益气活血法对脑出血炎症调节血管新生机制的影响

In terms of inflammation regulating angiogenesis mediated by NF-κB pathway,the purpose of this project is to further study the therapeutic mechanism of Qi-tonifying and stasis-eliminating therapy for intracerebral hemorrhage(ICH).Content:immunohistochemisty, Molecular biology,and the other technology would be used to observe temporal and spatial expression of classical/non-classical NF-κB pathway, angiogenic factors and the effect of Qi-tonifying and stasis-eliminating therapy on them.Inflammatory response would be estimated by Fluoro-Jade B staining, detection of MPO,and so on.Microvascular reconstruction would be evaluated by intraperitoneal injection of BrdU combined with immunofluorescence confocal microscopy and perfusion of FITC-dextran combined with confocal laser three dimensional reconstruction technology.Regular and three dimensional protein gel culture system and serum contained with traditional Chinese medicine could be used in cell culture.Then ,molecular biology and cell proliferation assay technology could be used to observe NF-κB pathway, cell proliferation, angiogenic factors and the formation of microvascular networks in proliferous cell induced by thrombin.Significance: by detecting index related with NF-κB pathway and angiogenic factors,maybe we can elucidate the mechanism for Qi-tonifying and stasis-eliminating therapy promoting the repair of damaged brain tissues in ICH,thus to provide new ideas for the modern research of the treatment.

本项目拟从NF-κB通路活化介导炎症调控血管新生的角度，进一步研究益气活血法治疗脑出血的机制。内容：运用免疫组织化学、分子生物学等技术观察脑出血后大鼠脑内NF-κB经典/非经典通路时空表达、血管新生因子及益气活血法的影响；Fluoro-Jade B染色、MPO检测等评价炎症反应；BrdU腹腔注射结合免疫荧光共聚焦以及FITC-dextran 灌注结合激光共聚焦三维血管重建技术评价损伤区的微血管系统重建；并运用常规和三维蛋白凝胶培养系统及含中药血清的干预，以分子生物和细胞增殖分析等技术，观察凝血酶诱导增殖的脑微血管内皮细胞NF-κB经典/非经典通路的表达、细胞增殖、血管新生因子及微血管系统形成的变化和益气活血法的作用。意义：通过检测NF-κB通路及血管新生相关因子，揭示益气活血法促进损伤组织修复，治疗脑出血的机理，为该治法的现代研究提供新的思路。

脑出血是脑卒中中的危重类型，病死率高、致残率高，预后很差，对家庭与社会都造成了严重负担。西医主张的对症支持治疗和颅内血肿清除术等治疗效果仍不理想。作为益气活血法的代表方剂，补阳还五汤（BYHWD）在临床被广泛应用于脑出血的治疗，且疗效确切，但其治疗机制有待进一步阐明。本研究构建胶原酶诱导的脑出血大鼠模型，通过神经功能评分及行为学实验等明确BYHWD在脑出血中的疗效；侧脑室注射BAY 11-7083阻断NF-κB经典通路，显示BYHWD通过调控NF-κB经典通路促进血管新生发挥作用；构建NIK-siRNAs病毒阻断NF-κB非经典通路,正反验证显示BYHWD通过调控NF-κB非经典通路抑制炎症发挥作用；细胞层面制备含药血清，构建凝血酶诱导的脑微血管内皮细胞增殖细胞模型进行体外验证。本研究从炎症与血管新生的角度，探索补阳还五汤治疗脑出血的作用机制，为该方在临床上的运用提供新的实验证据，更为中药复方研究提供新的思路。