脑外伤血脑屏障连接蛋白的氧化应激致损：大黄修复的吸收成分及其分子机制

Antioxidant strategy is the key of repairing blood brain barrier injury (BBB) following traumatic brain injury. The Chinese herb rhubarb originated from Chinese medicinal theory "Tong Fu Gong Xia" can perform the definite effects to treat traumatic brain injury by its multi-target effects. But the molecular mechanism of rhubarb in repairing BBB following TBI and the basis of its absorbed bioactive compounds are not well understood. We detected anthraquinones in cerebrospinal fluid of TBI rats model following oral rhubarb, and it is found that they could exert brain protection by anti-oxidation.The preliminary experiment suggested that the intervention of rhubarb and its absorbed bioactive compounds enhanced expression of Zonula Occludens-1(ZO-1) in TBI rats. On these grounds, we suppose that the molecular mechanism of traumatic brain injury protection by rhubarb and its absorbed bioactive compounds refer to the Blood Brain Barrier protection because of increased ZO-1 lever via NADPH oxidase/MAPK/NF-κB signal pathway. The research will start to determine the absorbed bioactive compounds from rhubarb into cerebrospinal fluid, then to apply the methods such as molecular biological skill and Liquid Chromatography with Mass Spectrometry, and combine with in vivo and vitro experiment, to study the molecular mechanism of rhubarb and its bioactive materials in performing blood brain barrier protection from the TBI- NADPH oxidase/MAPK/NF-κB signal pathway-BBB conjuction protein based on the whole body, cell and molecular lever, to elucidate compounds basis of TBI treatment, and to quantitate the contribution of absorbed compounds spectrum into cerebrospinal fluid to the original herb rhubarb, finally to open up a new way of research of brain protection by rhubarb.

抗氧化策略是修复颅脑外伤后血脑屏障（BBB）损伤的重中之重。"通腑攻下"中药大黄以其多靶点效应治疗颅脑外伤疗效确切，但不清楚其抗氧化修复BBB的物质基础及其分子机制。我们在颅脑外伤模型大鼠服用大黄后脑脊液中检测到蒽醌类成分，并发现其抗氧化脑保护作用。预实验显示大黄及吸收成分上调模型大鼠BBB闭锁小带蛋白-1（ZO-1）的表达。据此推测，大黄及吸收成分治疗颅脑外伤的分子机制涉及通过抑制NADPH氧化酶/MAPK/NF-κB途径上调ZO-1介导的BBB保护。本项目拟从检测大黄的脑脊液吸收成分出发，应用分子生物学、液质联用等技术，基于整体、细胞和分子水平三个层次，结合体内外实验，从颅脑外伤-NADPH氧化酶/MAPK/NF-κB通路-BBB连接蛋白途径研究大黄及吸收成分修复BBB的分子机制，以明确大黄治疗颅脑外伤的药效物质基础，定量药效物质对母药的疗效贡献度，开辟大黄脑保护分子机制研究的新途径。

西医药物对脑外伤（traumatic brain injury, TBI）的治疗效果有限。中药大黄以其“多靶点”药理作用显示出对TBI进行修复的满意疗效，但其治疗TBI的分子机制及其药效物质基础有待阐明。已知抗氧化策略是修复TBI后血脑屏障（blood-brain barrier, BBB）损伤的重中之重。为深入揭示大黄发挥TBI后BBB保护作用的分子机制及其药效物质基础，本研究对大黄吸收入靶器官-脑脊液生物吸收靶成分减轻TBI后BBB损伤/脑水肿程度的分子机制进行研究，以阐明大黄治疗TBI的颅内药效物质基础，明确BBB修复吸收成分发挥类似其母药大黄疗效的作用机理。本项目从检测大黄的脑脊液吸收成分出发，应用分子生物学、液质联用等技术，基于整体、细胞和分子水平三个层次，结合体内外实验，从TBI-NADPH氧化酶亚基gp91phox/ROS/MAPK ERK/MMP-9通路-BBB连接蛋白ZO-1途径研究大黄及吸收成分修复BBB的分子机制。通过本项目研究表明：基于超高效液相色谱-质谱/质谱联用检测方法分析显示：大黄酸是在TBI血脑屏障开放状态下服用大黄汤剂后唯一进入颅内的大黄蒽醌类成分；分子生物学实验研究结果证实：生物吸收靶成分大黄酸发挥了类似其母药大黄通过抑制NADPH氧化酶催化亚基gp91phox/ROS/MAPK ERK/MMP-9信号通路途径，上调BBB紧密连接蛋白ZO-1水平，从而发挥修复脑外伤后血脑屏障的药理作用。本研究为大黄“上病下取”和“归经入脑”治疗TBI提供了分子机制方面的现代科学内涵，开辟了大黄及其颅内药效物质脑保护作用机制研究的新途径，对中医药治疗脑外伤，发现治疗TBI的创新中药成分具有重要意义。