犀角地黄汤通过抑制神经元自噬保护脑缺血再灌注损伤的作用机理研究

Cerebral ischemia reperfusion injury is a difficult problem to be solved in stroke treatment. The recent studies have shown that autophagy in cerebral ischemia reperfusion can aggravate neuronal damage, and autophagy inhibition has been proposed as a new strategy for stroke treatment. Xijiao Dihuang Decoction (XDD), a empirical formula used by Chinese medicine master Zhou Zhongying, is proved to be effective in treating stroke, and could significantly inhibit cerebral ischemia reperfusion induced autophagy activation with decrease of Atg-5, Beclin-1 expression. However, it is not clear that whether XDD exhibit protective effect in cerebral ischemic reperfusion through inhibiting autophagy. Aiming to study the neuroprotective mechanism of XDD, we would firstly apply cell and animal models to confirm the protective effect of XDD on cerebral ischemia reperfusion injury. Secondly, immunofluorescence, RT-PCR, Western blot, are used to study the regulation effect of XDD on expression of autophagy-related key proteins and genes, revealing the relationship between the brain protection effect of XDD and autophagy; Lastly, regulation mechanism of XDD on neuronal autophagy is investigated using pathway inhibitor and RNA interference technology. The project would provide important scientific basis for clinical application and drug development of XDD, and propose a novel therapeutic strategy for stroke.

缺血再灌注损伤是脑卒中治疗亟待解决的难题。最新研究表明，自噬在缺血再灌注期可加重脑部神经细胞损伤，抑制自噬成为脑卒中新的治疗策略。犀角地黄汤是国医大师周仲瑛临床用于治疗脑卒中的验方，临床效果显著。课题组前期实验表明，该方可抑制脑缺血再灌注诱导的神经元自噬，降低Atg-5、Beclin-1表达。但自噬在犀角地黄汤保护脑缺血再灌注中的关键作用机理尚不明确。本项目拟通过细胞和动物模型验证犀角地黄汤对脑缺血再灌注损伤的保护作用；然后运用免疫荧光、RT-PCR、Western blot等细胞分子生物学技术，研究犀角地黄汤对细胞自噬途径关键分子及靶基因表达的影响，探讨犀角地黄汤神经保护作用与自噬的关系；并利用通路抑制剂、RNA干扰等技术研究调控神经元自噬的分子机理，为犀角地黄汤的临床应用及开发提供科学依据，亦为临床缺血性脑卒中治疗提供新的思路。

缺血再灌注损伤是脑卒中治疗亟待解决的难题。最新研究表明，自噬在缺血再灌注期可加重脑部神经细胞损伤，抑制自噬成为脑卒中新的治疗策略。犀角地黄汤是国医大师周仲瑛临床用于治疗脑卒中的验方，临床效果显著。课题组前期实验表明，该方可抑制脑缺血再灌注诱导的神经元自噬，降低Atg-5、Beclin-1表达。但自噬在犀角地黄汤保护脑缺血再灌注中的关键作用机理尚不明确。本项目通过体外神经细胞缺氧/复氧及大鼠脑缺血/再灌注模型对犀角地黄汤的神经保护作用进行多方面确证，发现犀角地黄汤可以改善大鼠的神经行为学，减轻细胞肿胀坏死及神经元的脱失，减少脑梗死体积；同时运用免疫荧光、电镜观察、Western blot等技术，从细胞和整体动物水平进一步研究发现犀角地黄汤抑制LC3-Ⅰ向LC3-Ⅱ转化及P62蛋白降解，减少自噬小体形成；促进mTOR和PI3K的表达，减低ERK磷酸化；自噬诱导剂雷帕霉素可加重脑缺血损伤，与犀角地黄汤联用能降低犀角地黄汤的神经保护作用，提示犀角地黄汤可能通过促进AKT-mTOR信号通路，抑制ERK磷酸化，从而抑制脑缺血的自噬改善脑缺血再灌注损伤。本研究从抑制神经元自噬的角度阐明犀角地黄汤保护脑缺血再灌注损伤的潜在机制，为临床脑卒中治疗提供一个新的思路，亦为后续犀角地黄汤临床应用及深入开发提供充分的科学依据和坚实的药理学基础。