脑微透析/激光散斑成像技术与PK/PD结合进行补阳还五汤"益气行血生新"机制的研究

Buyang Huanwu Decoction has a significant clinical effects in treating ischemic stroke .Our Previous research has shown that it has the function of nourishing qi, moving blood and promoting tissue regeneration. But the mechanism remains poorly understood. This project takes rat models of cerebral ischemia/reperfusion as objects. Using microdialysis accompaning with LC-MS to simultaneously measure active constituent in brain、amino acid neural transmitter of pharmacodynamic indexes in awake and freely-moving rats after repeat administration. The changes of blood flow of brain, morphology of cerebral microvascular and infarct area after administration were monitored in real time by laser speckle imaging(LSI) technique，combining with hemorheological index to clarify the mechanism of nourishing qi and blood from the PK-PD of the local cerebral perspective；The immunohistochemical method was applied to determine Brain Tissue Micrangium Density, the number of VEGF and bFGF positive cell and new neurons cell ,we probe into the role of promoting tissue regeneration from the perspective of Microvessel and neurons cell. we take advantage of Cells in vitro experiments to Explore the access of Buyang Huanwu Decoction→Improving blood supply to the brain→increasing the number of bFGF→Activation and proliferation of Blood vessels and neurons. This research reveals the mechanism of nourishing qi, moving blood and promoting tissue regeneration of Buyang Huanwu Decoction treating on ischemic stroke. It is to explain the scientific grounds of treating ischemic stroke by Buyang Huanwu Decoction， to lay foundation on the in-depth study of stabilizing efficient Chinese medicine preparation.

补阳还五汤治疗缺血性脑中风具有显著的临床疗效，本课题组前期研究发现其具有"益气行血生新"作用，而作用机制尚不明确。本项目以脑缺血再灌注大鼠模型为用药对象，运用微透析采样与LC-MS联用技术同步测定重复给药过程中清醒模型动物脑组织有效组分和氨基酸类神经递质等药效指标，配合激光散斑成像技术监测给药后动物脑部血流、微血管形态及脑梗死面积变化，以及血液流变学指标，从脑局部PK/PD角度阐明补阳还五汤益气行血机制；运用免疫组织化学法测定脑组织微血管密度、VEGF和bFGF阳性细胞数和新生神经元细胞数，从微血管及神经元细胞角度探讨补阳还五汤"生新"作用；利用体外细胞实验探究"补阳还五汤→改善脑部供血→促使bFGF数量增加→血管和神经元细胞活化增殖→修复脑中风损伤"通路。本研究将揭示补阳还五汤治疗脑中风的益气行血生新机理，阐明其治疗缺血性脑中风及后期康复的作用机制，为稳定有效中药制剂的深入研究奠定基础。

本课题在“气旺则血行，血行则新生”的中医理论指导下，对补阳还五汤促进脑中风康复的作用机制进行了探究，运用微透析采样与LC-MS联用技术进行重复给药过程中清醒动物模型脑组织的药物动力学研究，配合激光散斑成像技术监测给药后动物脑部血流、脑梗死体积变化，以及血液流变学指标，从脑局部PK/PD角度阐明补阳还五汤的益气行血机制；同时在细胞水平探讨补阳还五汤“生新”的作用机制。研究发现复方中黄芪的剂量与指标成分的脑靶向效率、脑梗死体积和含水量，以及灌注期间的脑血流量有一定联系；血液流变学指标的变化与脑缺血时间有关；补阳还五汤能提高OGD/R损伤后细胞的存活率，调节VEGF、bFGF的表达从而发挥其对脑中风的保护作用。通过该项目的研究，阐明了补阳还五汤有效成分的脑内过程与药理效应的关系，加深了对补阳还五汤治疗脑中风机理的认识，其作用机制为调控bFGF信号通路相关因子的表达。