GSS对IL-10激活Akt/mTOR/Atg通路调节慢性脑缺血细胞自噬的干预研究

Chronic cerebra ischemia plays a critical role in neurodegenerative disease progression and can cause permanent or progressive cognitive impairment. So far the pathogenesis of chronic cerebral ischemia remains unclear and the disease is clinically associated with unfavorable treatment outcome. Previous studies from our group and others revealed that excessive autophagy is involved in the pathological development of chronic cerebral ischemia. The IL-10/Akt/mTOR/Atg signal pathway has been known to negatively regulate autophagy. Our preliminary data showed that genistein-3'-sodium sulfonate(GSS) treatment improved the learning or memory function in rats with chronic cerebral ischemia and up-regulated and the Akt phosphorylation and mTOR phosphorylation in the hippocampus region. In addition, by studying the acute cerebral ischemia-reperfusion injury model, we found that GSS activated IL-10/IL-10R and the mTOR phosphorylation in the ischemic penumbra brain tissue, and down-regulated the expression of autophagy marker LC3B and Beclin-1. Hence, we hypothesize that GSS up-regulated IL-10,further activates the Akt/mTOR/Atg signal pathway to inhibit excessive autophagy, thereby improving the learning or memory impairment observed in chronic cerebral ischemia. In this project, we will utilize a variety of approaches, including behavior, morphology, pharmacology, biochemistry, molecular biology and electrophysiology, to fully investigate the mechanism by which GSS improves the cognitive impairment caused by chronic cerebral ischemia both in vitro and in vivo. Hopefully, our study can provide guidance for drug development in the field of rehabilitation therapy.

慢性脑缺血是神经退行性疾病过程中的重要环节，可引起持久或进展性认知功能损害，但其发病机制尚不明确，临床疗效也不理想。文献称，过度自噬参与了慢性脑缺血的病理过程，Akt/mTOR/Atg信号通路是自噬的负调控机制之一。项目组前期研究发现，染料木素磺酸钠（GSS）改善慢性脑缺血大鼠的认知功能，上调海马组织Akt及mTOR的磷酸化水平；提高急性脑缺血大鼠半损伤区脑组织IL-10与IL-10R表达，激活mTOR，减少自噬形成的标记性蛋白LC3B和Beclin-1的表达。结合“IL-10激活Akt/mTOR信号”的文献报道，我们假设，GSS通过上调IL-10激活Akt/mTOR/Atg信号通路负调控过度自噬，从而改善慢性脑缺血大鼠学习记忆能力。本项目拟采用行为学、形态学、药理学、生化与分子生物学及电生理学等方法，从离体及在体水平验证上述假说。该研究成果可为开发脑功能康复治疗药物提供理论与实践基础。

自噬在慢性脑缺血诱导的神经元损伤中的作用尚不明确。本研究从自噬角度出发，从在体与离体水平探讨染料木素磺酸钠(genistein-3'-sodium sulfonate,GSS)对慢性脑缺血大鼠海马功能的保护作用及机制。方法：分别采用双侧结扎大鼠颈总动脉（2-vesselocclusion,2-VO）缺血8W和氧糖剥夺再灌注（OGD/R）处理直接共培养HT22/BV2细胞制备慢性脑缺血损伤模型，运用Morris水迷宫实验、恐惧箱实验评价大鼠的学习记忆和空间记忆功能；HE染色、尼氏染色、免疫荧光标记及透射电镜等手段观察大鼠海马区神经元的损伤情况；运用iTRAQ技术、生物信息学和细胞干预等方法阐明GSS对2-VO大鼠的保护机制。结果：在缺血1，2，4，8W的进展中，大鼠出现学习记忆功能渐进性减退、自噬程度逐渐增强和线粒体损伤逐渐加重等特征，第8W至12W则维持相对稳定状态。GSS治疗8W后：①显著缩短大鼠平均逃避潜伏期、延长空间探索时间和增加穿越隐匿平台次数；增加大鼠恐惧训练后的僵立时间、僵立静止频率，并减少狂躁时间和狂躁频率；②有效改善大鼠海马CA1区神经元及尼氏小体的形态和数量，增强NeuN和iBa1荧光标记的阳性细胞数量与荧光强度，并减少自噬小体的数量；③下调Beclin-1、LC3B、P62和ULK-1和上调mTOR、Akt、P70s6k（磷酸化）蛋白(和/或)mRNA表达水平；④Rapa可以部分阻断GSS抑制p-P62蛋白的表达，但对GSS抑制Beclin-1和LC3B的阻断作用无明显差异；⑤显著升高海马组织中IL-10的蛋白表达水平。结论：慢性脑缺血可能诱导大鼠海马神经元出现自噬流甚至过度自噬，GSS可能通过上调IL-10，直接或间接激活Akt/mTOR通路抑制过度自噬对神经元的损伤，从而保护2-VO大鼠，mTORC1可能是GSS抑制过度自噬的关键靶点。