生地大黄汤通过HMGB1/TLR4/MMP-9信号通路保护急性脑出血时血脑屏障的机制研究

Acute intracerebral hemorrhage seriously threats human’s life and heath. Protecting blood-brain barrier during ICH is extremely important to maintain steady state of the survival micro environment of neurons. Results of our studies showed that Rehmannia and Rhubarb Decoction had curative effect on ICH, it protected blood-brain barrier, reduced cerebral edema and depressed the expression and activity of MMP-9. However, the mechanisms of rehmannia and rhubarb decoction how to regulate the expression of MMP-9 and protect blood-brain barrier are unknown. In this project, we hypothesize that during ICH Rehmannia and Rhubarb Decoction protects blood-brain barrier through HMGB1/TLR4/MMP-9 signaling pathway basing on the new knowledge that the activation of this signaling pathway plays an important role to damage blood-brain barrier. We hope to find the changes of molecular expression in HMGB1/TLR4/MMP-9 signaling pathway during ICH and the correlation between the changes and the degree of blood-brain barrier damage, and to prove the mechanisms of Rehmannia and Rhubarb Decoction protecting blood-brain barrier through HMGB1/TLR4/MMP-9 signaling pathway in vivo（ICH model of spontaneous hypertensive rats）and in vitro（HMGB1 induced model of astrocytes）. This study will provide a theoretical support and basis for the use of Rehmannia and Rhubarb Decoction, a famous ancient prescription, to treat ICH.

急性脑出血严重威胁人类生命健康，脑出血时保护血脑屏障对维持神经元生存微环境稳态极其重要。前期研究证实生地大黄汤治疗急性脑出血临床疗效明确，能保护血脑屏障，减轻脑水肿，降低MMP-9表达和活性。生地大黄汤如何调控MMP-9表达，保护血脑屏障的作用机制须进一步明确。本项目结合HMGB1/TLR4/MMP-9信号通路激活在血脑屏障破坏中起重要作用的的新认识，提出“生地大黄汤通过干预HMGB1/TLR4/MMP-9信号通路保护急性脑出血时血脑屏障”的假说。拟分别采用体内动物模型（自发性高血压大鼠脑出血模型）及体外细胞模型（HMGB1诱导星型胶质细胞模型），明确急性脑出血时HMGB1/TLR4/MMP-9信号通路分子表达变化与血脑屏障破坏程度相关性及生地大黄汤通过HMGB1/TLR4/MMP-9信号通路保护血脑屏障的作用机制，为古方生地大黄汤治疗急性脑出血提供理论支持和依据。

目的：观察脑出血时HMGB1/TLR4/MMP-9信号通路与血脑屏障破坏程度的相关性,及生地大黄汤通过HMGB1/TLR4/MMP-9信号通路对脑出血时血脑屏障的影响。方法：自发性高血压大鼠随机分为假手术组、模型组、生地大黄汤组、HMGB1抑制剂组、HMGB1抑制剂+生地大黄汤组、TLR4拮抗剂组和TLR4拮抗剂+生地大黄汤组。术后第3天取材，各组大鼠进行神经功能缺损评分；电镜、免疫荧光染色观察各组大鼠血脑屏障结构变化；脑含水量检测、伊文思蓝法、MRI检测观察各组大鼠血脑屏障功能变化；TUNEL染色、Nissl染色观察各组大鼠血肿周围脑组织病理学变化；免疫荧光双染观察各组大鼠血脑屏障处蛋白定位；Western blot 检测各组大鼠脑血肿周围组织HMGB1、TLR4、MMP-9、Claudin-5、Occludin、Zo-1蛋白分子的表达变化；细胞实验观察HMGB1诱导星形胶质细胞模型后各组TLR4、MMP-9蛋白mRNA表达，明胶酶法检测MMP-9活性。结果：造模后3d,脑出血模型血脑屏障结构破坏，脑含水量、血脑屏障通透性、脑水肿体积均升高，血肿周围脑组织细胞凋亡数升高、Nissl小体减少，血脑屏障结构蛋白及星形胶质细胞标志物表达减少，血肿周围脑组织HMGB1、TLR4、MMP-9蛋白表达水平均升高，Claudin-5、Occludin、Zo-1蛋白表达水平均降低；药物干预后，均得到不同缓解，差异具有统计学意义（P<0.05）。其中生地大黄汤组、HMGB1抑制剂组、HMGB1抑制剂+生地大黄汤组与模型组比较，血肿周围脑组织HMGB1、TLR4、MMP-9蛋白表达水平降低，而Claudin-5、Occludin、Zo-1蛋白表达水平升高，差异均有统计学意义（P<0.05）；TLR4拮抗剂组和TLR4拮抗剂+生地大黄汤组与模型组比较，血肿周围脑组织TLR4、MMP-9蛋白表达水平均降低，Claudin-5、Occludin、Zo-1蛋白表达水平均升高，差异均有统计学意义（P<0.05）。免疫荧光双染观察发现HMGB1、TLR4、MMP-9分别与Claudin-5融合表达。细胞实验HMGB1造模后TLR4、MMP-9 蛋白及mRNA表达上升，MMP-9活性增强。加用生地大黄汤或TLR4拮抗剂后TLR4、MMP-9 蛋白及mRNA表达下降，MMP-9活性抑制。