【作者】 刘芳；

【导师】 蔡光先；

【作者基本信息】 湖南中医药大学 ， 中医内科学， 2014， 博士

【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。更多还原

【Abstract】 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.更多还原