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Acupuncture ameliorates inflammatory response in a chronic unpredictable stress rat model of depression
Accepted Manuscript Title: Acupuncture ameliorates inflammatory response in a chronic unpredictable stress rat model of depression Author: Jun Lu Run-Hui Shao Shu-Ying Jin Li Hu Ya Tu Jian-You Guo PII: DOI: Reference:
S0361-9230(16)30304-5 http://dx.doi.org/doi:10.1016/j.brainresbull.2016.11.010 BRB 9121
To appear in:
Brain Research Bulletin
Received date: Revised date: Accepted date:
28-9-2016 5-11-2016 23-11-2016
Please cite this article as: Jun Lu, Run-Hui Shao, Shu-Ying Jin, Li Hu, Ya Tu, Jian-You Guo, Acupuncture ameliorates inflammatory response in a chronic unpredictable stress rat model of depression, Brain Research Bulletin http://dx.doi.org/10.1016/j.brainresbull.2016.11.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Acupuncture ameliorates inflammatory response in a chronic unpredictable stress rat model of depression Jun Lua, Run-Hui Shaoa, Shu-Ying Jinb, Li Huc, Ya Tua* , Jian-You Guod* a
College of Acupuncture-Moxibustion and Tui Na, Beijing University of Chinese
Medicine, Beijing, 100029, China b
Beijing Feng-Tai Pu-Huangyu Community Health Center, Beijing, 100075, China
c
Tangshan Feng-Run Hospital of TCM, Tangshan, 064000, China
d
Key Laboratory of Mental Health, Institute of Psychology, Chinese Academy of
Sciences, Beijing, 100101, China Corresponding Authors: Ya Tu and Jian-You Guo E-mail address: Ya Tu: [email protected]. Jian-You Guo: [email protected].
1
Highlights
Acupuncture treatment alleviated the depressive-like behavior in CUMS rats.
Acupuncture treatment decreased levels of iNOS and NO in the hippocampus and prefrontal cortex in CUMS rats.
Acupuncture treatment reduced expressions of COX-2 and PGE2 in the hippocampus and prefrontal cortex in CUMS rats.
Acupuncture treatment inhibited NF-κB activation in the hippocampus and prefrontal cortex in CUMS rats.
2
Abstract Depression is one of the most common psychiatric disorders. Chronic inflammatory response has been viewed as a key factor in depression. Acupuncture in Chinese medicine has been shown to be an effective treatment for depression. In the present study, we investigated the mechanism underlying antidepressant effect of acupuncture. The rats were subjected to chronic unpredictable mild stress (CUMS) for 28 days to induce depressive-like behaviors. Acupuncture treatment was applied once every other day during the 28-day stress period. The behavioral tests (body weight, sucrose consumption and locomotor activity) were performed. The expressions of nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and nuclear factor kappa B (NF-κB) were determined in the rat hippocampus and prefrontal cortex. CUMS induced depressive-like behavior in rats, which was alleviated by acupuncture treatment. The increased levels of NO, PGE2, iNOS and COX-2 induced by CUMS, were all significantly decreased in the hippocampus and prefrontal cortex by acupuncture. Moreover, acupuncture markedly inhibited the activation of NF-κB in rats. These findings showed that the antidepressant-like effect of acupuncture might be mediated by inhibition of inflammatory mediators via modulation of NF-κB in the brain regions. Key Words: acupuncture; depression; chronic unpredictable stress; inflammatory response
3
1. Introduction Depression is a common and debilitating psychiatric disorder in modern society, characterized by a pervasive low mood and loss of interest in usual activities[1]. Antidepressants dominate current treatment in clinical practice. There are three main kinds of classical antidepressants, including tricyclic antidepressants (TCA), selective serotonin reuptake inhibitors (SSRIs), and monoamine oxidase inhibitors (MAOIs). However, some of these drugs show undesirable side effects. Meanwhile, less than 50% of depression patients achieve remission with the available antidepressants[2]. Therefore, there is no doubt that alternative medicines with safety and efficacy are needed to treat depression. Acupuncture in traditional Chinese medicine has become a widely accepted alternative therapy. The specific effects of acupuncture on central nervous system are attracting more and more interest. For example, acupuncture plays an important role in protecting the central nervous system from neuronal damage[3,4]. Moreover, clinical trials have demonstrated that acupuncture could alleviate depressive symptoms[5-8], and modulate the corticostriatal reward circuitry in major depressive disorder[9]. Previous studies have shown that acupuncture could have antidepressant-like effect by modulating hypothalamic-pituitary-adrenal axis, enhancing hippocampal serotonin system[10], modulating dopaminergic neuroactivity[11], and reducing pro-inflammatory cytokines
[12].
In recent years, chronic inflammatory response has been viewed as a key factor in depression. The expressions of many inflammatory mediators, such as 4
pro-inflammatory
cytokines,
inducible
nitric
oxide
synthase
(iNOS)
and
cyclooxygenase-2 (COX-2), are regulated by nuclear factor kappa B (NF-κB)[13, 14]. NF-κB can be activated by a number of stimuli, including lipopolysaccharide or stress. After activation, NF-κB induces the transcription of the inflammatory mediators involved in inflammatory response[15, 16]. Therefore, NF-κB pathway play a vital role in regulating inflammatory responses in depression[17-19]. For example, it has been reported that chronic stress enhanced the activation of NF-κB[15, 20], and increased expression of COX-2[21, 22], as well as iNOS in the rat model of depression[23-26]. The increase of COX-2 and iNOS expression has also been found in depression patients [27]. However, the effect of acupuncture on the inflammatory mediators involved in depression remains unclear. The hippocampus and prefrontal cortex have been suggested to be the important brain regions associated with the mood regulation, cognitive function and memory. Clinical studies have indicated their functional and structural abnormalities in depression[28-30]. Therefore, in this study, we investigated the effect of acupuncture on inflammatory response in hippocampus and prefrontal cortex in a chronic unpredictable mild stress (CUMS) rat model of depression. Our results showed that acupuncture could attenuate inflammatory response in depression by inhibiting the key inflammation mediators via modulation of NF-κB activation in the brain regions. 2. Materials and Methods 2.1. Animals. Male Sprague-Dawley rats (180～200g) were obtained from Beijing Vital River Laboratories. Rats were kept in an air-conditioned room with a 12 h light/dark cycle with free access to food and water except when animals were 5
subjected to deprivation stressors as described in stress. The experiment procedures were approved by the Animal Care and Use Committee at Beijing University of Chinese Medicine. 2.2. Groups and Treatment. The rats were randomly divided into five groups (eight rats in each group): the control group was given no stress except general handling for 4 weeks; the CUMS group was exposed to CUMS for 4 weeks; the CUMS + Acu(Acu) group received acupuncture treatment once every other day during the 4-week stress period; the CUMS + Fluoxetine (Flu) group, used as a positive comparator for an antidepressant effect, was given Fluoxetine during the 4-week stress period. Fluoxetine was diluted in distilled water and orally given one hour before the stress exposure. 2.3. Chronic Unpredictable Mild Stress Procedure. Rats in stressed groups were exposed to CUMS after 1 week of acclimatization period under the housing conditions. The CUMS model was modified from the methods previously described[31, 32]. The rats were randomly exposed to various stressors for 4 weeks, and each stressor was administered once a week. The stressors applied included: deprivation of food (24 h, F), deprivation of water (24 h, W), cold swimming (4°C, 5 min, S), cage shaking (30 min,C), behavior restraint (3 h, R), tail pinch (2 min, T), and soiled bedding (24 h, B). The stress sequence was changed every week in order to make the stress procedure unpredictable (Fig.1). The control group was housed in a separate room, with free access to food and water. 2.4. Acupuncture treatment
and
drug
6
administration.
During acupuncture
administration, the rats were maintained within a cloth bag without anesthesia, with one forelimb taken out, similar to what we described previously[12]. Two points were selected: Baihui (GV20) and Neiguan (PC6). GV20 is located above the apex auriculate, on the midline of the head. PC6 is located between the tendons of m. palmarislongus and m. flexor carpi radialis, proximal to the transverse crease of the wrist. Sterilized disposable stainless steel needles (0.3*25mm, HuanQiu brand, Suzhou, China) were inserted obliquely as deep as 2-3 mm at Baihui (GV20) and Neiguan (PC6). The acupuncture treatment was manually manipulated by twisting the needles at a frequency of twice per second for 1 min, and then the needles were retained for 10 minutes. The rats received acupuncture treatment once every other day before the stress during the 4-week period. Fluoxetine (Zhejiang Regen Chemical Co., Ltd, China) was diluted in saline and administered daily by oral gavage at a dose of 10 mg/kg for 4 weeks (Fig.1). The dosage of 10 mg/kg for fluoxetine has been reported to show antidepressant action in previous work[33]. 2.5. Body weight. The body weight was measured at the beginning and the end of the experiment. 2.6. Sucrose intake test. The sucrose intake test was conducted according to methods described in literatures with minor modifications[31, 32]. Rats were exposed to 1% sucrose solution for 48 h without any water available. Then the test was carried out after 24h of water deprivation. The rats were given a 1h sucrose test. The sucrose consumption was measured by reweighing preweighed bottles of sucrose solution. The sucrose intake was performed at the beginning and the end of the experiment. 7
2.7. Open field test. At the end of the experiment, the open field test was performed. The apparatus consisted of a square arena 100×100 cm with 50 cm high wall. It was divided into 5×5 equal squares which had been drawn in the floor of the arena. A single rat was gently placed in the center of the floor in order to explore the arena for 5 min. The crossing numbers (defined as at least three paws in a square) and the rearing numbers (defined as the rat standing upright on its hind legs) were counted manually by two observers who were blind to the experiment. 2.8. Enzyme-linked immunosorbent assay (ELISA). After the behavioral tests, the rats were sacrificed and the brains were quickly removed on ice. The hippocampus and prefrontal cortex were isolated and put into chilled tubes in liquid nitrogen, then stored at -80C for further analysis. The concentrations of COX-2, iNOS and prostaglandin E2 (PGE2) were measured using commercially available ELISA kits (R&D Systems, USA) according to the manufacturer’s instructions. 2.9. Determination of nitric oxide (NO) content. The NO concentration in the hippocampus and prefrontal cortex was measured using commercially available nitrate reductase method kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions. 2.10. Western blot analysis. Half of the hippocampus and prefrontal cortex were isolated and put into chilled tubesin liquid nitrogen. The brain regions were homogenized and Western blot analysis was carried out as previously reported[22]. Briefly, primary antibodies for NF-κB p65 and β-actin at 1:1000 dilution (Santa Cruz Biotech Inc., CA, USA). A secondary antibody conjugated with horseradish 8
peroxidase was used. Immuno-reactivity was visualized by ECLreagent. NF-κB p65protein expression was quantified by densitometry using the Scion Image Beta 4.02 software and are shown as density relative to β-actin. 2.11. Statistical Analysis. Data were presented as means ± S.E.M. Differences among groups were examined using one-way ANOVA, followed by Newman-Keuls test. P< 0.05 was the accepted level of significance. 3. Results 3.1 Effects of acupuncture treatment on the body weight. As illustrated in Fig. 2,before the CUMS procedure, no differences in body weight were observed between groups (F3,28= 0.46, p>0.05), but a significant difference in body weight was observed between groups following 4 weeks of CUMS (F3,28= 23.79, p< 0.01). The body weight significantly decreased in the CUMS group compared with the control group(p< 0.01). Compared with the CUMS group, the body weight significantly increased in the acupuncture group (p<0.05), and the fluoxetine group also exhibited a significant increase in body weight (p<0.05). 3.2. Effects of acupuncture treatment on the sucrose consumption. As shown in Fig. 3, before the CUMS procedure, no differences in sucrose consumption were observed between groups (F3,28 = 0.09, p >0.05), but a significant difference in sucrose consumption was observed between groups following 4 weeks of CUMS (F3,28 = 7.15, p< 0.01). Stressed rats exhibited a reduction of sucrose consumption compared with controls after 4 weeks of stress exposure (p< 0.01). Rats that were subjected to CUMS and received acupuncture treatment exhibited a significant increase in sucrose consumption compared with rats that were subjected only to CUMS (p< 0.01). Sucrose consumption in the fluoxetine group was also 9
significantly higher compared with the CUMS group (p< 0.01). 3.3. Effects of acupuncture treatment on the locomotor activity. As shown in Fig. 4, a significant difference was observed between groups in the crossing numbers (F3,28 = 8.96, p< 0.01) and the rearing numbers (F3,28 = 7.76, p< 0.01) after CUMS. Acupuncture and fluoxetine treatment significantly reversed the stress-induced decrease in the number of crossings(both p< 0.01). Acupuncture and fluoxetine treatment also significantly increased the number of rearings (p< 0.01, p< 0.05 respectively). 3.4. Effects of acupuncture treatment on NO and PGE2 content in the hippocampus and prefrontal cortex. As shown in Fig.5, following 4 weeks of CUMS, there were significant differences in the content of NO (F3,28 =8.84, p< 0.01) and PGE2 (F3,28 =6.05, p< 0.01) in the hippocampus between groups. Compared with the control group, the content of NO and PGE2 significantly increased in the hippocampus in stressed rats (both p< 0.01). Acupuncture and fluoxetine treatment significantly decreased the production of NO (both p< 0.001), and PGE2 (both p< 0.01) in the hippocampus in CUMS rats. Significant differences were also observed between groups in the production of NO (F3,28 =6.04, p< 0.01) and PGE2 (F3,28 =6.50, p< 0.01) in the prefrontal cortex. CUMS significantly increased the production of NO and PGE2 in the prefrontal cortex compared with the control group (all p< 0.01). Acupuncture and fluoxetine treatment significantly decreased the content of NO (both p< 0.01), and PGE2 (both p< 0.01) in the prefrontal cortex in stressed rats. 3.5. Effects of acupuncture treatment on iNOS and COX-2 expression in the hippocampus and prefrontal cortex. As shown in Fig.6, we further detected protein levels of iNOS and COX-2 related 10
toNO and PGE2 synthesis respectively. A significant difference was observed between groups in the iNOS expression in the hippocampus (F3,28 =9.95, p< 0.01) and prefrontal cortex (F3,28 =13.52, p< 0.01). The expression of iNOS in the hippocampus and prefrontal cortex was significantly higher in CUMS group (both p< 0.01) compared with the control group. Acupuncture and fluoxetine treatment significantly reduced the expression of iNOS in the hippocampus (both p< 0.01) and prefrontal cortex (both p< 0.01) of CUMS rats. A significant difference was also observed between groups in the COX-2 expression in the hippocampus (F3,28 =8.36, p< 0.01) and prefrontal cortex (F3,28 =14.37, p< 0.01). The expression of COX-2 in the hippocampus and prefrontal cortex was also significantly higher in CUMS group (both p< 0.01) compared with the control group. Acupuncture and fluoxetine treatment significantly reduced the expression of COX-2 in the hippocampus (both p< 0.01) and prefrontal cortex (both p< 0.01) of stressed rats. 3.6. Effects of acupuncture treatment on NF-κB expression in the hippocampus and prefrontal cortex. As shown in Fig.7, we detected NF-κB, the major transcription factor involved in inflammatory processes, which induced iNOS and COX-2 expression. A significant difference was observed between groups in NF-κB expression in the hippocampus (F3,11= 8.04, p< 0.01) and prefrontal cortex (F3,11= 12.81, p< 0.01) after CUMS. Acupuncture treatment significantly downregulated the stress-induced increase in NF-κB expression in the hippocampus and prefrontal cortex (both p< 0.01). Fluoxetine treatment also significantly decreased NF-κB expression in the hippocampus and prefrontal cortex (both p< 0.01). 4. Discussion 11
The
present
study
demonstrates
that
acupuncture
treatment
exhibits
antidepressant-like effect in CUMS rat model of depression. Furthermore, we found that this effect might be mediated by inhibition of inflammatory mediators (iNOS, COX-2, NO, PGE2) via regulation of NF-κB in the hippocampus and prefrontal cortex. Mounting evidence has recently indicated that chronic inflammatory response plays an important role in the pathophysiology of depression[34, 35]. NF-κB has been considered to be a critical mediator of behavioral changes and inflammatory response of depression[20]. Clinical study has demonstrated that NF-κB expression was significantly increased in depression patients[36]. We found that the expression of NF-κB in the hippocampus was increased by CUMS, which was in line with previous findings[15, 20]. We also found that NF-κB expression was increased in the prefrontal cortex. In addition, we found that acupuncture treatment significantly downregulated NF-κB expression in these brain regions. It has been reported that continuous infusion of selective NF-κB inhibitor significantly alleviated chronic stress-induced depressive-like
behaviors
and
protected
stress-impaired
hippocampal
neurogenesis[20]. The antidepressant-like effect of acupuncture might be mediated through reducing activation of NF-κB. As a transcription factor, NF-κB has been regarded as a central regulator of a number of genes involved in inflammatory response, such as pro-inflammatory cytokines, iNOS and COX-2[13-15]. Previous study has found that acupuncture prevented the increase of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor 12
necrosis factor-α (TNF-α) in rat model of depression[12]. However, the effect of acupuncture on key inflammatory enzymes involved in depression is not clear. NOS is the enzyme which generates NO. There are three isoforms of NOS enzymes: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). iNOS is inflammation inducible, and closely involved in regulating pathological inflammatory processes[37]. Although it has been revealed that 6h acute immobilization stress increased nNOS activity in rats, 6h chronic immobilization stress of 21 days induced iNOS in rat neurons[25, 37]. Therefore, it is likely that chronic stress induced iNOS in rats. We found that CUMS increased iNOS and NO in the hippocampus and prefrontal cortex. Evidence suggested that iNOS may contribute to depressive-like behaviors in a chronic stress mouse model[27]. Our data support a link between iNOS, inflammatory response, and depressive-like behaviors in an animal model of depression. Moreover, we found that acupuncture treatment significantly reduced iNOS and NO in the hippocampus and prefrontal cortex of CUMS rats. A study indicated that pretreating the mice with an iNOS inhibitor decreased iNOS mRNA expression and reversed stress-induced depressive-like behaviors[26]. Therefore, it showed that acupuncture could alleviate inflammatory response in depression through inhibition of iNOS and NO in the brain. COX is the enzyme that converts arachidonic acid to PGE2. It exists in two subtypes, cyclooxygenase-1 (COX-1) and COX-2. COX-1 is expressed in nearly all tissues and produces PGs to maintain physiological functions, while COX-2 is induced in brain regions in response to stress or injury, and release large amounts of 13
PGs to induce inflammatory state[21]. COX-2 has been well studied for its role in depression. For example, a clinical study has shown that COX-2 mRNA expression was increased in patients with recurrent depression[27]. Our results showed that exposure to CUMS markedly increased COX-2 and PGE2 in the hippocampus and prefrontal cortex. This is consistent with previously published data concerning the role of COX-2 in animal model of depression[22]. In addition, we found acupuncture treatment significantly decreased COX-2 and PGE2 in the hippocampus and prefrontal cortex. Previous studies have shown that chronic treatment with COX-2 inhibitor celecoxib displayed the antidepressant-like effect and decreased COX-2 expression in CUMS rats[22]. Celecoxib also decreased pro-inflammatory cytokines in a rat model of depression[38-40]. Our results revealed that the antidepressant-like effect of acupuncture could be mediated by decreasing COX-2 and PGE2 in brain regions. 5. Conclusion In summary, acupuncture displayed the antidepressant-like effect in CUMS rat model of depression. Acupuncture could attenuate inflammatory response in depression by inhibiting the key inflammation mediators in NF-κB pathway in the brain regions. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgments This study was supported by the Grants from the National Natural Science 14
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Figure Legends Fig. 1. A schematic diagram of protocol design, including schedule of chronic unpredictable mild stress, acupuncture and drug treatment. Fig. 2. Body weight in the following groups (N= 8 per group): Control, chronic unpredictable mild stress (CUMS), CUMS + Acupuncture (Acu), CUMS + Fluoxetine (Flu). Data are means ± SEM. **P< 0.01 as compared with the control group, #P< 0.05 as compared with the CUMS group. Fig.3. Sucrose consumption in the following groups (N= 8 per group): Control, CUMS, CUMS + Acupuncture (Acu), CUMS + Fluoxetine (Flu). Data are means ± SEM. **P< 0.01 as compared with the control group, ##P< 0.01 as compared with the CUMS group. Fig.4. The locomotor activity of open field test in the following groups (N= 8 per group): Control, CUMS, CUMS + Acupuncture (Acu), CUMS + Fluoxetine (Flu). Data are means ± SEM. **P< 0.01 as compared with the control group, #P< 0.05, ##P< 0.01 as compared with the CUMS group. Fig.5. Hippocampus and prefrontal NO and PGE2 levels in the following groups (N= 8 per group): Control, CUMS, CUMS + Acupuncture (Acu), CUMS + Fluoxetine (Flu). A) Hippocampus levels of NO and PGE2. B) Prefrontal cortex level of NO and PGE2. Data are means ± SEM. **P< 0.01 as compared with the control group, ##
P< 0.01 as compared with the CUMS group.
Fig.6. Hippocampus and prefrontal cortex iNOS and COX-2 in the following groups (N= 8 per group): Control, CUMS, CUMS + Acupuncture (Acu), CUMS + Fluoxetine 19
(Flu). A) Hippocampus level of iNOS and COX-2. B) Prefrontal cortex level of iNOS and COX-2. Data are means ± SEM. **P< 0.01 as compared with the control group, ##P< 0.01 as compared with the CUMS group. Fig.7. NF-κB expression measured by Western blot in the following groups (N= 5 per group): Control, CUMS, CUMS + Acupuncture (Acu), CUMS + Fluoxetine (Flu). A) Hippocampus. B)Prefrontal cortex. Data are means ± SEM.
**
P< 0.01 as compared
with the control group, ##P< 0.01 as compared with the CUMS group.
20
Fig.1
21
Fig.2
22
Fig.3
23
Fig.4
24
Fig.5
25
Fig.6
26
Fig.7
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S0361-9230(16)30304-5 http://dx.doi.org/doi:10.1016/j.brainresbull.2016.11.010 BRB 9121
To appear in:
Brain Research Bulletin
Received date: Revised date: Accepted date:
28-9-2016 5-11-2016 23-11-2016
Please cite this article as: Jun Lu, Run-Hui Shao, Shu-Ying Jin, Li Hu, Ya Tu, Jian-You Guo, Acupuncture ameliorates inflammatory response in a chronic unpredictable stress rat model of depression, Brain Research Bulletin http://dx.doi.org/10.1016/j.brainresbull.2016.11.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Acupuncture ameliorates inflammatory response in a chronic unpredictable stress rat model of depression Jun Lua, Run-Hui Shaoa, Shu-Ying Jinb, Li Huc, Ya Tua* , Jian-You Guod* a
College of Acupuncture-Moxibustion and Tui Na, Beijing University of Chinese
Medicine, Beijing, 100029, China b
Beijing Feng-Tai Pu-Huangyu Community Health Center, Beijing, 100075, China
c
Tangshan Feng-Run Hospital of TCM, Tangshan, 064000, China
d
Key Laboratory of Mental Health, Institute of Psychology, Chinese Academy of
Sciences, Beijing, 100101, China Corresponding Authors: Ya Tu and Jian-You Guo E-mail address: Ya Tu: [email protected]. Jian-You Guo: [email protected].
1
Highlights
Acupuncture treatment alleviated the depressive-like behavior in CUMS rats.
Acupuncture treatment decreased levels of iNOS and NO in the hippocampus and prefrontal cortex in CUMS rats.
Acupuncture treatment reduced expressions of COX-2 and PGE2 in the hippocampus and prefrontal cortex in CUMS rats.
Acupuncture treatment inhibited NF-κB activation in the hippocampus and prefrontal cortex in CUMS rats.
2
Abstract Depression is one of the most common psychiatric disorders. Chronic inflammatory response has been viewed as a key factor in depression. Acupuncture in Chinese medicine has been shown to be an effective treatment for depression. In the present study, we investigated the mechanism underlying antidepressant effect of acupuncture. The rats were subjected to chronic unpredictable mild stress (CUMS) for 28 days to induce depressive-like behaviors. Acupuncture treatment was applied once every other day during the 28-day stress period. The behavioral tests (body weight, sucrose consumption and locomotor activity) were performed. The expressions of nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and nuclear factor kappa B (NF-κB) were determined in the rat hippocampus and prefrontal cortex. CUMS induced depressive-like behavior in rats, which was alleviated by acupuncture treatment. The increased levels of NO, PGE2, iNOS and COX-2 induced by CUMS, were all significantly decreased in the hippocampus and prefrontal cortex by acupuncture. Moreover, acupuncture markedly inhibited the activation of NF-κB in rats. These findings showed that the antidepressant-like effect of acupuncture might be mediated by inhibition of inflammatory mediators via modulation of NF-κB in the brain regions. Key Words: acupuncture; depression; chronic unpredictable stress; inflammatory response
3
1. Introduction Depression is a common and debilitating psychiatric disorder in modern society, characterized by a pervasive low mood and loss of interest in usual activities[1]. Antidepressants dominate current treatment in clinical practice. There are three main kinds of classical antidepressants, including tricyclic antidepressants (TCA), selective serotonin reuptake inhibitors (SSRIs), and monoamine oxidase inhibitors (MAOIs). However, some of these drugs show undesirable side effects. Meanwhile, less than 50% of depression patients achieve remission with the available antidepressants[2]. Therefore, there is no doubt that alternative medicines with safety and efficacy are needed to treat depression. Acupuncture in traditional Chinese medicine has become a widely accepted alternative therapy. The specific effects of acupuncture on central nervous system are attracting more and more interest. For example, acupuncture plays an important role in protecting the central nervous system from neuronal damage[3,4]. Moreover, clinical trials have demonstrated that acupuncture could alleviate depressive symptoms[5-8], and modulate the corticostriatal reward circuitry in major depressive disorder[9]. Previous studies have shown that acupuncture could have antidepressant-like effect by modulating hypothalamic-pituitary-adrenal axis, enhancing hippocampal serotonin system[10], modulating dopaminergic neuroactivity[11], and reducing pro-inflammatory cytokines
[12].
In recent years, chronic inflammatory response has been viewed as a key factor in depression. The expressions of many inflammatory mediators, such as 4
pro-inflammatory
cytokines,
inducible
nitric
oxide
synthase
(iNOS)
and
cyclooxygenase-2 (COX-2), are regulated by nuclear factor kappa B (NF-κB)[13, 14]. NF-κB can be activated by a number of stimuli, including lipopolysaccharide or stress. After activation, NF-κB induces the transcription of the inflammatory mediators involved in inflammatory response[15, 16]. Therefore, NF-κB pathway play a vital role in regulating inflammatory responses in depression[17-19]. For example, it has been reported that chronic stress enhanced the activation of NF-κB[15, 20], and increased expression of COX-2[21, 22], as well as iNOS in the rat model of depression[23-26]. The increase of COX-2 and iNOS expression has also been found in depression patients [27]. However, the effect of acupuncture on the inflammatory mediators involved in depression remains unclear. The hippocampus and prefrontal cortex have been suggested to be the important brain regions associated with the mood regulation, cognitive function and memory. Clinical studies have indicated their functional and structural abnormalities in depression[28-30]. Therefore, in this study, we investigated the effect of acupuncture on inflammatory response in hippocampus and prefrontal cortex in a chronic unpredictable mild stress (CUMS) rat model of depression. Our results showed that acupuncture could attenuate inflammatory response in depression by inhibiting the key inflammation mediators via modulation of NF-κB activation in the brain regions. 2. Materials and Methods 2.1. Animals. Male Sprague-Dawley rats (180～200g) were obtained from Beijing Vital River Laboratories. Rats were kept in an air-conditioned room with a 12 h light/dark cycle with free access to food and water except when animals were 5
subjected to deprivation stressors as described in stress. The experiment procedures were approved by the Animal Care and Use Committee at Beijing University of Chinese Medicine. 2.2. Groups and Treatment. The rats were randomly divided into five groups (eight rats in each group): the control group was given no stress except general handling for 4 weeks; the CUMS group was exposed to CUMS for 4 weeks; the CUMS + Acu(Acu) group received acupuncture treatment once every other day during the 4-week stress period; the CUMS + Fluoxetine (Flu) group, used as a positive comparator for an antidepressant effect, was given Fluoxetine during the 4-week stress period. Fluoxetine was diluted in distilled water and orally given one hour before the stress exposure. 2.3. Chronic Unpredictable Mild Stress Procedure. Rats in stressed groups were exposed to CUMS after 1 week of acclimatization period under the housing conditions. The CUMS model was modified from the methods previously described[31, 32]. The rats were randomly exposed to various stressors for 4 weeks, and each stressor was administered once a week. The stressors applied included: deprivation of food (24 h, F), deprivation of water (24 h, W), cold swimming (4°C, 5 min, S), cage shaking (30 min,C), behavior restraint (3 h, R), tail pinch (2 min, T), and soiled bedding (24 h, B). The stress sequence was changed every week in order to make the stress procedure unpredictable (Fig.1). The control group was housed in a separate room, with free access to food and water. 2.4. Acupuncture treatment
and
drug
6
administration.
During acupuncture
administration, the rats were maintained within a cloth bag without anesthesia, with one forelimb taken out, similar to what we described previously[12]. Two points were selected: Baihui (GV20) and Neiguan (PC6). GV20 is located above the apex auriculate, on the midline of the head. PC6 is located between the tendons of m. palmarislongus and m. flexor carpi radialis, proximal to the transverse crease of the wrist. Sterilized disposable stainless steel needles (0.3*25mm, HuanQiu brand, Suzhou, China) were inserted obliquely as deep as 2-3 mm at Baihui (GV20) and Neiguan (PC6). The acupuncture treatment was manually manipulated by twisting the needles at a frequency of twice per second for 1 min, and then the needles were retained for 10 minutes. The rats received acupuncture treatment once every other day before the stress during the 4-week period. Fluoxetine (Zhejiang Regen Chemical Co., Ltd, China) was diluted in saline and administered daily by oral gavage at a dose of 10 mg/kg for 4 weeks (Fig.1). The dosage of 10 mg/kg for fluoxetine has been reported to show antidepressant action in previous work[33]. 2.5. Body weight. The body weight was measured at the beginning and the end of the experiment. 2.6. Sucrose intake test. The sucrose intake test was conducted according to methods described in literatures with minor modifications[31, 32]. Rats were exposed to 1% sucrose solution for 48 h without any water available. Then the test was carried out after 24h of water deprivation. The rats were given a 1h sucrose test. The sucrose consumption was measured by reweighing preweighed bottles of sucrose solution. The sucrose intake was performed at the beginning and the end of the experiment. 7
2.7. Open field test. At the end of the experiment, the open field test was performed. The apparatus consisted of a square arena 100×100 cm with 50 cm high wall. It was divided into 5×5 equal squares which had been drawn in the floor of the arena. A single rat was gently placed in the center of the floor in order to explore the arena for 5 min. The crossing numbers (defined as at least three paws in a square) and the rearing numbers (defined as the rat standing upright on its hind legs) were counted manually by two observers who were blind to the experiment. 2.8. Enzyme-linked immunosorbent assay (ELISA). After the behavioral tests, the rats were sacrificed and the brains were quickly removed on ice. The hippocampus and prefrontal cortex were isolated and put into chilled tubes in liquid nitrogen, then stored at -80C for further analysis. The concentrations of COX-2, iNOS and prostaglandin E2 (PGE2) were measured using commercially available ELISA kits (R&D Systems, USA) according to the manufacturer’s instructions. 2.9. Determination of nitric oxide (NO) content. The NO concentration in the hippocampus and prefrontal cortex was measured using commercially available nitrate reductase method kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions. 2.10. Western blot analysis. Half of the hippocampus and prefrontal cortex were isolated and put into chilled tubesin liquid nitrogen. The brain regions were homogenized and Western blot analysis was carried out as previously reported[22]. Briefly, primary antibodies for NF-κB p65 and β-actin at 1:1000 dilution (Santa Cruz Biotech Inc., CA, USA). A secondary antibody conjugated with horseradish 8
peroxidase was used. Immuno-reactivity was visualized by ECLreagent. NF-κB p65protein expression was quantified by densitometry using the Scion Image Beta 4.02 software and are shown as density relative to β-actin. 2.11. Statistical Analysis. Data were presented as means ± S.E.M. Differences among groups were examined using one-way ANOVA, followed by Newman-Keuls test. P< 0.05 was the accepted level of significance. 3. Results 3.1 Effects of acupuncture treatment on the body weight. As illustrated in Fig. 2,before the CUMS procedure, no differences in body weight were observed between groups (F3,28= 0.46, p>0.05), but a significant difference in body weight was observed between groups following 4 weeks of CUMS (F3,28= 23.79, p< 0.01). The body weight significantly decreased in the CUMS group compared with the control group(p< 0.01). Compared with the CUMS group, the body weight significantly increased in the acupuncture group (p<0.05), and the fluoxetine group also exhibited a significant increase in body weight (p<0.05). 3.2. Effects of acupuncture treatment on the sucrose consumption. As shown in Fig. 3, before the CUMS procedure, no differences in sucrose consumption were observed between groups (F3,28 = 0.09, p >0.05), but a significant difference in sucrose consumption was observed between groups following 4 weeks of CUMS (F3,28 = 7.15, p< 0.01). Stressed rats exhibited a reduction of sucrose consumption compared with controls after 4 weeks of stress exposure (p< 0.01). Rats that were subjected to CUMS and received acupuncture treatment exhibited a significant increase in sucrose consumption compared with rats that were subjected only to CUMS (p< 0.01). Sucrose consumption in the fluoxetine group was also 9
significantly higher compared with the CUMS group (p< 0.01). 3.3. Effects of acupuncture treatment on the locomotor activity. As shown in Fig. 4, a significant difference was observed between groups in the crossing numbers (F3,28 = 8.96, p< 0.01) and the rearing numbers (F3,28 = 7.76, p< 0.01) after CUMS. Acupuncture and fluoxetine treatment significantly reversed the stress-induced decrease in the number of crossings(both p< 0.01). Acupuncture and fluoxetine treatment also significantly increased the number of rearings (p< 0.01, p< 0.05 respectively). 3.4. Effects of acupuncture treatment on NO and PGE2 content in the hippocampus and prefrontal cortex. As shown in Fig.5, following 4 weeks of CUMS, there were significant differences in the content of NO (F3,28 =8.84, p< 0.01) and PGE2 (F3,28 =6.05, p< 0.01) in the hippocampus between groups. Compared with the control group, the content of NO and PGE2 significantly increased in the hippocampus in stressed rats (both p< 0.01). Acupuncture and fluoxetine treatment significantly decreased the production of NO (both p< 0.001), and PGE2 (both p< 0.01) in the hippocampus in CUMS rats. Significant differences were also observed between groups in the production of NO (F3,28 =6.04, p< 0.01) and PGE2 (F3,28 =6.50, p< 0.01) in the prefrontal cortex. CUMS significantly increased the production of NO and PGE2 in the prefrontal cortex compared with the control group (all p< 0.01). Acupuncture and fluoxetine treatment significantly decreased the content of NO (both p< 0.01), and PGE2 (both p< 0.01) in the prefrontal cortex in stressed rats. 3.5. Effects of acupuncture treatment on iNOS and COX-2 expression in the hippocampus and prefrontal cortex. As shown in Fig.6, we further detected protein levels of iNOS and COX-2 related 10
toNO and PGE2 synthesis respectively. A significant difference was observed between groups in the iNOS expression in the hippocampus (F3,28 =9.95, p< 0.01) and prefrontal cortex (F3,28 =13.52, p< 0.01). The expression of iNOS in the hippocampus and prefrontal cortex was significantly higher in CUMS group (both p< 0.01) compared with the control group. Acupuncture and fluoxetine treatment significantly reduced the expression of iNOS in the hippocampus (both p< 0.01) and prefrontal cortex (both p< 0.01) of CUMS rats. A significant difference was also observed between groups in the COX-2 expression in the hippocampus (F3,28 =8.36, p< 0.01) and prefrontal cortex (F3,28 =14.37, p< 0.01). The expression of COX-2 in the hippocampus and prefrontal cortex was also significantly higher in CUMS group (both p< 0.01) compared with the control group. Acupuncture and fluoxetine treatment significantly reduced the expression of COX-2 in the hippocampus (both p< 0.01) and prefrontal cortex (both p< 0.01) of stressed rats. 3.6. Effects of acupuncture treatment on NF-κB expression in the hippocampus and prefrontal cortex. As shown in Fig.7, we detected NF-κB, the major transcription factor involved in inflammatory processes, which induced iNOS and COX-2 expression. A significant difference was observed between groups in NF-κB expression in the hippocampus (F3,11= 8.04, p< 0.01) and prefrontal cortex (F3,11= 12.81, p< 0.01) after CUMS. Acupuncture treatment significantly downregulated the stress-induced increase in NF-κB expression in the hippocampus and prefrontal cortex (both p< 0.01). Fluoxetine treatment also significantly decreased NF-κB expression in the hippocampus and prefrontal cortex (both p< 0.01). 4. Discussion 11
The
present
study
demonstrates
that
acupuncture
treatment
exhibits
antidepressant-like effect in CUMS rat model of depression. Furthermore, we found that this effect might be mediated by inhibition of inflammatory mediators (iNOS, COX-2, NO, PGE2) via regulation of NF-κB in the hippocampus and prefrontal cortex. Mounting evidence has recently indicated that chronic inflammatory response plays an important role in the pathophysiology of depression[34, 35]. NF-κB has been considered to be a critical mediator of behavioral changes and inflammatory response of depression[20]. Clinical study has demonstrated that NF-κB expression was significantly increased in depression patients[36]. We found that the expression of NF-κB in the hippocampus was increased by CUMS, which was in line with previous findings[15, 20]. We also found that NF-κB expression was increased in the prefrontal cortex. In addition, we found that acupuncture treatment significantly downregulated NF-κB expression in these brain regions. It has been reported that continuous infusion of selective NF-κB inhibitor significantly alleviated chronic stress-induced depressive-like
behaviors
and
protected
stress-impaired
hippocampal
neurogenesis[20]. The antidepressant-like effect of acupuncture might be mediated through reducing activation of NF-κB. As a transcription factor, NF-κB has been regarded as a central regulator of a number of genes involved in inflammatory response, such as pro-inflammatory cytokines, iNOS and COX-2[13-15]. Previous study has found that acupuncture prevented the increase of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor 12
necrosis factor-α (TNF-α) in rat model of depression[12]. However, the effect of acupuncture on key inflammatory enzymes involved in depression is not clear. NOS is the enzyme which generates NO. There are three isoforms of NOS enzymes: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). iNOS is inflammation inducible, and closely involved in regulating pathological inflammatory processes[37]. Although it has been revealed that 6h acute immobilization stress increased nNOS activity in rats, 6h chronic immobilization stress of 21 days induced iNOS in rat neurons[25, 37]. Therefore, it is likely that chronic stress induced iNOS in rats. We found that CUMS increased iNOS and NO in the hippocampus and prefrontal cortex. Evidence suggested that iNOS may contribute to depressive-like behaviors in a chronic stress mouse model[27]. Our data support a link between iNOS, inflammatory response, and depressive-like behaviors in an animal model of depression. Moreover, we found that acupuncture treatment significantly reduced iNOS and NO in the hippocampus and prefrontal cortex of CUMS rats. A study indicated that pretreating the mice with an iNOS inhibitor decreased iNOS mRNA expression and reversed stress-induced depressive-like behaviors[26]. Therefore, it showed that acupuncture could alleviate inflammatory response in depression through inhibition of iNOS and NO in the brain. COX is the enzyme that converts arachidonic acid to PGE2. It exists in two subtypes, cyclooxygenase-1 (COX-1) and COX-2. COX-1 is expressed in nearly all tissues and produces PGs to maintain physiological functions, while COX-2 is induced in brain regions in response to stress or injury, and release large amounts of 13
PGs to induce inflammatory state[21]. COX-2 has been well studied for its role in depression. For example, a clinical study has shown that COX-2 mRNA expression was increased in patients with recurrent depression[27]. Our results showed that exposure to CUMS markedly increased COX-2 and PGE2 in the hippocampus and prefrontal cortex. This is consistent with previously published data concerning the role of COX-2 in animal model of depression[22]. In addition, we found acupuncture treatment significantly decreased COX-2 and PGE2 in the hippocampus and prefrontal cortex. Previous studies have shown that chronic treatment with COX-2 inhibitor celecoxib displayed the antidepressant-like effect and decreased COX-2 expression in CUMS rats[22]. Celecoxib also decreased pro-inflammatory cytokines in a rat model of depression[38-40]. Our results revealed that the antidepressant-like effect of acupuncture could be mediated by decreasing COX-2 and PGE2 in brain regions. 5. Conclusion In summary, acupuncture displayed the antidepressant-like effect in CUMS rat model of depression. Acupuncture could attenuate inflammatory response in depression by inhibiting the key inflammation mediators in NF-κB pathway in the brain regions. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgments This study was supported by the Grants from the National Natural Science 14
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Figure Legends Fig. 1. A schematic diagram of protocol design, including schedule of chronic unpredictable mild stress, acupuncture and drug treatment. Fig. 2. Body weight in the following groups (N= 8 per group): Control, chronic unpredictable mild stress (CUMS), CUMS + Acupuncture (Acu), CUMS + Fluoxetine (Flu). Data are means ± SEM. **P< 0.01 as compared with the control group, #P< 0.05 as compared with the CUMS group. Fig.3. Sucrose consumption in the following groups (N= 8 per group): Control, CUMS, CUMS + Acupuncture (Acu), CUMS + Fluoxetine (Flu). Data are means ± SEM. **P< 0.01 as compared with the control group, ##P< 0.01 as compared with the CUMS group. Fig.4. The locomotor activity of open field test in the following groups (N= 8 per group): Control, CUMS, CUMS + Acupuncture (Acu), CUMS + Fluoxetine (Flu). Data are means ± SEM. **P< 0.01 as compared with the control group, #P< 0.05, ##P< 0.01 as compared with the CUMS group. Fig.5. Hippocampus and prefrontal NO and PGE2 levels in the following groups (N= 8 per group): Control, CUMS, CUMS + Acupuncture (Acu), CUMS + Fluoxetine (Flu). A) Hippocampus levels of NO and PGE2. B) Prefrontal cortex level of NO and PGE2. Data are means ± SEM. **P< 0.01 as compared with the control group, ##
P< 0.01 as compared with the CUMS group.
Fig.6. Hippocampus and prefrontal cortex iNOS and COX-2 in the following groups (N= 8 per group): Control, CUMS, CUMS + Acupuncture (Acu), CUMS + Fluoxetine 19
(Flu). A) Hippocampus level of iNOS and COX-2. B) Prefrontal cortex level of iNOS and COX-2. Data are means ± SEM. **P< 0.01 as compared with the control group, ##P< 0.01 as compared with the CUMS group. Fig.7. NF-κB expression measured by Western blot in the following groups (N= 5 per group): Control, CUMS, CUMS + Acupuncture (Acu), CUMS + Fluoxetine (Flu). A) Hippocampus. B)Prefrontal cortex. Data are means ± SEM.
**
P< 0.01 as compared
with the control group, ##P< 0.01 as compared with the CUMS group.
20
Fig.1
21
Fig.2
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Fig.3
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Fig.4
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Fig.5
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Fig.6
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Fig.7
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