节点文献
补阳还五精简方脑健片治疗脑缺血后脑损伤的谱效关系和药代动力学数学模型研究
【作者】 刘青萍；
【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：从整体动物、分子生物学、谱效关系和药代动力学四个方面着手,探讨脑健片抗脑缺血损伤的作用规律。通过建立局灶性脑缺血大鼠模型,研究补阳还五汤精简方脑健片对脑缺血大鼠脑损伤的保护作用及对大鼠海马组织周期蛋白CyclinB1和CyclinB2表达的影响；结合Matlab软件编程分析脑健片药效成分与脑损伤保护作用之间的谱效关系和药代动力学变化,探讨补阳还五汤全方和脑健片的药效物质基础及作用规律,并建立了补阳还五汤全方和脑健片的药代动力学数学模型,为脑健片临床应用的有效性提供科学的实验依椐。方法：采用大脑中动脉线栓法建立局灶性脑缺血大鼠模型,将动物随机分为假手术组、模型组、补阳还五汤组、脑健片高、中、低剂量组。称取各组大鼠1d到7d的体重,并将各组大鼠于7d做神经功能评分,处理后TTC计算其梗死面积；于1d、3d、7d、14d、28d取脑组织进行WB、RT-PCR实验,检测其海马组织周期蛋白Cyclin B1和CyclinB2表达。运用PCA和GRA法对实验结果进行分析,获得补阳还五汤和脑健片指纹图谱与神经功能评分、脑梗死面积、脑干湿比等药效指标和周期蛋白Cyclin B1和Cyclin B2之间关系,并基于曲线拟合法建立其药代动力学数学模型。结果：(1)结合卡方拟合优度检验和两组独立样本T检验法,运用matlab编程分析的结果：脑健片组与模型组相比较,大鼠体重、脑干湿重比值明显增加(P<0.05),脑梗死面积(P<0.05)明显降低,且脑健片中剂量组对脑缺血的治疗效果最佳,其疗效略优于补阳还五汤组(正常人体剂量)。(2)WB和RT-PCR检测结果表明：脑健片组与模型组相比较,能有效降低周期蛋白Cyclin B1和Cyclin B2表达,差异具有统计学意义(P<0.05),脑健片组和补阳还五汤全方组间对Cyclin B1和CyclinB2表达的调控作用没有显著差异(P>0.05)。(3)谱效关系研究结果显示：脑健片和补阳还五汤样品共有峰排名均大于其各自的特有峰,且保留时间为tR=20.649、6.474、26.404min的化学成分对神经功能评分、脑梗死面积、脑干湿比、周期蛋白CyclinB1和Cyclin B2的贡献度排在前五位。(4)药代动力学模型评价的结果显示：脑健片和补阳全方对阿魏酸与槀本内酯的吸收分布和消除无明显差异(P>0.05),但补阳全方中的洋川芎内酯A更容易被体内吸收、分布和消除。结论：(1)脑健片能有效改善神经功能症状、缩小脑梗死面积、减轻脑水肿,显著降低脑缺血模型大鼠海马组织cyclins B1、cyclins B2的表达,且其作用与补阳还五汤全方相当。(2)脑健片中指纹图谱保留时间为tR=20.649、6.474、26.404min的化学成分对调控脑缺血损伤和干预周期蛋白起到关键作用,推测是其主要药效物质基础,且其作用机制可能与海马组织神经细胞周期的调控作用有关。(3)脑健片和补阳还五汤对阿魏酸与槀本内酯的吸收、分布和消除无明显差异,但洋川芎内酯A的吸收分布和消除具有显著性差异,此种差异是否会影响脑健片的药效,有待进一步研究。(4) PCA-GRA方法能客观评价中药复方的谱效关系,是一种值得借鉴的好方法。(5)基于曲线拟合的方法,结合matlab科学工具编程,建立了符合脑健片和补阳还五汤全方中阿魏酸、洋川芎内酯A、桑本内酯三种成分的药代动力学模型,能有效、客观的评价药物在体内的吸收、分布和消除过程,且数学模型的拟合结果优于传统二室模型,为中药复方药代动力学研究提供了新思路。(6)脑健片在治疗脑缺血方面具有良好的优势,符合中风患者的需求,为指导临床合理用药提供了理论依据。
【Abstract】 Objective:From four aspects of Animal, Molecular Biology, spectrum-effect relationship and pharmacokinetics, study on the protective effect of cerebral ischemic injury.On the basis of the establishment of focal cerebral ischemia in rats, to study the protective effect of Naojian Tablet (the thin recipe of Buyanghuanwu), which is based on the method of benefiting vital energy, removing stasis and producing new, on cerebral ischemia injury in rat and the effect on the expression of hippocampal tissue cyclin, CyclinB1and CyclinB2. On this basis, combined with matlab software programming to analyze the spectrum-effect relationship between the effective component of Naojian Tablet and the protective effect on cerebral injury, as well as the pharmacokinetics,and to investigate the the material basis for efficacy and law of action of Buyanghuanwu recipe and Naojian Tablet, and to establish mathematical model fitting the spectrum-effect relationship and pharmacokinetics of Buyanghuanwu recipe and Naojian Tablet, providing scientific experiments basis for the effectiveness of clinical applications of Naojian Tablet.Methods:The rat model of focal cerebral ischemia was built using middle cerebral artery occlusion, and the animals were randomly divided into sham operation group, model group, Buyanghuanwu Decoction (BYHWD) group, the high, medium and low dose of Naojian Tablet groups. The rats of each group were weighed at1d to7d, and the neurological score of the rats of each group was evaluated at7d, which was used to calculate infarct size with TTC after processing. The brain tissue was taken at1d,3d,7d,14d,28d to run WB, RT-PCR experiments to test the expression of their hippocampus cyclin, Cyclin B1and Cyclin B2. The experimental results were analyzed with PCA and GRA method to obtain the relationship between the fingerprints of BYHWD and Naojian Tablet, the pharmacodynamic indexes such as neurological score, cerebral infarct size, and dry-wet ratio of brain, and the Cyclin B1and Cyclin B2, then the spectrum-effect relationship and pharmacokinetics mathematical model was established based on curve-fitting method.Results:(1) Compared with the model group, body weight of rats in Naojian Tablet group increased significantly (P<0.05), neurological score (P<0.05), infarct size (P<0.05) and brain dry-wet ratio (P<0.05) were significantly decreased, and the medium dose group of Naojian Tablet had the optimal therapeutic effect on cerebral ischemia, which efficacy is slightly better than BYHWD group (normal human dose).(2) The test results of WB and RT-PCR showed that, compared with the model group, the expression of Cyclin B1and Cyclin B2can be reduced effectively in Naojian Tablet group, the difference was statistically significant (P<0.05), the regulation of Naojian Tablet on cyclin was most obvious at7d (P<0.05); the expression of Cyclin B1and Cyclin B2had no significant difference between Naojian Tablet group and BYHWD group (P>0.05).(3) The study result of spectrum-effect relationship showed that, for the samples of Naojian Tablet and BYHWD, the ranking of common peaks was larger than their respective unique peaks, and the contributions of chemical components at the retention time tR=20.649,6.474and26.404min on neurological score, infarct size, brain dry-wet ratio as well as Cyclin B1and Cyclin B2were of the top five.(4) The results of evaluation of pharmacokinetics model showed that there was no significant difference (P>0.05) between Naojian Tablet and BYHWD on the absorption, distribution and elimination of ferulic acid and ligustilide, but the senkyunolide in BYHWD was more easily absorbed、distributed and eliminated in body.Conclusion:(1) Naojian Tablet can improve neurological symptoms, reduce infarct size, relieve cerebral edema effectively, confirming that Naojian Tablet has protective effect on cerebral ischemia injury, and its effect is equivalent to the whole receipt of BYHWD. 40(2) Naojian Tablet can significantly reduce the expression of hippocampus Cyclins B1and Cyclins B2in cerebral ischemia model rats, and its effect is equivalent to the whole receipt of BYHWD.(3) The chemical compositions of tR=20.649,6.474and26.404min in the fingerprinting spectrum of Naojian Tablet may play a key role in regulation of cerebral ischemic injury and intervention of cyclin, which were speculated as its primary efficacy material basis.(4) There is no significant difference between Naojian Tablet and BYHWD on the absorption, distribution and elimination of ferulic acid and ligustilide, but the absorption, distribution and elimination of senkyunolide A has significant difference, on which a follow-up in-depth investigation can be carried on.(5) The spectrum-effect relationship of traditional Chinese medicine compound can be objectively evaluated based on PCA-GRA method, which is a good method worth learning.(6) The pharmacokinetic mathematical model established based on curve fitting method combined with matlab programming can effectively and objectively evaluate the absorption, distribution and elimination of drug in body, providing a new thought for the follow-up study of pharmacokintrics.(7) Naojian Tablet have a good advantage in the treatment of cerebral ischemia, according with the needs of stroke patients, which provides a theoretical basis for indicating theclinical rational medication.