安宫牛黄丸调节细胞自噬对脑出血继发性损伤的修复作用及其机制研究

Treatment of secondary injury after cerebral hemorrhage is crucial for functional recovery of central nerve system. Apoptosis is involved in the secondary injury after cerebral hemorrhage. Whether AnGong NiuHuang Pill, which is effective in treating cerebral hemorrhage, can affect autophagy has not been reported. We will study the role of autophagy in the secondary injury after cerebral hemorrhage in the animal model induced by autologous blood injection in the brain. The rats will be divided into 6 groups: normal group, sham operated control group，model group，rapamycin plus AnGong NiuHuang Pill group, 3-MA plus AnGong NiuHuang Pill group and AnGong NiuHuang Pill group.We will evaluate the number of autophagic vascule and autolysosome at the different time point after intracerebral injection of autologous blood by electric microscopy, immunohistochemistry and confocal laser microscope. Expression of LC3I, LC3II, Beclin1 and Cathepsin D will be detected by immunohistochemistry. Based on these data we will elucidate the development of authphage after cerebral hemarrhage and the effect of AnGong NiuHuang Pill on autophage in treating cerebral hemorrhage, which will provide new evidence for treating cerebral hemorrhagic patients with AN Gong Niu Huang Pill.

防治继发性损伤是治疗脑出血的关键，细胞凋亡参与了脑出血的继发性损伤，而与细胞凋亡密切相关的自噬在脑出血发生后所起的作用及能否被安宫牛黄丸所调节，未见报道。安宫牛黄丸治疗脑出血疗效显著，但作用机制尚不明了。本课题通过脑内注射自体血建立大鼠脑出血模型，设立正常对照组、模型对照组、假手术组、安宫牛黄丸干预组、安宫牛黄丸+雷帕霉素干预组及安宫牛黄丸+3-甲基腺嘌呤霉素干预组，采用电镜、蛋白免疫印迹、免疫双标激光共聚焦显微镜检测不同时间点神经细胞增殖和凋亡、自噬体和自噬溶酶体、自噬相关蛋白LC3、LC3II/LC3I的比值及Beclin 1、Cathepsin D的表达，了解脑出血后自噬的发生和发展规律及其在脑出血过程中所起的作用，阐明安宫牛黄丸调节细胞自噬对脑出血继发性损伤的修复作用及其机制，为安宫牛黄丸治疗脑出血提供科学的理论基础和实验依据。

脑出血后继发损伤的病理生理机制尚不明确，造成现阶段对脑出血的治疗效果不理想，故防治继发性损伤是治疗脑出血的关键，自噬参与了脑出血的继发性损伤。安宫牛黄丸已广泛用于治疗脑出血。本课题应用电镜及免疫印记法检测脑出血后各组自噬相关蛋白LC3Ⅰ、LC3Ⅱ、Beclin 1、Cathepsin D表达水平及超微结构的变化，从而了解安宫牛黄丸对脑出血后自噬现象的影响。项目研究显示，脑出血后血肿周围脑组织中自噬途径被激活，表现为神经细胞内自噬泡及自噬蛋白表达增多；安宫牛黄丸通过提高自噬水平，适当延长自噬时间，从而对脑出血后血肿周围神经细胞起到保护作用，对神经功能的恢复有促进作用。在一定程度上为安宫牛黄丸治疗脑出血提供科学的理论基础和实验依据。