基于Rho/ROCK通路研究蛭龙活血通瘀胶囊对脑出血大鼠脑水肿的调控机制

Brain edema formation after intracerebral hemorrhage of brain tissue damage is an important factor of disease progression and also the focus of drug treatment. Zhilonghuoxuetongyu capsule is an interior hospital Chinese medicine prescription developed by the second affiliated hospital of Southwest Medical University, which showed desirable clinical efficiency to cure intracerebral hemorrhage. Our previous study showed that Zhilonghuoxuetongyu capsule can improve the blood-brain barrier permeability, inhibit apoptosis of neuron cell, relieve brain edema and sustain normal nerve function by inhibiting the gene expression of AQP - 4, MMP – 9 both at protein and mRNA level, suppressing the activation of PAR-1, but increasing the expression of TIMP-1 and HIF-α in the peri- hemorrhage tissue. Rho/ROCK pathways mediated cerebral edema formation provides a new research direction for the drug therapy of cerebral hemorrhage. To further uncover the mechanism by which Zhilonghuoxuetongyu capsule ameliorates intracerebral hemorrhage, we focused on Rho/ROCK pathway with tool of intracerebral hemorrhage rat model. By experimental techniques such as fluorescent immunostaining, western blot, real-time PCR, the therapy efficiency of Zhilonghuoxuetongyu capsule on intracerebral hemorrhage and its influence on Rho/ROCK signal will be deeply investigated in respect to pathomorphology, protein and gene expression. The clarification of disease treatment mechanism and target will provide the research foundation to facilitate the application of Zhilonghuoxuetongyu capsule as a novel medicine in intracerebral hemorrhage treatment.

脑出血后血肿周围水肿对脑组织造成损害是病情加重的重要因素，也是药物治疗的重点。蛭龙活血通瘀胶囊是西南医大附二院研制的院内制剂，临床运用发现对脑出血有明显疗效，研究表明，该药能下调脑出血大鼠血肿周围AQP-4、MMP-9，并上调TIMP-1蛋白及基因表达，改善血脑屏障通透性，抑制PAR-1活化，促进HIF-1α表达，抑制神经细胞凋亡，减轻脑水肿，改善神经功能。Rho/ROCK通路介导脑水肿形成为脑出血药物治疗提供了新的研究方向。为进一步探寻蛭龙活血通瘀胶囊改善脑出血的作用机制，本项目以Rho/ROCK通路为切入点，以脑出血大鼠为研究对象，运用免疫荧光、Western Blot、real-timePCR等技术，从病理形态学、蛋白及基因水平等层面研究蛭龙活血通瘀胶囊对脑出血的治疗作用和对Rho/ROCK通路的影响，阐明其治疗脑出血的分子机制和有效作用靶点，为开发治疗脑出血的中药新药奠定实验基础。

脑出血后血肿周围水肿对脑组织造成损害是疾病发展转归的关键因素，Rho/ROCK信号通路介导脑水肿的形成，为脑出血的药物研究提供了新方向。我们前期研究发现蛭龙活血通瘀胶囊能明显改善脑水肿，有效防治脑出血。本项目以Rho/ROCK通路为切入点，运用免疫组学、蛋白组学、基因组学等关键技术，从病理形态学、蛋白及基因水平等层面进一步研究该药对脑出血的治疗作用机制及对Rho/ROCK通路的影响。本研究中，我们采用大鼠自体血注入法制备脑出血模型，HE染色光镜下观察血肿周围组织水肿情况，干湿重法检测大鼠脑含水量及脑系数，神经功能评分评估大鼠神经功能，透射电镜观察大鼠脑出血损伤区血脑屏障超微结构，伊文思蓝（EB）法检测血脑屏障通透性，免疫组化及蛋白质印迹试验，实时荧光定量PCR法，系统研究该药对血脑屏障紧密连接蛋白以及Rho/ROCK通路上关键信号因子的影响。研究结果表明，该药能明显改善脑出血大鼠神经功能，降低脑含水量及脑系数，明显减轻脑组织水肿程度，使病理形态得到明显改善，能明显改善血脑屏障超微结构，降低血脑屏障的通透性，增加脑组织紧密连接蛋白的表达，并下调Rho/ROCK通路关键因子在脑内的表达。说明该药能通过抑制Rho/ROCK通路调节血脑屏障结构及功能，改善脑出血后脑水肿，起到脑保护作用。此外，我们用ELISA法检测脑出血大鼠血清炎症因子的浓度水平，证实了该药能明显降低脑出血大鼠血清炎症因子水平。这些研究结果为蛭龙活血通瘀胶囊用于脑出血的防治提供了科学依据，并为中药新药的研发奠定了实验基础。