含与不含朱砂雄黄的安宫牛黄丸对大鼠出血性脑损伤的保护作用及机理研究
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摘要
脑出血是指非外伤性脑实质内出血,属中医中风病之范畴。其患病人数约占所有脑卒中病人的20%~30%,住院死亡率往往高达43%~51%。且现在发病年龄呈年轻化的趋势,因此对脑出血的治疗和实验方面的研究日益受到关注。
对于脑出血的治疗,中医有着独特的疗效。传统医学认为脑出血为风火相煽,痰湿蕴盛,瘀血阻滞致气血逆乱,上犯于脑而发病。因此中医治疗拟予化痰通腑、解毒通络、泻火熄风开窍、活血化瘀等法。
安宫牛黄丸具有清热解毒、镇惊开窍之功效,在临床上对于脑出血的治疗有很好的疗效。方中朱砂、雄黄由于含有重金属化合物,历来是大家争议的焦点。中医理论认为:雄黄具有清热解毒、化瘀消积的功效,朱砂具有清心镇惊,安神解毒的功效。从二者的功效上看,与中医治疗出血性中风的治法相一致,因此北京市科委立专项(项目编号:H040230130712)探讨含与不含朱砂雄黄的安宫牛黄丸在药效上的区别,以明确朱砂、雄黄在安宫牛黄丸方中存在的意义。
本论文研究的主要内容包括两部分:第一部分采用大鼠脑出血损伤动物模型,从整体水平观察含与不含朱砂雄黄的安宫牛黄丸对大鼠脑出血损伤的保护作用并探讨其机制;第二部分从细胞水平观察含与不含朱砂雄黄的安宫牛黄丸含药血清和含药脑脊液对原代皮层神经元的保护作用并探讨其机制。
第一部分含与不含朱砂雄黄的安宫牛黄丸对大鼠脑出血损伤的保护作用及机制研究建立胶原酶诱导大鼠脑出血损伤动物模型,观察安宫牛黄丸对大鼠脑出血损伤的保护作用,并探讨其可能机制,同时以不含朱砂雄黄的安宫牛黄丸与之对比,观察去掉朱砂雄黄之后作用与全方是否存在差别。
1含与不含朱砂、雄黄的安宫牛黄丸对脑出血大鼠保护作用的研究
目的:研究安宫牛黄丸对脑出血模型大鼠神经症状、脑系数、脑含水量、全血的血液流变性、抗氧化能力、病理形态学的影响。同时观察不含朱砂雄黄的安宫牛黄丸与之是否存在差异。方法:采用尾状核注射胶原酶(TYPEⅣ)脑出血模型大鼠,于术后第3天进行神经病学症状评分;称重法测定脑系数;干湿重法测定脑含水量;生化试剂盒检测脑组织匀浆中LDH、SOD活性,MDA含量;血流变仪进行血液流变学检测;HE染色进行病理形态学观察。结果:(1)模型组动物神经功能症状评分、脑指数、脑含水量增加;红细胞变形性降低;LDH活性升高,SOD活性降低,MDA含量增加;损伤侧脑组织可见出血、水肿,血肿周边脑组织呈大片变性、坏死,神经细胞肿胀等明显病理形态学改变。
(2)安宫牛黄丸200mg/kg、400mg/kg可显著改善神经症状评分(与模型组比,p<0.01, p<0.05)、降低脑指数(与模型组比,p<0.01,p<0.05),提高红细胞变形性(与模型组比,p<0.05),提高SOD活性(与模型组比,p<0.05, p<0.01),降低MDA含量(与模型组比,p<0.01),降低LDH活性(与模型组比,p<0.01);缩小出血面积、减轻组织间隙水肿、修复血肿周围组织,减轻神经细胞肿胀。(3)不含朱砂雄黄的安宫牛黄丸两个剂量组改善动物神经功能障碍的作用不显著,小剂量提高红细胞变形性、降低脑指数作用不显著,与模型组相比未见显著差异。320mg/kg可明显降低脑组织含水量(与模型组比,p<0.05);提高SOD活性(与模型组比,p<0.05);降低LDH活性(与模型组比,p<0.05)。160mg/kg、320mg/kg可显著降低MDA含量(与模型组比,p<0.01, p<0.05);可缩小出血面积、促进血肿周围组织修复。结论:(1)安宫牛黄丸对脑出血模型大鼠有保护作用。可显著改善神经症状、降低脑指数、提高红细胞变形性,降低LDH活性、提高SOD活性、降低MDA含量,改善组织病理形态。(2)去朱砂、雄黄后改善神经症状作用有所减弱,两个剂量组与模型组相比均无显著性差异。降低脑指数、提高脑组织SOD活性,减少LDH含量和提高红细胞变形性指标上作用有所减弱,小剂量组与模型组相比无显著性差异。全方等效剂量对这些指标有显著改善作用,但两种药物等效剂量组间比较无显著性差异。
2含与不含朱砂雄黄的安宫牛黄丸对脑出血模型大鼠脑组织凋亡相关蛋白、血脑屏障通透性相关蛋白的影响
目的:探讨安宫牛黄丸对脑出血模型大鼠细胞凋亡、凋亡相关蛋白表达、以及血脑屏障通透性相关蛋白表达的影响。同时观察不含朱砂雄黄的安宫牛黄丸对这些蛋白表达的影响与之是否存在差异。方法:用免疫组化的方法观察脑出血模型大鼠出血侧细胞的凋亡(TUNEL染色)、凋亡相关蛋白的表达(Bcl-2,Bax)、血脑屏障通透性相关蛋白(MMP9、AQP4)的表达。结果:(1)模型大鼠TUNEL染色阳性表达增加,说明脑出血后脑组织细胞出现凋亡。Bax染色模型组阳性表达较假手术明显增加,MMP9、AQP4染色模型组阳性表达增加。(2)安宫牛黄丸400mg/kg可以显著减少TUNEL染色阳性表达(与模型组比,p<0.05),可促进Bcl-2的表达,安宫牛黄丸200mg/kg、400mg/kg剂量组可以减少AQP4的阳性表达(与模型组比,p<0.01),减少MMP9的阳性表达(与模型组比,p<0.01),减少Bax阳性表达(与模型组比,p<0.01)。(3)不含朱砂雄黄的安宫牛黄丸160mg/kg、320mg/kg可以减少MMP9(与模型组比,p<0.01)、AQP4的阳性表达(与模型组比,p<0.05),减少Bax的阳性表达(与模型组比,p<0.01,p<0.05)。两个剂量组促进Bcl-2表达的作用均不显著。结论:(1)安宫牛黄丸可以通过抗凋亡、影响血脑屏障通透性来发挥神经保护作用。(2)不含朱砂雄黄的安宫牛黄丸对血脑屏障通透性相关蛋白表达的影响与全方相近,对凋亡的抑制作用有所减弱,两个剂量组TUNEL染色阳性表达与模型组相比均无显著性差异。两个剂量组促进Bcl-2表达的作用均不显著。全方和简方等效剂量组间比较无显著性差异。
3含与不含朱砂雄黄的安宫牛黄丸对脑出血模型大鼠脑组织MAPK通路的影响
目的:探讨安宫牛黄丸对脑出血模型大鼠脑组织MAPK通路的影响,同时观察不含朱砂雄黄的安宫牛黄丸对脑出血模型大鼠MAPK通路的影响与之是否存在差异。方法:应用免疫组化的方法观察脑出血大鼠脑组织与MAPK通路活化相关蛋白—PKC、MAPK通路底物蛋白c-fos、c-jun的表达,用实时荧光定量RT-PCR的方法观察MAPK通路家族ERKmRNA、P38mRNA的表达,比较不含朱砂、雄黄的安宫牛黄丸对其表达的影响与安宫牛黄丸是否存在差异。结果:(1)出血损伤引起脑组织PKC、c-fos、c-jun蛋白表达增加,ERKmRNA表达减少,P38mRNA表达增加。(2)安宫牛黄丸200mg/kg、400mg/kg减少c-fos的表达(与模型组比,p<0.01)、减少c-jun的表达(与模型组比,p<0.01),减少PKC的表达(与模型组比,p<0.05),促进ERKmRNA表达(与模型组比,p<0.05)、减少P38mRNA的表达(与模型组比,p<0.01)。(3)不含朱砂雄黄的安宫牛黄丸两个剂量组也可减少c-fos、c-jun、PKC的阳性表达(与模型组比,p<0.01or p<0.05),促进ERKmRNA表达(与模型组比,p<0.05)。不含朱砂雄黄的安宫牛黄丸大剂量减少c-fos、c-jun、P38mRNA表达的作用显著低于安宫牛黄丸大剂量(与全方大剂量比,p<0.05),小剂量减少c-fos阳性表达的作用显著低于安宫牛黄丸小剂量(与全方小剂量比,p<0.05)。结论:(1)安宫牛黄丸可以通过影响MAPK通路发挥神经保护作用。(2)不含朱砂雄黄的安宫牛黄丸也可以通过影响MAPK通路发挥神经保护作用。其中减少c-fos、c-jun、P38mRNA的表达的作用明显弱于安宫牛黄丸组,在这些指标上,两种药物相应剂量之间进行组间比较,差异有统计学意义。朱砂雄黄与安宫牛黄丸对MAPK通路的调节作用可能有关。
第二部分含与不含朱砂雄黄的安宫牛黄丸对原代培养大鼠皮层神经元损伤的保护作用及机理的研究(血清、脑脊液药理)
建立缺糖缺氧诱导损伤、谷氨酸诱导损伤原代培养皮层神经细胞模型,观察含与不含朱砂雄黄的安宫牛黄丸含药血清、含药脑脊液对两种损伤细胞的保护作用,及对损伤细胞线粒体膜电位、细胞内钙离子浓度的影响。同时进行脑脊液、血清中砷、汞含量测定,探讨神经元保护与血清、脑脊液中砷、汞的关系。
4含与不含朱砂雄黄的安宫牛黄丸对体外培养的原代皮层神经元的保护作用
目的:观察含与不含朱砂雄黄的安宫牛黄丸含药血清、含药脑脊液对损伤神经细胞的保护作用是否存在差异,明确朱砂雄黄在全方中的作用。方法:采用缺糖缺氧诱导损伤细胞模型和谷氨酸诱导损伤细胞模型,用血清药理学和脑脊液药理学的方法观察含与不含朱砂雄黄的安宫牛黄丸含药血清、含药脑脊液对损伤神经元的保护作用。结果:Ⅰ抗缺糖缺氧损伤(1)缺糖缺氧损伤使细胞LDH漏出率增加,细胞活性降低。(2)安宫牛黄丸800mg/kg剂量组含药血清、20%添加比例含药脑脊液可显著升高细胞活性(与模型组比,p<0.05),降低LDH漏出率(与模型组比,p<0.01,p<0.05);400mg/kg剂量组含药血清、20%添加比例含药脑脊液,可以显著降低LDH漏出率(与模型组比,p<0.05)。(3)不含朱砂雄黄的安宫牛黄丸640mg/kg含药血清、640mg/kg,20%添加比例含药脑脊液可显著升高细胞活性(与模型组比,p<0.05),降低LDH漏出率(与模型组比,p<0.05)。Ⅱ抗谷氨酸损伤(1)谷氨酸200μmmol·L-1损伤细胞后,LDH漏出率明显增加、细胞活性下降。细胞中MDA含量升高、SOD活性降低。(2)安宫牛黄丸800mg/kg剂量组含药血清、800mg/kg剂量组10%、20%添加比例的脑脊液可以显著提高损伤细胞的活性(与模型组比,p<0.05,p<0.01,p<0.05),显著降低LDH漏出率(与模型组比,p<0.01,p<0.05),降低MDA含量(与模型组比,p<0.05),提高SOD活性(与模型组比,p<0.05)。400mg/kg剂量组含药血清可以显著降低损伤细胞LDH漏出率(与模型组比,p<0.05)。(3)不含朱砂雄黄的安宫牛黄丸640mg/kg剂量组含药血清可以显著提高损伤细胞的活性(与模型组比,p<0.05)、降低LDH漏出率(与模型组比,p<0.01),降低MDA含量(与模型组比,p<0.05),提高SOD活性(与模型组比,p<0.05)。不含朱砂雄黄的安宫牛黄丸640mg/kg剂量组20%添加比例的含药脑脊液可以降低损伤细胞的MDA含量(与模型组比,p<0.05),提高损伤细胞的SOD活性(与模型组比,p<0.05)。不含朱砂雄黄的安宫牛黄丸640mg/kg剂量组10%添加比例的含药脑脊液可提高损伤细胞的SOD活性(与模型组比,p<0.05),降低MDA含量作用不显著。结论:(1)安宫牛黄丸大剂量组含药血清、含药脑脊液可对抗缺糖缺氧损伤引起的细胞活性降低,两个剂量组含药血清含药脑脊液均可降低LDH漏出率。不含朱砂雄黄的安宫牛黄丸含药血清、含药脑脊液降低缺糖缺氧LDH漏出率作用有所减弱,小剂量含药血清含药脑脊液不能显著降低LDH漏出率。两种药物相应剂量含药血清、含药脑脊液组间比较无统计学意义。(2)安宫牛黄丸含药血清、含药脑脊液对谷氨酸诱导的神经元损伤有对抗作用。提高损伤细胞活性、降低损伤细胞LDH漏出率、降低MDA含量、提高SOD活性是其对抗谷氨酸诱导损伤的可能机制。不含朱砂雄黄的安宫牛黄丸含药血清、含药脑脊液提高损伤细胞活性、降低损伤细胞LDH漏出率、降低MDA含量的作用均有所减弱。两个剂量的含药脑脊液对损伤引起的细胞活性降低均无明显对抗作用,与模型组相比无统计学意义。小剂量含药血清、大剂量10%添加比例的脑脊液不能显著降低损伤细胞LDH漏出率,大剂量10%添加比例的脑脊液不能显著降低损伤细胞MDA含量,全方相应剂量的血清或脑脊液在相应指标上有显著改善作用。但两种药物相应剂量含药血清、含药脑脊液组间并无明显差异。
5含与不含朱砂雄黄的安宫牛黄丸对谷氨酸损伤神经元线粒体膜电位、细胞内钙离子浓度的影响
目的:观察安宫牛黄丸含药血清、含药脑脊液对损伤神经细胞的线粒体膜电位、细胞内钙离子浓度的影响。同时观察不含朱砂雄黄的安宫牛黄丸含药血清、含药脑脊液的作用,与之进行比较。方法:采用谷氨酸诱导损伤细胞模型,应用流式细胞技术测定线粒体膜电位和细胞内钙离子浓度,进行组间比较。结果:(1)细胞损伤后,线粒体膜电位下降,细胞内钙离子浓度增加。(2)安宫牛黄丸含药血清、含药脑脊液可显著改善线粒体膜电位的下降(与模型组比,p<0.01),安宫牛黄丸含药脑脊液、含药血清能对抗损伤造成的细胞内钙离子浓度升高(与模型组比,p<0.01, p<0.05)。(3)不含朱砂雄黄的安宫牛黄丸含药血清、含药脑脊液可显著改善线粒体膜电位的下降(与模型组比,p<0.01),不含朱砂雄黄的安宫牛黄丸含药脑脊液降低细胞内钙离子浓度的作用显著弱于安宫牛黄丸含药脑脊液(与全方组比,p<0.05)。含药血清对谷氨酸损伤引起的钙超载无明显对抗作用。结论:安宫牛黄丸可以升高损伤细胞的线粒体膜电位,降低损伤细胞内钙离子浓度;不含朱砂雄黄的安宫牛黄丸对损伤引起的膜电位降低有明显改善,不含朱砂雄黄的安宫牛黄丸含药脑脊液对细胞内钙超载的对抗作用较全方含药脑脊液明显减弱,朱砂雄黄可能与安宫牛黄丸抑制钙超载作用有关。
6含与不含朱砂雄黄的安宫牛黄丸含药血清、含药脑脊液中砷、汞含量的测定
目的:观察组方中朱砂、雄黄所含的砷、汞是否能够被吸收入血或穿透血脑屏障进入脑脊液,从而探讨安宫牛黄丸的脑保护作用与朱砂、雄黄之间是否有一定的关联。方法:采用电感耦合等离子质谱法测定安宫牛黄含药血清、含药脑脊液中砷、汞的含量。结果:(1)正常对照组大鼠和不含朱砂雄黄的安宫牛黄丸组大鼠血清、脑脊液中也可检测到微量的砷。正常对照组大鼠血清中砷含量为36.83ng/ml,脑脊液中砷含量为12.92 ng/ml,血中砷含量为脑脊液中砷含量的2.85倍。(2)不含朱砂雄黄的安宫牛黄丸组大鼠血清中砷含量40.12 ng/ml,脑脊液中砷含量16.34 ng/ml,血中砷含量为脑脊液中砷含量的2.45倍。(3)安宫牛黄丸组大鼠血清中砷含量为228.18 ng/ml,脑脊液中砷含量为43.37 ng/ml,血中砷含量为脑脊液中砷含量的5.26倍。(4)汞的含量在仪器检出限附近,未能精确测定,故论文暂不讨论,需采用更精确的测定方法来进行定量分析。结论:安宫牛黄丸含药血清、含药脑脊液中有砷、汞的存在,含与不含朱砂雄黄的安宫牛黄丸对损伤神经细胞的保护作用的差异与朱砂雄黄可能相关。
结语:
1.安宫牛黄丸对胶原酶诱导的脑出血损伤有明显保护作用,可以降低损伤动物神经症状评分,降低脑指数、提高红细胞变形性、降低脑组织MDA含量,LDH活性、提高SOD酶活性,改善损伤脑组织病理形态。
2.安宫牛黄丸通过减少MMP9、AQP4的表达改善血脑屏障通透性,减轻脑损伤;通过影响Bax、Bcl-2的表达、以及促进MAPK通路中ERKmRNA,抑制P38mRNA、c-fos、c-jun表达发挥抗凋亡的作用,起到神经保护作用。
3.安宫牛黄丸含药血清、含药脑脊液对缺糖缺氧诱导的原代皮层神经细胞损伤有一定的对抗,可以降低损伤细胞的LDH漏出率,提高细胞活性。对谷氨酸诱导的原代皮层神经细胞损伤有一定的对抗,可以降低LDH漏出率,提高细胞活性,通过降低MDA含量、升高SOD活性、提高线粒体膜电位、减轻钙超载保护损伤的神经元。
4.去朱砂雄黄后对脑出血损伤仍表现一定的保护作用,简方减少c-fos、c-jun、P38mRNA表达的作用显著弱于全方,两者相应剂量组进行组间比较,有显著性差异。在其他指标上改善作用较全方有所减弱。但两种药物相应剂量组之间比较无统计学差异。简方含药血清、含药脑脊液对缺糖缺氧损伤、谷氨酸损伤原代皮层神经元表现出一定的保护作用,简方含药脑脊液对钙超载的抑制作用较全方含药脑脊液明显减弱,二者进行组间比较,差异有统计学意义。在其他指标上,改善作用较全方含药血清、含药脑脊液有所减弱。但两种药物相应剂量组之间比较无统计学差异。
创新点:
1.从整体药效学、血清药理学、脑脊液药理学三个层面研究和分析含与不含朱砂雄黄的安宫牛黄丸对脑出血大鼠损伤的保护作用及差异,明确朱砂雄黄在全方中存在的意义。
2.通过含与不含朱砂雄黄的安宫牛黄丸对大鼠脑出血损伤保护作用机制的探讨,明确朱砂、雄黄可能与安宫牛黄丸抗凋亡、抑制钙超载作用有关。
3.首次报道含与不含朱砂雄黄的安宫牛黄丸对于MAPK信号转导通路的影响。
Intra-cerebral hemorrhage (ICH) is also called non-wounded cerebral hemorrhage. It belongs to one of the category of stoke. Among all the stroke patients, the number of ICH patients is going up to 20%~30%, and the hospital mortality has reached 43%~51%. Nowadays, the onset age of ICH tends to be younger, therefore, more and more attention is attracted to the research on prevention and therapy of ICH. Generally speaking, ICH is caused by the attack of both wind and fire, and the dampness is more one bad factor.
With regard to the therapy on ICH, Traditional Chinese Medicine (TCM) has its own unique curative effect. The basic principle of TCM is to apply different prescriotions to treat different patients, that is to say, disease and syndrome are taken into account all together when facing different patient.
Angongniuhuang pill (AGNHW), the fomous Chinese medicine has remarkable effects of removing heat and cleaning toxin, so it has good curative effect on ICH. But for a long time, it is argued whether it is necessary to add Cinnabaris and Realgar into the medicine because of their huge toxicity. Therefore, We design a series of experiments so as to clarify the difference of AGNHW with Cinnabaris and Realgar and AGNHW without Cinnabaris and Realgar (QZX-AGNHW). And the chief results are described as follows.
PART ONE : Protective effects and its mechanism on neurons in rats with ICH of AGNHW with or wihtout Cinnabaris and Realgar
1 Protective effects and its mechanism on neurons in rats with ICH of AGNHW containing Cinnabaris and Realgar or not
Objective : To study the effects of AGNHW on nerve symptoms, cerebral index cerebral edema, antioxidation capacity, blood rheology and morphological changes. At the same time, the effect of QZX-AGNHW and the difference between the two kinds of medicines are observed. Methods: Collagenase-Induced intra-cerebral hemorrhage model in rat caudate nucleus was applied. Nerve symptoms were scored at the fourth day after the operation, moreover, the other factors such as the cerebral index , the content of water , antioxidation capacity, blood rheology and morphological changes were tested at the same time. Results:
(1)In the vehicle group, nerve symptoms, content of water , cerebral index,content of MDA, activity of LDH increased greatly. (2)The dosage of 200mg/kg, 400mg/kg of AGNHW can improve significantly the symdrome (vs vehicle p<0.01or p<0.05) . In the vehicle group, the activity of SOD, erythrocyte deformability decreased greatly, on the contrary, the dosage of 200mg/kg, 400mg/kg of AGNHW increased these targets significantly ,( vs vehicle, p<0.01or p<0.05) . (3)Compared with AGNHW, the function of improving nerve symptoms and decreasing cerebral index of QZX- AGNHW was weakened. Conclusion:(1)AGNHW has protective effect on ICH, and it can improve nerve symptoms and content of water, also, decrease cerebral index, content of MDA and the activity of LDH. (2) The function of improving nerve symptoms and decreasing cerebral index of QZX-AGNHW was weakened.
2. Effects on the expressions of apoptosis - related proteinas BBB associated protein in rats with ICH of AGNHW containing Cinnabaris and Realgar or not Objective: To approach the effect of AGNHW on the apoptosis and apoptosis- related proteinas BBB associated with protein expressions. and compare the difference of the effects of AGNHW and QZX-AGNHW. Methods: Expressions of apoptisis (TUNEL), apoptisis -associated protein (Bcl-2,Bax)BBB associated protein (MMP9, AQP4)were observed by immunohistochcmistry of AGNHW on perihematomal isscue in rats with ICH. Results:(1)In the vehicle group, The expression of TUNEL, Bax, MMP9, AQP4 were increased, and the expressions of Bcl-2 deceased. (2) The dosage of 400mg/kg of AGNHW can decreased the expression of TUNEL, Bax, MMP9 (vs vehicle p<0.05). On the other hand, it can increase the espression of Bcl-2 (Vs vehicle, p<0.05). The dosage of 200mg/kg ,400mg/kg of AGNHW can decrease the expression of AQP4(Vs vehicle p<0.05, or p<0.01, respectively).(3)The dosage of 160mg/kg, 320mg/kg of QZX-AGNHW decrese the expression of MMP9, AQP4 (Vs vehicle, p<0.05 or p<0.05). Conclusion:(1)AGNHW protect neurons by affecting anti-apoptosis and related BBB permeability. (2)The function of related BBB permeability of QZX- AGNHW was similar to AGNHW, while the influence of anti-apoptosis decreased obviously.
3.Effects on MAPK signal transduction pathway in rats with ICH of AGNHW containing Cinnabaris and Realgar or not
Objective: To observe the effect of AGNHW and QZX-AGNHW on MAPK signal transduction pathway in rats with ICH, and compare their difference. Methods: Expression of the MAPK signal transduction pathway associated protein PKC、c-fos and c-jun was observed by immunohistochcmical. Expression of the MAPK signal transduction pathway family ERK、P38mRNA was observed by real time RT-PCR. Results: (1)The expression of PKC、c-fos、c-jun inceased after ICH injury, and the expressions of ERKmRNA deceased. However, the expression of P38 mRNA increased (Vs vehicle, p<0.01). (2) The dosage of 200mg/kg, 400mg/kg of AGNHW reduced the expression of c-fos, c-jun (Vs vehicle, p<0.01, p<0.05), The dosage of 400mg/kg of AGNHW reduced the expression of P38mRNA and PKC, and improve the expression of ERKmRNA(Vs vehicle, p<0.05). (3) The dosage of 160mg/kg, 320 mg/kg of QZX-AGNHW decrease c-fos expression(Vs vehicle, p<0.05 or p<0.01),and the dosage of 160mg/kg decrease PKC expression(Vs vehicle, p<0.05), the dosage of 320mg/kg decrease the expression of c-jun , P38mRNA, (Vs vehicle, p<0.01),and increase ERKmRNA expression (Vs vehicle, p<0.01). Conclusions: (1)Protective effects of AGNHW on neurons in rats with ICH was observed by influence activity of MAPK signal transduction pathway.
(2)The innfluence of QZX-AGNHW on MAPK signal transduction path-way was similar to AGNHW, but its function of decreasing expression of P38mRNA was weakened. Part two: Protective effects and its mechanism on primary cultured cortex neuron of AGNHW containing Cinnabaris and Realgar or not with serum or CSF containing drug
Protective effect of serum or CSF containing drug was observed on oxygen-glucose deprivation or GLU inducing cultured cortex neuron model. The influence of serum or CSF containing drug on MMP and [Ca~(2+)]i of GLU induced cultured cortex neuron model. Contents of determination Cinnaba and Realgar in serum or CSF containing drug were determined. 4 Protective effects and its mechanism on primary cultured cortex neuron of AGNHW containing Cinnabaris and Realgar or not with serum or CSF containing drug
Objective: To compare the protective effects of AGNHW and QZX-AGNHW with serum or CSF containing drug. Methods: Using cell damage models induced by oxygen/glucose deprivation and GLU,the serum and cerebrospinal fluid pharmacological methods were determined, in order to observe the function of cinnabar and realgar. Results: (1) The damage of oxygen-glucose deprivation increased the leakage rate of LDH. (2)observe morphocytological chages and measure the LDH leak rate , cytoactive , intracellular concentration of MDA and SOD. In the cells injured by the oxygen/glucose deprivation,LDH leak rate was significantly increased, while the cytoactivty was inhibited. These changes were blocked effectively by serum and cerebrospinal fluid containing AGNHW, especialy by the dose of 800mg/kg,and the cerebrospinal fluid with the additive proportion of 20% is better than the same dose. The result indicated that the effection of AGNHW is better than that of QZX-AGNHW. After cells were injured by GLU,LDH leak rate was also significantly increased,meanwhile, the cytoactive was inhibited,and morphocytologic changes were observed obviously. AGNHW had the significant protective actions when the dose reached 640mg/kg,and the cerebro spinal fluid with the additive proportion of 20% was better using the same dose. Conclusion: AGNHW has significant effects of protecting cells.
5 The effects on mitochondria membrane potential and intracellular Ca~(2+) concentration of rat cortical neurons injured by GLU of AGNHW and QZX-AGNHW
Objective: To observe the effects of AGNHW and QZX-AGNHW on mitochondria membrane potential and intracellular Ca~(2+) concentration of rat cortical neurons injured by GLU. Methods: Using the method of flow cytometry(FCM),mitochondria membrane potential and intracellular Ca~(2+) concentration of rat cortical neurons were measured, then the effects of AGNHW and QZX-AGNHW were compared on the basis of the above data. Results: In rats whose cortical neurons were injured by GLU,mitochondria membrane potential was observed to decrease,and the concentration of intracellular Ca~(2+) raised. The value of mitochondria membrane potential could be increased by AGNHW and QZX-AGNHW containing serum and cerebrospinal fluid. While the concentration of intracellular Ca~(2+) reduced evidently when acted by AGNHW containing cerebrospinal fluid and QZX-AGNHW containing serum and cerebrospinal fluid. Conclusion: AGNHW and QZX-AGNHW had the similar effects, which produced the protective effects on injuryed neurons through stabilizing mitochondria membrane potential and blocking calcium overload.
6 Determination of Concentration of cinnabar and realgar in serum and CSF of AGNHW and QZX-AGNHW
Objective: To determine whether As and Hg contained in cinnabar and realgar were absorbed into blood or cerebrospinal fluid (CSF) through BBB. And further to analyze if cinnabar and realgar play an important role in the protective effect of AGNHW. Methods: Inductively Coupled Plasma-mass was used to determine the concentration of As and Hg in serum and cerebrospinal fluid . Results:( 1) As in AGNHW could be tested in blood and might be absorbed into the CSF through BBB. The content of As in serum is 5.26 times that of As in CSF.(2) Trace As could be tested in serum and CSF of normal control group and QZX-AGNHW group.(3) The content of Hg was very near the limit of detection of instrument, so it could not be detected exactly. that is to say, the content of Hg need more accurate methods to be detected. Conclusion: Both Serum and CSF Contaning Drug of AGNHW contained As and Hg,the difference of neuro protective effect of AGNHW and QZX-AGNHW had relationship with As and Hg.
In summary, AGNHW alleviated the neurologic symptoms of rats after ICH; amelioration morphology changes induced by ICH; reduce cerebral index , improve erythrocyte deformability, all these results show its satisfactory protective effect to experimental ICH. The main mechanism of effect may as follows: It can relieve the injury induced by the toxic product such as excitatory amino acids(EAA), MDA and free redical. In addition, partially inhibit calcium overload induced by EAA, stabilize the mitochondria membrane potential. It has the function of anti-apoptosis by increasing the express of Bcl-2,and decreasing the express of Bax. It can surpression the expression of c-fos,c-jun,PKC,elevate the mRNA expression of ERK and decrease the mRNA expression of P38.their specific receptor. It can surpression the expression of AQP4,MMP9.Compaired with AGNHW , the function of QZX- AGNHW of increasing nerve symptoms, decreasing cerebral index was weakened .And the function of anti-apoptosis of QZX- AGNHW is not notable.
In this experiment ,we studied the protective effect of AGNHW and QZX- AGNHW to ICH rats.
1 In this study, the protective function of AGNHW and QZX-AGNHW on rats injured with ICH was analyzed. Accordingly entirety pharmacodynamics,serum phamacology and CSF phamacology , the effective differences of the two kinds of medicines were compared. The results show that cinnabar and realgar play an important role in the total prescription.
2 Based on the experiments of the mechanism analysis, AGNHW and QZX-AGNHW have the similar effects, which produce the protective effects on injuryed neurons through anti-apoptosis and blocking calcium overload.
3 For the first time, it is reported that the effection of AGNHW and QZX-AGNHW on the pathway of signal transconduction of MAPK.
对于脑出血的治疗,中医有着独特的疗效。传统医学认为脑出血为风火相煽,痰湿蕴盛,瘀血阻滞致气血逆乱,上犯于脑而发病。因此中医治疗拟予化痰通腑、解毒通络、泻火熄风开窍、活血化瘀等法。
安宫牛黄丸具有清热解毒、镇惊开窍之功效,在临床上对于脑出血的治疗有很好的疗效。方中朱砂、雄黄由于含有重金属化合物,历来是大家争议的焦点。中医理论认为:雄黄具有清热解毒、化瘀消积的功效,朱砂具有清心镇惊,安神解毒的功效。从二者的功效上看,与中医治疗出血性中风的治法相一致,因此北京市科委立专项(项目编号:H040230130712)探讨含与不含朱砂雄黄的安宫牛黄丸在药效上的区别,以明确朱砂、雄黄在安宫牛黄丸方中存在的意义。
本论文研究的主要内容包括两部分:第一部分采用大鼠脑出血损伤动物模型,从整体水平观察含与不含朱砂雄黄的安宫牛黄丸对大鼠脑出血损伤的保护作用并探讨其机制;第二部分从细胞水平观察含与不含朱砂雄黄的安宫牛黄丸含药血清和含药脑脊液对原代皮层神经元的保护作用并探讨其机制。
第一部分含与不含朱砂雄黄的安宫牛黄丸对大鼠脑出血损伤的保护作用及机制研究建立胶原酶诱导大鼠脑出血损伤动物模型,观察安宫牛黄丸对大鼠脑出血损伤的保护作用,并探讨其可能机制,同时以不含朱砂雄黄的安宫牛黄丸与之对比,观察去掉朱砂雄黄之后作用与全方是否存在差别。
1含与不含朱砂、雄黄的安宫牛黄丸对脑出血大鼠保护作用的研究
目的:研究安宫牛黄丸对脑出血模型大鼠神经症状、脑系数、脑含水量、全血的血液流变性、抗氧化能力、病理形态学的影响。同时观察不含朱砂雄黄的安宫牛黄丸与之是否存在差异。方法:采用尾状核注射胶原酶(TYPEⅣ)脑出血模型大鼠,于术后第3天进行神经病学症状评分;称重法测定脑系数;干湿重法测定脑含水量;生化试剂盒检测脑组织匀浆中LDH、SOD活性,MDA含量;血流变仪进行血液流变学检测;HE染色进行病理形态学观察。结果:(1)模型组动物神经功能症状评分、脑指数、脑含水量增加;红细胞变形性降低;LDH活性升高,SOD活性降低,MDA含量增加;损伤侧脑组织可见出血、水肿,血肿周边脑组织呈大片变性、坏死,神经细胞肿胀等明显病理形态学改变。
(2)安宫牛黄丸200mg/kg、400mg/kg可显著改善神经症状评分(与模型组比,p<0.01, p<0.05)、降低脑指数(与模型组比,p<0.01,p<0.05),提高红细胞变形性(与模型组比,p<0.05),提高SOD活性(与模型组比,p<0.05, p<0.01),降低MDA含量(与模型组比,p<0.01),降低LDH活性(与模型组比,p<0.01);缩小出血面积、减轻组织间隙水肿、修复血肿周围组织,减轻神经细胞肿胀。(3)不含朱砂雄黄的安宫牛黄丸两个剂量组改善动物神经功能障碍的作用不显著,小剂量提高红细胞变形性、降低脑指数作用不显著,与模型组相比未见显著差异。320mg/kg可明显降低脑组织含水量(与模型组比,p<0.05);提高SOD活性(与模型组比,p<0.05);降低LDH活性(与模型组比,p<0.05)。160mg/kg、320mg/kg可显著降低MDA含量(与模型组比,p<0.01, p<0.05);可缩小出血面积、促进血肿周围组织修复。结论:(1)安宫牛黄丸对脑出血模型大鼠有保护作用。可显著改善神经症状、降低脑指数、提高红细胞变形性,降低LDH活性、提高SOD活性、降低MDA含量,改善组织病理形态。(2)去朱砂、雄黄后改善神经症状作用有所减弱,两个剂量组与模型组相比均无显著性差异。降低脑指数、提高脑组织SOD活性,减少LDH含量和提高红细胞变形性指标上作用有所减弱,小剂量组与模型组相比无显著性差异。全方等效剂量对这些指标有显著改善作用,但两种药物等效剂量组间比较无显著性差异。
2含与不含朱砂雄黄的安宫牛黄丸对脑出血模型大鼠脑组织凋亡相关蛋白、血脑屏障通透性相关蛋白的影响
目的:探讨安宫牛黄丸对脑出血模型大鼠细胞凋亡、凋亡相关蛋白表达、以及血脑屏障通透性相关蛋白表达的影响。同时观察不含朱砂雄黄的安宫牛黄丸对这些蛋白表达的影响与之是否存在差异。方法:用免疫组化的方法观察脑出血模型大鼠出血侧细胞的凋亡(TUNEL染色)、凋亡相关蛋白的表达(Bcl-2,Bax)、血脑屏障通透性相关蛋白(MMP9、AQP4)的表达。结果:(1)模型大鼠TUNEL染色阳性表达增加,说明脑出血后脑组织细胞出现凋亡。Bax染色模型组阳性表达较假手术明显增加,MMP9、AQP4染色模型组阳性表达增加。(2)安宫牛黄丸400mg/kg可以显著减少TUNEL染色阳性表达(与模型组比,p<0.05),可促进Bcl-2的表达,安宫牛黄丸200mg/kg、400mg/kg剂量组可以减少AQP4的阳性表达(与模型组比,p<0.01),减少MMP9的阳性表达(与模型组比,p<0.01),减少Bax阳性表达(与模型组比,p<0.01)。(3)不含朱砂雄黄的安宫牛黄丸160mg/kg、320mg/kg可以减少MMP9(与模型组比,p<0.01)、AQP4的阳性表达(与模型组比,p<0.05),减少Bax的阳性表达(与模型组比,p<0.01,p<0.05)。两个剂量组促进Bcl-2表达的作用均不显著。结论:(1)安宫牛黄丸可以通过抗凋亡、影响血脑屏障通透性来发挥神经保护作用。(2)不含朱砂雄黄的安宫牛黄丸对血脑屏障通透性相关蛋白表达的影响与全方相近,对凋亡的抑制作用有所减弱,两个剂量组TUNEL染色阳性表达与模型组相比均无显著性差异。两个剂量组促进Bcl-2表达的作用均不显著。全方和简方等效剂量组间比较无显著性差异。
3含与不含朱砂雄黄的安宫牛黄丸对脑出血模型大鼠脑组织MAPK通路的影响
目的:探讨安宫牛黄丸对脑出血模型大鼠脑组织MAPK通路的影响,同时观察不含朱砂雄黄的安宫牛黄丸对脑出血模型大鼠MAPK通路的影响与之是否存在差异。方法:应用免疫组化的方法观察脑出血大鼠脑组织与MAPK通路活化相关蛋白—PKC、MAPK通路底物蛋白c-fos、c-jun的表达,用实时荧光定量RT-PCR的方法观察MAPK通路家族ERKmRNA、P38mRNA的表达,比较不含朱砂、雄黄的安宫牛黄丸对其表达的影响与安宫牛黄丸是否存在差异。结果:(1)出血损伤引起脑组织PKC、c-fos、c-jun蛋白表达增加,ERKmRNA表达减少,P38mRNA表达增加。(2)安宫牛黄丸200mg/kg、400mg/kg减少c-fos的表达(与模型组比,p<0.01)、减少c-jun的表达(与模型组比,p<0.01),减少PKC的表达(与模型组比,p<0.05),促进ERKmRNA表达(与模型组比,p<0.05)、减少P38mRNA的表达(与模型组比,p<0.01)。(3)不含朱砂雄黄的安宫牛黄丸两个剂量组也可减少c-fos、c-jun、PKC的阳性表达(与模型组比,p<0.01or p<0.05),促进ERKmRNA表达(与模型组比,p<0.05)。不含朱砂雄黄的安宫牛黄丸大剂量减少c-fos、c-jun、P38mRNA表达的作用显著低于安宫牛黄丸大剂量(与全方大剂量比,p<0.05),小剂量减少c-fos阳性表达的作用显著低于安宫牛黄丸小剂量(与全方小剂量比,p<0.05)。结论:(1)安宫牛黄丸可以通过影响MAPK通路发挥神经保护作用。(2)不含朱砂雄黄的安宫牛黄丸也可以通过影响MAPK通路发挥神经保护作用。其中减少c-fos、c-jun、P38mRNA的表达的作用明显弱于安宫牛黄丸组,在这些指标上,两种药物相应剂量之间进行组间比较,差异有统计学意义。朱砂雄黄与安宫牛黄丸对MAPK通路的调节作用可能有关。
第二部分含与不含朱砂雄黄的安宫牛黄丸对原代培养大鼠皮层神经元损伤的保护作用及机理的研究(血清、脑脊液药理)
建立缺糖缺氧诱导损伤、谷氨酸诱导损伤原代培养皮层神经细胞模型,观察含与不含朱砂雄黄的安宫牛黄丸含药血清、含药脑脊液对两种损伤细胞的保护作用,及对损伤细胞线粒体膜电位、细胞内钙离子浓度的影响。同时进行脑脊液、血清中砷、汞含量测定,探讨神经元保护与血清、脑脊液中砷、汞的关系。
4含与不含朱砂雄黄的安宫牛黄丸对体外培养的原代皮层神经元的保护作用
目的:观察含与不含朱砂雄黄的安宫牛黄丸含药血清、含药脑脊液对损伤神经细胞的保护作用是否存在差异,明确朱砂雄黄在全方中的作用。方法:采用缺糖缺氧诱导损伤细胞模型和谷氨酸诱导损伤细胞模型,用血清药理学和脑脊液药理学的方法观察含与不含朱砂雄黄的安宫牛黄丸含药血清、含药脑脊液对损伤神经元的保护作用。结果:Ⅰ抗缺糖缺氧损伤(1)缺糖缺氧损伤使细胞LDH漏出率增加,细胞活性降低。(2)安宫牛黄丸800mg/kg剂量组含药血清、20%添加比例含药脑脊液可显著升高细胞活性(与模型组比,p<0.05),降低LDH漏出率(与模型组比,p<0.01,p<0.05);400mg/kg剂量组含药血清、20%添加比例含药脑脊液,可以显著降低LDH漏出率(与模型组比,p<0.05)。(3)不含朱砂雄黄的安宫牛黄丸640mg/kg含药血清、640mg/kg,20%添加比例含药脑脊液可显著升高细胞活性(与模型组比,p<0.05),降低LDH漏出率(与模型组比,p<0.05)。Ⅱ抗谷氨酸损伤(1)谷氨酸200μmmol·L-1损伤细胞后,LDH漏出率明显增加、细胞活性下降。细胞中MDA含量升高、SOD活性降低。(2)安宫牛黄丸800mg/kg剂量组含药血清、800mg/kg剂量组10%、20%添加比例的脑脊液可以显著提高损伤细胞的活性(与模型组比,p<0.05,p<0.01,p<0.05),显著降低LDH漏出率(与模型组比,p<0.01,p<0.05),降低MDA含量(与模型组比,p<0.05),提高SOD活性(与模型组比,p<0.05)。400mg/kg剂量组含药血清可以显著降低损伤细胞LDH漏出率(与模型组比,p<0.05)。(3)不含朱砂雄黄的安宫牛黄丸640mg/kg剂量组含药血清可以显著提高损伤细胞的活性(与模型组比,p<0.05)、降低LDH漏出率(与模型组比,p<0.01),降低MDA含量(与模型组比,p<0.05),提高SOD活性(与模型组比,p<0.05)。不含朱砂雄黄的安宫牛黄丸640mg/kg剂量组20%添加比例的含药脑脊液可以降低损伤细胞的MDA含量(与模型组比,p<0.05),提高损伤细胞的SOD活性(与模型组比,p<0.05)。不含朱砂雄黄的安宫牛黄丸640mg/kg剂量组10%添加比例的含药脑脊液可提高损伤细胞的SOD活性(与模型组比,p<0.05),降低MDA含量作用不显著。结论:(1)安宫牛黄丸大剂量组含药血清、含药脑脊液可对抗缺糖缺氧损伤引起的细胞活性降低,两个剂量组含药血清含药脑脊液均可降低LDH漏出率。不含朱砂雄黄的安宫牛黄丸含药血清、含药脑脊液降低缺糖缺氧LDH漏出率作用有所减弱,小剂量含药血清含药脑脊液不能显著降低LDH漏出率。两种药物相应剂量含药血清、含药脑脊液组间比较无统计学意义。(2)安宫牛黄丸含药血清、含药脑脊液对谷氨酸诱导的神经元损伤有对抗作用。提高损伤细胞活性、降低损伤细胞LDH漏出率、降低MDA含量、提高SOD活性是其对抗谷氨酸诱导损伤的可能机制。不含朱砂雄黄的安宫牛黄丸含药血清、含药脑脊液提高损伤细胞活性、降低损伤细胞LDH漏出率、降低MDA含量的作用均有所减弱。两个剂量的含药脑脊液对损伤引起的细胞活性降低均无明显对抗作用,与模型组相比无统计学意义。小剂量含药血清、大剂量10%添加比例的脑脊液不能显著降低损伤细胞LDH漏出率,大剂量10%添加比例的脑脊液不能显著降低损伤细胞MDA含量,全方相应剂量的血清或脑脊液在相应指标上有显著改善作用。但两种药物相应剂量含药血清、含药脑脊液组间并无明显差异。
5含与不含朱砂雄黄的安宫牛黄丸对谷氨酸损伤神经元线粒体膜电位、细胞内钙离子浓度的影响
目的:观察安宫牛黄丸含药血清、含药脑脊液对损伤神经细胞的线粒体膜电位、细胞内钙离子浓度的影响。同时观察不含朱砂雄黄的安宫牛黄丸含药血清、含药脑脊液的作用,与之进行比较。方法:采用谷氨酸诱导损伤细胞模型,应用流式细胞技术测定线粒体膜电位和细胞内钙离子浓度,进行组间比较。结果:(1)细胞损伤后,线粒体膜电位下降,细胞内钙离子浓度增加。(2)安宫牛黄丸含药血清、含药脑脊液可显著改善线粒体膜电位的下降(与模型组比,p<0.01),安宫牛黄丸含药脑脊液、含药血清能对抗损伤造成的细胞内钙离子浓度升高(与模型组比,p<0.01, p<0.05)。(3)不含朱砂雄黄的安宫牛黄丸含药血清、含药脑脊液可显著改善线粒体膜电位的下降(与模型组比,p<0.01),不含朱砂雄黄的安宫牛黄丸含药脑脊液降低细胞内钙离子浓度的作用显著弱于安宫牛黄丸含药脑脊液(与全方组比,p<0.05)。含药血清对谷氨酸损伤引起的钙超载无明显对抗作用。结论:安宫牛黄丸可以升高损伤细胞的线粒体膜电位,降低损伤细胞内钙离子浓度;不含朱砂雄黄的安宫牛黄丸对损伤引起的膜电位降低有明显改善,不含朱砂雄黄的安宫牛黄丸含药脑脊液对细胞内钙超载的对抗作用较全方含药脑脊液明显减弱,朱砂雄黄可能与安宫牛黄丸抑制钙超载作用有关。
6含与不含朱砂雄黄的安宫牛黄丸含药血清、含药脑脊液中砷、汞含量的测定
目的:观察组方中朱砂、雄黄所含的砷、汞是否能够被吸收入血或穿透血脑屏障进入脑脊液,从而探讨安宫牛黄丸的脑保护作用与朱砂、雄黄之间是否有一定的关联。方法:采用电感耦合等离子质谱法测定安宫牛黄含药血清、含药脑脊液中砷、汞的含量。结果:(1)正常对照组大鼠和不含朱砂雄黄的安宫牛黄丸组大鼠血清、脑脊液中也可检测到微量的砷。正常对照组大鼠血清中砷含量为36.83ng/ml,脑脊液中砷含量为12.92 ng/ml,血中砷含量为脑脊液中砷含量的2.85倍。(2)不含朱砂雄黄的安宫牛黄丸组大鼠血清中砷含量40.12 ng/ml,脑脊液中砷含量16.34 ng/ml,血中砷含量为脑脊液中砷含量的2.45倍。(3)安宫牛黄丸组大鼠血清中砷含量为228.18 ng/ml,脑脊液中砷含量为43.37 ng/ml,血中砷含量为脑脊液中砷含量的5.26倍。(4)汞的含量在仪器检出限附近,未能精确测定,故论文暂不讨论,需采用更精确的测定方法来进行定量分析。结论:安宫牛黄丸含药血清、含药脑脊液中有砷、汞的存在,含与不含朱砂雄黄的安宫牛黄丸对损伤神经细胞的保护作用的差异与朱砂雄黄可能相关。
结语:
1.安宫牛黄丸对胶原酶诱导的脑出血损伤有明显保护作用,可以降低损伤动物神经症状评分,降低脑指数、提高红细胞变形性、降低脑组织MDA含量,LDH活性、提高SOD酶活性,改善损伤脑组织病理形态。
2.安宫牛黄丸通过减少MMP9、AQP4的表达改善血脑屏障通透性,减轻脑损伤;通过影响Bax、Bcl-2的表达、以及促进MAPK通路中ERKmRNA,抑制P38mRNA、c-fos、c-jun表达发挥抗凋亡的作用,起到神经保护作用。
3.安宫牛黄丸含药血清、含药脑脊液对缺糖缺氧诱导的原代皮层神经细胞损伤有一定的对抗,可以降低损伤细胞的LDH漏出率,提高细胞活性。对谷氨酸诱导的原代皮层神经细胞损伤有一定的对抗,可以降低LDH漏出率,提高细胞活性,通过降低MDA含量、升高SOD活性、提高线粒体膜电位、减轻钙超载保护损伤的神经元。
4.去朱砂雄黄后对脑出血损伤仍表现一定的保护作用,简方减少c-fos、c-jun、P38mRNA表达的作用显著弱于全方,两者相应剂量组进行组间比较,有显著性差异。在其他指标上改善作用较全方有所减弱。但两种药物相应剂量组之间比较无统计学差异。简方含药血清、含药脑脊液对缺糖缺氧损伤、谷氨酸损伤原代皮层神经元表现出一定的保护作用,简方含药脑脊液对钙超载的抑制作用较全方含药脑脊液明显减弱,二者进行组间比较,差异有统计学意义。在其他指标上,改善作用较全方含药血清、含药脑脊液有所减弱。但两种药物相应剂量组之间比较无统计学差异。
创新点:
1.从整体药效学、血清药理学、脑脊液药理学三个层面研究和分析含与不含朱砂雄黄的安宫牛黄丸对脑出血大鼠损伤的保护作用及差异,明确朱砂雄黄在全方中存在的意义。
2.通过含与不含朱砂雄黄的安宫牛黄丸对大鼠脑出血损伤保护作用机制的探讨,明确朱砂、雄黄可能与安宫牛黄丸抗凋亡、抑制钙超载作用有关。
3.首次报道含与不含朱砂雄黄的安宫牛黄丸对于MAPK信号转导通路的影响。
Intra-cerebral hemorrhage (ICH) is also called non-wounded cerebral hemorrhage. It belongs to one of the category of stoke. Among all the stroke patients, the number of ICH patients is going up to 20%~30%, and the hospital mortality has reached 43%~51%. Nowadays, the onset age of ICH tends to be younger, therefore, more and more attention is attracted to the research on prevention and therapy of ICH. Generally speaking, ICH is caused by the attack of both wind and fire, and the dampness is more one bad factor.
With regard to the therapy on ICH, Traditional Chinese Medicine (TCM) has its own unique curative effect. The basic principle of TCM is to apply different prescriotions to treat different patients, that is to say, disease and syndrome are taken into account all together when facing different patient.
Angongniuhuang pill (AGNHW), the fomous Chinese medicine has remarkable effects of removing heat and cleaning toxin, so it has good curative effect on ICH. But for a long time, it is argued whether it is necessary to add Cinnabaris and Realgar into the medicine because of their huge toxicity. Therefore, We design a series of experiments so as to clarify the difference of AGNHW with Cinnabaris and Realgar and AGNHW without Cinnabaris and Realgar (QZX-AGNHW). And the chief results are described as follows.
PART ONE : Protective effects and its mechanism on neurons in rats with ICH of AGNHW with or wihtout Cinnabaris and Realgar
1 Protective effects and its mechanism on neurons in rats with ICH of AGNHW containing Cinnabaris and Realgar or not
Objective : To study the effects of AGNHW on nerve symptoms, cerebral index cerebral edema, antioxidation capacity, blood rheology and morphological changes. At the same time, the effect of QZX-AGNHW and the difference between the two kinds of medicines are observed. Methods: Collagenase-Induced intra-cerebral hemorrhage model in rat caudate nucleus was applied. Nerve symptoms were scored at the fourth day after the operation, moreover, the other factors such as the cerebral index , the content of water , antioxidation capacity, blood rheology and morphological changes were tested at the same time. Results:
(1)In the vehicle group, nerve symptoms, content of water , cerebral index,content of MDA, activity of LDH increased greatly. (2)The dosage of 200mg/kg, 400mg/kg of AGNHW can improve significantly the symdrome (vs vehicle p<0.01or p<0.05) . In the vehicle group, the activity of SOD, erythrocyte deformability decreased greatly, on the contrary, the dosage of 200mg/kg, 400mg/kg of AGNHW increased these targets significantly ,( vs vehicle, p<0.01or p<0.05) . (3)Compared with AGNHW, the function of improving nerve symptoms and decreasing cerebral index of QZX- AGNHW was weakened. Conclusion:(1)AGNHW has protective effect on ICH, and it can improve nerve symptoms and content of water, also, decrease cerebral index, content of MDA and the activity of LDH. (2) The function of improving nerve symptoms and decreasing cerebral index of QZX-AGNHW was weakened.
2. Effects on the expressions of apoptosis - related proteinas BBB associated protein in rats with ICH of AGNHW containing Cinnabaris and Realgar or not Objective: To approach the effect of AGNHW on the apoptosis and apoptosis- related proteinas BBB associated with protein expressions. and compare the difference of the effects of AGNHW and QZX-AGNHW. Methods: Expressions of apoptisis (TUNEL), apoptisis -associated protein (Bcl-2,Bax)BBB associated protein (MMP9, AQP4)were observed by immunohistochcmistry of AGNHW on perihematomal isscue in rats with ICH. Results:(1)In the vehicle group, The expression of TUNEL, Bax, MMP9, AQP4 were increased, and the expressions of Bcl-2 deceased. (2) The dosage of 400mg/kg of AGNHW can decreased the expression of TUNEL, Bax, MMP9 (vs vehicle p<0.05). On the other hand, it can increase the espression of Bcl-2 (Vs vehicle, p<0.05). The dosage of 200mg/kg ,400mg/kg of AGNHW can decrease the expression of AQP4(Vs vehicle p<0.05, or p<0.01, respectively).(3)The dosage of 160mg/kg, 320mg/kg of QZX-AGNHW decrese the expression of MMP9, AQP4 (Vs vehicle, p<0.05 or p<0.05). Conclusion:(1)AGNHW protect neurons by affecting anti-apoptosis and related BBB permeability. (2)The function of related BBB permeability of QZX- AGNHW was similar to AGNHW, while the influence of anti-apoptosis decreased obviously.
3.Effects on MAPK signal transduction pathway in rats with ICH of AGNHW containing Cinnabaris and Realgar or not
Objective: To observe the effect of AGNHW and QZX-AGNHW on MAPK signal transduction pathway in rats with ICH, and compare their difference. Methods: Expression of the MAPK signal transduction pathway associated protein PKC、c-fos and c-jun was observed by immunohistochcmical. Expression of the MAPK signal transduction pathway family ERK、P38mRNA was observed by real time RT-PCR. Results: (1)The expression of PKC、c-fos、c-jun inceased after ICH injury, and the expressions of ERKmRNA deceased. However, the expression of P38 mRNA increased (Vs vehicle, p<0.01). (2) The dosage of 200mg/kg, 400mg/kg of AGNHW reduced the expression of c-fos, c-jun (Vs vehicle, p<0.01, p<0.05), The dosage of 400mg/kg of AGNHW reduced the expression of P38mRNA and PKC, and improve the expression of ERKmRNA(Vs vehicle, p<0.05). (3) The dosage of 160mg/kg, 320 mg/kg of QZX-AGNHW decrease c-fos expression(Vs vehicle, p<0.05 or p<0.01),and the dosage of 160mg/kg decrease PKC expression(Vs vehicle, p<0.05), the dosage of 320mg/kg decrease the expression of c-jun , P38mRNA, (Vs vehicle, p<0.01),and increase ERKmRNA expression (Vs vehicle, p<0.01). Conclusions: (1)Protective effects of AGNHW on neurons in rats with ICH was observed by influence activity of MAPK signal transduction pathway.
(2)The innfluence of QZX-AGNHW on MAPK signal transduction path-way was similar to AGNHW, but its function of decreasing expression of P38mRNA was weakened. Part two: Protective effects and its mechanism on primary cultured cortex neuron of AGNHW containing Cinnabaris and Realgar or not with serum or CSF containing drug
Protective effect of serum or CSF containing drug was observed on oxygen-glucose deprivation or GLU inducing cultured cortex neuron model. The influence of serum or CSF containing drug on MMP and [Ca~(2+)]i of GLU induced cultured cortex neuron model. Contents of determination Cinnaba and Realgar in serum or CSF containing drug were determined. 4 Protective effects and its mechanism on primary cultured cortex neuron of AGNHW containing Cinnabaris and Realgar or not with serum or CSF containing drug
Objective: To compare the protective effects of AGNHW and QZX-AGNHW with serum or CSF containing drug. Methods: Using cell damage models induced by oxygen/glucose deprivation and GLU,the serum and cerebrospinal fluid pharmacological methods were determined, in order to observe the function of cinnabar and realgar. Results: (1) The damage of oxygen-glucose deprivation increased the leakage rate of LDH. (2)observe morphocytological chages and measure the LDH leak rate , cytoactive , intracellular concentration of MDA and SOD. In the cells injured by the oxygen/glucose deprivation,LDH leak rate was significantly increased, while the cytoactivty was inhibited. These changes were blocked effectively by serum and cerebrospinal fluid containing AGNHW, especialy by the dose of 800mg/kg,and the cerebrospinal fluid with the additive proportion of 20% is better than the same dose. The result indicated that the effection of AGNHW is better than that of QZX-AGNHW. After cells were injured by GLU,LDH leak rate was also significantly increased,meanwhile, the cytoactive was inhibited,and morphocytologic changes were observed obviously. AGNHW had the significant protective actions when the dose reached 640mg/kg,and the cerebro spinal fluid with the additive proportion of 20% was better using the same dose. Conclusion: AGNHW has significant effects of protecting cells.
5 The effects on mitochondria membrane potential and intracellular Ca~(2+) concentration of rat cortical neurons injured by GLU of AGNHW and QZX-AGNHW
Objective: To observe the effects of AGNHW and QZX-AGNHW on mitochondria membrane potential and intracellular Ca~(2+) concentration of rat cortical neurons injured by GLU. Methods: Using the method of flow cytometry(FCM),mitochondria membrane potential and intracellular Ca~(2+) concentration of rat cortical neurons were measured, then the effects of AGNHW and QZX-AGNHW were compared on the basis of the above data. Results: In rats whose cortical neurons were injured by GLU,mitochondria membrane potential was observed to decrease,and the concentration of intracellular Ca~(2+) raised. The value of mitochondria membrane potential could be increased by AGNHW and QZX-AGNHW containing serum and cerebrospinal fluid. While the concentration of intracellular Ca~(2+) reduced evidently when acted by AGNHW containing cerebrospinal fluid and QZX-AGNHW containing serum and cerebrospinal fluid. Conclusion: AGNHW and QZX-AGNHW had the similar effects, which produced the protective effects on injuryed neurons through stabilizing mitochondria membrane potential and blocking calcium overload.
6 Determination of Concentration of cinnabar and realgar in serum and CSF of AGNHW and QZX-AGNHW
Objective: To determine whether As and Hg contained in cinnabar and realgar were absorbed into blood or cerebrospinal fluid (CSF) through BBB. And further to analyze if cinnabar and realgar play an important role in the protective effect of AGNHW. Methods: Inductively Coupled Plasma-mass was used to determine the concentration of As and Hg in serum and cerebrospinal fluid . Results:( 1) As in AGNHW could be tested in blood and might be absorbed into the CSF through BBB. The content of As in serum is 5.26 times that of As in CSF.(2) Trace As could be tested in serum and CSF of normal control group and QZX-AGNHW group.(3) The content of Hg was very near the limit of detection of instrument, so it could not be detected exactly. that is to say, the content of Hg need more accurate methods to be detected. Conclusion: Both Serum and CSF Contaning Drug of AGNHW contained As and Hg,the difference of neuro protective effect of AGNHW and QZX-AGNHW had relationship with As and Hg.
In summary, AGNHW alleviated the neurologic symptoms of rats after ICH; amelioration morphology changes induced by ICH; reduce cerebral index , improve erythrocyte deformability, all these results show its satisfactory protective effect to experimental ICH. The main mechanism of effect may as follows: It can relieve the injury induced by the toxic product such as excitatory amino acids(EAA), MDA and free redical. In addition, partially inhibit calcium overload induced by EAA, stabilize the mitochondria membrane potential. It has the function of anti-apoptosis by increasing the express of Bcl-2,and decreasing the express of Bax. It can surpression the expression of c-fos,c-jun,PKC,elevate the mRNA expression of ERK and decrease the mRNA expression of P38.their specific receptor. It can surpression the expression of AQP4,MMP9.Compaired with AGNHW , the function of QZX- AGNHW of increasing nerve symptoms, decreasing cerebral index was weakened .And the function of anti-apoptosis of QZX- AGNHW is not notable.
In this experiment ,we studied the protective effect of AGNHW and QZX- AGNHW to ICH rats.
1 In this study, the protective function of AGNHW and QZX-AGNHW on rats injured with ICH was analyzed. Accordingly entirety pharmacodynamics,serum phamacology and CSF phamacology , the effective differences of the two kinds of medicines were compared. The results show that cinnabar and realgar play an important role in the total prescription.
2 Based on the experiments of the mechanism analysis, AGNHW and QZX-AGNHW have the similar effects, which produce the protective effects on injuryed neurons through anti-apoptosis and blocking calcium overload.
3 For the first time, it is reported that the effection of AGNHW and QZX-AGNHW on the pathway of signal transconduction of MAPK.
引文
1 Andersong CS,Jamrozik KD,Burvill PW. Determining the incidence of Different subtype of stroke:result from the Perth Community Stroke study. 1989-1990. Med Aust, 1993, 158(2):85-89
2 Andersong CS,Jamrozik KD,Burvill PW. Determining the incidence of Different subtype of stroke:result from the Perth Community Stroke study. 1989-1990. Med Aust, 1993, 158(2):85-89
3 Andersong CS,Jamrozik KD,Burvill PW. Determining the incidence of Different subtype of stroke:result from the Perth Community Stroke study. 1989-1990. Med Aust, 1993, 158(2):85-89
4 Thrift Beauchamp NJ,Barker PB, WangP Y, et al. Imaging of acute cerebral ischemia. Radiology,1999,212(2):
5 Northern Manhattan Stroke,Teaching resources. Glial intercellular wave 2005(290):tr19.
6 Wu MF, Pang ZP, Zhuo M,et al.Prolonged membrane potential depolarization in cingulated pyramidal cell safter digit a mputation in adult rats.Mol Pain,2005,1:23.
7 Wijdicks EF, Fulgham JR. Acute fatal deterioration in putaminal hemorrhage. Stroke, 1995,26(10):1953-1955.
8 Kobayashi T. The roles of mitochondrial permeability transition in brain ischema. Hokkaido lgaku Zasshi,2000,75(4):243-252.
9 Lee KR, Colon GP, BetzAL,et al.Edema from intracerebral hemorrhage:the role of thrombin. Neurosurg,1996,84(1):91-96.
10 Heather C,et al.Intracerebral infusion of thromb in a sacause of braine dema. Neurosurg,1995,83(6):1045-1050
11 BrottT, Broderick J,Kothari R,et al.Early hemorrhage growth in patients with intracerebral hemorrhage.Stroke,1997,28(1):1
12 张昱,刘多三.高血压脑出血病理组织学改变与 CT 影像对照研究.中风与神经疾病杂志,1996,3:134~137
13 张昱,姜明辉,刘多三.实验性脑出血在不同时期脑电和病理组织学变化的研究.中风与神经疾病杂志,1997,4:138~140
14 张昱.脑出血的病理改变及病理生理过程.中国现代神经疾病杂志,2005,5(2)75~79
15 Mayer S A. Ultra-early hemostatic therapy for intracerebral hemorrhage. Stroke,2003,34:224
16 Fujii Y, Takeeuchi S, Sasaki O,et al. Multivariate analysis of preditors of hematoma enlargementin spontaneous intracerebral hemorrhage. Stroke,1998,29(6):1160
17 Xi G, Keep R F,HuaY,et al.Attenuation of thrombin-induced braine dema by cerebral thrombin preconditioning.Stroke,1999,30(6):1247-1255.
18 Wagner KR, Keep RF,et al.Role of blood clotformation on early edema development after experimental intracerebral hemorrhage.Stroke,1998,29(12):2580-2586.
19 Wagner KR ,XiG,HuaY,et al.Lobar intracerebral hemorrhage model in pigs:rapide dema deelopment in perihematomal white matter.Stroke,1996,27(3):4902497.
20 刘庆新,宋月雁,邓玉琴,等.不同剂量甘露醇对脑出血患者血小板 CD62p 和 CD42b 表达的影响,中风与神经疾病杂志,2005,22(6):554~555
21 Vaughan PJ,Pike CJ,Cotman CW,et al.Thromb ineceptor activation protects neurons and astro cytes from cell death produced by environmental insults.Neurosci,1995,15(7Pt2):5389-5401.
22 Roseberg,Matz PG,Weinstein PR,SharpFR .Heme oxygenase and heat shock protein 70 induction in glia and neurons through out rat brain after experimental intracerebral hemorrhage. Neurosurgery,1997,40(1):152-162.
23 Del Bigio MR,Yan HJ,Buist R,et al.Experimental intracerebral hemorrhage in rats: Magneticreas on anceimaging and histopatho logical correlates.Stroke,1996,27(9):2312-2320.
24 Heiss W D,KrachtL W,Thiel A,et al.Penumbral probability threshold sofcortical flumaze nilbinding and blood flowpredicting tissue outcome in patient switch erebral ischaemia.Brain,2001,124:20-29.
25 Yang GY ,Betz AL,Chenevert TL,et al.Experiment alintra cerebral hemorrhage : relationship between brainedema,blood flow,andblood-brain barrier permeability in rats. Neurosurg, 1994, 81 (1):93-102.
26 Beauchamp NJ, Barker PB,Wang PY,et al.Imaging of acute cerebral ischemia.Radiology,1999,212(2):307-324.
27 Carhuapoma JR, Wang PY, Beauchamp NJ,et al.Diffusion-weighted MRI and proton MR spectroscopic imaging in the study of secondary neuronal injury after intracerebral hemorrhage.Stroke,2000,31:726-732.
28 Mendelow AD,Machanisms of ischemic brain damage with intercerebrual hemorrhage Stroke,1993,24,1115~1117
29 Siddique MS,Fornandes HM,Wooldridge TD,et al.Reversible ischemia around intracerbral hemorrhage:a single-emission computerized tomography study. Neurosurg, 2002,96(4):7362741.
30 Bhasin RR,Xi G,Hua Y,et al.Experimental intracerebral hemorrhage:effect of lysed erythrocytes on brain edeme and blood-brain barrier permeability. Acta Neuro chinir Suppl,2002,81:249-251.
31 Xue M, Del Bigio MR.Intracortical hemorrhage injury in rats:relationship between blood fraction sand brain cell death.Stroke,2000,3(7):1721-1727.
32 林秀瑾,田少华.蛛网膜下腔出血患者脑血流改变.中华老年心脑血管病杂志,2004(6)3:159
33 崔元孝,张清华,刘兆孔.等.蛛网膜下腔出血患者血中纤溶活性变化的临床意义. 山东医药,2005,1:5~7
34 张学勤,王阳,秦朝晖,等. 检测脑梗死患者血小板膜表面 P-选择素与纤维蛋白原受体的临床意义. 北京医学 2005,27(2):92~94
35 Vaughan PJ,Pike CJ,Cotman CW,et al.Thrombin receptor activation protects neuron sandastro cytes from cell death produced by environmental insults.Neurosci,1995,15:538925401.
36 Matsushita K,Meng W,Wang X,et al.Evidence for apoptosis after intercerebral hemorrhage in rats triatum. Cereb Blood FlowMetab,2000,20:396-404.
37 Qureshi Al, Suri MF, Ostrow PT,ET Al. Apoptosis as a form of cell death in tracerebral hemorrhage.Neurosurgury,2003 52,1041~1047
38 MacManus ,HuaY, XiG ,Keep RF,et al.Complement activation in the brain after experimental intracerebral hemorrhage. J Neurosurg,2000,92:1016-1022.
39 MacManus ,HuaY, XiG ,Keep RF,et al.Complement activation in the brain after experimental intracerebral hemorrhage. J Neurosurg,2000,92:1016-1022.
40 Xue M,Del Bigio MR.Acute tissue damage after injections of thrombin and plasmin into rat sriatum.Stroke,2001,32:2164-2169.
41 Tremblay R, Chakravarthy B, Hewitt K, et al. Pharmacologic reduction of mean arterial ressure does not adversely affect regional cerebral blood flow and intracranial pressure in experimental. Neurosci, 2000, 20(19):7183-92
42 Zhang C, Shen W, Zhang G. Thrombin receptor activation protects neuron sandastro cytes from cell death produced by environmental insults Neurosci Lett , 2002,328(3):265-8
43 Dingledine R, Borges K, Bowie CD, et al. Evidence for apoptosis after intercerebral hemorrhage in rats triatum. Cereb Blood FlowMetab .Pharmacol Rev, 1999 ,51(1):7-16
44 Matsumoto S. Friberg H , Ferrand-Drake M , et al. Experimental intracerebral hemorrhage in rats: Magneticreas on anceimaging and histopatho logical correlates Cere Blood Metab, 1999, 19(7):736-741
45 Paschen W , Doutheil. Acute tissue damage after injections of thrombin and plasmin into rat sriatum Cere Blood Metab, 1999,19(1):1-18
46 Connor,Schwab BL,Guerini D, Didszun, C, et al. Apoptosis as a form of cell death in tracerebral hemorrhage .Cell Death Differ,2002,9(8):818-31
47 Ghribi O,,Herman MM, Savory.Acute tissue damage after injections of thrombin and plasmin into rat sriatum. Neurosci Lett, 2002,324(3):217-21
48 Blomgren K, Zhu C, Wang X, et al. Evidence for apoptosis after intercerebral hemorrhage in rats triatum .Biol Chem,2001,276(13):10191-8
49 Ma J, Endres M, Moskowitz MA. Br. Apoptosis as a form of cell death in tracerebral hemorrhage.Pharmacol, 1998,124(4):756-762
50 Nakagawa T, Zhu H, Morishima N, et al . Acute tissue damage after injections of thrombin and plasmin into rat sriatum.Nature ,2000,403(6765):98-103
51 Krajewski S, Krajewska M, Ellerby LM, et al. Thrombin receptor activation protects neuron sandastro cytes from cell death produced by environmental insults .Proc Natl Acad Sci USA,1999 ,96(10):5752-7
52 Berke ,Shibasaki F, Hallin U, Uchino H. Evidence for apoptosis after intercerebral hemorrhage in rats triatum .Biochem (Tokyo),2002,131(1):1-15
53 Min Chen, Dong-Jun Won, Stan Krajewski , et al. Biology of ischemic cerebral cell death. Prog Cardiovasc Dis .Biol Chem,2002,277(32):29181-29186
54 Suprynowicz ,Xie J. Guo Q, Zhu H, et al. Dynamics of polymor phonuclear leukocyte accumulation in acute cerebral infarction and the ircorrelation with brain tissue damage .Brain Res,2000,82(1-2):107-13
55 Miettinen S, Roivainen R, Keinanen R, et al. Ischemic stroke is associated with a systemic increase of blood mononuclear cells expressing interleukin-8 mRNA . Neurosci, 1996,16(19): 36-45
56 Ziemka-Nalecz M, Zalewska T, Zajac H, et al. Apoptosis as a form of cell death in tracerebral hemorrhage. Neurochem Int,2003,42(3):5-14
57 Scu ,Bastianetto S, Zheng WH, Quirion R. Inhibition of human lung cancer cell growth by the peroxisome proliferator-activated recrptor-gamma agonists through induction of a poptosis. Neurochem,2000,74(6): 68-77
58 Parnham M, Sies H. Brain protection by resveratrol and fenofibrate against stroke requires peroxisome proliferator-activated receptor alpha in mice. Expert opin Investig Drugs, 2000,9(3):07-19
59 Gong C, Bopulis N,Qian,et al.Intracerebral hemorrhage-induced neuronal dearh, Neurosurger -y , 2001,48(4):875~880
60 Guohua, Heiss WD,KrachtLW, Thiel A,et al.Penumbral probability thres holds of cortical flumaze nilbinding and blood flow predicting tissue outcome in patients with cerebral ischaemia.Brain,2001,124:20-29.
61 Bereczki D.Liu M,Prado G F,et al.Cochrane report:Asystematic review of mannitol therapy for acute ischemic stroke and cerebral parenchymal hemorrhage.Stroke,2000,31(11):2719~22
62 Bereczki D,Liu M,doPrado G F,et al. Mannitol for acute stroke.Cochran Data base Syst Rev,2001,(1):1153
63 Popa G,Amaireh M,Dinu M,et al.Acetazolamide therapy evaluation in haemorrhagic stroke.Rom .Neurol Psychiatry,1995,33(2):145~155
64 Rosenberg G A,Estrada EY.Atrial natriuretic peptide blocks hemorrhagi brain edema after 4-hour delay in rats. Stroke, 1995,26(5):847~7
65 Qureshi AI,Wilson DA,Hanley DF,et al.Pharmacologic reduction of mean arterial ressure does not adversely affect regional cerebral blood flow and intracranial pressure in experimental intracerebral hemorrhage. Crit Care Med 1999,27(5):965~971
66 Powers WJ,Zazulia AR, Videen TO,et al.Autoregulation of cerebral blood flow surrounding acute (6to22hours) intracerebral hemorrhage. Neurology 2001,57(1):18~24
67 Lyden PD. Jackson-Friedman C,Lonzo-DoktorL. Madical therapy for intrace-rebral hematoma with the gamma-aminobutyric acid-A agonist muscimol Strok,1997,28(2):387~391
68 Lyden P,Shin C,Jackson-Friedman C,et al.Effect of ganaxolone in a rodent model of cerebral hematoma.Stroke,2000,31(1):169~175
69 Clark W,Gunion-Rinker L, Lessov N,et al. Citiconine treatment for experimental in tracerebral hemorrhage in mice.Stroke, 1998,29(10):2136~40
70 Bullock MR,Merchant RE,Carmack CA,et al.An open-label study of CP-101,606 in subjects with a severe traumatic head injury or spontaneous intracerebral hemorrhage. AnnN YA cad Sci, 1999,890:51~58
71 Mayne M, FotheringhamJ,Yan H,et al.Adenosine A-A receptor activation reduces proinflammatory events and decreases cell death following intracere-bral hemorrhage.Ann Neurol,2001,49(6):727~735
72 DelBigio MR,YanHJ,XueM.Intracerebral in fusion of a second-generation ciliary neurotrophic factor reduces neuronal loss in rats triatum following experimental in tracerebral hemorrhage. JNeurol Sci,2001,192(1~2):53~59
73 PeelingJ,Yan H J,CorbettD,et al.Effect of FK2506 on inflammation and behavioral outcome following intracerebral hemorrhage in rat..Exp Neurol 2001,167(2):341~347
74 Peeling J,DelBigioMR,CorbettD,et al.Efficacy of disodium4-[(tert-bu-tylimino)methyl] benzene-1,3-disulfonate N-oxide(NXY2059), a free radical trapping agent,in a rat model of hemorrhagic stroke.Neuro pharmacology 2001,40(3):433~9
75 Mohadijer M, Braus DF,MyersA,et al.CT-stereotactic fibrinolysis of spon-taneous intracerebral hematomas.NeurosurgRev,1992,15(2):105~110
76 Miller DW,Barnett GH,Kormos DW,et al.Stereotactically guided throm-Bolysis of deep cerebral hemorrhage:preliminary results.Cleve Clin J Med1993,60(4):321~324
77 杨军,潘树茂,刘运祥,等.基因工程链激酶在鼠脑内血肿模型中局部应用的实验研究.中国现代医学杂志,1999,10;9(10):16~17
78 Schaller C,Rohde V,HasslerW. Local thrombolytic treatment of spontaneous intracerebral hemorrhage with plasminogen activator.Indication and Limits Nervenarzt, 1995, 66(4): 75 ~ 81
79 Deinsberger W, VogelJ,Fuchs C,et al.Fibrinolysis and aspiration of experimental intracerebral hematoma reduces the volume of ischemic braininrats Neurol Res,1999,21(5):517~23
80 贾宝祥,孙仁泉,左鸿,等. 穿刺射流及液化技术治疗高血压脑出血的初步报告.中国神经精神病杂志.1996,22:233
81 郭岩,张文斌,刘劲瑞.等.尿激酶加自体血清治疗重症脑室出血的应用.中华神经外科杂志,1999,15:236
82 Teerntra OP,Evers SM,Lodder ,et al.Stereotactic treatment of intracerebral hematoma by means of a plasminogen activatoria multicenter randomized trial.Stroke,2003,34:968
83 Xue M,Bigio M,Frepe K,et al.Acute tissue damage after injections of thrombin and plasmin into rat striatum.Stroke,2001,32(9):2164~2169
1 顾宁,周仲英.通下法治疗急性脑出血研究进展.中国中医急诊,2000,9(5):227-229
2 林亚明,任继学.破瘀醒神法在出血性中风急性期的应用.中医杂志,1992,33(5):251
3 黄培新,刘茂才.中医专科专病临床诊治丛书·神经科专病临床诊治.北京:人民卫生出版社,2000,407-411
4 郑国庆,黄培新.中风病阴阳为纲辨治源流.中华医史杂志, 2003,33(4):227-230
5 李 求 兵 . 浅 述 “ 血 证 论 ” 治 血 四 法 对 中 风 脑 出 血 治 疗 的 指 导 意 义 . 北 京 中医,2003,22(6):17-18
6 陈胜,陈武进.应用“治血四法”探讨出血性中风的治疗.甘肃中医,1995,8(6):1-2
7 汤克仁,等.复方丹参液静滴治疗急性高血压脑出血 30 例.山东中医杂志,1987;5:32
8 汤克仁,等.复方丹参液静滴治疗急性高血压脑出血 30 例.山东中医杂志,1987;5:32
9 翁维良.血瘀证及活血化瘀中西医结合研究新进展.中西医结合杂志,1987;7(3):190
10 谢道珍,等.脑血康治疗高血压脑出血的临床与实验研究.中西医结合杂志,1988;8(6):341
11 梁清华,等.平肝熄风汤对脑溢血患者日常生活活动能力的影响.湖南医科大学学报,1994;19(6):513
12 梁清华,等.脑溢安颗粒剂与汤剂治疗肝阳化风型急性脑出血临床疗效比较.湖南医科大学学报,1995;20(3):215
13 周绍华.出血性中风的中医治疗.实用中西医结合杂志,1992;5(3):139
14 许振亚.出血性中风的证治经验.中医杂志,1993;3(1):17
15 薛金发,等.祛瘀通下法治疗脑溢血 33 例.江西中医药,1995;26(4):19
16 虢周科,等.脑出血的病因病机及辨证论治规律探讨.陕西中医,1992;13(11):496
17 李克玲,黄启福,严京,等.中风脑得平冲剂对大鼠实验性脑血肿诱发脑水肿的影响.北京中医药大学学报,1997,20(3):35.
18 李克玲,黄启福,朱陵群,等.中风脑得平冲剂对实验性兔脑血肿的作用.北京中医药大学学报,1996,19(4):22.
19 李克玲,黄启福,张丽萍,等.醒脑健神胶囊对实验性脑血肿诱发大鼠脑水肿的作用.中国中医急症,1997,6(2):82.
20 刘莉,钱家骏,胡加跃,等.醒脑健神胶囊清开灵注射液对急性脑出血大鼠脑含水量、离子含量及自由基代谢的影响.北京中医药大学学报,1997,20(1):38.
21 李克玲,黄启福,蒋玉凤,等.清开灵注射液治疗家兔脑血肿的实验研究.中国中西医结合杂志,1997,17(2):91.
22 陈绍宏,张晓云,刘永家,等.逐瘀化痰口服液治疗急性脑出血的临床及实验研究.中国中医急症,1995,4(2):58.
23 黄世敬,黄启富,孙塑伦,等.救脑宁注射液对实验性脑出血大鼠脑水肿及脂质过氧化的影响.北京中医药大学学报,1998,21(2):38.
24 杨万章,万中民,李明富,等.逐瘀化痰汤等三方对大鼠脑出血脑组织 MDA、Ca2+、Fe2+影响的实验研究.北京中医药大学学报,1997,20(6):35.
25 胡随瑜,金益强,王勇华,等.脑溢安颗粒剂主要药效学研究.中药新药与临床 药理,1997,8(3):150.
26 李德伟,任恩发,许红,等.中药中风脑得平冲剂对脑出血大鼠尾壳核壳—脑啡肽(L-ENK)含量影响的实验研究.中国中医基础医学杂志,1996,2(1):39.
27 白丽敏,孙红梅,朱培纯,等.醒脑健神丹对急性脑出血大鼠脑内神经肽 Y 含量的影响.中国中医基础医学杂志,1995,1(4):33.
28 蒋玉凤,朱陵群,黄启福,等.清开灵注射液对 SHRSP 出血性中风海马区兴奋性氨基酸含量的影响.中国中医急症,1997,6(5):219.
29 钱家骏,刘莉,庞鹤,等.醒脑健神胶囊、清开灵注射液对急性脑出血大鼠血浆 ET、CK 及 VWF 水平的影响北京中医药大学学报,1997,20(2):25.
30 孙红梅,白丽敏,朱培纯,等.醒脑健神胶囊对实验性脑出血大鼠脑内 β-内啡肽(β-EP)影响的实验研究.中国中医基础医学杂志,1997,3(6):17.
31 黄培新,刘茂才,陈根成,等.毛冬青甲素对高血压性大鼠脑内血肿及脑微血管超微结构的影响.中国中医急症,1997,6(3):127.
32 林霞,潘其民,金益强,等.平肝熄风汤对出血性中风大鼠神经病理变化的影响.湖南医科大学学报,1994,19(3):204.
33 王玉良,唐可欣,王益光,等.牛黄熄风胶囊治疗脑出血大鼠的体感诱发电位观察.潍坊医学院学报,1997,19(2):86.
34 杨万章,万中民.逐瘀化痰汤、三生饮、抵挡汤对实验大鼠脑出血后 EEG 变化的研究.北京中医药大学学报,1996,19(3):64.
35 黄海青,朱敬荣,开国维.114 例出血性中风中医证型及脑部 CT 的研究.南京中医药大学学报,1996,12(3):15.
36 丁沧清 , 智慧 , 邢海燕 , 等 . 脑出血的临床证型与影像指标初探 . 辽 宁 中 医 杂志,1995,22(11):486.
37 梁清华,黎杏群,李家邦,等.出血性中风病人的脑 CT、中医辨证与血液流变学的关系探讨湖南医科大学学报,1995,20(6):554.
38 魏丹,张东友,王金娥.脑出血中医临床证型与头颅 CT 表现的相关性.中医药研究,1999,15(3):26.
39 肖小华,舒晓春,刘元章,等.中风 CT 扫描、血液流变学与辨证的研究.江西中医学院学报,1995,7(2):2.
40 周庆芳,刘华,许存森.出血性中风中医证型与颅脑核磁共振检查关系的研究.浙江中医杂志,1995,30(12):550.
41 孔 炳 耀 , 李 小 敏 . 中 风 急 性 期 血 清 血 脂 脂 蛋 白 亚 组 分 与 辨 证 关 系 . 辽 宁 中 医 杂志,1997,24(5):199.
1 张昱.脑出血的病理改变及病理生理过程.中国现代神经疾病杂志,2005 5(2):75~78
2 Rosenberg GA,Mun-Bryces,Wesley M.et al.collagenase-induced intracerebral hemorrhage in rats.Stroke,1990,21:801～811
3 Alexis NE . Neurobehavioral consequences of induced spreading depression following photothrombotic middle cerebral artery occlusion.BrainRes,1996,(7):273～282
4 张震中,吕安坤,张燕.脑出血大鼠模型的血液流变学研究.中华实用中西医杂志,2004,4(17):3673~3675
5 隋邦森.脑血管疾病 MR、CT、DSA 与临床.北京:人民卫生出版社,1991:196~1971
6 李俐涛,尹静,张祥建,等.脑出血模型.河北医科大学学报,27(5):496~499
7 王永炎.今日中医内科学.北京:人民卫生出版社,2000,63~65
8 杨菊芬.血液流变学对脑出血与脑梗塞的诊断价值.遵义医学院学报,2000,23(2):156
9 侯玉立.血液流变测定在脑出血与脑梗塞中的应用.山西医科大学学报, 2001,32(3):226-227
10 杨舒,朱雯梅,李晓萍.脑出血与脑梗塞患者血液流变学及凝血功能的改变.中华实用医学, 2002, 4(23) :93-94.
11 Chesney J A,Kondoh T, Conrad J A,et al.Collagenase induced intrastriatal hemorrhage in rats results in long-term locomotor deficits.Stroke,1995,26(2):312-317
12 夏鹰,陈衔城,季耀东.等.脑出血血肿周围脑组织的糖代谢、氧自由基及血脑屏障的变化与脑水肿形成的关系.复旦学报(医学版),2002,3(2):119~223
13 Stroke Therapy Academic Industry Roundtable(STAIR).Recommendations for standards regarding preclinical neu-roprotective and restorative drug development. Stroke, 1999, 30:2752~2758
1 Gong C,Boulis N,Qian I,et al.intracerebral hemorrhage-induced neuronal death.Neurosurgery, 2001,48,875~882
2 Johnsen SP, Pedersen L, Friis S,et al.Nonaspirin nonsteroidal anti-inflammatory drugs and risk of hospitalization for intracerebral hemorrhage:population based case control study . Stroke, 2003, 34:387~391
3 成样林,赵成三,汪华.丙戊酸钠对戊四氮致大鼠海马 Bax 和 Bcl-2 表达的影响.临床神经病学杂志,2004,17(5):351~353
4 Mendelow AD.Mechanisms of ischemic braindamage with intracerebral hemorrhage. stroke, 1993, 24:1115-1117
5 Rosenberg GA,Navratil M,Barone F.proteolytic cascade enzymes increase in focal cerebral ischemia off gerbils.Neurochem Res,1996,16;360
6 Kobayashi H,Minami S,Itoh S,et al.Aquaporin subtypes inratcerebral microvessels.Neurosci Lett, 2001, 297(3):163-166.
7 石善溶.免疫组织化学技术.成都:四川科技出版社
8 纪小龙.免疫组织化学新编.北京:人民军医出版社
9 Johnasson AC, Visse E, Widegren B, et al. Computerized image analysis as a tool to quantify infiltrating leukocytes: A compariaon between highand low magnification images. J Histochemisry and Cytochemistry, 2001;49(9):1073-1079
10 O’Leary TJ. Standardization in immunohistochemistry. Appl Immunohistochem Mol Morphol, 2001;9(1):3-8
11 杨继文,贺大权,张秋才.等.脑出血的病理损伤机制.脑与神经疾病杂志,2005,13(6):470-471
12 Belayev L, Busto R, Zhao W, etal. Quantitative evaluation of blood-brain barrier permeability following middle cerebral artery occlusion in rats. Brain Res. 1996, 739(1-2); 88.
13 Betz AL, Coester HC. Effect of steroids on edema and sodium uptake of the brain during focal ischemia in rats. Stroke. 1990, 21(8); 1199.
14 Steller H, Mechanisms and genes of cellular suicide. Science, 1995, 267: 1445～1449
15 Hochenkery DM, Oltval ZN, Yin XM, et al.Bcl-2 fuctions in an antioxidant pathway to prevent apoptosis.Cell, 1993, 75: 241～251
16 Oltvai ZN, Milliman CL. Korsmeyer SJ. Bcl-2 heterodimerizes in vivo wih a conserbed homolog, bax,thataccelerates programed cell death. Cell, 1993,74:609～615
17 Stanley JK. Regulators of cell death. TIG, 1995, 11(3): 1011
1 许川山,刘志君,唐建民,等.低强度激光促细胞增殖效应与丝裂原激活蛋白激酶信号传导途径.中国激光医学杂志,2000,(2):47-48
2 周志琦,刘强.真核生物的 MAPK 级联信号传递途径.生物化学与生物物理进展,1998,25(6):496-503
3 Ferrer I, Friguls B, Dalfo E, et a l. Early modifications in the expression of mitogen activated protein kinase (MAPK/ERK) , stress-activated kinases SAPK/JNK and p38, and their phosphorylated substrates following focal cerebral ischemia. Acta Neuropathol (Berl) , 2003, 105 (5) : 425-437.
4 Xia ZG, Martin D, Joel B, et al.Opposing effets of ERK and JNK P38MAP kinase on apoptosis.Science,1995,270:1326-1331.
5 王凌, 李大金, 朱影.脱氢表雄酮经不依赖于雌、雄激素受体的 MAPK 信号途径抑制成骨细胞凋亡.分子细胞生物学报,39(4) 365-372
6 赵婷,侯孟君,肖勇梅,等.ERK1/2 和 P38 信号通路在瘦素诱导巨噬细胞 TNF-α表达中的作用.中山大学学报(医学科学版),2006(9):500-505
7 赵婷,侯孟君,肖勇梅,等.ERK1/2 和 P38 信号通路在瘦素诱导巨噬细胞 TNF-α表达中的作用.中山大学学报(医学科学版),2006(9):500-505
8 Gutcher J Webb PR, Anderson NG. The isoform-specific regulation of apotosis by protein kinase C.Cell Mol Life Sci,2003,6:1061-1070
9 黄河清,陈康宁,屠永华,等.培养神经元缺 PKC 活性变化与细胞凋亡关系的研究,第三军医大学学报,2002,12:1399-1401
10 Simi A , Ingelman-Sundberg M , T indberg N. Neuroprotective agent chlomethiazole attenuates c-fos, c-jun, and AP-1 activation through inhibition of p38 MAP kinase. Cereb Blood Flow M etab, 2000, 20 (7) : 1077-1088.
1 获原幸夫等.血清添加法を用し新ぃ实验系こよゐ汉方剂の研究.细谷英夫,山雄一:最新の汉方药理-汉方药の科学ち验证と展望.Tokyo:ExceptaMedia,Ltd.1988
2 杨彦芳;王玉芹.中药复方血清药理学方法规范化探讨.中国中西医结合杂志 2000. 20(5):380~382.
3 梅建勋,张伯礼,陆 融。中药脑脊液药理学研究方法的初建,中草药 2000,31(7):523~526
4 洪庆涛,唐一鹏.新生大鼠大脑皮层神经细胞的体外原代培养.神经解剖学杂志,1994,10(3):259.
5 张挺 , 于鸿儒 , 杨菁 , 等 . SD 乳鼠海马神经细胞原代培养方法的探讨 . 锦州医学院学报,2005,26(5):47-49
6 刘娜,左萍萍,周帆,张放,等.连二亚硫酸钠致 PC12 和 NG108 15 细胞拟缺血损伤研究. 中国药理学通报,1998;14(6):525-529
7 聂亚雄,黎杏群,梁清华.脑溢安含药血清对谷氨酸致培养大鼠皮层神经元损伤的保护作用.湖南医科大学学报, 2002,27(5):429-431
8 李仪奎,吴健宇.血清药理试验中采血时间的通法方案.中国药理学通报,1999;15(6):569-70
9 王金华,叶祖光,梁爱华,等.安宫牛黄丸中砷、汞在正常和脑缺血模型大鼠体内的吸收与分布研究.中国中药杂志,2003,7:639-643
1 Ferri KF,Kroemer G.Mitochondria-the suicide organelles,Bioessay,2001,23(2):111-115
2 谭勇,梁清华,李霞玲,等.平肝熄风汤对脑出血大鼠海马线粒体膜电位的影响.中南大学学报(医学版),2004,29(1):35-381
3 高春锦,赵立明,夏成清,等.葛环高压氧对 CO 中毒大鼠海马中钙离子、cGMP 及血浆 NO含量的影响.中华航海医学杂志,1999,6(4): 226-229
4 Kiyoshi K, Hisao Y. Mild hypothermia revived counter measure against ischemic neuronal damages.Neuroscience research, 1998, 32:103～117
5 Nakayama R, Yano T, Ushijima K, et al. Effects of dantrolene on extracellular glutamate concentration and neuronal death in the rat hippocampal CAI region subjected to transient ischemia. Anesthesiology,2002, 96(3): 705～710
6 朱陵群,范吉平,黄启福,等.人参皂甙 1 对大鼠胎鼠海马神经细胞凋亡的影响.中国病理生理杂志,2001,17(12):1229-12311
7 管增伟,王盛兰,李勇,等.凋亡细胞细胞膜和线粒体的动态变化.卫生研究,2000,29(2):83-86
8 Harry I, Michael FB, Tsuyoschi S,et al. Nitricoxide production and perivascular tyrosine nitration in brain after carbonmonoxid poisoningintherats.JClinInvest,1996,97(10):2260-2267.
1 张丽敏.全血中 16 种微量元素的微波消解电感耦合等离子体质谱测定法.中华劳动卫生职业病杂志,2006,24(1):50~51
2 王刚,陈荣达,林炳承,等.中药中微量元素测定的研究进展.药物分析杂志,2002,22(2):151
3 邱招钗,杨妙峰,何鹰,等.用ICP-MS法测定四个不同地区泽泻种微量元素的含量.福建分析测试,2003,12(2):1739.
4 陈浩,梁沛,胡斌,等.电感耦合等离子体原子发射光谱/质谱法在中药微量元素及形态分析中的应用.光谱学与光谱分析,2002,22(6):1019.
5 高雅,李更生,赵唯贤,等.反反正正论朱砂.中国中医药信息杂志,1999,6(12):42~43.
6 郝红缨.四硫化四砷对急性早幼粒细胞白血病细胞株 NB4 的凋亡作用.中国药理学与毒理学杂志,2002,16(1):37
7 陈思宇.雄黄诱导 K562 细胞调亡中 Bcl-2 和 Bax 的表达.第四军医大学学报, 2002, 23(9):790.
8 刘延方 , 等 . 硫化砷诱导 NB4 细胞调亡及细胞周期阻滞的研究 . 中华血液学杂志,2000,21(12):647.
9 张昌,等.硫化砷对 K562/ADM 细胞 HSP70 蛋白表达的影响. 中国癌症杂志,2001,11(1):33
10 周霭祥.青黄散治疗慢粒 25 例近期疗效观察.中国中西医结合杂志,1981, 1(1):16.
11 陈思宇.雄黄对急性早幼粒细胞白血病细胞诱导调亡中和促进分化的双重作用.西安交通大学学报(医学版),2002,23(4):401.
12 Deng Y., et al. Size effects of realgar particles on apoptosis in a human umbilical vein endothelial cell line BEC-304. Pharmacological Research, 2001, 44 (6) :513.
2 Andersong CS,Jamrozik KD,Burvill PW. Determining the incidence of Different subtype of stroke:result from the Perth Community Stroke study. 1989-1990. Med Aust, 1993, 158(2):85-89
3 Andersong CS,Jamrozik KD,Burvill PW. Determining the incidence of Different subtype of stroke:result from the Perth Community Stroke study. 1989-1990. Med Aust, 1993, 158(2):85-89
4 Thrift Beauchamp NJ,Barker PB, WangP Y, et al. Imaging of acute cerebral ischemia. Radiology,1999,212(2):
5 Northern Manhattan Stroke,Teaching resources. Glial intercellular wave 2005(290):tr19.
6 Wu MF, Pang ZP, Zhuo M,et al.Prolonged membrane potential depolarization in cingulated pyramidal cell safter digit a mputation in adult rats.Mol Pain,2005,1:23.
7 Wijdicks EF, Fulgham JR. Acute fatal deterioration in putaminal hemorrhage. Stroke, 1995,26(10):1953-1955.
8 Kobayashi T. The roles of mitochondrial permeability transition in brain ischema. Hokkaido lgaku Zasshi,2000,75(4):243-252.
9 Lee KR, Colon GP, BetzAL,et al.Edema from intracerebral hemorrhage:the role of thrombin. Neurosurg,1996,84(1):91-96.
10 Heather C,et al.Intracerebral infusion of thromb in a sacause of braine dema. Neurosurg,1995,83(6):1045-1050
11 BrottT, Broderick J,Kothari R,et al.Early hemorrhage growth in patients with intracerebral hemorrhage.Stroke,1997,28(1):1
12 张昱,刘多三.高血压脑出血病理组织学改变与 CT 影像对照研究.中风与神经疾病杂志,1996,3:134~137
13 张昱,姜明辉,刘多三.实验性脑出血在不同时期脑电和病理组织学变化的研究.中风与神经疾病杂志,1997,4:138~140
14 张昱.脑出血的病理改变及病理生理过程.中国现代神经疾病杂志,2005,5(2)75~79
15 Mayer S A. Ultra-early hemostatic therapy for intracerebral hemorrhage. Stroke,2003,34:224
16 Fujii Y, Takeeuchi S, Sasaki O,et al. Multivariate analysis of preditors of hematoma enlargementin spontaneous intracerebral hemorrhage. Stroke,1998,29(6):1160
17 Xi G, Keep R F,HuaY,et al.Attenuation of thrombin-induced braine dema by cerebral thrombin preconditioning.Stroke,1999,30(6):1247-1255.
18 Wagner KR, Keep RF,et al.Role of blood clotformation on early edema development after experimental intracerebral hemorrhage.Stroke,1998,29(12):2580-2586.
19 Wagner KR ,XiG,HuaY,et al.Lobar intracerebral hemorrhage model in pigs:rapide dema deelopment in perihematomal white matter.Stroke,1996,27(3):4902497.
20 刘庆新,宋月雁,邓玉琴,等.不同剂量甘露醇对脑出血患者血小板 CD62p 和 CD42b 表达的影响,中风与神经疾病杂志,2005,22(6):554~555
21 Vaughan PJ,Pike CJ,Cotman CW,et al.Thromb ineceptor activation protects neurons and astro cytes from cell death produced by environmental insults.Neurosci,1995,15(7Pt2):5389-5401.
22 Roseberg,Matz PG,Weinstein PR,SharpFR .Heme oxygenase and heat shock protein 70 induction in glia and neurons through out rat brain after experimental intracerebral hemorrhage. Neurosurgery,1997,40(1):152-162.
23 Del Bigio MR,Yan HJ,Buist R,et al.Experimental intracerebral hemorrhage in rats: Magneticreas on anceimaging and histopatho logical correlates.Stroke,1996,27(9):2312-2320.
24 Heiss W D,KrachtL W,Thiel A,et al.Penumbral probability threshold sofcortical flumaze nilbinding and blood flowpredicting tissue outcome in patient switch erebral ischaemia.Brain,2001,124:20-29.
25 Yang GY ,Betz AL,Chenevert TL,et al.Experiment alintra cerebral hemorrhage : relationship between brainedema,blood flow,andblood-brain barrier permeability in rats. Neurosurg, 1994, 81 (1):93-102.
26 Beauchamp NJ, Barker PB,Wang PY,et al.Imaging of acute cerebral ischemia.Radiology,1999,212(2):307-324.
27 Carhuapoma JR, Wang PY, Beauchamp NJ,et al.Diffusion-weighted MRI and proton MR spectroscopic imaging in the study of secondary neuronal injury after intracerebral hemorrhage.Stroke,2000,31:726-732.
28 Mendelow AD,Machanisms of ischemic brain damage with intercerebrual hemorrhage Stroke,1993,24,1115~1117
29 Siddique MS,Fornandes HM,Wooldridge TD,et al.Reversible ischemia around intracerbral hemorrhage:a single-emission computerized tomography study. Neurosurg, 2002,96(4):7362741.
30 Bhasin RR,Xi G,Hua Y,et al.Experimental intracerebral hemorrhage:effect of lysed erythrocytes on brain edeme and blood-brain barrier permeability. Acta Neuro chinir Suppl,2002,81:249-251.
31 Xue M, Del Bigio MR.Intracortical hemorrhage injury in rats:relationship between blood fraction sand brain cell death.Stroke,2000,3(7):1721-1727.
32 林秀瑾,田少华.蛛网膜下腔出血患者脑血流改变.中华老年心脑血管病杂志,2004(6)3:159
33 崔元孝,张清华,刘兆孔.等.蛛网膜下腔出血患者血中纤溶活性变化的临床意义. 山东医药,2005,1:5~7
34 张学勤,王阳,秦朝晖,等. 检测脑梗死患者血小板膜表面 P-选择素与纤维蛋白原受体的临床意义. 北京医学 2005,27(2):92~94
35 Vaughan PJ,Pike CJ,Cotman CW,et al.Thrombin receptor activation protects neuron sandastro cytes from cell death produced by environmental insults.Neurosci,1995,15:538925401.
36 Matsushita K,Meng W,Wang X,et al.Evidence for apoptosis after intercerebral hemorrhage in rats triatum. Cereb Blood FlowMetab,2000,20:396-404.
37 Qureshi Al, Suri MF, Ostrow PT,ET Al. Apoptosis as a form of cell death in tracerebral hemorrhage.Neurosurgury,2003 52,1041~1047
38 MacManus ,HuaY, XiG ,Keep RF,et al.Complement activation in the brain after experimental intracerebral hemorrhage. J Neurosurg,2000,92:1016-1022.
39 MacManus ,HuaY, XiG ,Keep RF,et al.Complement activation in the brain after experimental intracerebral hemorrhage. J Neurosurg,2000,92:1016-1022.
40 Xue M,Del Bigio MR.Acute tissue damage after injections of thrombin and plasmin into rat sriatum.Stroke,2001,32:2164-2169.
41 Tremblay R, Chakravarthy B, Hewitt K, et al. Pharmacologic reduction of mean arterial ressure does not adversely affect regional cerebral blood flow and intracranial pressure in experimental. Neurosci, 2000, 20(19):7183-92
42 Zhang C, Shen W, Zhang G. Thrombin receptor activation protects neuron sandastro cytes from cell death produced by environmental insults Neurosci Lett , 2002,328(3):265-8
43 Dingledine R, Borges K, Bowie CD, et al. Evidence for apoptosis after intercerebral hemorrhage in rats triatum. Cereb Blood FlowMetab .Pharmacol Rev, 1999 ,51(1):7-16
44 Matsumoto S. Friberg H , Ferrand-Drake M , et al. Experimental intracerebral hemorrhage in rats: Magneticreas on anceimaging and histopatho logical correlates Cere Blood Metab, 1999, 19(7):736-741
45 Paschen W , Doutheil. Acute tissue damage after injections of thrombin and plasmin into rat sriatum Cere Blood Metab, 1999,19(1):1-18
46 Connor,Schwab BL,Guerini D, Didszun, C, et al. Apoptosis as a form of cell death in tracerebral hemorrhage .Cell Death Differ,2002,9(8):818-31
47 Ghribi O,,Herman MM, Savory.Acute tissue damage after injections of thrombin and plasmin into rat sriatum. Neurosci Lett, 2002,324(3):217-21
48 Blomgren K, Zhu C, Wang X, et al. Evidence for apoptosis after intercerebral hemorrhage in rats triatum .Biol Chem,2001,276(13):10191-8
49 Ma J, Endres M, Moskowitz MA. Br. Apoptosis as a form of cell death in tracerebral hemorrhage.Pharmacol, 1998,124(4):756-762
50 Nakagawa T, Zhu H, Morishima N, et al . Acute tissue damage after injections of thrombin and plasmin into rat sriatum.Nature ,2000,403(6765):98-103
51 Krajewski S, Krajewska M, Ellerby LM, et al. Thrombin receptor activation protects neuron sandastro cytes from cell death produced by environmental insults .Proc Natl Acad Sci USA,1999 ,96(10):5752-7
52 Berke ,Shibasaki F, Hallin U, Uchino H. Evidence for apoptosis after intercerebral hemorrhage in rats triatum .Biochem (Tokyo),2002,131(1):1-15
53 Min Chen, Dong-Jun Won, Stan Krajewski , et al. Biology of ischemic cerebral cell death. Prog Cardiovasc Dis .Biol Chem,2002,277(32):29181-29186
54 Suprynowicz ,Xie J. Guo Q, Zhu H, et al. Dynamics of polymor phonuclear leukocyte accumulation in acute cerebral infarction and the ircorrelation with brain tissue damage .Brain Res,2000,82(1-2):107-13
55 Miettinen S, Roivainen R, Keinanen R, et al. Ischemic stroke is associated with a systemic increase of blood mononuclear cells expressing interleukin-8 mRNA . Neurosci, 1996,16(19): 36-45
56 Ziemka-Nalecz M, Zalewska T, Zajac H, et al. Apoptosis as a form of cell death in tracerebral hemorrhage. Neurochem Int,2003,42(3):5-14
57 Scu ,Bastianetto S, Zheng WH, Quirion R. Inhibition of human lung cancer cell growth by the peroxisome proliferator-activated recrptor-gamma agonists through induction of a poptosis. Neurochem,2000,74(6): 68-77
58 Parnham M, Sies H. Brain protection by resveratrol and fenofibrate against stroke requires peroxisome proliferator-activated receptor alpha in mice. Expert opin Investig Drugs, 2000,9(3):07-19
59 Gong C, Bopulis N,Qian,et al.Intracerebral hemorrhage-induced neuronal dearh, Neurosurger -y , 2001,48(4):875~880
60 Guohua, Heiss WD,KrachtLW, Thiel A,et al.Penumbral probability thres holds of cortical flumaze nilbinding and blood flow predicting tissue outcome in patients with cerebral ischaemia.Brain,2001,124:20-29.
61 Bereczki D.Liu M,Prado G F,et al.Cochrane report:Asystematic review of mannitol therapy for acute ischemic stroke and cerebral parenchymal hemorrhage.Stroke,2000,31(11):2719~22
62 Bereczki D,Liu M,doPrado G F,et al. Mannitol for acute stroke.Cochran Data base Syst Rev,2001,(1):1153
63 Popa G,Amaireh M,Dinu M,et al.Acetazolamide therapy evaluation in haemorrhagic stroke.Rom .Neurol Psychiatry,1995,33(2):145~155
64 Rosenberg G A,Estrada EY.Atrial natriuretic peptide blocks hemorrhagi brain edema after 4-hour delay in rats. Stroke, 1995,26(5):847~7
65 Qureshi AI,Wilson DA,Hanley DF,et al.Pharmacologic reduction of mean arterial ressure does not adversely affect regional cerebral blood flow and intracranial pressure in experimental intracerebral hemorrhage. Crit Care Med 1999,27(5):965~971
66 Powers WJ,Zazulia AR, Videen TO,et al.Autoregulation of cerebral blood flow surrounding acute (6to22hours) intracerebral hemorrhage. Neurology 2001,57(1):18~24
67 Lyden PD. Jackson-Friedman C,Lonzo-DoktorL. Madical therapy for intrace-rebral hematoma with the gamma-aminobutyric acid-A agonist muscimol Strok,1997,28(2):387~391
68 Lyden P,Shin C,Jackson-Friedman C,et al.Effect of ganaxolone in a rodent model of cerebral hematoma.Stroke,2000,31(1):169~175
69 Clark W,Gunion-Rinker L, Lessov N,et al. Citiconine treatment for experimental in tracerebral hemorrhage in mice.Stroke, 1998,29(10):2136~40
70 Bullock MR,Merchant RE,Carmack CA,et al.An open-label study of CP-101,606 in subjects with a severe traumatic head injury or spontaneous intracerebral hemorrhage. AnnN YA cad Sci, 1999,890:51~58
71 Mayne M, FotheringhamJ,Yan H,et al.Adenosine A-A receptor activation reduces proinflammatory events and decreases cell death following intracere-bral hemorrhage.Ann Neurol,2001,49(6):727~735
72 DelBigio MR,YanHJ,XueM.Intracerebral in fusion of a second-generation ciliary neurotrophic factor reduces neuronal loss in rats triatum following experimental in tracerebral hemorrhage. JNeurol Sci,2001,192(1~2):53~59
73 PeelingJ,Yan H J,CorbettD,et al.Effect of FK2506 on inflammation and behavioral outcome following intracerebral hemorrhage in rat..Exp Neurol 2001,167(2):341~347
74 Peeling J,DelBigioMR,CorbettD,et al.Efficacy of disodium4-[(tert-bu-tylimino)methyl] benzene-1,3-disulfonate N-oxide(NXY2059), a free radical trapping agent,in a rat model of hemorrhagic stroke.Neuro pharmacology 2001,40(3):433~9
75 Mohadijer M, Braus DF,MyersA,et al.CT-stereotactic fibrinolysis of spon-taneous intracerebral hematomas.NeurosurgRev,1992,15(2):105~110
76 Miller DW,Barnett GH,Kormos DW,et al.Stereotactically guided throm-Bolysis of deep cerebral hemorrhage:preliminary results.Cleve Clin J Med1993,60(4):321~324
77 杨军,潘树茂,刘运祥,等.基因工程链激酶在鼠脑内血肿模型中局部应用的实验研究.中国现代医学杂志,1999,10;9(10):16~17
78 Schaller C,Rohde V,HasslerW. Local thrombolytic treatment of spontaneous intracerebral hemorrhage with plasminogen activator.Indication and Limits Nervenarzt, 1995, 66(4): 75 ~ 81
79 Deinsberger W, VogelJ,Fuchs C,et al.Fibrinolysis and aspiration of experimental intracerebral hematoma reduces the volume of ischemic braininrats Neurol Res,1999,21(5):517~23
80 贾宝祥,孙仁泉,左鸿,等. 穿刺射流及液化技术治疗高血压脑出血的初步报告.中国神经精神病杂志.1996,22:233
81 郭岩,张文斌,刘劲瑞.等.尿激酶加自体血清治疗重症脑室出血的应用.中华神经外科杂志,1999,15:236
82 Teerntra OP,Evers SM,Lodder ,et al.Stereotactic treatment of intracerebral hematoma by means of a plasminogen activatoria multicenter randomized trial.Stroke,2003,34:968
83 Xue M,Bigio M,Frepe K,et al.Acute tissue damage after injections of thrombin and plasmin into rat striatum.Stroke,2001,32(9):2164~2169
1 顾宁,周仲英.通下法治疗急性脑出血研究进展.中国中医急诊,2000,9(5):227-229
2 林亚明,任继学.破瘀醒神法在出血性中风急性期的应用.中医杂志,1992,33(5):251
3 黄培新,刘茂才.中医专科专病临床诊治丛书·神经科专病临床诊治.北京:人民卫生出版社,2000,407-411
4 郑国庆,黄培新.中风病阴阳为纲辨治源流.中华医史杂志, 2003,33(4):227-230
5 李 求 兵 . 浅 述 “ 血 证 论 ” 治 血 四 法 对 中 风 脑 出 血 治 疗 的 指 导 意 义 . 北 京 中医,2003,22(6):17-18
6 陈胜,陈武进.应用“治血四法”探讨出血性中风的治疗.甘肃中医,1995,8(6):1-2
7 汤克仁,等.复方丹参液静滴治疗急性高血压脑出血 30 例.山东中医杂志,1987;5:32
8 汤克仁,等.复方丹参液静滴治疗急性高血压脑出血 30 例.山东中医杂志,1987;5:32
9 翁维良.血瘀证及活血化瘀中西医结合研究新进展.中西医结合杂志,1987;7(3):190
10 谢道珍,等.脑血康治疗高血压脑出血的临床与实验研究.中西医结合杂志,1988;8(6):341
11 梁清华,等.平肝熄风汤对脑溢血患者日常生活活动能力的影响.湖南医科大学学报,1994;19(6):513
12 梁清华,等.脑溢安颗粒剂与汤剂治疗肝阳化风型急性脑出血临床疗效比较.湖南医科大学学报,1995;20(3):215
13 周绍华.出血性中风的中医治疗.实用中西医结合杂志,1992;5(3):139
14 许振亚.出血性中风的证治经验.中医杂志,1993;3(1):17
15 薛金发,等.祛瘀通下法治疗脑溢血 33 例.江西中医药,1995;26(4):19
16 虢周科,等.脑出血的病因病机及辨证论治规律探讨.陕西中医,1992;13(11):496
17 李克玲,黄启福,严京,等.中风脑得平冲剂对大鼠实验性脑血肿诱发脑水肿的影响.北京中医药大学学报,1997,20(3):35.
18 李克玲,黄启福,朱陵群,等.中风脑得平冲剂对实验性兔脑血肿的作用.北京中医药大学学报,1996,19(4):22.
19 李克玲,黄启福,张丽萍,等.醒脑健神胶囊对实验性脑血肿诱发大鼠脑水肿的作用.中国中医急症,1997,6(2):82.
20 刘莉,钱家骏,胡加跃,等.醒脑健神胶囊清开灵注射液对急性脑出血大鼠脑含水量、离子含量及自由基代谢的影响.北京中医药大学学报,1997,20(1):38.
21 李克玲,黄启福,蒋玉凤,等.清开灵注射液治疗家兔脑血肿的实验研究.中国中西医结合杂志,1997,17(2):91.
22 陈绍宏,张晓云,刘永家,等.逐瘀化痰口服液治疗急性脑出血的临床及实验研究.中国中医急症,1995,4(2):58.
23 黄世敬,黄启富,孙塑伦,等.救脑宁注射液对实验性脑出血大鼠脑水肿及脂质过氧化的影响.北京中医药大学学报,1998,21(2):38.
24 杨万章,万中民,李明富,等.逐瘀化痰汤等三方对大鼠脑出血脑组织 MDA、Ca2+、Fe2+影响的实验研究.北京中医药大学学报,1997,20(6):35.
25 胡随瑜,金益强,王勇华,等.脑溢安颗粒剂主要药效学研究.中药新药与临床 药理,1997,8(3):150.
26 李德伟,任恩发,许红,等.中药中风脑得平冲剂对脑出血大鼠尾壳核壳—脑啡肽(L-ENK)含量影响的实验研究.中国中医基础医学杂志,1996,2(1):39.
27 白丽敏,孙红梅,朱培纯,等.醒脑健神丹对急性脑出血大鼠脑内神经肽 Y 含量的影响.中国中医基础医学杂志,1995,1(4):33.
28 蒋玉凤,朱陵群,黄启福,等.清开灵注射液对 SHRSP 出血性中风海马区兴奋性氨基酸含量的影响.中国中医急症,1997,6(5):219.
29 钱家骏,刘莉,庞鹤,等.醒脑健神胶囊、清开灵注射液对急性脑出血大鼠血浆 ET、CK 及 VWF 水平的影响北京中医药大学学报,1997,20(2):25.
30 孙红梅,白丽敏,朱培纯,等.醒脑健神胶囊对实验性脑出血大鼠脑内 β-内啡肽(β-EP)影响的实验研究.中国中医基础医学杂志,1997,3(6):17.
31 黄培新,刘茂才,陈根成,等.毛冬青甲素对高血压性大鼠脑内血肿及脑微血管超微结构的影响.中国中医急症,1997,6(3):127.
32 林霞,潘其民,金益强,等.平肝熄风汤对出血性中风大鼠神经病理变化的影响.湖南医科大学学报,1994,19(3):204.
33 王玉良,唐可欣,王益光,等.牛黄熄风胶囊治疗脑出血大鼠的体感诱发电位观察.潍坊医学院学报,1997,19(2):86.
34 杨万章,万中民.逐瘀化痰汤、三生饮、抵挡汤对实验大鼠脑出血后 EEG 变化的研究.北京中医药大学学报,1996,19(3):64.
35 黄海青,朱敬荣,开国维.114 例出血性中风中医证型及脑部 CT 的研究.南京中医药大学学报,1996,12(3):15.
36 丁沧清 , 智慧 , 邢海燕 , 等 . 脑出血的临床证型与影像指标初探 . 辽 宁 中 医 杂志,1995,22(11):486.
37 梁清华,黎杏群,李家邦,等.出血性中风病人的脑 CT、中医辨证与血液流变学的关系探讨湖南医科大学学报,1995,20(6):554.
38 魏丹,张东友,王金娥.脑出血中医临床证型与头颅 CT 表现的相关性.中医药研究,1999,15(3):26.
39 肖小华,舒晓春,刘元章,等.中风 CT 扫描、血液流变学与辨证的研究.江西中医学院学报,1995,7(2):2.
40 周庆芳,刘华,许存森.出血性中风中医证型与颅脑核磁共振检查关系的研究.浙江中医杂志,1995,30(12):550.
41 孔 炳 耀 , 李 小 敏 . 中 风 急 性 期 血 清 血 脂 脂 蛋 白 亚 组 分 与 辨 证 关 系 . 辽 宁 中 医 杂志,1997,24(5):199.
1 张昱.脑出血的病理改变及病理生理过程.中国现代神经疾病杂志,2005 5(2):75~78
2 Rosenberg GA,Mun-Bryces,Wesley M.et al.collagenase-induced intracerebral hemorrhage in rats.Stroke,1990,21:801～811
3 Alexis NE . Neurobehavioral consequences of induced spreading depression following photothrombotic middle cerebral artery occlusion.BrainRes,1996,(7):273～282
4 张震中,吕安坤,张燕.脑出血大鼠模型的血液流变学研究.中华实用中西医杂志,2004,4(17):3673~3675
5 隋邦森.脑血管疾病 MR、CT、DSA 与临床.北京:人民卫生出版社,1991:196~1971
6 李俐涛,尹静,张祥建,等.脑出血模型.河北医科大学学报,27(5):496~499
7 王永炎.今日中医内科学.北京:人民卫生出版社,2000,63~65
8 杨菊芬.血液流变学对脑出血与脑梗塞的诊断价值.遵义医学院学报,2000,23(2):156
9 侯玉立.血液流变测定在脑出血与脑梗塞中的应用.山西医科大学学报, 2001,32(3):226-227
10 杨舒,朱雯梅,李晓萍.脑出血与脑梗塞患者血液流变学及凝血功能的改变.中华实用医学, 2002, 4(23) :93-94.
11 Chesney J A,Kondoh T, Conrad J A,et al.Collagenase induced intrastriatal hemorrhage in rats results in long-term locomotor deficits.Stroke,1995,26(2):312-317
12 夏鹰,陈衔城,季耀东.等.脑出血血肿周围脑组织的糖代谢、氧自由基及血脑屏障的变化与脑水肿形成的关系.复旦学报(医学版),2002,3(2):119~223
13 Stroke Therapy Academic Industry Roundtable(STAIR).Recommendations for standards regarding preclinical neu-roprotective and restorative drug development. Stroke, 1999, 30:2752~2758
1 Gong C,Boulis N,Qian I,et al.intracerebral hemorrhage-induced neuronal death.Neurosurgery, 2001,48,875~882
2 Johnsen SP, Pedersen L, Friis S,et al.Nonaspirin nonsteroidal anti-inflammatory drugs and risk of hospitalization for intracerebral hemorrhage:population based case control study . Stroke, 2003, 34:387~391
3 成样林,赵成三,汪华.丙戊酸钠对戊四氮致大鼠海马 Bax 和 Bcl-2 表达的影响.临床神经病学杂志,2004,17(5):351~353
4 Mendelow AD.Mechanisms of ischemic braindamage with intracerebral hemorrhage. stroke, 1993, 24:1115-1117
5 Rosenberg GA,Navratil M,Barone F.proteolytic cascade enzymes increase in focal cerebral ischemia off gerbils.Neurochem Res,1996,16;360
6 Kobayashi H,Minami S,Itoh S,et al.Aquaporin subtypes inratcerebral microvessels.Neurosci Lett, 2001, 297(3):163-166.
7 石善溶.免疫组织化学技术.成都:四川科技出版社
8 纪小龙.免疫组织化学新编.北京:人民军医出版社
9 Johnasson AC, Visse E, Widegren B, et al. Computerized image analysis as a tool to quantify infiltrating leukocytes: A compariaon between highand low magnification images. J Histochemisry and Cytochemistry, 2001;49(9):1073-1079
10 O’Leary TJ. Standardization in immunohistochemistry. Appl Immunohistochem Mol Morphol, 2001;9(1):3-8
11 杨继文,贺大权,张秋才.等.脑出血的病理损伤机制.脑与神经疾病杂志,2005,13(6):470-471
12 Belayev L, Busto R, Zhao W, etal. Quantitative evaluation of blood-brain barrier permeability following middle cerebral artery occlusion in rats. Brain Res. 1996, 739(1-2); 88.
13 Betz AL, Coester HC. Effect of steroids on edema and sodium uptake of the brain during focal ischemia in rats. Stroke. 1990, 21(8); 1199.
14 Steller H, Mechanisms and genes of cellular suicide. Science, 1995, 267: 1445～1449
15 Hochenkery DM, Oltval ZN, Yin XM, et al.Bcl-2 fuctions in an antioxidant pathway to prevent apoptosis.Cell, 1993, 75: 241～251
16 Oltvai ZN, Milliman CL. Korsmeyer SJ. Bcl-2 heterodimerizes in vivo wih a conserbed homolog, bax,thataccelerates programed cell death. Cell, 1993,74:609～615
17 Stanley JK. Regulators of cell death. TIG, 1995, 11(3): 1011
1 许川山,刘志君,唐建民,等.低强度激光促细胞增殖效应与丝裂原激活蛋白激酶信号传导途径.中国激光医学杂志,2000,(2):47-48
2 周志琦,刘强.真核生物的 MAPK 级联信号传递途径.生物化学与生物物理进展,1998,25(6):496-503
3 Ferrer I, Friguls B, Dalfo E, et a l. Early modifications in the expression of mitogen activated protein kinase (MAPK/ERK) , stress-activated kinases SAPK/JNK and p38, and their phosphorylated substrates following focal cerebral ischemia. Acta Neuropathol (Berl) , 2003, 105 (5) : 425-437.
4 Xia ZG, Martin D, Joel B, et al.Opposing effets of ERK and JNK P38MAP kinase on apoptosis.Science,1995,270:1326-1331.
5 王凌, 李大金, 朱影.脱氢表雄酮经不依赖于雌、雄激素受体的 MAPK 信号途径抑制成骨细胞凋亡.分子细胞生物学报,39(4) 365-372
6 赵婷,侯孟君,肖勇梅,等.ERK1/2 和 P38 信号通路在瘦素诱导巨噬细胞 TNF-α表达中的作用.中山大学学报(医学科学版),2006(9):500-505
7 赵婷,侯孟君,肖勇梅,等.ERK1/2 和 P38 信号通路在瘦素诱导巨噬细胞 TNF-α表达中的作用.中山大学学报(医学科学版),2006(9):500-505
8 Gutcher J Webb PR, Anderson NG. The isoform-specific regulation of apotosis by protein kinase C.Cell Mol Life Sci,2003,6:1061-1070
9 黄河清,陈康宁,屠永华,等.培养神经元缺 PKC 活性变化与细胞凋亡关系的研究,第三军医大学学报,2002,12:1399-1401
10 Simi A , Ingelman-Sundberg M , T indberg N. Neuroprotective agent chlomethiazole attenuates c-fos, c-jun, and AP-1 activation through inhibition of p38 MAP kinase. Cereb Blood Flow M etab, 2000, 20 (7) : 1077-1088.
1 获原幸夫等.血清添加法を用し新ぃ实验系こよゐ汉方剂の研究.细谷英夫,山雄一:最新の汉方药理-汉方药の科学ち验证と展望.Tokyo:ExceptaMedia,Ltd.1988
2 杨彦芳;王玉芹.中药复方血清药理学方法规范化探讨.中国中西医结合杂志 2000. 20(5):380~382.
3 梅建勋,张伯礼,陆 融。中药脑脊液药理学研究方法的初建,中草药 2000,31(7):523~526
4 洪庆涛,唐一鹏.新生大鼠大脑皮层神经细胞的体外原代培养.神经解剖学杂志,1994,10(3):259.
5 张挺 , 于鸿儒 , 杨菁 , 等 . SD 乳鼠海马神经细胞原代培养方法的探讨 . 锦州医学院学报,2005,26(5):47-49
6 刘娜,左萍萍,周帆,张放,等.连二亚硫酸钠致 PC12 和 NG108 15 细胞拟缺血损伤研究. 中国药理学通报,1998;14(6):525-529
7 聂亚雄,黎杏群,梁清华.脑溢安含药血清对谷氨酸致培养大鼠皮层神经元损伤的保护作用.湖南医科大学学报, 2002,27(5):429-431
8 李仪奎,吴健宇.血清药理试验中采血时间的通法方案.中国药理学通报,1999;15(6):569-70
9 王金华,叶祖光,梁爱华,等.安宫牛黄丸中砷、汞在正常和脑缺血模型大鼠体内的吸收与分布研究.中国中药杂志,2003,7:639-643
1 Ferri KF,Kroemer G.Mitochondria-the suicide organelles,Bioessay,2001,23(2):111-115
2 谭勇,梁清华,李霞玲,等.平肝熄风汤对脑出血大鼠海马线粒体膜电位的影响.中南大学学报(医学版),2004,29(1):35-381
3 高春锦,赵立明,夏成清,等.葛环高压氧对 CO 中毒大鼠海马中钙离子、cGMP 及血浆 NO含量的影响.中华航海医学杂志,1999,6(4): 226-229
4 Kiyoshi K, Hisao Y. Mild hypothermia revived counter measure against ischemic neuronal damages.Neuroscience research, 1998, 32:103～117
5 Nakayama R, Yano T, Ushijima K, et al. Effects of dantrolene on extracellular glutamate concentration and neuronal death in the rat hippocampal CAI region subjected to transient ischemia. Anesthesiology,2002, 96(3): 705～710
6 朱陵群,范吉平,黄启福,等.人参皂甙 1 对大鼠胎鼠海马神经细胞凋亡的影响.中国病理生理杂志,2001,17(12):1229-12311
7 管增伟,王盛兰,李勇,等.凋亡细胞细胞膜和线粒体的动态变化.卫生研究,2000,29(2):83-86
8 Harry I, Michael FB, Tsuyoschi S,et al. Nitricoxide production and perivascular tyrosine nitration in brain after carbonmonoxid poisoningintherats.JClinInvest,1996,97(10):2260-2267.
1 张丽敏.全血中 16 种微量元素的微波消解电感耦合等离子体质谱测定法.中华劳动卫生职业病杂志,2006,24(1):50~51
2 王刚,陈荣达,林炳承,等.中药中微量元素测定的研究进展.药物分析杂志,2002,22(2):151
3 邱招钗,杨妙峰,何鹰,等.用ICP-MS法测定四个不同地区泽泻种微量元素的含量.福建分析测试,2003,12(2):1739.
4 陈浩,梁沛,胡斌,等.电感耦合等离子体原子发射光谱/质谱法在中药微量元素及形态分析中的应用.光谱学与光谱分析,2002,22(6):1019.
5 高雅,李更生,赵唯贤,等.反反正正论朱砂.中国中医药信息杂志,1999,6(12):42~43.
6 郝红缨.四硫化四砷对急性早幼粒细胞白血病细胞株 NB4 的凋亡作用.中国药理学与毒理学杂志,2002,16(1):37
7 陈思宇.雄黄诱导 K562 细胞调亡中 Bcl-2 和 Bax 的表达.第四军医大学学报, 2002, 23(9):790.
8 刘延方 , 等 . 硫化砷诱导 NB4 细胞调亡及细胞周期阻滞的研究 . 中华血液学杂志,2000,21(12):647.
9 张昌,等.硫化砷对 K562/ADM 细胞 HSP70 蛋白表达的影响. 中国癌症杂志,2001,11(1):33
10 周霭祥.青黄散治疗慢粒 25 例近期疗效观察.中国中西医结合杂志,1981, 1(1):16.
11 陈思宇.雄黄对急性早幼粒细胞白血病细胞诱导调亡中和促进分化的双重作用.西安交通大学学报(医学版),2002,23(4):401.
12 Deng Y., et al. Size effects of realgar particles on apoptosis in a human umbilical vein endothelial cell line BEC-304. Pharmacological Research, 2001, 44 (6) :513.