http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–8 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.967783
ORIGINAL ARTICLE
Antidepressant-like effects of magnesium lithospermate B in a rat model of chronic unpredictable stress Wei Quan1*, Feihu Liu1*, Yan Zhang1*, Congmin Xie1, Bin Wu1, Jipeng Yin2, Lei Wang1, Wei Zhang3, Xiaohong Zhang1, and Qiangju Wu1
Pharmaceutical Biology Downloaded from informahealthcare.com by Nyu Medical Center on 04/12/15 For personal use only.
1
Xi’an Mental Health Center, Institute of Mental Health, Xi’an Medical University, Xi’an, China, 2State Key Laboratory of Cancer Biology, Xijing Hospital, Fourth Military Medical University, Xi’an, China, and 3Department of Medical Statistics, School of Preventive Medicine, Fourth Military Medical University, Xi’an, China Abstract
Keywords
Context: Magnesium lithospermate B (MLB), an active polyphenol acid of Danshen [Radix Salviae miltiorrhizae (Labiatae)], shows neuroprotective and anti-inflammatory effects in vivo and in vitro. Objective: We hypothesized that MLB might exert antidepressant-like effects by targeting the neuroinflammatory signals. Materials and methods: Sprague-Dawley rats were subjected to the chronic unpredictable stress (CUS) protocol. Rats in the control group received no CUS during the whole experiment. In the model group, rats were exposed to CUS for 7 weeks. From the beginning of the 5th week, model group rats were randomly grouped and subjected to different treatments. In the experiment, control and model group rats were intraperitoneally (i.p.) injected with saline. MLB was dissolved in saline to give a final concentration, and the rats were injected (i.p.) with 15, 30, or 60 mg/kg MLB once a day for 3 weeks. Results: MLB administration significantly reduced: (1) the immobility time in the forced swimming test (19 s, p50.05); (2) the immobility time in the tail suspension test (76.3 s, p50.05); (3) the corticosterone (CORT) concentrations in the serum (21.7 nmol/L, p40.05); (4) the pro-inflammatory cytokine levels in the serum – TNF-a (92.1 pg/ml, p50.05), IL-1b (86.9 pg/ ml, p50.05), and IL-6 (93.8 pg/ml, p50.05); (5) pro-inflammatory cytokine levels in tissue – TNF-a (3.2 pg/mg protein, p50.05), IL-1b (1.5 pg/mg protein, p40.05), and IL-6 (6.3 pg/mg protein, p50.05); and (6) phospho-NF-kB (1.6, p50.05) and phospho-IkB-a (0.4, p50.05) expression in tissue. Discussion and conclusion: The results suggested that MLB might exert therapeutic actions on depression-like behavior and the HPA axis hyperactivity in CUS rats, and the mechanisms underlying the antidepressant-like effects of MLB might be mediated by regulation of the expression of NF-kB and IkB-a in rats.
Forced swimming test, nuclear factor kappa B, pro-inflammatory cytokines, tail suspension test
Introduction Depression is a major mental disorder. According to recent epidemiological surveys, the lifetime incidence of depression is between 10% and 15% (Lepine & Briley, 2011). Currently, antidepressant drugs used in the treatment of major depressive disorders are believed to act on the central monoaminergic systems, mainly 5-hydroxytryptamine (5-HT) and noradrenergic synaptic neurotransmissions (Pazos, 2013). Selective serotonin reuptake inhibitors and noradrenaline reuptake inhibitors are effective in treating most depressive episodes, but about one-third of these patients show only partial or no *These authors contributed equally to this work. Correspondence: Xiaohong Zhang, Xi’an Mental Health Center, Institute of Mental Health, Xi’an Medical University, Xi’an 710061, China. Tel: +86 29 85551351. E-mail: [email protected]; Qiangju Wu, Xi’an Mental Health Center, Institute of Mental Health, Xi’an Medical University, Xi’an, 710061, China. E-mail: [email protected]
History Received 2 May 2014 Revised 19 August 2014 Accepted 28 August 2014 Published online 10 April 2015
response to the treatment (Taylor et al., 2011). The classical antidepressants based on the monoamine hypothesis of depression have been challenged due to their clinical limitations and adverse effects (Berton & Nestler, 2006; Hansen et al., 2005). Therefore, the development of more effective antidepressants is of great interest. Traditional Chinese medicine (TCM) has been widely used for the treatment of depression (Li et al., 2008; Zhang et al., 2007). Several ancient herb formula prescriptions designated to treat depressive-like symptoms are still frequently prescribed in China either alone or with conventional antidepressants (Guo et al., 2009; Molina-Hernandez et al., 2008; Tesch, 2003). Magnesium lithospermate B [MLB, also named salvianolic acid B (Sal B) magnesium salt, or magnesium tanshinoate B, Figure 1], a derivative of the caffeic acid tetramer and the major soluble ingredient in danshen [Radix Salviae miltiorrhizae, (Labiatae)], has been shown to induce several medicinal effects, such as vasodilating blood vessels,
2
W. Quan et al
Pharm Biol, Early Online: 1–8
Table 1. Schedule of chronic mild stress procedures. Day 1 2 3 4 5 6
Pharmaceutical Biology Downloaded from informahealthcare.com by Nyu Medical Center on 04/12/15 For personal use only.
Figure 1. Structure of MLB. Molecular formula: C38H28MgO16; molecular weight: 740.90. MLB, magnesium lithospermate B.
decreasing blood pressure, acting as an antioxidant, and performing free radical scavenging activities (Liu et al., 2009; Paik et al., 2011; Qu et al., 2011; Zhang et al., 2004). Recently, MLB was suggested to be responsible for antiischemic neuroprotection by its effective inhibition of the Na+/K+-ATPase (Tzen et al., 2007). The ability of MLB to cross the blood–brain barrier (BBB) (Liu et al., 2006) and protect SH-SY5Y cells against MPP(+)-induced apoptosis by relieving oxidative stress and modulating the apoptotic process of neural stem cells (Zeng et al., 2010) has also been shown. Notably, MLB could ameliorate learning and memory dysfunctions induced by cerebral transient ischemia (Du et al., 2000). In light of these previous findings, we hypothesize that MLB might play an important role in the pathogenesis of depression. In addition, our present study has established that MLB could attenuate phospho-NF-kB levels in myocardial tissues, and this decrease in phospho-NF-kB was associated with a decrease in tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), and interleukin-6 (IL-6) in the serum (Quan et al., 2013). As we already know, inflammatory mechanisms play a role in the cause of major depressive disorders (Raedler, 2011). According to the cytokine hypothesis of depression, inflammatory cytokines act on neural circuits to evoke the behavioral and physiological changes observed in depression (Piser, 2010). The elevated systemic levels of TNF-a, IL-1b, and IL-6 were found to be reversed by antidepressants (Berton & Nestler, 2006). This has raised the potential of exploiting cytokine-regulated pathways in the development of novel antidepressants. Together, these data support the possibility that MLB may exert antidepressant-like activity by targeting the neuroinflammatory signals, and this might provide a new therapeutic target for the treatment of depression.
Materials and methods Animals Adult male Sprague–Dawley rats (220–240 g, 10–12 weeks old) were obtained from the experimental animal center [Animal Certificate of Conformity: SCXK-(Army) 2007-007] of the FMMU, Xi’an 710032, China. All animals were cared according to the guidelines of National Institutes of the Health Guide for the Care and Use of Laboratory Animals.
7
Stressors
Time
10 h crowded cage (5–6 rats per cage) a.m.–p. m. 15 min forced swim (22 C) p.m. 15 h wet bedding 4 h restraint 24 h food deprivation a.m. 5 min cold swim (4 C) p.m. 4 h restraint 24 h water deprivation a.m. 2 h cold stress (15 C) a.m. 4 h restraint p.m. 4 h restraint a.m. 2 min tail pinch p.m.
Chemicals and reagents MLB (molecular formula: C36H28MgO16, molecular weight: 740.90, purity495%) was provided by Xi’an Honson Biotechnology Co., Ltd (Xi’an, China). IL-1b (limit detection: 10 pg/ml, variability coefficients: 59.8%), IL-6 (limit detection: 16 pg/ml, variability coefficients: 510%), and TNF-a (limit detection: 16 pg/ml, variability coefficients: 510%) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Westang Reagent Company (Shanghai, China). The corticosterone (CORT) ELISA kit was purchased from Cusabio Biotech (Wuhan, China). IMI (imipramine) and western blot reagents were purchased from Sigma (St. Louis, MO). Anti-phospho-IkB-a, anti-phospho-NF-kB, and antib-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Experimental protocols The chronic unpredictable stress (CUS) protocol was modified from the procedures described by Katz (1981) and Willner et al. (1987). The protocol consisted of a variety of sequential stressors applied randomly every day for 7 weeks (Table 1). Rats in the control group received no CUS during the whole experiment. In the model group, rats were exposed to CUS for 7 weeks. From the beginning of the 5th week, model group rats were randomly grouped and subjected to different treatments (Figure 2). Part 1: To investigate the antidepressant-like activity of MLB, rats were divided randomly into four large groups: control (n ¼ 8), model (n ¼ 8), MLB (n ¼ 8), and IMI groups (n ¼ 8). Part 2: To explore the underlying therapeutic mechanism of MLB, rats were randomly divided into three groups (n ¼ 8 in each subgroup): control, model, and MLB groups. In the Part 1 and Part 2 experiments, control and model rats were intraperitoneally (i.p.) injected with saline. MLB was dissolved in saline to give three different final concentrations, and rats were injected (i.p.) with 15, 30, and 60 mg/kg MLB once a day for 3 weeks. Open-field test The open-field test (OFT) was carried out at the end of 1st, 4th, and 7th week. The open-field apparatus consisted of a
Antidepressant-like effects of MLB
DOI: 10.3109/13880209.2014.967783
3
Pharmaceutical Biology Downloaded from informahealthcare.com by Nyu Medical Center on 04/12/15 For personal use only.
Figure 2. Study design. Experimental groups and protocols for the antidepressant-like effects by MLB in rats. MLB, magnesium lithospermate B; CUS, chronic unpredictable stress.
square wooden arena (100 cm 100 cm 40 cm) with a black surface covering the inside walls. The floor of the wooden arena was divided equally into 25 squares marked with black lines. During the test, the rats were placed individually into the center of the arena and allowed to explore freely. The number of crossings (squares crossed with all paws) was recorded during a test period of 5 min. This apparatus was cleaned with a detergent and dried after occupancy by each rat (Rodrigues et al., 1996). Forced swimming test (FST) The forced swimming test (FST) was carried out at the end of 7th week. Rats were forced to swim in a glass cylinder (diameter 26 cm and height 60 cm) containing water at a depth of 35 cm and a temperature of 25 C. The water was changed after each test. Three types of behaviors were monitored for 5 min: immobility, swimming, and climbing. Immobility was defined as floating with no activity other than those necessary to keep the head above the water. Swimming was defined as active movement throughout the cylinder, including crossing into another quadrant. Climbing was defined as upwarddirected movement of the forepaws against the cylinder walls (Porsolt et al., 1977). Tail suspension test The tail suspension test (TST) was carried out at the end of 7th week. The tail suspension test was performed as previously described (Svenningsson et al., 2007). Antidepressant-like behavior is identified as decreased immobility of the rats. Briefly, 30 min after the last drug treatment, the rats were suspended by the tail from a ledge with adhesive tape (approximately 1 cm from the tip of the tail). The distance between the tip of the nose of the rat and the floor was approximately 5 cm. Rats were partitioned to avoid interference during the test. Immobility was defined as the absence of
movement and was scored over a 5 min trial by an observer blinded to the drug treatment. Measurement of CORT in the serum The measurement of CORT in serum was carried out at the end of 7th week. The blood samples were prepared by centrifugation of the collected blood samples (1000 rpm for 15 min) and then stored at 80 C for biochemical determinations. The serum levels of CORT were measured using ELISA kits according to the manufacturer’s instructions. Measurement of IL-1b, IL-6, and TNF-a in the serum In brief, the blood sample was prepared as previously described. The levels of IL-1b, IL-6, and TNF-a in the serum were measured using ELISA kits according to the manufacturer’s instructions. Measurement of tissue IL-1b, IL-6, and TNF-a The measurement of tissue IL-1b, IL-6, and TNF-a levels was carried out at the end of 7th week. The brains were removed, and then the hypothalamus and the hippocampus were dissected out of the brain and placed onto an ice cold plate. All the tissue samples were quickly frozen and stored in a deep freezer at 80 C until assayed. The tissue samples were weighed and then homogenized with an ultrasonic disruptor in ice cold saline. The sample was centrifuged at 1000 rpm for 20 min at 4 C, and the supernatant was removed into a tube for detection. The levels of IL-1b, IL-6, and TNF-a in the tissue were measured using ELISA kits according to the manufacturer’s instructions. Western blot analysis Western blot analysis was carried out at the end of 7th week. Proteins were extracted from brain tissue and protein
4
W. Quan et al
concentrations were determined. The total nuclear protein 50 mg was resolved on a 15% SDS-polyacrylamide gel. The fractionated proteins were electrophoretically transferred to an immobilon polyvinylidene difuride membrane and probed with rabbit polyclone anti-phospho-NF-kB and anti-phosphoIkB-a at overnight at 4 C followed by incubation with the corresponding secondary antibodies at room temperature for 2 h as previously described. The blots were visualized with the ECL-Plus reagent (GE Healthcare, Piscataway, NJ). In all immunoblotting experiments, the blots were reprobed with an anti-b-actin antibody to control protein loading.
Pharmaceutical Biology Downloaded from informahealthcare.com by Nyu Medical Center on 04/12/15 For personal use only.
Statistical analysis First, we checked whether the data were normally distributed. If the data were normally distributed, we planned to use a parametric analysis method, and if not, we planned to conduct a nonparametric statistical analysis. All the data showed normal distribution; therefore, we conducted a parametric analysis. Continuous variables that approximated the normal distribution were expressed as the mean ± standard deviation (SD). Comparison between groups was subjected to ANOVA, followed by Tukey’s multiple comparison tests. A level of p50.05 was considered to be statistically significant. The statistical analyses were performed using GraphPad Prism software Version 5.01 (GraphPad Inc., LaJolla, CA).
Pharm Biol, Early Online: 1–8
Effects of MLB on the FST The FST and the TST are behavioral despair tests useful for the evaluation of antidepressant drugs (Shirayama et al., 2002). To examine the antidepressant-like effects of MLB, we first performed the FST in rats. In the FST, rats given MLB at 60 mg/kg (F(5,42) ¼ 6.394, p50.05, 94.0 ± 10.2 s versus 113.3 ± 12.4 s, Figure 4) exhibited a significantly shorter duration of immobility compared with the model group. ANOVA showed no significant differences in the FST results between the other two MLB groups, or any significant differences between the MLB-treated (60 mg/kg) and IMItreated rats (F(5,42) ¼ 6.394, p40.05, 94.0 ± 10.2 s versus 88.1 ± 17.0 s, Figure 4). These results suggest that MLB stimulates an antidepressant effect that is similar to that of IMI during the FST. Effects of MLB on the TST Next, we performed the TST in rats. The data demonstrated that the immobility time was significantly decreased in rats treated with MLB at the doses of 30 and 60 mg/kg compared with saline-treated rats (F(5,42) ¼ 47.73, p50.05, 131.6 ± 23.3 s versus 207.9 ± 15.4 s, Figure 4). There were no
Results Effects of MLB on the OFT The effect of CUS on the general activity of the rats and the effect of MLB on the models were examined during the OFT. The test showed that 4 weeks of CUS lead to a significant decrease in the number of crossings (F(5,42) ¼ 21.34, p50.05, 31 ± 5 versus 75 ± 9, Figure 3). After treatment for 3 weeks with MLB or IMI, the reduced number of crossings in the model group was significantly reversed during the OFT (F(5,42) ¼ 21.34, p50.05, 25 ± 9, 35 ± 7 versus 14 ± 8, Figure 3), compared with the model group.
Figure 3. Effects of CUS in rats and antidepressant-like effects of MLB in rats during the OFT. MLB, magnesium lithospermate B; OFT, openfield test. Values are expressed as the mean ± S.D. n ¼ 8. Significance was determined by ANOVA followed by Tukey’s test. #p50.05 versus the control group, *p50.05 versus the model group.
Figure 4. Antidepressant-like effects of MLB in rats during the (A) FST and (B) TST. MLB, magnesium lithospermate B; FST, forced swimming test; TST, tail suspension test. Values are expressed as the mean ± S.D. n ¼ 8. Significance was determined by ANOVA followed by Tukey’s test. #p50.05 versus the control group, *p50.05 versus the model group.
DOI: 10.3109/13880209.2014.967783
significant differences between the MLB (60 mg/kg)treated group and the IMI-treated group (F(5,42) ¼ 47.73, p40.05, 131.6 ± 23.3 s versus 118.4 ± 13.5 s, Figure 4). In conclusion, MLB reduced the immobility time in the rats during the TST.
Antidepressant-like effects of MLB
5
Effects of MLB on CORT levels in serum To confirm our behavioral results, we subsequently assessed the activity of the HPA axis in the rats. In agreement with this hypothesis, the CUS rats displayed hyperactivity of the HPA axis in the present study, as indicated by the elevation of serum CORT concentrations. Serum CORT concentrations of the CUS rats were significantly higher than those of the control rats (F(2,21) ¼ 5.93, p50.05, 108.5 ± 37.5 nmol/L versus 65.3 ± 13.5 nmol/L, Figure 5). Treatment with MLB decreased the elevated CORT level (F(2,21) ¼ 5.93, p40.05, 86.8 ± 17.8 nmol/L versus 108.5 ± 37.3 nmol/L, Figure 5).
Pharmaceutical Biology Downloaded from informahealthcare.com by Nyu Medical Center on 04/12/15 For personal use only.
Effects of MLB on IL-1b, IL-6, and TNF-a levels in serum
Figure 5. Effects of MLB on CORT levels in the serum. MLB, magnesium lithospermate B; CORT, corticosterone. Values are expressed as the mean ± S.D. n ¼ 8. Significance was determined by ANOVA followed by Tukey’s test. #p50.05 versus the control group, *p50.05 versus the model group.
Figure 6. Effects of MLB on (A) IL-1b, (B) IL-6, and (C) TNF-a levels in serum. MLB, magnesium lithospermate B; IL-1b, interleukin 1b; IL-6, interleukin 6; TNF-a, tumor necrosis factor-a. Values are expressed as the mean ± S.D. n ¼ 8. Significance was determined by ANOVA followed by Tukey’s test. # p50.05 versus the control group, *p50.05 versus the model group.
Accumulating evidence indicates that pro-inflammatory cytokines such as TNF-a, IL-1b, and IL-6 play a critical role in the pathogenesis of depression (O’Donovan, 2014). In this study, we found that the levels of TNF-a, IL-1b, and IL-6 in serum were significantly elevated in the model group (TNF-a: F(2,21) ¼ 88.93, p50.05, 181.9 ± 24.1 pg/ml versus 74.2 ± 13.9 pg/ml; IL-1b: F(2,21) ¼ 69.76, p50.05, 198.2 ± 22.0 pg/ml versus 84.8 ± 12.1 pg/ml; IL-6: F(2,21) ¼ 54.99, p50.05, 193.6 ± 24.7 pg/ml versus 105.2 ± 18.4 pg/ ml, Figure 6), but markedly decreased with the administration of MLB (60 mg/kg) (TNF-a: F(2,21) ¼ 88.93, p50.05, 89.8 ± 11.9 pg/ml versus 181.9 ± 24.1 pg/ml; IL-1b: F(2,21) ¼ 69.76, p50.05, 111.3 ± 24.0 pg/ml versus 198.2 ± 22.0 pg/ml;
6
W. Quan et al
Pharm Biol, Early Online: 1–8
Pharmaceutical Biology Downloaded from informahealthcare.com by Nyu Medical Center on 04/12/15 For personal use only.
Figure 7. Effects of MLB on the tissue levels of (A) IL-1b, (B) IL-6, and (C) TNF-a. MLB, magnesium lithospermate B; IL-1b, interleukin 1b; IL-6, interleukin 6; TNF-a, tumor necrosis factor-a. Values are expressed as the mean ± S.D. n ¼ 8. Significance was determined by ANOVA followed by Tukey’s test. # p50.05 versus the control group, *p50.05 versus the model group.
IL-6: F(2,21) ¼ 54.99, p50.05, 99.8 ± 16.1 pg/ml versus 193.6 ± 24.7 pg/ml, Figure 6). Effects of MLB on the tissue levels of TNF-a, IL-1b, and IL-6 The tissue levels of TNF-a, IL-1b, and IL-6 were very low in the control group, but markedly increased in the model group (TNF-a: F(2,21) ¼ 17.00, p50.05, 12.2 ± 3.2 pg/mg protein versus 4.9 ± 2.3 pg/mg protein; IL-1b: F(2,21) ¼ 6.965, p50.05, 17.9 ± 6.2 pg/mg protein versus 10.1 ± 2.2 pg/mg protein; IL-6: F(2,21) ¼ 10.57, p50.05, 21.7 ± 5.7 pg/mg protein versus 11.7 ± 3.1 pg/mg protein, Figure 7). MLB attenuated the tissue levels of TNF-a, IL-1b, and IL-6 (TNF-a: F(2,21) ¼ 17.00, p50.05, 9.0 ± 1.9 pg/mg protein versus 12.2 ± 3.2 pg/mg protein; IL-1b: F(2,21) ¼ 6.965, p50.05, 16.4 ± 3.9 pg/mg protein versus 17.9 ± 6.2 pg/mg protein; IL-6: F(2,21) ¼ 10.57, p50.05, 15.4 ± 4.1 pg/mg protein versus 21.7 ± 5.7 pg/mg protein, Figure 7). These changes in the tissue were consistent with the pro-inflammatory cytokine results seen in the serum. One interpretation of these results could be that MLB prevented depression through the regulation of inflammatory responses. Effects of MLB on phosphorylated proteins that are involved in inflammation Furthermore, exploring the mechanism of action involved in inflammation is important. Activation of NF-kB in the
pathogenesis of depression is well accepted. Therefore, the expression of phospho-IkB-a and phospho-NF-kB was determined by western blot analysis. Phospho-IkB-a and phospho-NF-kB were highly expressed in the model group (p-IkB-a: F(2,9) ¼ 214.0, p50.05, 2.2 ± 0.3 versus 1.0 ± 0.0; p-NF-kB: F(2,9) ¼ 214.0, p50.05, 3.0 ± 0.3 versus 1.0 ± 0.0, Figure 8), but were inhibited by treatment with MLB (p-IkB-a: F(2,9) ¼ 214.0, p50.05, 1.8 ± 0.2 versus 2.2 ± 0.3; p-NF-kB: F(2,9) ¼ 214.0, p50.05, 1.4 ± 0.3 versus 3.0 ± 0.3, Figure 8).
Discussion TCM is widely believed to be beneficial, but, for the most part, neither the active components nor their molecular targets are well defined. Therefore, understanding the active component(s) and the mechanism(s) of action can make TCM more acceptable (Sandur et al., 2007). Recently, studies have pointed to the possibility that MLB may act on the neuroinflammatory signals that lead to antidepressant-like effects. However, the particular effects and underlying mechanisms have yet to be elucidated. The FST and the TST are behavioral despair tests useful for probing the pathological mechanism of depression and for the evaluation of antidepressant drugs (Feng et al., 2012). These tests are also well established for screening new, potent antidepressant drugs in rats. The characteristic behavior scored in both tests is termed immobility, which reflects a behavioral state of despair (learned helplessness), as seen in
Antidepressant-like effects of MLB
DOI: 10.3109/13880209.2014.967783
7
Pharmaceutical Biology Downloaded from informahealthcare.com by Nyu Medical Center on 04/12/15 For personal use only.
Figure 8. Effects of MLB on phosphorylated proteins that are involved in inflammation. (A) Phosphorylation of IkB-a and NF-kB was analyzed by western blot. (B) Graphs showing the protein ratio of phospo-IkBa/b-actin and phospo-NF-kB/b-actin. MLB, magnesium lithospermate B; NF-kB, nuclear factor kappa B. Values are expressed as the mean ± S.D. n ¼ 8. Significance was determined by ANOVA followed by Tukey’s test. # p50.05 versus the sham group, *p50.05 versus the control group.
human depression (Lucki et al., 2001). To investigate whether MLB can produce chronic changes in depression-related behavior during the FST and the TST, we treated rats for 7 weeks with a variety of sequential stressors applied randomly every day. MLB caused a reduction in the immobility time during the FST and the TST. The results presented here show, to our knowledge for the first time, that MLB is effective in producing significant antidepressant-like activity when assessed during the FST and the TST. The results of the behavioral investigation proved the antidepressant-like effect of MLB at the tested doses. To confirm our behavioral results, we subsequently assessed the activity of the HPA axis in the rats. The stress response is mediated by the HPA system, and the HPA axis is considered to be the ‘‘final common pathway’’ in the pathogenesis of depression (Swaab et al., 2005). In agreement with this hypothesis, the CUS rats in the present study displayed hyperactivity of the HPA axis as indicated by the elevation of serum CORT concentrations. Consistent with the results of the behavior tests, this change was reversed by treatment with MLB, indicating the regulatory effect of MLB on the HPA axis dysfunction. MLB was reported to protect against the ischemia/ reperfusion-induced injury in the rat brain in vivo and in vitro (Quan et al., 2014; Song et al., 2014). MLB has also been demonstrated to inhibit lipopolysaccharide-induced expression of inflammatory molecules (Zhao et al., 2013). Inhibition of neuroinflammation may represent a novel mechanism of action for antidepressant treatment (Wong et al., 2008). Several recent studies have reported that some natural products such as omega-3 fatty acids and curcumin induced antidepressant effects through the modulation of oxidative reactions and inflammatory cytokine production in the brain (Lin et al., 2011; Su, 2009; Xu et al., 2005). The results of our and other previous studies led us to hypothesize that MLB may act as an antidepressant via regulating inflammatory responses. To further clarify the antidepressant mechanisms of MLB, pro-inflammatory cytokines, including TNF-a, IL-1b, and IL-6, were investigated in this study. A previous study demonstrated that the elevated systemic levels of pro-inflammatory cytokines are decreased by antidepressants (Yang et al., 2011). Our data show that the levels of TNF-a, IL-1b, and IL-6 in the serum increased in the model group, but the increase of these inflammatory cytokines was significantly decreased with MLB treatment, which suggests that the antidepressant-like effects of MLB could be attributed to the suppression of inflammatory responses via inhibition of
pro-inflammatory cytokines. This notion was supported by the alteration of TNF-a, IL-1b, and IL-6 in brain tissue. Further studies were conducted to investigate the possible mechanism in our study, such as signal transduction pathways underlying the antidepressant-like effects of MLB. Given that NF-kB is a ubiquitously expressed transcription factor that has a significant role in both the inflammatory response and the cell survival in the pathogenesis of depression (Lee et al., 2012), our study was centered on the NF-kB signaling pathway. NF-kB is sequestered in the cytoplasm in an inactive form by its association with IkB-a, which prevents NF-kB from translocating into the nucleus (Hinz & Scheidereit, 2014). However, activation of NF-kB signaling results from the phosphorylation-induced proteolytic degradation of IkB-a. Free NF-kB then translocates to the nucleus, where it induces transcription of a variety of genes, including TNF-a and IL-6. Our study indicates that MLB could attenuate phospho-IkB-a and phospho-NF-kB levels in heart tissue, and this was associated with a decrease in TNF-a, IL-1b, and IL-6 in the serum. The results indicate that the anti-inflammatory effect and the antidepressant-like effect of MLB relied heavily on the inhibition of the NF-kB signaling pathway. Undoubtedly, our research has a number of limitations. Although MLB clearly reduced the inflammatory markers and alleviated depression significantly, our results could not directly prove that MLB exerted its protection solely through an anti-inflammatory effect. More compelling evidence should be provided to support our current hypothesis.
Conclusion In our study, treatment with MLB significantly improved the depression-like behaviors and the HPA axis hyperactivity in CUS rats. Moreover, MLB also significantly decreased the levels of TNF-a, IL-6, and IL-1b. Based on our results, we demonstrated that MLB had therapeutic effects on the CUSinduced depression model in rats, and these effects might be related, in large part, to the suppression of the inflammatory response via inhibition of NF-kB activation.
Declaration of interest The authors report that they have no conflicts of interest. This study was supported by the National Natural Science Foundation of China (No. 81202938), Science and Technology Research Development Plan Project of Shaanxi Province (No. 2011KJXX65).
8
W. Quan et al
Pharmaceutical Biology Downloaded from informahealthcare.com by Nyu Medical Center on 04/12/15 For personal use only.
References Berton O, Nestler EJ. (2006). New approaches to antidepressant drug discovery: Beyond monoamines. Nat Rev Neurosci 7:137–51. Du GH, Qiu Y, Zhang JT. (2000). Salvianolic acid B protects the memory functions against transient cerebral ischemia in mice. J Asian Nat Prod Res 2:145–52. Feng Y, You Z, Yan S, et al. (2012). Antidepressant-like effects of salvianolic acid B in the mouse forced swim and tail suspension tests. Life Sci 90:1010–14. Guo JY, Li CY, Ruan YP, et al. (2009). Chronic treatment with celecoxib reverses chronic unpredictable stress-induced depressive-like behavior via reducing cyclooxygenase-2 expression in rat brain. Eur J Pharmacol 612:54–60. Hansen RA, Gartlehner G, Lohr KN, et al. (2005). Efficacy and safety of second-generation antidepressants in the treatment of major depressive disorder. Ann Intern Med 143:415–26. Hinz M, Scheidereit C. (2014). The IkappaB kinase complex in NFkappaB regulation and beyond. EMBO Rep 15:46–61. Katz RJ. (1981). Animal models and human depressive disorders. Neurosci Biobehav Rev 5:231–46. Lee YH, Kim SH, Kim Y, et al. (2012). Inhibitory effect of the antidepressant imipramine on NF-kappaB-dependent CXCL1 expression in TNFalpha-exposed astrocytes. Int Immunopharmacol 12: 547–55. Lepine JP, Briley M. (2011). The increasing burden of depression. Neuropsychiatr Dis Treat 7:3–7. Li LT, Wang SH, Ge HY, et al. (2008). The beneficial effects of the herbal medicine Free and Easy Wanderer Plus (FEWP) and fluoxetine on post-stroke depression. J Altern Complement Med 14:841–6. Lin TY, Lu CW, Wang CC, et al. (2011). Curcumin inhibits glutamate release in nerve terminals from rat prefrontal cortex: Possible relevance to its antidepressant mechanism. Prog Neuropsychopharmacol Biol Psychiatry 35:1785–93. Liu CS, Cheng Y, Hu JF, et al. (2006). Comparison of antioxidant activities between salvianolic acid B and Ginkgo biloba extract (EGb761). Acta Pharmacol Sin 27:1137–45. Liu X, Chen R, Shang Y, et al. (2009). Superoxide radicals scavenging and xanthine oxidase inhibitory activity of magnesium lithospermate B from Salvia miltiorrhiza. J Enzyme Inhib Med Chem 24:663–8. Lucki I, Dalvi A, Mayorg AJ. (2001). Sensitivity to the effects of pharmacologically selective antidepressants in different strains of mice. Psychopharmacology 155:315–22. Molina-Hernandez M, Tellez-Alcantara NP, Perez-Garcia J, et al. (2008). Desipramine or glutamate antagonists synergized the antidepressantlike actions of intra-nucleus accumbens infusions of minocycline in male Wistar rats. Prog Neuropsychopharmacol Biol Psychiatry 32: 1660–6. O’Donovan A. (2014). Inflammation and depression: Unraveling the complex interplay in inflammatory bowel disease. J Pediatr Gastroenterol Nutr 58:541–2. Paik YH, Yoon YJ, Lee HC, et al. (2011). Antifibrotic effects of magnesium lithospermate B on hepatic stellate cells and thioacetamide-induced cirrhotic rats. Exp Mol Med 43:341–9. Pazos A. (2013). Future trends in the development of new antidepressant drugs. Curr Pharm Des 20:3717. Piser TM. (2010). Linking the cytokine and neurocircuitry hypotheses of depression: A translational framework for discovery and development of novel anti-depressants. Brain Behav Immun 24:515–24. Porsolt RD, Bertin A, Jalfre M. (1977). Behavioral despair in mice: A primary screening test for antidepressants. Arch Int Pharmacodyn Ther 229:327–36. Qu J, Ren X, Hou RY, et al. (2011). The protective effect of magnesium lithospermate B against glucose-induced intracellular oxidative damage. Biochem Biophys Res Commun 411:32–9. Quan W, Wei G, Zhou D, et al. (2013). Magnesium lithospermate B reduces myocardial ischemia/reperfusion injury in rats via regulating the inflammation response. Pharm Biol 51:1355–62.
Pharm Biol, Early Online: 1–8
Quan W, Wu B, Bai Y, et al. (2014). Magnesium lithospermate B improves myocardial function and prevents simulated ischemia/ reperfusion injury-induced H9c2 cardiomyocytes apoptosis through Akt-dependent pathway. J Ethnopharmacol 151:714–21. Raedler TJ. (2011). Inflammatory mechanisms in major depressive disorder. Curr Opin Psychiatry 24:519–25. Rodrigues AL, Rocha JB, Mello CF, Souza DO. (1996). Effect of perinatal lead exposure on rat behaviour in open-field and two-way avoidance tasks. Pharmacol Toxicol 79:150–6. Sandur SK, Pandey MK, Sung B, et al. (2007). Curcumin, demethoxycurcumin, bisdemethoxycurcumin, tetrahydrocurcumin and turmerones differentially regulate anti-inflammatory and anti-proliferative responses through a ROS-independent mechanism. Carcinogenesis 28:1765–73. Shirayama Y, Chen AC, Nakagawa S, et al. (2002). Brain-derived neurotrophic factor produces antidepressant effects in behavioral models of depression. J Neurosci 22:3251–61. Song S, Liu W, Liu F, et al. (2014). Magnesium lithospermate B reduces inflammatory response in a mouse model of hepatic ischemiareperfusion injury. Cell Biochem Biophys 69:347–55. Su KP. (2009). Biological mechanism of antidepressant effect of omega-3 fatty acids: How does fish oil act as a mind–body interface? Neurosignals 17:144–52. Svenningsson P, Tzavara ET, Qi H, et al. (2007). Biochemical and behavioral evidence for antidepressant-like effects of 5-HT6 receptor stimulation. J Neurosci 27:4201–9. Swaab DF, Bao AM, Lucassen PJ. (2005). The stress system in the human brain in depression and neurodegeneration. Ageing Res Rev 4: 141–94. Taylor D, Meader N, Bird V, et al. (2011). Pharmacological interventions for people with depression and chronic physical health problems: Systematic review and meta-analyses of safety and efficacy. Br J Psychiatry 198:179–88. Tesch BJ. (2003). Herbs commonly used by women: An evidence-based review. Am J Obstet Gynecol 188:44–55. Tzen JT, Jinn TR, Chen YC, et al. (2007). Magnesium lithospermate B possesses inhibitory activity on Na+, K+-ATPase and neuroprotective effects against ischemic stroke. Acta Pharmacol Sin 28:609–15. Willner P, Towell A, Sampson D, et al. (1987). Reduction of sucrose preference by chronic unpredictable mild stress, and its restoration by a tricyclic antidepressant. Psychopharmacology 93:358–64. Wong ML, Dong C, Maestre-Mesa J, Licinio J. (2008). Polymorphisms in inflammation-related genes are associated with susceptibility to major depression and antidepressant response. Mol Psychiatry 13: 800–12. Xu Y, Ku BS, Yao HY, et al. (2005). Antidepressant effects of curcumin in the forced swim test and olfactory bulbectomy models of depression in rats. Pharmacol Biochem Behav 82:200–6. Yang JJ, Zhou ZQ, Yang C. (2011). Letter to the editor: Does ketamine exert a fast-acting antidepressant effect via inhibition of pro-inflammatory cytokines? Psychol Med 41:1787–9. Zeng G, Tang T, Wu HJ, et al. (2010). Salvianolic acid B protects SHSY5Y neuroblastoma cells from 1-methyl-4-phenylpyridiniuminduced apoptosis. Biol Pharm Bull 33:1337–42. Zhang Y, Akao T, Nakamura N, et al. (2004). Magnesium lithospermate B is excreted rapidly into rat bile mostly as methylated metabolites, which are potent antioxidants. Drug Metab Dispos 32: 752–7. Zhang ZJ, Kang WH, Li Q, Tan QR. (2007). The beneficial effects of the herbal medicine Free and Easy Wanderer Plus (FEWP) for mood disorders: Double-blind, placebo-controlled studies. J Psychiatr Res 41:828–36. Zhao M, Chen YH, Dong XT, et al. (2013). Folic acid protects against lipopolysaccharide-induced preterm delivery and intrauterine growth restriction through its anti-inflammatory effect in mice. PLoS One 8: e82713.
ORIGINAL ARTICLE
Antidepressant-like effects of magnesium lithospermate B in a rat model of chronic unpredictable stress Wei Quan1*, Feihu Liu1*, Yan Zhang1*, Congmin Xie1, Bin Wu1, Jipeng Yin2, Lei Wang1, Wei Zhang3, Xiaohong Zhang1, and Qiangju Wu1
Pharmaceutical Biology Downloaded from informahealthcare.com by Nyu Medical Center on 04/12/15 For personal use only.
1
Xi’an Mental Health Center, Institute of Mental Health, Xi’an Medical University, Xi’an, China, 2State Key Laboratory of Cancer Biology, Xijing Hospital, Fourth Military Medical University, Xi’an, China, and 3Department of Medical Statistics, School of Preventive Medicine, Fourth Military Medical University, Xi’an, China Abstract
Keywords
Context: Magnesium lithospermate B (MLB), an active polyphenol acid of Danshen [Radix Salviae miltiorrhizae (Labiatae)], shows neuroprotective and anti-inflammatory effects in vivo and in vitro. Objective: We hypothesized that MLB might exert antidepressant-like effects by targeting the neuroinflammatory signals. Materials and methods: Sprague-Dawley rats were subjected to the chronic unpredictable stress (CUS) protocol. Rats in the control group received no CUS during the whole experiment. In the model group, rats were exposed to CUS for 7 weeks. From the beginning of the 5th week, model group rats were randomly grouped and subjected to different treatments. In the experiment, control and model group rats were intraperitoneally (i.p.) injected with saline. MLB was dissolved in saline to give a final concentration, and the rats were injected (i.p.) with 15, 30, or 60 mg/kg MLB once a day for 3 weeks. Results: MLB administration significantly reduced: (1) the immobility time in the forced swimming test (19 s, p50.05); (2) the immobility time in the tail suspension test (76.3 s, p50.05); (3) the corticosterone (CORT) concentrations in the serum (21.7 nmol/L, p40.05); (4) the pro-inflammatory cytokine levels in the serum – TNF-a (92.1 pg/ml, p50.05), IL-1b (86.9 pg/ ml, p50.05), and IL-6 (93.8 pg/ml, p50.05); (5) pro-inflammatory cytokine levels in tissue – TNF-a (3.2 pg/mg protein, p50.05), IL-1b (1.5 pg/mg protein, p40.05), and IL-6 (6.3 pg/mg protein, p50.05); and (6) phospho-NF-kB (1.6, p50.05) and phospho-IkB-a (0.4, p50.05) expression in tissue. Discussion and conclusion: The results suggested that MLB might exert therapeutic actions on depression-like behavior and the HPA axis hyperactivity in CUS rats, and the mechanisms underlying the antidepressant-like effects of MLB might be mediated by regulation of the expression of NF-kB and IkB-a in rats.
Forced swimming test, nuclear factor kappa B, pro-inflammatory cytokines, tail suspension test
Introduction Depression is a major mental disorder. According to recent epidemiological surveys, the lifetime incidence of depression is between 10% and 15% (Lepine & Briley, 2011). Currently, antidepressant drugs used in the treatment of major depressive disorders are believed to act on the central monoaminergic systems, mainly 5-hydroxytryptamine (5-HT) and noradrenergic synaptic neurotransmissions (Pazos, 2013). Selective serotonin reuptake inhibitors and noradrenaline reuptake inhibitors are effective in treating most depressive episodes, but about one-third of these patients show only partial or no *These authors contributed equally to this work. Correspondence: Xiaohong Zhang, Xi’an Mental Health Center, Institute of Mental Health, Xi’an Medical University, Xi’an 710061, China. Tel: +86 29 85551351. E-mail: [email protected]; Qiangju Wu, Xi’an Mental Health Center, Institute of Mental Health, Xi’an Medical University, Xi’an, 710061, China. E-mail: [email protected]
History Received 2 May 2014 Revised 19 August 2014 Accepted 28 August 2014 Published online 10 April 2015
response to the treatment (Taylor et al., 2011). The classical antidepressants based on the monoamine hypothesis of depression have been challenged due to their clinical limitations and adverse effects (Berton & Nestler, 2006; Hansen et al., 2005). Therefore, the development of more effective antidepressants is of great interest. Traditional Chinese medicine (TCM) has been widely used for the treatment of depression (Li et al., 2008; Zhang et al., 2007). Several ancient herb formula prescriptions designated to treat depressive-like symptoms are still frequently prescribed in China either alone or with conventional antidepressants (Guo et al., 2009; Molina-Hernandez et al., 2008; Tesch, 2003). Magnesium lithospermate B [MLB, also named salvianolic acid B (Sal B) magnesium salt, or magnesium tanshinoate B, Figure 1], a derivative of the caffeic acid tetramer and the major soluble ingredient in danshen [Radix Salviae miltiorrhizae, (Labiatae)], has been shown to induce several medicinal effects, such as vasodilating blood vessels,
2
W. Quan et al
Pharm Biol, Early Online: 1–8
Table 1. Schedule of chronic mild stress procedures. Day 1 2 3 4 5 6
Pharmaceutical Biology Downloaded from informahealthcare.com by Nyu Medical Center on 04/12/15 For personal use only.
Figure 1. Structure of MLB. Molecular formula: C38H28MgO16; molecular weight: 740.90. MLB, magnesium lithospermate B.
decreasing blood pressure, acting as an antioxidant, and performing free radical scavenging activities (Liu et al., 2009; Paik et al., 2011; Qu et al., 2011; Zhang et al., 2004). Recently, MLB was suggested to be responsible for antiischemic neuroprotection by its effective inhibition of the Na+/K+-ATPase (Tzen et al., 2007). The ability of MLB to cross the blood–brain barrier (BBB) (Liu et al., 2006) and protect SH-SY5Y cells against MPP(+)-induced apoptosis by relieving oxidative stress and modulating the apoptotic process of neural stem cells (Zeng et al., 2010) has also been shown. Notably, MLB could ameliorate learning and memory dysfunctions induced by cerebral transient ischemia (Du et al., 2000). In light of these previous findings, we hypothesize that MLB might play an important role in the pathogenesis of depression. In addition, our present study has established that MLB could attenuate phospho-NF-kB levels in myocardial tissues, and this decrease in phospho-NF-kB was associated with a decrease in tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), and interleukin-6 (IL-6) in the serum (Quan et al., 2013). As we already know, inflammatory mechanisms play a role in the cause of major depressive disorders (Raedler, 2011). According to the cytokine hypothesis of depression, inflammatory cytokines act on neural circuits to evoke the behavioral and physiological changes observed in depression (Piser, 2010). The elevated systemic levels of TNF-a, IL-1b, and IL-6 were found to be reversed by antidepressants (Berton & Nestler, 2006). This has raised the potential of exploiting cytokine-regulated pathways in the development of novel antidepressants. Together, these data support the possibility that MLB may exert antidepressant-like activity by targeting the neuroinflammatory signals, and this might provide a new therapeutic target for the treatment of depression.
Materials and methods Animals Adult male Sprague–Dawley rats (220–240 g, 10–12 weeks old) were obtained from the experimental animal center [Animal Certificate of Conformity: SCXK-(Army) 2007-007] of the FMMU, Xi’an 710032, China. All animals were cared according to the guidelines of National Institutes of the Health Guide for the Care and Use of Laboratory Animals.
7
Stressors
Time
10 h crowded cage (5–6 rats per cage) a.m.–p. m. 15 min forced swim (22 C) p.m. 15 h wet bedding 4 h restraint 24 h food deprivation a.m. 5 min cold swim (4 C) p.m. 4 h restraint 24 h water deprivation a.m. 2 h cold stress (15 C) a.m. 4 h restraint p.m. 4 h restraint a.m. 2 min tail pinch p.m.
Chemicals and reagents MLB (molecular formula: C36H28MgO16, molecular weight: 740.90, purity495%) was provided by Xi’an Honson Biotechnology Co., Ltd (Xi’an, China). IL-1b (limit detection: 10 pg/ml, variability coefficients: 59.8%), IL-6 (limit detection: 16 pg/ml, variability coefficients: 510%), and TNF-a (limit detection: 16 pg/ml, variability coefficients: 510%) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Westang Reagent Company (Shanghai, China). The corticosterone (CORT) ELISA kit was purchased from Cusabio Biotech (Wuhan, China). IMI (imipramine) and western blot reagents were purchased from Sigma (St. Louis, MO). Anti-phospho-IkB-a, anti-phospho-NF-kB, and antib-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Experimental protocols The chronic unpredictable stress (CUS) protocol was modified from the procedures described by Katz (1981) and Willner et al. (1987). The protocol consisted of a variety of sequential stressors applied randomly every day for 7 weeks (Table 1). Rats in the control group received no CUS during the whole experiment. In the model group, rats were exposed to CUS for 7 weeks. From the beginning of the 5th week, model group rats were randomly grouped and subjected to different treatments (Figure 2). Part 1: To investigate the antidepressant-like activity of MLB, rats were divided randomly into four large groups: control (n ¼ 8), model (n ¼ 8), MLB (n ¼ 8), and IMI groups (n ¼ 8). Part 2: To explore the underlying therapeutic mechanism of MLB, rats were randomly divided into three groups (n ¼ 8 in each subgroup): control, model, and MLB groups. In the Part 1 and Part 2 experiments, control and model rats were intraperitoneally (i.p.) injected with saline. MLB was dissolved in saline to give three different final concentrations, and rats were injected (i.p.) with 15, 30, and 60 mg/kg MLB once a day for 3 weeks. Open-field test The open-field test (OFT) was carried out at the end of 1st, 4th, and 7th week. The open-field apparatus consisted of a
Antidepressant-like effects of MLB
DOI: 10.3109/13880209.2014.967783
3
Pharmaceutical Biology Downloaded from informahealthcare.com by Nyu Medical Center on 04/12/15 For personal use only.
Figure 2. Study design. Experimental groups and protocols for the antidepressant-like effects by MLB in rats. MLB, magnesium lithospermate B; CUS, chronic unpredictable stress.
square wooden arena (100 cm 100 cm 40 cm) with a black surface covering the inside walls. The floor of the wooden arena was divided equally into 25 squares marked with black lines. During the test, the rats were placed individually into the center of the arena and allowed to explore freely. The number of crossings (squares crossed with all paws) was recorded during a test period of 5 min. This apparatus was cleaned with a detergent and dried after occupancy by each rat (Rodrigues et al., 1996). Forced swimming test (FST) The forced swimming test (FST) was carried out at the end of 7th week. Rats were forced to swim in a glass cylinder (diameter 26 cm and height 60 cm) containing water at a depth of 35 cm and a temperature of 25 C. The water was changed after each test. Three types of behaviors were monitored for 5 min: immobility, swimming, and climbing. Immobility was defined as floating with no activity other than those necessary to keep the head above the water. Swimming was defined as active movement throughout the cylinder, including crossing into another quadrant. Climbing was defined as upwarddirected movement of the forepaws against the cylinder walls (Porsolt et al., 1977). Tail suspension test The tail suspension test (TST) was carried out at the end of 7th week. The tail suspension test was performed as previously described (Svenningsson et al., 2007). Antidepressant-like behavior is identified as decreased immobility of the rats. Briefly, 30 min after the last drug treatment, the rats were suspended by the tail from a ledge with adhesive tape (approximately 1 cm from the tip of the tail). The distance between the tip of the nose of the rat and the floor was approximately 5 cm. Rats were partitioned to avoid interference during the test. Immobility was defined as the absence of
movement and was scored over a 5 min trial by an observer blinded to the drug treatment. Measurement of CORT in the serum The measurement of CORT in serum was carried out at the end of 7th week. The blood samples were prepared by centrifugation of the collected blood samples (1000 rpm for 15 min) and then stored at 80 C for biochemical determinations. The serum levels of CORT were measured using ELISA kits according to the manufacturer’s instructions. Measurement of IL-1b, IL-6, and TNF-a in the serum In brief, the blood sample was prepared as previously described. The levels of IL-1b, IL-6, and TNF-a in the serum were measured using ELISA kits according to the manufacturer’s instructions. Measurement of tissue IL-1b, IL-6, and TNF-a The measurement of tissue IL-1b, IL-6, and TNF-a levels was carried out at the end of 7th week. The brains were removed, and then the hypothalamus and the hippocampus were dissected out of the brain and placed onto an ice cold plate. All the tissue samples were quickly frozen and stored in a deep freezer at 80 C until assayed. The tissue samples were weighed and then homogenized with an ultrasonic disruptor in ice cold saline. The sample was centrifuged at 1000 rpm for 20 min at 4 C, and the supernatant was removed into a tube for detection. The levels of IL-1b, IL-6, and TNF-a in the tissue were measured using ELISA kits according to the manufacturer’s instructions. Western blot analysis Western blot analysis was carried out at the end of 7th week. Proteins were extracted from brain tissue and protein
4
W. Quan et al
concentrations were determined. The total nuclear protein 50 mg was resolved on a 15% SDS-polyacrylamide gel. The fractionated proteins were electrophoretically transferred to an immobilon polyvinylidene difuride membrane and probed with rabbit polyclone anti-phospho-NF-kB and anti-phosphoIkB-a at overnight at 4 C followed by incubation with the corresponding secondary antibodies at room temperature for 2 h as previously described. The blots were visualized with the ECL-Plus reagent (GE Healthcare, Piscataway, NJ). In all immunoblotting experiments, the blots were reprobed with an anti-b-actin antibody to control protein loading.
Pharmaceutical Biology Downloaded from informahealthcare.com by Nyu Medical Center on 04/12/15 For personal use only.
Statistical analysis First, we checked whether the data were normally distributed. If the data were normally distributed, we planned to use a parametric analysis method, and if not, we planned to conduct a nonparametric statistical analysis. All the data showed normal distribution; therefore, we conducted a parametric analysis. Continuous variables that approximated the normal distribution were expressed as the mean ± standard deviation (SD). Comparison between groups was subjected to ANOVA, followed by Tukey’s multiple comparison tests. A level of p50.05 was considered to be statistically significant. The statistical analyses were performed using GraphPad Prism software Version 5.01 (GraphPad Inc., LaJolla, CA).
Pharm Biol, Early Online: 1–8
Effects of MLB on the FST The FST and the TST are behavioral despair tests useful for the evaluation of antidepressant drugs (Shirayama et al., 2002). To examine the antidepressant-like effects of MLB, we first performed the FST in rats. In the FST, rats given MLB at 60 mg/kg (F(5,42) ¼ 6.394, p50.05, 94.0 ± 10.2 s versus 113.3 ± 12.4 s, Figure 4) exhibited a significantly shorter duration of immobility compared with the model group. ANOVA showed no significant differences in the FST results between the other two MLB groups, or any significant differences between the MLB-treated (60 mg/kg) and IMItreated rats (F(5,42) ¼ 6.394, p40.05, 94.0 ± 10.2 s versus 88.1 ± 17.0 s, Figure 4). These results suggest that MLB stimulates an antidepressant effect that is similar to that of IMI during the FST. Effects of MLB on the TST Next, we performed the TST in rats. The data demonstrated that the immobility time was significantly decreased in rats treated with MLB at the doses of 30 and 60 mg/kg compared with saline-treated rats (F(5,42) ¼ 47.73, p50.05, 131.6 ± 23.3 s versus 207.9 ± 15.4 s, Figure 4). There were no
Results Effects of MLB on the OFT The effect of CUS on the general activity of the rats and the effect of MLB on the models were examined during the OFT. The test showed that 4 weeks of CUS lead to a significant decrease in the number of crossings (F(5,42) ¼ 21.34, p50.05, 31 ± 5 versus 75 ± 9, Figure 3). After treatment for 3 weeks with MLB or IMI, the reduced number of crossings in the model group was significantly reversed during the OFT (F(5,42) ¼ 21.34, p50.05, 25 ± 9, 35 ± 7 versus 14 ± 8, Figure 3), compared with the model group.
Figure 3. Effects of CUS in rats and antidepressant-like effects of MLB in rats during the OFT. MLB, magnesium lithospermate B; OFT, openfield test. Values are expressed as the mean ± S.D. n ¼ 8. Significance was determined by ANOVA followed by Tukey’s test. #p50.05 versus the control group, *p50.05 versus the model group.
Figure 4. Antidepressant-like effects of MLB in rats during the (A) FST and (B) TST. MLB, magnesium lithospermate B; FST, forced swimming test; TST, tail suspension test. Values are expressed as the mean ± S.D. n ¼ 8. Significance was determined by ANOVA followed by Tukey’s test. #p50.05 versus the control group, *p50.05 versus the model group.
DOI: 10.3109/13880209.2014.967783
significant differences between the MLB (60 mg/kg)treated group and the IMI-treated group (F(5,42) ¼ 47.73, p40.05, 131.6 ± 23.3 s versus 118.4 ± 13.5 s, Figure 4). In conclusion, MLB reduced the immobility time in the rats during the TST.
Antidepressant-like effects of MLB
5
Effects of MLB on CORT levels in serum To confirm our behavioral results, we subsequently assessed the activity of the HPA axis in the rats. In agreement with this hypothesis, the CUS rats displayed hyperactivity of the HPA axis in the present study, as indicated by the elevation of serum CORT concentrations. Serum CORT concentrations of the CUS rats were significantly higher than those of the control rats (F(2,21) ¼ 5.93, p50.05, 108.5 ± 37.5 nmol/L versus 65.3 ± 13.5 nmol/L, Figure 5). Treatment with MLB decreased the elevated CORT level (F(2,21) ¼ 5.93, p40.05, 86.8 ± 17.8 nmol/L versus 108.5 ± 37.3 nmol/L, Figure 5).
Pharmaceutical Biology Downloaded from informahealthcare.com by Nyu Medical Center on 04/12/15 For personal use only.
Effects of MLB on IL-1b, IL-6, and TNF-a levels in serum
Figure 5. Effects of MLB on CORT levels in the serum. MLB, magnesium lithospermate B; CORT, corticosterone. Values are expressed as the mean ± S.D. n ¼ 8. Significance was determined by ANOVA followed by Tukey’s test. #p50.05 versus the control group, *p50.05 versus the model group.
Figure 6. Effects of MLB on (A) IL-1b, (B) IL-6, and (C) TNF-a levels in serum. MLB, magnesium lithospermate B; IL-1b, interleukin 1b; IL-6, interleukin 6; TNF-a, tumor necrosis factor-a. Values are expressed as the mean ± S.D. n ¼ 8. Significance was determined by ANOVA followed by Tukey’s test. # p50.05 versus the control group, *p50.05 versus the model group.
Accumulating evidence indicates that pro-inflammatory cytokines such as TNF-a, IL-1b, and IL-6 play a critical role in the pathogenesis of depression (O’Donovan, 2014). In this study, we found that the levels of TNF-a, IL-1b, and IL-6 in serum were significantly elevated in the model group (TNF-a: F(2,21) ¼ 88.93, p50.05, 181.9 ± 24.1 pg/ml versus 74.2 ± 13.9 pg/ml; IL-1b: F(2,21) ¼ 69.76, p50.05, 198.2 ± 22.0 pg/ml versus 84.8 ± 12.1 pg/ml; IL-6: F(2,21) ¼ 54.99, p50.05, 193.6 ± 24.7 pg/ml versus 105.2 ± 18.4 pg/ ml, Figure 6), but markedly decreased with the administration of MLB (60 mg/kg) (TNF-a: F(2,21) ¼ 88.93, p50.05, 89.8 ± 11.9 pg/ml versus 181.9 ± 24.1 pg/ml; IL-1b: F(2,21) ¼ 69.76, p50.05, 111.3 ± 24.0 pg/ml versus 198.2 ± 22.0 pg/ml;
6
W. Quan et al
Pharm Biol, Early Online: 1–8
Pharmaceutical Biology Downloaded from informahealthcare.com by Nyu Medical Center on 04/12/15 For personal use only.
Figure 7. Effects of MLB on the tissue levels of (A) IL-1b, (B) IL-6, and (C) TNF-a. MLB, magnesium lithospermate B; IL-1b, interleukin 1b; IL-6, interleukin 6; TNF-a, tumor necrosis factor-a. Values are expressed as the mean ± S.D. n ¼ 8. Significance was determined by ANOVA followed by Tukey’s test. # p50.05 versus the control group, *p50.05 versus the model group.
IL-6: F(2,21) ¼ 54.99, p50.05, 99.8 ± 16.1 pg/ml versus 193.6 ± 24.7 pg/ml, Figure 6). Effects of MLB on the tissue levels of TNF-a, IL-1b, and IL-6 The tissue levels of TNF-a, IL-1b, and IL-6 were very low in the control group, but markedly increased in the model group (TNF-a: F(2,21) ¼ 17.00, p50.05, 12.2 ± 3.2 pg/mg protein versus 4.9 ± 2.3 pg/mg protein; IL-1b: F(2,21) ¼ 6.965, p50.05, 17.9 ± 6.2 pg/mg protein versus 10.1 ± 2.2 pg/mg protein; IL-6: F(2,21) ¼ 10.57, p50.05, 21.7 ± 5.7 pg/mg protein versus 11.7 ± 3.1 pg/mg protein, Figure 7). MLB attenuated the tissue levels of TNF-a, IL-1b, and IL-6 (TNF-a: F(2,21) ¼ 17.00, p50.05, 9.0 ± 1.9 pg/mg protein versus 12.2 ± 3.2 pg/mg protein; IL-1b: F(2,21) ¼ 6.965, p50.05, 16.4 ± 3.9 pg/mg protein versus 17.9 ± 6.2 pg/mg protein; IL-6: F(2,21) ¼ 10.57, p50.05, 15.4 ± 4.1 pg/mg protein versus 21.7 ± 5.7 pg/mg protein, Figure 7). These changes in the tissue were consistent with the pro-inflammatory cytokine results seen in the serum. One interpretation of these results could be that MLB prevented depression through the regulation of inflammatory responses. Effects of MLB on phosphorylated proteins that are involved in inflammation Furthermore, exploring the mechanism of action involved in inflammation is important. Activation of NF-kB in the
pathogenesis of depression is well accepted. Therefore, the expression of phospho-IkB-a and phospho-NF-kB was determined by western blot analysis. Phospho-IkB-a and phospho-NF-kB were highly expressed in the model group (p-IkB-a: F(2,9) ¼ 214.0, p50.05, 2.2 ± 0.3 versus 1.0 ± 0.0; p-NF-kB: F(2,9) ¼ 214.0, p50.05, 3.0 ± 0.3 versus 1.0 ± 0.0, Figure 8), but were inhibited by treatment with MLB (p-IkB-a: F(2,9) ¼ 214.0, p50.05, 1.8 ± 0.2 versus 2.2 ± 0.3; p-NF-kB: F(2,9) ¼ 214.0, p50.05, 1.4 ± 0.3 versus 3.0 ± 0.3, Figure 8).
Discussion TCM is widely believed to be beneficial, but, for the most part, neither the active components nor their molecular targets are well defined. Therefore, understanding the active component(s) and the mechanism(s) of action can make TCM more acceptable (Sandur et al., 2007). Recently, studies have pointed to the possibility that MLB may act on the neuroinflammatory signals that lead to antidepressant-like effects. However, the particular effects and underlying mechanisms have yet to be elucidated. The FST and the TST are behavioral despair tests useful for probing the pathological mechanism of depression and for the evaluation of antidepressant drugs (Feng et al., 2012). These tests are also well established for screening new, potent antidepressant drugs in rats. The characteristic behavior scored in both tests is termed immobility, which reflects a behavioral state of despair (learned helplessness), as seen in
Antidepressant-like effects of MLB
DOI: 10.3109/13880209.2014.967783
7
Pharmaceutical Biology Downloaded from informahealthcare.com by Nyu Medical Center on 04/12/15 For personal use only.
Figure 8. Effects of MLB on phosphorylated proteins that are involved in inflammation. (A) Phosphorylation of IkB-a and NF-kB was analyzed by western blot. (B) Graphs showing the protein ratio of phospo-IkBa/b-actin and phospo-NF-kB/b-actin. MLB, magnesium lithospermate B; NF-kB, nuclear factor kappa B. Values are expressed as the mean ± S.D. n ¼ 8. Significance was determined by ANOVA followed by Tukey’s test. # p50.05 versus the sham group, *p50.05 versus the control group.
human depression (Lucki et al., 2001). To investigate whether MLB can produce chronic changes in depression-related behavior during the FST and the TST, we treated rats for 7 weeks with a variety of sequential stressors applied randomly every day. MLB caused a reduction in the immobility time during the FST and the TST. The results presented here show, to our knowledge for the first time, that MLB is effective in producing significant antidepressant-like activity when assessed during the FST and the TST. The results of the behavioral investigation proved the antidepressant-like effect of MLB at the tested doses. To confirm our behavioral results, we subsequently assessed the activity of the HPA axis in the rats. The stress response is mediated by the HPA system, and the HPA axis is considered to be the ‘‘final common pathway’’ in the pathogenesis of depression (Swaab et al., 2005). In agreement with this hypothesis, the CUS rats in the present study displayed hyperactivity of the HPA axis as indicated by the elevation of serum CORT concentrations. Consistent with the results of the behavior tests, this change was reversed by treatment with MLB, indicating the regulatory effect of MLB on the HPA axis dysfunction. MLB was reported to protect against the ischemia/ reperfusion-induced injury in the rat brain in vivo and in vitro (Quan et al., 2014; Song et al., 2014). MLB has also been demonstrated to inhibit lipopolysaccharide-induced expression of inflammatory molecules (Zhao et al., 2013). Inhibition of neuroinflammation may represent a novel mechanism of action for antidepressant treatment (Wong et al., 2008). Several recent studies have reported that some natural products such as omega-3 fatty acids and curcumin induced antidepressant effects through the modulation of oxidative reactions and inflammatory cytokine production in the brain (Lin et al., 2011; Su, 2009; Xu et al., 2005). The results of our and other previous studies led us to hypothesize that MLB may act as an antidepressant via regulating inflammatory responses. To further clarify the antidepressant mechanisms of MLB, pro-inflammatory cytokines, including TNF-a, IL-1b, and IL-6, were investigated in this study. A previous study demonstrated that the elevated systemic levels of pro-inflammatory cytokines are decreased by antidepressants (Yang et al., 2011). Our data show that the levels of TNF-a, IL-1b, and IL-6 in the serum increased in the model group, but the increase of these inflammatory cytokines was significantly decreased with MLB treatment, which suggests that the antidepressant-like effects of MLB could be attributed to the suppression of inflammatory responses via inhibition of
pro-inflammatory cytokines. This notion was supported by the alteration of TNF-a, IL-1b, and IL-6 in brain tissue. Further studies were conducted to investigate the possible mechanism in our study, such as signal transduction pathways underlying the antidepressant-like effects of MLB. Given that NF-kB is a ubiquitously expressed transcription factor that has a significant role in both the inflammatory response and the cell survival in the pathogenesis of depression (Lee et al., 2012), our study was centered on the NF-kB signaling pathway. NF-kB is sequestered in the cytoplasm in an inactive form by its association with IkB-a, which prevents NF-kB from translocating into the nucleus (Hinz & Scheidereit, 2014). However, activation of NF-kB signaling results from the phosphorylation-induced proteolytic degradation of IkB-a. Free NF-kB then translocates to the nucleus, where it induces transcription of a variety of genes, including TNF-a and IL-6. Our study indicates that MLB could attenuate phospho-IkB-a and phospho-NF-kB levels in heart tissue, and this was associated with a decrease in TNF-a, IL-1b, and IL-6 in the serum. The results indicate that the anti-inflammatory effect and the antidepressant-like effect of MLB relied heavily on the inhibition of the NF-kB signaling pathway. Undoubtedly, our research has a number of limitations. Although MLB clearly reduced the inflammatory markers and alleviated depression significantly, our results could not directly prove that MLB exerted its protection solely through an anti-inflammatory effect. More compelling evidence should be provided to support our current hypothesis.
Conclusion In our study, treatment with MLB significantly improved the depression-like behaviors and the HPA axis hyperactivity in CUS rats. Moreover, MLB also significantly decreased the levels of TNF-a, IL-6, and IL-1b. Based on our results, we demonstrated that MLB had therapeutic effects on the CUSinduced depression model in rats, and these effects might be related, in large part, to the suppression of the inflammatory response via inhibition of NF-kB activation.
Declaration of interest The authors report that they have no conflicts of interest. This study was supported by the National Natural Science Foundation of China (No. 81202938), Science and Technology Research Development Plan Project of Shaanxi Province (No. 2011KJXX65).
8
W. Quan et al
Pharmaceutical Biology Downloaded from informahealthcare.com by Nyu Medical Center on 04/12/15 For personal use only.
References Berton O, Nestler EJ. (2006). New approaches to antidepressant drug discovery: Beyond monoamines. Nat Rev Neurosci 7:137–51. Du GH, Qiu Y, Zhang JT. (2000). Salvianolic acid B protects the memory functions against transient cerebral ischemia in mice. J Asian Nat Prod Res 2:145–52. Feng Y, You Z, Yan S, et al. (2012). Antidepressant-like effects of salvianolic acid B in the mouse forced swim and tail suspension tests. Life Sci 90:1010–14. Guo JY, Li CY, Ruan YP, et al. (2009). Chronic treatment with celecoxib reverses chronic unpredictable stress-induced depressive-like behavior via reducing cyclooxygenase-2 expression in rat brain. Eur J Pharmacol 612:54–60. Hansen RA, Gartlehner G, Lohr KN, et al. (2005). Efficacy and safety of second-generation antidepressants in the treatment of major depressive disorder. Ann Intern Med 143:415–26. Hinz M, Scheidereit C. (2014). The IkappaB kinase complex in NFkappaB regulation and beyond. EMBO Rep 15:46–61. Katz RJ. (1981). Animal models and human depressive disorders. Neurosci Biobehav Rev 5:231–46. Lee YH, Kim SH, Kim Y, et al. (2012). Inhibitory effect of the antidepressant imipramine on NF-kappaB-dependent CXCL1 expression in TNFalpha-exposed astrocytes. Int Immunopharmacol 12: 547–55. Lepine JP, Briley M. (2011). The increasing burden of depression. Neuropsychiatr Dis Treat 7:3–7. Li LT, Wang SH, Ge HY, et al. (2008). The beneficial effects of the herbal medicine Free and Easy Wanderer Plus (FEWP) and fluoxetine on post-stroke depression. J Altern Complement Med 14:841–6. Lin TY, Lu CW, Wang CC, et al. (2011). Curcumin inhibits glutamate release in nerve terminals from rat prefrontal cortex: Possible relevance to its antidepressant mechanism. Prog Neuropsychopharmacol Biol Psychiatry 35:1785–93. Liu CS, Cheng Y, Hu JF, et al. (2006). Comparison of antioxidant activities between salvianolic acid B and Ginkgo biloba extract (EGb761). Acta Pharmacol Sin 27:1137–45. Liu X, Chen R, Shang Y, et al. (2009). Superoxide radicals scavenging and xanthine oxidase inhibitory activity of magnesium lithospermate B from Salvia miltiorrhiza. J Enzyme Inhib Med Chem 24:663–8. Lucki I, Dalvi A, Mayorg AJ. (2001). Sensitivity to the effects of pharmacologically selective antidepressants in different strains of mice. Psychopharmacology 155:315–22. Molina-Hernandez M, Tellez-Alcantara NP, Perez-Garcia J, et al. (2008). Desipramine or glutamate antagonists synergized the antidepressantlike actions of intra-nucleus accumbens infusions of minocycline in male Wistar rats. Prog Neuropsychopharmacol Biol Psychiatry 32: 1660–6. O’Donovan A. (2014). Inflammation and depression: Unraveling the complex interplay in inflammatory bowel disease. J Pediatr Gastroenterol Nutr 58:541–2. Paik YH, Yoon YJ, Lee HC, et al. (2011). Antifibrotic effects of magnesium lithospermate B on hepatic stellate cells and thioacetamide-induced cirrhotic rats. Exp Mol Med 43:341–9. Pazos A. (2013). Future trends in the development of new antidepressant drugs. Curr Pharm Des 20:3717. Piser TM. (2010). Linking the cytokine and neurocircuitry hypotheses of depression: A translational framework for discovery and development of novel anti-depressants. Brain Behav Immun 24:515–24. Porsolt RD, Bertin A, Jalfre M. (1977). Behavioral despair in mice: A primary screening test for antidepressants. Arch Int Pharmacodyn Ther 229:327–36. Qu J, Ren X, Hou RY, et al. (2011). The protective effect of magnesium lithospermate B against glucose-induced intracellular oxidative damage. Biochem Biophys Res Commun 411:32–9. Quan W, Wei G, Zhou D, et al. (2013). Magnesium lithospermate B reduces myocardial ischemia/reperfusion injury in rats via regulating the inflammation response. Pharm Biol 51:1355–62.
Pharm Biol, Early Online: 1–8
Quan W, Wu B, Bai Y, et al. (2014). Magnesium lithospermate B improves myocardial function and prevents simulated ischemia/ reperfusion injury-induced H9c2 cardiomyocytes apoptosis through Akt-dependent pathway. J Ethnopharmacol 151:714–21. Raedler TJ. (2011). Inflammatory mechanisms in major depressive disorder. Curr Opin Psychiatry 24:519–25. Rodrigues AL, Rocha JB, Mello CF, Souza DO. (1996). Effect of perinatal lead exposure on rat behaviour in open-field and two-way avoidance tasks. Pharmacol Toxicol 79:150–6. Sandur SK, Pandey MK, Sung B, et al. (2007). Curcumin, demethoxycurcumin, bisdemethoxycurcumin, tetrahydrocurcumin and turmerones differentially regulate anti-inflammatory and anti-proliferative responses through a ROS-independent mechanism. Carcinogenesis 28:1765–73. Shirayama Y, Chen AC, Nakagawa S, et al. (2002). Brain-derived neurotrophic factor produces antidepressant effects in behavioral models of depression. J Neurosci 22:3251–61. Song S, Liu W, Liu F, et al. (2014). Magnesium lithospermate B reduces inflammatory response in a mouse model of hepatic ischemiareperfusion injury. Cell Biochem Biophys 69:347–55. Su KP. (2009). Biological mechanism of antidepressant effect of omega-3 fatty acids: How does fish oil act as a mind–body interface? Neurosignals 17:144–52. Svenningsson P, Tzavara ET, Qi H, et al. (2007). Biochemical and behavioral evidence for antidepressant-like effects of 5-HT6 receptor stimulation. J Neurosci 27:4201–9. Swaab DF, Bao AM, Lucassen PJ. (2005). The stress system in the human brain in depression and neurodegeneration. Ageing Res Rev 4: 141–94. Taylor D, Meader N, Bird V, et al. (2011). Pharmacological interventions for people with depression and chronic physical health problems: Systematic review and meta-analyses of safety and efficacy. Br J Psychiatry 198:179–88. Tesch BJ. (2003). Herbs commonly used by women: An evidence-based review. Am J Obstet Gynecol 188:44–55. Tzen JT, Jinn TR, Chen YC, et al. (2007). Magnesium lithospermate B possesses inhibitory activity on Na+, K+-ATPase and neuroprotective effects against ischemic stroke. Acta Pharmacol Sin 28:609–15. Willner P, Towell A, Sampson D, et al. (1987). Reduction of sucrose preference by chronic unpredictable mild stress, and its restoration by a tricyclic antidepressant. Psychopharmacology 93:358–64. Wong ML, Dong C, Maestre-Mesa J, Licinio J. (2008). Polymorphisms in inflammation-related genes are associated with susceptibility to major depression and antidepressant response. Mol Psychiatry 13: 800–12. Xu Y, Ku BS, Yao HY, et al. (2005). Antidepressant effects of curcumin in the forced swim test and olfactory bulbectomy models of depression in rats. Pharmacol Biochem Behav 82:200–6. Yang JJ, Zhou ZQ, Yang C. (2011). Letter to the editor: Does ketamine exert a fast-acting antidepressant effect via inhibition of pro-inflammatory cytokines? Psychol Med 41:1787–9. Zeng G, Tang T, Wu HJ, et al. (2010). Salvianolic acid B protects SHSY5Y neuroblastoma cells from 1-methyl-4-phenylpyridiniuminduced apoptosis. Biol Pharm Bull 33:1337–42. Zhang Y, Akao T, Nakamura N, et al. (2004). Magnesium lithospermate B is excreted rapidly into rat bile mostly as methylated metabolites, which are potent antioxidants. Drug Metab Dispos 32: 752–7. Zhang ZJ, Kang WH, Li Q, Tan QR. (2007). The beneficial effects of the herbal medicine Free and Easy Wanderer Plus (FEWP) for mood disorders: Double-blind, placebo-controlled studies. J Psychiatr Res 41:828–36. Zhao M, Chen YH, Dong XT, et al. (2013). Folic acid protects against lipopolysaccharide-induced preterm delivery and intrauterine growth restriction through its anti-inflammatory effect in mice. PLoS One 8: e82713.
