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1、1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
2、囊组大鼠接受连续3 周的 CMS，造模期间，乌灵胶囊按 100 mg/(kgd)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
3、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2Recently, great progresses have been made in understanding the pathogenesis of depression, such as the creation of the chronic unpredictable mild stress (CMS) model1 an
4、d the hypothesis that hippocampal brain derived neurotrophic factor (BDNF) down regulation and dysfunctional neurogenesis may lead to the depressed behaviors2. Substantial research evidence indicated that stress plays an important role in the pathogenesis of depression3, but its mechanism still need
5、s 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 spot4 to deeply explore. In vitro and in vivo
6、experimental evidence suggests that gap junctions, formed by connexins (Cxs) between neurons and/or astrocytes, contribute to the neural generation and recovery after lesion5. Cx43, as a specific connexin, is mainly located in astrocytes and is known to participate in various developmental stages of
7、 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 traditiona
8、l 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 administe
9、red in combination with mirtazapine than mirtazapine alone6, 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,
10、expressions of BDNF and Cx43 were also detected to explore the mechanisms of Wuling Capsule in antidepression.1 Materials and methods1.1 Animals and groupingIn this study, 45 adult male Sprague Dawley rats from the Animal Center of Zhejiang University (cleaning rank, animal license No. SYXK2005 0072
11、), with the weight range from 240 to 280 g, were randomly divided 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 individuall
12、y. 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 (
13、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.31.2 CMS modelingTwenty four hours after the basal sucrose preference
14、, 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 study7 with a minor modification. One of the following stressors was adm
15、inistered 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
16、 (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 testingSucrose preference has been proposed to detect the levels of anhedonia and the proced
