乌灵胶囊增加慢性不可预见性温和应激大鼠海马连接蛋白43的表达改善神经再生

　　　　　　 作者：李德强， 李旭娟， 段金凤， 蔡巍

【摘要】 　　目的：探讨乌灵胶囊对慢性不可预见性温和应激(chronic unpredictable mild stress， CMS)抑郁模型大鼠海马齿状回脑源性神经营养因子(brainderived neurotrophic factor， BDNF)、神经再生，以及连接蛋白43(connexin 43， Cx43)表达的影响。方法：45只成年雄性SpragueDawley大鼠随机分入对照组(n=15)、模型组(n=15)和乌灵胶囊组(n=15)。模型组和乌灵胶囊组大鼠接受连续3周的CMS，造模期间，乌灵胶囊按100 mg/(kg·d)加入乌灵胶囊组大鼠的膳食中，连续治疗21 d。采用糖水偏爱实验评价大鼠的抑郁程度。抑郁行为测试结束后，取双侧海马组织，用免疫组织化学法检测BDNF与5溴脱氧尿苷(5bromodeoxyuridine，BrdU)的表达；用逆转录聚合酶链反应和蛋白印迹法检测海马中Cx43 mRNA与蛋白表达。结果：慢性应激大鼠齿状回BDNF阳性表达、新生细胞数及Cx43 mRNA和蛋白表达水平均较正常大鼠明显下降。乌灵胶囊治疗后，CMS大鼠抑郁行为得到改善，异常的海马神经再生恢复，Cx43 mRNA和蛋白表达变得正常，但BDNF表达无明显改变。结论：乌灵胶囊可增加CMS模型大鼠海马神经再生及改善抑郁症状，其机制可能与增加Cx43表达有关，与BDNF无关。

【关键词】 慢性不可预见性温和应激; 脑源性神经营养因子; 5溴脱氧尿苷; 连接蛋白; 大鼠

　　Recently， great progresses have been made in understanding the pathogenesis of depression， such as the creation of the chronic unpredictable mild stress (CMS) model［1］ and the hypothesis that hippocampal brainderived neurotrophic factor (BDNF) downregulation and dysfunctional neurogenesis may lead to the depressed behaviors［2］. Substantial research evidence indicated that stress plays an important role in the pathogenesis of depression［3］， but its mechanism still needs to be exactly elucidated， especially in making clear what substrates are involved and how these pertinent substrates mediate stressinduced inhibition of hippocampal neurogenesis. This topic has currently caused more attention and has become a new hot spot［4］ to deeply explore. In vitro and in vivo experimental evidence suggests that gap junctions， formed by connexins (Cxs) between neurons and/or astrocytes， contribute to the neural generation and recovery after lesion［5］. Cx43， as a specific connexin， is mainly located in astrocytes and is known to participate in various developmental stages of cell proliferation， including neuronal migration， axonal elongation， axon pruning， dendrite morphogenesis and synaptic maturation. However， the relationship between Cx43 expression in hippocampus and abnormal neurogenesis induced by CMS is still unclear so far. Wuling Capsule， a compound traditional Chinese herbal medicine， has been used in clinic for many years， and has been proved to be potent to fully improve the signs of insomnia and cognitive deficits since its introduction in 1999. Recently， a more powerful response rate of antidepression had been shown when Wuling Capsule was administered in combination with mirtazapine than mirtazapine alone［6］， which indicates that the pharmacological action of Wuling Capsule is worthy of further study. In this research， we examined the effects of Wuling Capsule on depressionlike behaviors in rats subjected to CMS. And hippocampal neurogenesis， expressions of BDNF and Cx43 were also detected to explore the mechanisms of Wuling Capsule in antidepression.

　　1 Materials and methods

　　1.1 Animals and grouping

　　In this study， 45 adult male SpragueDawley rats from the Animal Center of Zhejiang University (cleaningrank， animal license No. SYXK20050072)， with the weight range from 240 to 280 g， were randomly pided into three groups: control group (n=15)， untreated group (n=15) and Wuling group (n=15). After basal sucrose preference test， all rats except those in the control group were subjected to threeweek CMS procedure and housed inpidually. Meanwhile， Wuling powder (Xyloria， with the purity quotient 98.5%， purchased from Zhejiang Jolly Pharmaceutical Co.， LTD， batch No. 20090201) was mixed with feedstuff and daily administered consecutively to the rats in the Wuling group at a dose of 100 mg/kg body weight for 21 days. The placebo (amylum parvule) was given (100 mg/kg) to rats in the control group and untreated group. Seven， fourteen and twentyone days after the beginning of CMS procedure， sucrose preference test was performed to access the depression level.

　　1.2 CMS modeling

　　Twentyfour hours after the basal sucrose preference， rats in the untreated group and Wuling group were subjected to the CMS procedure to induce the core depression symptom. The CMS protocol was designed to maximize the unpredictable nature of the stressors according to previous study［7］ with a minor modification. One of the following stressors was administered daily (in random order) over a period of 3 weeks: fasting food deprivation for 20 h; water deprivation for 17 h; swimming at 4 ℃ for 5 min; heat stress (40 ℃) for 5 min; 45° cage tilt for 17 h; shaker stress (horizontal shakes at high speed) for 10 min; restraint stress for 2 h; soiled bedding (200 mL water in 100 g sawdust bedding) for 5 h; persistent illumination (light for 17 h); tail pinch for 2 min; and intermittent white noise for 5 min. Immediately after each stress session， the rats were returned to the single room and maintained in standard conditions until the next session of the CMS regime.

　　1.3 Sucrose preference testing

　　Sucrose preference has been proposed to detect the levels of anhedonia and the procedures had been described in details［8］. Rats were housed inpidually with two bottles containing either 1% sucrose solution or tap water (daily initial time: 20:00 to 21:00) with standard lab chow available continuously. The basal sucrose preference was determined prior to CMS procedure， and the other three times of sucrose preference were calculated under the similar conditions (twobottle test， 2hour period) on every weekend since the beginning of the CMS procedure. Sucrose preference was evaluated as sucrose uptake rate， namely， ratio of volume of sucrose consumption to volume of sucrose consumption plus tap water consumption.

　　1.4 Sample processing

　　After the last behavioral evaluation， all rats were intraperitoneally injected with 5bromodeoxyuridine (BrdU) (Sigma) at a dose of 100 mg/kg body weight， and 2 hours later， BrdU would become a component of the new synthesized DNA. All rats were sacrificed with an intravenous overdose of 10% chloral hydrate (3 mL/kg) and the bilateral hippocampuses were isolated. The left was placed in 4% paraformaldehyde solution for 24 h for analysis of BDNF expression and the right was preserved at －60 ℃ for the freezedsection staining and the analysis of Cx43 mRNA and protein expressions.

　　1.5 Hippocampal immunohistochemical assay

　　1.5.1 Expression of BDNF protein

　　Immunohistochemical method was performed under the same conditions and was guided seriously in the conventional steps of the streptavidinbiotin peroxidase complex (SABC) staining in each group in order to ensure the accuracy of immunohistochemical staining. SABCkit (BOSTER Corp) was applied for BDNF immunohistochemistry. In brief mention， after dewaxing and hydration， sagittal paraffin sections of hippocampus with the thickness of 3 μm were covered by 5% bull serum albumin for 20 min， followed by sequential incubation with rabbit antimouse BDNF antibody (1︰200， Sigma) at 4 ℃ overnight. Subsequently， biotinylated goat antirabbit IgG was added to the slide for 2 h at 37 ℃ followed by incubation with SABC. Then， brain slices were colored with 3，3′diaminobenzidine and counterstained with hematoxylin， dehydrated， cleared， and mounted.

　　1.5.2 Expression of BrdU protein

　　Coronal frozen sections (50 μm thick) of hippocampus were used to analyze the neurogenesis. The sections were treated sequentially with 0.3% hydrogen peroxide in phosphate buffered solution (PBS) for 30 min and 10% normal goat serum in 0.05 mol/L PBS for 30 min. They were then incubated with diluted goat antimouse BrdU monoclonal antibody (Sigma， 1︰200) overnight at room temperature and subsequently exposed to biotinylated goat antirabbit IgG and SABC for 2 h (37 ℃).

　　1.5.3 Count of the immunohistochemical products

　　The sections were mounted in neutral resin following dehydration and visualized under an Olympus optical microscope (Olympus Shanghai Trading， China). The numbers of BDNFpostive cells and BrdUpostive particles were counted in 400× magnification and analyzed with Visiopharm software in the same part of the dentate gyrus (DG).

　　1.6 Detection of Cx43 protein expression by Western blotting

　　The process of Western blotting was performed accoding to previous study［9］. In short， protein extracts were lysed in a lysis buffer (0.05% leupeptin， 50 mmol/L TrisHCl， 5 mmol/L EDTA， 150 mmol/L NaCl， 1% deoxycholic acid， 0.1% SDS， 1% Triton X100). The samples were centrifuged (11 500×g， 4 ℃) for 30 min and supernatants were preserved for protein quantification. The aliquots of total proteins were separated on 12.5% sodium dodecyl sulfatepolyacrylamide gels and transferred to a nitrocellulose membrane. Membranes were saturated for 30 min in PBS containing 0.1% Tween and 5% nonfat dried milk and subsequently incubated overnight with either antiCx43 monoclonal antibody (BD Transduction Laboratories， USA， 1︰1 000) or antiactin antibody (PharMingen， San Jose， USA， 1︰500). After sequential washes， membranes were incubated with SABC for 1 h. Specific signals were identically visualized with enhanced chemiluminescence and exposed to hyperfilm photograph (AmershamPharmacia， Buckinghamshire， UK). The expression level of Cx43 protein was quantified by measuring the values of optical density (OD) at 260 nm.

　　1.7 Detection of Cx43 mRNA expression by reverse transcriptionpolymerase chain reaction

　　The samples of 2 μg total RNA were extracted by TRIzol reagent (Gibco Brl， Rockville， Massachusetts， USA) and reversely transcribed into cDNA based on a typical protocol［10］. Briefly， Cx43 mRNA expression was determined through a semiquantitative polymerase chain reaction (PCR) with glyceraldehyde phosphated dehydrogenase (GAPDH) as a reference. Reverse transcriptionPCR (RTPCR) primers were specifically designed with the assistance of a computer. Forward primer sequence of Cx43: 5′CAT CTT CAT GCT GGT GGT GT3′; reverse primer sequence: 5′TAG TTC GCC CAG TTT TGC TC3′; GAPDH forward primer: 5′GCG CCT GGT CAC CAG GGC TGC TT3′; reverse: 5′TGC CGA AGT GGT CGT GGA TGA CCT3′. The sizes of products including Cx43 (283 bp) and GAPDH (465 bp) were adequate to match and incorporate target genes. The PCR reaction mixture (specific primers， reaction buffer， reverse transcriptase and dNTP) was submitted to 30 amplification cycles. The PCR cycling contained the following steps: preincubation for 2 min at 37 ℃， denaturation for 5 min at 94 ℃， annealing for 45 s at 57 ℃， elongation for 10 min at 72 ℃ and 45 ℃ for 15 s. Then， 2% agarose gel electrophoresis was applied to confirm the mRNA expression of RTPCR products (10 μL). The signal intensities were quantified by using Dolphin software (Wealtec Corp).

　　1.8 Statistical analysis

　　All data were expressed as x±s. Multigroup comparisons were analyzed by oneway ANOVA and StudentNewmanKeuls q test was used to compare the differences between two groups with software SPSS 12.0. In the analysis， P value less than 0.05 was considered statistically significant.

　　2 Results

　　2.1 Outcomes of sucrose preference testing

　　Low sucrose preference was found in the CMS rats， the difference was statistically significant (P&<0.05) as compared with rats in the control group at the same time point. After Wuling Capsule treatment， the low sucrose preference was increased and returned to normal level in the rats subjected to CMS (see Figure 1).

　　2.2 BDNF expression in DG

　　The expressions of BDNF in DG in three groups were observed under an optical microscope. As shown in Figure 2， a large number of brown patches in the sections demonstrated the positive expression of BDNF， which was mainly located in the cytoplasm of progenitor neurons in subgranular zone and also appeared in nerve processes. BDNF expression was higher in the control group than in the other two groups. For further comparisons of the BDNFpositive cells in three groups， number of BDNFpositive cells in the Wuling group or the untreated group was significantly lower than that in the control group (P&<0.05). However， no statistical difference was found between the untreated group and the Wuling group， which indicates that Wuling Capsule can not improve the low BDNF expression in rats exposed to CMS.

　　2.3 BrdU expression in DG

　　BrdU， as a thymine affinis agent， may competitively incorporate to the new synthesized DNA， so the positive expressions always represent the newgenerated neural cells. It has been proposed for long time to be a marker of neurogenesis. In this research newborn neurons were marked by black particles in nuclei， from which we may verify the process of neurogenesis. As shown in Figure 3， the number of newborn cells in hippocampus of the untreated group was significantly lower than that of the control group， but this low neurogenesis induced by CMS became normal after the Wuling Capsule treatment.

　　2.4 Cx43 mRNA and protein expressions

　　Results of expressions of Cx43 mRNA and protein were listed in Figure 4 and 5. mRNA and protein expressions of Cx43 in the untreated group were significantly decreased (P&<0.05) as compared with the control group. After Wuling Capsule treatment， the low expressions of Cx43 mRNA and protein were significantly increased， and recovered to the normal level， with significant difference (P&<0.05) from the untreated group.

　　3 Discussion

　　Many researches［1113］ have found that the atrophy of hippocampus is ubiquitous in CMSmodel animals or patients with depression， and antidepressants may relieve the symptoms of depression by increasing the neurogenesis and restoring the volume of pathological hippocampus. Therefore， one concept， being interiorized to most researchers is that the occurrence of depression may be related to the abnormal hippocampal plasticity. It is convincing that the neurogenesis is maintained throughout the life of the animal in hippocampus. The neural progenitor cells originate initially from the subgranular zone of DG. Along with the processes of proliferation and differentiation， the newgenerated cells migrate gradually into the CA3 area and eventually link to pyramidal neurons to integrate information into the cortexhippocampal loop to regulate emotion and cognitive function. It has reported that a variety of factors could influence the regulation of hippocampal neurogenesis， including neurotransmitters， hormones， growth factors and external environmental factors such as stress， social isolation， substance abuse and physical exercise［1416］. Neurogenesis may be detected dynamically by labeling the newgenerated nucleus with BrdU. Two hours after the injection， BrdU incorporated to the newly synthesized DNA sequences. Along with the latter stepwise cell differentiation， this marker of neurogenesis will occur eventually in various target neurons. Therefore， expression level of BrdU may reflect the number of newborn neurons. As stated above， a variety of factors may influence the hippocampal neurogenesis. Chronic unpredictable mild stress model was induced based on the aforementioned theory， which has been proposed to induce core symptoms of depression， anhedonia［17］ with the representations of excessive decrease in reward sensitivity and increase in harm avoidance. In this research， it was further manifested that CMS significantly led to the low stimulation reward， i.e. decreased sucrose preference was found in CMS rats. After Wuling Capsule treatment， the excessive lower sucrose preference was improved showing that Wuling Capsule may relieve symptoms of depression. Wuling powder， the main ingredient of Wuling Capsule， increases the activity of glutamate decarboxylase， and results in the strong capacity in promoting glutamate to be uptaken quickly and increases the synthesis of gammaaminobutyric acid to improve brain function. This is the probable antidepressant mechanism of Wuling Capsule.

　　We found significantly decreased expressions of BrdU and BDNF in the hippocampal DG in CMS rats. After treatment of Wuling Capsule， the abnormal neurogenesis returned to normal， but the expression of BDNF did not change in CMS rats. The relationship between the incidence of depression and the abnormal neurogenesis was also confirmed by these results， which was consistent with the former studies［18］. Now， researchers generally agreed that increasing the expression of BDNF would help improving the remodeling of neurogenesis in hippocampus. In the present study， the numbers of BDNFpositive cells and BrdUpostive cells in the untreated group were decreased as compared with the control group. Furthermore， BDNF expression products were located mainly in subgranular zone of the hippocampal DG where the newborn cells originated from. Additionally large， hyperchromatic nuclei with mitotic figures were seen in the majority of BDNFpositive neurons. All the abovementioned findings revealed the intimate relations between BDNF and neurogenesis. The finding in this report that chronic unpredictable mild stress decreased BDNF expression in the DG was consistent with previous studies［19］. The causes may be due to the effects of chronic stressors on the hypothalamuspituitaryadrenal axis and immune cytokines［20］. It was easy to conclude that the intermediation in BDNF and neurogenesis was related to the depressed behaviors in CMS rats， but we found an interesting manifestation in this research that the low sucrose preference and the abnormal neurogenesis became entirely normal after Wuling Capsule treatment， whereas， the decreased BDNF expression was remained at low level. So， we conclude that other substrates may be involved in the antidepressant effect of Wuling Capsule.

　　Gap junction in the brain may synchronize neuronal activity of neuron and glial cells by two means: (1) participating in the regulation of brain metabolism and homeostasis including ATP and glutamate［21］; (2) influencing developmental neural stem cells involving in hippocampal structure plasticity. For example， Cx43 gap junctions can provide an activitydependent intercellular pathway to regulate the energy transfer［22］， especially by αamino3hydroxy5methyl4isoxazolepropionic acid receptor activity to deliver energetic metabolites from blood vessels to distal neurons. Animal model studies have suggested that Cx43 involved in the pathogenesis of Parkinson’s disease［23］ and epilepsy［24］. Gap junction is known to be associated with structural plasticity in the central nervous system. Cx43， as a predominant gap junction protein， and specific to neurons as other connexins is mainly expressed in premitotic radial glial cells and mature astrocytes. Cx43deficient mouse showed the smaller hippocampus， cortex， and cerebellum further verified the relationship between Cx43 and neural plasticity［25］. The role of Cx43 might be involved in many aspects of brain physiology， including intercellular communication， the release of neuroactive substances， neural and glial proliferation and migration. In this research， some cryptic correlations between Cx43 and the neurogenesis appeared， for the same variation rhythm was found in rats when subjected to CMS or in drug intervention. Wuling Capsule promoted the expression of Cx43 in hippocampus， relieved depressionlike behavior and enhanced neurogenesis. Since this preliminary research only unveiled a hidden correlation in the changes of Cx43 expression and neurogenesis， we could not definitely draw a conclusion that the Cx43 is the exact target of Wuling Capsule. However， a few clues in this report have reminded us that the accurate mechanism on the actions of Wuling Capsule needs further investigation.

　　In summary， Wuling Capsule can restore hippocampal abnormal neurogenesis induced by chronic stressors not through the promotion of BDNF expression， but by enhancing the expression of Cx43， which contributes to the reduction of depressed behaviors.

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