CN107441468A - 中药组方水提物在制备预防及治疗老年痴呆药物中的应用 - Google Patents
中药组方水提物在制备预防及治疗老年痴呆药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种中药组方水提物在制备预防及治疗老年痴呆药物中的应用。本发明采用多种中药提取物组合作为制备预防及治疗老年痴呆药物,它能改善Aβ25‑35诱导的学习记忆功能减退,具有作为预防及治疗老年痴呆药物的应用价值。特别是其疗效与阳性药多奈哌齐相当,给药后可明显减轻神经元损伤,阻遏神经元数量的减少,发挥神经保护作用。本发明材料来源广泛,易于获取,用于制备药物简单易行、治疗效果好。
Description
技术领域
本发明涉及药学领域,尤其是一种中药组方水提物在制备预防及治疗老年痴呆药物中的应用。
背景技术
阿尔茨海默病(Alzheimer’s disease,AD)是以渐进性记忆丢失、认知功能减退为特征的神经退行性疾病,是老年人发生痴呆的主要类型[1]。AD典型的病理学特征为:①Tau蛋白过度磷酸化形成神经原纤维缠结(不溶性成对螺旋丝);②β-淀粉样蛋白(β-amyloid,Aβ)沉积形成的老年斑(senile plaque,SP);③神经元变性及部分突触丢失。AD患者的临床表现为慢性记忆力丢失、认知、精神及运动障碍。全世界目前约有4600万痴呆患者,研究调查发现预计在2050年痴呆患者的人数将会增加到1亿3500万。
AD的病因复杂,目前其发病机制未被完全阐明,其机制可能涉及神经炎症、基因突变、线粒体功能障碍、氧化应激等多方面。在众多AD发病的学说中,最为大家所认可的仍然是Aβ级联学说。该学说表明,Aβ是β-分泌酶和γ-分泌酶有序水解β-淀粉样前体蛋白(β-amyloid precursor protein,APP)而得;Aβ的聚集和沉积(特别是Aβ1-42)是AD发病机制中的主要事件,可引发神经毒性和神经变性。在生理状态下,脑内的Aβ生成和清除是保持动态平衡状态,但当此平衡被打破后,会导致Aβ的异常沉积,而影响脑内细胞的生理活动,诱发系列的复杂反应,包括神经炎症反应、递质缺失、代谢紊乱等,进而引发神经元变性、死亡,最终出现学习、认知功能减退,导致AD的发生。Aβ沉积于脑实质部分,可诱导胶质细胞的活化,活化后的胶质细胞又可释放大量的炎性细胞因子,如白细胞介素-1β(inkerleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor‐α,TNF-α)、环氧合酶-2(cyclooxygenase-2,COX-2)等[6,18]。并且,这些炎症因子可增加脑内APP的含量及增强γ-分泌酶的活性,使得Aβ的来源增加,即增加了脑内Aβ的含量,以上过程形成一个恶性循环,加速AD的进程。另外,研究发现过度沉积于脑内的Aβ可诱导神经元凋亡,进一步可导致学习记忆功能减退。在细胞凋亡进程中,B淋巴细胞瘤-2(B-cell lymphoma-2,bcl-2)基因家族成员与凋亡执行蛋白酶起着至关重要的作用,前者是细胞凋亡信号转到途径中的重要调节因子,包括功能相互对立的凋亡调控相关蛋白:1.抗凋亡蛋白(以Bcl-2蛋白为主),2促凋亡蛋白[Bcl-2相关X蛋白(以Bcl-2 Assaciated X protein,Bax)为主];后者主要是含半胱氨酸的天冬氨酸蛋白酶3(cysteinyl aspartate specific proteinase,caspase3),且caspase3是介导细胞凋亡的关键蛋白酶。而且,研究证实可通过调节凋亡信号因子Bax和Bcl-2的蛋白水平,改善Aβ1-40诱导的类AD大鼠模型的学习记忆功能减退。综上所述,抑制神经炎症反应和抗神经元凋亡可作为治疗AD的策略。
目前,AD患者的临床用药有胆碱酯酶抑制剂多奈哌齐、加兰他敏等,N-甲基-D-天冬氨酸(NMDA)受体拮抗药盐酸美金刚等,但由于疗效有限,价格昂贵,毒副作用大等原因,使得临床使用有限。由于AD的病因复杂,治疗AD缺乏理想的药物,所以针对多因素影响的复杂性疾病,中医药的多靶点可能比单靶点具有优势。我国也有学者致力于中药治疗AD的研究。关于AD,中医理论认为,肾主骨生髓主脑,脑为髓海,真气之所聚,所聚之真气下降,以激发肾气,推动脏腑功能活动。肾中精气不足,则髓海失养,髓海空虚则致脑转耳鸣、健忘、失忆、振颤等诸证。就髓海空虚治宜滋补肾气,肾气得充,肾阴肾阳得补,髓海得充,诸证自除。肝藏血,肾藏精,精血互相滋生和相互转化,故养肝、滋肝同时具有补充肾精之作用。而本实验用药中药组方水提物(CZ2HF)的主要成分是淫羊藿(仙灵脾)、仙茅、巴戟天、枸杞、玄参、石菖蒲、桂枝、干姜。本方立意为治疗脑髓失养,出现失忆等。方剂中君药淫羊藿辛、甘,温,入肝肾、补命门,补阴虚而助阳,使肝肾之阴得补,肾阳得温。臣药仙茅入足少阴,兼足厥阴经血分,助命火,益阳道,明耳目;巴戟天强阴益精;枸杞子兼厥阴经血分,益肾水之阳,补肝经之阴。佐药玄参入足少阴肾,以管领诸气,上下肃清而不浊,能壮水以制君臣药温燥之性;桂枝气薄,温经通脉,引药上行;石菖蒲入手少阴、足厥阴经气分,宣五脏,通九窍,以助温阳之性上达神明、外布周身。使药干姜辛,热,能引血药入血分,气药入气分。全方共凑填精生髓,温阳补肾之功。髓海得充,诸证皆除。现代药理学研究发现,淫羊藿植物的主要活性成分之一淫羊藿苷可下调Tg2576转基因小鼠脑内Aβ1-40、Aβ1-42水平改善学习记忆功能障碍;在自发性高血压大鼠模型中,还可通过线粒体凋亡途径阻止高血压诱导的心肌细胞凋亡。淫羊藿植物的另一活性成分淫羊藿次苷II可下调TNF-α、COX-2、IL-1β等炎症因子及抗神经元凋亡作用改善Aβ25-35诱导的学习记忆减退。巴戟天的活性成分之一巴戟素可改善0.5%D-半乳糖诱导的学习记忆障碍;石菖蒲的活性成分之一β-细辛醚可下调APP/PS1转基因小鼠脑内的Aβ1-42及RAGE蛋白含量;还可通过CaMKII/CREB/Bcl-2信号通路抑制体外和APP/PS1小鼠脑内的神经元凋亡。然而,由植物淫羊藿、仙茅、巴戟天、石菖蒲等组成的中药复方CZ2HF能否通过降低神经炎症反应及抑制神经元凋亡而改善Aβ25-35诱导的大鼠学习记忆功能减退,尚不明确。因此,本研究通过双侧海马注入Aβ25-35建立大鼠学习记忆功能减退模型,观察CZ2HF对该模型大鼠学习记忆功能的作用,继而探索其对该模型神经炎症及神经元凋亡的影响,为治疗AD的后续研究提供基础药理学依据。
发明内容
本发明的目的是:提供一种中药组方水提物在制备预防及治疗老年痴呆药物中的应用,它能明显减轻由于Aβ25-35诱导的学习记忆功能减退并改善海马神经元的损伤,可作为预防及治疗老年痴呆药物。
本发明是这样实现的:中药组方水提物在制备预防及治疗老年痴呆药物中的应用,该组方的组成包括淫羊藿、仙茅、巴戟天、石菖蒲、枸杞、玄参、桂枝及干姜,淫羊藿、仙茅、巴戟天、石菖蒲、枸杞、玄参、桂枝、干姜的质量比例为9:9:9:9:9:9:5:5;将上述药物按照上述比例进行混合后,放入双蒸水中冷浸60min;然后每次加水量为药材质量的7倍进行煎煮,水沸腾后改为文火,每次煎煮1.5h,煎煮3次;将每次煎煮的药液混合,再用旋转蒸发器浓缩药液,后放入-20℃冰箱中,凝固后放入冷冻干燥机中,除去水分,使成为干粉,密封,在4℃下冷藏备用,获得中药组方水提物。
将上述的中药组方水提物与药学上可接受的辅料制备成药学上可接受的剂型。
所述的药学上可接受的剂型包括片剂,胶囊剂,口服液或颗粒剂。
为了验证本发明的实验效果,进行了以下体内实验:
1 材料及方法
1.1 主要实验材料
1.1.1 实验动物
98只健康雄性Sprague-Dawlry(SD)大鼠(体重250-300g),购自中国人民解放军第三军医大学实验动物中心,许可证号:SCXK-(军)2012-0011。饲养于SPF级动物房(5只/笼),环境温度为22±1℃、湿度60%±2%,光照时间为8:00点至20:00点,黑暗时间为20:00至早上8:00,自由饮食。适应性饲养1周。
1.1.2 主要仪器
1.1.3 主要药品和试剂
1.2 方法
1.2.1 动物分组
98只健康雄性Sprague Dawley(SD)大鼠(体重:250-300g),随机分为7组,14只/组:假手术组(sham)、假手术给药组(sham+CZ2HF 400mg/kg)、模型组(Aβ25-35)、CZ2HF低剂量组(Aβ25-35+CZ2HF100mg/kg)、CZ2HF中剂量组(Aβ25-35+CZ2HF 200mg/kg)、CZ2HF高剂量组(Aβ25-35+CZ2HF 400mg/kg)及阳性药多奈哌齐组(Aβ25-35+donepezil 1.0mg/kg)。
1.2.2 Aβ25-35溶液的配置
Aβ25-35 1mg,溶于500μl生理盐水,配置成2.0μg/μl溶液,37℃孵育4天,使其成为聚集态,毒性增强[31]。
1.2.3 CZ2HF的制备
CZ2HF中药材全部购买于遵义医学院附属医院,全部药材经遵义医学院药学院生药学教研室主任杨建文教授鉴定。煎煮法:将中药药材放入双蒸水中冷浸60min,煎煮时每次加水量为药材的7倍,沸腾后改为文火,每次煎煮1.5h,煎煮3次。每次煎煮药液混合。用旋转蒸发器,浓缩药液,后放入-20℃冰箱,凝固后放入冷冻干燥机中,除去水分,使成为干粉,密封,放置于4℃冰箱,备用。
1.2.4 模型的制备及给药
全部SD大鼠经腹腔注入2%戊巴比妥钠(3.0ml/kg)麻醉后,进行手术。首先将大鼠脑颅平位固定在大鼠脑立体定位仪上,然后找准海马进针位置,用钝头针与颅骨上打孔;海马进针坐标为:以前囟为零点,前囟后3.5mm,中线旁开2.5mm,颅骨下3.5mm。5min内用微量注射器于双侧海马各匀速注入Aβ25-35溶液5μl,注射器内溶液全部打出后,待针于脑内停留5min后缓慢退出注射器;假手术组与假手术给药组同法注入等体积生理盐水[31]。手术后第一天开始灌胃给药,CZ2HF低、中、高及阳性药组大鼠分别给予CZ2HF100mg/kg、200mg/kg、400mg/kg及多奈哌齐1.0mg/kg;模型组与假手术组大鼠给予的是等体积的双蒸水,连续给予17天。
1.2.5 Morris水迷宫
手术后给药的第11天进行为期6天的Morris水迷宫实验,由前1-5天的定向航行实验加最后一天的空间探索实验两个部分构成。前1-5天的定向航行实验是每天将各组大鼠面向池壁分别从3个象限(3个固定的入水点)放入水中,同时通过视频采集、软件分析记录系统(Topscan)自动实时录像分析,分别记录所有大鼠在120s内寻找到隐藏平台的时间,此时间即为逃避潜伏期(平台在水面下2cm深处)。假若在120s内大鼠还没找到隐藏于水面下的平台,人为将大鼠引上平台上停留20s,则120s记为此大鼠的逃避潜伏期;如若大鼠在120s内任一时间找到平台,则此时间为该大鼠的逃避潜伏期。空间探索实验中,首先撤去隐匿于水下平台,将所有大鼠面向池壁于相同入水点轻放入水,然后通过自动分析记录系统纪录分析120s内大鼠的目标象限停留时间百分比。最后根据分析各组大鼠在定向航行实验中的逃避潜伏期(学习过程)及空间探索实验中的目标象限停留时间百分比(认知过程)判断大鼠的学习记忆功能。
1.2.6 标本制备
全部SD大鼠在进行Mirros水迷宫行为学实验检测结束后,腹腔注射(100g大鼠注入0.3ml)2%戊巴比妥钠进行麻醉。每组随机取5只大鼠,大鼠板上仰卧位固定,手术剪剪开胸腔同时暴露心脏,左手拿镊子固定心脏,右手将钝头针管从心尖插入主动脉,另外用止血钳固定针尖同时剪开右心耳,慢慢匀速注入PBS液(约200ml)至心耳不再流出红色液体,继续缓慢匀速注入4%多聚甲醛溶液(约200ml),直到头颈部、四肢、尾巴僵硬后,断头剥离大鼠大脑,放入预冷的PBS溶液中洗掉血液,于滤纸上绑好大脑同时贴好标签;立即放入4%多聚甲醛溶液中浸泡7天,取出用石蜡包埋,切片后做苏木素-伊红染色(hematoxylin andeosin,HE)、尼氏染色(Nissl)、原位末端转移酶标记技术(TdT-mediated dUTP nick-endlabeling,TUNEL,TUNEL)染色;剩余大鼠麻醉后直接断头,剥离大脑脑,于冰袋上分离出左、右侧海马组织,分别放入已标记1.5mLEP管中,放-80℃冰箱保存,用于后续实验指标检测。
1.2.7 苏木素-伊红染色(HE染色)
将切好的石蜡脑片(约3mm)粘贴于载玻片上,经二甲苯和梯度乙醇脱蜡:二甲苯I、二甲苯II各1次,每次10min;拿出后按顺序无水乙醇、95%乙醇、90%乙醇、80%乙醇、70乙醇(各5min),双蒸水洗5min;苏木素溶液12min,双蒸水洗3min;1%盐酸酒精分化3s,流水冲洗3min;复染0.5%伊红溶液2.5min,双蒸水5min;梯度乙醇脱水:95%乙醇I、95%乙醇II各2min、无水乙醇2min、二甲苯I 2min,二甲苯II 2min,放置于通风橱内(约3min),待载玻片上无液体后滴加中性树胶封片。观察海马神经元损伤情况。每组每只大鼠选择大脑切片3张,观察CA1区神经元损伤情况(400倍镜下拍摄3个不同的视野)。
1.2.8 Nissl染色
脑片脱蜡按1.2.7步骤后,脑片上加入1%甲苯胺蓝溶液(100μl/片),60℃,10min,然后自来水冲洗5min;按步骤1.2.7脱水、封片;每组的每只大鼠选择大脑切片3张,观察CA1区神经元损伤情况(400倍镜下拍摄3个不同的视野),计数并统计。
1.2.9 TUNEL染色
按1.2.7步骤给脑片脱蜡后,双蒸水洗5min×2次;浸泡于避光的3%H2O2溶液内15min(灭活内源性酶),PBS洗5min×3次;将脑片放在暗湿盒中,每张脑片加40μL 20μg/mL的Proteinase K工作液,放37℃烘箱,15min,PBS洗5min×3次;暗湿盒内的每张切片滴加40μLTUNEL反应混合工作液(荧光素:TdT Enzyme=9:1),放37℃烘箱,60min,PBS洗5min×3次;每张脑片滴加40μL Converter-POD工作液,放37℃烘箱,30min,PBS洗5min×3次;每张脑片滴加DAB显色液50μL,于光学显微镜下观察脑片颜色,约40-60s/张,染色后立即放入水中终止染色,双蒸水冲洗10min;苏木素复染5-10s,双蒸水洗3min;1%盐酸乙醇分化3s,双蒸水冲洗8min;按步骤1.2.7脱水、封片;每组每只大鼠选择大脑切片3张,观察CA1区神经元损伤情况(400倍镜下拍摄3个不同的视野)。1.2.10免疫印迹实验(Western blot,WB)及相关试剂配制1)8%、10%、12%分离胶及5%浓缩胶的配制:
表1 不同浓度的分离胶成分(单位:mL)
表2 5%浓度的浓缩胶成分(单位:mL)
2)10%过硫酸铵(ammonium persulfate,AP)的配置
称取0.1g AP溶于800μL双蒸水中,待AP溶解后加双蒸水补足至1mL,涡旋混匀后分装成300μL/管,–20℃保存,备用。
3)10×电转缓冲液的配置
加入800mL双蒸水搅拌并超声,待其充分溶解后加双蒸水补足至1000mL,搅拌混匀后放置于4℃冷藏室保存,备用。电转时使用的1×电转缓冲液按比例配置(10×电转液:甲醇:双蒸水=1:2:7)4)10×电泳缓冲液的配置
取双蒸水800mL然后超声并搅拌,待其充分溶解后,加双蒸水补足至1000mL,室温保存,备用。实验时使用的1×电泳缓冲液按比例配置(双蒸水:10×电泳缓冲液=9:1)。
5)20%Tween20的配置
取20mL Tween20加入双蒸水50mL,搅拌混匀后,加双蒸水补足至100mL,4℃冷藏室保存,备用。
6)10×TBS缓冲液的配置
取800mL双蒸水搅拌同时超声,待其充分溶解后,加双蒸水补足至1000mL,玻璃棒搅拌混匀后4℃保存。使用的1×TBS缓冲液按比例配置(双蒸水:10×TBS缓冲液=9:1)
7)TBST缓冲液
1000mL的1×TBS中加入20%吐温-20 2.4mL,用玻璃棒搅拌混匀后,1000mL约加入4mL浓HCl调pH值,约7.0~7.8,现配现用。
8)5%封闭牛奶的配置
称取10g脱脂奶粉然后加入180mL TBST溶液溶解,再加入TBST补足至200mL,-20℃冰箱保存,备用。
1.2.11 主要操作步骤:
按本课题组以往Western blot实验方法进行[33]。包括提取蛋白、测定样本总蛋白浓度、蛋白变性、点样、电泳、转膜(半干转)、TBST洗涤、牛奶封闭、TBST洗涤、一抗反应、TBST洗涤、二抗反应、TBST洗涤、ECL显色等。一抗稀释度为:COX-2(1:1000)、TNF-α(1:2000)、IL-1β(1:1000)、Aβ1-42(1:2000)、IκBα(1:2000)NF-κB p65(1:1000)、p-NF-κB p65(1:1000)、Bax(1:500)、Bcl-2(1:500)、pro-caspase 3(1:1000)、active-caspase 3(1:1000),二抗稀释度为1:2000;显色后,采用Quantity One系统拍照并分析。
1.2.12 统计学分析
所有数据均采用SPSS17.0统计软件分析,用均数±标准误表示。其中,水迷宫实验结果采用重复测量数据的方差分析方法,先通过球形检验分析数据,当P>0.05,接受球形检验分析,不拒绝球形检验假设;当P<0.05,则采用Greenhouse-Geisser法进行校正分析;定向航行实验中,各时间点每组间的比较采用多元方差分析。其他数据均采用单因素方差分析,方差齐用LSD检验,P<0.05,P<0.01为差异有统计学意义。
2 结果
2.1 CZ2HF对Aβ25-35诱导的大鼠学习记忆功能减退的影响
为了探索CZ2HF是否能够减轻Aβ25-35诱导的大鼠空间学习记忆减退,本实验利用Morris水迷宫法检测大鼠的空间学习记忆能力。手术后第11天,进行Morris水迷宫实验(图1B),在前1-5天的定向航行试验中,各组大鼠找到隐藏平台的时间逐渐缩短(逃避潜伏期),分析数据采用重复数据测量,球形检验P=0.042,小于0.05,不满足球形检验假说,所以采用Greenhouse-Geisser法进行较正分析,分析结果为时间因素有统计学意义[F(3.618,307.501)=130.340,P<0.01],结果说明时间因素对大鼠逃避潜伏期缩短起作用;分组主效应有统计学差异[F(6,85)=6.768,P<0.01],说明分组因素对大鼠逃避潜伏期缩短起作用;时间因素和分组因素的交互作用无统计学意义[F(21.706,307.501)=0.485,P=0.976],说明分组因素及时间因素无交互影响。第3天、第4天、第5天开始模型组大鼠逃避潜伏期较其余组大鼠逃避潜伏期明显增加(P<0.01,P<0.05,P<0.01),模型给药组(给予CZ2HF 100、200、400mg/kg)及阳性药(多奈哌齐1.0mg/kg)后大鼠逃避潜伏期明显缩短(P<0.05)(图1B)。第6天的空间探索实验中[F(6,85)=2.217,P<0.05],模型组大鼠较假手术组大鼠目标象限停留时间百分比明显降低,模型给药组大鼠目标象限停留时间百分比较模型组大鼠明显增加(P<0.05)(图1C)。以上结果说明大鼠双侧海马注入Aβ25-35可诱导大鼠学习记忆减退,而给与CZ2HF 100、200、400mg/kg及多奈哌齐1.0mg/kg可缩短逃避潜伏期及增加目标象限停留时间百分比。提示,CZ2HF和多奈哌齐可以改善Aβ25-35诱导的大鼠学习记忆功能减退。
2.2 CZ2HF对Aβ25-35诱导的大鼠海马神经元损伤的影响
通过HE染色法观察CZ2HF对大鼠的海马神经元形态学的影响。结果如图2所示:假手术组及假手术给药组海马组织CA1区神经元排列整齐、细胞核和细胞质染色均一、边缘清晰(图2A、图2B);模型组大鼠海马组织神经元排列紊乱、大量神经元出现核固缩(图2C)。然而模型给药组CZ2HF 100、200、400mg/kg及阳性药多奈哌齐1.0mg/kg大鼠海马较模型组海马神经元细胞结构完整且排列较为整齐。以上结果说明双侧海马注入Aβ25-35片段诱导的大鼠学习记忆减退模型中,大鼠海马组织出现损伤情况,而给予CZ2HF和多奈哌齐后可明显减轻此情况。
2.3 CZ2HF对Aβ25-35诱导的大鼠海马神经元数量的影响
通过Nissl染色法观察CZ2HF对大鼠双侧海马注入Aβ25-35片段诱导的海马存活神经元数量。结果如图3所示:假手术组及假手术给药组海马组织CA1区神经元排列整齐、细胞核和细胞质染色均一,细胞结构完整清晰(图3A的a、b图);模型组大鼠海马组织大量细胞的细胞结构不完整、活细胞密度低、神经元排列紊乱,存活神经元数量减少(图3A)。然而,与模型组相比CZ2HF100、200、400mg/kg组及阳性药多奈哌齐1.0mg/kg组大鼠海马神经元结构较为完整且排列较为整齐、结构完整清晰的细胞数量增加(图3A的d、e、f、g)。
2.4 CZ2HF对Aβ25-35诱导的大鼠海马神经元凋亡的影响
通过TUNEL染色法观察CZ2HF对大鼠双侧海马注入Aβ25-35片段诱导的海马神经元凋亡的影响。结果如图4所示:假手术组及假手术给药组海马组织未见凋亡阳性染色细胞(棕褐色)(图4A的a、b图),模型组大鼠海马组织有大量的阳性细胞染色(图4A的c图)。然而,与模型组相比,CZ2HF100、200、400mg/kg组及阳性药多奈哌齐1.0mg/kg组大鼠海马神经元凋亡细胞数明显减少(图4的d、e、f、g图)。
2.5 CZ2HF对Aβ25-35诱导的大鼠海马Aβ1-42蛋白含量的影响
Western blot法检测各组大鼠大脑海马组织Aβ1-42蛋白含量。经单因素方差分析,Aβ1-42[F(6,14)=6.283,P<0.01]蛋白含量有统计学差异(图5),模型组海马Aβ1-42蛋白水平较假手术组明显升高;与模型组相比,CZ2HF 200mg/kg、400mg/kg与多奈哌齐1.0mg/kg可显著降低Aβ1-42蛋白水平。提示,CZ2HF和多奈哌齐可阻遏Aβ25-35诱导的Aβ1-42蛋白水平上调。
2.6 CZ2HF对大鼠海马COX-2、TNF-α、IL-1β蛋白水平的影响
Western blot法检测各组大鼠大脑海马组织COX-2、TNF-α、IL-1β蛋白水平。经单因素方差分析,COX-2[F(6,14)=2.96,P<0.05]、TNF-α[F(6,14)=6.082,P<0.01]、IL-1β[F(6,14)=7.802,P<0.01]蛋白水平均有差异(图6),模型组海马组织中COX-2、TNF-α、IL-1β蛋白水平较假手术组明显升高;与模型组相比,CZ2HF 200mg/kg、400mg/kg与多奈哌齐1.0mg/kg组显著降低COX-2、TNF-α、IL-1β蛋白水平。
2.7 CZ2HF对Aβ25-35诱导的海马组织Bax、Bcl-2蛋白水平的影响
Western blot法检测各组大鼠大脑海马组织Bax、Bcl-2蛋白水平。经单因素方差分析,Bax[F(6,14)=2.955,P<0.05]、Bcl-2[F(6,14)=3.802,P<0.05]蛋白水平均有差异(图7);模型组海马组织中Bax蛋白水平较假手术组明显上调(图7B)、而Bcl-2蛋白水平较假手术组明显下调(图7C);然而,与模型组相比,CZ2HF 200mg/kg、400mg/kg组与多奈哌齐1.0mg/kg组显著下调Bax蛋白水平和上调Bcl-2蛋白水平(图7)。由此可见CZ2HF和多奈哌齐可阻遏Aβ25-35诱导的Bax/Bcl-2比值上升(图7D)。
2.8 CZ2HF对Aβ25-35诱导的大鼠海马组织中caspase 3活化的影响
Western blot法检测各组大鼠大脑海马组织pro-caspase3、active-caspase 3蛋白水平。经单因素方差分析,pro-caspase 3[F(6,14)=9.026,P<0.01]、active-caspase 3[F(6,14)=7.867,P<0.01]蛋白水平均有差异(图8),模型组海马组织active-caspase3蛋白水平较假手术组明显升高,pro-caspase 3蛋白水平较假手术组明显降低;然而与模型组相比,CZ2HF低中高剂量组与阳性药组可显著调节pro-caspase 3、active-caspase 3蛋白水平。其中CZ2HF200mg/kg、400mg/kg和多奈哌齐1.0mg/kg可下调active-caspase3的蛋白水平、上调pro-caspase 3蛋白水平。提示,CZ2HF及多奈哌齐可阻遏蛋白酶caspase 3的活化。
2.9 CZ2HF对Aβ25-35诱导的海马组织IκB-α蛋白的降解和NF-κB磷酸化的影响
Western blot法检测各组大鼠大脑海马组织IκB-α、NF-κB p65、p-NF-κB p65蛋白水平。经单因素方差分析,各组大鼠海马组织中IκB-α蛋白水平[F(6,14)=13.850,P<0.05]和NF-κB p65的磷酸化水平[F(6,14)=3.829,P<0.05]存在差异(图9),模型组海马组织中IκB-α蛋白水平较假手术组显著降低,NF-κB p65蛋白的磷酸化水平较假手术组明显升高;与模型组相比,CZ2HF 100mg/kg、200mg/kg、400mg/kg与多奈哌齐1.0mg/kg组显著降低NF-κB p65蛋白的磷酸化水平。由此可得,CZ2HF和多奈哌齐可以阻遏Aβ25-35诱导的NF-κB p65的磷酸化。
3 讨论
Aβ沉积于脑内对AD的病理进程有促进作用。并且,前期研究证实双侧海马注入Aβ25-35可建立AD学习记忆减退模型,此模型已被广泛应用于AD的研究。因此,本研究通过双侧海马注入Aβ25-35建立大鼠学习记忆功能减退模型,并观察CZ2HF对该大鼠模型的影响。Morris水迷宫被广泛应用于学习记忆和新药筛选、评价等领域,是研究AD动物模型行为学的经典实验。本研究Morris水迷宫实验结果发现,大鼠双侧海马注入Aβ25-35后,其逃避潜伏期较假手术组显著延长,目标象限停留时间百分比明显降低,说明AD大鼠学习记忆功能减退模型制备成功。盐酸多奈哌齐是临床上治疗轻、中度AD的经典药物,能明显改善动物模型及AD患者的学习记忆减退,所以本实验采用盐酸多奈哌齐作为阳性药。实验中多奈哌齐1.0mg/kg明显改善了Aβ25-35诱导的大鼠学习记忆功能减退。与以往研究结果一致。与多奈哌齐相似,CZ2HF可明显改善Aβ25-35诱导的大鼠学习记忆功能减退,提示CZ2HF在治疗AD方面有潜在的临床应用价值。
学习记忆储存于海马体中,海马神经元损伤或缺失可导致学习记忆减退,其已成为AD的典型病理变化之一。本研究发现双侧海马注入Aβ25-35后大鼠出现学习记忆功能减退现象,推测可能与海马神经元损伤或缺失有关。因此,本实验先通过HE染色观察海马神经元的形态;再进行尼氏染色观察存活神经元的数量。HE染色法的原理是先用碱性染料(苏木素)将细胞核染成蓝色,而酸性染料(伊红)将细胞浆染成红色,从而可清晰地观察神经元的形态,生理状态的细胞着色较前,而在衰老或受刺激的细胞胞浆会深染。尼氏小体(神经细胞体的特征结构之一)是神经细胞合成蛋白质的主要场所,也是神经细胞功能活性的形态指标,可通过尼氏染色(甲苯胺蓝法)法进行检测。尼氏染色法是利用甲苯胺蓝溶液能把嗜碱性的尼氏体染成蓝色,可在显微镜下观察尼氏体形态。正常情况下,海马组织在HE染色和尼氏染色下,海马神经元排列整齐、细胞结构清晰完整、细胞呈圆型;而当神经元损伤或尼氏小体受刺激后,神经元排列紊乱、细胞结构不清晰不完整即为细胞变性、细胞出现皱缩深染;神经元排列紊乱、活神经元数量减少甚至消失。在本实验中,Aβ25-35诱导的大鼠模型出现海马神经元排列紊乱、细胞变性且深染,即神经元已明显受损,且存活神经元数量减少,与以往研究一致。CZ2HF和阳性药多奈哌齐连续给药可明显减轻神经元损伤,阻遏神经元数量的减少,发挥神经保护作用。
众所周知,Aβ沉积于脑内可加速AD的进程。而Aβ来源于APP的有序水解,在该过程中形成了39-43个氨基酸的Aβ片段包括Aβ1-40、Aβ1-42,其中Aβ1-42的神经毒性最强。而且,在Aβ25-35诱导的大鼠学习记忆减退模型中发现下调Aβ1-42及BDNF蛋白水平而减轻大鼠学习记忆减退。本研究发现,Aβ25-35诱导的学习记忆减退模型大鼠脑内Aβ1-42水平显著上调,与上述研究一致。然而,有文献报道,多奈哌齐可下调脑内Aβ1-42蛋白水平,本实验结果亦证实了这一发现。值得关注的是,CZ2HF连续给药明显下调Aβ1-42蛋白水平,且在400mg/kg时与多奈哌齐类似。
神经炎症是指在中枢神经系统中炎症反应继发到神经元损伤,且在AD的发生发展中有促进作用。研究表明,在中枢神经系统中,Aβ1-42的上调可激活胶质细胞释放炎症因子COX-2、IL-1β、TNF-α等,其表达上调时,可加重学习记忆障碍,加快AD进程。而炎症因子TNF-α、IL-1β可增加β-分泌酶活性和APP含量,从而增加了Aβ1-42水平,形成一个恶性循环,加重认知功能障碍。在Aβ25-35诱导的大鼠学习记忆减退的模型中,我们发现Aβ1-42水平升高的同时,TNF-α、IL-1β、COX-2蛋白表达明显增加;而CZ2HF及多奈哌齐可明显下调TNF-α、IL-1β和COX-2蛋白水平。由此可见,CZ2HF缓解Aβ25-35诱导的学习记忆减退、海马神经元的损伤与下调Aβ1-42蛋白水平、降低炎症相关蛋白TNF-α、IL-1β、COX-2的过度表达有关。
神经元凋亡是神经元缺失的主要方式之一,在AD的发生和发展过程中也扮演了关键角色。本实验为进一步探索Aβ25-35诱导的大鼠学习记忆功能减退模型中神经元缺失的原因,通过TUNEL染色观察海马神经元的凋亡情况。TUENL染色法是在末端脱氧核苷酸转移酶的催化下基因组DNA断裂暴露的3’-OH末端可以被dUTP标记,呈现棕褐色(正常或者正在增殖的细胞几乎没有断裂的DNA,即不能被染成棕褐色),可通过显微镜观察到棕褐色的凋亡神经元。本实验结果发现,双侧海马注入Aβ25-35后可导致海马神经元出现大量的棕褐色阳性细胞,与既往研究一致。而CZ2HF及多奈哌齐可显著减少神经元的凋亡。在细胞凋亡进程中,Bcl-2蛋白家族中的促凋亡蛋白Bax和抗凋亡蛋白Bcl-2在神经元凋亡进程中起着至关重要的作用。神经元内Bax蛋白的上调可引发线粒体凋亡途径,即Bax与线粒体结合,使细胞色素c释放到细胞质,活化caspase级联反应,引起细胞凋亡。然而,Caspase酶原并没有生物学活性,需被活化后才可启动细胞程序性死亡,其中caspase3是细胞凋亡过程中关键的终末剪切酶,是细胞凋亡的执行者。有研究表明,Aβ脑内的沉积可诱发神经元凋亡,且与caspase3的活化及增加Bax/Bcl-2比值有关。在Aβ25-35诱导的大鼠模型中,我们也发现了Bax蛋白表达上调和Bcl-2表达降低,同时pro-caspase3蛋白表达降低而active-caspase3的活化增加,进一步证实了Aβ过度表达可诱发细胞凋亡。重要的是,CZ2HF明显降低Bax/Bcl-2比值,减少caspase3的活化。综上所述,CZ2HF可通过降低Bax/Bcl-2比值,阻遏caspase3活化发挥抗神经元凋亡的作用,从而增加神经元存活数量,缓解学习记忆损伤。
NF-κB是重要的核转录因子,NF-κB的活化参与细胞免疫炎症反应的调节和凋亡因子的表达。生理状态下,NF-κB位于胞浆内,在细胞质内IκB和NF-κB结合(使得NF-κB不能进入细胞核内调控相关的靶基因);当细胞受到刺激后,IκB磷酸化并降解,NF-κB激活,并迅速转移至细胞核内中与相应的靶基因结合,调控相关蛋白的表达。文献报道,激活NF-κB途径可诱发炎症反应释放炎症因子IL-1β、TNF-α等,并可诱导细胞凋亡。在Aβ25-35诱导的PC12细胞中,可通过上调Bcl-2蛋白水平,下调Bax蛋白水平,同时降低p-NF-κB p65的水平,而抑制PC12细胞的凋亡,发挥细胞保护作用。本实验结果发现,大鼠双侧海马注射Aβ25-35能激活NF-κB引起大量神经元损伤及缺失,而CZ2HF及多奈哌齐能抑制大鼠海马NF-κB的激活,从而发挥神经元保护作用。
4 结论
在本实验条件下,CZ2HF可明显减轻Aβ25-35诱导的大鼠学习记忆功能减退并改善海马神经元的损伤,其机制与其抗炎、抗凋亡有关。
本发明采用多种中药提取物组合作为制备预防及治疗老年痴呆药物,它能改善Aβ25-35诱导的学习记忆功能减退,具有作为预防及治疗老年痴呆药物的应用价值。特别是疗效和阳性药多奈哌齐相当,可明显减轻神经元损伤,阻遏神经元数量的减少,发挥神经保护作用。本发明材料来源广泛,易于获取,用于制备药物简单易行、治疗效果好。
附图说明
图1为CZ2HF对Aβ25-35诱导的大鼠学习记忆减退的影响;
图1中:A.实验设计;B.逃避潜伏期;C.目标象限停留时间百分比;D.游泳速度C+L:CZ2HF 100mg/kg;C+M:CZ2HF 200mg/kg;C+H:CZ2HF 400mg/kg;Don:Donepezil 1.0mg/kg。*P<0.05和**P<0.01vs Aβ25-35;#P<0.05和##P<0.01vs Aβ25-35;
图2为CZ2HF对Aβ25-35诱导的大鼠海马神经元损伤的影响;
图2中:A:假手术组;B:假手术+CZ2HF 400mg/kg;C:Aβ25-35;D:CZ2HF 100mg/kg;E:CZ2HF 200mg/kg;F:CZ2HF 400mg/kg;G:多奈哌齐1.0mg/kg。
图3 CZ2HF对Aβ25-35诱导的大鼠海马存活神经元的影响;
图3中:A:尼氏染色代表图片(×400,标尺=50μm);B:存活神经元统计图a:假手术组;b:假手术+CZ2HF 400mg/kg;c:Aβ25-35;d:CZ2HF 100mg/kg;e:CZ2HF 200mg/kg;f:CZ2HF 400mg/kg;g:多奈哌齐1.0mg/kg。C+L:CZ2HF 100mg/kg;C+M:CZ2HF 200mg/kg;C+H:CZ2HF 400mg/kg;Don:Donepezil 1.0mg/kg。**P<0.01vs sham;##P<0.01vs Aβ25-35。
图4CZ2HF对Aβ25-35诱导的大鼠海马神经元凋亡的影响;
图4中:A:TUNEL染色代表图片(×400,标尺=50μm);B:各组细胞凋亡数a:假手术组;b:假手术给CZ2HF400mg/kg;c:Aβ25-35;d:CZ2HF 100mg/kg;e:CZ2HF 200mg/kg;f:CZ2HF 400mg/kg;g:多奈哌齐1.0mg/kg。**P<0.01vs sham;##P<0.01vs Aβ25-35;
图5CZ2HF对Aβ25-35诱导的大鼠海马Aβ1-42蛋白含量的影响;
图5中:A:蛋白表达的代表性条带;B:Aβ1-42蛋白表达量。C+L:CZ2HF 100mg/kg;C+M:CZ2HF 200mg/kg;C+H:CZ2HF 400mg/kg;Don:Donepezil 1.0mg/kg;**P<0.01vs sham;#P<0.05vs Aβ25-35,
图6 CZ2HF对大鼠海马COX-2、TNF-α、IL-1β蛋白水平的影响;
图6中:注:A:蛋白表达的代表性条带;B:COX-2蛋白相对表达量;C:IL-1β蛋白相对表达;D:TNF-α蛋白相对表达量。C+L:CZ2HF100mg/kg;C+M:CZ2HF 200mg/kg;C+H:CZ2HF400mg/kg;Don:Donepezil 1.0mg/kg**<0.01vs sham;#P<0.05,##P<0.05vs Aβ25-35;
图7CZ2HF对Aβ25-35诱导的大鼠海马Bax、Bcl-2蛋白水平的影响;
图7中:注:A:蛋白表达的代表性条带;B:Bax蛋白水平统计图;C:Bcl-2蛋白水平统计图;D:Bax/Bcl-2蛋白比值统计图。C+L:CZ2HF100mg/kg;C+M:CZ2HF 200mg/kg;C+H:CZ2HF400mg/kg;Don:Donepezil 1.0mg/kg*P<0.05、**P<0.01vs sham;#P<0.05、##P<0.01vs Aβ25-35;
图8 CZ2HF对Aβ25-35诱导的大鼠海马pro-caspase 3、active-caspase 3水平的影响;
图8中:A:蛋白表达的代表性条带;B:pro-caspase3蛋白水平统计图;C:active-caspase3蛋白水平统计图。C+L:CZ2HF 100mg/kg;C+M:CZ2HF 200mg/kg;C+H:CZ2HF 400mg/kg;Don:Donepezil 1.0mg/kg**P<0.01vs sham;#P<0.05,##P<0.01vsAβ25-35;
图9CZ2HF对Aβ25-35诱导的大鼠海马IκB-α、NF-κB p65、p-NF-κB p65蛋白水平的影响;
图9中:A:蛋白表达的代表性条带;B:IκB-α蛋白水平;C:p-NF-κB p65蛋白水平。C+L:CZ2HF 100mg/kg;C+M:CZ2HF 200mg/kg;C+H:CZ2HF 400mg/kg;Don:Donepezil 1.0mg/kg**P<0.05vs sham;#P<0.05,##P<0.01vs Aβ25-35 group。
具体实施方式
本发明的实施例1:该组方的组成包括淫羊藿、仙茅、巴戟天、石菖蒲、枸杞、玄参、桂枝及干姜,淫羊藿、仙茅、巴戟天、石菖蒲、枸杞、玄参、桂枝、干姜的质量比例为9:9:9:9:9:9:5:5;将上述药物按照上述比例进行混合后,放入双蒸水中冷浸60min;然后每次加水量为药材质量的7倍进行煎煮,水沸腾后改为文火,每次煎煮1.5h,煎煮3次;将每次煎煮的药液混合,再用旋转蒸发器浓缩药液,后放入-20℃冰箱中,凝固后放入冷冻干燥机中,除去水分,使成为干粉,密封,在4℃下冷藏备用,获得中药组方水提物。
取中药组方提取物5g、药用淀粉75g、微晶纤维素20g,将药用淀粉先干燥,过120目筛,再与淫羊藿苷、微晶纤维素混合,过两次120目筛,填入硬胶囊中,制成1000粒本发明胶囊。每粒硬胶囊含主药成分0.5mg。
本发明的实施例2:取中药组方水提物5g、羟丙甲纤维素6g、羧甲淀粉钠10g,微晶纤维素8g,乳糖115g、淀粉50g,硬脂酸镁1g;将主药与辅料充分混匀后投入高速搅拌机中,喷雾加水适量,整粒,水分控制在3~4%,然后压片,制成1000片,包薄膜衣。
本发明的实施例3:取中药组方水提物5g,加入400ml聚乙二醇200溶解,再加入适量蒸馏水进行稀释,然后再加入适量蔗糖并调整体积至1000ml,搅匀,过滤,灌装成10ml或20ml每只,灭菌包装。
Claims (4)
1.一种中药组方水提物在制备预防及治疗老年痴呆药物中的应用,其特征在于:该组方的组成包括淫羊藿、仙茅、巴戟天、石菖蒲、枸杞、玄参、桂枝及干姜,淫羊藿、仙茅、巴戟天、石菖蒲、枸杞、玄参、桂枝、干姜的质量比例为9:9:9:9:9:9:5:5;将上述药物按照上述比例进行混合后,放入双蒸水中冷浸60min;然后每次加水量为药材质量的7倍进行煎煮,水沸腾后改为文火,每次煎煮1.5h,煎煮3次;将每次煎煮的药液混合,再用旋转蒸发器浓缩药液,后放入-20℃冰箱中,凝固后放入冷冻干燥机中,除去水分,使成为干粉,密封,在4℃下冷藏备用,获得中药组方水提物。
2.根据权利要求1所述的中药组方水提物在制备预防及治疗老年痴呆药物中的应用,其特征在于:中药组方水提物作为唯一有效成分。
3.根据权利要求1所述的中药组方水提物在制备预防及治疗老年痴呆药物中的应用,其特征在于:将中药组方水提物与药学上可接受的辅料制备成药学上可接受的剂型。
4.根据权利要求3所述的一种中药组方水提物在制备预防及治疗老年痴呆药物中的应用,其特征在于:所述的药学上可接受的剂型包括片剂,胶囊剂,口服液或颗粒剂。
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