肝硬化组织高表达的多巴胺（DA）与轻微肝性脑病（MHE）发病的相关性研究

The mechanism of Minimal hepatic encephalopathy (MHE) resulted from liver cirrhosis is not clear，most of people consider that it is associated with the increased blood ammonia. We find the levels of DA were higher than normal level in the blood of a liver cirrhosis patient and tissues of liver cirrhosis rats. Then using MHE rat model, intravenous injection of DA rat model, we find the cognitive disorder, the increased levels of DA in serum and hippocampus. Therefore, we speculate that "DA highly expressed in cirrhotic liver enters into the brain by the circulation of the blood, and inhibits the learning and memory ability (glutamate-NO-cGMP… cGMP-PKG-CREB-Bdnf/c-fos/Nr4a2 pathway), leading to MHE". In order to confirm the speculation, using the rat model of MHE , the pairwise co-culture system of original cirrhotic liver cell/ neurons /astrocytes, treatment of DA/COMT and their inhibitor, we study that DA enter into brain and has effect on the cognition by the technology of morris water maze, open field, Y-maze, elevated plus maze, isotopic tracing, HPLC; study that DA has effect on the learning and memory pathway by the technology of immunofluorescent double staining, immunoblotting, HPLC-MS, Cell patch clamp technology, Gene transfection / silence; may provide new explanation of pathogenesis of MHE.

目前，肝硬化导致轻微肝性脑病（MHE）的机理尚不清楚，大多数人认为与血氨增高相关。但我们发现1例肝硬化病人血液中与大鼠肝硬化组织中多巴胺（DA）水平较正常高；MHE大鼠与DA 静脉注射大鼠均表现出认知障碍，血浆和海马组织中DA明显升高。因此我们推测"肝硬化组织表达升高的DA，经血液循环入脑影响学习记忆功能（Glu-NO-cGMP…cGMP-PKG-CREB-Bdnf/c-fos/Nr4a2通路），导致MHE发病"。为进一步证实此推测，我们通过大鼠MHE模型、原代硬化肝细胞/神经元/星形胶质细胞两两共培养体系，及DA/COMT及其抑制剂等干预，采用同位素示踪、HPLC、行为学测试等技术研究MHE大鼠肝硬化组织中高表达DA经血液循环入脑及DA的升高影响MHE发病；免疫荧光双染、免疫印迹、液质联用、细胞膜片钳、基因转染/沉默等技术研究DA影响MHE发病的分子机制。为MHE发病机理提供新的解释。

本项目旨在探讨轻微肝性脑病（MHE）发病的具体分子机制。.我们首先明确肝硬化组织中高DA与MHE发病的关系。并探讨DA诱导MHE发病的分子机制与Glu-NO-cGMP、 Shh信号通路、JAK2/STAT3/ChAT/M1 mAChR等多条信号通路有关，及星形胶质细胞中的蛋白质酪氨酸硝基化（PTN）和释放的TNF-α也参与DA诱导的MHE发病。此外，我们进一步明确星形胶质细胞分泌的Wnt5a与MHE发病的关系。结果显示， MHE大鼠肝组织中调节DA代谢的COMT抑制剂的表达升高， DA在MHE大鼠肝组织、血浆、脑组织中蓄积，升高的DA诱导MHE大鼠学习记忆障碍；Glu-NO-cGMP通路、JAK2/STAT3/ChAT/M1 mAChR通路和Shh信号通路在MHE大鼠脑组织中均失活，DA通过抑制Glu-NO-cGMP通路、JAK2/STAT3/ChAT/M1 mAChR通路和Shh/NTs信号通路活性诱导MHE发病；MHE大鼠脑组织中发生蛋白质酪氨酸硝基化并且TNF-α水平升高，DA通过促进星形胶质细胞蛋白质酪氨酸硝基化及表达和分泌TNF-α诱导MHE发病； Wnt5a在MHE大鼠脑组织中表达降低，Wnt5a介导的CaMK Ⅱ – CREB-NTs 信号通路在MHE大鼠中失活，Wnt5a介导的CaMK Ⅱ – CREB-NTs通路的失活诱导MHE发病。本项目所获得的研究结果为肝硬化病人并发MHE的发病机理提供更新的理论解释，并为MHE的治疗提供更全面的理论依据及相关靶标。