微泡空化效应增强川芎嗪抗缺血再灌注损伤的物理/分子机制及声学调控研究

The recanalization of the ischemic region has been recognized to be crucial to the succussful treatment for the ischemic stroke with high incidence, high morbidity and high mortality. However, ischemia/reperfusion (I/R) might result in unrecoverable brain cell damage. Thus, there is urgent demands on exploring alterative clinic strategy to achieve effective ischemic recanalization with minimized cell damage. In the present project, systematic studies will be performed to investigate the physical mechanisms and acoustic modulation of enhanced tetramethylpyrazine (TMP) protection of cerebral I/R injury facilitated by ultrasound-induced microbubble cavitation activities, which is important for exploring new type neuro-protective drugs and therapeutic targets. Glutamate-induced toxicity to pheochromocytoma (PC12) cells will be established first to model I/R injury. The bioavailability of TMP could be improved due to the microbubble-mediated cavitation activity induced by ultrasound sonication, which would be helpful for enhancing the treatment efficacy of TMP in anti-cardiovascular/cerebrovascular diseases. Immunohistochemistry assay, enzymatic rate assay, enzyme-linked immunosorbent assay and real-time polymerase chain reaction were performed to examine changes in superoxide dismutase, lactate dehydrogenase, inflammatory factors and apoptosis, in an effort to provide insight into the biomolecular mechanisms underlying the neuroprotective effects of TMP. Passive cavitation detection, particle image of velocity system and scanning electron imaging will be used to quantitatively measure the inertial cavitation dosage (ICD), microstreaming field intensity induced by microbubble stable cavitation (SC) and the sizes of sonoporation pores generated in the cell membranes, respectively. The correlation between enhanced neuroprotetive effects of TMP and ICD/SC-induced microstreaming field intensity will be explored systemically. The results of this project will be helpful for optimizing TMP’s treatment effects on I/R injury by modulating acoustic excitation parameters, while minimizing its cytotoxity and other side-effect in clinic.

缺血性脑卒中的治疗中缺血再灌注会导致脑细胞损伤。本项目拟研究微泡空化效应增强川芎嗪抗脑缺血再灌注损伤的物理/分子机制及声学调控，探索新的神经保护药物及治疗靶点。建立谷氨酸损伤PC12细胞模型以模拟脑组织缺血再灌注损伤，从氧化应激、炎症反应、细胞抗凋亡等方面探讨川芎嗪抗缺血再灌注脑损伤的分子机制；基于被动空化探测系统检测微泡瞬态空化剂量、基于粒子速度成像系统测量稳态空化泡微流场产生的剪切力、基于扫描电镜观察不同超声作用模式下微泡空化效应产生的细胞膜声穿孔、基于多种生化分析手段来探索如何利用不同强度和不同作用模式的超声联合微泡提高川芎嗪的生物利用度，减小生物毒性；研究微泡瞬态空化剂量及稳态空化微流场强度与细胞膜声穿孔、川芎嗪抗缺血再灌注脑损伤疗效及生物毒性之间的相关性，进一步阐明不同强度及模式下超声联合微泡增强川芎嗪抗缺血再灌注损伤的物理机制，优化声学调控参数，为指导临床治疗新方案奠定基础。

本项目研究微泡空化效应增强川芎嗪抗脑缺血再灌注损伤的物理/分子机制及声学调控。按计划开展的工作包括：（1）首先研究川芎嗪减轻脑缺血再灌注损伤的疗效及分子机制；（2）构建超声细胞作用系统，优化超声作用参数。体外实验通过细胞活力、细胞迁移、细胞活性氧水平、谷胱甘肽、凋亡蛋白（caspase-8,-9,-3）以及相关因子（缺氧诱导因子-1α,p53,MDM2）探讨了超声增强川芎嗪减轻PC12细胞谷氨酸损伤的分子机制；（3）摸索超声对大鼠血脑屏障开放的最适参数，发现超声微泡可显著增强川芎嗪改善脑缺血再灌注损伤；（4）体外实验证实超声微泡增强川芎嗪对OGD模型PC12细胞的保护作用及机制；（5）进行超声导波抑菌的研究，明确其声学机制是基于导波传播带来的机械振动，分析原型装置的传播模式、频率、振幅等与抑菌效果的关系，研究不同振动幅度的导波对气管插管表面菌膜形成过程的抑制效果；（6）发现超声可增强铜绿假单胞菌生物膜对庆大霉素的敏感性，并干扰与细菌代谢、运动、抗生素抗性、毒性和生物膜形成相关基因的表达；（7）明确微泡在低强度脉冲超声激励下产生的声微流运动及剪切应力显著增加三维藻酸钙凝胶支架材料的孔隙率和通透性；（8）研究新颖的生物标志物miR-106b-5p在诊断、治疗急性缺血性脑卒中的应用，体内外实验阐明miR-106b-5p经由凋亡以及氧化损伤参与脑缺血再灌注损伤机理；（9）研究miR-199a-5p和膜微囊蛋白Caveolin-1在缺血性脑卒中患者及MCAO模型大鼠外周免疫组织的表达及临床意义；（10）鉴定MCAO模型大鼠脑组织和血清代谢物表达谱，分析差异代谢物可能参与的代谢通路；（11）研究化橘红柚皮苷对大鼠脑缺血再灌注损伤的疗效及Nrf2/HO-1和JAK2/STAT3信号通路的影响，深入探讨化橘红柚皮苷对脑缺血再灌注损伤神经保护机理。.本项目共发表SCI论文7篇，中文4篇，申请2项发明专利。在项目的支持下，共有3名硕士研究生从事该方向的研究，其中2名硕士已毕业，并获硕士学位。本项目团队成员获得3项国家自然科学基金项目的资助。积极参与学术会议，并做研究成果汇报。