基于内皮β-catenin信号通路在血脑屏障中的作用研究“通腑醒脑”法改善肝性脑病的机制

In recent years, it has been found that the blood-brain barrier plays a key role in the pathogenesis of hepatic encephalopathy, the regulation of blood-brain barrier may be a good way to prevent and cure hepatic encephalopathy. Based on a large number of previous studies, it was found that the occurrence and development of hepatic encephalopathy is closely related to the permeability of the blood-brain barrier. Endothelial β-catenin signaling pathway is necessary to maintain the integrity of blood-brain barrier and central nervous system homeostasis in adults. Rhubarb decoction has a good clinical effect on patients with hepatic encephalopathy. We hypothesize that rhubarb decoction may improve the function of blood-brain barrier by targeting endothelial β-catenin signaling pathway to treat hepatic encephalopathy. To observe the effects of rhubarb decoction on endothelial β-catenin signaling pathway and downstream Occludin,Claudin signal transduction in rat model of hepatic encephalopathy and blood-brain barrier permeability change cell model. It will contribute to clarify the molecular mechanism of rhubarb decoction in promoting tight junctional protein secretion and improving the function of blood-brain barrier by regulating endothelial β-catenin signaling pathway in treatment of hepatic encephalopathy, provide a strong evidence to treat hepatic encephalopathy by “dredge intestines and restore consciousness” strategy of the traditional Chinese medicine.

近年来研究发现血脑屏障在肝性脑病发病过程中起着关键作用，对其调控或是防治肝性脑病的良策。本项目基于大量前期研究发现，肝性脑病的发生发展与血脑屏障通透性密切相关，内皮β-catenin信号通路是维持成人血脑屏障完整性和中枢神经系统稳态所必需的，大黄煎剂对肝性脑病患者具有很好的临床疗效，提出“大黄煎剂可能通过靶向内皮β-catenin信号通路来改善血脑屏障功能从而治疗肝性脑病”的工作假说。拟通过建立肝性脑病大鼠模型和血脑屏障通透性改变细胞模型，从整体和细胞水平观察大黄煎剂对内皮β-catenin信号通路及其下游Occludin、Claudin信号转导的影响，系统探讨大黄煎剂通过调控内皮β-catenin信号通路促进紧密连接蛋白分泌、改善血脑屏障功能治疗大黄煎剂的分子机制，为进一步推广大黄煎剂“通腑醒脑”治法奠定基础，为肝性脑病的防治提供新思路。

肝性脑病（hepatic encephalopathy，HE）是肝硬化最严重的并发症，其发生率高、预后差，是导致肝病患者死亡的主要原因之一。目前，HE的确切发病机制尚不明确，且缺乏有效防治手段，严重威胁患者生命。临床研究表明，大黄煎剂治疗HE具有不错的临床疗效。本课题以CHE小鼠为动物模型，以小鼠内皮细胞和星形胶质细胞共培养为细胞模型，采用神经行为学、生物免疫学等技术，从蛋白、细胞和整体水平，以Wnt/β-catenin信号通路为中心研究了大黄煎剂治疗HE的机制。本项目首先采用UPLC-MS指纹图谱对大黄煎剂单味药中的化学成分进行分析，共确定8种有效成分。本研究结果表明：①以CHE小鼠为研究对象，大黄煎剂可明显改善小鼠肝脏和脑组织细胞结构，缓解组织损伤；大黄煎剂可明显改善CHE小鼠神经功能损伤和行为异常，抑制血管通透性损伤和结构破坏，并抑制海马组织Wnt/β-catenin通路活化。②将小鼠脑内皮细胞bEnd.3与星形胶质细胞C8-D1A共培养，用NH4Cl处理建立血脑屏障损伤模型，选择合适浓度大黄煎剂提取液干预细胞。大黄煎剂可显著增加细胞模型中紧密连接蛋白（ZO-1、Occludin、Claudin5）表达，并明显降低Wnt/β-catenin通路表达水平。大黄煎剂可缓解HE肝脏和脑组织损伤，保护血脑屏障，抑制Wnt/β-catenin通路活化。本研究丰富了“腑气不通，脑窍蒙蔽”中医病机和“通腑醒神，泄毒降浊”中医治则的科学内涵，初步明确了Wnt/β-catenin信号通路在CHE中的调控机制以及大黄煎剂的干预作用机制。