甘青铁线莲中新的活性成分APG激活GSK-3β/Nrf2通路减轻脑缺血/再灌注损伤的机制研究

Ischemic stroke is a major disease which has seriously jeopardized human health.At present, there are no ideal clinical treatments or measures. The process of discovering novel efficient and safe drugs was more urgent. The applicant obtained the active ingredient named APG from the Tibet herbal medicine Clematis tangutica. And found for the first time that APG has significantly reduced cerebral ischemia/reperfusion injury, reduced infarct size and improved neurological behavior score. The result of pre-experiment showed that APG pretreatment increased GSK-3β phosphorylation after reperfusion on ischemic injury, decreased endogenous ROS levels, increased T-SOD activities, and inhibited cell apoptosis. This suggests that APG may activated GSK-3β/Nrf2 pathway, accommodated ischemia reperfusion-mediated oxidative stress, exerted a protection on the ischemic brain. Thus, we planned to apply the Gene Transfection, Gene Silencing, Flow Cytometer and Immunohistochemisty method and technic to explore the potential role of GSK-3β/Nrf2 pathway in brain protection of APG on the animal, cellular and molecular levels. This project provide theoretical basis for APG treating ischemic stroke in clinical in the future

缺血性脑卒中是严重危害人类健康的重大疾病。目前临床尚无理想的治疗药物或措施。寻找新型高效安全、作用机制清晰的脑卒中防治药物是治疗该疾病的迫切需求。申请者前期从传统藏药甘青铁线莲中获得一种黄酮苷类活性成分APG，首次发现APG能够显著降低大鼠脑中动脉栓塞(MCAO)造成的缺血再灌注损伤，减少脑梗死面积，改善神经行为学评分。预实验研究发现APG干预能够通过增加脑缺血损伤后GSK-3β磷酸化，降低内源性ROS水平，增加T-SOD的活性，抑制细胞凋亡。提示APG可能通过激活GSK-3β/Nrf2通路，调节缺血/再灌注损伤介导的氧化应激，进而发挥脑保护作用。因此，本项目拟在前期工作的基础上，采用基因转染技术，基因沉默技术，流式细胞术、免疫组化、免疫电镜技术等方法，从整体、细胞、分子水平阐明APG的脑保护机制，为其治疗缺血性脑卒中的临床应用提供理论依据。

脑中风严重威胁人类生命健康，已成为全世界范围内引起人类死亡和残疾的第二大常见死因，且发病率有逐年增多趋势。从传统中医药或民族药中寻找新型安全有效、作用机制清晰的治疗脑卒中的活性成分，使中风患者能够得到及时、有效的治疗及干预，促进健康恢复是目前临床研究的热点问题。申请者前期从传统藏药甘青铁线莲中获得一种黄酮苷类活性成分APG，首次发现APG干预具有明显的神经保护作用，并且可以上调脑缺血损伤后GSK-3β磷酸化的表达，抑制细胞凋亡。那么APG是否通过激活GSK-3β/Nrf2通路，调节缺血/再灌注损伤介导的氧化应激，进而发挥脑保护作用。本课题证实在体和离体实验中，APG均能上调缺血在灌注损伤后GSK-3β磷酸化和Nrf2蛋白的表达，抑制神经元细胞凋亡。体内试验结果显示：基因敲除Nrf2蛋白能够逆转APG诱导的抗氧化损伤作用；PI3K抑制剂Wortmannin和LY294002均可抑制GSK-3β磷酸化，逆转APG的脑保护作用。体外实验结果显示利用腺病毒转染、siRNA和抑制剂等干涉的方法分别下调Nrf2和p-GSK-3β的表达，结果发现下调Nrf2和p-GSK-3β均能逆转APG的脑保护作用。上述结果提示GSK-3β/Nrf2在APG诱导的脑缺血耐受，抑制神经元细胞凋亡过程中发挥了重要作用。为开发缺血性脑损伤防治策略提供新思路。