Effect of diallyl disulfide on lipopolysaccharide-induced depression-like behavior in mice

Background: Depression is associated with high levels of pro-inflammatory cytokines and oxidative markers. Inhibition of neuroinflammation and oxidative stress is beneficial for depression prevention and/or therapy. Diallyl disulfide (DADS), an active compound in garlic oil, has been shown to inhibit neuroinflammation and oxidative stress. The purpose of this study is to investigate the role and mechanism of DADS in lipopolysacharide (LPS)-induced depression-like behaviors in mice. Methods: We used behavioral tests and biochemical analysis to illustrate the role and mechanism of DADS in depression regulation. Results: Similarly to imipramine (10 mg/kg), a clinical antidepressant, DADS (40 or 80 mg/kg), which was administered 1 h before LPS treatment (pre-LPS) or 1.5 h and 23.5 h after LPS treatment (post-LPS), prevented and reversed the LPS (100 μg/kg)-induced increase in immobility time in the tail suspension test (TST) and forced swim test (FST) in mice. Mechanistic studies revealed that DADS pre-treatment or post-treatment at the dose of 40 and 80 mg/kg prevented and reversed (i) the LPS-induced increases in interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and nitric oxide (NO) levels in the hippocampus and prefrontal cortex, (ii) the LPS-induced increases in contents of malondialdehyde (MDA), a parameter reflecting high levels of oxidative stress, as well as (iii) the LPS-induced decreases in contents of GSH, a marker reflecting weakened anti-oxidative ability, in both hippocampus and prefrontal cortexes in mice. Conclusions: DADS is comparable to imipramine in effectively ameliorating LPS-induced depression-like behaviors in mice, providing a potential value for DADS in prevention and/or therapy of depression. In

Depression is a common mental disease, causing severe economic and social burdens [1].
The pathogenesis of depression is related to various factors, such as adverse social stress, heredity, and the decrease in monoamine levels in the brain [2]. On the basis of the latter factor, researchers have raised a monoamine dysfunction hypothesis of depression, and according to this hypothesis researchers have developed several antidepressants, such as the serotonin reuptake inhibitor and the monoamine inhibitor. However, these antidepressants usually need several weeks to produce therapeutic effects [3], and approximately one-third of patients do not respond to these drugs [4]. Further, these antidepressants may evoke suicide attempt and suicide in some cases [5]. It is therefore necessary to search novel drugs for depression regulation, including depression prevention and depression treatment.
Besides monoamine dysfunction, many other mechanisms, such as the impairment of hippocampal neurogenesis and glutamatergic neurotransmission, the dysfunction of the neuroendocrine system, and the impairment of the brain-derived neurotrophic factor (BDNF) signaling, have been raised to explain the pathogenesis of depression [6][7][8].
Increasing evidence has also revealed that there is a close relationship between immune activation and depression, thereby making the neuroinflammatory hypothesis be a new focus [9][10][11][12][13]. In depressed patients, pro-inflammatory cytokines, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and nitric oxide (NO), have been observed repeatedly to be increased in serum [14,15]. Systemic administration of lipopolysaccharide (LPS), a classical activator of the immune cell, triggers depression-like behaviors in humans and rodents via inducing severe peripheral and central inflammatory responses [16,17]. Using compounds which can inhibit neuroinflammatory response could be a potential strategy for depression prevention and/or therapy. Indeed, this hypothesis has been supported by a lot of evidence. For example, some pre-clinical compounds which possess anti-inflammatory properties have been confirmed to be capable of ameliorating depression-like behavior in rodents induced by acute immune stress or the other chronic stresses, such as the chronic unpredictable stress and the chronic social defeat stress [16][17][18][19], and some clinical anti-inflammatory drugs, such as nonsteroidal antiinflammatory drugs (NSAIDs) and minocycline, can suppress depressive symptoms in patients [20,21]. Further, the traditional antidepressants, including the paroxetine, fluoxetine, and imipramine, have been reported to reduce pro-inflammatory responses in the brain in rodents [22][23][24]. Mechanistic studies have shown that the pro-inflammatory cytokine triggers depression onset likely through promoting oxido-nitrosative stress in the hippocampus and prefrontal cortex [16,25]. Interruption with neuroinflammatory response along with oxido-nitrosative stress may be an important strategy for depression prevention and/or therapy.
Garlic is a flavoring agent for diet in daily life. It has great antimicrobial effects against genera Pseudomonas, Streptococcus, and Staphylococcus [26,27]. Garlic and/or garlicderived compounds can also display preventive and/or therapeutic activities in central nervous system (CNS) disorders, such as headache [28], Parkinson's disease [29], and Alzheimer's disease [30]. In a rat diabetic model, garlic can ameliorate anxiety-and depressive-like behaviors via inhibiting oxidative stress [31]. Further, garlic extracts are capable of alleviating LPS-triggered oxidative stress and neuroinflammatory response in BV-2 microglia [32]. Diallyl disulfide (DADS) is an active compound derived from garlic oil.
It can inhibit LPS-induced neutrophil infiltration and damage in rat intestines [33], suppress inflammation and apoptosis resistance in human Barrett's epithelial cells [34], attenuate airway inflammation via inhibiting IL-6/IL-1β production in LPS-stimulated macrophages and animals [35], and alleviate LPS-induced osteoclastogenesis via reducing pro-inflammatory cytokine production [36]. We speculate that DADS may regulate LPS-induced depression-like behavior in mice via alteration of the neuroinflammatory and oxidative status of the brain. In order to investigate this hypothesis, we evaluated the changes in depression-like behaviors in mice administered with DADS in a pre-or post-LPS model, and also evaluated whether the depression regulation effect of DADS is associated with the changes in parameters reflecting the neuroinflammatory response and oxidative stress of the mice brain (hippocampus and prefrontal cortex) following a pre-or post-LPS administration, such as IL-1β, NO, malondialdehyde (MDA), and reduced glutathione (GSH).

Materials And Methods
Materials DADS was purchased from Sigma and dissolved in dimethyl sulfoxide (DMSO). Imipramine is the product of MedChem Express (Princeton, NJ, USA). They were prepared as stock solutions and stored at -20°C.

Animals
Male C57BL6/J mice (6−8 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China), and were housed 5 per cage for 1 week with free access to food and water under standard conditions (five mice per cage), which includes 12-h of light/dark cycle, lights on from 07:00 to 19:00, 55 ± 10% of relative humidity, and 23 ± 1°C of temperature. Behavioral experiments, which were approved by the University Animal Ethics Committee of Nantong University (Permit Number: 2110836), were carried out during the light phase, according to internationally accepted guidelines for the use of animals in toxicology as adopted by the Society of Toxicology in 1999.
Pharmacological treatments DADS and LPS were given intraperitoneally. Imipramine (10 mg/kg) was used as a positive control. In the pre-treatment model, the mice in separated groups were administered with

Measurement of MDA and GSH levels
To assess the oxidative stress status in hippocampus and prefrontal cortex, the MDA and GSH levels were determined using method of Jangra et al. 43 and Beutler et al. 44, respectively. The estimation of MDA and GSH were expressed as μmol/g of wet tissue. The measurement of brain protein was according to the Lowry method and was expressed as μmol/mg of protein concentration 45.

Statistical analysis
All analyses were performed using SPSS 13.0 software (SPSS Inc., USA), and data are presented as mean ± SEM. Differences between mean values were evaluated using oneway or two-way analysis of variance (ANOVA). Bonferroni's post hoc test was applied to assess isolated comparisons. P < 0.05 was considered statistically significant.

Effect of DADS pre-treatment on LPS-induced depression-like behavior in mice
In order to determine whether DADS can prevent LPS-induced depression-like behavior, the mice were pretreated with DADS (40 or 80 mg/kg, i.p.) at 1 h before LPS injection (100 μg/kg, i.p., Fig. 1A). For TST, a two-way ANOVA revealed significant effects for LPS stimulation (F 1,72 = 5.38, P < 0.05), drug pre-treatment (F 3,72 = 4.69, P < 0.01), and LPS × drug interaction (F 3,72 = 11.68, P < 0.001). For FST, the two-way ANOVA revealed significant effects for LPS stimulation (F 1,72 = 6.94, P < 0.05), drug pre-treatment (F 3,72 = 2.79, P < 0.05), and LPS × drug interaction (F 3,72 = 3.08, P < 0.05). Post hoc analysis showed that DADS pre-treatment at the dose of 40 and 80 mg/kg markedly prevented the LPS-induced increase in immobility time in the TST and FST in mice. Similarly, imipramine pre-treatment (10 mg/kg) also prevented the LPS-induced in immobility time in the TST and FST in mice (Fig. 1B, C). The OFT results showed that both DADS and imipramine pretreatment did not affect the locomotor activity in mice with or without LPS treatment (no significant effects for LPS stimulation (F 1,72 = 0.34, P = 0.56), drug pre-treatment (F 3,72 = 0.34, P = 0.79), and LPS × drug interaction (F 3,72 = 0.19, P = 0.91), Fig. 1D). These results indicate that DADS has a prophylactic effect on LPS-induced depression-like behavior in mice. 9 We next evaluated the inhibitory effect of DADS on LPS-induced depression-like behavior in mice in a post-treatment model, in which the mice were administered with DADS (40 or 80 mg/kg, i.p.) at either 1.5 or 23.5 h after LPS treatment (100 μg/kg, i.p., Fig. 2A). For TST, a two-way ANOVA revealed significant effects for drug post-treatment (F 3,72 = 4.76, P < 0.05) and LPS × drug interaction (F 3,72 = 5.09, P < 0.001), but not for LPS stimulation These results indicate that DADS post-treatment can reverse LPS-induced depression-like behavior in mice.

Effect of DADS pre-or post-treatment on LPS-induced increase in IL-1β and TNF-α levels in the hippocampus and prefrontal cortex
The systemic inflammation induced by LPS could lead to pro-inflammatory cytokine production in the CNS, among which IL-1β and TNF-α was closely related to the induction of depression-like behavior in mice. In this study, we evaluated the change of IL-1β and TNF-α levels in the hippocampus and prefrontal cortex in both pre-and post-treatment models. In the pre-treatment model, a one-way ANOVA for IL-1β (hippocampus: F 3,36 = 10 8.72, P < 0.001; prefrontal cortex: F 3, 36 = 15.74, P < 0.001) and TNF-α (hippocampus: F 3,36 = 7.95, P < 0.001; prefrontal cortex: F 3,36 = 5.09, P < 0.001), and post hoc analysis showed that both imipramine pre-treatment (10 mg/kg) and DADS pre-treatment at the dose of 40 and 80 mg/kg prevented the LPS (100 μg/kg)-induced increase in IL-1β (Fig. 3A,   B) and TNF-α (Fig. 4A, B) (Fig. 5C) and prefrontal cortex (Fig. 5D). Our results showed that DADS pre-treatment (1 h before a single LPS injection) prevented while DADS post-treatment (1.5 h and 23.5 h after a single LPS injection) reversed the LPS-induced depression-like behavior in mice, suggesting that in the present experimental model a relatively short-term drug exposure is enough for DADS to induce depression regulation effect. LPS is an endotoxin which is produced endogenously via gut microbiota dysfunction [46]. The increased LPS would enter into the blood via the impaired gut barrier and activate the peripheral and central immune system, thereby inducing sickness and depression-like behavior in both rodents and humans [16,47,48]. The acute behavioral response to endotoxin exposure, including the sickness and depression-like behavior, is well-known to cope with environmental challenges. However, repeated or chronic endotoxin exposure may induce an accumulated behavioral response, which has been shown to be tightly associated with the progression of chronic brain damage [49,50]. Suppression of behavioral abnormalities upon endotoxin challenge may help prevent the occurrence or progression of chronic brain damage. Our results demonstrate that DADS supplementation may be a potential strategy in accord with that purpose because both DADS pre-treatment and post-treatment were found to affect LPS-induced increase in immobility time in the TST and FST. Furthermore, since DADS is mainly extracted from 13 garlic oil, our results may show a great significance for the application of DADS and DADScontaining garlic or the other edible plants in depression prevention and therapy, especially at the condition of endotoxin presence. The gastrointestinal system, at all times, faces various detrimental challenges, such as Escherichia coli infection, food poisoning, and intestinal cold. These detrimental factors would induce endotoxin accumulation, which may promote the occurrence of repeated behavioral damages. Daily intake of DADS or DADS-containing plants may help suppress the potential damage in the brain induced by gut-derived endotoxin. On the basis of these contexts, the significance of the present study should be strengthened particularly.

Effect of DADS pre-or post-treatment on LPS-induced alterations in markers reflecting the oxidative and anti-oxidative status of the hippocampus and prefrontal cortex
Besides the classical monoamine dysfunction hypothesis, the neuroinflammatory hypothesis has been raised to explain the pathogenesis of depression in the past decades.
This hypothesis appears to be a new focus for depression study in recent years because the actively involvement of neuroinflammation in depression onset and/or progression has been supported by more and more studies [14,15,19,48,51,52]. For example, the overaccumulation of pro-inflammatory cytokines in the blood and/or brain have been observed repeatedly in depressed patients and animals [14,15,19], and stimulation of animals with pro-inflammatory cytokines, such as IL-1β and TNF-α, can induce typical depression-like behaviors [51,52]. Researchers have observed that peoples who accept a low dose of endotoxin immune (24 h) develop sickness and depressive behaviors [48]. Moreover, some clinical available anti-inflammatory drugs, such as NSAIDs and minocycline, can produce obvious antidepressant effects in human individuals and rodent models [20,21,53].
Therefore, inhibition of neuroinflammation may be a potential mechanism for the antidepressant effect of the clinical and pre-clinical drugs. Our results showed that DADS pre-treatment markedly prevented while DADS post-treatment reversed the LPS-induced increases in IL-1β and TNF-α levels in the hippocampus and prefrontal cortex in mice, suggesting inhibition of IL-1β and TNF-α production in the brain may mediate the pharmacological effect of DADS in depression prevention and therapy. Further clarifying the mechanism for the regulatory effect of DADS pretreatment and post-treatment on IL-1β and TNF-α expression and/or release in conditions of endotoxin exposure may help develop DADS as a preventive and/or therapeutic drug for depression induced by endotoxin as well as endotoxin-producing factors.
In the neuroinflammation hypothesis, the oxidative/nitrosative stress constitutes an important aspect of depression pathogenesis [54,55]. The activated immune system in the brain first induces a pro-inflammatory cascade, which includes a tremendous production of various pro-inflammatory cytokines, such as IL-1β and TNF-α, and then the increased pro-inflammatory cytokines promote the production of reactive oxygen species via enhancing nitrite synthesis and release [56]. The increased nitrite together with reactive oxygen species subsequently causes oxido-nitrosative stress which leads to behavioral abnormalities [57]. This hypothesis was evidenced further by our present findings that markers reflecting oxidative/nitrosative stress, such as MDA and nitrite, in the hippocampus and prefrontal cortex were increased markedly by a single LPS injection, and meanwhile the levels of GSH in the hippocampus and prefrontal cortex were reduced by LPS injection. Reversing the harmful changes upon endotoxin exposure would be beneficial for depression prevention and/or therapy. DADS administration may be a potential strategy for that purpose because both DADS pre-treatment and post-treatment were found to attenuate the LPS-induced increase in MDA and nitrite levels as well as the LPS-induced decrease in GSH levels in the hippocampus and prefrontal cortex.
In depression regulation, most anti-inflammatory compounds require a relatively long time to produce significant regulatory effects [16,37]. However, in the present study we found that an acute treatment is enough for DADS to produce prophylactic and therapeutic effects on LPS-induced depression-like behaviors in mice. What's the reason for this phenomenon? We noticed that in a previous study by Mello et al. (2013), researchers have reported a similar effect for doxycycline, an antibiotic used in clinic [39]. In that study, the preventive and reversing effects of imipramine and doxycycline on LPS-induced depression-like behavior in mice are considered to be associated with their anti-microbial activities [39]. It is well-known that the change in microbiota in the brain-gut axis is tightly linked with depression pathogenesis [58], and some antidepressants, such as ketamine and IL-6 receptor antagonist, have been shown to rapidly ameliorate depressionlike behavior and alter the composition of the gut microbiota in depressed animals [59,60]. Given that DADS can inhibit bacteria growth [26,27], it is reasonable to speculate that the herein observed prophylactic and reversing effect of DADS on LPS-induced depression-like behavior in mice may be mediated by its inhibitory effect on gut bacteria. This hypothesis should be clarified clearly in order to explain why DADS benefits the human health.

Conclusion
This study was designed to search drugs that prevent and/or rescue behavioral abnormalities induced by endotoxin exposure, and shows for the first time that similarly to imipramine both DADS pre-and post-treatment ameliorate LPS-induced depression-like behavior in mice, suggesting that daily supplementation of DADS or DADS-containing garlic might be an adjuvant method for the prevention of brain damages induced by repeated endotoxin exposure, a condition which could be observed in various pathological conditions, such as gut bacterial toxicosis.

Declarations
Availability of supporting data 16 All data generated or analyzed during this study are included in this work.