found that AβOs induced the neuroinflammation and oxidative stress in the rat brain. SF exhibited both anti-inflammation and anti-oxidative stress, protected the serotonergic system, and modulated the depressive-like behavior in rats. The mechanism that underlies the antidepressant-like effects of SF may also be attributed to its known antioxidant and anti-inflammatory properties.

Psychiatric and behavioral symptoms particularly depression commonly accompany the AD. In order to assess depressive-like behaviors, OFT, SPT, and FST was performed. We observed AβOs i.c.v injection induced depressive-like behavior in rats indicated by decreased the horizontal activity, vertical activity, and locomotion ratio in OFT [15]. AβOs i.c.v injection also could increase immobility time and decrease the number of jump in the FST and reduced the sucrose consumption in SPT, which were related to behavioral despair and anhedonia, respectively [16]. As memory deficit is the main clinical symptom of AD, we investigated the impact of AβOs i.c.v injection on the memory of rats using the MWM task. The results showed that memory dysfunction was measurable in the AβOs group. Together, these results indicate that treatment with AβOs have an impact on memory, learning, and mood in rats. In the study, we also observed prominent anxiolytic effects in rats inducing by AβOs. OFT has been taken up to investigate exploratory and as anxiety index in rodents. The results showed that the time in the center of the open-field are significant decreased in the AβOs group compared with the control group. Anxiety was reduced by treatment with SF as evidenced by increased the time spent in the central zone. Unfortunately, we mainly focused on the depressive-like behavior of rats, and did not investigate the ‘cued’ version of the MWM in the study, which is a limitation of the study design.

Increasing evidence suggests that oxidative stress and neuroinflammation play significant roles in disease progression, particularly in cellular and tissue damage, which is closely related to neurodegenerative conditions in the brain [17, 18]. However, whether oxidative stress and neuroinflammation are involved in the onset of neuropsychiatric symptoms of dementia remains unclear. In the present study, MDA levels were significantly increased, GSH levels were markedly decreased in the brain of AβOs i.c.v injection rats compared with the control rats, which means that AβOs i.c.v injection induced oxidative stress in the brain [19]. One study described that activated microglia could increase the expression of many inflammatory markers, including TNF-α and IL-1β, which could induce neuronal apoptosis, interfere with intracellular calcium homeostasis, and suppress the long-term potentiation [20, 21]. Our results showed that IL-1β and TNF-α concentrations in the brain of rats marked increase after AβOs i.c.v. injection. It is reasonable to infer that AβOs i.c.v injection causes cognitive decline and depressive-like behavioral changes in rats by inducing oxidative stress and neuroinflammatory responses in the brain.

Serotonin (5-HT) is one of the most extensively studied neurotransmitters in the central nervous system (CNS) regulating multiple physiological functions [22]. TPH is the rate-limiting enzyme in 5-HT synthesis, and is identified two isoforms, TPH1 and TPH2, both with unique characteristics and have demonstrated tissue specificity with TPH1 mainly located in the pineal gland and peripheral tissues such as gut, spleen, and thymus, whereas TPH2 is predominantly found in the brainstem [23, 24]. The projections from the raphe nuclei, where 5-HT is produced, are widespread across the CNS, including ascending to the cerebral cortex, thalamus, hypothalamus, and basal ganglia, and descending to the brainstem and spinal cord [25]. SERT is primarily located on serotonergic nerve terminals and axons in the adult brain, and its density is considered to be a representative marker of the integrity of the serotonergic neuron [26]. The current results established that AβOs damaged the serotonergic neuron and SERT density. It is possible that AβOs induced inflammation and oxidative stress modulating the activity of TPH involved in 5-HT metabolism. Recent studies have shown that the cytokine could reduce 5-HT levels by inhibiting tryptophan hydroxylase (TPH) mRNA in the brain, and the hyperactive inflammatory response system could induce enhanced tryptophan hydroxylase breakdown [27, 28]. 5-HT metabolism dysfunction is widely involved in the depression but apart from 5-HT, dopaminergic and noradrenergic systems also play vital roles in the treatment of depression [29]. Interestingly, Kuhn’s study found that TPH2 depletion did not affect mouse sucrose preference, implicating non-serotonin-dependent pathways [30]. Therefore, future research needs to investigate these intriguing relationships between neurotransmitters and depressive-like behavior. Interestingly, the correlation analysis between the expression of serotonergic system in the brain with behavioral findings showed that the immobility time and expression of TPH2 are negatively correlated, the immobility time and expression of SERT are not correlated. This might be because there are only six samples in each group. We will verify the correlation of brain serotonergic system and behavioral findings in the future study.

SF is an isothiocyanate compound which functions as an antioxidant activating the Nrf2/ARE pathway [31, 32]. Aside from being an activator, SF has also shown various biological functions, including the inhibition of the microglial cell activation and the subsequent inflammatory cascades against brain immune cells [9, 33]. SF can release Nrf2 from Keap1 by directly interacting with Keap1; the released Nrf2 can then translocate to the nucleus and bind to ARE sequences, thus increase the transcription of the phase II enzymes that can resist the injury of oxidative stress and inflammation [34, 35]. Previous studies have found that SF could inhibit Aβ induced inflammation and reduce oxidative stress injury in an animal model [36, 37]. In our study, we found that SF could effectively reduce AβOs induced neuroinflammation and oxidative stress response, as indicated by decreased MDA, IL-1β, and TNF-α, and increased GSH in the brain of rats, improving the memory dysfunction and the depressive-like behavior.

The current findings establish the link of AβOs to memory impairment and depressive-like behavior in rats, and provide molecular mechanistic support to clinical evidence connecting AD with depressive disorder. The impact of AβOs on mood, learning, and memory could likely be attributed to the activation of inflammatory and oxidative stress pathways and to the downregulation of the serotonergic axis. SF, a compound that has both antioxidant and anti-inflammatory activities, can be a useful therapeutic strategy in the treatment and protection of not only cognitive symptoms, but also psychological symptoms such as depression in dementia.

Footnotes

ACKNOWLEDGMENTS

This study was supported by the National Key R&D Program of China (No. 2017YFC1310103); the Key Project of the National Natural Science Foundation of China (81530036); the National Key Scientific Instrument and Equipment Development Project (31627803); Mission Program of Beijing Municipal Administration of Hospitals (SML20150801); Beijing Scholars Program; Beijing Brain Initiative from Beijing Municipal Science & Technology Commission (Z161100000216137); Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support (No.ZYLX201837).

Authors’ disclosures available online ().

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