基于破血化瘀法的脑出血方干预脑血肿吸收机制的研究

Intracerebral hemorrhage (ICH) is a disease with high mortality and high disability rates, which was caused by the toxic effects of hematoma and degradation products on brain tissue. Therefore, it is the key to accelerate the absorption of hematoma for the treatment of ICH. Modern medical research has proved that the treatments of surgical evacuation of hematoma and internal medicine have no good effects against hematoma. Based on Poxuehuayu Method, which was proposed by Ren Jixue, a master of Chinese medicine, we founded Naochuxue Prescription. Our clinical studies and animal experiments have confirmed that the method could promote hematoma absorption and improve neurological deficit. However, the mechanism and material basis of Naochuxue Prescription still remains unclear. Previous studies also indicated that monarch drug, leech, had a better protective effect on injured glial cells and damaged nerve cells. Based on our existing results, we could elucidate the regulation of intracerebral hemorrhage on endogenous clearance of red blood cells and their degradation products used multiple kinds of techniques, such as CRISPR/Cas9 knockout skill and immunohistochemical staining for the key molecules, CD36, CD47, and erythrocyte degradation products. Furthermore, we could analyze effective ingredients of monarch drug, leech from Naochuxue Prescription using HPLC and gel chromatography to identify the material basis of promoting blood absorption. This project could provide a reliable experimental basis for the treatment of ICH using Poxuehuayu Method to guide clinical application.

脑出血(ICH)病理损害来自于血肿的占位效应及其降解产物的毒性作用，血肿是脑出血后一系列损伤的始动环节，加快血肿吸收是治疗的关键。研究证明，外科血肿清除手术及内科保守治疗均未有较好的治疗效果。在国医大师任继学提出的破血化瘀法治疗脑出血的基础上，我们制定了脑出血方。大量临床研究及动物试验均证实，本法能够促进血肿吸收，改善神经功能缺损，但其作用机制及物质基础尚不清楚。前期研究也发现，本方君药水蛭对损伤的小胶质细胞和神经细胞均有较好的保护作用。在已有研究的基础上，本课题拟在脑出血动物和细胞模型上，采用CRISPR/Cas9基因敲除、免疫组化等技术阐明脑出血方对关键分子CD36、CD47及红细胞降解产物介导的血肿内源性清除的调控作用。同时，应用高效液相、凝胶色谱等方法分析脑出血方君药水蛭的有效成分，进而确定其发挥作用的物质基础。本项目将为破血化瘀法治疗脑出血提供更可靠的实验依据进而指导临床。

脑出血（Intracerebral Hemorrhage，ICH）占全部脑卒中比率的10%～15%，3个月内的病死率为20%～30%，约40%的存活患者遗留明显残疾，给患者家庭及社会造成了沉重的负担。.脑出血的病理损害来自于血肿的占位效应及其降解产物的毒性作用，加快血肿吸收是治疗关键。小胶质细胞为颅内巨噬细胞，脑出血后被血肿产物激活，具有吞噬清除血肿及分泌炎性因子双重作用。CD47能够抑制小胶质细胞吞噬红细胞；CD36介导小胶质细胞的吞噬作用，其中PPARγ、Nrf2对CD36能够促进CD36表达，从而促进血肿清除；TLR4、NF-κB等为小胶质细胞释放的炎症因子，在损伤神经细胞的同时抑制CD36表达，加重神经损伤。国医大师任继学原创性提出了应用破血化瘀法治疗脑出血，并被证实可加快血肿吸收，疗效确切。但其作用机制与尚未明确，为解决以上关键问题，本研究采用了胶原酶诱导建立大鼠脑出血模型、红细胞与小胶质细胞共培养构建体外模型，并应用以破血化瘀法化裁的脑出血方进行实验干预。.结果表明：脑出血方可促进小胶质细胞吞噬清除红细胞，从而促进血肿吸收；脑出血方可上调CD36信号通路相关靶点如PPARγ、Nrf2，同时下调CD47、TLR4、NF-κB等蛋白表达，并应用CD36、CD47抑制剂反向验证其靶点作用，进而阐明脑出血方通过以上激活信号通路促进脑出血后的血肿吸收；脑出血方还可抑制TLR4/MYD88炎症通路，减轻脑出血应激后继发的炎症损伤，最终改善神经功能缺损。本项目的研究为破血化瘀法治疗脑出血提供了更可靠的实验依据，进一步指导临床用药，使更多的脑出血患者在药物治疗方面获益，相对节约医疗成本，创造良好的社会效益和经济效益。