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Involvement of nitric oxide (NO) signaling pathway in the antidepressant action of the total flavonoids extracted from Xiaobuxin-Tang
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Involvement of nitric oxide (NO) signaling pathway in the antidepressant action of the total flavonoids extracted from Xiaobuxin-Tang
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Li-Ming Zhang a,1 , Heng-Lin Wang b,1 , Nan Zhao a , Hong-Xia Chen a , Yun-Feng Li a,∗∗ , You-Zhi Zhang a,∗ a b
Department of New Drug Evaluation, Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Haidian, Beijing 100850, China Department of Anesthesiology, 309 Hospital of PLA, 17 Heishanhu Road, Haidian, Beijing 100091, China
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h i g h l i g h t s • XBXT-2 presented antidepressant-like effects in the mouse forced swim test (FST). • The antidepressant effect of XBXT-2 was reversed by pretreatment with l-arginine. • 7-Nitroindazole potentiated the antidepressant effect of XBXT-2.
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Article history: Received 17 December 2013 Received in revised form 27 March 2014 Accepted 24 April 2014 Available online xxx
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Keywords: Forced swim test Antidepressant-like effect Nitric oxide Xiaobuxin-Tang
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1. Introduction
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Our previous studies have demonstrated that the total flavonoids (XBXT-2) isolated from the extract of Xiaobuxin-Tang (XBXT), a traditional Chinese herbal decoction, exerted antidepressant-like effects. Recently, accumulating studies have suggested that l-arginine-NO is implicated in the regulation of depression. Therefore, the aim of current study attempts to explore the involvement of l-arginine-NO pathway in the antidepressant-like effect of XBXT-2 in the mouse forced swim test (FST). Our results showed that the antidepressant-like action of XBXT-2 (100 mg/kg, i.g.) was reversed by pretreatment with l-arginine (a nitric oxide precursor, 750 mg/kg, i.p.). While co-administration of aminoguanidine (a specific inducible NOS inhibitor, 40, 80 mg/kg, i.p.) and sub-effective dose of XBXT-2 (50 mg/kg, i.g.) did not significantly alter the immobility in FST. In contrast, combined administration of 7-nitroindazole (a specific neuronal NOS inhibitor, 50 mg/kg, i.p.) potentiated the antidepressant-like effect of non-effective doses of XBXT-2 (50 mg/kg, i.g.). Meanwhile, NO modulators were devoid of any locomotor effects on the animals. In conclusion, the antidepressant-like action of XBXT-2 may be involvement of NO signaling pathway. © 2014 Published by Elsevier Ireland Ltd.
Depression is a serious and burdensome psychiatric illness that is exacerbated by its recurrent nature. Although the monoamine
Abbreviations: ANOVA, analysis of variance; eNOS, endothelial NOS; FST, forced swim test; iNOS, inducible NOS; NMDA, N-methyl-d-aspartate; nNOS, neuronal NOS; NO, nitric oxide; NOS, nitric oxide synthase; SSRIs, selective serotonin reuptake inhibitors; TCM, traditional Chinese medicine; XBXT, Xiaobuxin-Tang; XBXT-2, the total flavonoids isolated from the extract of XBXT. Q2 ∗ Corresponding author. Tel.: +86 1066874606. ∗∗ Corresponding author. E-mail addresses: [email protected] (Y.-F. Li), [email protected], [email protected] (Y.-Z. Zhang). 1 These authors contributed equally to this study.
hypothesis has been the mainstay of depression research for many years, the underlying pathophysiological mechanism of depression remains obscure. It has been suggested nitric oxide (NO) was involved in the pathophysiology of major depression [7] [11]. Decreasing the levels or blocking the synthesis of NO in brain can induce antidepressant-like effects, thus implicating the role of NO in the pathophysiology of major depression [5]. The level of plasma NO metabolites had been found to be significantly higher in suicidal patients than in nonsuicidal psychiatric patients or in normal control subjects [10]. Various nitric oxide synthase (NOS) inhibitors viz. l-NAME (a non-selective NOS inhibitor) or 7-nitroindazole (7-NI, a selective neuronal NOS inhibitor) are known to display antidepressant-like activity [12]. In addition, some clinical evidence also supported the possible role of elevated NO functioning in depression. Paroxetine, a selective serotonin reuptake inhibitor
http://dx.doi.org/10.1016/j.neulet.2014.04.039 0304-3940/© 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: L.-M. Zhang, et al., Involvement of nitric oxide (NO) signaling pathway in the antidepressant action of the total flavonoids extracted from Xiaobuxin-Tang, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.039
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(SSRI), is a NOS inhibitor which has encouraged the researchers to explore more about NO in the pathogenesis of major depression [8]. Xiaobuxin-Tang (XBXT), comprising Haematitum, Flos Inulae, Folium Phyllostachydis Henonis and Semen Sojae Preparatum, four Chinese medicines, was originally recorded in the silk scroll manuscript of “Fuxinjue Zangfu Yongyao Fayao”, written one thousand years ago and discovered in Mogao Caves of Dunhuang. The three ingredients of XBXT (Flos Inulae, Folium Phyllostachydis Henonis and Semen Sojae Preparatum) all contain a large amount of flavonoids. And during the process of extraction, the flavonoids may react with Haematitum, the fourth ingredient of XBXT, to formsome flavone complexes. Our previous study has demonstrated that the total flavonoids (XBXT-2) isolated from XBXT exerted antidepressant-like effects in the behavioral despair models [13]. Given this background, we hypothesized that NO signaling pathway might be involvement in the antidepressant action of XBXT-2. We confirmed our hypothesis by using forced swim test as the animal model of behavioral despair. In this context, we used l-arginine (a precursor of NO, l-Arg), 7-NI, aminoguanidine (a specific inducible NOS inhibitor, AMG) as different NO modulators. These agents were administered prior to XBXT-2 and the doses of all these modulators have previously been reported by other laboratory or established in our laboratory.
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2. Materials and methods
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2.1. Animals
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Male ICR mice (18 ± 2 g) were purchased from the Beijing Vital Laboratory Animal Technology Company. Animals were maintained under standard conditions of controlled temperature (23 ± 1 ◦ C), humidity (45%), and lighting (12-h light/dark cycle). Food pellets and water were freely available. The experimental procedures were approved by the local Committee on Animal Care and Use, and all efforts were made to minimize animal suffering and reduce the number of animals used for the experiments.
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2.2. Drugs and treatments
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Fig. 1. XBXT-2 reduces immobility time in the mouse FST. Data represent the mean + S.E.M. of 8–10 animals per group. Values were analyzed by One Way Analysis of Variance (ANOVA) followed by Dunnett’s tests. *P < 0.05, **P < 0.05 vs. vehicle control.
To investigate the possible involvement of the l-arginine-NO pathway in the anti-immobility effects of XBXT-2 in the FST, mice were pre-treated with l-Arg (350, 700 mg/kg, i.p., a precursor of NO) 30 min before XBXT-2 (100 mg/kg, i.g.) and 60 min thereafter, animals were challenged with forced swim test. Desipramine (DIM, SIGMA, 15 mg/kg), a tricyclic antidepressant, was i.g. administered as a positive control. In another set of experiments, we investigated the effects of the combined administration of AMG (40, 80 mg/kg, i.p.) or 7-NI (25, 50 mg/kg, i.p.) with sub-effective dose of XBXT-2 (50 mg/kg, i.g.). XBXT-2 or vehicle was administered 10 min after NOS inhibitor and a further 50 min were allowed to elapse before the animals were tested in the forced swim test. 2.4. Behavioral analysis
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Traditional Chinese medicines Haematitum, Flos Inulae, Folium Phyllostachydis Henonis and Semen Sojae Preparatum were purchased from Beijing Tongrentang Drugstore (Beijing, China) and were identified by Prof. Lian-Sheng Shen (School of Chinese Medicine, Beijing University of Chinese Medicine) as calcined product of ochery hematite, flowers of Inula japonica Thunb., leafs of Phyllostachys nigra (Lodd.) Munro var. henonis (Mitf.) Stapf ex Rendle, fermented product of Glycine max (L.) Merr., respectively. The voucher specimens (Nos. 02002, 02003, 02004, 02005, respectively) are deposited in the Laboratory of Phytochemistry, Beijing Institute of Pharmacology and Toxicology, China. The extraction of XBXT-2 was carried out according to the method of An [2]. And using lutin as standard substance, the apparent flavones content of the extract was determined as 76.03% by colorimetric method. All chemicals were purchased from Sigma (St. Louis, USA). lArginine, AMG, and XBXT-2 were dissolved in saline. 7-NI was prepared in 20% DMSO (v/v). All treatments were administered by intragastric gavage (i.g.) or intraperitoneally (i.p.) in a volume of 2 ml/kg.
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2.3. Pharmacological treatment
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Firstly, we studied the effect of different doses of XBXT-2 (50, 100 and 200 mg/kg) or NO modulators (l-Arg, 7-NI, AMG) for determination of the effective and sub-effective doses of them.
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2.4.1. Open-field test: locomotor activity In order to rule out any non-specific locomotor effect of XBXT-2 on antidepressant-like activity, mice were assessed the locomotor activity in an open-field test, immediately before the forced swim test. A mouse was placed in the corner of a plastic box (the base was divided into equal sectors, 36 cm × 29 cm × 23 cm) for a 5-min acclimation; the number of crossings (crossing the sector with four Table 1 Effect of NO modulators in the mouse forced swim test. Drug
Dose (mg/kg)
Duration of immobility
Saline l-Arg
– 350 700 1400
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± ± ± ±
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Saline AMG
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Saline 7-NI
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Data represent the mean ± S.E.M. of 6 mice/group. Separate saline controls are presented for each experiment. Values were analyzed by One Way Analysis of Variance (ANOVA) followed by Dunnett’s tests. * P < 0.05. ** P < 0.05 vs. saline.
Please cite this article in press as: L.-M. Zhang, et al., Involvement of nitric oxide (NO) signaling pathway in the antidepressant action of the total flavonoids extracted from Xiaobuxin-Tang, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.039
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Fig. 2. (A) l-Arginine (l-arg) antagonizes the action of XBXT-2 in the mouse FST. (B) Effects of l-arg, XBXT-2, and their combination on locomotion. l-Arg (350, 700 mg/kg, i.p.) was administered 30 min before the treatment with XBXT-2 and after further 60 min, animals were challenged with forced swim test. Data represent the mean + S.E.M. of 8–10 animals per group. Values were analyzed by Two Way Analysis of Variance (ANOVA) followed by Bonferroni post-tests. *P < 0.05 vs. vehicle/vehicle-treated control; # P < 0.05 vs. vehicle/XBXT-2 (100 mg/kg).
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paws) and rearings (raising the forepaws) in the next 5 min was then recorded. The light inside the apparatus was maintained at minimum to avoid anxiety behavior. After each test the apparatus was cleaned with a solution of 10% ethanol to hide animal clues.
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2.4.2. Forced swim test (FST) Immediately after open-field test, mice were individually placed in an open cylindrical container (diameter 10 cm, height 25 cm) containing 19 cm of water at 25 ± 1 ◦ C for 6 min. The immobility time (characterized by slow movements necessary to avoid drowning) was measured during the last 4-min period by a trained observer that was blind to the treatment condition.
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2.5. Data analysis
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Data were statistically examined using one-way or two-way analysis of variance (ANOVA). Post hoc comparisons between individual groups were performed by Dunnett’s test. Data were expressed as the mean values + S.E.M. Differences were considered to be statistically significant when P < 0.05.
3. Results
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3.1. Effect of XBXT-2 or NO modulators on forced-swimming test
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Desipramine, a positive control, caused a reduction in the immobility time in FST at a dose of 30 mg/kg. XBXT-2 (100, 200 mg/kg, i.g.) also significantly decreased the immobility time of mice in FST, showing an antidepressant-like effect (Fig. 1). Treatment with the NO synthase substrate, l-Arg alone (350–1400 mg/kg) did not modify the behavior of animals in forced swimming test. Both AMG (120 mg/kg) and 7-NI (50, 100 mg/kg), given by i.p. route, decreased immobility time in the FST.(Table 1). 3.2. Effect of NO modulators on anti-immobility effect of XBXT-2 on forced-swimming test For further determination of NO role in antidepressant-like effect of XBXT-2, l-Arg was applied as a NO precursor. Two-way ANOVA showed significant effect of l-Arg [F(2,53) = 7.108, P < 0.05], XBXT-2 [F(1,53) = 13.97, P < 0.001] and a l-Arg × XBXT-2 interaction
Please cite this article in press as: L.-M. Zhang, et al., Involvement of nitric oxide (NO) signaling pathway in the antidepressant action of the total flavonoids extracted from Xiaobuxin-Tang, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.039
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Fig. 3. (A) Effect of co-administration of non-effective doses of aminoguanidine (AMG) on antidepressant-like effect of XBXT-2. (B) Effects of AMG, XBXT-2, and their combination on locomotion. XBXT-2 or vehicle was administered 10 min after AMG (40, 80 mg/kg, i.p.) and after further 50 min, the duration of immobility in FST during the last 4 min of the test was recorded. Data represent the mean + S.E.M. of 8–10 animals per group. Values were analyzed by Two Way Analysis of Variance (ANOVA) followed by Bonferroni post-tests.
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[F(2,53) = 6.949, P < 0.05]. As it was determined in Fig. 2A, treatment with l-Arg alone (350, 700 mg/kg) did not modify the behavior of animals in FST. XBXT-2 (100 mg/kg) significantly decreased the immobility time (P < 0.05 vs. vehicle group), and pretreatment with l-Arg (350 mg/kg) did not counteract the antidepressant effect of XBXT-2 (100 mg/kg), while higher dose of l-Arg (700 mg/kg) could reverse its effect (P < 0.05 vs. XBXT-2 group). Administration of l-Arg alone or in combination with XBXT-2 did not affect the locomotor activity in the open-field (Fig. 2B). To investigate the possible interaction of inducible NOS inhibition in anti-immobility effect of XBXT-2, effect of AMG was determined in FST in combination with XBXT-2. Two-way ANOVA showed no significant effects of AMG [F(2,53) = 0.1548, P > 0.05], XBXT-2 [F(1,53) = 0.7592, P > 0.05] and a AMG × XBXT-2 interaction [F(2,53) = 0.06484, P > 0.05]. As shown in Fig. 3A, administration of AMG (40, 80 mg/kg) alone or in combination with non-effective dose of XBXT-2 (50 mg/kg) did not significantly change the immobility time in FST, or alter the locomotor activity in the open field test (Fig. 3B). To investigate the possible interaction of neuronal NOS inhibition in anti-immobility effect of XBXT-2, we further evaluated whether co-administration of 7-NI and non-effective doses of XBXT-2 (50 mg/kg) had any effect on the immobility time in FST. Two-way ANOVA showed significant effects of 7-NI [F(2,53) = 41.88,
P < 0.001], XBXT-2 [F(1,53) = 32.03, P < 0.05] and a 7-NI × XBXT2 interaction [F(2,53) = 4.568, P < 0.001]. The results depicted in Fig. 4A showed that when 7-NI (50 mg/kg) was administered concomitantly with sub-active dose of XBXT-2 (50 mg/kg), the immobility time of mice in the FST was significantly decreased (P < 0.05) compared with vehicle/vehicle treated animals without affecting locomotor activity in the open field test (Fig. 4B).
4. Discussion In the present study, the antidepressant action of XBXT-2 was attenuated by pretreatment with l-Arg and the action of noneffective dose of XBXT-2 was potentiated by sub-effective doses of 7-NI, a neuronal NOS inhibitor, but not by sub-effective doses of AMG, an inducible NOS inhibitor. The present results firstly showed that XBXT-2 given orally was effective in producing significant antidepressant-like effects, when assessed in the forced swim test, confirming previous results from our group [1–3,13]. Moreover, our findings also demonstrated that XBXT-2 at dose of 100 mg/kg was more efficacious than that of 200 mg/kg, which was also in line with our behavioral results which showed that XBXT-2 exhibited an inverse U-shaped dose-response curve in acute and subchronic models [3].
Please cite this article in press as: L.-M. Zhang, et al., Involvement of nitric oxide (NO) signaling pathway in the antidepressant action of the total flavonoids extracted from Xiaobuxin-Tang, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.039
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Fig. 4. (A) 7-NI augments the action of XBXT-2 in the mouse FST. (B) Effects of 7-NI, XBXT-2, and their combination on locomotion. XBXT-2 or vehicle was administered 10 min after 7-NI (25, 50 mg/kg, i.p.) and after further 50 min, the duration of immobility in FST during the last 4 min of the test was recorded. Data represent the mean + S.E.M. of 8–10 animals per group. Values were analyzed by Two Way Analysis of Variance (ANOVA) followed by Bonferroni post-tests. *P < 0.05 vs. vehicle/vehicle-treated control; # P < 0.05 vs. vehicle/XBXT-2 (50 mg/kg).
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Having established both effective and sub-effective doses of XBXT-2 in mouse forced swim test, we further extended our study to explore the role of NO pathway in its antidepressant action. In this present study, we have employed 350, 750 mg/kg, i.p. dose of l-Arg that did not affect the immobility period in mouse forced swim test. It was in line with previous reports that showed l-Arg in a dose range of 750–1000 mg/kg does not affect the immobility period in mouse forced swim test. The antidepressant action of XBXT-2 was prevented by the pretreatment of l-Arg suggesting that the antidepressant-effect of XBXT-2 may be dependent on, at least in part, the inhibition of NO synthesis. Our results are in accordance with previous studies that pre-treatment with l-Arg counteracted the antidepressant-like effect of imipramine, venlafaxine and duloxetine in the forced swimming test [9]. To further confirm our hypothesis, we then combined noeffective dose of XBXT-2 with selective neuronal NOS inhibitors. The neuronal NOS is the more abundant isoform in the brain under normal circumstances. Pre-clinical and clinical findings, such as increased expression of neuronal NOS in limbic regions of stressed animals and depressed patients have helped to link neuronal NOSmediated NO synthesis to the neurobiology of depression. Data presented here demonstrated that combination of 7-NI (50 mg/kg) with XBXT-2 (50 mg/kg) further augmented the antidepressant
response. Our results are in line with results that showed an increased anti-immobility effect after combined administration of 7-NI and venfalaxine, duloxetine [14] and fluoxetine [9]. The antidepressant-like effect of AMG was evident at the smallest dose tested (100 mg/kg). This is in agreement with previous works describing that AMG attenuates stress induced effects in a dose range of 50–100 mg/kg. While co-administration of subeffective doses of AMG and XBXT-2 did not significantly alter the immobility in the forced swim test, which suggested demonstrated that calcium-insensitive NOS enzyme (inducible NOS) probably might not be involved in the antidepressant-like effect of XBXT2 and the calcium-dependent constitutive NOS enzymes (neuronal NOS) are probably involved in this mechanism. Although, the FST is generally recommended for a screen of antidepressant-like effects, drugs enhancing motor activity may give a ‘false’ positive effect in this model, decreasing immobility time by stimulating locomotor activity. Therefore, a relevant question that should be answered is whether the results observed in the FST could be influenced by differences in locomotor behavior. As showed in the present study, XBXT-2 and/or NO modulators showed no stimulatory effect on locomotor activity, ruling out the hypothesis that a psycho-stimulant effect might be responsible for the behavior effects observed in the current study.
Please cite this article in press as: L.-M. Zhang, et al., Involvement of nitric oxide (NO) signaling pathway in the antidepressant action of the total flavonoids extracted from Xiaobuxin-Tang, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.039
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It has been demonstrated that NO also modulates the serotoninergic system. The majority of serotonergic neurons in the raphe nucleus contain neuronal NOS. Several in vitro studies have demonstrated that NO modulates the extracellular levels of serotonin in the central nervous system. In addition, NO can inactivate the rate limiting enzyme in the synthesis of 5-HT, tryptophan hydroxylase [4]. Also, fluoxetine, a selective serotonin reuptake inhibitor, could decrease striatal NO production induced by N-methyl-daspartate (NMDA) injection and downregulate nNOS expression in the hippocampus region of the brain [6]. Further, NAN-190 (a selective 5-HT1A serotonin receptor antagonist) has been found to up-regulate the hippocampal neuronal NOS expression. Furthermore, it was also shown that the antidepressant-like behavioral effects of both l-NA and 7-NI were dependent on endogenous serotonin. Interestingly, our previous study has also shown that administration of the XBXT-2 exerted significant antidepressantlike effect in chronically mildly stressed rats, possibly mediated by serotonergic activation. Thus, these studies demonstrated the inter-relation between NO and serotonin might be involved in the pharmacological effects of XBXT-2. It is important to point out that the behavioral results presented in this report were obtained through the use of a single animal model, the forced swim test. Therefore, it will be important in future studies to validate the pattern of effects presented here using other behavioral paradigms such as learned helplessness, chronic mild stress and social defeat stress models.
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5. Conclusion
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Together with our previous results, it is reasonable to speculate that l-arginine-NO may be involved in the antidepressant-like effect of XBXT-2, and further experiments are necessary to clarify the exact molecular mechanisms underlying the behavioral effects of XBXT-2 and the interplay between NO and serotonin. Furthermore, these data raise the possibility that inhibition of NO synthesis could be used as a strategy to enhance the clinical efficacy of serotonergic antidepressant. Acknowledgements This study was supported by the National Natural Science Foundation of China (Nos. 81001653, 81302726, 81274117 and
81173036), the National Key New Drug Creation Program (Nos. 2012ZX09102101-004 and 2012ZX09J12201-004) and the 12th fine-year army science and technology project (No. CWS12J084). References [1] L. An, Y.Z. Zhang, X.M. Liu, N.J. Yu, H.X. Chen, N. Zhao, L. Yuan, Y.F. Li, Total flavonoids extracted from Xiaobuxin-Tang on the hyperactivity of hypothalamic–pituitary–adrenal axis in chronically stressed rats, Evid. Based Complement. Altern. Med. 2011 (2011) 367619. [2] L. An, Y.Z. Zhang, N.J. Yu, X.M. Liu, N. Zhao, L. Yuan, H.X. Chen, Y.F. Li, The total flavonoids extracted from Xiaobuxin-Tang up-regulate the decreased hippocampal neurogenesis and neurotrophic molecules expression in chronically stressed rats, Prog. Neuro-Psychopharmacol. Biol. Psychiatry 32 (2008) 1484–1490. [3] L. An, Y.Z. Zhang, N.J. Yu, X.M. Liu, N. Zhao, L. Yuan, Y.F. Li, Role for serotonin in the antidepressant-like effect of a flavonoid extract of Xiaobuxin-Tang, Pharmacol. Biochem. Behav. 89 (2008) 572–580. [4] M. Breard, M.A. Sari, Y. Frapart, J.L. Boucher, C. Ducrocq, C. Grillon, The endogenous neurotransmitter, serotonin, modifies neuronal nitric oxide synthase activities, Free Radic. Res. 41 (2007) 413–423. [5] W.E. Chrapko, P. Jurasz, M.W. Radomski, N. Lara, S.L. Archer, J.M. Le Melledo, Decreased platelet nitric oxide synthase activity and plasma nitric oxide metabolites in major depressive disorder, Biol. Psychiatry 56 (2004) 129–134. [6] F. Crespi, The selective serotonin reuptake inhibitor fluoxetine reduces striatal in vivo levels of voltammetric nitric oxide (NO): a feature of its antidepressant activity? Neurosci. Lett. 470 (2010) 95–99. [7] A. Dhir, S.K. Kulkarni, Nitric oxide and major depression, Nitric Oxide: Biol. Chem./Off. J. Nitric Oxide Soc. 24 (2011) 125–131. [8] M.S. Finkel, F. Laghrissi-Thode, B.G. Pollock, J. Rong, Paroxetine is a novel nitric oxide synthase inhibitor, Psychopharmacol. Bull. 32 (1996) 653–658. [9] M. Krass, G. Wegener, E. Vasar, V. Volke, The antidepressant action of imipramine and venlafaxine involves suppression of nitric oxide synthesis, Behav. Brain Res. 218 (2011) 57–63. [10] J. Moreno, E. Gaspar, G. Lopez-Bello, E. Juarez, S. Alcazar-Leyva, E. GonzalezTrujano, L. Pavon, N. Alvarado-Vasquez, Increase in nitric oxide levels and mitochondrial membrane potential in platelets of untreated patients with major depression, Psychiatry Res. 209 (2013) 447–452. [11] A. Spiacci Jr., F. Kanamaru, F.S. Guimaraes, R.M. Oliveira, Nitric oxide-mediated anxiolytic-like and antidepressant-like effects in animal models of anxiety and depression, Pharmacol. Biochem. Behav. 88 (2008) 247–255. [12] F. Yildiz, B.F. Erden, G. Ulak, T. Utkan, N. Gacar, Antidepressant-like effect of 7nitroindazole in the forced swimming test in rats, Psychopharmacology (Berl) 149 (2000) 41–44. [13] Y.Z. Zhang, Y.F. Li, N.J. Yu, L. Yuan, Y.M. Zhao, W.B. Xiao, Z.P. Luo, Antidepressantlike effects of the ethanolic extract of Xiaobuxin-Tang, a traditional Chinese herbal prescription in animal models of depression, Chin. Med. J. (Engl.) 120 (2007) 1792–1796. [14] A.D. Zomkowski, D. Engel, M.P. Cunha, N.H. Gabilan, A.L. Rodrigues, The role of the NMDA receptors and l-arginine-nitric oxide-cyclic guanosine monophosphate pathway in the antidepressant-like effect of duloxetine in the forced swimming test, Pharmacol. Biochem. Behav. 103 (2012) 408– 417.
Please cite this article in press as: L.-M. Zhang, et al., Involvement of nitric oxide (NO) signaling pathway in the antidepressant action of the total flavonoids extracted from Xiaobuxin-Tang, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.039
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Involvement of nitric oxide (NO) signaling pathway in the antidepressant action of the total flavonoids extracted from Xiaobuxin-Tang
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Li-Ming Zhang a,1 , Heng-Lin Wang b,1 , Nan Zhao a , Hong-Xia Chen a , Yun-Feng Li a,∗∗ , You-Zhi Zhang a,∗ a b
Department of New Drug Evaluation, Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Haidian, Beijing 100850, China Department of Anesthesiology, 309 Hospital of PLA, 17 Heishanhu Road, Haidian, Beijing 100091, China
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h i g h l i g h t s • XBXT-2 presented antidepressant-like effects in the mouse forced swim test (FST). • The antidepressant effect of XBXT-2 was reversed by pretreatment with l-arginine. • 7-Nitroindazole potentiated the antidepressant effect of XBXT-2.
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Article history: Received 17 December 2013 Received in revised form 27 March 2014 Accepted 24 April 2014 Available online xxx
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Keywords: Forced swim test Antidepressant-like effect Nitric oxide Xiaobuxin-Tang
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1. Introduction
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Our previous studies have demonstrated that the total flavonoids (XBXT-2) isolated from the extract of Xiaobuxin-Tang (XBXT), a traditional Chinese herbal decoction, exerted antidepressant-like effects. Recently, accumulating studies have suggested that l-arginine-NO is implicated in the regulation of depression. Therefore, the aim of current study attempts to explore the involvement of l-arginine-NO pathway in the antidepressant-like effect of XBXT-2 in the mouse forced swim test (FST). Our results showed that the antidepressant-like action of XBXT-2 (100 mg/kg, i.g.) was reversed by pretreatment with l-arginine (a nitric oxide precursor, 750 mg/kg, i.p.). While co-administration of aminoguanidine (a specific inducible NOS inhibitor, 40, 80 mg/kg, i.p.) and sub-effective dose of XBXT-2 (50 mg/kg, i.g.) did not significantly alter the immobility in FST. In contrast, combined administration of 7-nitroindazole (a specific neuronal NOS inhibitor, 50 mg/kg, i.p.) potentiated the antidepressant-like effect of non-effective doses of XBXT-2 (50 mg/kg, i.g.). Meanwhile, NO modulators were devoid of any locomotor effects on the animals. In conclusion, the antidepressant-like action of XBXT-2 may be involvement of NO signaling pathway. © 2014 Published by Elsevier Ireland Ltd.
Depression is a serious and burdensome psychiatric illness that is exacerbated by its recurrent nature. Although the monoamine
Abbreviations: ANOVA, analysis of variance; eNOS, endothelial NOS; FST, forced swim test; iNOS, inducible NOS; NMDA, N-methyl-d-aspartate; nNOS, neuronal NOS; NO, nitric oxide; NOS, nitric oxide synthase; SSRIs, selective serotonin reuptake inhibitors; TCM, traditional Chinese medicine; XBXT, Xiaobuxin-Tang; XBXT-2, the total flavonoids isolated from the extract of XBXT. Q2 ∗ Corresponding author. Tel.: +86 1066874606. ∗∗ Corresponding author. E-mail addresses: [email protected] (Y.-F. Li), [email protected], [email protected] (Y.-Z. Zhang). 1 These authors contributed equally to this study.
hypothesis has been the mainstay of depression research for many years, the underlying pathophysiological mechanism of depression remains obscure. It has been suggested nitric oxide (NO) was involved in the pathophysiology of major depression [7] [11]. Decreasing the levels or blocking the synthesis of NO in brain can induce antidepressant-like effects, thus implicating the role of NO in the pathophysiology of major depression [5]. The level of plasma NO metabolites had been found to be significantly higher in suicidal patients than in nonsuicidal psychiatric patients or in normal control subjects [10]. Various nitric oxide synthase (NOS) inhibitors viz. l-NAME (a non-selective NOS inhibitor) or 7-nitroindazole (7-NI, a selective neuronal NOS inhibitor) are known to display antidepressant-like activity [12]. In addition, some clinical evidence also supported the possible role of elevated NO functioning in depression. Paroxetine, a selective serotonin reuptake inhibitor
http://dx.doi.org/10.1016/j.neulet.2014.04.039 0304-3940/© 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: L.-M. Zhang, et al., Involvement of nitric oxide (NO) signaling pathway in the antidepressant action of the total flavonoids extracted from Xiaobuxin-Tang, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.039
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(SSRI), is a NOS inhibitor which has encouraged the researchers to explore more about NO in the pathogenesis of major depression [8]. Xiaobuxin-Tang (XBXT), comprising Haematitum, Flos Inulae, Folium Phyllostachydis Henonis and Semen Sojae Preparatum, four Chinese medicines, was originally recorded in the silk scroll manuscript of “Fuxinjue Zangfu Yongyao Fayao”, written one thousand years ago and discovered in Mogao Caves of Dunhuang. The three ingredients of XBXT (Flos Inulae, Folium Phyllostachydis Henonis and Semen Sojae Preparatum) all contain a large amount of flavonoids. And during the process of extraction, the flavonoids may react with Haematitum, the fourth ingredient of XBXT, to formsome flavone complexes. Our previous study has demonstrated that the total flavonoids (XBXT-2) isolated from XBXT exerted antidepressant-like effects in the behavioral despair models [13]. Given this background, we hypothesized that NO signaling pathway might be involvement in the antidepressant action of XBXT-2. We confirmed our hypothesis by using forced swim test as the animal model of behavioral despair. In this context, we used l-arginine (a precursor of NO, l-Arg), 7-NI, aminoguanidine (a specific inducible NOS inhibitor, AMG) as different NO modulators. These agents were administered prior to XBXT-2 and the doses of all these modulators have previously been reported by other laboratory or established in our laboratory.
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2. Materials and methods
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2.1. Animals
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Male ICR mice (18 ± 2 g) were purchased from the Beijing Vital Laboratory Animal Technology Company. Animals were maintained under standard conditions of controlled temperature (23 ± 1 ◦ C), humidity (45%), and lighting (12-h light/dark cycle). Food pellets and water were freely available. The experimental procedures were approved by the local Committee on Animal Care and Use, and all efforts were made to minimize animal suffering and reduce the number of animals used for the experiments.
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2.2. Drugs and treatments
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Fig. 1. XBXT-2 reduces immobility time in the mouse FST. Data represent the mean + S.E.M. of 8–10 animals per group. Values were analyzed by One Way Analysis of Variance (ANOVA) followed by Dunnett’s tests. *P < 0.05, **P < 0.05 vs. vehicle control.
To investigate the possible involvement of the l-arginine-NO pathway in the anti-immobility effects of XBXT-2 in the FST, mice were pre-treated with l-Arg (350, 700 mg/kg, i.p., a precursor of NO) 30 min before XBXT-2 (100 mg/kg, i.g.) and 60 min thereafter, animals were challenged with forced swim test. Desipramine (DIM, SIGMA, 15 mg/kg), a tricyclic antidepressant, was i.g. administered as a positive control. In another set of experiments, we investigated the effects of the combined administration of AMG (40, 80 mg/kg, i.p.) or 7-NI (25, 50 mg/kg, i.p.) with sub-effective dose of XBXT-2 (50 mg/kg, i.g.). XBXT-2 or vehicle was administered 10 min after NOS inhibitor and a further 50 min were allowed to elapse before the animals were tested in the forced swim test. 2.4. Behavioral analysis
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Traditional Chinese medicines Haematitum, Flos Inulae, Folium Phyllostachydis Henonis and Semen Sojae Preparatum were purchased from Beijing Tongrentang Drugstore (Beijing, China) and were identified by Prof. Lian-Sheng Shen (School of Chinese Medicine, Beijing University of Chinese Medicine) as calcined product of ochery hematite, flowers of Inula japonica Thunb., leafs of Phyllostachys nigra (Lodd.) Munro var. henonis (Mitf.) Stapf ex Rendle, fermented product of Glycine max (L.) Merr., respectively. The voucher specimens (Nos. 02002, 02003, 02004, 02005, respectively) are deposited in the Laboratory of Phytochemistry, Beijing Institute of Pharmacology and Toxicology, China. The extraction of XBXT-2 was carried out according to the method of An [2]. And using lutin as standard substance, the apparent flavones content of the extract was determined as 76.03% by colorimetric method. All chemicals were purchased from Sigma (St. Louis, USA). lArginine, AMG, and XBXT-2 were dissolved in saline. 7-NI was prepared in 20% DMSO (v/v). All treatments were administered by intragastric gavage (i.g.) or intraperitoneally (i.p.) in a volume of 2 ml/kg.
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2.3. Pharmacological treatment
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Firstly, we studied the effect of different doses of XBXT-2 (50, 100 and 200 mg/kg) or NO modulators (l-Arg, 7-NI, AMG) for determination of the effective and sub-effective doses of them.
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2.4.1. Open-field test: locomotor activity In order to rule out any non-specific locomotor effect of XBXT-2 on antidepressant-like activity, mice were assessed the locomotor activity in an open-field test, immediately before the forced swim test. A mouse was placed in the corner of a plastic box (the base was divided into equal sectors, 36 cm × 29 cm × 23 cm) for a 5-min acclimation; the number of crossings (crossing the sector with four Table 1 Effect of NO modulators in the mouse forced swim test. Drug
Dose (mg/kg)
Duration of immobility
Saline l-Arg
– 350 700 1400
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± ± ± ±
21.57 29.85 39.41 27.58
Saline AMG
– 40 80 120
190.5 184.33 179.17 164.67
± ± ± ±
15.96 16.09 15.94 17.78*
– 25 50 100
188 178.67 153.67 140.5
± ± ± ±
21.40 22.88 16.73* 22.71**
Saline 7-NI
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Data represent the mean ± S.E.M. of 6 mice/group. Separate saline controls are presented for each experiment. Values were analyzed by One Way Analysis of Variance (ANOVA) followed by Dunnett’s tests. * P < 0.05. ** P < 0.05 vs. saline.
Please cite this article in press as: L.-M. Zhang, et al., Involvement of nitric oxide (NO) signaling pathway in the antidepressant action of the total flavonoids extracted from Xiaobuxin-Tang, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.039
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Fig. 2. (A) l-Arginine (l-arg) antagonizes the action of XBXT-2 in the mouse FST. (B) Effects of l-arg, XBXT-2, and their combination on locomotion. l-Arg (350, 700 mg/kg, i.p.) was administered 30 min before the treatment with XBXT-2 and after further 60 min, animals were challenged with forced swim test. Data represent the mean + S.E.M. of 8–10 animals per group. Values were analyzed by Two Way Analysis of Variance (ANOVA) followed by Bonferroni post-tests. *P < 0.05 vs. vehicle/vehicle-treated control; # P < 0.05 vs. vehicle/XBXT-2 (100 mg/kg).
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paws) and rearings (raising the forepaws) in the next 5 min was then recorded. The light inside the apparatus was maintained at minimum to avoid anxiety behavior. After each test the apparatus was cleaned with a solution of 10% ethanol to hide animal clues.
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2.4.2. Forced swim test (FST) Immediately after open-field test, mice were individually placed in an open cylindrical container (diameter 10 cm, height 25 cm) containing 19 cm of water at 25 ± 1 ◦ C for 6 min. The immobility time (characterized by slow movements necessary to avoid drowning) was measured during the last 4-min period by a trained observer that was blind to the treatment condition.
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2.5. Data analysis
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Data were statistically examined using one-way or two-way analysis of variance (ANOVA). Post hoc comparisons between individual groups were performed by Dunnett’s test. Data were expressed as the mean values + S.E.M. Differences were considered to be statistically significant when P < 0.05.
3. Results
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3.1. Effect of XBXT-2 or NO modulators on forced-swimming test
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Desipramine, a positive control, caused a reduction in the immobility time in FST at a dose of 30 mg/kg. XBXT-2 (100, 200 mg/kg, i.g.) also significantly decreased the immobility time of mice in FST, showing an antidepressant-like effect (Fig. 1). Treatment with the NO synthase substrate, l-Arg alone (350–1400 mg/kg) did not modify the behavior of animals in forced swimming test. Both AMG (120 mg/kg) and 7-NI (50, 100 mg/kg), given by i.p. route, decreased immobility time in the FST.(Table 1). 3.2. Effect of NO modulators on anti-immobility effect of XBXT-2 on forced-swimming test For further determination of NO role in antidepressant-like effect of XBXT-2, l-Arg was applied as a NO precursor. Two-way ANOVA showed significant effect of l-Arg [F(2,53) = 7.108, P < 0.05], XBXT-2 [F(1,53) = 13.97, P < 0.001] and a l-Arg × XBXT-2 interaction
Please cite this article in press as: L.-M. Zhang, et al., Involvement of nitric oxide (NO) signaling pathway in the antidepressant action of the total flavonoids extracted from Xiaobuxin-Tang, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.039
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Fig. 3. (A) Effect of co-administration of non-effective doses of aminoguanidine (AMG) on antidepressant-like effect of XBXT-2. (B) Effects of AMG, XBXT-2, and their combination on locomotion. XBXT-2 or vehicle was administered 10 min after AMG (40, 80 mg/kg, i.p.) and after further 50 min, the duration of immobility in FST during the last 4 min of the test was recorded. Data represent the mean + S.E.M. of 8–10 animals per group. Values were analyzed by Two Way Analysis of Variance (ANOVA) followed by Bonferroni post-tests.
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[F(2,53) = 6.949, P < 0.05]. As it was determined in Fig. 2A, treatment with l-Arg alone (350, 700 mg/kg) did not modify the behavior of animals in FST. XBXT-2 (100 mg/kg) significantly decreased the immobility time (P < 0.05 vs. vehicle group), and pretreatment with l-Arg (350 mg/kg) did not counteract the antidepressant effect of XBXT-2 (100 mg/kg), while higher dose of l-Arg (700 mg/kg) could reverse its effect (P < 0.05 vs. XBXT-2 group). Administration of l-Arg alone or in combination with XBXT-2 did not affect the locomotor activity in the open-field (Fig. 2B). To investigate the possible interaction of inducible NOS inhibition in anti-immobility effect of XBXT-2, effect of AMG was determined in FST in combination with XBXT-2. Two-way ANOVA showed no significant effects of AMG [F(2,53) = 0.1548, P > 0.05], XBXT-2 [F(1,53) = 0.7592, P > 0.05] and a AMG × XBXT-2 interaction [F(2,53) = 0.06484, P > 0.05]. As shown in Fig. 3A, administration of AMG (40, 80 mg/kg) alone or in combination with non-effective dose of XBXT-2 (50 mg/kg) did not significantly change the immobility time in FST, or alter the locomotor activity in the open field test (Fig. 3B). To investigate the possible interaction of neuronal NOS inhibition in anti-immobility effect of XBXT-2, we further evaluated whether co-administration of 7-NI and non-effective doses of XBXT-2 (50 mg/kg) had any effect on the immobility time in FST. Two-way ANOVA showed significant effects of 7-NI [F(2,53) = 41.88,
P < 0.001], XBXT-2 [F(1,53) = 32.03, P < 0.05] and a 7-NI × XBXT2 interaction [F(2,53) = 4.568, P < 0.001]. The results depicted in Fig. 4A showed that when 7-NI (50 mg/kg) was administered concomitantly with sub-active dose of XBXT-2 (50 mg/kg), the immobility time of mice in the FST was significantly decreased (P < 0.05) compared with vehicle/vehicle treated animals without affecting locomotor activity in the open field test (Fig. 4B).
4. Discussion In the present study, the antidepressant action of XBXT-2 was attenuated by pretreatment with l-Arg and the action of noneffective dose of XBXT-2 was potentiated by sub-effective doses of 7-NI, a neuronal NOS inhibitor, but not by sub-effective doses of AMG, an inducible NOS inhibitor. The present results firstly showed that XBXT-2 given orally was effective in producing significant antidepressant-like effects, when assessed in the forced swim test, confirming previous results from our group [1–3,13]. Moreover, our findings also demonstrated that XBXT-2 at dose of 100 mg/kg was more efficacious than that of 200 mg/kg, which was also in line with our behavioral results which showed that XBXT-2 exhibited an inverse U-shaped dose-response curve in acute and subchronic models [3].
Please cite this article in press as: L.-M. Zhang, et al., Involvement of nitric oxide (NO) signaling pathway in the antidepressant action of the total flavonoids extracted from Xiaobuxin-Tang, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.039
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Fig. 4. (A) 7-NI augments the action of XBXT-2 in the mouse FST. (B) Effects of 7-NI, XBXT-2, and their combination on locomotion. XBXT-2 or vehicle was administered 10 min after 7-NI (25, 50 mg/kg, i.p.) and after further 50 min, the duration of immobility in FST during the last 4 min of the test was recorded. Data represent the mean + S.E.M. of 8–10 animals per group. Values were analyzed by Two Way Analysis of Variance (ANOVA) followed by Bonferroni post-tests. *P < 0.05 vs. vehicle/vehicle-treated control; # P < 0.05 vs. vehicle/XBXT-2 (50 mg/kg).
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Having established both effective and sub-effective doses of XBXT-2 in mouse forced swim test, we further extended our study to explore the role of NO pathway in its antidepressant action. In this present study, we have employed 350, 750 mg/kg, i.p. dose of l-Arg that did not affect the immobility period in mouse forced swim test. It was in line with previous reports that showed l-Arg in a dose range of 750–1000 mg/kg does not affect the immobility period in mouse forced swim test. The antidepressant action of XBXT-2 was prevented by the pretreatment of l-Arg suggesting that the antidepressant-effect of XBXT-2 may be dependent on, at least in part, the inhibition of NO synthesis. Our results are in accordance with previous studies that pre-treatment with l-Arg counteracted the antidepressant-like effect of imipramine, venlafaxine and duloxetine in the forced swimming test [9]. To further confirm our hypothesis, we then combined noeffective dose of XBXT-2 with selective neuronal NOS inhibitors. The neuronal NOS is the more abundant isoform in the brain under normal circumstances. Pre-clinical and clinical findings, such as increased expression of neuronal NOS in limbic regions of stressed animals and depressed patients have helped to link neuronal NOSmediated NO synthesis to the neurobiology of depression. Data presented here demonstrated that combination of 7-NI (50 mg/kg) with XBXT-2 (50 mg/kg) further augmented the antidepressant
response. Our results are in line with results that showed an increased anti-immobility effect after combined administration of 7-NI and venfalaxine, duloxetine [14] and fluoxetine [9]. The antidepressant-like effect of AMG was evident at the smallest dose tested (100 mg/kg). This is in agreement with previous works describing that AMG attenuates stress induced effects in a dose range of 50–100 mg/kg. While co-administration of subeffective doses of AMG and XBXT-2 did not significantly alter the immobility in the forced swim test, which suggested demonstrated that calcium-insensitive NOS enzyme (inducible NOS) probably might not be involved in the antidepressant-like effect of XBXT2 and the calcium-dependent constitutive NOS enzymes (neuronal NOS) are probably involved in this mechanism. Although, the FST is generally recommended for a screen of antidepressant-like effects, drugs enhancing motor activity may give a ‘false’ positive effect in this model, decreasing immobility time by stimulating locomotor activity. Therefore, a relevant question that should be answered is whether the results observed in the FST could be influenced by differences in locomotor behavior. As showed in the present study, XBXT-2 and/or NO modulators showed no stimulatory effect on locomotor activity, ruling out the hypothesis that a psycho-stimulant effect might be responsible for the behavior effects observed in the current study.
Please cite this article in press as: L.-M. Zhang, et al., Involvement of nitric oxide (NO) signaling pathway in the antidepressant action of the total flavonoids extracted from Xiaobuxin-Tang, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.039
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It has been demonstrated that NO also modulates the serotoninergic system. The majority of serotonergic neurons in the raphe nucleus contain neuronal NOS. Several in vitro studies have demonstrated that NO modulates the extracellular levels of serotonin in the central nervous system. In addition, NO can inactivate the rate limiting enzyme in the synthesis of 5-HT, tryptophan hydroxylase [4]. Also, fluoxetine, a selective serotonin reuptake inhibitor, could decrease striatal NO production induced by N-methyl-daspartate (NMDA) injection and downregulate nNOS expression in the hippocampus region of the brain [6]. Further, NAN-190 (a selective 5-HT1A serotonin receptor antagonist) has been found to up-regulate the hippocampal neuronal NOS expression. Furthermore, it was also shown that the antidepressant-like behavioral effects of both l-NA and 7-NI were dependent on endogenous serotonin. Interestingly, our previous study has also shown that administration of the XBXT-2 exerted significant antidepressantlike effect in chronically mildly stressed rats, possibly mediated by serotonergic activation. Thus, these studies demonstrated the inter-relation between NO and serotonin might be involved in the pharmacological effects of XBXT-2. It is important to point out that the behavioral results presented in this report were obtained through the use of a single animal model, the forced swim test. Therefore, it will be important in future studies to validate the pattern of effects presented here using other behavioral paradigms such as learned helplessness, chronic mild stress and social defeat stress models.
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5. Conclusion
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Together with our previous results, it is reasonable to speculate that l-arginine-NO may be involved in the antidepressant-like effect of XBXT-2, and further experiments are necessary to clarify the exact molecular mechanisms underlying the behavioral effects of XBXT-2 and the interplay between NO and serotonin. Furthermore, these data raise the possibility that inhibition of NO synthesis could be used as a strategy to enhance the clinical efficacy of serotonergic antidepressant. Acknowledgements This study was supported by the National Natural Science Foundation of China (Nos. 81001653, 81302726, 81274117 and
81173036), the National Key New Drug Creation Program (Nos. 2012ZX09102101-004 and 2012ZX09J12201-004) and the 12th fine-year army science and technology project (No. CWS12J084). References [1] L. An, Y.Z. Zhang, X.M. Liu, N.J. Yu, H.X. Chen, N. Zhao, L. Yuan, Y.F. Li, Total flavonoids extracted from Xiaobuxin-Tang on the hyperactivity of hypothalamic–pituitary–adrenal axis in chronically stressed rats, Evid. Based Complement. Altern. Med. 2011 (2011) 367619. [2] L. An, Y.Z. Zhang, N.J. Yu, X.M. Liu, N. Zhao, L. Yuan, H.X. Chen, Y.F. Li, The total flavonoids extracted from Xiaobuxin-Tang up-regulate the decreased hippocampal neurogenesis and neurotrophic molecules expression in chronically stressed rats, Prog. Neuro-Psychopharmacol. Biol. Psychiatry 32 (2008) 1484–1490. [3] L. An, Y.Z. Zhang, N.J. Yu, X.M. Liu, N. Zhao, L. Yuan, Y.F. Li, Role for serotonin in the antidepressant-like effect of a flavonoid extract of Xiaobuxin-Tang, Pharmacol. Biochem. Behav. 89 (2008) 572–580. [4] M. Breard, M.A. Sari, Y. Frapart, J.L. Boucher, C. Ducrocq, C. Grillon, The endogenous neurotransmitter, serotonin, modifies neuronal nitric oxide synthase activities, Free Radic. Res. 41 (2007) 413–423. [5] W.E. Chrapko, P. Jurasz, M.W. Radomski, N. Lara, S.L. Archer, J.M. Le Melledo, Decreased platelet nitric oxide synthase activity and plasma nitric oxide metabolites in major depressive disorder, Biol. Psychiatry 56 (2004) 129–134. [6] F. Crespi, The selective serotonin reuptake inhibitor fluoxetine reduces striatal in vivo levels of voltammetric nitric oxide (NO): a feature of its antidepressant activity? Neurosci. Lett. 470 (2010) 95–99. [7] A. Dhir, S.K. Kulkarni, Nitric oxide and major depression, Nitric Oxide: Biol. Chem./Off. J. Nitric Oxide Soc. 24 (2011) 125–131. [8] M.S. Finkel, F. Laghrissi-Thode, B.G. Pollock, J. Rong, Paroxetine is a novel nitric oxide synthase inhibitor, Psychopharmacol. Bull. 32 (1996) 653–658. [9] M. Krass, G. Wegener, E. Vasar, V. Volke, The antidepressant action of imipramine and venlafaxine involves suppression of nitric oxide synthesis, Behav. Brain Res. 218 (2011) 57–63. [10] J. Moreno, E. Gaspar, G. Lopez-Bello, E. Juarez, S. Alcazar-Leyva, E. GonzalezTrujano, L. Pavon, N. Alvarado-Vasquez, Increase in nitric oxide levels and mitochondrial membrane potential in platelets of untreated patients with major depression, Psychiatry Res. 209 (2013) 447–452. [11] A. Spiacci Jr., F. Kanamaru, F.S. Guimaraes, R.M. Oliveira, Nitric oxide-mediated anxiolytic-like and antidepressant-like effects in animal models of anxiety and depression, Pharmacol. Biochem. Behav. 88 (2008) 247–255. [12] F. Yildiz, B.F. Erden, G. Ulak, T. Utkan, N. Gacar, Antidepressant-like effect of 7nitroindazole in the forced swimming test in rats, Psychopharmacology (Berl) 149 (2000) 41–44. [13] Y.Z. Zhang, Y.F. Li, N.J. Yu, L. Yuan, Y.M. Zhao, W.B. Xiao, Z.P. Luo, Antidepressantlike effects of the ethanolic extract of Xiaobuxin-Tang, a traditional Chinese herbal prescription in animal models of depression, Chin. Med. J. (Engl.) 120 (2007) 1792–1796. [14] A.D. Zomkowski, D. Engel, M.P. Cunha, N.H. Gabilan, A.L. Rodrigues, The role of the NMDA receptors and l-arginine-nitric oxide-cyclic guanosine monophosphate pathway in the antidepressant-like effect of duloxetine in the forced swimming test, Pharmacol. Biochem. Behav. 103 (2012) 408– 417.
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