逍遥散对慢性应激损伤大鼠海马神经细胞的影响及作用机制研究
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摘要
研究目的和意义
大量临床及实验研究证实,慢性应激在疾病发生、发展过程中扮演重要角色,但慢性应激引起或诱发疾病的确切机制尚未完全明了,因而目前关于抗应激损伤的治疗措施也存在靶向不确定或单一、副作用明显等缺陷,大大限制了该类药物的临床应用。中药复方逍遥散由宋代太医局制定颁布,临床常用于治疗郁证相关疾病,近千年的临床实践证明其疏肝解郁、健脾和营之功效显著,现代实验研究也已证实,逍遥散能够改善或削除心理应激引起的学习记忆能力下降和抑郁、焦虑等症状,具有明确的镇静、镇痛、抗惊厥、抗焦虑及抗慢性抑郁作用。本研究即在总结以往慢性应激损伤机制、逍遥散抗应激损伤相关研究的基础上,提出逍遥散抗慢性应激损伤机制的假说,并围绕这一假说,以体外培养的大鼠海马神经细胞为模型,展开一系列相关实验研究,以期探明慢性应激损伤及逍遥散抗慢性应激损伤可能存在的某种中枢机制。
研究方法
以体外原代培养的Wistar乳鼠(新生24小时内)海马神经细胞为模型,以正常大鼠血清、慢性应激大鼠模型血清、逍遥散治疗血清以及谷氨酸(Glu)为干预因素,以N-甲基-D-天门冬氨酸受体(NR)非选择性抑制剂地佐环平(MK-801)为工具药,研究慢性应激状态下以及逍遥散治疗对大鼠海马神经细胞存活率、NR各亚其mRNA的表达、海马神经细胞内游离钙离子浓度、糖皮质激素受体蛋白表达的影响,探讨中药复方逍遥散抗慢性应激损伤的作用以及可能存在的中枢作用机制。
1大鼠海马神经细胞体外原代培养与鉴定：无菌条件下急性分离新生24小时内的Wistar大鼠乳鼠海马组织,用胰蛋白酶消化与机械吹打相结合的方法分离海马神经细胞,以含血清的培养基种植、无血清饲养液维持饲养,神经微管相关蛋白-2(Map-2)鉴定神经元纯度。
2慢性应激模型大鼠血清、逍遥散治疗血清的制备：实验动物随机分为3组：正常对照组(每天每只灌胃生理盐水2ml,不进行应激刺激)、慢性应激模型组(每天每只灌胃生理盐水2ml后,进行应激刺激)、逍遥散组(每天每只灌胃逍遥散水煎剂2ml/后,进行应激刺激),采用Cart的多相性应激模型并加以改进,应激源包括：足底电击(电压30V,电击频率0.1Hz,每次持续1s,共30次)、冰水游泳(4℃左右,5min)、禁水(24h)、禁食(24h)、束缚(4h),每天不定时随机安排一种应激刺激,避免相邻两天出现重复,避免形成规律性及造成大鼠适应性,连续21天。于末次给药1-2h后麻醉条件下腹主动脉采血,血液以3000rpm离心,分离血清,每组血清分别混合后于56℃水浴中灭活30min。置超净台内,无菌条件下以0.22μm滤器抽滤除菌,按1ml分装并标记,-20℃冰箱保存备用。
3分组处理后Glu-NR-Ca2+-cAMP-iGR信号通路相关指标检测：细胞培养至第8d分为7组：(1)空白对照组(正常细胞,不做任何处理)、(2)正常血清对照组、(3)正常血清+Glu组、(4)模型血清+Glu组、(5)模型血清+Glu+MK801组、(6)逍遥散治疗血清+Glu组、(7)逍遥散治疗血清+Glu+MK801组。每组均先以制备的血清或和MK-801预处理20min,再加入Glu作用30min。以常规MTT法检测各组细胞存活情况；采用Taqme.n荧光探针定量PCR法测定各组Ct值,2-△△Ct相对定量法计算NR各亚基表达量相对值；在同一时间段内,通过激光共聚焦显微镜技术对模拟慢性应激微环境下以及逍遥散治疗血清干预后大鼠海马神经细胞内游离钙离子浓度进行检测；采用免疫印迹法检测慢性应激大鼠模型血清以及逍遥散治疗血清干预体外培养的海马神经细胞后,其胞内糖皮质激素受体(iGR)表达情况。
研究结果
1培养8天,神经元胞体清晰,结构完整,神经元纯度最高达到85.71%,平均为75.32%。
2各组细胞存活率相当。第(3)组(正常血清+Glu组)与第(4)组(模型血清+Glu)细胞存活率相对较低,其余各组无统计学差异。
3模型血清与谷氨酸模拟的慢性应激微环境下神经细胞存活率明显降低,海马神经细胞NR各亚基mRNA出现不同程度的表达上调,细胞内Ca2+浓度显著升高,iGR表达明显下调。
4在拟慢性应激作用前对细胞进行MK-801预处理,即阻滞NR后,细胞存活率与空白对照组比较无统计学差异,NR2AmRNA、NR2BmRNA表达水平明显降低,但尚未达到正常水平,胞内Ca2+浓度虽有明显降低,但未能更显著地改善Ca2+内流增加的状况,也未能完全阻断iGR的表达下调。
5逍遥散治疗血清干预后,细胞存活率有升高趋势,NR2AmRNA表达明显上调,NR2BmRNA表达水平较慢性应激组显著降低,而NR1mRNA表达无明显下降,神经细胞内Ca2+浓度显著降低,iGR的表达下调有所改善,但与空白组比较还存在差异。
6 MK-801与逍遥散治疗血清共同作用后,细胞存活率与空白组比较无显著性差异,NR2AmRNA、NR2BmRNA表达量接近正常,Glu未能引发Ca2+内流增加,与空白对照组无统计学差异,其对抗iGR含量降低的作用倾向更加显著。研究结论
1以新生24h内Wistar乳鼠为供体,采用含10%胎牛血清的DMEM/F12培养基种植,以Neurobasal配合B27进行无血清饲养的原代大鼠海马神经细胞培养法,无需阿糖胞苷纯化细胞,培养至第8d可以获得纯度最大值达到85.71%,平均为75.32%的神经元,能够满足纯度要求不太高,而细胞量较大的实验需要,是适合进行多种细胞水平研究的良好模型。
2使用慢性应激动物模型血清加谷氨酸能够模拟慢性应激状态,可以导致NR过度激活从而出现细胞内钙超载,继而引起iGR合成减少,对海马神经元造成了类似于慢性应激损伤的病理结果。
3逍遥散治疗血清能够纠正NR各亚基表达失衡的状态,促进机体恢复或保持NR2A与NR2B的正常比例,维持胞内钙稳态,对抗慢性应激导致的iGR表达下降,对维持海马神经细胞的稳定起到一定作用,从而发挥了保护神经元、对抗慢性应激损伤的作用。
4 MK-801不能完全阻滞本实验模拟的慢性应激微环境引起的钙超载和iGR表达量下降,还存在通过Glu-NR以外其他信号转导通路的共同作用。与逍遥散治疗血清共同作用后,除MK-801抑制NR激活外,逍遥散治疗血清发挥了保持NR正常数量及活性,抑制Ca2+超载,维持胞内钙稳态,对抗iGR表达减少,从而保护神经元免受兴奋性毒性损伤的作用,这种保护性作用可能与其中某些有效成分阻滞了包括Glu-NR-Ca2+-iGR在内的多种信号通路有关。
小结
慢性应激大鼠模型血清与Glu共同作用能够模拟慢性应激状态,可以导致NR过度激活从而出现细胞内钙超载,继而引起iGR合成减少,对海马神经元造成了类似于慢性应激损伤的病理结果。逍遥散治疗血清能够纠正NR各亚基表达失衡的状态,促进机体恢复或保持NR2A与NR2B的正常比例,维持胞内钙稳态,对抗慢性应激导致的iGR表达下降,对维持海马神经细胞的稳定起到一定作用,在慢性应激条件下发挥了保护神经元、对抗慢性应激损伤的效应。MK-801工具药的使用使我们了解到逍遥散治疗血清可能从包括Glu-NR-Ca2+-cAMP-iGR的多种信号途径发挥这种抗慢性应激损伤作用。
Research purpose and sense
Many clinical and experimental studies confirmed that chronic stress play an important role in occurrence and development of many diseases, but the exact mechanism has not yet been fully explained. Thus currently, the treatment measures of anti-stress have many defects, such as uncertain or a single target, obvious side effects, and so on that greatly limited the clinical application of these drugs. Xiao Yao San as a Chinese herbal formula is an official prescription promulgated in the Pharmacopoeia of the Imperial Medical Bureau in the Song Dynasty, and it has been commonly used in the treatment of depression related diseases until today. Nearly a thousand years of clinical practice proved that it has the effects of coursing the liver and resolving depression, fortifying the spleen and supplementing the blood. Modern clinical and experimental studies have confirmed that the mental stress-induced learning and memory decline, depression, anxiety and other symptoms can be improved or eliminated by treating with Xiao Yao San, it has exact effects of sedation, analgesia, anticonvulsant, anti-anxiety, and anti-depression. In this study, hypothesis on mechanism of anti-chronic stress of Xiao Yao San has been proposed on the basis of analysis based on related research. And around this hypothesis, a series of experiments was conducted to investigate the possible central mechanism of the anti-chronic stress of Xiao Yao San, in the process of that the cultured rat hippocampal neurons were used as model.
Research methods
To establish a primary culture method of hippocampal neurons of new born rats, prepare serum of rats suffered with chronic stress, serum of chronic stress rats treated with Xiao Yao San, and the serum of normal control group, and use dizocilpine as a tool for drug, then treated each group with different factors according to the experimental design, and study the effects of the serum of chronic stress rat model and the serum treated with Xiao Yao San on the cell viability, expression of NMDA receptor subunits'mRNA, intracellular free Ca2+ concentration and the expression of intracellular glucocorticoid receptor (iGR) in the cultured hippocampal neurons of rats.
1 Hippocampal neurons of newborn rats within 24 hours were isolated by the method of trypsin digestion combining with that of mechanical dispersion, which were planted with medium containing 10% fetal bovine serum, and maintained feeding with serum-free medium. Identification of neurons and its purity were conducted by neural microtubule-associated protein (Map-2).
2 30 rats were randomly divided into 3 groups:Normal control group (saline 2ml/d, ig, not to be stimulated by stress), chronic stress model group (saline 2ml/d, ig, and 1 hour after, each to be stimulated by stress), chronic stress model treated with Xiao Yao San group (Xiao Yao San decoction 2ml/d, ig, and 1 hour after, each to be stimulated by stress). The multi-stress model by Cart was improved and used in this experiment. Stressors used in this experiment include electric shock through feet (30V,0. 1Hz, lsec stimuli and lsec interval each time,30times/d), ice water swimming (4℃or so,5min), prohibition cf water (24h), fasting (24h), binding (4h). Each stressor was assigned randomly everyday, the same stressor in adjacent days and regularity of the stimulaticn or adaptability of the rats must be avoided. Modeling was lasted for 21 days. Blood of each group were collected by artery puncture through abdominal aorta after administration in 1 to 2 hours on the last day of modeling, then separated serum by centrifugation at 3000 rpm, serum of each group were mixed respectively, and inactivated in water bath at 56℃for 30min, then filtrated bacteria by using filter of 0.22μm under sterile conditions. Packed serum of each group into lml per tube and marked clearly and then stored at-20℃in refrigerator.
3 Hippocampal neurons were divided into 7 groups after cultured for 8 days: (1)control group (normal cells, no treatment), (2)normal serum control group, (3)normal serum+glutamate group, (4)chronic stress model serum+glutamate group, (5)chronic stress model serum+glutamate+MK-801 group, (6)Xiao Yao San treatment serum+glutamate group, (7)Xiao Yao San treatment serum+ glutamate+MK-801 group. Each group was pretreated with serum or together with MK-801 for 20min, then after that added glutamate for the following 30min. Cell viability of each group was detected by MTT assay. The methods of Taqman fluorescence probe real-time quantitative PCR and relative quantification were used to determine the expression of NMDAR subunits mRNA. Intracellular free Ca2+ concentration in cultured hippocampal neurons in the simulated micro-environment of chronic stress and after intervention with the serum treated with Xiao Yao San were detected by confocal laser microscope at the same period of time. The methods of Western blot and relative quantification was used to determine the protein expression of iGR in the cultured rat hippocampal nerve cells after being conducted by the serum of chronic stress rat model and the serum treated with Xiao Yao San.
Results
1 The structures of nerve cells were clear and integral, and the purity of the neurons is up to 85.71% after cultured for 8 days.
2 Hippocampal neurons viability of the normal serum+glutamate group and the chronic stress model serum+glutamate group were relatively low, and there were no significant differences in other groups. Hippocampal neurons viability in each group was approximately the same, which can meet the needs of follow-up assay.
3 Neurons viability was affected obviously, NR1, NR2A, NR2B mRNA were all increased in different degrees, concentration of free Ca2+ intracellular increased significantly, and the protein expression of iGR was decreased significantly when micro-environment of chronic stress simulated by glutamate and serum of rat model with chronic stress was effected on the cultured hippocampal neurons.
4 When cultured hippocampal neurons were prepared with MK-801, viability of the cultured nerve cells could be improved markedly, NR2A and NR2B mRNA were significantly decreased, concentration of free Ca2+ intracellular in cultured rat hippocampal neurons was decreased, but MK-801 has failed to improve the situation of increased Ca2+ influx and the downregulation of iGR expression.
5 Neurons viability showed a trend of increasing, the expression of NR2A mRNA was upregulated significantly but that of NR2B mRNA was downregulated and NR1mRNA expression was not significantly decreased, concentration of free Ca2+ intracellular hadn't increased significantly and chronic stress-induced downregulation of iGR could be against significantly though there still were differences between the group with control group. When serum of chronic stress treated with Xiao Yao San was effected on the neurons.
6 When MK-801 and serum of chronic stress treated with Xiao Yao San were used together, hippocampal neurons viability and expression of NR2A and NR2B mRNA were closer to normal, and glutamate had failed to lead to Ca2+ overload, chronic stress-induced downregulation of iGR could be significant(?)y antagonized and there was no significant difference between the group with control group. Conclusion
1 No need to strictly control the digestion time and used the Ara-C to purify cells, neurons with good condition and high purity were obtained.
2 The state of chronic stess micro-environment could be simulated by using serum of chronic stress rat model together with glutamate on the cultured hippocampal neurons, which caused the similar pathological results with that of chronic stress.
3 Imbalance state of NR subunits under chronic stress could be corrected, and the normal ratio of NR2A AND NR2B could be maintained by using serum of chronic stress treated with Xiao Yao San, and it had effects of inhibiting Ca2+ overload and antagonizing chronic stress-induced downregulation of iGR in hippocampal neurons.
4 Ca2+ overload and downregulation of iGR induced by chronic stress could not be completely blocked by using MK-801, there might be some other signaling pathways other than that of Glu-NR that interacted in the processes. Besides the effect of inhibition the activation of NR caused by MK-801, serum of chronic stress treated with Xiao Yao San played a role in neurons stability maintenance, and protected neurons against injury induced by chronic stress when MK-801 and serum of chronic stress treated with Xiao Yao San were used together. It may work through a variety of signaling pathways including Glu-NR-Ca2+-iGR to maintain the steady-state of the neurons, and then to protect neurons from the neurotoxic effects of excitatory.
Summary
Micro-environment of chronic stress around neurons in vivo could be simulated by serum of chronic stress rats together with glutamate, which could cause excessive activation of NR and calcium overload intracellular, subsequently reduced synthesis of iGR, and ultimately led to damage of hippocampal neurons and pathological results similar to chronic stress. Xiao Yao San treatment serum could correct the imbalance state of the NR subunits mRNA expression, maintain the normal proportion of NR2A and NR2B and calcium homeostasis intracellular, and keep iGR from decreased expression caused by chronic stress. It played a role in maintaining stability of hippocampal nerve cells, and protected neurons against the effects of chronic stress. Application of MK-801 made it clear that Xiao Yao San may work through multiple signaling pathways including that of Glu-NR-Ca2+-cAMP-iGR.
大量临床及实验研究证实,慢性应激在疾病发生、发展过程中扮演重要角色,但慢性应激引起或诱发疾病的确切机制尚未完全明了,因而目前关于抗应激损伤的治疗措施也存在靶向不确定或单一、副作用明显等缺陷,大大限制了该类药物的临床应用。中药复方逍遥散由宋代太医局制定颁布,临床常用于治疗郁证相关疾病,近千年的临床实践证明其疏肝解郁、健脾和营之功效显著,现代实验研究也已证实,逍遥散能够改善或削除心理应激引起的学习记忆能力下降和抑郁、焦虑等症状,具有明确的镇静、镇痛、抗惊厥、抗焦虑及抗慢性抑郁作用。本研究即在总结以往慢性应激损伤机制、逍遥散抗应激损伤相关研究的基础上,提出逍遥散抗慢性应激损伤机制的假说,并围绕这一假说,以体外培养的大鼠海马神经细胞为模型,展开一系列相关实验研究,以期探明慢性应激损伤及逍遥散抗慢性应激损伤可能存在的某种中枢机制。
研究方法
以体外原代培养的Wistar乳鼠(新生24小时内)海马神经细胞为模型,以正常大鼠血清、慢性应激大鼠模型血清、逍遥散治疗血清以及谷氨酸(Glu)为干预因素,以N-甲基-D-天门冬氨酸受体(NR)非选择性抑制剂地佐环平(MK-801)为工具药,研究慢性应激状态下以及逍遥散治疗对大鼠海马神经细胞存活率、NR各亚其mRNA的表达、海马神经细胞内游离钙离子浓度、糖皮质激素受体蛋白表达的影响,探讨中药复方逍遥散抗慢性应激损伤的作用以及可能存在的中枢作用机制。
1大鼠海马神经细胞体外原代培养与鉴定：无菌条件下急性分离新生24小时内的Wistar大鼠乳鼠海马组织,用胰蛋白酶消化与机械吹打相结合的方法分离海马神经细胞,以含血清的培养基种植、无血清饲养液维持饲养,神经微管相关蛋白-2(Map-2)鉴定神经元纯度。
2慢性应激模型大鼠血清、逍遥散治疗血清的制备：实验动物随机分为3组：正常对照组(每天每只灌胃生理盐水2ml,不进行应激刺激)、慢性应激模型组(每天每只灌胃生理盐水2ml后,进行应激刺激)、逍遥散组(每天每只灌胃逍遥散水煎剂2ml/后,进行应激刺激),采用Cart的多相性应激模型并加以改进,应激源包括：足底电击(电压30V,电击频率0.1Hz,每次持续1s,共30次)、冰水游泳(4℃左右,5min)、禁水(24h)、禁食(24h)、束缚(4h),每天不定时随机安排一种应激刺激,避免相邻两天出现重复,避免形成规律性及造成大鼠适应性,连续21天。于末次给药1-2h后麻醉条件下腹主动脉采血,血液以3000rpm离心,分离血清,每组血清分别混合后于56℃水浴中灭活30min。置超净台内,无菌条件下以0.22μm滤器抽滤除菌,按1ml分装并标记,-20℃冰箱保存备用。
3分组处理后Glu-NR-Ca2+-cAMP-iGR信号通路相关指标检测：细胞培养至第8d分为7组：(1)空白对照组(正常细胞,不做任何处理)、(2)正常血清对照组、(3)正常血清+Glu组、(4)模型血清+Glu组、(5)模型血清+Glu+MK801组、(6)逍遥散治疗血清+Glu组、(7)逍遥散治疗血清+Glu+MK801组。每组均先以制备的血清或和MK-801预处理20min,再加入Glu作用30min。以常规MTT法检测各组细胞存活情况；采用Taqme.n荧光探针定量PCR法测定各组Ct值,2-△△Ct相对定量法计算NR各亚基表达量相对值；在同一时间段内,通过激光共聚焦显微镜技术对模拟慢性应激微环境下以及逍遥散治疗血清干预后大鼠海马神经细胞内游离钙离子浓度进行检测；采用免疫印迹法检测慢性应激大鼠模型血清以及逍遥散治疗血清干预体外培养的海马神经细胞后,其胞内糖皮质激素受体(iGR)表达情况。
研究结果
1培养8天,神经元胞体清晰,结构完整,神经元纯度最高达到85.71%,平均为75.32%。
2各组细胞存活率相当。第(3)组(正常血清+Glu组)与第(4)组(模型血清+Glu)细胞存活率相对较低,其余各组无统计学差异。
3模型血清与谷氨酸模拟的慢性应激微环境下神经细胞存活率明显降低,海马神经细胞NR各亚基mRNA出现不同程度的表达上调,细胞内Ca2+浓度显著升高,iGR表达明显下调。
4在拟慢性应激作用前对细胞进行MK-801预处理,即阻滞NR后,细胞存活率与空白对照组比较无统计学差异,NR2AmRNA、NR2BmRNA表达水平明显降低,但尚未达到正常水平,胞内Ca2+浓度虽有明显降低,但未能更显著地改善Ca2+内流增加的状况,也未能完全阻断iGR的表达下调。
5逍遥散治疗血清干预后,细胞存活率有升高趋势,NR2AmRNA表达明显上调,NR2BmRNA表达水平较慢性应激组显著降低,而NR1mRNA表达无明显下降,神经细胞内Ca2+浓度显著降低,iGR的表达下调有所改善,但与空白组比较还存在差异。
6 MK-801与逍遥散治疗血清共同作用后,细胞存活率与空白组比较无显著性差异,NR2AmRNA、NR2BmRNA表达量接近正常,Glu未能引发Ca2+内流增加,与空白对照组无统计学差异,其对抗iGR含量降低的作用倾向更加显著。研究结论
1以新生24h内Wistar乳鼠为供体,采用含10%胎牛血清的DMEM/F12培养基种植,以Neurobasal配合B27进行无血清饲养的原代大鼠海马神经细胞培养法,无需阿糖胞苷纯化细胞,培养至第8d可以获得纯度最大值达到85.71%,平均为75.32%的神经元,能够满足纯度要求不太高,而细胞量较大的实验需要,是适合进行多种细胞水平研究的良好模型。
2使用慢性应激动物模型血清加谷氨酸能够模拟慢性应激状态,可以导致NR过度激活从而出现细胞内钙超载,继而引起iGR合成减少,对海马神经元造成了类似于慢性应激损伤的病理结果。
3逍遥散治疗血清能够纠正NR各亚基表达失衡的状态,促进机体恢复或保持NR2A与NR2B的正常比例,维持胞内钙稳态,对抗慢性应激导致的iGR表达下降,对维持海马神经细胞的稳定起到一定作用,从而发挥了保护神经元、对抗慢性应激损伤的作用。
4 MK-801不能完全阻滞本实验模拟的慢性应激微环境引起的钙超载和iGR表达量下降,还存在通过Glu-NR以外其他信号转导通路的共同作用。与逍遥散治疗血清共同作用后,除MK-801抑制NR激活外,逍遥散治疗血清发挥了保持NR正常数量及活性,抑制Ca2+超载,维持胞内钙稳态,对抗iGR表达减少,从而保护神经元免受兴奋性毒性损伤的作用,这种保护性作用可能与其中某些有效成分阻滞了包括Glu-NR-Ca2+-iGR在内的多种信号通路有关。
小结
慢性应激大鼠模型血清与Glu共同作用能够模拟慢性应激状态,可以导致NR过度激活从而出现细胞内钙超载,继而引起iGR合成减少,对海马神经元造成了类似于慢性应激损伤的病理结果。逍遥散治疗血清能够纠正NR各亚基表达失衡的状态,促进机体恢复或保持NR2A与NR2B的正常比例,维持胞内钙稳态,对抗慢性应激导致的iGR表达下降,对维持海马神经细胞的稳定起到一定作用,在慢性应激条件下发挥了保护神经元、对抗慢性应激损伤的效应。MK-801工具药的使用使我们了解到逍遥散治疗血清可能从包括Glu-NR-Ca2+-cAMP-iGR的多种信号途径发挥这种抗慢性应激损伤作用。
Research purpose and sense
Many clinical and experimental studies confirmed that chronic stress play an important role in occurrence and development of many diseases, but the exact mechanism has not yet been fully explained. Thus currently, the treatment measures of anti-stress have many defects, such as uncertain or a single target, obvious side effects, and so on that greatly limited the clinical application of these drugs. Xiao Yao San as a Chinese herbal formula is an official prescription promulgated in the Pharmacopoeia of the Imperial Medical Bureau in the Song Dynasty, and it has been commonly used in the treatment of depression related diseases until today. Nearly a thousand years of clinical practice proved that it has the effects of coursing the liver and resolving depression, fortifying the spleen and supplementing the blood. Modern clinical and experimental studies have confirmed that the mental stress-induced learning and memory decline, depression, anxiety and other symptoms can be improved or eliminated by treating with Xiao Yao San, it has exact effects of sedation, analgesia, anticonvulsant, anti-anxiety, and anti-depression. In this study, hypothesis on mechanism of anti-chronic stress of Xiao Yao San has been proposed on the basis of analysis based on related research. And around this hypothesis, a series of experiments was conducted to investigate the possible central mechanism of the anti-chronic stress of Xiao Yao San, in the process of that the cultured rat hippocampal neurons were used as model.
Research methods
To establish a primary culture method of hippocampal neurons of new born rats, prepare serum of rats suffered with chronic stress, serum of chronic stress rats treated with Xiao Yao San, and the serum of normal control group, and use dizocilpine as a tool for drug, then treated each group with different factors according to the experimental design, and study the effects of the serum of chronic stress rat model and the serum treated with Xiao Yao San on the cell viability, expression of NMDA receptor subunits'mRNA, intracellular free Ca2+ concentration and the expression of intracellular glucocorticoid receptor (iGR) in the cultured hippocampal neurons of rats.
1 Hippocampal neurons of newborn rats within 24 hours were isolated by the method of trypsin digestion combining with that of mechanical dispersion, which were planted with medium containing 10% fetal bovine serum, and maintained feeding with serum-free medium. Identification of neurons and its purity were conducted by neural microtubule-associated protein (Map-2).
2 30 rats were randomly divided into 3 groups:Normal control group (saline 2ml/d, ig, not to be stimulated by stress), chronic stress model group (saline 2ml/d, ig, and 1 hour after, each to be stimulated by stress), chronic stress model treated with Xiao Yao San group (Xiao Yao San decoction 2ml/d, ig, and 1 hour after, each to be stimulated by stress). The multi-stress model by Cart was improved and used in this experiment. Stressors used in this experiment include electric shock through feet (30V,0. 1Hz, lsec stimuli and lsec interval each time,30times/d), ice water swimming (4℃or so,5min), prohibition cf water (24h), fasting (24h), binding (4h). Each stressor was assigned randomly everyday, the same stressor in adjacent days and regularity of the stimulaticn or adaptability of the rats must be avoided. Modeling was lasted for 21 days. Blood of each group were collected by artery puncture through abdominal aorta after administration in 1 to 2 hours on the last day of modeling, then separated serum by centrifugation at 3000 rpm, serum of each group were mixed respectively, and inactivated in water bath at 56℃for 30min, then filtrated bacteria by using filter of 0.22μm under sterile conditions. Packed serum of each group into lml per tube and marked clearly and then stored at-20℃in refrigerator.
3 Hippocampal neurons were divided into 7 groups after cultured for 8 days: (1)control group (normal cells, no treatment), (2)normal serum control group, (3)normal serum+glutamate group, (4)chronic stress model serum+glutamate group, (5)chronic stress model serum+glutamate+MK-801 group, (6)Xiao Yao San treatment serum+glutamate group, (7)Xiao Yao San treatment serum+ glutamate+MK-801 group. Each group was pretreated with serum or together with MK-801 for 20min, then after that added glutamate for the following 30min. Cell viability of each group was detected by MTT assay. The methods of Taqman fluorescence probe real-time quantitative PCR and relative quantification were used to determine the expression of NMDAR subunits mRNA. Intracellular free Ca2+ concentration in cultured hippocampal neurons in the simulated micro-environment of chronic stress and after intervention with the serum treated with Xiao Yao San were detected by confocal laser microscope at the same period of time. The methods of Western blot and relative quantification was used to determine the protein expression of iGR in the cultured rat hippocampal nerve cells after being conducted by the serum of chronic stress rat model and the serum treated with Xiao Yao San.
Results
1 The structures of nerve cells were clear and integral, and the purity of the neurons is up to 85.71% after cultured for 8 days.
2 Hippocampal neurons viability of the normal serum+glutamate group and the chronic stress model serum+glutamate group were relatively low, and there were no significant differences in other groups. Hippocampal neurons viability in each group was approximately the same, which can meet the needs of follow-up assay.
3 Neurons viability was affected obviously, NR1, NR2A, NR2B mRNA were all increased in different degrees, concentration of free Ca2+ intracellular increased significantly, and the protein expression of iGR was decreased significantly when micro-environment of chronic stress simulated by glutamate and serum of rat model with chronic stress was effected on the cultured hippocampal neurons.
4 When cultured hippocampal neurons were prepared with MK-801, viability of the cultured nerve cells could be improved markedly, NR2A and NR2B mRNA were significantly decreased, concentration of free Ca2+ intracellular in cultured rat hippocampal neurons was decreased, but MK-801 has failed to improve the situation of increased Ca2+ influx and the downregulation of iGR expression.
5 Neurons viability showed a trend of increasing, the expression of NR2A mRNA was upregulated significantly but that of NR2B mRNA was downregulated and NR1mRNA expression was not significantly decreased, concentration of free Ca2+ intracellular hadn't increased significantly and chronic stress-induced downregulation of iGR could be against significantly though there still were differences between the group with control group. When serum of chronic stress treated with Xiao Yao San was effected on the neurons.
6 When MK-801 and serum of chronic stress treated with Xiao Yao San were used together, hippocampal neurons viability and expression of NR2A and NR2B mRNA were closer to normal, and glutamate had failed to lead to Ca2+ overload, chronic stress-induced downregulation of iGR could be significant(?)y antagonized and there was no significant difference between the group with control group. Conclusion
1 No need to strictly control the digestion time and used the Ara-C to purify cells, neurons with good condition and high purity were obtained.
2 The state of chronic stess micro-environment could be simulated by using serum of chronic stress rat model together with glutamate on the cultured hippocampal neurons, which caused the similar pathological results with that of chronic stress.
3 Imbalance state of NR subunits under chronic stress could be corrected, and the normal ratio of NR2A AND NR2B could be maintained by using serum of chronic stress treated with Xiao Yao San, and it had effects of inhibiting Ca2+ overload and antagonizing chronic stress-induced downregulation of iGR in hippocampal neurons.
4 Ca2+ overload and downregulation of iGR induced by chronic stress could not be completely blocked by using MK-801, there might be some other signaling pathways other than that of Glu-NR that interacted in the processes. Besides the effect of inhibition the activation of NR caused by MK-801, serum of chronic stress treated with Xiao Yao San played a role in neurons stability maintenance, and protected neurons against injury induced by chronic stress when MK-801 and serum of chronic stress treated with Xiao Yao San were used together. It may work through a variety of signaling pathways including Glu-NR-Ca2+-iGR to maintain the steady-state of the neurons, and then to protect neurons from the neurotoxic effects of excitatory.
Summary
Micro-environment of chronic stress around neurons in vivo could be simulated by serum of chronic stress rats together with glutamate, which could cause excessive activation of NR and calcium overload intracellular, subsequently reduced synthesis of iGR, and ultimately led to damage of hippocampal neurons and pathological results similar to chronic stress. Xiao Yao San treatment serum could correct the imbalance state of the NR subunits mRNA expression, maintain the normal proportion of NR2A and NR2B and calcium homeostasis intracellular, and keep iGR from decreased expression caused by chronic stress. It played a role in maintaining stability of hippocampal nerve cells, and protected neurons against the effects of chronic stress. Application of MK-801 made it clear that Xiao Yao San may work through multiple signaling pathways including that of Glu-NR-Ca2+-cAMP-iGR.
引文
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