CN117860860A - 一种中药组合物在制备保护神经的药物中的应用 - Google Patents
一种中药组合物在制备保护神经的药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种中药组合物在制备保护神经的药物中的应用,属于生物医药技术领域。所述中药组合物按重量份计,包括以下原料:黄芪425份、人参120份、天麻170份、丹参170份、红花85份、葛根340份、川芎170份、石菖蒲170份、郁金85份、水蛭85份、乌梢蛇170份和冰片10份。本发明构建了大鼠永久性大脑中动脉阻塞模型,通过动物实验证实,由上述中药组合物制备成的药物,通过调节AMPK‑ULK1‑Beclin1/BINP3L信号通路抑制神经元自噬,改善神经功能,减轻脑组织损伤,进而发挥神经保护作用,可用于制备神经保护药物。
Description
技术领域
本发明涉及生物医药技术领域,特别是涉及一种中药组合物在制备保护神经的药物中的应用。
背景技术
脑卒中是指各种原因引起的脑血管狭窄、闭塞或破裂,最终导致急性脑血液循环障碍。缺血性脑卒中是最常见的类型,病死率和致残率均较高。神经细胞变性坏死是导致缺血性脑卒中患者死亡和病残的关键因素,最大程度保护正常神经细胞是治疗的重要环节。因此,神经功能修复是脑血管病急需攻克的一个科学难题。近二十年来,世界上许多国家对脑血管病的基础研究投入了大量人力和物力,然而收效甚微,神经保护的防治策略亟待进一步加强。缺血性脑血管病占脑血管病发病的80%左右,目前在缺血性脑血管病的药物研究中,除了组织型纤溶酶原激活剂(tPA)之外,其他药物的临床实验几乎全部失败。而tPA受严格的时间窗限制,接受治疗的患者极其有限,仅有不到5%的患者能接受此治疗疗法。中药往往通过多靶点或协同效应增强对病症的治疗效果,临床应用价值显著。因此,开发安全有效的神经保护和神经修复的中药治疗药物,对于防治或减轻神经损伤病情,促进神经功能恢复,降低致死率和致残率,具有重要意义。
蛭蛇通络胶囊是陕西健民制药有限公司开发研制的治疗动脉粥样硬化性血栓性脑梗死的中药新药,由人参、黄芪、天麻、丹参、红花、葛根、川芎、石菖蒲、郁金、水蛭、冰片及乌梢蛇等组成,具有补气养血、开窍醒神、活血化淤以及祛风通络等功效,能够预防和治疗动脉血栓的形成,进而治疗脑梗死。然而该中药组合物是否能够有效地保护和修复神经,并未见相关报道。
发明内容
本发明的目的是提供一种中药组合物在制备保护神经的药物中的应用,以解决上述现有技术存在的问题,本发明通过动物实验表明,所述中药组合物通过调节AMPK-ULK1-Beclin1/BINP3L信号通路抑制神经元自噬,改善神经功能,减轻脑组织损伤,进而发挥神经保护作用。
为实现上述目的,本发明提供了如下方案:
本发明提供一种中药组合物在制备保护神经的药物中的应用,所述中药组合物按重量份计,包括以下原料:
黄芪425份、人参120份、天麻170份、丹参170份、红花85份、葛根340份、川芎170份、石菖蒲170份、郁金85份、水蛭85份、乌梢蛇170份和冰片10份。
进一步地,所述中药组合物通过抑制神经元自噬,发挥保护神经的作用。
进一步地,所述中药组合物通过调节AMPK-ULK1-Beclin1/BINP3L信号通路抑制神经元自噬。
进一步地,所述神经元为缺血缺氧的神经元。
进一步地,所述药物还包括药学上能接受的辅料。
进一步地,所述药物的剂型包括胶囊剂。
本发明公开了以下技术效果:
本发明提供了一种中药组合物在制备保护神经的药物中的应用,由该中药组合物制备的产品名称为“蛭蛇通络胶囊”,由陕西健民制药有限公司生产。本发明构建了大鼠永久性大脑中动脉阻塞模型,通过病理染色、炎症、氧化应激、血管再生和神经元自噬等相关指标进行评价,结果表明,蛭蛇通络胶囊抑制氧化应激和炎症反应,促进血管新生,通过调节AMPK-ULK1-Beclin1/BINP3L信号通路抑制神经元自噬,改善神经功能,减轻脑组织损伤,最终发挥神经保护作用。
本发明证明了由上述中药组合物制备的药物对缺血缺氧的神经细胞具有明显的保护作用,可用于制备神经保护药物。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为蛭蛇通络胶囊对MCAO大鼠神经功能的影响,其中,n=10/组,**代表与假手术组比较,P<0.01;##代表与MCAO模型组比较,P<0.01;
图2为ELISA法检测蛭蛇通络胶囊对MCAO大鼠血清炎症因子IL-1α、IL-6、IL-17和TNF-α表达的影响,其中,n=10/组,*代表与假手术组比较,P<0.05;**代表与假手术组比较,P<0.01;#代表与MCAO模型组比较,P<0.05;##代表与MCAO模型组比较,P<0.01;
图3为TTC染色测定蛭蛇通络胶囊对MCAO大鼠脑梗死面积的影响,其中A为各实验组脑组织TTC染色图,B为各实验组脑组织梗死面积统计条图,n=6/组;*代表与假手术组比较,P<0.05;**代表与假手术组比较,P<0.01;#代表与MCAO模型组比较,P<0.05;##代表与MCAO模型组比较,P<0.01;
图4为HE染色显示蛭蛇通络胶囊对MCAO大鼠脑皮层组织结果的影响(标尺100μm,放大倍数×200倍),其中A为假手术组,B为MCAO模型组,C为蛭蛇通络胶囊低剂量组,D为蛭蛇通络胶囊中剂量组,E为蛭蛇通络胶囊高剂量组,F为金纳多组,n=5/组;
图5为Nissl染色显示蛭蛇通络胶囊对MCAO大鼠脑皮层组织的影响(标尺100μm,放大倍数×200倍),其中,A为假手术组,B为MCAO模型组,C为蛭蛇通络胶囊低剂量组,D为蛭蛇通络胶囊中剂量组,E为蛭蛇通络胶囊高剂量组,F为金纳多组,n=5/组;
图6为免疫组化法检测蛭蛇通络胶囊对MCAO大鼠缺血半暗带区皮层神经元Beclin-1蛋白表达的影响(标尺100μm,放大倍数×200倍),其中A为假手术组,B为MCAO模型组,C为蛭蛇通络胶囊低剂量组,D为蛭蛇通络胶囊中剂量组,E为蛭蛇通络胶囊高剂量组,F为金纳多组,n=5/组;
图7为免疫组化法检测蛭蛇通络胶囊对MCAO大鼠脑缺血半暗带区皮层BNIP3L蛋白表达的影响(标尺100μm,放大倍数×200倍),其中A为假手术组,B为MCAO模型组,C为蛭蛇通络胶囊低剂量组,D为蛭蛇通络胶囊中剂量组,E为蛭蛇通络胶囊高剂量组,F为金纳多组,n=5/组;
图8为免疫组化法检测蛭蛇通络胶囊对MCAO大鼠缺血半暗区皮质VEGF蛋白表达的影响(×标尺100μm,放大倍数×200倍),其中A为假手术组,B为MCAO模型组,C为蛭蛇通络胶囊低剂量组,D为蛭蛇通络胶囊中剂量组,E为蛭蛇通络胶囊高剂量组,F为金纳多组,n=5/组;
图9为免疫组化法检测蛭蛇通络胶囊对MCAO大鼠缺血半暗区皮质BDNF蛋白表达的影响(标尺100μm,放大倍数×200倍),其中A为假手术组,B为MCAO模型组,C为蛭蛇通络胶囊低剂量组,D为蛭蛇通络胶囊中剂量组,E为蛭蛇通络胶囊高剂量组,F为金纳多组,n=5/组;
图10为蛭蛇通络胶囊对MCAO大鼠梗死侧脑组织氧化应激相关指标SOD(A)和GSH-Px(B)的影响,n=6/组;其中,**代表与假手术组比较,P<0.01;#代表与MCAO模型组比较,P<0.05;##代表与MCAO模型组比较,P<0.01;
图11为Western blotting法检测蛭蛇通络胶囊对MCAO大鼠梗死侧脑组织自噬相关AMPK-ULK1-Beclin1/BINP3L信号通路表达的影响,n=3/组;其中,A为Westernblotting凝胶电泳图,B为自噬相关AMPK-ULK1-Beclin1/BINP3L信号通路相对表达量统计图,*代表与假手术组比较,P<0.01;**代表与假手术组比较,P<0.01;#代表与MCAO模型组比较,P<0.05;##代表与MCAO模型组比较,P<0.01。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值,以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
实施例
一、实验材料
1.实验动物
SPF级雄性SD大鼠,120只,体重(220-250)g,购自斯贝福(北京)生物科技股份有限公司,许可证号SCXK(京)2019-0010,实验动物质量合格证号No.110324211103175027。实验期间大鼠饲养于中国中医科学院中药研究所动物房,实验动物使用许可证SCXK(京)2019-0003。动物房为屏障系统,人工光照,12小时明暗周期,温度控制在(20-22)℃范围,相对湿度为(40-70)%,换气次数15次/h。
2.实验药品及试剂
本发明用于实验的药物,有效成分为中药组合物,原料组方按照重量份计,为:黄芪425份、人参120份、天麻170份、丹参170份、红花85份、葛根340份、川芎170份、石菖蒲170份、郁金85份、水蛭85份、乌梢蛇170份和冰片10份。所述药物及其制备方法已公开在申请号为200610105114.8,发明名称为“一种治疗中风病的药物及其制备工艺”的专利文件中。所述药物名称为“蛭蛇通络胶囊”,国药准字号Z20090658,由陕西健民制药有限公司提供,批号2011106。
阳性对照药为金纳多EGb 761(Ginaton,进口药品注册证号H20140768),由Dr.Willmar Schwabe GmbH&Co.KG(德国威玛舒培博士药厂)生产,批号8530120,有效成分为银杏叶提取物。
大鼠源IL-1α(批号19033946)、IL-1β(批号20033937)、IL-6(批号18033935)、IL-10(批号104033934)、IL-17(批号J23033933)、TNF-α(批号J29035736)和谷胱甘肽过氧化物酶(GSH-Px,批号I07091452)ELISA试剂盒均购于武汉华美生物科技有限公司;戊巴比妥钠(批号20191119)购于美国Sigma公司(分装);超氧化物歧化酶(SOD,批号20210714)检测试剂盒购于南京建成生物工程研究所;高效RIPA组织/细胞裂解液(批号20200928)、4%多聚甲醛(批号20200522)、2,3,5-三苯基氯化四氮唑(TTC,批号520C032)和丙二醛(malondialdehyde,MDA,批号20210709)检测试剂盒购于北京索莱宝生物科技有限公司;BCA蛋白浓度测定试剂盒(批号RK240545)购于美国Pierce公司;Beclin-1抗体(货号sc-48381)和VEGF抗体(sc-7269)购于美国Santa Cruz公司;p-AMPK抗体(货号2537S)、BNIP3L抗体(货号12396S)和GAPDH抗体(货号5174S)购于美国Cell Signaling Technology公司;BDNF抗体(货号ab108319)购于美国Abcam公司;AMPK抗体(货号66536-1-Ig)和p-ULK1抗体(货号29006-1-AP)购于美国Proteintech公司;ULK1抗体(货号bs-3602R)购于北京博奥森生物技术有限公司。
3.实验仪器
美国Becton-Dickinson SpectraMax M5多功能酶标仪;宁波新芝生物科技股份有限公司SCNTZ-48高通量组织研磨器;黄石市恒丰医疗器械有限公司FCD-3000系列电热恒温鼓风干燥箱;上海精密科学仪器有限公司FA2204B电子天平。
二、实验方法
1.模型构建
采用Zea-Longa改良线栓法构建永久性大脑中动脉脑阻塞(MCAO)模型,戊巴比妥钠(1%,0.55mL/100g)腹腔注射麻醉,仰卧位固定于手术台上,剪除颈部鼠毛,碘伏消毒。取颈正中作切口,切开皮肤和浅筋膜,钝性分离肌肉,充分游离左侧颈总、颈内和颈外动脉,结扎颈外动脉及颈总动脉近心端,动脉夹夹闭颈内动脉,在颈外动脉近心端距离其在颈总动脉分叉约4.0mm处用眼科剪剪一小口,将尼龙线(直径为0.3mm)插入颈内动脉中,至感到有轻微阻力以阻断大脑中动脉入口(进线长度距离分叉处约为20.0mm),固定尼龙线后,将切口逐层缝合关闭。
术后6h采用Zea-Longa法对大鼠进行神经功能缺损评分,具体标准如下,0分:无神经功能障碍;1分:轻度神经功能缺损(右侧前爪无法完全伸展);2分:中度神经功能缺损(向右侧转圈,追尾);3分:严重神经功能缺损(行走时向右侧倾斜);4分:意识丧失,无法行走。(1~3)分视为造模成功,可纳入实验组。观察用药前后按神经功能缺损评分的评分值的变化情况,分值越低表示治疗效果越理想。
2.剂量确定
蛭蛇通络胶囊临床人(60kg)使用剂量0.5g/粒,4粒/次,3次/日,疗程4周,共计6.0g/天,相当于0.1g/kg/day,换算成相应的大鼠剂量(200g)为0.625g/kg/day,作为本实验的低剂量。按2倍递增,1.25g/kg/d与2.50g/kg/d分别作为本实验的中剂量和高剂量。
阳性对照药金纳多(ginaton,银杏叶提取物片)临床人(60kg)使用剂量40mg/片,(1~2)片/次,(2~3)次/日,相当于4.0mg/kg/day,换算成相应的大鼠剂量(200g)为2.5mg/kg/day,作为本实验的剂量。
3.实验分组
造模成功的大鼠,按神经功能缺损评分随机分为6组,每组21只。
(1)假手术组(Sham):取颈正中作切口,充分游离右侧颈总动脉、颈内动脉、颈外动脉,将切口逐层缝合关闭。
(2)模型组(MCAO):实验第一天制备MCAO模型,手术6h后,灌胃等体积去离子水,每日1次,连续7天。
(3)蛭蛇通络胶囊低(ZSTL 0.63)、中(ZSTL 1.25)、高(ZSTL 2.50)剂量组:实验第一天制备MCAO模型,手术6h后,灌胃给与不同浓度蛭蛇通络胶囊(0.63、1.25和2.50g/kg/day,分别对应ZSTL 0.63组、ZSTL 1.25组和ZSTL 2.50组),每日1次,连续7天。
(4)阳性药物组:实验第一天制备MCAO模型,手术6h后,灌胃给与金纳多(2.50mg/kg/day),每日1次,连续7天。
4.检测指标
4.1神经功能
实验第7天,采用Zea-Longa法对各组大鼠神经功能缺损程度进行评分,具体标准同“1.模型构建”中所述。
4.2炎症因子
实验第8天腹主动脉取血,3000rpm/min离心10min,取上清,ELISA法检测炎症因子IL-1α、IL-1β、IL-6、IL-10、IL-17和TNF-α的表达。
4.3脑组织染色
(1)TTC染色
小心解剖出大脑后,应用大鼠脑模具自视交叉水平切成2mm厚的连续冠状切片5片。置于1%TTC染液中,37℃条件下,避光孵育30min,不断翻动切片保证其均匀着色。在4%多聚甲醛中放置染色后的切片,固定24h后拍照。根据Image-Pro Plus软件计算脑梗死体积,依据公式:脑梗死体积比=(正常侧脑组织体积-脑梗死侧正常脑组织)/(正常脑组织体积)×100%。
(2)苏木精-伊红(hematoxylin-eosin,HE)染色
将脑组织在脑中线2mm的位置(近脑梗死侧),按照Ashwal法平均法纵向均分,用刀片在冰面上均分成3片,舍弃边上两片,取中间一片脑组织,把整个脑片看做1个时钟面,在2点和8点连线,此处切开后与垂直线(12点和6点连线)之间的脑组织就是所需标本,置于4%多聚甲醛固定,保存备用。石蜡包埋切片等,HE染色观察脑部缺血半暗区组织病理形态变化。
(3)尼氏(Nissl)染色
取脑组织,在多聚甲醛中固定24h,先后在20%和30%的蔗糖溶液中脱水,切片,尼氏染色,取缺血侧皮质区,高倍镜(×200倍)算各区完整的锥体细胞数,每张切片计数3个区域,连续取7张脑片,取平均数为该脑梗死侧皮质区的存活细胞数。
4.4线粒体自噬
取脑组织块,石蜡包埋,切片机切成厚度4μm的组织片,脱蜡至水。室温下在3%H2O2中孵育,PBS冲洗,切片放入枸橼酸盐缓冲液。用PBS冲洗5min,滴加一抗,过夜。滴加二抗,室温孵育30min,PBS冲洗。DAB显色5~10min。蒸馏水冲洗。苏木素复染1min,水洗,脱水至二甲苯,中性树胶封片,应用病理图像分析系统对脑部缺血半暗带组织表达自噬蛋白Beclin1和BNIP3L的阳性细胞数,进行分析和拍照。
4.5血管再生
实验步骤同“4.4线粒体自噬”,免疫组化法检测脑部缺血半暗区组织血管内皮生长因子(vascular endothelial growth factor,VEGF)和脑源性神经生长因子(brain-derived nerve growth factor,BDNF)的表达情况。
4.6氧化应激
取梗死侧脑组织,加入含蛋白酶抑制剂的高效RIPA组织裂解液,超声制备组织匀浆液,12000g/min离心15min,取上清,BCA蛋白浓度测定试剂盒检测总蛋白浓度。生化试剂盒结合酶标仪检测氧化应激相关分子超氧化物歧化酶(superoxide dismutase,SOD)的活性。ELISA法测定氧化应激相关分子谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)的表达水平。
4.7线粒体自噬信号通路
取梗死侧脑组织,加入含蛋白酶抑制剂的高效RIPA裂解液,超声充分裂解组织,提取总蛋白,BCA蛋白浓度测定试剂盒检测总蛋白浓度,Western boltting法检测线粒体自噬信号通路AMPK-ULK1-Beclin1/BINP3L的表达。
5.数据处理
所有数据均采用均数±标准差表示,采用SPSS20.0软件进行统计学处理,多组间比较采用单因素方差分析,任意两组间比较采用t检验,P<0.05为差异具有统计学意义。
三、实验结果
1.蛭蛇通络胶囊对MCAO大鼠神经功能的影响
实验第1天术后6h和第7天对大鼠进行神经功能缺损评分,结果表明实验第1天时,与假手术组比较,MCAO模型组大鼠神经功能评分均升高(P<0.01);与MCAO模型组比较,蛭蛇通络胶囊低、中、高剂量组与金纳多组大鼠神经功能评分无统计学差异。实验第7天时,与MCAO模型组比较,蛭蛇通络胶囊低、中、高剂量组与金纳多组大鼠神经功能评分均下降(图1)。
2.蛭蛇通络胶囊对MCAO大鼠血清炎症因子的影响
采用ELISA法对血清炎症因子IL-1α、IL-1β、IL-6、IL-10、IL-17和TNF-α的表达进行检测,研究结果表明,与假手术组比较,模型组IL-1α、IL-6、IL-17和TNF-α的表达水平均明显升高;与MCAO模型组比较,蛭蛇通络胶囊低、中和高剂量组均可显著降低IL-1α、IL-6和TNF-α的表达;蛭蛇通络胶囊中和高剂量组均可显著降低IL-17的表达(图2)。阳性对照药金纳多也可以降低上述炎症因子的表达。
3.蛭蛇通络胶囊对MCAO大鼠脑组织结构的影响
3.1TTC染色观察蛭蛇通络胶囊对MCAO大鼠脑梗死面积的影响
TTC染色结果显示假手术组大鼠脑组织呈均匀淡红色,无脑梗死病灶。与假手术组比较,MCAO模型组大鼠脑梗死部位被染成白色,涉及到大脑皮层、海马区、丘脑和纹状体,梗死面积增大(P<0.01);与MCAO模型组相比,蛭蛇通络胶囊不同剂量组脑梗死面积均明显降低,且具有统计学差异,以高剂量组梗死灶最少(图3)。阳性对照药金纳多也可减少脑梗死面积。
3.2HE染色观察蛭蛇通络胶囊对MCAO大鼠缺血半暗区皮层组织结构的影响
HE染色结果显示假手术组脑皮层组织结构正常,神经元排列整齐;MCAO模型组缺血半暗区皮层神经元排列紊乱,大量坏死的神经元,存活的神经元明显缩小;与模型组相比较,蛭蛇通络胶囊中、高剂量组大脑皮层神经元排列较整齐,仅有部分神经元排列散乱,少部分神经元细胞出现坏死征象,大部分神经元体积大于模型组。各剂量组缺血半暗区皮层组织结构均有不同程度好转,以高剂量组效果最为显著(图4)。金纳多组缺血半暗区皮层组织结构也趋于好转。
3.3Nissl染色观察蛭蛇通络胶囊对MCAO大鼠缺血半暗区皮层组织结构的影响
Nissl染色结果显示假手术组大鼠脑皮层神经元排列整齐,核仁清晰,细胞核周围尼氏体颗粒多。与假手术组相比较,MCAO模型组大鼠缺血半暗区皮层神经元排列紊乱,形态不完整,细胞核皱缩,尼氏小体数量减少。与MCAO模型组相比较,蛭蛇通络胶囊中和高剂量组缺血半暗区皮层神经元结构损伤程度逐渐降低,且具有统计学差异(图5)。金纳多组缺血半暗区皮层组织结构也趋于好转。
4.蛭蛇通络胶囊对MCAO大鼠缺血半暗区皮质神经元自噬的影响
免疫组化法检测MCAO大鼠缺血半暗区皮层神经元自噬相关蛋白Beclin1和BNIP3L的表达,结果表明假手术组脑皮层神经元Beclin1和BNIP3L蛋白均无表达;MCAO模型组缺血半暗区皮层神经元Beclin1和BNIP3L蛋白表达均明显增多,阳性呈棕色;与MCAO模型组相比较,蛭蛇通络胶囊中、高剂量组缺血半暗区皮层神经元Beclin1和BNIP3L蛋白表达均逐渐减少,以高剂量组最为显著(图6和图7)。阳性对照药金纳多着色率降低,表明其也可抑制上述两种蛋白的表达。
5.蛭蛇通络胶囊对MCAO大鼠缺血半暗区皮质血管新生的影响
免疫组化法检测MCAO大鼠缺血半暗区皮层VEGF和BDNF蛋白的表达,结果表明假手术组皮质VEGF和BDNF蛋白均正常表达;MCAO模型组缺血半暗区皮质VEGF和BDNF蛋白表达均明显降低,阳性呈棕色;与MCAO模型组比较,蛭蛇通络胶囊中和高剂量组缺血半暗区皮质VEGF和BDNF蛋白表达均逐渐增多,以高剂量组最为显著(图8和9)。阳性对照药金纳多着色率升高,表明其也可促进上述两种蛋白的表达。
6.蛭蛇通络胶囊对MCAO大鼠梗死侧脑组织氧化应激的影响
利用生化试剂盒和ELISA试剂盒分别检测MCAO大鼠梗死侧脑组织氧化应激相关指标SOD和GSH-Px活力的变化,结果表明与假手术组比较,MCAO模型组脑组织SOD和GSH-Px活力均显著降低(P<0.01);与MCAO模型组比较,蛭蛇通络胶囊不同剂量组SOD活力明显升高(P<0.05),蛭蛇通络胶囊中和高剂量组GSH-Px活力均明显升高(图10)。金纳多也可以升高SOD和GSH-Px活力(P<0.01)。
7.蛭蛇通络胶囊对MCAO大鼠梗死侧脑组织自噬相关AMPK-ULK1-Beclin1/BINP3L信号通路表达的影响
采用Western blotting法检测MCAO大鼠梗死侧脑组织线粒体自噬相关蛋白AMPK、ULK1、Beclin1和BINP3L的表达,结果表明与假手术组比较,MCAO模型组p-AMPK/AMPK、p-ULK1/ULK1、Beclin1和BINP3L蛋白的表达明显升高。与MCAO模型组比较,蛭蛇通络胶囊中和高剂量组p-AMPK/AMPK和p-ULK1/ULK1的蛋白表达均明显降低;蛭蛇通络胶囊不同剂量组Beclin1和BINP3L的蛋白表达均明显降低(图11)。金纳多也可抑制AMPK-ULK1-Beclin1/BINP3L信号通路的表达。
四、实验结论
1.利用Zea-Longa改良线栓法构建大鼠大脑中动脉脑阻塞(MCAO)模型,TTC染色、HE染色和尼氏染色结果均表明模型构建成功。
2.MCAO大鼠神经功能评分升高;血清炎症因子如IL-1α、IL-6、IL-17和TNF-α水平均升高。TTC染色显示脑梗死部位染成白色,涉及到皮层、海马区、丘脑及纹状体;HE染色显示缺血半暗区皮层神经元排列紊乱,大量的神经元坏死,存活的神经元明显缩小;尼氏染色显示缺血半暗区皮层神经元形态不完整,细胞核皱缩,尼氏小体数量减少。缺血半暗区皮层神经元自噬相关蛋白Beclin1和BINP3L表达增多,以及血管新生相关蛋白VEGF和BDNF表达减少。梗死侧脑组织氧化应激相关分子SOD和GSH-Px活力均降低;梗死侧脑组织线粒体自噬相关信号通路AMPK-ULK1-Beclin1/BINP3L表达升高。
3.采用治疗性灌胃给药的方式,蛭蛇通络胶囊改善MCAO大鼠神经功能;抑制血清炎症因子如IL-1α、IL-6、IL-17和TNF-α的水平;减轻脑组织损伤,TTC染色显示脑梗死体积减少,HE和尼氏染色均显示缺血半暗区皮层组织结构有明显好转。抑制缺血半暗区皮层神经元自噬相关蛋白Beclin1和BINP3L的表达;促进缺血半暗区皮层VEGF和BDNF蛋白的表达;升高梗死侧脑组织SOD和GSH-Px活力;下调梗死侧脑组织线粒体自噬相关蛋白AMPK、ULK1、Beclin1和BINP3L的表达。
4.动物实验研究结果表明,蛭蛇通络胶囊抑制氧化应激和炎症反应,促进血管新生,通过调节AMPK-ULK1-Beclin1/BINP3L信号通路抑制神经元自噬,改善神经功能,减轻脑组织损伤,发挥神经保护作用。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (6)
1.一种中药组合物在制备保护神经的药物中的应用,其特征在于,所述中药组合物按重量份计,包括以下原料:
黄芪425份、人参120份、天麻170份、丹参170份、红花85份、葛根340份、川芎170份、石菖蒲170份、郁金85份、水蛭85份、乌梢蛇170份和冰片10份。
2.根据权利要求1所述的应用,其特征在于,所述中药组合物通过抑制神经元自噬,发挥保护神经的作用。
3.根据权利要求2所述的应用,其特征在于,所述中药组合物通过调节AMPK-ULK1-Beclin1/BINP3L信号通路抑制神经元自噬。
4.根据权利要求2所述的应用,其特征在于,所述神经元为缺血缺氧的神经元。
5.根据权利要求1所述的应用,其特征在于,所述药物还包括药学上能接受的辅料。
6.根据权利要求1所述的应用,其特征在于,所述药物的剂型包括胶囊剂。
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