脑健片对脑缺血损伤的干预及其主要药效成分的药代动力学研究
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摘要
目的从体外、体内两方面探讨脑健片对脑缺血损伤的干预作用,并研究脑健片主要药效成分的药代动力学特征,为后期新药申报工作奠定基础。
方法体外研究以0.3mM的H202与PC12细胞共同作用2h,成功复制脑缺血体外氧化应激细胞模型,MTT法考察脑健片及补阳还五汤不同拆方对该模型的保护作用,酶联免疫法检测细胞培养液中L-LDH的含量和胞内GSH的含量,流式细胞术检测胞内ROS含量,细胞免疫荧光法检测胞内CDK5和Tau蛋白的表达；体内研究以MCAO大鼠为动物模型,将SD大鼠随机分为假手术组、MCAO模型组、补阳还五汤干预组(灌胃：3.15g/kg)和脑健片干预组(灌胃：2.41g/kg),采用免疫组化法和RT-PCR法考察脑健片及补阳还五汤对模型大鼠海马组织中CDK5. CDK4/CyclinD1表达的调控；采用HPLC-DAD法检测药效成分阿魏酸、洋川芎内酯A和槀本内酯在脑健片中的含量,并采用血药浓度法研究脑健片及补阳还五汤单次灌胃给药颈静脉插管大鼠的药代动力学特征。结果(1)体外实验表明脑健片及补阳还五汤在0.2～1.0mg/ml浓度范围内均能有效抑制H20z对PC12细胞的损伤作用,可有效降低模型细胞培养液中L-LDH的含量,增加胞内GSH,降低胞内ROS,可能通过降低模型细胞内CDK5和Tau蛋白的表达对体外氧化应激细胞模型发挥保护作用；(2)体内实验结果表明脑缺血损伤模型大鼠海马组织中,三种蛋白的表达上调,提示脑缺血损伤将激活CDK5和细胞周期信号通路,脑健片干预后的3、7、14、28d均能显著降低三种蛋白的表达(p<0.05),补阳还五汤亦表现出相同的作用机制,两者之间无显著性差异(p>0.05)；(3) HPLC-DAD法能实现对脑健片中盐酸川芎嗪、阿魏酸、洋川芎内酯A和藁本内酯的同时分离和含量测定,为脑健片的质量控制提供依据；阿魏酸、洋川芎内酯A、藁本内酯的代谢过程均符合二室模型,为该药的临床给药剂量和二次给药时间提供参考,但三种药效成分在补阳还五汤和脑健片中的药代动力学参数存在一定差异,脑健片中各有效成分的代谢规律表现出自身特点。结论本论文证实在传统经典名方基础上筛选出的脑健片处方是合理的,初步验证了脑健片处方以黄芪、川芎、地龙以10：3：1的比例入药是合理的,应验了“君臣佐使”的中药方剂学原理,并通过生物相容性实验验证了脑健片的“高效低毒”。脑健片可能通过CDK5和细胞周期信号通路对神经细胞发挥保护作用,验证了中药经由多通路、多靶点发挥药效的观点。药代实验结果提示中药复方及剂型的改变需重新对其药效成分的体内药代动力学过程进行研究。
Based on the theories of modern medicine and traditional Chinese medicine, the purpose of this study was to investigate the prevention and its mechanism of NaoJian tablet during focal ischemic brain injury condition in vivo/in vitro.
In vitro studies, MTT, ELISA, FCM and IF methods were used to determine the prevention effect of NaoJian Tablet and Buyang Huanwu Decoction by means of focal ischemic brain injury cell model, induced by treating PC12cell with0.3mM H2O2for2h. The result suggested that both of the two tested subjects could effectively protect PC12cell from impairment induced by H2O2in the range of0.2～1.0mg/mL. Moreover, we found not only the reduction of L-LDH in cell culture medium but also inhibition of the basal cellular ROS and CDK5/Tau levels following exposure to NaoJian Tablet from Buyang Huanwu Decoction, Conversely, the increasing cellular GSH content been detected. The all results reveal the reasonable of selection of NaoJian Tablet. Moreover, the decomposing experiment of NaoJian Tablet show that Astragalus membranaceus (Fisch.) Bunge, Rhizoma Chuanxiong, and Pheretima aspergillum (E.Perrier) in the ratio of10:3:1is consistent with the principle of science of traditional Chinese formula. The concentration main bioactive suhbstance such as ligustrazine, ferulic acid, senkyunolide and ligustilide in NaoJian Tablet was determined simultaneously by use of HPLC-DAD method for its overall quality evaluation.
In vivo studies, immunohistochemistry, RT-PCR and Western blot were applied to determine the expression of CDK5、CDK4/CyclinD1in the hippocampus tissues in MCAO rat by treating with NaoJian Tablet and Buyang Huanwu Decoction. The result indicated that NaoJian Tablet (ig,2.41g/kg) could significantly decrease the levels of CDK5、CDK4/CyclinD1in hippocampus tissues in MACO rat after treated by3d,7d,14d,28d (p<0.05). The similar mechanism was observed in the Buyang Huanwu Decoction treated MACO rat (p>0.05). The above-mentioned in vivo studies suggested that NaoJian Tablet maybe play its prevention effect on nerve cells via its acting on CDK5signaling and cell cycle pathway, which verified the multi-target and multi active pathways efficiency of tradition Chinese medicine in treating diverse disease. On the other hand, the processes of metabolism and disposition of several active components in NaoJian Tablet and Buyang Huanwu Decoction were analyzed and characterized using HPLC-DAD methods. The data show that all of the three tested compounds were fitted to two-compartment metabolic model. The three tested compounds in NaoJian Tablet maintained its own metabolism regularity owing to the discrepancy in pharmacokinetic parameters with Buyang Huanwu Decoction.
The present study mainly focus on the preclinical research of NaoJian Tablet. The results lay a foundation on the registration application of it. Simultaneously, it can conduct as a reference of research approach for traditional Chinese medicine.
方法体外研究以0.3mM的H202与PC12细胞共同作用2h,成功复制脑缺血体外氧化应激细胞模型,MTT法考察脑健片及补阳还五汤不同拆方对该模型的保护作用,酶联免疫法检测细胞培养液中L-LDH的含量和胞内GSH的含量,流式细胞术检测胞内ROS含量,细胞免疫荧光法检测胞内CDK5和Tau蛋白的表达；体内研究以MCAO大鼠为动物模型,将SD大鼠随机分为假手术组、MCAO模型组、补阳还五汤干预组(灌胃：3.15g/kg)和脑健片干预组(灌胃：2.41g/kg),采用免疫组化法和RT-PCR法考察脑健片及补阳还五汤对模型大鼠海马组织中CDK5. CDK4/CyclinD1表达的调控；采用HPLC-DAD法检测药效成分阿魏酸、洋川芎内酯A和槀本内酯在脑健片中的含量,并采用血药浓度法研究脑健片及补阳还五汤单次灌胃给药颈静脉插管大鼠的药代动力学特征。结果(1)体外实验表明脑健片及补阳还五汤在0.2～1.0mg/ml浓度范围内均能有效抑制H20z对PC12细胞的损伤作用,可有效降低模型细胞培养液中L-LDH的含量,增加胞内GSH,降低胞内ROS,可能通过降低模型细胞内CDK5和Tau蛋白的表达对体外氧化应激细胞模型发挥保护作用；(2)体内实验结果表明脑缺血损伤模型大鼠海马组织中,三种蛋白的表达上调,提示脑缺血损伤将激活CDK5和细胞周期信号通路,脑健片干预后的3、7、14、28d均能显著降低三种蛋白的表达(p<0.05),补阳还五汤亦表现出相同的作用机制,两者之间无显著性差异(p>0.05)；(3) HPLC-DAD法能实现对脑健片中盐酸川芎嗪、阿魏酸、洋川芎内酯A和藁本内酯的同时分离和含量测定,为脑健片的质量控制提供依据；阿魏酸、洋川芎内酯A、藁本内酯的代谢过程均符合二室模型,为该药的临床给药剂量和二次给药时间提供参考,但三种药效成分在补阳还五汤和脑健片中的药代动力学参数存在一定差异,脑健片中各有效成分的代谢规律表现出自身特点。结论本论文证实在传统经典名方基础上筛选出的脑健片处方是合理的,初步验证了脑健片处方以黄芪、川芎、地龙以10：3：1的比例入药是合理的,应验了“君臣佐使”的中药方剂学原理,并通过生物相容性实验验证了脑健片的“高效低毒”。脑健片可能通过CDK5和细胞周期信号通路对神经细胞发挥保护作用,验证了中药经由多通路、多靶点发挥药效的观点。药代实验结果提示中药复方及剂型的改变需重新对其药效成分的体内药代动力学过程进行研究。
Based on the theories of modern medicine and traditional Chinese medicine, the purpose of this study was to investigate the prevention and its mechanism of NaoJian tablet during focal ischemic brain injury condition in vivo/in vitro.
In vitro studies, MTT, ELISA, FCM and IF methods were used to determine the prevention effect of NaoJian Tablet and Buyang Huanwu Decoction by means of focal ischemic brain injury cell model, induced by treating PC12cell with0.3mM H2O2for2h. The result suggested that both of the two tested subjects could effectively protect PC12cell from impairment induced by H2O2in the range of0.2～1.0mg/mL. Moreover, we found not only the reduction of L-LDH in cell culture medium but also inhibition of the basal cellular ROS and CDK5/Tau levels following exposure to NaoJian Tablet from Buyang Huanwu Decoction, Conversely, the increasing cellular GSH content been detected. The all results reveal the reasonable of selection of NaoJian Tablet. Moreover, the decomposing experiment of NaoJian Tablet show that Astragalus membranaceus (Fisch.) Bunge, Rhizoma Chuanxiong, and Pheretima aspergillum (E.Perrier) in the ratio of10:3:1is consistent with the principle of science of traditional Chinese formula. The concentration main bioactive suhbstance such as ligustrazine, ferulic acid, senkyunolide and ligustilide in NaoJian Tablet was determined simultaneously by use of HPLC-DAD method for its overall quality evaluation.
In vivo studies, immunohistochemistry, RT-PCR and Western blot were applied to determine the expression of CDK5、CDK4/CyclinD1in the hippocampus tissues in MCAO rat by treating with NaoJian Tablet and Buyang Huanwu Decoction. The result indicated that NaoJian Tablet (ig,2.41g/kg) could significantly decrease the levels of CDK5、CDK4/CyclinD1in hippocampus tissues in MACO rat after treated by3d,7d,14d,28d (p<0.05). The similar mechanism was observed in the Buyang Huanwu Decoction treated MACO rat (p>0.05). The above-mentioned in vivo studies suggested that NaoJian Tablet maybe play its prevention effect on nerve cells via its acting on CDK5signaling and cell cycle pathway, which verified the multi-target and multi active pathways efficiency of tradition Chinese medicine in treating diverse disease. On the other hand, the processes of metabolism and disposition of several active components in NaoJian Tablet and Buyang Huanwu Decoction were analyzed and characterized using HPLC-DAD methods. The data show that all of the three tested compounds were fitted to two-compartment metabolic model. The three tested compounds in NaoJian Tablet maintained its own metabolism regularity owing to the discrepancy in pharmacokinetic parameters with Buyang Huanwu Decoction.
The present study mainly focus on the preclinical research of NaoJian Tablet. The results lay a foundation on the registration application of it. Simultaneously, it can conduct as a reference of research approach for traditional Chinese medicine.
引文
[1]前十位死亡原因.世界卫生组织实况报道,2011年6月,第310号.
[2]世界卒中日已成为了人类死亡的第二大元凶http://www.06news.com/ szxw/2012-10-29/423.html
[3]GAN Zhao-ru, SANG Li-nan. Research Progress of Cascade by Cerebral ischemia-reperfusion Injury [J]. Medical Recapitulate,2009,15(1):43-45.
[4]Waldowski K, Seniow J, Lesniak M, et al. Effect of low-frequency repetitive transcranial magnetic stimulation on naming abilities in early-stroke aphasic patients:a prospective, randomized, double-blind sham-controlled study [J]. Scientific World Journal,2012:518568. doi:10.1100/2012/518568.
[5]Duangjit S, Muangpaisan W, Chotinaiwattarakul W, et al. Functional Recovery at 3 Months in Stroke Patients Not Receiving Thrombolytic Therapy:The Comparison Between Patients Arriving Earlier and Later Than 4.5 Hours [J]. J Stroke Cerebrovasc Dis,2012,28(12):1052-57.
[6]Sanberg PR, Eve DJ, Metcalf C, et al. Advantages and challenges of alternative sources of adult-derived stem cells for brain repair in stroke [J]. Prog Brain Res,2012,201: 99-117.
[7]Sinden JD, Vishnubhatla I, Muir KW. Prospects for stem cell-derived therapy in stroke[J]. Prog Brain Res,2012,201:119-67.
[8]杨艺敏.Notch信号通路对大鼠脑缺血后神经再生的调控及辛伐他汀与神经再生关系的实验研究[D].吉林大学博士学位论文,2010.3.
[9]Wang Z, Huang W, Zuo Z. Perioperative aspirin improves neurological outcome after focal brain ischemia possibly via inhibition of Notch 1 in rat [J]. J Neuroinflammation, 2014,11(1):56-60.
[10]Zhang L, Dong LY, Li YJ, et al. miR-21 represses FasL in microglia and protects against microglia-mediated neuronal cell death following hypoxia/ischemia [J]. Glia,2012,60(12):1888-95.
[11]Fernandes D J, Ravenhall C E, Harris T, et al. Contribution of the p38 MAPK signalling pathway to proliferation in human cultured airway smooth muscle cells is mitogen-specific[J]. Br J Pharmaco,2004,142:1182-90.
[12]Engel F B, Schebesta M, Duong M T, et al. p38 MAPK kinase inhibition enables proliferation of adult mammalian cardiomyocytes [J]. Genes Dev,2005,19(10): 1175-87.
[13]Osuga H, Osuga S, Wang FH, et al. Cyclin-depedent kinases as a therapeutic target for stroke [J]. Proc Natl Acad Sci USA,2000,97(18):10254-59.
[14]Castro-Alvarez JF, Gutierrez-Vargas J, Darnaudery M, et al. ROCK inhibition prevents tau hyperphosphorylation and p25/CDK5 increase after global cerebral ischemia [J]. Neurosci,2011,125(3):465-72.
[15]Slimone DG, Vilen M, Farid A, et al. Cell cycle inhibition provides neuroprotection and reduces glial proliferation and scar formation after traumatic brain injury [J]. PNAS, 2005,102(23):8333-38.
[16]张贵斌,徐运兰,王伟,等.细胞周期调控对大鼠局灶性脑缺血后迟发性神经元死亡的影响[J].中风与神经疾病杂志,2005,22(6)：495-97.
[17]谭斌.浅谈祖国医学对中风病的认识[J].中医中药,2009,28(33)：91-93.
[18]王清任.医林改错[M].上海：上海科技出版社,1966.31.
[19]张均克,邱幸凡,张六通,等.论祛瘀生新促血管新生的理论基础[J].湖北中医药大学学报,2010,12(5)：47-48.
[20]金远林,王海燕,邱幸凡,等.祛瘀生新法对脑梗死大鼠治疗性血管新生作用及机制研究[J].中国中医急症,2006,15(6)：632-33,638.
[21]张金生.中医“祛瘀生新”理论内涵新解[J].辽宁中医杂志,2009,36(5)：704-05.
[22]韩经丹,王珏,王永炎,等.“毒损脑络”与脑缺血-再灌注损伤病机关系探讨[J].中国中医基础医学杂志,2012,18(8)：823-29.
[23]Zhang Yun-ke. Influences of Buyanghuanwu decoction and its decomposed formulas on cell apoptosis and expressions of Bcl-2 and Bax protein in rats withcerebral ischemia /reperfusion [J]. Chinese Journal of Clinical Rehabilitation,2006,10 (43):196-99.
[24]邓常青,贺福元,唐映红,等.补阳还五汤及其有效部位组方对沙鼠脑缺血再灌注后海马CA1区超微结构的影响[J].中药药理与临床,2000,16(3)：7-9.
[25]邓凯文,贺福元,刘文龙,等.RP-HPLC法测定大鼠血浆中补阳还五汤及总苷中芍药苷的含量及药代动力学研究[J].中药材,2010,33(11)：1768-70.
[26]贺福元,邓凯文,刘文龙,等.中药复方药物动力学总量统计矩法的实验验证研究[J].中国中药杂志,2013,38(2)：253-62.
[27]贺福元,邓凯文,刘文龙,等.中药复方网络药理学及其动力学关键技术问题的 探讨[C].首届全国方剂组成原理高峰论坛论文集,2012,28(5)：256-59.
[28]李再新,吴小红,贺福元,等.补阳还五汤中川芎嗪的药代动力学研究[J].中国药业,2007,16(18)：23-24.
[29]贺福元,刘文龙,贺庆平等.反相高效液相色谱法测定大鼠血浆中补阳还五汤总生物碱中川芎嗪的含量及药代动力学研究[J].中国药物与临床,2005,5(12)：920-22.
[30]尹天雷,李雅,李鹤白,等.补阳还五汤对脑缺血模型大鼠脑损伤的保护作用[J].世界中医药,2007,2(4)：241-42.
[31]刘芳,白雪松,刘柏炎,等.补阳还五汤对脑缺血大鼠碱性成纤维细胞生长因子的影响[J].中国中西医结合急救杂志,2008,15(1)：9-12.
[32]俞悦,易健,蔡光先,等.Caveolin-1敲除对C57BL/6小鼠繁殖性能及子代离乳前体质量的影响[J].湖南中医药大学学报,2011,31(11)：7-9.
[33]刘柏炎,蔡光先,刘维,等.补阳还五汤对大鼠局灶性脑缺血后血管内皮生长因子及其受体Flk1的影响[J].中草药,2007,38(3)：394-96.
[34]周赛男.补阳还五汤对大鼠局灶性脑缺血后神经细胞增殖及脑组织TLR2.TLR4、 NF-k-B、MyD88表达的影响[D].湖南中医药大学博士论文.2012,5.
[35]Yi J, Huang X, Yu Y, Cai GX, Liu BY. Effect of Buyang Huanwu decoction on interleukin-1β and tumor necrosis factor-a expression in rats after cerebral infarction [J]. Zhongguo Wei Zhong Bing Ji Jiu Yi Xue.2010,22(10):599-601.
[36]Zhao LD, Wang JH, Jin GR, et al. Neuroprotective effect of Buyang Huanwu decoction against focal cerebral ischemia/reperfusion injury in rats--time window and mechanism [J]. J Ethnopharmacol,2012,140(2):339-44.
[37]Li TJ, Qiu Y, Rui YC, Li TZ, et al. DNA microarray analysis of protective mechanism of buyang huanwu decoction on focal cerebral ischemia/reperfusion in rats [J]. Zhongguo Zhong Yao Za Zhi,2004,29(6):559-63.
[38]李淑贞,杨桂染,杨艳梅等.补阳还五汤及拆方预处理对MIRI家兔心功能的影响[J].西南国防医药,2010,20(1)：13-16.
[39]崔翰博,韩涛,张东钰,等.补阳还五汤及拆方对代谢综合征模型大鼠胰岛素、TXA2水平的影响[J].山东中医药大学学报,2011,35(5)：446-47.
[40]邓常青,贺福元,唐映红,等.补阳还五汤及其有效部位组方对沙鼠脑缺血再灌注后海马CA1区超微结构的影响[J].中药药理与临床,2000,16(3)：7-9.
[41]Spicer Z, Millhorn D E. Oxygen sensing in neuroendocrine cells and other cell types: pheochromocytoma (PC 12) cells as an experimental model [J]. Endocr Pathol,2003, 14(4):277.
[42]Hou CW, Chen YL, Chuang SH, et al. Protective effect of a sesamin derivative,3-bis (3-methoxybenzyl) butane-1,4-diol on ischemic and hypoxic neuronal injury [J]. J Biomed Sci.2014,21(1):15. doi:10.1186/1423-0127-21-15.
[43]. Guo H, Shen X, Xu Y, et al. The effect of activin A on signal transduction pathways in PC12 cells subjected to oxygen and glucose deprivation [J]. Int J Mol Med,2014, 33(1):135-41.
[44]Lin CY, Wang N, Zhang YF, et al. Neuroprotective effect of muscone on glutamate-induced apoptosis in PC 12 cells via antioxidant and Ca2+antagonism [J]. Neurochem Int,2014,14. doi:10.1016/j.neuint.2014.03.003.
[45]Yang R, Cui HJ, Wang H, et al. N-Stearoyltyrosine Protects Against Glutamate-Induced Oxidative Toxicity by an Apoptosis-Inducing Factor (AIF)-Mediated Caspase-Independent Cell Death Pathway [J]. J Pharmacol Sci,2014,124(2):169-79.
[46]胡海燕,席东泽,雷磊,等.清心开窍方含药脑脊液对谷氨酸致PC12细胞损伤的保护机制[J].中国中药杂志,2013,38(12)：1997-2000.
[47]Ma S, Liu X, Xun Q, Zhang X. Neuroprotective Effect of Ginkgolide K against H2O2-Induced PC 12 Cell Cytotoxicity by Ameliorating Mitochondrial Dysfunction and Oxidative Stress [J]. Biol Pharm Bull,2014,37(2):217-25.
[48]Wu FJ, Xue Y, Liu XF, et al. The protective effect of eicosapentaenoic acid-enriched phospholipids from sea cucumber Cucumaria frondosa on oxidative stress in PC 12 cells and SAMP8 mice [J]. Neurochem Int,2014,64:9-17.
[49]Rizak J, Tan H, Zhu H, et al. Chronic treatment with the mood stabilizing drug lithium up-regulates nuclear factor E2-related factor 2 in rat pheochromocytoma PC12 cells in vitro [J]. Neuroscience, doi:10.1016/j.2013.10.036.
[50]富苏,范吉平,王新志.通络化痰胶囊对H202所致的PC12细胞凋亡的保护作用及机制[J].中国实验方剂学杂志,2013,19(12)：188-93.
[51]唐春萍,陈雪梅.不同提取工艺对补阳还五汤药理作用的影响[J].中成药,1999,21(7)：360-62.
[52]陈红海.补阳还五汤及其拆方抗脑缺血损伤作用的比较研究[D].河北医科大学硕士论文,2011.10.
[53]谢裕华,陈超.不同黄茂剂量的补阳还五汤对中风后遗症患者外周血ESR PCV水平的影响[J].新中医,2007,39(6)：23-24.
[54]Swanson R A. A Semiautomatod method for measuring brain infarct volume [J]. J ceveb Blood Flow Metab,1990,10(2):293-95.
[55]LIU X G, XU L N. A rat middle cerebral artery thrombosis model for evaluation of thrombolytie and amithrunbotis agents [J]. Acta Pharm Sin,1995,30(9):662-67.
[56]Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats [J]. Stroke,1989,20(1):84-91.
[57]Meyerson M, Enders G H, Wu C L, et al. A family of human cdc2-related protein kinases [J]. EMBO J,1992,11(8):2909-17.
[58]Patrick GN, Zukerberg L, Nikolic M, et al. Conversion of p35 to p25 deregulates CDK5 activity and promotes neurodegeneration [J]. Nature,1999,402:615-22.
[59]Lee M S, Kwon Y T, Li M W, et al. Neurotoxicity induces cleavage of p35 to p25 by calpain [J]. Nature,2000,405(18):360-64.
[60]Green SL, Kulp KS, Vulliet R. Cyclin-dependent protein kinase 5 activity increases in rat brain following ischemia [J]. Neurochem Int.1997,31(4):617-23.
[61]Wang J, Liu S, Fu Y, et al. Cdk5 activation induces hippocampal CA1 cell death by directly phosphorylating NMDA receptors [J]. Nat Neurosci,2003,6(10):1039-47.
[62]Weishaupt JH, Neusch C, Bahr M. Cyclin-dependent kinase 5 (CDK5) and neuronal cell death [J]. Cell Tissue Res,2003,312(1):1-8.
[63]Rashidian J, Iyirhiaro G, AleyasinH, et al. Multiple cyclin-dependent kinases signals are critical mediators of ischemia-hypoxic neuronal death in vitro and in vivo [J]. PNAS, 2005,102(39):14080-85.
[64]Slimone DG, Vilen M, Farid A, et al. Cell cycle inhibition provides neuroprotection and reduces glial proliferation and scar formation after traumatic brain injury [J]. PNAS, 2005,102(23):8333-38.
[65]Menn B, Bach S, Blevins TL, et al. Delayed treatment with systemic (S)-roscovitine provides neuroprotection and inhibits in vivo CDK5 activity increase in animal stroke models [J]. PLoS One,2010,5(8):e12117.
[66]Small DL, Monette R, Foumier M C, et al. Characterization of cyclin D1 expression in a rat global model of cerebral ischemia [J]. Brain Res,2001,923(1):26-37.
[67]Boutillier A L, Kienlen-Campard P, Loeffler J P. Depolarization regulates cyclin D1 degradation and neuronal apoptosis:a hypothesis about the role of the ubiquitin/ proteasome signaling pathway [J]. Eur J Neurosci,1999,11(3):441-47.
[68]Timsit S, Rivera S, Ouaghi P, et al. Increased cyclin D1 in vulnerable neurons in the hippocampus after ischemia and epilepsy:a modulator of in vivo programmed cell death [J]. Eur J Neurosci,1999,11(1):263-78.
[69]柳耀泉,张维杰.局灶性脑缺血对细胞周期蛋白D1/CDK4基因表达的影响[J].中华神经医学杂志,2007,6(8)：770-72.
[70]陈华先,丁旭东,朱舟,等.脑缺血上调大鼠神经元和星形胶质细胞CDK4的表达[J].神经损伤与功能重建,2008,3(4)：221-23.
[71]乔海兵,尚君璟.小鼠脑缺血/再灌注后细胞周期相关蛋白在神经元的表达规律[J].山西医科大学学报,2013,44(9)：671-74.
[72]游哲斌,张洁,张德贤,等.黄连素对脑缺血再灌注后细胞cyclin D1、CDK4表达的影响[J].中华神经医学杂志,2008,7(8)：788-92.
[73]田代真一.“血清药理学”和“血清药化学[J].代东洋医学,1992,13(1)：113-117.
[74]罗国安,王义明.中药复方的化学研究体系[J].世界科学技术——中药现代化,1999,1(1)：16-19.
[75]薛燕.中药复方霰弹理论[M].北京：中国环境科学出版社,1996.102-103.
[76]黄熙.方剂体内/血清成分谱与靶成分概念的提出及意义[J].第四军医大学学报,1999,20(4)：277-279.
[77]黄罗生,郭健新,刘咏梅,等.HPLC测定阿魏酸含量的探讨[J].中成药,2004,26(2)：134-136.
[78]吕海涛,王艳丽,王英芹.当归提取液中阿魏酸稳定性研究[J].中成药,2008,30(10)：1555.
[79]周浓,杨敏,白志川,段宝忠,姜北等.HPLC测定不同来源川楝子中阿魏酸的含量[J].中国实验方剂学杂志,2012,18(3)：84-87.
[80]肖静,王正荣,罗红琳.川芎嗪对脑循环和脑血管的作用[J].中药药理与临床,1994,10(3)：28-29.
[81]李秋怡,干国平,刘焱文.川芎的化学成分及药理研究进展[J].时珍国医国药,2006,17(7)：1298-99.
[82]谭载友,江涛,唐春萍,等.阿魏酸川芎嗪的抗血小板聚集作用[J].中国新药杂志,2003,12(7)：529-31.
[83]王雁南,杜俊蓉.藁本内酯对脑缺血再灌注大鼠的作用及其抗炎机制的研究[J].华西药学杂志,2010,25(6)：685-89.
[84]吴平丽,刘雯,张继全,等.川芎中洋川芎内酯A和Z-蒿本内酯的HPLC法测定[J].中国医药工业杂志,2010,41(4)：290-92.
[85]李桂生,马成俊,李香玉,等.藁本内酯稳定性研究及异构化产物的GC-MS分析[J].中草药,2000,31(6)：405-07.
[86]李中文,孙兆华,张慧.超临界C02萃取当归挥发油的研究及藁本内酯的HPLC法测定[J].齐鲁药事,2007,26(6)：36-37.
[87]Zhang X Z, Xiao H B, Xu Q, et al. Characterization of phthalides in Ligusticum chuanxiong by liquid chromatographic-atmospheric pressure chemical ionization-mass spectrometry [J]. J Chromatogr Sci,2003,41(8):428-32.
[88]谢雁呜,王建农,贺福元,等.中药群体药代动力学专家共识(征求意见稿)[J].中国中药杂志,2013,38(18)：2937-42.
[89]冯彬彬,张建海,张继芬,等.不同配伍对川芎嗪体内药代动力学影响[J].中国中药杂志,2012,37(9)：1311-14.
[90]刘晓东,薛玉英,谢林等.大鼠灌胃川芎、当归及其复方中阿魏酸的药代动力学[J].中国药科大学学报,2003,34(5)：448-51.
[91]潘莹.大鼠口服川芎和通脉方后阿魏酸的药动学比较[J].中国实验方剂学杂志,2011,17(4)：193-96.
[92]欧阳臻,潘璐琳,汤建明.阿魏酸、川芎及复方脑得生中阿魏酸在大鼠体内的药动学研究[J].中国中药杂志,2010,2：226-28.
[93]王岚,成龙,李慧,等.川芎和黄芪合用在心肌缺血大鼠体内藁本内酯血药浓度测定及药代动力学研究[J].中国实验方剂学杂志,2010,16(15)：158-61.
1 Zhang Yun-ke. Influences of Buyanghuanwu decoction and its decomposed formulas on cell apoptosis and expressions of Bcl-2 and Bax protein in rats withcerebral ischemia /reperfusion[J]. Chinese Journal of Clinical Rehabilitation,2006,10 (43):196-199.
2莫孙炼,郑有顺.补阳还五汤拆方对实验性脑缺血损伤大鼠脑组织皮层梗塞面积及血浆内皮素的影响[J].中药药理与临床,1997,13(1)：1-3.
3赵燕玲,曲友直.补阳还五汤及拆方对脑缺血后髓过氧化物酶活性及神经细胞凋亡的影响[J].中国中医急症,2007,16(3)：317-318.
4张林,孙宏伟,马贤德等.不同黄芪剂量的补阳还五汤对局灶性脑缺血大鼠血清IL-1,IL-6, IL-10的影响[J].中国实验方剂学杂志,2009,15(10)：62-64.
5邓常青,贺福元,唐映红等.补阳还五汤及其有效部位组方对沙鼠脑缺血再灌注后海马CA1区超微结构的影响[J].中药药理与临床,2000,16(3)：7-9.
6 Lu JF, Ii CX, Muteliefu. Efects of B YHW Decoction and Its Efective Constituents on the Fluidity of the Cell M embrane in a Stroke-Modeled Rat Brain[J]. Chinese Pharmaceutical Sciences,2002,11(4):132-137.
7周赛男.补阳还五汤对大鼠局灶性脑缺血后神经细胞增殖及脑组织TLR2、TLR4、 NF-k-B、MyD88表达的影响[D].湖南中医药大学博士论文.2012,5.
8李淑贞,杨桂染,杨艳梅等.补阳还五汤及拆方预处理对MIRI家兔心功能的影响[J].西南国防医药,2010,20(1)：13-16.
9崔翰博,韩涛,张东钰,鞠翡翡.补阳还五汤及拆方对代谢综合征模型大鼠胰岛素、TXA2水平的影响[J].山东中医药大学学报,2011,35(5)：446-447.
10张鹞年.补阳还五汤中用不同剂量的黄芪治疗脑梗塞恢复期108例对比观察[J].上海中医药杂志,1997,7：10-11.
11谢裕华,陈超.不同黄茂剂量的补阳还五汤对中风后遗症患者外周血ESR PCV及水平的影响[J].新中医,2007,39(6)：23-24.
12杨文斌,马世民,王艳萍等.应用不同剂量黄芪的补阳还五汤治疗慢性脑供血不足临床观察[J].中国中医药信息杂志,2011,18(5)：7-9.
13徐杰男,阙华发,唐汉钧等.不同黄芪剂量补阳还五汤促进大鼠慢性难愈性创面修复愈合的实验研究[J].浙江中西医结合杂志,2009,19(9)：534-536.
14闫润红,王世,闫志芳.不同黄芪剂量的补阳还五汤对“气虚血瘀”家兔血粘度的影响[J].中药药理与临床,1999,15(1)：7-9.
15陈红海.补阳还五汤及其拆方抗脑缺血损伤作用的比较研究[D].河北医科大学硕士论文,2011.10.
16林桂涛,尹宁宁,马承严.灰色系统理论与均匀设计结合在补阳还五汤处方筛选中的应用[J].中国实验方剂学杂志,1997,3(4)：16-20.
17姚晖,吴勇杰,张继平等.补阳还五汤体外对家兔血小板PAF受体活性影响的拆方研究[J].新中医,2005,37(12)：82-84.
18朱伟,王学美.黄芪剂量对补阳还五汤中芍药苷、阿魏酸含量的影响[J].中国实验方剂学杂志,2006,12(3)：11-14.
19张珊珊,贺福元,胥新元.补阳还五汤及单味药材多糖成分含量的比较研究[J].湖南中药学院学报,2001,21(1)：13-15.
20李晨旭,卢号芬,古力努尔等.补阳还五汤及其有效成分体外给药对脑细胞膜流动性的影响[J].中国药学杂志,2001,36(8)：528-531.
21陈瑞芬,邓常青,陈北阳等.补阳还五汤四类有效部位对大鼠脑缺血再灌注后Caspase表达的作用[J].中医药导报,2009,15(6)：4-8.
22李花,陈刚,邓常清.补阳还五汤及其有效组分和三七总皂苷含药血浆抗血小板衍生生长因子诱导的血管平滑肌细胞增殖的作用[J].中西医结合学报,2009,7(11)：1078-108.
[2]世界卒中日已成为了人类死亡的第二大元凶http://www.06news.com/ szxw/2012-10-29/423.html
[3]GAN Zhao-ru, SANG Li-nan. Research Progress of Cascade by Cerebral ischemia-reperfusion Injury [J]. Medical Recapitulate,2009,15(1):43-45.
[4]Waldowski K, Seniow J, Lesniak M, et al. Effect of low-frequency repetitive transcranial magnetic stimulation on naming abilities in early-stroke aphasic patients:a prospective, randomized, double-blind sham-controlled study [J]. Scientific World Journal,2012:518568. doi:10.1100/2012/518568.
[5]Duangjit S, Muangpaisan W, Chotinaiwattarakul W, et al. Functional Recovery at 3 Months in Stroke Patients Not Receiving Thrombolytic Therapy:The Comparison Between Patients Arriving Earlier and Later Than 4.5 Hours [J]. J Stroke Cerebrovasc Dis,2012,28(12):1052-57.
[6]Sanberg PR, Eve DJ, Metcalf C, et al. Advantages and challenges of alternative sources of adult-derived stem cells for brain repair in stroke [J]. Prog Brain Res,2012,201: 99-117.
[7]Sinden JD, Vishnubhatla I, Muir KW. Prospects for stem cell-derived therapy in stroke[J]. Prog Brain Res,2012,201:119-67.
[8]杨艺敏.Notch信号通路对大鼠脑缺血后神经再生的调控及辛伐他汀与神经再生关系的实验研究[D].吉林大学博士学位论文,2010.3.
[9]Wang Z, Huang W, Zuo Z. Perioperative aspirin improves neurological outcome after focal brain ischemia possibly via inhibition of Notch 1 in rat [J]. J Neuroinflammation, 2014,11(1):56-60.
[10]Zhang L, Dong LY, Li YJ, et al. miR-21 represses FasL in microglia and protects against microglia-mediated neuronal cell death following hypoxia/ischemia [J]. Glia,2012,60(12):1888-95.
[11]Fernandes D J, Ravenhall C E, Harris T, et al. Contribution of the p38 MAPK signalling pathway to proliferation in human cultured airway smooth muscle cells is mitogen-specific[J]. Br J Pharmaco,2004,142:1182-90.
[12]Engel F B, Schebesta M, Duong M T, et al. p38 MAPK kinase inhibition enables proliferation of adult mammalian cardiomyocytes [J]. Genes Dev,2005,19(10): 1175-87.
[13]Osuga H, Osuga S, Wang FH, et al. Cyclin-depedent kinases as a therapeutic target for stroke [J]. Proc Natl Acad Sci USA,2000,97(18):10254-59.
[14]Castro-Alvarez JF, Gutierrez-Vargas J, Darnaudery M, et al. ROCK inhibition prevents tau hyperphosphorylation and p25/CDK5 increase after global cerebral ischemia [J]. Neurosci,2011,125(3):465-72.
[15]Slimone DG, Vilen M, Farid A, et al. Cell cycle inhibition provides neuroprotection and reduces glial proliferation and scar formation after traumatic brain injury [J]. PNAS, 2005,102(23):8333-38.
[16]张贵斌,徐运兰,王伟,等.细胞周期调控对大鼠局灶性脑缺血后迟发性神经元死亡的影响[J].中风与神经疾病杂志,2005,22(6)：495-97.
[17]谭斌.浅谈祖国医学对中风病的认识[J].中医中药,2009,28(33)：91-93.
[18]王清任.医林改错[M].上海：上海科技出版社,1966.31.
[19]张均克,邱幸凡,张六通,等.论祛瘀生新促血管新生的理论基础[J].湖北中医药大学学报,2010,12(5)：47-48.
[20]金远林,王海燕,邱幸凡,等.祛瘀生新法对脑梗死大鼠治疗性血管新生作用及机制研究[J].中国中医急症,2006,15(6)：632-33,638.
[21]张金生.中医“祛瘀生新”理论内涵新解[J].辽宁中医杂志,2009,36(5)：704-05.
[22]韩经丹,王珏,王永炎,等.“毒损脑络”与脑缺血-再灌注损伤病机关系探讨[J].中国中医基础医学杂志,2012,18(8)：823-29.
[23]Zhang Yun-ke. Influences of Buyanghuanwu decoction and its decomposed formulas on cell apoptosis and expressions of Bcl-2 and Bax protein in rats withcerebral ischemia /reperfusion [J]. Chinese Journal of Clinical Rehabilitation,2006,10 (43):196-99.
[24]邓常青,贺福元,唐映红,等.补阳还五汤及其有效部位组方对沙鼠脑缺血再灌注后海马CA1区超微结构的影响[J].中药药理与临床,2000,16(3)：7-9.
[25]邓凯文,贺福元,刘文龙,等.RP-HPLC法测定大鼠血浆中补阳还五汤及总苷中芍药苷的含量及药代动力学研究[J].中药材,2010,33(11)：1768-70.
[26]贺福元,邓凯文,刘文龙,等.中药复方药物动力学总量统计矩法的实验验证研究[J].中国中药杂志,2013,38(2)：253-62.
[27]贺福元,邓凯文,刘文龙,等.中药复方网络药理学及其动力学关键技术问题的 探讨[C].首届全国方剂组成原理高峰论坛论文集,2012,28(5)：256-59.
[28]李再新,吴小红,贺福元,等.补阳还五汤中川芎嗪的药代动力学研究[J].中国药业,2007,16(18)：23-24.
[29]贺福元,刘文龙,贺庆平等.反相高效液相色谱法测定大鼠血浆中补阳还五汤总生物碱中川芎嗪的含量及药代动力学研究[J].中国药物与临床,2005,5(12)：920-22.
[30]尹天雷,李雅,李鹤白,等.补阳还五汤对脑缺血模型大鼠脑损伤的保护作用[J].世界中医药,2007,2(4)：241-42.
[31]刘芳,白雪松,刘柏炎,等.补阳还五汤对脑缺血大鼠碱性成纤维细胞生长因子的影响[J].中国中西医结合急救杂志,2008,15(1)：9-12.
[32]俞悦,易健,蔡光先,等.Caveolin-1敲除对C57BL/6小鼠繁殖性能及子代离乳前体质量的影响[J].湖南中医药大学学报,2011,31(11)：7-9.
[33]刘柏炎,蔡光先,刘维,等.补阳还五汤对大鼠局灶性脑缺血后血管内皮生长因子及其受体Flk1的影响[J].中草药,2007,38(3)：394-96.
[34]周赛男.补阳还五汤对大鼠局灶性脑缺血后神经细胞增殖及脑组织TLR2.TLR4、 NF-k-B、MyD88表达的影响[D].湖南中医药大学博士论文.2012,5.
[35]Yi J, Huang X, Yu Y, Cai GX, Liu BY. Effect of Buyang Huanwu decoction on interleukin-1β and tumor necrosis factor-a expression in rats after cerebral infarction [J]. Zhongguo Wei Zhong Bing Ji Jiu Yi Xue.2010,22(10):599-601.
[36]Zhao LD, Wang JH, Jin GR, et al. Neuroprotective effect of Buyang Huanwu decoction against focal cerebral ischemia/reperfusion injury in rats--time window and mechanism [J]. J Ethnopharmacol,2012,140(2):339-44.
[37]Li TJ, Qiu Y, Rui YC, Li TZ, et al. DNA microarray analysis of protective mechanism of buyang huanwu decoction on focal cerebral ischemia/reperfusion in rats [J]. Zhongguo Zhong Yao Za Zhi,2004,29(6):559-63.
[38]李淑贞,杨桂染,杨艳梅等.补阳还五汤及拆方预处理对MIRI家兔心功能的影响[J].西南国防医药,2010,20(1)：13-16.
[39]崔翰博,韩涛,张东钰,等.补阳还五汤及拆方对代谢综合征模型大鼠胰岛素、TXA2水平的影响[J].山东中医药大学学报,2011,35(5)：446-47.
[40]邓常青,贺福元,唐映红,等.补阳还五汤及其有效部位组方对沙鼠脑缺血再灌注后海马CA1区超微结构的影响[J].中药药理与临床,2000,16(3)：7-9.
[41]Spicer Z, Millhorn D E. Oxygen sensing in neuroendocrine cells and other cell types: pheochromocytoma (PC 12) cells as an experimental model [J]. Endocr Pathol,2003, 14(4):277.
[42]Hou CW, Chen YL, Chuang SH, et al. Protective effect of a sesamin derivative,3-bis (3-methoxybenzyl) butane-1,4-diol on ischemic and hypoxic neuronal injury [J]. J Biomed Sci.2014,21(1):15. doi:10.1186/1423-0127-21-15.
[43]. Guo H, Shen X, Xu Y, et al. The effect of activin A on signal transduction pathways in PC12 cells subjected to oxygen and glucose deprivation [J]. Int J Mol Med,2014, 33(1):135-41.
[44]Lin CY, Wang N, Zhang YF, et al. Neuroprotective effect of muscone on glutamate-induced apoptosis in PC 12 cells via antioxidant and Ca2+antagonism [J]. Neurochem Int,2014,14. doi:10.1016/j.neuint.2014.03.003.
[45]Yang R, Cui HJ, Wang H, et al. N-Stearoyltyrosine Protects Against Glutamate-Induced Oxidative Toxicity by an Apoptosis-Inducing Factor (AIF)-Mediated Caspase-Independent Cell Death Pathway [J]. J Pharmacol Sci,2014,124(2):169-79.
[46]胡海燕,席东泽,雷磊,等.清心开窍方含药脑脊液对谷氨酸致PC12细胞损伤的保护机制[J].中国中药杂志,2013,38(12)：1997-2000.
[47]Ma S, Liu X, Xun Q, Zhang X. Neuroprotective Effect of Ginkgolide K against H2O2-Induced PC 12 Cell Cytotoxicity by Ameliorating Mitochondrial Dysfunction and Oxidative Stress [J]. Biol Pharm Bull,2014,37(2):217-25.
[48]Wu FJ, Xue Y, Liu XF, et al. The protective effect of eicosapentaenoic acid-enriched phospholipids from sea cucumber Cucumaria frondosa on oxidative stress in PC 12 cells and SAMP8 mice [J]. Neurochem Int,2014,64:9-17.
[49]Rizak J, Tan H, Zhu H, et al. Chronic treatment with the mood stabilizing drug lithium up-regulates nuclear factor E2-related factor 2 in rat pheochromocytoma PC12 cells in vitro [J]. Neuroscience, doi:10.1016/j.2013.10.036.
[50]富苏,范吉平,王新志.通络化痰胶囊对H202所致的PC12细胞凋亡的保护作用及机制[J].中国实验方剂学杂志,2013,19(12)：188-93.
[51]唐春萍,陈雪梅.不同提取工艺对补阳还五汤药理作用的影响[J].中成药,1999,21(7)：360-62.
[52]陈红海.补阳还五汤及其拆方抗脑缺血损伤作用的比较研究[D].河北医科大学硕士论文,2011.10.
[53]谢裕华,陈超.不同黄茂剂量的补阳还五汤对中风后遗症患者外周血ESR PCV水平的影响[J].新中医,2007,39(6)：23-24.
[54]Swanson R A. A Semiautomatod method for measuring brain infarct volume [J]. J ceveb Blood Flow Metab,1990,10(2):293-95.
[55]LIU X G, XU L N. A rat middle cerebral artery thrombosis model for evaluation of thrombolytie and amithrunbotis agents [J]. Acta Pharm Sin,1995,30(9):662-67.
[56]Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats [J]. Stroke,1989,20(1):84-91.
[57]Meyerson M, Enders G H, Wu C L, et al. A family of human cdc2-related protein kinases [J]. EMBO J,1992,11(8):2909-17.
[58]Patrick GN, Zukerberg L, Nikolic M, et al. Conversion of p35 to p25 deregulates CDK5 activity and promotes neurodegeneration [J]. Nature,1999,402:615-22.
[59]Lee M S, Kwon Y T, Li M W, et al. Neurotoxicity induces cleavage of p35 to p25 by calpain [J]. Nature,2000,405(18):360-64.
[60]Green SL, Kulp KS, Vulliet R. Cyclin-dependent protein kinase 5 activity increases in rat brain following ischemia [J]. Neurochem Int.1997,31(4):617-23.
[61]Wang J, Liu S, Fu Y, et al. Cdk5 activation induces hippocampal CA1 cell death by directly phosphorylating NMDA receptors [J]. Nat Neurosci,2003,6(10):1039-47.
[62]Weishaupt JH, Neusch C, Bahr M. Cyclin-dependent kinase 5 (CDK5) and neuronal cell death [J]. Cell Tissue Res,2003,312(1):1-8.
[63]Rashidian J, Iyirhiaro G, AleyasinH, et al. Multiple cyclin-dependent kinases signals are critical mediators of ischemia-hypoxic neuronal death in vitro and in vivo [J]. PNAS, 2005,102(39):14080-85.
[64]Slimone DG, Vilen M, Farid A, et al. Cell cycle inhibition provides neuroprotection and reduces glial proliferation and scar formation after traumatic brain injury [J]. PNAS, 2005,102(23):8333-38.
[65]Menn B, Bach S, Blevins TL, et al. Delayed treatment with systemic (S)-roscovitine provides neuroprotection and inhibits in vivo CDK5 activity increase in animal stroke models [J]. PLoS One,2010,5(8):e12117.
[66]Small DL, Monette R, Foumier M C, et al. Characterization of cyclin D1 expression in a rat global model of cerebral ischemia [J]. Brain Res,2001,923(1):26-37.
[67]Boutillier A L, Kienlen-Campard P, Loeffler J P. Depolarization regulates cyclin D1 degradation and neuronal apoptosis:a hypothesis about the role of the ubiquitin/ proteasome signaling pathway [J]. Eur J Neurosci,1999,11(3):441-47.
[68]Timsit S, Rivera S, Ouaghi P, et al. Increased cyclin D1 in vulnerable neurons in the hippocampus after ischemia and epilepsy:a modulator of in vivo programmed cell death [J]. Eur J Neurosci,1999,11(1):263-78.
[69]柳耀泉,张维杰.局灶性脑缺血对细胞周期蛋白D1/CDK4基因表达的影响[J].中华神经医学杂志,2007,6(8)：770-72.
[70]陈华先,丁旭东,朱舟,等.脑缺血上调大鼠神经元和星形胶质细胞CDK4的表达[J].神经损伤与功能重建,2008,3(4)：221-23.
[71]乔海兵,尚君璟.小鼠脑缺血/再灌注后细胞周期相关蛋白在神经元的表达规律[J].山西医科大学学报,2013,44(9)：671-74.
[72]游哲斌,张洁,张德贤,等.黄连素对脑缺血再灌注后细胞cyclin D1、CDK4表达的影响[J].中华神经医学杂志,2008,7(8)：788-92.
[73]田代真一.“血清药理学”和“血清药化学[J].代东洋医学,1992,13(1)：113-117.
[74]罗国安,王义明.中药复方的化学研究体系[J].世界科学技术——中药现代化,1999,1(1)：16-19.
[75]薛燕.中药复方霰弹理论[M].北京：中国环境科学出版社,1996.102-103.
[76]黄熙.方剂体内/血清成分谱与靶成分概念的提出及意义[J].第四军医大学学报,1999,20(4)：277-279.
[77]黄罗生,郭健新,刘咏梅,等.HPLC测定阿魏酸含量的探讨[J].中成药,2004,26(2)：134-136.
[78]吕海涛,王艳丽,王英芹.当归提取液中阿魏酸稳定性研究[J].中成药,2008,30(10)：1555.
[79]周浓,杨敏,白志川,段宝忠,姜北等.HPLC测定不同来源川楝子中阿魏酸的含量[J].中国实验方剂学杂志,2012,18(3)：84-87.
[80]肖静,王正荣,罗红琳.川芎嗪对脑循环和脑血管的作用[J].中药药理与临床,1994,10(3)：28-29.
[81]李秋怡,干国平,刘焱文.川芎的化学成分及药理研究进展[J].时珍国医国药,2006,17(7)：1298-99.
[82]谭载友,江涛,唐春萍,等.阿魏酸川芎嗪的抗血小板聚集作用[J].中国新药杂志,2003,12(7)：529-31.
[83]王雁南,杜俊蓉.藁本内酯对脑缺血再灌注大鼠的作用及其抗炎机制的研究[J].华西药学杂志,2010,25(6)：685-89.
[84]吴平丽,刘雯,张继全,等.川芎中洋川芎内酯A和Z-蒿本内酯的HPLC法测定[J].中国医药工业杂志,2010,41(4)：290-92.
[85]李桂生,马成俊,李香玉,等.藁本内酯稳定性研究及异构化产物的GC-MS分析[J].中草药,2000,31(6)：405-07.
[86]李中文,孙兆华,张慧.超临界C02萃取当归挥发油的研究及藁本内酯的HPLC法测定[J].齐鲁药事,2007,26(6)：36-37.
[87]Zhang X Z, Xiao H B, Xu Q, et al. Characterization of phthalides in Ligusticum chuanxiong by liquid chromatographic-atmospheric pressure chemical ionization-mass spectrometry [J]. J Chromatogr Sci,2003,41(8):428-32.
[88]谢雁呜,王建农,贺福元,等.中药群体药代动力学专家共识(征求意见稿)[J].中国中药杂志,2013,38(18)：2937-42.
[89]冯彬彬,张建海,张继芬,等.不同配伍对川芎嗪体内药代动力学影响[J].中国中药杂志,2012,37(9)：1311-14.
[90]刘晓东,薛玉英,谢林等.大鼠灌胃川芎、当归及其复方中阿魏酸的药代动力学[J].中国药科大学学报,2003,34(5)：448-51.
[91]潘莹.大鼠口服川芎和通脉方后阿魏酸的药动学比较[J].中国实验方剂学杂志,2011,17(4)：193-96.
[92]欧阳臻,潘璐琳,汤建明.阿魏酸、川芎及复方脑得生中阿魏酸在大鼠体内的药动学研究[J].中国中药杂志,2010,2：226-28.
[93]王岚,成龙,李慧,等.川芎和黄芪合用在心肌缺血大鼠体内藁本内酯血药浓度测定及药代动力学研究[J].中国实验方剂学杂志,2010,16(15)：158-61.
1 Zhang Yun-ke. Influences of Buyanghuanwu decoction and its decomposed formulas on cell apoptosis and expressions of Bcl-2 and Bax protein in rats withcerebral ischemia /reperfusion[J]. Chinese Journal of Clinical Rehabilitation,2006,10 (43):196-199.
2莫孙炼,郑有顺.补阳还五汤拆方对实验性脑缺血损伤大鼠脑组织皮层梗塞面积及血浆内皮素的影响[J].中药药理与临床,1997,13(1)：1-3.
3赵燕玲,曲友直.补阳还五汤及拆方对脑缺血后髓过氧化物酶活性及神经细胞凋亡的影响[J].中国中医急症,2007,16(3)：317-318.
4张林,孙宏伟,马贤德等.不同黄芪剂量的补阳还五汤对局灶性脑缺血大鼠血清IL-1,IL-6, IL-10的影响[J].中国实验方剂学杂志,2009,15(10)：62-64.
5邓常青,贺福元,唐映红等.补阳还五汤及其有效部位组方对沙鼠脑缺血再灌注后海马CA1区超微结构的影响[J].中药药理与临床,2000,16(3)：7-9.
6 Lu JF, Ii CX, Muteliefu. Efects of B YHW Decoction and Its Efective Constituents on the Fluidity of the Cell M embrane in a Stroke-Modeled Rat Brain[J]. Chinese Pharmaceutical Sciences,2002,11(4):132-137.
7周赛男.补阳还五汤对大鼠局灶性脑缺血后神经细胞增殖及脑组织TLR2、TLR4、 NF-k-B、MyD88表达的影响[D].湖南中医药大学博士论文.2012,5.
8李淑贞,杨桂染,杨艳梅等.补阳还五汤及拆方预处理对MIRI家兔心功能的影响[J].西南国防医药,2010,20(1)：13-16.
9崔翰博,韩涛,张东钰,鞠翡翡.补阳还五汤及拆方对代谢综合征模型大鼠胰岛素、TXA2水平的影响[J].山东中医药大学学报,2011,35(5)：446-447.
10张鹞年.补阳还五汤中用不同剂量的黄芪治疗脑梗塞恢复期108例对比观察[J].上海中医药杂志,1997,7：10-11.
11谢裕华,陈超.不同黄茂剂量的补阳还五汤对中风后遗症患者外周血ESR PCV及水平的影响[J].新中医,2007,39(6)：23-24.
12杨文斌,马世民,王艳萍等.应用不同剂量黄芪的补阳还五汤治疗慢性脑供血不足临床观察[J].中国中医药信息杂志,2011,18(5)：7-9.
13徐杰男,阙华发,唐汉钧等.不同黄芪剂量补阳还五汤促进大鼠慢性难愈性创面修复愈合的实验研究[J].浙江中西医结合杂志,2009,19(9)：534-536.
14闫润红,王世,闫志芳.不同黄芪剂量的补阳还五汤对“气虚血瘀”家兔血粘度的影响[J].中药药理与临床,1999,15(1)：7-9.
15陈红海.补阳还五汤及其拆方抗脑缺血损伤作用的比较研究[D].河北医科大学硕士论文,2011.10.
16林桂涛,尹宁宁,马承严.灰色系统理论与均匀设计结合在补阳还五汤处方筛选中的应用[J].中国实验方剂学杂志,1997,3(4)：16-20.
17姚晖,吴勇杰,张继平等.补阳还五汤体外对家兔血小板PAF受体活性影响的拆方研究[J].新中医,2005,37(12)：82-84.
18朱伟,王学美.黄芪剂量对补阳还五汤中芍药苷、阿魏酸含量的影响[J].中国实验方剂学杂志,2006,12(3)：11-14.
19张珊珊,贺福元,胥新元.补阳还五汤及单味药材多糖成分含量的比较研究[J].湖南中药学院学报,2001,21(1)：13-15.
20李晨旭,卢号芬,古力努尔等.补阳还五汤及其有效成分体外给药对脑细胞膜流动性的影响[J].中国药学杂志,2001,36(8)：528-531.
21陈瑞芬,邓常青,陈北阳等.补阳还五汤四类有效部位对大鼠脑缺血再灌注后Caspase表达的作用[J].中医药导报,2009,15(6)：4-8.
22李花,陈刚,邓常清.补阳还五汤及其有效组分和三七总皂苷含药血浆抗血小板衍生生长因子诱导的血管平滑肌细胞增殖的作用[J].中西医结合学报,2009,7(11)：1078-108.