运用中医肝主疏泄理论治疗卒中后抑郁症的研究
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摘要
卒中后抑郁症(Post-stroke depression，PSD)是指发生于卒中后经量表测定证实有抑郁情绪或处于抑郁的状态，是脑卒中的重要并发症之一，发生率为25％～80％，大部分为轻—中度。卒中病人的活动障碍常导致抑郁症的发生，而抑郁症又可影响卒中病人的康复，形成恶性循环。PSD作为脑卒中后继发的一种情感障碍，除可造成情感上的痛苦外，还减慢肢体功能和认知功能恢复，并可使病死率升高，严重影响着病人生活质量和预后。目前中西医学工作者对PSD的发病机制和临床治疗都进行了系统全面的研究，并取得了显著进展。由于传统的及目前新型的抗抑郁药物(西药)均有不同程度的毒副作用，成瘾性以及禁忌症等缺陷，严重影响了临床的治疗依从性，因此运用中医药治疗PSD越来越得到关注，研究也越来越深入。
本研究探讨了中医肝主疏泄与PSD的关系，并结合以往的研究工作基础上，在本研究中，以肝主疏泄理论为指导，运用调肝治法方药(JWSNS)治疗PSD，并进一步探讨其作用机理，从而为今后的中药新药研发提供科学的依据。
第一部分临床研究
1临床资料与方法
1．1研究对象
1．1．1病例来源所有病例均来自2003年10月-2006年10月来我院就诊的门诊和住院患者，共103例。
1．1．2纳入标准
(1)首发脑卒中后2周内，意识清楚，无智力障碍，能配合检查。
(2)所有病例均符合中华神经科学会1995年全国第四界脑血管病学术会议通过的脑出血、脑梗死的诊断标准，并经1～3次的头颅CT或MRI检查确诊。符合《中医内科学》(第5版)中风、郁证辨证。
(3)所有患者在接受药物治疗前，汉密顿抑郁量表(HAMD)总分＞17，神经功能缺损评分(SSS)＞16分，日常生活活动能力(ADL)评价采用Barthel指数评价表，Barthel指数＜70分。
(4)首诊1周内未服用抗抑郁制剂。2周内无使用单胺氧化酶抑制剂，治疗中不合并使用其他抗抑郁药和抗精神病药。近3个月内无使用过甘草、皮质激素、促肾上腺皮质激素(ACTH)或类似物。
(5)入组前血、尿、粪常规及肝、肾功能正常，无严重心、肝、肾疾病史，无精神疾病史及癫痫病史，无药物过敏史；排除老年良性记忆减退、老年性痴呆和神经衰弱。
1．2分组及治疗方法
1．2．1分组
所有病例均为第一次发病后1个月内，按就诊顺序采用抽签法随机分为2组。治疗组52例，男32例，女20例，年龄38岁～68岁，平均40.21±8.57岁；脑出血28例，脑梗死8例，脑栓塞10例，蛛网膜下腔出血6例。对照组51例，男31例，女20例，年龄37岁～69岁，平均41.53±7.92岁；脑出血27例，脑梗死6例，脑栓塞12例，蛛网膜下腔出血6例。治疗前经HAMD评分17～30，平均20.42±1.82／17～31，20.16±2.51；神经功能缺损选用神经功能缺损评分16～45分，平均(25.3±4.2)分／16～45分，24.2±5.4；Barthel指数0～70分，平均(26.1±6.8)分／0～70分，25.1±7.2。两组年龄、性别、病因、病程、抑郁程度、神经功能缺损程度及Barthel指数均无明显差异(P＞0.05)。另设健康组50例，均来自医院体检健康人群，男30例，女20例，年龄36岁～67岁，平均40.44±8.72岁，在年龄、性别构成方面与对照组和治疗组无差异。
1．2．2治疗与护理方法
两组患者均给予口服喜得镇1日3次，1次1mg。高血压、糖尿病患者给予降压降糖治疗。同时采取以Bobath疗法为主的神经肌肉促通技术进行肢体功能训练，每日1次，每次50min。在此基础上，两组分别采用以下治疗方法。治疗组：采用加味四逆散(JWSNS)为主进行治疗。JWSNS组成及剂量：柴胡5g、白芍15g、枳壳6g、枸杞子15g、山栀5g、干地黄18g、石决明30g。每日1剂，水煎服，早晚各1次。气虚明显者加黄芪、党参：肾精不足者加何首乌、枸杞子；肾阳亏虚者加巴戟天、淫羊藿；痰热盛者加制半夏、黄连：阴虚火旺者加知母、丹皮；心神不宁、失眠较重者加合欢花、生龙骨、生牡蛎，瘀血甚者加桃仁、红花。对照组：采用百忧解(盐酸氟西汀，美国lilly公司生产，批号208151)进行治疗，每次20mg，每日1次，晨起服。基础护理措施：护理人员应以开朗的情绪与患者交流，观察患者的言谈举止，洞察患者的心理活动，加强对患者行为与思维的监控；以积极开朗的情绪去影响患者，唤起患者的兴趣，对患者在感情上予以支持、理解及同情，以获得患者的信任，引导患者倾吐心中的不满和郁结，使其内心的郁闷得到宣泄；鼓励患者户外活动，还可开展适宜的娱乐活动，使患者在活动中忘记烦恼；饮食宜清淡且避免过饱，忌肥腻，忌烟酒，宜以玫瑰花、菊花等代茶饮；病区环境布置雅静，可给予轻音乐等。以上两组均以4周为1个疗程，共治疗2个疗程。疗程结束后观察统计疗效。治疗前、治疗第4、8周末分别查血、小便、大便常规及肝、肾功能、心电图。并进行HAMD评分。
1．3观察指标及疗效评价
1．3．1中医疗效评价标准
参考1994年6月国家中医药管理局公布的《中医病症诊断疗效标准》制定。显效：症状消失，情绪正常。有效：症状减轻，情绪基本稳定。无效：症状情绪均无改善。
1．3．2量表评定标准
对两组患者于治疗前和治疗后4周、8周后进行HAMD、神经功能缺损程度评分(SSS)和日常生活活动能力(ADL)评定(Barthel指数)。
1．3．2．1根据汉密尔顿抑郁量表(HAMD)评定抑郁症状选择24项症状与体征按5级评分法进行评分，记录治疗前后两组患者评分的变化，结合《临床疾病诊断依据治愈好转标准》，分为临床治愈、显效、有效、无效四级。临床治愈：HAMD评分变化大于90％者；显效：HAMD评分变化在90％～75％之间；有效：HAMD评分变化在75％～50％之间：无效：HAMD评分变化小于50％。
1．3．2．2神经缺损评分(SSS)参照1995年第四届脑血管病会议通过的“卒中患者临床神经功能缺损程度评分标准”。
1．3．2．3日常生活活动能力(ADL)采用Barthel指数记分法。记录治疗前后评分的变化按脑血管疾病的疗效评定标准判定脑血管病的疗效。疗效标准采用Radit分析，评分变化采用t检验。
量表评定由两名受过训练的医师共同评定，一次性检验的相关系数(r=0.92～0.98)。分别于治疗前和治疗后第4、第8周评定疗效。
1．3．3血清NE、5-HT水平测定采用Hitachi公司产荧光分光仪，分别于治疗前、后测定血清NE和5-HT水平。
1．3．4血浆皮质醇水平检测受试者均于8∶00AM于安静、空腹状态下抽取肘静脉血，采用双抗体夹心放射免疫法测定血浆皮质醇。
1．3．5事件相关电位P300(event-related potential P300，P300)测定采用意大利安培MK15型诱发电位仪。
1．3．6副反应情况副反应情况采用量表(TESS)评定，于治疗前及治疗4周、8周末评定。
1．4统计学分析：所有结果以(?)±S表示，采用SPSS 12.0统计软件包处理全部数据，组间比较采用成组设计的t检验，治疗前后比较采用配对t检验。计数资料采用X~2检验。
2结果
2．1临床疗效比较治疗组52例，显效36例(69.23％)，有效6例(11.54％)，无效10例(19.23％)，总有效率(80.77％)。对照组51例，显效27例(52.94％)，有效8例(15.69％)，无效16例(31.37％)，总有效率68.63％。治疗组总有效率与对照组比较有显著性差异(P＜0.05)，治疗组优于对照组。
2．2量表评定比较
2．2．1汉密顿抑郁量表(HAMD)评分治疗组与对照组在治疗前HAMD积分基本一致，治疗组在服用JWSNS4周和8周后，HAMD评分较治疗前显著性下降，与治疗前比较，差异均有显著性意义(P＜0.01)。两组HAMD评分均随治疗时间延长逐步下降，提示两药均有明显抗抑郁作用。两组治疗后比较，差异有显著性意义(P＜0.01)，治疗组优于对照组。
2．2．2神经功能缺损(SSS)评分治疗组与对照组在治疗前神经功能缺损程度评分基本一致。治疗组治疗4周和8周后，神经功能缺损评分与治疗前相比明显下降，对照组则下降不明显，表明JWSNS可明显加快神经功能缺损程度的改善。
2．2．3日常生活活动能力(ADL)评分(Barthel指数)治疗组与对照组在治疗前ADL能力评分基本一致。治疗组在治疗4周和8周后，ADL能力评分即明显提高。而此时对照组的ADL能力评分尚无明显变化，表明加味四逆汤可以提高患者的日常生活活动能力。
2．3血清NE、5-HT水平变化治疗前，两组患者血清NE、5-HT水平均显著低于正常值(P＜0.01)。疗程结束后，两组血清NE、5-HT水平与治疗前比较显著上升(P＜0.01)，并且两组血清NE、5-HT水平均随治疗时间的延长逐步上升，提示两药均有明显抗抑郁作用。两组治疗后比较，差异有显著性意义(P＜0.01)，治疗组优于对照组。
2．4血浆皮质醇水平的变化治疗前，两组患者血浆皮质醇水平均显著高于健康组(P＜0.01)。疗程结束后，两组血浆皮质醇与治疗前比较显著降低(P＜0.01)，并随治疗时间的延长逐步下降，两组治疗后比较，差异有显著性意义(P＜0.01)，治疗组优于对照组。
2．5事件相关电位P300的变化PSD患者与健康人相比，P300成分中N2和P3波潜伏期延长，P3波幅降低，两组有显著性差异(P＜0.01)，而其它波潜伏期及波幅两组相比，两组相比未见显著性差异(P＞0.05)。两组疗程结束后，P300成分中N2和P3波潜伏期缩短，P3波幅升高，与治疗前相比均有显著性差异(P＜0.05)，但两组治疗后比较，无显著性差异(P＞0.05)。
2．6副反应情况两组药物不良反应的出现均以治疗初期的1～2周内明显。两组间治疗后TESS项次数比较，经X~2检验差异有显著性(P＜0.01)。两组病人血、尿和大便常规以及肝、肾功能，心电图，心肌酶均无异常变化。
3讨论
JWSNS临床治疗PSD有确切的效果，总有效率为80.77％，优于西药百忧解。表现为可明显降低HAMD评分，降低神经功能缺损(SSS)评分，加快神经功能缺损程度的改善；显著升高日常生活活动能力(ADL)评分(Barthel指数)，提高PSD患者的日常生活活动能力。此外，在临床应用的不良反应方面，我们的观察结果表明，JWSNS没有明显的副反应，与西药相比，具有安全、长效和稳效的优势的特点，显示出临床应用的广阔前景。对本研究所获得的临床观察结果进行分析，我们认为JWSNS治疗PSD的疗效作用机理涉及到以下几方面：1．显著升高改善PSD患者血清NE、5-HT的低下状况，抑制单胺类神经递质的重摄取；2．降低血浆皮质醇水平，调节下丘脑—垂体—肾上腺轴功能。3．改善患者的大脑感知容量，增加大脑执行功能，提高对外界信息的加工和对行为的调节能力，改善PSD患者的认知功能。
第二部分实验研究
1材料与方法
1．1实验动物
SPF级雄性Wistar大鼠，2月龄，体重220～270g，南方医科大学实验动物中心提供，动物合格证号：2002-009。大鼠自由饮水与进食，在光暗周期为12h，温度为23±2℃的安静环境适应一周。
1．2实验药物及制备
中药加味四逆散(JWSNS)组成：柴胡5g、白芍15g、枳壳6g、枸杞子15g、山栀5g、干地黄18g、石决明30g。药材由本校第一附属医院药房提供，经药剂科鉴定均为纯正药材。将中药制成粗粉，首煎将中药粗粉置8倍温水中浸泡0.5h，沸腾后文火煎煮4h，注意均匀搅拌，取汁后经三层纱布过滤；第二次和第三次煎煮均分别以6倍水文火煎煮2h，同样方法取汁，合并三次药液，置水浴箱内浓缩至含生药1.69g／mL。药液常温冷却后，置4℃冰箱内保存备用。西药：盐酸氟西汀胶囊(美国lilly公司生产，批号208151)，临用时20mg氟西汀胶囊粉末溶于30mL蒸馏水并混匀，为0.75mg／kg(成人剂量的6.25倍)给大鼠灌胃。
1．3主要仪器和试剂(略)
1．4实验分组
大鼠自由饮水与进食，在光暗周期为12h，温度为23±2℃的安静环境适应性喂养一周，以敞箱实验(Open-field test)作行为学筛选，敞箱实验水平无能无力和垂直运动总得分低于30分或高于120分的动物予以剔除。将80只大鼠按体重以SPSS统计软件进行随机选取10只大鼠作为正常对照组，其余用于制备大脑中动脉阻塞(Middle cerebral artery occlusion，MCAO)大鼠模型及行伪手术。将造模后存活的大鼠(除去伪手术组10只)按上述方法随机分为3组，即模型组、氟西汀组、JWSNS组。
1．5模型制备方法
1．5．1电凝法制备MCAO大鼠模型
1．5．2卒中后抑郁大鼠模型的复合制备MCAO大鼠在清醒后移入小鼠笼内进行28天孤养，在卒中后第8天(神经功能缺损已基本恢复)相继给予21天共9种温和不可预测的应激刺激。
1．6检测指标及方法
1．6．1一般状况观察
每天观察各组大鼠的精神、进食、饮水情况；大便质地；切口是否有感染表现；是否有死亡大鼠，记录死亡时间并作尸解以查明死因；每周测一次体重。
1．6．2局灶性脑缺血大鼠神经功能缺损评定大鼠清醒后24小时内采用Longa5分对大鼠神经功能缺损进行评分。1．6．3大鼠行为学观察
1．6．3．1糖水消耗实验于卒中后第28天评定。大鼠首先训练饮用1％蔗糖水，即在开始的48小时内用1％蔗糖水代替自来水，然后给予1％蔗糖水饮用，并计算24小时的饮用量。
1．6．3．2敞箱实验
1．6．4大鼠脑内单胺类递质(NE、5-HT、DA)测定荧光分光光度法。NE、5-HT和DA的具体含量计算：(样品管A值—空白管A值)／(标准管A值—空白管A值)×0.2×1.5×(1／脑组织重量g)=()u／g。
1．6．5大鼠海马cAMP含量、PKA及PKC活性检测cAMP含量测定：放免法。PKA、PKC活性的测定：放射性同位素法。
1．6．6大鼠海马P-CREB表达的检测免疫组织化学法，病理图像分析系统分析统计海马P-CREB阳性表达的光密度和面密度。
1．7给药剂量与方法
总灌胃给药时间为22天，从卒中后第8天开始，灌胃次数每天1次，每周测体重1次。正常对照组、模型组以及伪手术组灌胃等量蒸馏水。盐酸氟西汀组灌胃剂量0.75mg／kg，JWSNS组每次灌胃2ml(每ml含生药1.69g)。
1．8统计学处理所有结果采用以(?)±S表示，实验数据用SPSS12.0统计软件包处理，组间数据比较采用单因素方差分析，其中方差齐者采用LSD法检验，方差不齐者采用Dunnett'sT3法检验。个别不符合正态分布数据则采用非参数检验。两组间的均数比较采用配对T检验。
2结果
2．1各组大鼠造模及死亡情况术后4周内共死亡12只大鼠。在行MCAO手术时死亡5只，其中3只死于术中大出血，2只死于麻醉意外。复合造模期间死亡2只大鼠。在应激处理7天中死亡2只，1只在电应激处理后死亡，1只死因不明。在应激处理后到处死前死亡5只大鼠，其中因灌胃不当死亡3只，肺部感染死亡2只。在所有死亡大鼠中正常对照组死亡1只，伪手术组无死亡大鼠。64只大鼠制备MCAO模型，死亡5只，2只神经功能缺损评分为0，造模成功率为82.3％。PSD造模成功大鼠为51只，造模成功率为79.7％
2．2手术大鼠神经功能缺损评分结果MCAO大鼠在麻醉清醒后，除2只未出现神经功能缺损，评分为0外，其余均呈现左侧不同程度的神经缺失症状，评分为2.61±0.59。伪手术组无神经功能缺损表现。
2．3各组大鼠体重变化的情况与正常对照组比较，在卒中7天后，各组大鼠体重增长均降低，但未发现显著性差异：模型组、西药治疗组、中药治疗组在卒中后第14天到卒中后第28天的体重都显著低于正常对照组(P＜0.05或0.01)：伪手术组与对照组无统计学差异。与模型组比较：西药治疗组、中药治疗组在卒中后第28天显著高于模型组(P＜0.05)。此外，模型组体重增长明显减慢，各治疗组均有加快大鼠体重增长的趋势。结果表明：复合造模处理可以显著减慢大鼠体重增长。各治疗组均有加快大鼠体重增长的趋势，在卒中后第28天显著高于模型组。
2．4大鼠行为学观察结果
2．4．1糖水消耗实验结果与正常对照组比较，模型组、伪手术组和各治疗组的糖水消耗量均显著降低(P＜0.05或0.01)。与模型组比较，各治疗组糖水消耗量均显著增加(P＜0.05或0.01)。复合造模处理可使大鼠糖水消耗量显著降低。各治疗组均能显著增加PSD大鼠的糖水消耗量，提示药物可抑制PSD大鼠兴趣感下降，抑制抑郁行为的发生。
2．4．2敞箱实验结果与正常对照组比较：卒中后7天模型组及药物组水平运动有显著下降(P＜0.05)，垂直运动各组间无显著差异；卒中后14天、21天、28天模型组、伪手术组及各药物组水平运动与垂直运动均有显著下降(P＜0.05或0.01)。与模型组比较：卒中后7天垂直运动各组间无显著差异；卒中后14天伪手术组垂直、水平活动，中药治疗组的水平活动显著增高(P＜0.05)；卒中后21天、28天伪手术组、西药治疗组、中药治疗组的水平和垂直活动均显著增高(P＜0.05或0.01)。此外，空白对照组每次敞箱活动评定均无显著变化，所有手术大鼠在1周后活动出现明显下降，各药物组大鼠的活动呈现先下降后上升的趋势。由此可见：复合造模处理可引起大鼠出现敞箱行为异常(水平活动和垂直活动的降低)，提示其对外界新鲜环境的探究性行为和自发活动降低。而各药物组对PSD大鼠探究性行为和自发活动有显著改善作用。
2．5大鼠脑内单胺类递质测定结果
2．5．1大鼠脑内NE测定结果与正常对照组比较，模型组、各药物治疗组左、右侧额顶皮层和脑干的NE含量显著下降(P＜0.05或0.01)。与模型组比较：伪手术组、各药物治疗组左、右侧额项皮层及脑干的NE含量均显著增高(P＜0.05或0.01)。结果表明，复合造模处理可引起大鼠脑内NE显著降低，而各药物治疗组均能显著增加PSD大鼠脑内NE含量。
2．5．2大鼠脑内5-HT测定结果与正常对照组比较，模型组及各药物组的左、右侧额顶皮层及脑干的5-HT含量均有显著下降(P＜0.05或0.01)，伪手术组有所降低但无统计学意义。与模型组比较：伪手术组、各药物组的左、右侧额顶皮层及脑干的5-HT含量均有显著增加(P＜0.05，P＜0.01)。结果表明，复合造模处理可引起大鼠脑内5-HT显著降低，而各药物组均能显著增加PSD大鼠脑内5-HT含量。
2．5．3大鼠脑内DA测定结果与正常对照组比较，模型组和各药物组的左、右侧额顶皮层的DA均显著下降(P＜0.05或0.01)；与模型组比较，伪手术组、氟西汀组右侧额顶皮层的DA均显著增高(P＜0.05)；伪手术组和氟西汀组左侧额顶皮层的DA显著增高(P＜0.05)；氟西汀组脑干DA的含量显著增高(P＜0.05)。中药治疗组的左、右侧额顶皮层和脑干DA含量均有增加(P＜0.05)。结果表明，复合造模处理可引起大鼠脑内DA含量显著降低，氟西汀和JWSNS均能显著地增加左、右侧额顶皮层以及脑干DA的含量。
2．6大鼠海马cAMP含量、PKA及PKC活性变化情况模型组和伪手术组大鼠皮质及海马cAMP含量明显降低，与正常组相比，有统计学意义(P＜0.01)，西药治疗组和JWSNS组大鼠皮质及海马cAMP含量均较模型组明显升高(P＜0.01)，两组之间无明显差别。模型组和伪手术组大鼠皮质及海马PKA活性明显降低，与正常组相比有统计学意义(P＜0.05)。西药治疗组及JWSNS组大鼠皮质及海马PKA活性较模型组明显升高(P＜0.05)，两组之间无明显差别。模型组和伪手术组大鼠皮质及海马PKC活性明显升高，与正常组相比有统计学意义(P＜0.01)。西药治疗组及JWSNS组大鼠皮质及海马PKC活性较模型组明显降低(P＜0.01)，两组之间无明显差别。
2．2大鼠海马P-CREB表达的变化P-CREB在正常大鼠海马广泛表达，对海马结构CA1区进行观察，CA1区锥体细胞层阳性核表达为主，深棕色，胞质和突起也有表达，但着色较浅；模型组和伪手术组海马CA1区阳性细胞较正常少，阳性细胞出现空泡状或萎缩状，染色浅，面密度和光密度降低(P＜0.05或0.01)，西药组和中药组阳性细胞较模型组增加，染色加深，面密度和光密度值增高(P＜0.05)。
5讨论
本实验结果表明，复合造模处理可以显著减慢大鼠体重增长，故体重增长缓慢可能是大鼠PSD的一个表现。西药氟西汀和中药JWSNS均有加快大鼠体重增长的趋势，在卒中后第28天显著高于模型组。糖水消耗实验结果表明，PSD大鼠糖水消耗量显著降低，西药氟西汀和中药JWSNS均能显著增加PSD大鼠的糖水消耗量(P＜0.05或0.01)，提示药物可抑制PSD大鼠兴趣感下降，抑制抑郁行为的发生。敞箱实验结果表明，复合造模处理可引起大鼠出现敞箱行为异常(水平活动和垂直活动的降低)，提示其对外界新鲜环境的探究性行为和自发活动降低。而氟西汀和JWSNS对PSD大鼠探究性行为和自发活动均有显著改善作用。在本实验中，与正常对照组比较，模型组大鼠左、右侧额顶皮层和脑干的NE、5-HT、DA含量显著下降(P＜0.05或0.01)，可能与脑卒中病灶损伤NE、DA、5-HT到大脑皮质的投射纤维，继之引起皮质神经元递质合成减少有关。氟西汀和JWSNS均能显著增加PSD大鼠双侧皮层及脑干的NE、5-HT和DA的含量(P＜0.05或P＜0.01)。本实验结果表明，PSD模型组大鼠皮质及海马cAMP含量及PKA活性明显降低，而PKC活性明显升高，提示卒中后抑郁模型大鼠脑皮质及海马受体后cAMP-PKA信号通路系统下调而PKC信号通路上调，氟西汀和JWSNS具有改善和调节第二信使及蛋白激酶的作用，提示药物的抗抑郁作用可能与调节受体后cAMP-PKA及PKC信号通路有关。此外，本实验研究结果显示，PSD存在脑前皮质及海马P-CREB表达减少的现象。其机制可能是cAMP-PKA通路可直接使CREB磷酸化而激活，PSD模型大鼠存在神经元功能障碍，导致脑内单胺递质受体功能障碍及cAM P-PKA通路功能的下降，由此导致CREB功能下调。氟西汀和JWSNS对PSD的干预作用与增加CREB的表达有关，但具体的作用途径还需在今后的工作中探讨。
第三部分研究结论
一、以肝主疏泄理论为指导，运用调肝方药加味四逆散(JWSNS)防治PSD具有科学的理论依据。
二、JWSNS临床治疗PSD有确切的效果，总有效率为80.77％，优于西药百忧解。表现为可明显降低HAMD评分，降低神经功能缺损(SSS)评分，加快神经功能缺损程度的改善；显著升高日常生活活动能力(ADL)评分(Barthel指数)，提高PSD患者的日常生活活动能力。此外，在临床应用的不良反应方面，我们的研究结果表明，JWSNS没有明显的副反应，与西药相比，具有安全、长效和稳效的优势的特点，显示出临床应用的广阔前景。对本研究所获得的临床观察结果进行分析，我们认为JWSNS治疗PSD的疗效作用机理涉及到以下几方面：1．显著升高改善PSD患者血清NE、5-HT的低下状况，抑制单胺类神经递质的重摄取；2．降低血浆皮质醇水平，调节下丘脑—垂体—肾上腺轴功能。3．改善患者的大脑感知容量，增加大脑执行功能，提高对外界信息的加工和对行为的调节能力，改善PSD患者的认知功能。
三、JWSNS均能显著增加PSD大鼠的糖水消耗量，抑制PSD大鼠兴趣感下降，抑制抑郁行为的发生；对PSD大鼠探究性行为和自发活动均有显著改善作用。JWSNS均能显著增加PSD大鼠双侧皮层及脑干的NE、5-HT和DA的含量；改善和调节第二信使及蛋白激酶的作用，其抗抑郁作用可能与调节受体后cAMP-PKA及PKC信号通路有关。此外，JWSNS对PSD的干预作用与增加CREB的表达有关。
Title: Research on treatment to post-stroke depression under the direction of theory of "The liver controlling dispersion" in Chinese medicine
Specialty: Diagnostics of Chinese Medicine
Candidate: Gao Min
Tutor: Professor Chen Qun
Post-stroke depression (PSD) means having depressive emotion or in the station of depression after stroke and having been confirmed by related scales. It is one of the important complications of stroke and the incidence rate is 25%-80%, mostly manifested light-midrange. The activity disorder of PSD patients can often induce the happening of depressive disorder and the last one can affect the recovery from stroke and form infernal circle. As a kind of secondary affective disorder after stroke, the PSD can step down the functional recovery of limbs and recognition, increase the case fatality ratio and affect the patients' quality of life and prognosis seriously except can cause emotional pain. At present, worker of both Chinese and western medicines have make systematical research to the pathogenesis and clinical therapy to the PSD and have make notable advancement. Making use of the Chinese medicine to treat the PSD get more and more attention and the research get more and more profound for the side-effect, addiction and contraindication of traditional and new type anti-depressive medicines (western medicines).
The research approached the relationship between the theory of liver controlling dispersion and PSD. Directed by the theory of liver controlling dispersion and combined the forward research foundation, the research make use of the regulating "liver" therapy herbs-JWSNS to treat the PSA and approach the mechanism of action and provide scientific base to manufacture and develop new Chinese medicines in the future.
Part One Clinical Researches
1 Clinical data and Methods
1.1 Research objects
1.1.1 Source of the case All the 103 cases came from our capital from October, 2003 to October,2006.
1.1.2 Internalization criterion
(1) Patients with clear awareness, no dysgnosia and can go with examination in 2 weeks at the first post-stroking.
(2) all the cases are consistent with the diagnostic criteria of brain hemorrhage and cerebral infarction which passed by the 4th national CVD academic meeting in 1995 and the stroke and depression differentiation of symptoms and signs of internal medicine of TCM, and get final diagnosis by 1～3 times skull CT or MRI.
(3) Before the drug treatment, all patients have such characters: Hamilton depression rating scale (HAMD) score＞17, neurological impairment score (SSS)＞16, Barthel index＜70(Barthel index evaluation scale is used to evaluate activities of daily living(ADL))
(4) Taking no anti-depression preparation in 1 weeks at the first diagnosis. Taking no monoamine oxidase inhibitor in 2 weeks and no other anti-depression preparation and antipsychotic drug. Taking no licorice root, adrenal cortex hormone, adrenocorticotrop (h)ic hormone (ACTH) or analog.
(5)Normal routine of blood, urine and stool and normal function of liver and kidney, no serious medical record of heart, liver, kidney, mental disorder, epilepsy and drug allergy; Depleting innocent failure of memory of old ages, senile dementia and neurastheria before searching.
1.2 Subgroup and therapy
1.2.1 Subgroup
All the cases are divided into 2 groups at random in the 1st month after onset. 52 cases in the therapy group: male:32, female:20, age:38～68 years old, the mean is 40.21±8.57; 28 cases of apoplexy, 8 cases of lacunar infarction, 10 cases of cerebral embolism, 6 cases of subarachnoid hemorrhage.51 cases in control group: male:31, female:20, age:37～69 years old, the mean is 41.53±7.92,27 cases of apoplexy, 6 cases of lacunar infarction, 12 cases of cerebral embolism, 6 cases of subarachnoid hemorrhage. Before treatment, the HAMD score is 17～30, mean is 20.42±1.82/17～31, 20.16±2.51. The SSS score is 16～45, mean is (25.3±4.2)分/16～45分, 24.2±5.4. The Barthel index is 0～70, mean is (26.1±6.8)分/0～70分, 25.1±7.2. There have no statistical difference between the 2 groups in age, sex, etiological factor, course of disease, depressive degree, neurologic impairment degree and Barthel index (P＞0.05). 50 cases in healthy group, all of them came from the healthy peoples of health examination: male:30, female:20, age:36～67 years old, the mean is 40.44±8.72, and there have no statistical difference between the control group and therapy group in age and sex construction.
1.2.2 Therapy and nursing methods
The 2 groups were given orally hydergine 1mg/time, Tid. The hypertension and diabetic were given the treatment of decrease the blood pressure and blood sugar. In the same time, the technique of nerve-muscle unobstructed especially the Bobath therapy were used to train the limbs function, QD, 50min/time. In the base, the 2 groups were given the blow treating methods: the therapy group: JWSNS, composition and dosage: Bupleurum Root 5 g、White Peony Root 15g、Bitter Orange 6g、Wolfberry Fruit 15g、Capejasmine Fruit 5g、Rehmannia Root 18g、Concha haliotidis 30g. 1 dosage per day, decocted in water for oral dose and drink in the morning and evening, milkvetch root and codonopsis pilosula were added for serious deficiency of vital energy, polygoni muliflori and barbary wolfberry fruit for deficiency of kidney-essence, morindae officinalis and epimedii for deficiency of kidney-YANG, processing pinelliae and Coptis chinensis for sputum-fire, common anemarrhena rhizome and peony root bark for asthenic yin causing excessive pyrexia, flos albiziae, crude fossilia ossis mastoid and crude Ostreae testa for serious insomnia, peach seed and Carthamus tinctorius for stagnant blood. The control group: Fluoxetine (fluoxertine hydrochloride caps, lilly company, USA, Batch No208151), QD, 20mg/time and ate in the morning. Basic nursing: nursing staff communicated with the patients with broad-minded, observed the talk and behavior, perspect the mentation and strengthen the control of behavior and emotions. Affected the patients with broad-minded, evoked the interests of them and provide support, apprehension and sympathy in emotion, achieve the trust and conduct them to pour out their heart, encouraged out door activity, avoided fat and oily food and smoke and wine, drank rosae and chrysanthemi flos tea. The endemic area is calm and has light music. 4 weeks as one course of treatment and we make 2 courses of treatment. Observe and statistics the therapeutic effect. Detecting the routine of blood, urine and stool and function of liver and kidney pretherapy and the 4th and the 8th week of the cause of the therapy and scored HAMD.
1.3 Index surveyed and evaluation of therapeutic effect
1.3.1 Evaluation criterion
Referencing Diagnostic therapeutic effect criterion of TCM diseases, which were draw up by the Nation TCM Administration in June, 1994, we institute the evaluation criterion. Excellence: symptom disappeared, normal emotion. Utility: symptom decreased, basic stabilization of emotion. Invalid: no improvement of both symptom and emotion.
1.3.2 Evaluation criterion of the scale
HAMD, SSS and ADL were evaluated of the 2 groups of patients at the 4th week and 8th week of pre-treatment and post-treatment.
1.3.2.1 Evaluating the depressive symptom based on HAMD scale Score 24 kinds symptoms and signs with 5 grades methods and noting the score change of the 2groups of patients pre-tretment and post-treatment. Combining the Diagnostic base, healing well (HW) and improvement criterion of clinical diseases, the results are classified into HW, excellence, utility and invalid. HW: the change of HAMD score exceed 90%; Excellence: the change of HAMD score between 90%～75%; Utility: the change of HAMD score between 75%～50%; Invalid: the change of HAMD scores below 50%.
1.3.2.2 SSS score Referencing the Clinical SSS criterion of stroke patients, which was passed in the 4th CVD meeting in 1995.
1.3.2.3 ADL Barthel index scoring method. Noting the score change of pre-treatment and post-treatment and assessing the effect of CVD basing on the therapeutic effect evaluation criterion of CVD. Therapeutic effect criterion is analyzed with Radit analysis and score change with t-test.
2 trained doctors evaluate the Scale at the same time; therapeutic evaluation is carried out at the 4th and 8th week of pre-treatment and post-treatment.
1.3.3 Detection of serum NE and 5-HT Fluorescence spectroscope, Hitachi Company. Detect serum NE and 5-HT of pre-treatment post-treatment.
1.3.4 Detection of plasma cortisol collecting ulnar vein blood at 8:00AM of the subjects with calm and empty stomach station and detecting the plasma cortisol with double antibody sandwich radio-immunization.
1.3.5 Detection of event-related potential P300 Evoked potential evoked potential, ampere MK15, Italy
1.3.6 Station of side effect Evaluating the station of side effect with scale (TESS) at the 4th week and 8th week before the treating and in the treating course.
1.4 Statistics analysis All results are expressed byx±s. The data are handled by SPSS 12.0 statistical package, in which t-test are used to group comparison, paired t-test to comparison of pre-treatment and post-treatment, X~2 test to numeration data.
2 Results
2.1 Comparison of clinical therapeutic effect therapy group52 examples: excellence 36 examples (69.23%), utility 6 examples (11.54%), invalid 10 examples (19.23%), total effective rate 80.77%. Control group 51 examples: excellence 27 examples (52.94%), utility 8 examples (15.69%), invalid 16 examples (31.37%), total effective rate 68.63%. There have significant difference between the therapy group and control group in total effective rate (P＜0.05), the therapy group is outstrip control group.
2.2 Comparison of the scale evaluation
2.2.1 Score of HAMD scale The HAMD score are coincidence between the therapy group and control group. Compared with pre-treatment, The HAMD score of the therapy group significantly decreased after taking JWSNS for 4 weeks and 8 weeks and has significant difference(P＜0.01). The score of both group decreased with the treating, which indicated that the two medicines have obviously anti-depression effect. There have significant difference between the 2 groups after treatment (P＜0.01) and the therapy group is outstrip the control group.
2.2.2 SSS score The SSS score are coincidence between the therapy group and control group. Compared with pre-treatment, The SSS score of the therapy group significantly decreased after taking JWSNS for 4 weeks and 8 weeks while which of the control group didn't, which manifested that JWSNS can significantly improve the neurologic impairment.
2.2.3 ADL score (Barthel index) The ADL score are coincidence between the therapy group and control group. Compared with pre-treatment, The ADL score of the therapy group significantly increased after taking JWSNS for 4 weeks and 8 weeks while which of the control group didn't, which manifested that JWSNS can improve the activities of daily living.
2.3 change of NE and 5-HT in serum The levels of NE and 5-HT in serum in both 2 groups are significantly decreased before treatment comparing with normal value (P＜0.01). After the course of treatment, the levels of NE and 5-HT in serum in both 2 groups are significantly increased (P＜0.01). With the prolonged treating time, the levels of NE and 5-HT increased gradually, which indicates that the 2 kinds of medicines have effect of anti-depression. There have significant difference between the 2 groups after treatment (P＜0.01) and the therapy group is outstrip the control group.
2.4 Change of plasm cortisol The levels of cortisol in both 2 groups are significantly increased before treatment comparing with normal value (P＜0.01). After the course of treatment, the levels of cortisol in both 2 groups are significantly decreased (P＜0.01) and the levels increased gradually with the prolonged treating time. There have significant difference between the 2 groups after treatment(P＜0.01) and the therapy group is outstrip the control group.
2.5 Change of event-related potential P300 Compared with healthy people, the PSD patients have such characters: prolonged latent period of N2 wave and P3 wave in P300, decreased P3 wave amplitude, be different significantly from the healthy people (P＜0.01), while the other waves have no such condition (P＞0.05). After the course of treatment, the latent period of N2 wave and P3 wave in P300 shorten and P3 wave amplitude heighten of the 2 groups, there exist difference between pre-tratment and post-treatment(P＜0.05), but there have no difference between the 2 groups after treatment(P＞0.05).
2.6 Side effects The side effects emerged mostly in the first 1～2 weeks of the course of treatment. There exist significant difference in the times of TESS item between the 2 groups post-treatment(P＜0.01). There have no change in Normal routine of blood, urine and stool, function of liver and kidney and electrocardiogram and creatase.
3 Discussions
JWSNS have exact effect to PSD and the total effective rate is 80.77％, which is outstripped of Fluoxetine. The effect manifests that decrease the HAMD score and SSS score, improve the neurologic impairment, increase the ADL score and improve the activities of daily living. We found that in the side effect, JWSNS have no distinct side effect and have characters of safety, prolonged and steady actions, which manifest wide prospect of clinical application. According to the result, we think the curative effect mechanism of action of JWSNS to PSD is involve in the blow: 1. Significant increase the level of serum NE and 5-HT, refrain the reuptake of monoamine neurotransmitter; 2. Decrease the plasma cortisol and regulate the function of HPA; 3. Improve the brain capacity and function of sensation and perception and execute function, increase the progress of brain to outside message and regulating function of brain to behavior.
Part Two Experimental Researches
1 Materials and Methods
1.1 Experimental animals
Wistar rats, grade SPF, male, 2 months old, 220-270g in weight. Offered by animals' centre in Southern medical university, certification no: 2002-009. the rats can drink water and take food in freedom and acclimatize for 1 week, where the light-dark cycle is 12h, temperature is 23±2℃and calm.
1.2 Experimental medicines and preparation
Composition and dosage of JWSNS: Bupleurum Root 5g、White Peony Root 15 g、Bitter Orange 6 g、Wolfberry Fruit 15 g、Capejasmine Fruit 5 g、Rehmannia Root 18 g、Concha haliotidis 30 g. The herbs are bought from the first affiliated hospitalme of Guangzhou university of TCM and appraised as pure crude herbs by the drug department. Take the traditional Chinese medicine into coarse powder and soaked in eight multiple warm water for 0.5h, simmering decoction for 4h after boiled. Attention: even mixed and filtered with three-tier gauze; the 2nd and 3rd decotation with 6 multiple water for 2h. Merged the thrice physic liquor and concentrated to 1.69g/ml and preserved it in refrigerator at 4 centigrade after being cooled. Western medicine: fluoxertine hydrochloride caps(lilly company, USA, Batch No208151), dissolving 20mg fluoxertine hydrochloride caps in to 30ml distilled water and misce bene, intragastric administrating rats at a dose of 0.75mg/kg (6.25 multiple adult dosage).
1.3 The main equipment and agent(omitting)
1.4 The subgroup
The rats can drink water and take food in freedom and acclimatize for 1 week, where the light-dark cycle is 12h, temperature is 23±2℃and calm. Open-field test were used to sieve rats. The total score between 30～120 of level movement and vertical movement are qualified. 10 of the 80 rats are chosen as control group by SPSS 12.0 statistical package according to weight, the others are chosen to prepare Middle cerebral artery occlusion(MCAO) model and artificial operation. The living rats(remove 10 rats of artificial operation) are divided into 3 groups in random: model group, fluoxetine group and JWSNS group.
1.5 Preparative method of model
1.5.1 Preparation of rat MCAO model with electric coagulation
1.5.2 Complex preparation of rat PSD model Shift the MCAO rats in mouse cages after awake and breed alone for 28days. In the 8th days after stroke(neurological impairment have recover on the whole), 9 kinds of mild and unpredictable stress stimulus will be given for 21 days.
1.6 Index and methods
1.6.1 Common condition
Observe the mind, food taking and drinking water conditions, the stool texture, whether have infected and died rats, note the time of death and find out the death cause by dead body dissection, measure the weight every week.
1.6.2 SSS score of focal cerebral ischemia rats Longa5 is used to score the SSS after the rats awake in 24h.
1.6.3 Observation of rats ethology
1.6.3.1 The Sucrose Consumptions test The test are preceding in the 28th days after stroke. First, the rats are trained to drink 1％cane sugar solution, which means in the first 48h 1％cane sugar solution are used to take the place of the tap water, then giving 1％cane sugar solution to rats and calculating the drinking weight of 24h.
1.6.3.2 Open-field test
1.6.4 Detection of the monoamine transmitter(NE、5-HT、DA) of rat' brain Fluorospectrophotometry. Calculation the concrete contents of NE, 5-HT and DA: (values of the sample tube A—values of the vacant tube A)/(values of the standard A—values of the vacant tube A)×0.2×1.5×(1/weight of the brain tissue g)=( )U/g。
1.6.5 Detection of the contents of cAMP, the activity of PKA and PKC of rat'hippocampus Detection of the contents of cAMP: radio-immunity method. Detection of the activity of PKA and PKC: radio active isotope method.
1.6.6 Detection of the expression of P-CREB in rat'hippoeampus Analyzing and statistics optical density and areas density of expression of P-CREB with immunohistochemical method and patho-image analytical system.
1.7 The administrating dosage and methods
The time of the administration is 22d, ltime/d of intragastric administration from the 8th day after stroke and measure weight 1 time/week. The control group, model group and artificial operation group are given the same volume of distilled water. The dosage of fluoxertine hydrochloride group is 0.75mg/kg and JWSNS group 3.38 g/d to 1 rat.
1.8 Statistical treatment All results are expressed by (?)±s. The data are handled by SPSS 12.0 statistical package. ANOVE are used to group comparison, in which LSD are to regular variance and Dunnett'sT3 for irregular variance. Non-parameter test are used to data with no normal distribution. Paired t-test is used to group comparison of mean.
2 Results
2.1 Model and death conditions of rats 12 rats died in 4 weeks after the operation: 5 died in the course of the operation, 3 died for hemorrhoea, 2for anesthetic accident and 2 died in the course of the complex model. 2 died in 7 days of stress process: 1 died for electro-stress and 1 have no exact reason. After the stress process finished, 5 died before executing: 3 for mal-intragastric administration and 2 for pulmonary infection. 1 died of the control group and none of artificial operation group. 5 died in the 64 rats which are used to make model of MCAO, 2 scored 0 in SSS, the achievement ratio of making model is 82.3％. 51 rats succeeded in making model the achievement ratio is 79.7％.
2.2 SSS score of operation rats All the MCAO rats show the left neurological impairment of different degree and the score is 2.61±0.59 except 2 rats after awake. There have no neurological impairment in artificial operation group.
2.3 Change of rats weight Compared with the control group, the other groups manifested weight increased, but the difference were not distinct; the weight of the model group, fluoxetine group and JWSNS group lower significantly than the control group between the 14th day to 28th day after stroke(P＜0.05 or 0.01), but there had no difference between the artificial operation group and the control group(P＜0.05). In addition, the rats weight in model group increased slowly while those treatment groups had the tendency of increase weight. The results manifested that complex model preparation can significant step down the weight growth, while those treatment groups have the adverse effect and the difference is significantly comparing to the model group.
2.4 Results of the rats ethnology
2.4.1 Results of the sucrose Consumptions test The sucrose consumption significantly decreased in model group, artificial operation group and the treatment groups compared with that in the control group (P＜0.05 or 0.01). The sucrose consumption significant increased in the treatment group compared with that in the model group (P＜0.05 or 0.01). Complex model preparation can significantly decrease the sucrose consumption, while the treatment groups can improve the condition which indicates that the medicines can depress the PSD rat's interest and the occurrence of depressive action.
2.4.2 Results of the open-field test Compared with the control group, the model group and the treatment groups manifested that the movement of level decreased significantly in the 7th day after the stroke (P＜0.05) but the vertical movement didn't change. The level and vertical movement decreased significantly in the 14th, the 21st and 28th day after stroke of the model group, artificial operation group and the treatment groups (P＜0.05 or 0.01). Compared with the model group, there's no difference of the vertical movement in the 7th day after stroke but have significant difference of the level and vertical movement in the 14th day of artificial operation group and level movement of JWSNS group(P＜0.05). In the 21st day and 28th the both movement significantly increased of the artificial operation group and the treatment groups(P＜0.05 or 0.01). In addition, the open-field test didn't change of the control group but the movement decreased in the operation rats 1 week later and the rats in the treatment groups manifested the tendency of descend first and ascend last. Conclusion: Complex model preparation can make the open-field behavior disorder(decrease the level and vertical movement), which indicate that probe behavior and spontaneous movement decreased, while the treatment groups can significantly improve the disorder.
2.5 Result of the monoamine transmitter of rats' brain
2.5.1 Result of the NE of rats' brain Compared with the control group, the model group and the treatment groups manifested that the NE decreased significantly in the left and right cortex of forehead and brain stem(P＜0.05 or 0.01). Compared with the model group, the artificial operation group and the treatment groups manifested that the NE increased significantly in the left and right cortex of forehead and brain stem (P＜0.05 or 0.01). Results indicate that complex model preparation can make the NE decreased while the medicines can improve the change.
2.5.2 Result of the 5-HT of rats' brain Compared with the control group, the model group and the treatment groups manifested that the 5-HT decreased significantly in the left and right cortex of forehead and brain stem (P＜0.05 or 0.01) and the artificial operation group decreased also but have no statistical meanings. Compared with the model group, the artificial operation group and the treatment groups manifested that the 5-HT increased significantly in the left and right cortex of forehead and brain stem(P＜0.05 or 0.01). Results indicate that complex model preparation can make the 5-HT decreased while the medicines can improve the change.
2.5.3 Result of the DA of rats' brain Compared with the control group, the model group and the treatment groups manifested that the DA decreased significantly in the left and right cortex of forehead(P＜0.05 or 0.01). Compared with the model group, the artificial operation group and the fluoxetine group manifested that the DA increased significantly in the right cortex of forehead(P＜0.05) and the fluoxetine group manifested that the DA increased significantly in the brain item(P＜0.05); the JWSNS group manifested that the DA increased significantly in the right cortex of forehead and the brain item(P＜0.05). Results indicate that complex model preparation can make the DA decreased while the medicines can improve the change.
2.6 Result of the cAMP contents and activity of PKA and PKC in rats' hippocampus Compared with the control group, the model group and the artificial operation group manifested that the cAMP contents decreased significantly (P＜0.01) and the fluoxetine group and the JWSNS group manifested that the cAMP contents increased significantly in the cortex and hippocampus (P＜0.01) and there's no difference between the 2 treatment groups. Compared with the control group, the model group and the artificial operation group manifested that the activity of PKA decreased significantly in the cortex and hippocampus (P＜0.05) and the fluoxetine group and the JWSNS group manifested that the activity of PKA increased significantly in the cortex and hippocampus (P＜0.05) and there's no difference between the 2 treatment groups. Compared with the control group, the model group and the artificial operation group manifested that the activity of PKC increased significantly (P＜0.01)and the fluoxetine group and the JWSNS group manifested that the activity of PKC decreased significantly in the cortex and hippocampus (P＜0.01) and there's no difference between the 2 treatment groups.
2.7 Result of the P-CREB expression in rats' hippocampus The P-CREB expression is broad in the normal rats hippocampus and in the CA1 realm, the expression mostly lie in nuclear of the pyramidal layer the color is deep brown and some expression lie in the kytoplasm and ecphyma but the color is faint. In the model group and artificial operation group, the masculine cells are fewer and show vacuolus or atrophia and faint color, the area density and light density are lower (P＜0.05 or 0.01), while the 2 treatment groups have the opposite manifestation (P＜0.05)
5 Discussions
The results indicate that the complex model preparation can significantly decrease the growth of weight, so the slowly weight growth is one of the manifestation of PSD rats. Fluoxetine and JWSNS can increase the weight and super than the model group in the 28th day after stroke. The results of the sucrose consumption test indicate that the sucrose consumption significantly decreased in PSD rats and the fluoxetine and JWSNS can improve the condition (P＜0.05 or 0.01), which indicate that the medicines can depress the PSD rats interest and the occurrence of depressive action. The results of the open-field test indicate that the complex model preparation can make the open-field behavior disorder (decrease the level and vertical movement), which indicate that probe behavior and spontaneous movement decreased, while the 2 kinds of medicines can significantly improve the disorder. Compared with the control group, the contents of NE, 5-HT and DA significantly decreased in model group in the left and right cortex of forehead and brain stem (P＜0.05 or 0.01), maybe this is related to the lessen cortical neuron transmitter synthesis induced by the stroke injury the projection fibers of NE, 5-HT and DA to cerebral cortex., while the 2 kinds of medicines can significantly improve the tendency (P＜0.05 or 0.01). The model group manifested that the cAMP contents and the activity of PKA decreased significantly (P＜0.01) while the activity of PKC increased which indicate that the cAMP-PKA signal iter system down regulated and the PKC up regulated in PSD rats, the 2 kinds of medicines have the effect of improve and regulate the second messenger and protien kinase, which indicate that the depressive effect of the medicines maybe related to the regulation of acceptor of cAMP-PKA and PKC signal iters. The PSD rats showed the P-CREB expression decreased in cortex and hippocampus, the mechanism maybe is that cAMP-PKA signal iter can directly make the CREB ptiosphorylation and activation. There exist neuronal functional impairment in PSD rats, for which the monoamine transmitter receptor and the cAMP-PKA signal iter have functional impairment, which induced the downregulation of CREB function. The intervention of the 2 kinds of medicines to PSD are related to the increased expression of CREB, but the concrete effective routes are still need to be probed in the late research.
Part Three Conclusions
1. Making use of the regulating "liver" therapy herbs-JWSNS to prevent and cure the PSA have scientific theoretical base directed by the theory of liver controlling dispersion.
2. JWSNS have exact effect to PSD and the total effective rate is 80.77%, which is outstripped of Fluoxetine. The effect manifests that decrease the HAMD score and SSS score, improve the neurological impairment, increase the ADL score and improve the activities of daily living. We found that in the side effect, JWSNS have no distinct side effect and have characters of safety, prolonged and steady actions, which manifest wide prospect of clinical application. According to the result, we think the curative effect mechanism of action of JWSNS to PSD is involve in the blow: 1. Significant increase the level of serum NE and 5-HT, refrain the reuptake of monoamine neurotransmitter; 2. Decrease the plasma cortisol and regulate the function of HPA; 3. Improve the brain capacity and function of sensation and perception and execute function, increase the progress of brain to outside message and regulating function of brain to behavior.
3. JWSNS can significantly increase the sucrose consumption of PSD rats and depress the interest and the occurrence of depressive action and improve the probe behavior and spontaneous movement. JWSNS can significantly increase the contents of NE, 5-HT and DA and improve and regulate the second messenger and protien kinase, which indicate that the depressive effect maybe related to the regulation of acceptor of cAMP-PKA and PKC signal iters. In addition, the intervention of JWSNS to PSD is related to the increased expression of CREB.
本研究探讨了中医肝主疏泄与PSD的关系，并结合以往的研究工作基础上，在本研究中，以肝主疏泄理论为指导，运用调肝治法方药(JWSNS)治疗PSD，并进一步探讨其作用机理，从而为今后的中药新药研发提供科学的依据。
第一部分临床研究
1临床资料与方法
1．1研究对象
1．1．1病例来源所有病例均来自2003年10月-2006年10月来我院就诊的门诊和住院患者，共103例。
1．1．2纳入标准
(1)首发脑卒中后2周内，意识清楚，无智力障碍，能配合检查。
(2)所有病例均符合中华神经科学会1995年全国第四界脑血管病学术会议通过的脑出血、脑梗死的诊断标准，并经1～3次的头颅CT或MRI检查确诊。符合《中医内科学》(第5版)中风、郁证辨证。
(3)所有患者在接受药物治疗前，汉密顿抑郁量表(HAMD)总分＞17，神经功能缺损评分(SSS)＞16分，日常生活活动能力(ADL)评价采用Barthel指数评价表，Barthel指数＜70分。
(4)首诊1周内未服用抗抑郁制剂。2周内无使用单胺氧化酶抑制剂，治疗中不合并使用其他抗抑郁药和抗精神病药。近3个月内无使用过甘草、皮质激素、促肾上腺皮质激素(ACTH)或类似物。
(5)入组前血、尿、粪常规及肝、肾功能正常，无严重心、肝、肾疾病史，无精神疾病史及癫痫病史，无药物过敏史；排除老年良性记忆减退、老年性痴呆和神经衰弱。
1．2分组及治疗方法
1．2．1分组
所有病例均为第一次发病后1个月内，按就诊顺序采用抽签法随机分为2组。治疗组52例，男32例，女20例，年龄38岁～68岁，平均40.21±8.57岁；脑出血28例，脑梗死8例，脑栓塞10例，蛛网膜下腔出血6例。对照组51例，男31例，女20例，年龄37岁～69岁，平均41.53±7.92岁；脑出血27例，脑梗死6例，脑栓塞12例，蛛网膜下腔出血6例。治疗前经HAMD评分17～30，平均20.42±1.82／17～31，20.16±2.51；神经功能缺损选用神经功能缺损评分16～45分，平均(25.3±4.2)分／16～45分，24.2±5.4；Barthel指数0～70分，平均(26.1±6.8)分／0～70分，25.1±7.2。两组年龄、性别、病因、病程、抑郁程度、神经功能缺损程度及Barthel指数均无明显差异(P＞0.05)。另设健康组50例，均来自医院体检健康人群，男30例，女20例，年龄36岁～67岁，平均40.44±8.72岁，在年龄、性别构成方面与对照组和治疗组无差异。
1．2．2治疗与护理方法
两组患者均给予口服喜得镇1日3次，1次1mg。高血压、糖尿病患者给予降压降糖治疗。同时采取以Bobath疗法为主的神经肌肉促通技术进行肢体功能训练，每日1次，每次50min。在此基础上，两组分别采用以下治疗方法。治疗组：采用加味四逆散(JWSNS)为主进行治疗。JWSNS组成及剂量：柴胡5g、白芍15g、枳壳6g、枸杞子15g、山栀5g、干地黄18g、石决明30g。每日1剂，水煎服，早晚各1次。气虚明显者加黄芪、党参：肾精不足者加何首乌、枸杞子；肾阳亏虚者加巴戟天、淫羊藿；痰热盛者加制半夏、黄连：阴虚火旺者加知母、丹皮；心神不宁、失眠较重者加合欢花、生龙骨、生牡蛎，瘀血甚者加桃仁、红花。对照组：采用百忧解(盐酸氟西汀，美国lilly公司生产，批号208151)进行治疗，每次20mg，每日1次，晨起服。基础护理措施：护理人员应以开朗的情绪与患者交流，观察患者的言谈举止，洞察患者的心理活动，加强对患者行为与思维的监控；以积极开朗的情绪去影响患者，唤起患者的兴趣，对患者在感情上予以支持、理解及同情，以获得患者的信任，引导患者倾吐心中的不满和郁结，使其内心的郁闷得到宣泄；鼓励患者户外活动，还可开展适宜的娱乐活动，使患者在活动中忘记烦恼；饮食宜清淡且避免过饱，忌肥腻，忌烟酒，宜以玫瑰花、菊花等代茶饮；病区环境布置雅静，可给予轻音乐等。以上两组均以4周为1个疗程，共治疗2个疗程。疗程结束后观察统计疗效。治疗前、治疗第4、8周末分别查血、小便、大便常规及肝、肾功能、心电图。并进行HAMD评分。
1．3观察指标及疗效评价
1．3．1中医疗效评价标准
参考1994年6月国家中医药管理局公布的《中医病症诊断疗效标准》制定。显效：症状消失，情绪正常。有效：症状减轻，情绪基本稳定。无效：症状情绪均无改善。
1．3．2量表评定标准
对两组患者于治疗前和治疗后4周、8周后进行HAMD、神经功能缺损程度评分(SSS)和日常生活活动能力(ADL)评定(Barthel指数)。
1．3．2．1根据汉密尔顿抑郁量表(HAMD)评定抑郁症状选择24项症状与体征按5级评分法进行评分，记录治疗前后两组患者评分的变化，结合《临床疾病诊断依据治愈好转标准》，分为临床治愈、显效、有效、无效四级。临床治愈：HAMD评分变化大于90％者；显效：HAMD评分变化在90％～75％之间；有效：HAMD评分变化在75％～50％之间：无效：HAMD评分变化小于50％。
1．3．2．2神经缺损评分(SSS)参照1995年第四届脑血管病会议通过的“卒中患者临床神经功能缺损程度评分标准”。
1．3．2．3日常生活活动能力(ADL)采用Barthel指数记分法。记录治疗前后评分的变化按脑血管疾病的疗效评定标准判定脑血管病的疗效。疗效标准采用Radit分析，评分变化采用t检验。
量表评定由两名受过训练的医师共同评定，一次性检验的相关系数(r=0.92～0.98)。分别于治疗前和治疗后第4、第8周评定疗效。
1．3．3血清NE、5-HT水平测定采用Hitachi公司产荧光分光仪，分别于治疗前、后测定血清NE和5-HT水平。
1．3．4血浆皮质醇水平检测受试者均于8∶00AM于安静、空腹状态下抽取肘静脉血，采用双抗体夹心放射免疫法测定血浆皮质醇。
1．3．5事件相关电位P300(event-related potential P300，P300)测定采用意大利安培MK15型诱发电位仪。
1．3．6副反应情况副反应情况采用量表(TESS)评定，于治疗前及治疗4周、8周末评定。
1．4统计学分析：所有结果以(?)±S表示，采用SPSS 12.0统计软件包处理全部数据，组间比较采用成组设计的t检验，治疗前后比较采用配对t检验。计数资料采用X~2检验。
2结果
2．1临床疗效比较治疗组52例，显效36例(69.23％)，有效6例(11.54％)，无效10例(19.23％)，总有效率(80.77％)。对照组51例，显效27例(52.94％)，有效8例(15.69％)，无效16例(31.37％)，总有效率68.63％。治疗组总有效率与对照组比较有显著性差异(P＜0.05)，治疗组优于对照组。
2．2量表评定比较
2．2．1汉密顿抑郁量表(HAMD)评分治疗组与对照组在治疗前HAMD积分基本一致，治疗组在服用JWSNS4周和8周后，HAMD评分较治疗前显著性下降，与治疗前比较，差异均有显著性意义(P＜0.01)。两组HAMD评分均随治疗时间延长逐步下降，提示两药均有明显抗抑郁作用。两组治疗后比较，差异有显著性意义(P＜0.01)，治疗组优于对照组。
2．2．2神经功能缺损(SSS)评分治疗组与对照组在治疗前神经功能缺损程度评分基本一致。治疗组治疗4周和8周后，神经功能缺损评分与治疗前相比明显下降，对照组则下降不明显，表明JWSNS可明显加快神经功能缺损程度的改善。
2．2．3日常生活活动能力(ADL)评分(Barthel指数)治疗组与对照组在治疗前ADL能力评分基本一致。治疗组在治疗4周和8周后，ADL能力评分即明显提高。而此时对照组的ADL能力评分尚无明显变化，表明加味四逆汤可以提高患者的日常生活活动能力。
2．3血清NE、5-HT水平变化治疗前，两组患者血清NE、5-HT水平均显著低于正常值(P＜0.01)。疗程结束后，两组血清NE、5-HT水平与治疗前比较显著上升(P＜0.01)，并且两组血清NE、5-HT水平均随治疗时间的延长逐步上升，提示两药均有明显抗抑郁作用。两组治疗后比较，差异有显著性意义(P＜0.01)，治疗组优于对照组。
2．4血浆皮质醇水平的变化治疗前，两组患者血浆皮质醇水平均显著高于健康组(P＜0.01)。疗程结束后，两组血浆皮质醇与治疗前比较显著降低(P＜0.01)，并随治疗时间的延长逐步下降，两组治疗后比较，差异有显著性意义(P＜0.01)，治疗组优于对照组。
2．5事件相关电位P300的变化PSD患者与健康人相比，P300成分中N2和P3波潜伏期延长，P3波幅降低，两组有显著性差异(P＜0.01)，而其它波潜伏期及波幅两组相比，两组相比未见显著性差异(P＞0.05)。两组疗程结束后，P300成分中N2和P3波潜伏期缩短，P3波幅升高，与治疗前相比均有显著性差异(P＜0.05)，但两组治疗后比较，无显著性差异(P＞0.05)。
2．6副反应情况两组药物不良反应的出现均以治疗初期的1～2周内明显。两组间治疗后TESS项次数比较，经X~2检验差异有显著性(P＜0.01)。两组病人血、尿和大便常规以及肝、肾功能，心电图，心肌酶均无异常变化。
3讨论
JWSNS临床治疗PSD有确切的效果，总有效率为80.77％，优于西药百忧解。表现为可明显降低HAMD评分，降低神经功能缺损(SSS)评分，加快神经功能缺损程度的改善；显著升高日常生活活动能力(ADL)评分(Barthel指数)，提高PSD患者的日常生活活动能力。此外，在临床应用的不良反应方面，我们的观察结果表明，JWSNS没有明显的副反应，与西药相比，具有安全、长效和稳效的优势的特点，显示出临床应用的广阔前景。对本研究所获得的临床观察结果进行分析，我们认为JWSNS治疗PSD的疗效作用机理涉及到以下几方面：1．显著升高改善PSD患者血清NE、5-HT的低下状况，抑制单胺类神经递质的重摄取；2．降低血浆皮质醇水平，调节下丘脑—垂体—肾上腺轴功能。3．改善患者的大脑感知容量，增加大脑执行功能，提高对外界信息的加工和对行为的调节能力，改善PSD患者的认知功能。
第二部分实验研究
1材料与方法
1．1实验动物
SPF级雄性Wistar大鼠，2月龄，体重220～270g，南方医科大学实验动物中心提供，动物合格证号：2002-009。大鼠自由饮水与进食，在光暗周期为12h，温度为23±2℃的安静环境适应一周。
1．2实验药物及制备
中药加味四逆散(JWSNS)组成：柴胡5g、白芍15g、枳壳6g、枸杞子15g、山栀5g、干地黄18g、石决明30g。药材由本校第一附属医院药房提供，经药剂科鉴定均为纯正药材。将中药制成粗粉，首煎将中药粗粉置8倍温水中浸泡0.5h，沸腾后文火煎煮4h，注意均匀搅拌，取汁后经三层纱布过滤；第二次和第三次煎煮均分别以6倍水文火煎煮2h，同样方法取汁，合并三次药液，置水浴箱内浓缩至含生药1.69g／mL。药液常温冷却后，置4℃冰箱内保存备用。西药：盐酸氟西汀胶囊(美国lilly公司生产，批号208151)，临用时20mg氟西汀胶囊粉末溶于30mL蒸馏水并混匀，为0.75mg／kg(成人剂量的6.25倍)给大鼠灌胃。
1．3主要仪器和试剂(略)
1．4实验分组
大鼠自由饮水与进食，在光暗周期为12h，温度为23±2℃的安静环境适应性喂养一周，以敞箱实验(Open-field test)作行为学筛选，敞箱实验水平无能无力和垂直运动总得分低于30分或高于120分的动物予以剔除。将80只大鼠按体重以SPSS统计软件进行随机选取10只大鼠作为正常对照组，其余用于制备大脑中动脉阻塞(Middle cerebral artery occlusion，MCAO)大鼠模型及行伪手术。将造模后存活的大鼠(除去伪手术组10只)按上述方法随机分为3组，即模型组、氟西汀组、JWSNS组。
1．5模型制备方法
1．5．1电凝法制备MCAO大鼠模型
1．5．2卒中后抑郁大鼠模型的复合制备MCAO大鼠在清醒后移入小鼠笼内进行28天孤养，在卒中后第8天(神经功能缺损已基本恢复)相继给予21天共9种温和不可预测的应激刺激。
1．6检测指标及方法
1．6．1一般状况观察
每天观察各组大鼠的精神、进食、饮水情况；大便质地；切口是否有感染表现；是否有死亡大鼠，记录死亡时间并作尸解以查明死因；每周测一次体重。
1．6．2局灶性脑缺血大鼠神经功能缺损评定大鼠清醒后24小时内采用Longa5分对大鼠神经功能缺损进行评分。1．6．3大鼠行为学观察
1．6．3．1糖水消耗实验于卒中后第28天评定。大鼠首先训练饮用1％蔗糖水，即在开始的48小时内用1％蔗糖水代替自来水，然后给予1％蔗糖水饮用，并计算24小时的饮用量。
1．6．3．2敞箱实验
1．6．4大鼠脑内单胺类递质(NE、5-HT、DA)测定荧光分光光度法。NE、5-HT和DA的具体含量计算：(样品管A值—空白管A值)／(标准管A值—空白管A值)×0.2×1.5×(1／脑组织重量g)=()u／g。
1．6．5大鼠海马cAMP含量、PKA及PKC活性检测cAMP含量测定：放免法。PKA、PKC活性的测定：放射性同位素法。
1．6．6大鼠海马P-CREB表达的检测免疫组织化学法，病理图像分析系统分析统计海马P-CREB阳性表达的光密度和面密度。
1．7给药剂量与方法
总灌胃给药时间为22天，从卒中后第8天开始，灌胃次数每天1次，每周测体重1次。正常对照组、模型组以及伪手术组灌胃等量蒸馏水。盐酸氟西汀组灌胃剂量0.75mg／kg，JWSNS组每次灌胃2ml(每ml含生药1.69g)。
1．8统计学处理所有结果采用以(?)±S表示，实验数据用SPSS12.0统计软件包处理，组间数据比较采用单因素方差分析，其中方差齐者采用LSD法检验，方差不齐者采用Dunnett'sT3法检验。个别不符合正态分布数据则采用非参数检验。两组间的均数比较采用配对T检验。
2结果
2．1各组大鼠造模及死亡情况术后4周内共死亡12只大鼠。在行MCAO手术时死亡5只，其中3只死于术中大出血，2只死于麻醉意外。复合造模期间死亡2只大鼠。在应激处理7天中死亡2只，1只在电应激处理后死亡，1只死因不明。在应激处理后到处死前死亡5只大鼠，其中因灌胃不当死亡3只，肺部感染死亡2只。在所有死亡大鼠中正常对照组死亡1只，伪手术组无死亡大鼠。64只大鼠制备MCAO模型，死亡5只，2只神经功能缺损评分为0，造模成功率为82.3％。PSD造模成功大鼠为51只，造模成功率为79.7％
2．2手术大鼠神经功能缺损评分结果MCAO大鼠在麻醉清醒后，除2只未出现神经功能缺损，评分为0外，其余均呈现左侧不同程度的神经缺失症状，评分为2.61±0.59。伪手术组无神经功能缺损表现。
2．3各组大鼠体重变化的情况与正常对照组比较，在卒中7天后，各组大鼠体重增长均降低，但未发现显著性差异：模型组、西药治疗组、中药治疗组在卒中后第14天到卒中后第28天的体重都显著低于正常对照组(P＜0.05或0.01)：伪手术组与对照组无统计学差异。与模型组比较：西药治疗组、中药治疗组在卒中后第28天显著高于模型组(P＜0.05)。此外，模型组体重增长明显减慢，各治疗组均有加快大鼠体重增长的趋势。结果表明：复合造模处理可以显著减慢大鼠体重增长。各治疗组均有加快大鼠体重增长的趋势，在卒中后第28天显著高于模型组。
2．4大鼠行为学观察结果
2．4．1糖水消耗实验结果与正常对照组比较，模型组、伪手术组和各治疗组的糖水消耗量均显著降低(P＜0.05或0.01)。与模型组比较，各治疗组糖水消耗量均显著增加(P＜0.05或0.01)。复合造模处理可使大鼠糖水消耗量显著降低。各治疗组均能显著增加PSD大鼠的糖水消耗量，提示药物可抑制PSD大鼠兴趣感下降，抑制抑郁行为的发生。
2．4．2敞箱实验结果与正常对照组比较：卒中后7天模型组及药物组水平运动有显著下降(P＜0.05)，垂直运动各组间无显著差异；卒中后14天、21天、28天模型组、伪手术组及各药物组水平运动与垂直运动均有显著下降(P＜0.05或0.01)。与模型组比较：卒中后7天垂直运动各组间无显著差异；卒中后14天伪手术组垂直、水平活动，中药治疗组的水平活动显著增高(P＜0.05)；卒中后21天、28天伪手术组、西药治疗组、中药治疗组的水平和垂直活动均显著增高(P＜0.05或0.01)。此外，空白对照组每次敞箱活动评定均无显著变化，所有手术大鼠在1周后活动出现明显下降，各药物组大鼠的活动呈现先下降后上升的趋势。由此可见：复合造模处理可引起大鼠出现敞箱行为异常(水平活动和垂直活动的降低)，提示其对外界新鲜环境的探究性行为和自发活动降低。而各药物组对PSD大鼠探究性行为和自发活动有显著改善作用。
2．5大鼠脑内单胺类递质测定结果
2．5．1大鼠脑内NE测定结果与正常对照组比较，模型组、各药物治疗组左、右侧额顶皮层和脑干的NE含量显著下降(P＜0.05或0.01)。与模型组比较：伪手术组、各药物治疗组左、右侧额项皮层及脑干的NE含量均显著增高(P＜0.05或0.01)。结果表明，复合造模处理可引起大鼠脑内NE显著降低，而各药物治疗组均能显著增加PSD大鼠脑内NE含量。
2．5．2大鼠脑内5-HT测定结果与正常对照组比较，模型组及各药物组的左、右侧额顶皮层及脑干的5-HT含量均有显著下降(P＜0.05或0.01)，伪手术组有所降低但无统计学意义。与模型组比较：伪手术组、各药物组的左、右侧额顶皮层及脑干的5-HT含量均有显著增加(P＜0.05，P＜0.01)。结果表明，复合造模处理可引起大鼠脑内5-HT显著降低，而各药物组均能显著增加PSD大鼠脑内5-HT含量。
2．5．3大鼠脑内DA测定结果与正常对照组比较，模型组和各药物组的左、右侧额顶皮层的DA均显著下降(P＜0.05或0.01)；与模型组比较，伪手术组、氟西汀组右侧额顶皮层的DA均显著增高(P＜0.05)；伪手术组和氟西汀组左侧额顶皮层的DA显著增高(P＜0.05)；氟西汀组脑干DA的含量显著增高(P＜0.05)。中药治疗组的左、右侧额顶皮层和脑干DA含量均有增加(P＜0.05)。结果表明，复合造模处理可引起大鼠脑内DA含量显著降低，氟西汀和JWSNS均能显著地增加左、右侧额顶皮层以及脑干DA的含量。
2．6大鼠海马cAMP含量、PKA及PKC活性变化情况模型组和伪手术组大鼠皮质及海马cAMP含量明显降低，与正常组相比，有统计学意义(P＜0.01)，西药治疗组和JWSNS组大鼠皮质及海马cAMP含量均较模型组明显升高(P＜0.01)，两组之间无明显差别。模型组和伪手术组大鼠皮质及海马PKA活性明显降低，与正常组相比有统计学意义(P＜0.05)。西药治疗组及JWSNS组大鼠皮质及海马PKA活性较模型组明显升高(P＜0.05)，两组之间无明显差别。模型组和伪手术组大鼠皮质及海马PKC活性明显升高，与正常组相比有统计学意义(P＜0.01)。西药治疗组及JWSNS组大鼠皮质及海马PKC活性较模型组明显降低(P＜0.01)，两组之间无明显差别。
2．2大鼠海马P-CREB表达的变化P-CREB在正常大鼠海马广泛表达，对海马结构CA1区进行观察，CA1区锥体细胞层阳性核表达为主，深棕色，胞质和突起也有表达，但着色较浅；模型组和伪手术组海马CA1区阳性细胞较正常少，阳性细胞出现空泡状或萎缩状，染色浅，面密度和光密度降低(P＜0.05或0.01)，西药组和中药组阳性细胞较模型组增加，染色加深，面密度和光密度值增高(P＜0.05)。
5讨论
本实验结果表明，复合造模处理可以显著减慢大鼠体重增长，故体重增长缓慢可能是大鼠PSD的一个表现。西药氟西汀和中药JWSNS均有加快大鼠体重增长的趋势，在卒中后第28天显著高于模型组。糖水消耗实验结果表明，PSD大鼠糖水消耗量显著降低，西药氟西汀和中药JWSNS均能显著增加PSD大鼠的糖水消耗量(P＜0.05或0.01)，提示药物可抑制PSD大鼠兴趣感下降，抑制抑郁行为的发生。敞箱实验结果表明，复合造模处理可引起大鼠出现敞箱行为异常(水平活动和垂直活动的降低)，提示其对外界新鲜环境的探究性行为和自发活动降低。而氟西汀和JWSNS对PSD大鼠探究性行为和自发活动均有显著改善作用。在本实验中，与正常对照组比较，模型组大鼠左、右侧额顶皮层和脑干的NE、5-HT、DA含量显著下降(P＜0.05或0.01)，可能与脑卒中病灶损伤NE、DA、5-HT到大脑皮质的投射纤维，继之引起皮质神经元递质合成减少有关。氟西汀和JWSNS均能显著增加PSD大鼠双侧皮层及脑干的NE、5-HT和DA的含量(P＜0.05或P＜0.01)。本实验结果表明，PSD模型组大鼠皮质及海马cAMP含量及PKA活性明显降低，而PKC活性明显升高，提示卒中后抑郁模型大鼠脑皮质及海马受体后cAMP-PKA信号通路系统下调而PKC信号通路上调，氟西汀和JWSNS具有改善和调节第二信使及蛋白激酶的作用，提示药物的抗抑郁作用可能与调节受体后cAMP-PKA及PKC信号通路有关。此外，本实验研究结果显示，PSD存在脑前皮质及海马P-CREB表达减少的现象。其机制可能是cAMP-PKA通路可直接使CREB磷酸化而激活，PSD模型大鼠存在神经元功能障碍，导致脑内单胺递质受体功能障碍及cAM P-PKA通路功能的下降，由此导致CREB功能下调。氟西汀和JWSNS对PSD的干预作用与增加CREB的表达有关，但具体的作用途径还需在今后的工作中探讨。
第三部分研究结论
一、以肝主疏泄理论为指导，运用调肝方药加味四逆散(JWSNS)防治PSD具有科学的理论依据。
二、JWSNS临床治疗PSD有确切的效果，总有效率为80.77％，优于西药百忧解。表现为可明显降低HAMD评分，降低神经功能缺损(SSS)评分，加快神经功能缺损程度的改善；显著升高日常生活活动能力(ADL)评分(Barthel指数)，提高PSD患者的日常生活活动能力。此外，在临床应用的不良反应方面，我们的研究结果表明，JWSNS没有明显的副反应，与西药相比，具有安全、长效和稳效的优势的特点，显示出临床应用的广阔前景。对本研究所获得的临床观察结果进行分析，我们认为JWSNS治疗PSD的疗效作用机理涉及到以下几方面：1．显著升高改善PSD患者血清NE、5-HT的低下状况，抑制单胺类神经递质的重摄取；2．降低血浆皮质醇水平，调节下丘脑—垂体—肾上腺轴功能。3．改善患者的大脑感知容量，增加大脑执行功能，提高对外界信息的加工和对行为的调节能力，改善PSD患者的认知功能。
三、JWSNS均能显著增加PSD大鼠的糖水消耗量，抑制PSD大鼠兴趣感下降，抑制抑郁行为的发生；对PSD大鼠探究性行为和自发活动均有显著改善作用。JWSNS均能显著增加PSD大鼠双侧皮层及脑干的NE、5-HT和DA的含量；改善和调节第二信使及蛋白激酶的作用，其抗抑郁作用可能与调节受体后cAMP-PKA及PKC信号通路有关。此外，JWSNS对PSD的干预作用与增加CREB的表达有关。
Title: Research on treatment to post-stroke depression under the direction of theory of "The liver controlling dispersion" in Chinese medicine
Specialty: Diagnostics of Chinese Medicine
Candidate: Gao Min
Tutor: Professor Chen Qun
Post-stroke depression (PSD) means having depressive emotion or in the station of depression after stroke and having been confirmed by related scales. It is one of the important complications of stroke and the incidence rate is 25%-80%, mostly manifested light-midrange. The activity disorder of PSD patients can often induce the happening of depressive disorder and the last one can affect the recovery from stroke and form infernal circle. As a kind of secondary affective disorder after stroke, the PSD can step down the functional recovery of limbs and recognition, increase the case fatality ratio and affect the patients' quality of life and prognosis seriously except can cause emotional pain. At present, worker of both Chinese and western medicines have make systematical research to the pathogenesis and clinical therapy to the PSD and have make notable advancement. Making use of the Chinese medicine to treat the PSD get more and more attention and the research get more and more profound for the side-effect, addiction and contraindication of traditional and new type anti-depressive medicines (western medicines).
The research approached the relationship between the theory of liver controlling dispersion and PSD. Directed by the theory of liver controlling dispersion and combined the forward research foundation, the research make use of the regulating "liver" therapy herbs-JWSNS to treat the PSA and approach the mechanism of action and provide scientific base to manufacture and develop new Chinese medicines in the future.
Part One Clinical Researches
1 Clinical data and Methods
1.1 Research objects
1.1.1 Source of the case All the 103 cases came from our capital from October, 2003 to October,2006.
1.1.2 Internalization criterion
(1) Patients with clear awareness, no dysgnosia and can go with examination in 2 weeks at the first post-stroking.
(2) all the cases are consistent with the diagnostic criteria of brain hemorrhage and cerebral infarction which passed by the 4th national CVD academic meeting in 1995 and the stroke and depression differentiation of symptoms and signs of internal medicine of TCM, and get final diagnosis by 1～3 times skull CT or MRI.
(3) Before the drug treatment, all patients have such characters: Hamilton depression rating scale (HAMD) score＞17, neurological impairment score (SSS)＞16, Barthel index＜70(Barthel index evaluation scale is used to evaluate activities of daily living(ADL))
(4) Taking no anti-depression preparation in 1 weeks at the first diagnosis. Taking no monoamine oxidase inhibitor in 2 weeks and no other anti-depression preparation and antipsychotic drug. Taking no licorice root, adrenal cortex hormone, adrenocorticotrop (h)ic hormone (ACTH) or analog.
(5)Normal routine of blood, urine and stool and normal function of liver and kidney, no serious medical record of heart, liver, kidney, mental disorder, epilepsy and drug allergy; Depleting innocent failure of memory of old ages, senile dementia and neurastheria before searching.
1.2 Subgroup and therapy
1.2.1 Subgroup
All the cases are divided into 2 groups at random in the 1st month after onset. 52 cases in the therapy group: male:32, female:20, age:38～68 years old, the mean is 40.21±8.57; 28 cases of apoplexy, 8 cases of lacunar infarction, 10 cases of cerebral embolism, 6 cases of subarachnoid hemorrhage.51 cases in control group: male:31, female:20, age:37～69 years old, the mean is 41.53±7.92,27 cases of apoplexy, 6 cases of lacunar infarction, 12 cases of cerebral embolism, 6 cases of subarachnoid hemorrhage. Before treatment, the HAMD score is 17～30, mean is 20.42±1.82/17～31, 20.16±2.51. The SSS score is 16～45, mean is (25.3±4.2)分/16～45分, 24.2±5.4. The Barthel index is 0～70, mean is (26.1±6.8)分/0～70分, 25.1±7.2. There have no statistical difference between the 2 groups in age, sex, etiological factor, course of disease, depressive degree, neurologic impairment degree and Barthel index (P＞0.05). 50 cases in healthy group, all of them came from the healthy peoples of health examination: male:30, female:20, age:36～67 years old, the mean is 40.44±8.72, and there have no statistical difference between the control group and therapy group in age and sex construction.
1.2.2 Therapy and nursing methods
The 2 groups were given orally hydergine 1mg/time, Tid. The hypertension and diabetic were given the treatment of decrease the blood pressure and blood sugar. In the same time, the technique of nerve-muscle unobstructed especially the Bobath therapy were used to train the limbs function, QD, 50min/time. In the base, the 2 groups were given the blow treating methods: the therapy group: JWSNS, composition and dosage: Bupleurum Root 5 g、White Peony Root 15g、Bitter Orange 6g、Wolfberry Fruit 15g、Capejasmine Fruit 5g、Rehmannia Root 18g、Concha haliotidis 30g. 1 dosage per day, decocted in water for oral dose and drink in the morning and evening, milkvetch root and codonopsis pilosula were added for serious deficiency of vital energy, polygoni muliflori and barbary wolfberry fruit for deficiency of kidney-essence, morindae officinalis and epimedii for deficiency of kidney-YANG, processing pinelliae and Coptis chinensis for sputum-fire, common anemarrhena rhizome and peony root bark for asthenic yin causing excessive pyrexia, flos albiziae, crude fossilia ossis mastoid and crude Ostreae testa for serious insomnia, peach seed and Carthamus tinctorius for stagnant blood. The control group: Fluoxetine (fluoxertine hydrochloride caps, lilly company, USA, Batch No208151), QD, 20mg/time and ate in the morning. Basic nursing: nursing staff communicated with the patients with broad-minded, observed the talk and behavior, perspect the mentation and strengthen the control of behavior and emotions. Affected the patients with broad-minded, evoked the interests of them and provide support, apprehension and sympathy in emotion, achieve the trust and conduct them to pour out their heart, encouraged out door activity, avoided fat and oily food and smoke and wine, drank rosae and chrysanthemi flos tea. The endemic area is calm and has light music. 4 weeks as one course of treatment and we make 2 courses of treatment. Observe and statistics the therapeutic effect. Detecting the routine of blood, urine and stool and function of liver and kidney pretherapy and the 4th and the 8th week of the cause of the therapy and scored HAMD.
1.3 Index surveyed and evaluation of therapeutic effect
1.3.1 Evaluation criterion
Referencing Diagnostic therapeutic effect criterion of TCM diseases, which were draw up by the Nation TCM Administration in June, 1994, we institute the evaluation criterion. Excellence: symptom disappeared, normal emotion. Utility: symptom decreased, basic stabilization of emotion. Invalid: no improvement of both symptom and emotion.
1.3.2 Evaluation criterion of the scale
HAMD, SSS and ADL were evaluated of the 2 groups of patients at the 4th week and 8th week of pre-treatment and post-treatment.
1.3.2.1 Evaluating the depressive symptom based on HAMD scale Score 24 kinds symptoms and signs with 5 grades methods and noting the score change of the 2groups of patients pre-tretment and post-treatment. Combining the Diagnostic base, healing well (HW) and improvement criterion of clinical diseases, the results are classified into HW, excellence, utility and invalid. HW: the change of HAMD score exceed 90%; Excellence: the change of HAMD score between 90%～75%; Utility: the change of HAMD score between 75%～50%; Invalid: the change of HAMD scores below 50%.
1.3.2.2 SSS score Referencing the Clinical SSS criterion of stroke patients, which was passed in the 4th CVD meeting in 1995.
1.3.2.3 ADL Barthel index scoring method. Noting the score change of pre-treatment and post-treatment and assessing the effect of CVD basing on the therapeutic effect evaluation criterion of CVD. Therapeutic effect criterion is analyzed with Radit analysis and score change with t-test.
2 trained doctors evaluate the Scale at the same time; therapeutic evaluation is carried out at the 4th and 8th week of pre-treatment and post-treatment.
1.3.3 Detection of serum NE and 5-HT Fluorescence spectroscope, Hitachi Company. Detect serum NE and 5-HT of pre-treatment post-treatment.
1.3.4 Detection of plasma cortisol collecting ulnar vein blood at 8:00AM of the subjects with calm and empty stomach station and detecting the plasma cortisol with double antibody sandwich radio-immunization.
1.3.5 Detection of event-related potential P300 Evoked potential evoked potential, ampere MK15, Italy
1.3.6 Station of side effect Evaluating the station of side effect with scale (TESS) at the 4th week and 8th week before the treating and in the treating course.
1.4 Statistics analysis All results are expressed byx±s. The data are handled by SPSS 12.0 statistical package, in which t-test are used to group comparison, paired t-test to comparison of pre-treatment and post-treatment, X~2 test to numeration data.
2 Results
2.1 Comparison of clinical therapeutic effect therapy group52 examples: excellence 36 examples (69.23%), utility 6 examples (11.54%), invalid 10 examples (19.23%), total effective rate 80.77%. Control group 51 examples: excellence 27 examples (52.94%), utility 8 examples (15.69%), invalid 16 examples (31.37%), total effective rate 68.63%. There have significant difference between the therapy group and control group in total effective rate (P＜0.05), the therapy group is outstrip control group.
2.2 Comparison of the scale evaluation
2.2.1 Score of HAMD scale The HAMD score are coincidence between the therapy group and control group. Compared with pre-treatment, The HAMD score of the therapy group significantly decreased after taking JWSNS for 4 weeks and 8 weeks and has significant difference(P＜0.01). The score of both group decreased with the treating, which indicated that the two medicines have obviously anti-depression effect. There have significant difference between the 2 groups after treatment (P＜0.01) and the therapy group is outstrip the control group.
2.2.2 SSS score The SSS score are coincidence between the therapy group and control group. Compared with pre-treatment, The SSS score of the therapy group significantly decreased after taking JWSNS for 4 weeks and 8 weeks while which of the control group didn't, which manifested that JWSNS can significantly improve the neurologic impairment.
2.2.3 ADL score (Barthel index) The ADL score are coincidence between the therapy group and control group. Compared with pre-treatment, The ADL score of the therapy group significantly increased after taking JWSNS for 4 weeks and 8 weeks while which of the control group didn't, which manifested that JWSNS can improve the activities of daily living.
2.3 change of NE and 5-HT in serum The levels of NE and 5-HT in serum in both 2 groups are significantly decreased before treatment comparing with normal value (P＜0.01). After the course of treatment, the levels of NE and 5-HT in serum in both 2 groups are significantly increased (P＜0.01). With the prolonged treating time, the levels of NE and 5-HT increased gradually, which indicates that the 2 kinds of medicines have effect of anti-depression. There have significant difference between the 2 groups after treatment (P＜0.01) and the therapy group is outstrip the control group.
2.4 Change of plasm cortisol The levels of cortisol in both 2 groups are significantly increased before treatment comparing with normal value (P＜0.01). After the course of treatment, the levels of cortisol in both 2 groups are significantly decreased (P＜0.01) and the levels increased gradually with the prolonged treating time. There have significant difference between the 2 groups after treatment(P＜0.01) and the therapy group is outstrip the control group.
2.5 Change of event-related potential P300 Compared with healthy people, the PSD patients have such characters: prolonged latent period of N2 wave and P3 wave in P300, decreased P3 wave amplitude, be different significantly from the healthy people (P＜0.01), while the other waves have no such condition (P＞0.05). After the course of treatment, the latent period of N2 wave and P3 wave in P300 shorten and P3 wave amplitude heighten of the 2 groups, there exist difference between pre-tratment and post-treatment(P＜0.05), but there have no difference between the 2 groups after treatment(P＞0.05).
2.6 Side effects The side effects emerged mostly in the first 1～2 weeks of the course of treatment. There exist significant difference in the times of TESS item between the 2 groups post-treatment(P＜0.01). There have no change in Normal routine of blood, urine and stool, function of liver and kidney and electrocardiogram and creatase.
3 Discussions
JWSNS have exact effect to PSD and the total effective rate is 80.77％, which is outstripped of Fluoxetine. The effect manifests that decrease the HAMD score and SSS score, improve the neurologic impairment, increase the ADL score and improve the activities of daily living. We found that in the side effect, JWSNS have no distinct side effect and have characters of safety, prolonged and steady actions, which manifest wide prospect of clinical application. According to the result, we think the curative effect mechanism of action of JWSNS to PSD is involve in the blow: 1. Significant increase the level of serum NE and 5-HT, refrain the reuptake of monoamine neurotransmitter; 2. Decrease the plasma cortisol and regulate the function of HPA; 3. Improve the brain capacity and function of sensation and perception and execute function, increase the progress of brain to outside message and regulating function of brain to behavior.
Part Two Experimental Researches
1 Materials and Methods
1.1 Experimental animals
Wistar rats, grade SPF, male, 2 months old, 220-270g in weight. Offered by animals' centre in Southern medical university, certification no: 2002-009. the rats can drink water and take food in freedom and acclimatize for 1 week, where the light-dark cycle is 12h, temperature is 23±2℃and calm.
1.2 Experimental medicines and preparation
Composition and dosage of JWSNS: Bupleurum Root 5g、White Peony Root 15 g、Bitter Orange 6 g、Wolfberry Fruit 15 g、Capejasmine Fruit 5 g、Rehmannia Root 18 g、Concha haliotidis 30 g. The herbs are bought from the first affiliated hospitalme of Guangzhou university of TCM and appraised as pure crude herbs by the drug department. Take the traditional Chinese medicine into coarse powder and soaked in eight multiple warm water for 0.5h, simmering decoction for 4h after boiled. Attention: even mixed and filtered with three-tier gauze; the 2nd and 3rd decotation with 6 multiple water for 2h. Merged the thrice physic liquor and concentrated to 1.69g/ml and preserved it in refrigerator at 4 centigrade after being cooled. Western medicine: fluoxertine hydrochloride caps(lilly company, USA, Batch No208151), dissolving 20mg fluoxertine hydrochloride caps in to 30ml distilled water and misce bene, intragastric administrating rats at a dose of 0.75mg/kg (6.25 multiple adult dosage).
1.3 The main equipment and agent(omitting)
1.4 The subgroup
The rats can drink water and take food in freedom and acclimatize for 1 week, where the light-dark cycle is 12h, temperature is 23±2℃and calm. Open-field test were used to sieve rats. The total score between 30～120 of level movement and vertical movement are qualified. 10 of the 80 rats are chosen as control group by SPSS 12.0 statistical package according to weight, the others are chosen to prepare Middle cerebral artery occlusion(MCAO) model and artificial operation. The living rats(remove 10 rats of artificial operation) are divided into 3 groups in random: model group, fluoxetine group and JWSNS group.
1.5 Preparative method of model
1.5.1 Preparation of rat MCAO model with electric coagulation
1.5.2 Complex preparation of rat PSD model Shift the MCAO rats in mouse cages after awake and breed alone for 28days. In the 8th days after stroke(neurological impairment have recover on the whole), 9 kinds of mild and unpredictable stress stimulus will be given for 21 days.
1.6 Index and methods
1.6.1 Common condition
Observe the mind, food taking and drinking water conditions, the stool texture, whether have infected and died rats, note the time of death and find out the death cause by dead body dissection, measure the weight every week.
1.6.2 SSS score of focal cerebral ischemia rats Longa5 is used to score the SSS after the rats awake in 24h.
1.6.3 Observation of rats ethology
1.6.3.1 The Sucrose Consumptions test The test are preceding in the 28th days after stroke. First, the rats are trained to drink 1％cane sugar solution, which means in the first 48h 1％cane sugar solution are used to take the place of the tap water, then giving 1％cane sugar solution to rats and calculating the drinking weight of 24h.
1.6.3.2 Open-field test
1.6.4 Detection of the monoamine transmitter(NE、5-HT、DA) of rat' brain Fluorospectrophotometry. Calculation the concrete contents of NE, 5-HT and DA: (values of the sample tube A—values of the vacant tube A)/(values of the standard A—values of the vacant tube A)×0.2×1.5×(1/weight of the brain tissue g)=( )U/g。
1.6.5 Detection of the contents of cAMP, the activity of PKA and PKC of rat'hippocampus Detection of the contents of cAMP: radio-immunity method. Detection of the activity of PKA and PKC: radio active isotope method.
1.6.6 Detection of the expression of P-CREB in rat'hippoeampus Analyzing and statistics optical density and areas density of expression of P-CREB with immunohistochemical method and patho-image analytical system.
1.7 The administrating dosage and methods
The time of the administration is 22d, ltime/d of intragastric administration from the 8th day after stroke and measure weight 1 time/week. The control group, model group and artificial operation group are given the same volume of distilled water. The dosage of fluoxertine hydrochloride group is 0.75mg/kg and JWSNS group 3.38 g/d to 1 rat.
1.8 Statistical treatment All results are expressed by (?)±s. The data are handled by SPSS 12.0 statistical package. ANOVE are used to group comparison, in which LSD are to regular variance and Dunnett'sT3 for irregular variance. Non-parameter test are used to data with no normal distribution. Paired t-test is used to group comparison of mean.
2 Results
2.1 Model and death conditions of rats 12 rats died in 4 weeks after the operation: 5 died in the course of the operation, 3 died for hemorrhoea, 2for anesthetic accident and 2 died in the course of the complex model. 2 died in 7 days of stress process: 1 died for electro-stress and 1 have no exact reason. After the stress process finished, 5 died before executing: 3 for mal-intragastric administration and 2 for pulmonary infection. 1 died of the control group and none of artificial operation group. 5 died in the 64 rats which are used to make model of MCAO, 2 scored 0 in SSS, the achievement ratio of making model is 82.3％. 51 rats succeeded in making model the achievement ratio is 79.7％.
2.2 SSS score of operation rats All the MCAO rats show the left neurological impairment of different degree and the score is 2.61±0.59 except 2 rats after awake. There have no neurological impairment in artificial operation group.
2.3 Change of rats weight Compared with the control group, the other groups manifested weight increased, but the difference were not distinct; the weight of the model group, fluoxetine group and JWSNS group lower significantly than the control group between the 14th day to 28th day after stroke(P＜0.05 or 0.01), but there had no difference between the artificial operation group and the control group(P＜0.05). In addition, the rats weight in model group increased slowly while those treatment groups had the tendency of increase weight. The results manifested that complex model preparation can significant step down the weight growth, while those treatment groups have the adverse effect and the difference is significantly comparing to the model group.
2.4 Results of the rats ethnology
2.4.1 Results of the sucrose Consumptions test The sucrose consumption significantly decreased in model group, artificial operation group and the treatment groups compared with that in the control group (P＜0.05 or 0.01). The sucrose consumption significant increased in the treatment group compared with that in the model group (P＜0.05 or 0.01). Complex model preparation can significantly decrease the sucrose consumption, while the treatment groups can improve the condition which indicates that the medicines can depress the PSD rat's interest and the occurrence of depressive action.
2.4.2 Results of the open-field test Compared with the control group, the model group and the treatment groups manifested that the movement of level decreased significantly in the 7th day after the stroke (P＜0.05) but the vertical movement didn't change. The level and vertical movement decreased significantly in the 14th, the 21st and 28th day after stroke of the model group, artificial operation group and the treatment groups (P＜0.05 or 0.01). Compared with the model group, there's no difference of the vertical movement in the 7th day after stroke but have significant difference of the level and vertical movement in the 14th day of artificial operation group and level movement of JWSNS group(P＜0.05). In the 21st day and 28th the both movement significantly increased of the artificial operation group and the treatment groups(P＜0.05 or 0.01). In addition, the open-field test didn't change of the control group but the movement decreased in the operation rats 1 week later and the rats in the treatment groups manifested the tendency of descend first and ascend last. Conclusion: Complex model preparation can make the open-field behavior disorder(decrease the level and vertical movement), which indicate that probe behavior and spontaneous movement decreased, while the treatment groups can significantly improve the disorder.
2.5 Result of the monoamine transmitter of rats' brain
2.5.1 Result of the NE of rats' brain Compared with the control group, the model group and the treatment groups manifested that the NE decreased significantly in the left and right cortex of forehead and brain stem(P＜0.05 or 0.01). Compared with the model group, the artificial operation group and the treatment groups manifested that the NE increased significantly in the left and right cortex of forehead and brain stem (P＜0.05 or 0.01). Results indicate that complex model preparation can make the NE decreased while the medicines can improve the change.
2.5.2 Result of the 5-HT of rats' brain Compared with the control group, the model group and the treatment groups manifested that the 5-HT decreased significantly in the left and right cortex of forehead and brain stem (P＜0.05 or 0.01) and the artificial operation group decreased also but have no statistical meanings. Compared with the model group, the artificial operation group and the treatment groups manifested that the 5-HT increased significantly in the left and right cortex of forehead and brain stem(P＜0.05 or 0.01). Results indicate that complex model preparation can make the 5-HT decreased while the medicines can improve the change.
2.5.3 Result of the DA of rats' brain Compared with the control group, the model group and the treatment groups manifested that the DA decreased significantly in the left and right cortex of forehead(P＜0.05 or 0.01). Compared with the model group, the artificial operation group and the fluoxetine group manifested that the DA increased significantly in the right cortex of forehead(P＜0.05) and the fluoxetine group manifested that the DA increased significantly in the brain item(P＜0.05); the JWSNS group manifested that the DA increased significantly in the right cortex of forehead and the brain item(P＜0.05). Results indicate that complex model preparation can make the DA decreased while the medicines can improve the change.
2.6 Result of the cAMP contents and activity of PKA and PKC in rats' hippocampus Compared with the control group, the model group and the artificial operation group manifested that the cAMP contents decreased significantly (P＜0.01) and the fluoxetine group and the JWSNS group manifested that the cAMP contents increased significantly in the cortex and hippocampus (P＜0.01) and there's no difference between the 2 treatment groups. Compared with the control group, the model group and the artificial operation group manifested that the activity of PKA decreased significantly in the cortex and hippocampus (P＜0.05) and the fluoxetine group and the JWSNS group manifested that the activity of PKA increased significantly in the cortex and hippocampus (P＜0.05) and there's no difference between the 2 treatment groups. Compared with the control group, the model group and the artificial operation group manifested that the activity of PKC increased significantly (P＜0.01)and the fluoxetine group and the JWSNS group manifested that the activity of PKC decreased significantly in the cortex and hippocampus (P＜0.01) and there's no difference between the 2 treatment groups.
2.7 Result of the P-CREB expression in rats' hippocampus The P-CREB expression is broad in the normal rats hippocampus and in the CA1 realm, the expression mostly lie in nuclear of the pyramidal layer the color is deep brown and some expression lie in the kytoplasm and ecphyma but the color is faint. In the model group and artificial operation group, the masculine cells are fewer and show vacuolus or atrophia and faint color, the area density and light density are lower (P＜0.05 or 0.01), while the 2 treatment groups have the opposite manifestation (P＜0.05)
5 Discussions
The results indicate that the complex model preparation can significantly decrease the growth of weight, so the slowly weight growth is one of the manifestation of PSD rats. Fluoxetine and JWSNS can increase the weight and super than the model group in the 28th day after stroke. The results of the sucrose consumption test indicate that the sucrose consumption significantly decreased in PSD rats and the fluoxetine and JWSNS can improve the condition (P＜0.05 or 0.01), which indicate that the medicines can depress the PSD rats interest and the occurrence of depressive action. The results of the open-field test indicate that the complex model preparation can make the open-field behavior disorder (decrease the level and vertical movement), which indicate that probe behavior and spontaneous movement decreased, while the 2 kinds of medicines can significantly improve the disorder. Compared with the control group, the contents of NE, 5-HT and DA significantly decreased in model group in the left and right cortex of forehead and brain stem (P＜0.05 or 0.01), maybe this is related to the lessen cortical neuron transmitter synthesis induced by the stroke injury the projection fibers of NE, 5-HT and DA to cerebral cortex., while the 2 kinds of medicines can significantly improve the tendency (P＜0.05 or 0.01). The model group manifested that the cAMP contents and the activity of PKA decreased significantly (P＜0.01) while the activity of PKC increased which indicate that the cAMP-PKA signal iter system down regulated and the PKC up regulated in PSD rats, the 2 kinds of medicines have the effect of improve and regulate the second messenger and protien kinase, which indicate that the depressive effect of the medicines maybe related to the regulation of acceptor of cAMP-PKA and PKC signal iters. The PSD rats showed the P-CREB expression decreased in cortex and hippocampus, the mechanism maybe is that cAMP-PKA signal iter can directly make the CREB ptiosphorylation and activation. There exist neuronal functional impairment in PSD rats, for which the monoamine transmitter receptor and the cAMP-PKA signal iter have functional impairment, which induced the downregulation of CREB function. The intervention of the 2 kinds of medicines to PSD are related to the increased expression of CREB, but the concrete effective routes are still need to be probed in the late research.
Part Three Conclusions
1. Making use of the regulating "liver" therapy herbs-JWSNS to prevent and cure the PSA have scientific theoretical base directed by the theory of liver controlling dispersion.
2. JWSNS have exact effect to PSD and the total effective rate is 80.77%, which is outstripped of Fluoxetine. The effect manifests that decrease the HAMD score and SSS score, improve the neurological impairment, increase the ADL score and improve the activities of daily living. We found that in the side effect, JWSNS have no distinct side effect and have characters of safety, prolonged and steady actions, which manifest wide prospect of clinical application. According to the result, we think the curative effect mechanism of action of JWSNS to PSD is involve in the blow: 1. Significant increase the level of serum NE and 5-HT, refrain the reuptake of monoamine neurotransmitter; 2. Decrease the plasma cortisol and regulate the function of HPA; 3. Improve the brain capacity and function of sensation and perception and execute function, increase the progress of brain to outside message and regulating function of brain to behavior.
3. JWSNS can significantly increase the sucrose consumption of PSD rats and depress the interest and the occurrence of depressive action and improve the probe behavior and spontaneous movement. JWSNS can significantly increase the contents of NE, 5-HT and DA and improve and regulate the second messenger and protien kinase, which indicate that the depressive effect maybe related to the regulation of acceptor of cAMP-PKA and PKC signal iters. In addition, the intervention of JWSNS to PSD is related to the increased expression of CREB.
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