头穴透刺调控RhoA-ROCK通路促进脑出血大鼠神经血管稳态重构的机制研究

NVU(neurovascular unit) is the new research focus of stroke. Rho/Rho-kinase signaling pathway plays a role of "molecular witch" ,after intracerebral hemorrhage， it damaged interconnection of NVU different structures, so Rho A -ROCK signaling pathway is the target for treatment. Observe rats' neurological behavior after cerebral hemorrhage continuously. Pathological changes of NVU around hematoma was observed By TEM. The neuron degeneration and microglia activation were observed after Fluoro-jade C staining or OX-6 staining respectively by fluorescence microscope. The expression of claudin-5,occludin,ZO-1 related to vascular endothelial permeability and the expression of NGF,GAP-43 related to neural regeneration were detected by immunohistochemistry. The genes content of the protein mentioned above were detected by RT-PCR. As the same time, analysis the correlation to RhoA/ ROCK. Confirm that scalp penetration acupuncture can regulates Rho/ Rock signaling pathway to improve TJ (tight junction) of microvascular endothelial cells,protect BBB(blood-brain barrier), reduce the degree of encephaledema, promote nerve regeneration. Compared with Fasudil, scalp penetration acupuncture had targeting effect significantly. Scalp penetration acupuncture has whole-regulation-effect, that reconstruct cerebral hemorrhage rat's neural-vascular steadystate, so that provided the new evidences to research on the mechanism of NVU damage and protection.

神经血管单元NVU是中风病新的研究焦点。脑出血后RhoA- ROCK信号被激活，导致NVU的各组分相互联系遭到破坏，因此使"神经血管稳态重构"关键治疗靶点。动态观察脑出血后神经行为学变化；透射电镜观察血肿周围组织NVU的病理变化；荧光显微镜观察Fluoro-jade C、OX-6染色后神经元变性、小胶质细胞激活情况；免疫组化、RT-PCR等检测血管内皮紧密连接(claudin-5、occludin、ZO-1)、神经再生 (NGF、GAP-43) 相关蛋白及其基因；分析与RhoA/ ROCK通路及其下游信号分子MLC/ F-actin的关系。证实头穴透刺能调控RhoA/ ROCK通路，改善微血管内皮细胞紧密连接，保护血脑屏障，减轻脑水肿和促进神经再生；与信号通路抑制剂比较，更具有靶向性。头穴透刺对NVU具有整体调节效应，神经血管稳态重构为整体研究神经元损伤及保护机制，寻找治疗的新靶点提供依据

目的：探讨头穴透刺调控RhoA-ROCK通路促进脑出血大鼠神经血管稳态重塑的机制。方法：Wistar 大鼠300只被分为假手术、模型、针刺、药物、针药结合组，注血法制备脑出血模型。观察脑出血后6h、24h、3d、7d动物神经行为学；电镜下血管内皮、血脑屏障、神经元、神经胶质细胞等超微结构；免疫组化、western blot检测血管内皮claudin-5、occludin、ZO-1、神经再生NGF、GAP-43、小胶质细胞活化OX-42、信号通路RhoA、ROCK、MLC、F-actin蛋白；PCR检测基因表达；干湿重法测定脑含水量；伊文氏兰检测血脑屏障完整性；Fluoro-jade C染色荧光显微镜观察神经元变性情况。结果：（1）脑出血后RhoA通路及其下游ROCK、MLC蛋白及其mRNA， F-actin mRNA被激活；RhoA激酶抑制剂、针刺、针药结合均能下调RhoA及其下游ROCK、MLC蛋白及其mRNA表达；针药结合调节作用更为明显（P<0.05）；法舒地尔、针刺、针药结合对F-actin mRNA 无调节作用（P>0.05）。（2）脑出血后血肿周围脑组织血管内皮通透性相关claudin-5、occludin、ZO-1蛋白及其mRNA下降，24h、3h下降更为明显（P<0.05）；伊文氏蓝含量、脑组织含水量升高；药物、针刺、针药结合均能调节claudin-5、occludin、ZO-1蛋白及其mRNA，针药结合作用明显（P<0.05）。（3）脑出血后神经再生相关蛋白NGF、GAP-43下降；针药、药物、针刺均能上调NGF、GAP-43蛋白及mRNA 表达，针药结合优于针刺组和药物组。（4）脑出血后血肿周围OX-42阳性小胶质细胞增多，针药、药物、针刺均能下调OX-42表达，针药结合优于针刺组和药物组。（5）脑出血后FJC 染色神经元减少，针药、药物、针刺均能保护神经元，针药结合优于针刺组和药物组。结论：（1）RhoA通路及其下游ROCK、MLC蛋白参与脑出血病理损害，是重要治疗靶点。（2）脑出血后神经血管单元稳态打破，神经元、血管内皮、小胶质细胞各组分均遭到破坏是神经功能损害最主要机制。（3）针刺能抑制RhoA通路保护神经血管单元各组分的结构和功能。（4）针药结合能抑制RhoA通路激活促进神经修复，具有叠加和协同效应，能重塑脑出血后神经血管稳态。