Curcumin Ameliorates Chronic Mild Stress-Induced Depressive-Like Behavior via HMGB1/TLR4/NF-κB Signaling Pathway

Background Depression, one of the most frequently-occurring psychiatric disorders worldwide, is a signi�cant in�ammatory disorder. The polyphenol curcumin (Cur), which is extracted from Curcuma longa, has marked anti-inammatory and anti ‐ oxidative effects against in�ammatory diseases. However, whether Cur has antidepressant effects and the possible mechanisms, are unclear. The present study aimed to assess Cur’s bene�cial effects on depressive-like behaviors using a chronic unpredictable mild stress (CUMS) model and its possible molecular mechanisms. Methods


Background
Among psychiatric disorders, depression is one of the most frequently-occurring, and is clinically characterized by feelings of guilt, cognitive dysfunction, body disturbances, and suicidal tendencies [1].
The World Health Organization (WHO) estimates that depression could become the second leading cause of disability after heart disease by 2030 [2,3].The pathophysiological symptoms of depression include de ciencies of in ammatory responses, cellular proliferation, dysregulated secretion of cytokines, and neuroplasticity [4].In ammation has been reported as an essential molecular mechanism of neuropsychiatric disorders, such as major depression [5,6].Depressive-like behavior might be closely related to elevation of in ammatory cytokines driven by oxygen stress, such as tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6), and Interleukin-1 beta (IL-1β) [7,8], which are also closely associated with depressive symptoms [9,10].Therefore, decreasing proin ammatory cytokine levels is an important method to treat depression [8,11,12].The identi cation of the optimal anti-in ammatory drugs for depression has become an urgent problem and requires further investigation.
In recent years, Chinese herbal extracts have been used as adjuvant therapy for neuroin ammatory diseases due to their anti-in ammatory activities [13][14][15].For example, the protective effects of Baicalin, apigenin, and Tanshinone IIA (Tan IIA) have been reported [8,16,17].Rhizome extracts of Curcuma longa Linnéa provide curcumin, a highly pleiotropic compound that interacts with in ammatory cytokines [18], which has been used as a remedy on to treat chronic in ammatory conditions, including cancers, chronic anterior uveitis, arthritis, pancreatitis, and in ammatory bowel disease [19][20][21][22].In the treatment of these chronic in ammatory diseases, curcumin demonstrates marked anti-in ammatory and anti-oxidation effects.In vitro and in vivo studies showed that curcumin might act as a TNF blocker by binding directly to TNF [23,24].Further investigation showed that curcumin alleviates oxidative stress and in ammation during the modulation of lung carcinogenesis via nuclear factor erythroid-2 related factor 2 (NRF2)-kelch like ECH associated protein 1 (KEAP1) signaling [25].Recent research demonstrated a certain therapeutic effect of curcumin on depression; however, the speci c mechanism remains unclear.
The highly conserved non-histone nuclear protein, high-mobility group box-1 (HMGB1), is a member of the damage-associated molecular patterns (DAMPs) or alarmins group of proteins [26,27].When cells are necrotic or damaged, HMGB1 in the nucleus can be passively released, which induces pro-in ammatory cytokines secretion by macrophages [28].Meanwhile, HMGB1 could be actively secreted by activated immune cells in response to in ammatory signals [29].Excess HMGB1 in the extracellular space binds with many receptors on the cell surface, such as Toll-like receptor 4 (TLR4) [30], and plays a crucial part in chronic and acute in ammation pathogenesis (e.g., in autoimmune diseases, atherosclerosis, cancer, and sepsis) [26].Increased HMGB1 levels in the central and peripheral nervous systems were observed in mice displaying symptoms of depression, leading to the proposal that that HMGB1 drives depression resulting from chronic stress [31,32].Moreover, in ammation mediated by HMGB1 in depressive-like behaviors might involve an imbalance between neurotoxic and neuroprotective factors, dysregulated kynurenine signaling, proin ammatory cytokines, and the Nod-like receptor family pyrin domain containing three (NLRP3) in ammasome [33].
Our previous experiment showed that Curcumin (Cur) reduced HMGB1 expression of in a rat model of chronic unpredictable mild stress (CUMS).Therefore, we hypothesized that Cur might ameliorate the induction of depressive-like behaviors by CUMS through the regulation of HMGB1 expression.Based on this hypothesis, we detected hippocampal TLR4/NF-kB pathway activation, in ammatory cytokines, markers of oxidative stress, and depressive-like behaviors to investigate the possible molecular mechanisms.

Animals
Sprague Dawley (SD) rats (male; approximately 6 to eight weeks old; 200 ± 20 g) were obtained from the Experimental Animal Center of Air Force Military Medical University (Xi'an, China).The animals were reared under conditions of 25 ± 2°C, light/dark cycle = 12 hours, and ad libitum access to water and food.The animal experiments were carried out acording to The National Institutes of Health guidelines for care and use of Laboratory Animals and were approved by the relevant ethics committee.

Grouping and treatment
Rats (n = 60) were assigned randomly to six groups: Control group, CUMS group, CUMS + Cur (50 mg/kg) group, CUMS + Cur (100 mg/kg) group, CUMS + Fluoxetine (Flu, antidepression drug) (20 mg/kg) group, and CUMS + Glycyrrhizic Acid (Gly, HMGB1 inhibitor) (10 mg/kg) group, with ten rats in each group.Rats in the control group were maintained under normal conditions.Rats in CUMS groups were subjected to CUMS according to the protocol of a previous study [34][35][36], and were scheduled randomly and changed weekly (Table 1).Cur was dissolved cocoa buffer at different concentrations; the other drugs were dissolved in water.All drugs were administered once per day between 08:00 and 09:00 h.The experimental procedure is shown in Fig. 1A.After a 7-day adaption period, all rats, except the control group, were exposed to CUMS for 28 days.Drugs were administrated to the speci c groups and behavior tests were assessed 2 weeks after drug administration (between 09:00 and 12:00 h).The cage is tilted 12 hours The cage is soiled 12 hours

Sucrose preference test (SPT)
A previously described protocol was used to carry out the SPT [37].Before the test, all rats were acclimated to a 1%, w/v sucrose solution for 24 h as a training period.After 23 h of deprivation of food and water, the rats were fed with water and 1% sucrose solution at the same time.The consumption of water and sucrose solution in the following 1 h were recorded.According to the ratio of sucrose solution consumption to total liquid, the sugar solution preference was obtained.

Forced swimming test (FST)
A previously described protocol was used to carry out the FST, with a minor change [38].In brief, individually, the rats were forced to swim in a glass cylinder (50 cm height ⋅ 20 cm diameter) containing water (30 cm height, 24 ± 2°C) for ten mins.A trained technician, who was blinded to the groups, recorded the rat's immobility time during the last 4 mins, which was de ned as the time that rats relaxed with no movement except keep their head above water.

Tail suspension test (TST)
A previously described protocol was used to carry out the TST, with a minor change [5].Rats were suspended individually via their tails using adhesive tape (approximately 50 cm above the oor).The test lasted for 6 mins.The immobility time was recorded during the last 4 mins by a trained technician who was blinded to groups, which was de ned as the time that rat relaxed without any struggling movemet.

Determination of oxidative stress markers and in ammatory cytokines in the hippocampus
To measure the oxidative stress markers and in ammatory cytokines, rat hippocampi were excised, homogenized in 1.0% KCl, centrifuged at 4°C for 10 min, and the supernatant retained.Enzyme-linked immunosorbent assay (ELISA) kits were used to determine the levels of pyruvate dehydrogenase (PDH), superoxide dismutase (SOD), glutathione (GSH), reactive oxygen species (ROS), IL-1β, IL-6, and TNF-α, following the manufacturer's protocols.

Immuno uorescence staining
After the behavioral tests, intracardial perfusion was used to sacri ce the rats.The hippocampi were excised and a cryostat (CM1800, Leica, Wetzlar, Germany) was used to prepare 20 µm longitudinal

Results
Cur alleviated depressive-like behavior in CUMSs exposed rats

Cur effects on sucrose consumption
Compared with that in the control group, the sucrose consumption in the CUMS group decreased signi cantly.Administration of Cur (50, 100 mg/kg), Flu (20 mg/kg), and Gly (10 mg/kg) increased the sucrose consumption signi cantly compared with that in the CUMS group (Fig. 1B).No signi cant difference was detected among the control, Cur (50, 100 mg/kg), Flu (20 mg/kg), and Gly (10 mg/kg) groups.

Effects of Cur on the immobility time in FST and TST
As shown in Fig. 1C and Fig. 1D, compared with the control group, a signi cant increase in the immobility time was observed in the CUMS group in the FST and TST tests.Administration of Cur (50, 100 mg/kg), Flu (20 mg/kg), and Gly (10 mg/kg) signi cantly decreased the immobility time compared with that in the CUMS group.The immobility times were not signi cantly different among the control, Cur (50, 100 mg/kg) Flu (20 mg/kg), and Gly (10 mg/kg) groups.

Statistical analysis
All data are shown as the mean ± the standard error of the mean (SEM).The data were analyzed using one way analysis of variance (ANOVA) together with Student-Newman-Keuls (SNK-q) test in SPSS 22.0 IBM Corp., Armonk, NY, USA).Statistical signi cance was indicated by P < 0.05.
Cur inhibited the level of oxidative stress in hippocampus exposed to CUMS As shown in Fig. 2, ROS expression increased signi cantly in the CUMS group compared with that in the control group.Meanwhile, GSH, SOD, and PDH activities were reduced signi cantly in the CUMS group compared with those in the control group.By contrast, treatment with Cur (50, 100 mg/kg), Flu (20 mg/kg), and Gly (10 mg/kg) decreased the hippocampal ROS generation and increased the activities of GSH, SOD, and PDH to varying degrees compared with those in the CUMS group.These activities and ROS levels were not signi cant different among the control, Cur (50, 100 mg/kg), Flu (20 mg/kg), and Gly (10 mg/kg) groups.
Cur reduced the in ammatory cytokine levels in the hippocampus exposed to CUMS As shown in Fig. 3A-3F, in ammatory cytokine levels, including TNF-α, IL-6, and IL-1β, increased signi cantly in the CUMS group compared with those in the control group.Cur (100 mg/kg), Flu (20 mg/kg), and Gly (10 mg/kg) treatment resulted in decreased in ammatory cytokine levels in the hippocampus to varying degrees compared with those in the CUMS group.There was no signi cant difference among the control, Cur (100 mg/kg), Flu (20 mg/kg), and Gly (10 mg/kg) groups.There was no signi cant difference between the Cur (50 mg/kg) and the CUMS groups.
As shown in Fig. 3G, HMGB1, IL-6, and IL-1β levels increased in the hippocampal cytoplasm of the CUMS group compared with those in the control group.Contrastingly, the Cur (50 and 100 mg/kg), Flu (20 mg/kg), and Gly (10 mg/kg) treatments resulted in varying degrees of reduction in the levels of these cytokines in the hippocampus compared with those in the CUMS group.Their levels among the Cur (100 mg/kg), Flu (20 mg/kg), and Gly (10 mg/kg) groups were not signi cantly different.
Cur regulated oxidative stress and in ammatory cytokines via NFκB/ HMGB1/ TLR4 signaling As shown in Fig. 4, compared with those in the control group, the CUMS group had increased levels of p-NF-κB p65, TLR4, and HMGB1, while the treatment with Cur (50 and 100 mg/kg), Flu (20 mg/kg), and Gly (10 mg/kg) dramatically restored their levels, to varying degrees.Among the control, Cur (50 and 100 mg/kg), Flu (20 mg/kg), and Gly (10 mg/kg) groups, there were no signi cant differences.

Discussion
Depressive disorder has a prevalence of 4.4% worldwide [39].Therefore, the discovery of new therapeutic targets to treat depressive disorder is imperative.Recent research suggests that the pathogenesis of depression involves neuroin ammation and oxidative stress injury [17], which is an important direction of anti-depressive therapy [8,11,12].Curcumin has powerful anti-in ammatory and antioxidation effects in chronic in ammatory disease; however, Cur's effects on depression and its mechanism are unclear.The results of the present study showed that CUMS-induced depressive-like behavior in rats could be ameliorated by treatment with Cur.Furthermore, Cur treatment exhibited antidepressant effects by ameliorating oxidative stress and neuroin ammation in the hippocampus, and the underlying mechanisms might be associated the HMGB1/TLR4/NF-κB pathway.
Patients with depression generally display many behavioral abnormalities, such as low mood, slow thinking, and cognitive function damage.Thus, the potential antidepressive activity of a drug are frequently assessed using behavioral experiments.The CUMS model is a recognized animal model of stress depression [34][35][36].It is reported that rats exposed to CUMS displayed phenotypes resembling depression and showed long-term changes to motivational states, anhedonia, and behavioral coping, which could be improved by treatment with antidepressants [40].Our results showed that the rats in the CUMS group showed signi cantly decreased sugar preference and increased immobility time in the FST and TST tests.Cur administration displayed antidepressive effects in CUMS-induced rats, as did treatment with Flu and Gly.
The vital roles of in ammatory cytokines and oxidative stress in chronic stressinduced psychiatric disorders are well documented [8,41].In the pathogenesis of a depressive disorder, upon chronic stress, the balance between antioxidant and oxidant factors shifts, resulting in the production of excess in ammatory cytokines and ROS [42].Many depressants act via their anti-in ammatory and antioxidative effects [43].Cur, a natural antioxidant, can capture or scavenge free radicals directly because of the phenolic hydroxyl group in its structure [44].Cur can exert a neuroprotective effect by inhibiting oxidative stress through the NRF2/protein kinase B (AKT) pathway, thereby preventing brain injury [45].In TNF-α-treated HaCaT cells, Cur was observed to inhibit IL-1β and IL-6 expression via the NF-κB and MAPK pathways [46].Herein, we revealed that Cur treatment increased antioxidant enzyme activities (PDH, SOD, and GSH), and decreased in ammatory factor expression (IL-6, TNF-α and IL-1β).These results agree with those of previous studies that demonstrated curcumin's anti-depressive effects acting via its antiin ammatory and anti-oxidative activities in CUMS.
The highly conserved nuclear protein HMGB1 is distributed widely in mammalian cells [47].HMBG1 has become a hotspot of in ammation research because of its pro-in ammatory effect, acting as a strong mediator of in ammation linking infection and tissue damage [33].In CUMS mice, the hippocampus [48] cerebral cortex [32] expressed high levels of HMGB1.Depression-like behavior can be induced by stress via NFκB/TLR4/HMGB1 signaling in the hippocampus [48].In animal models, a reduced sucrose preference and prolonged immobility in TST and FST indicate that HMGB1 mediation of in ammation induces depressive-like behaviors, which is consistent with the results of our previous study.In addition, western blotting analysis revealed that treatment with Cur reversed the high hippocampal HMGB1 levels and those of its downstream molecules, p-NF-κB p65, and TLR4, in CUMS rats similarly to Gly treatment.Thus, we concluded that HMGB1/TLR4/NF-κB signaling mediates the protection provide by Cur on the in ammatory cytokines, oxidative stress, and depressive-like behavior in rats exposed to CUMS.