基于氧化应激和NF-κB信号通路的破血化瘀、填精补髓法靶向Nrf2治疗脑出血损伤机制研究

The clinical diagnosis and treatment practice for the treatment of cerebral hemorrhage based on the method of activating blood and removing stasis and supplementing essence and marrow has been approved by the State Administration of Traditional Chinese Medicine and promoted throughout the country. However, the treatment mechanism is not clear. The method of activating blood and removing stasis and supplementing essence and marrow can reduce secondary injury after cerebral hemorrhage by regulating the NF-κB signaling pathway based on the previous research. It is reported that Nrf2 phosphorylation itself could induce the expression of SOD2 expression and involve in the inflammation regulations through its ligand keap1 by controlling the NF-κB signaling pathway so that to promote the proliferation and differentiation of NSCs. Therefore, our study takes Nrf2 as the target and establishes cerebral hemorrhage model of gene knockout mice to observe the influence on the change rule of correlation factor of oxidative stress and the Nrf2 signaling pathways by applying Immunofluorescence, RT-PCR and other experimental techniques in different time periods by the method of activating blood and removing stasis and supplementing essence and marrow. It proves that the method can regulate Nrf2 signaling pathways and inhibit inflammatory reaction and oxidative stress hypothesis by discussing the treatment on molecular mechanisms of secondary damage of cerebral hemorrage. It also makes it clear that Nrf2 is the target for treatment, which provide reliable evidence for theoretical basis and clinical application of interpretation of “marrow deficiency and toxin loss” theory of hemorrhagic stroke.

以破血化瘀、填精补髓法为主制定的治疗脑出血临床诊疗规范已通过国家中医药管理局论证并全国推广，但治疗机制尚不明确。前期工作发现，破血化瘀、填精补髓法可调节NF-κB信号通路减轻脑出血后继发性损伤。据报道，Nrf2自身磷酸化可诱导SOD2等表达，同时还通过其配体keap1调节NF-κB信号通路参与炎症调节，从而促进NSCs增殖和分化。因此，本项目以Nrf2为切入点，拟建立Nrf2基因敲除小鼠脑出血模型，应用免疫荧光、RT-PCR等技术，观察不同时间点破血化瘀、填精补髓法对Nrf2基因敲除小鼠脑出血后NF-κB信号通路和氧化应激相关因子变化规律的影响，探讨该法治疗脑出血继发性损伤的分子机制，证实破血化瘀、填精补髓法可通过靶向调控Nrf2信号通路抑制炎症反应和抗氧化应激的假说，Nrf2为该法防治脑出血的新靶点，为阐释出血性中风“髓虚毒损”理论本质和临床应用提供可靠依据。

破血化瘀、填精补髓法是在“八五重点攻关课题”的临床研究基础上，立足于国医大师任继学的中医“脑髓理论”，结合王永炎院士提出的“毒损脑络”病机提出的脑出血治疗方法，在临床已取得确切疗效。以本法为主制定的临床诊疗规范已通过国家中医药管理局组织的专家委员会论证，其作为出血性中风的治疗方案已在临床推广，有关该法作用机制的研究已经初步开展，但作用机制尚不清楚。本项目建立了Nrf2基因敲除小鼠脑出血继发性损伤模型，以Nrf2为切入点，采用神经功能学评分、神经行为学、脑含水量、HE、免疫组化、Western blot等方法，从氧化应激和炎症角度探讨了破血化瘀、填精补髓法中药汤剂改善Nrf2-/-小鼠脑出血继发性损伤的规律，揭示破血化瘀、填精补髓法防御神经细胞损伤的分子机制，阐释“髓虚毒损”所致出血性中风的理论本质。实验结果表明，小鼠脑出血后的继发损伤表现从1d、3d、5d、7d四个时间点看，3d病情表现最为显著，本项目的研究对象确定为3d脑出血小鼠。脑含水量和HE检测结果显示，Nrf2基因缺失小鼠在脑出血后继发性损伤加重，水肿程度、脑组织病理改变明显，炎症反应和氧化应激表达增强。破血化瘀、填精补髓中药汤剂治疗脑出血急性期可能是调节Keap1-Nrf2/HO-1途径相关蛋白的表达，进一步调节NF-κB信号通路的上游和下游因子，减轻炎症因子TNF-α、IL-6、IL-1和抗氧化应激酶抗氧化酶SOD2、GSH、CAT等的表达，从而防治脑出血后继发性损伤。本项目的完成，为深入探讨破血化瘀、填精补髓法治疗脑出血的科学内涵提供了一定的科学思路。另外，从分子水平研究破血化瘀、填精补髓法治疗脑出血的机制，为该法治疗脑出血提供更为翔实的现代医学证据，对于临床中药开发，提高治疗的有效性都具有重要的意义。