基于对NogoA-NgR/Rho-ROCK通路调节的清脑益智方促神经元突触重塑治疗VD的机制研究

Synaptic remodeling is the research fronts of neurodegenerative diseases including vascular dementia. Qingnao Yizhi fomula is based on the vascular dementia's pathology theory "poison injures brain collaterals",clinical trial has proved that Qingnao Yizhi fomula could ameliorate the cognitive function of vascular dementia patients,there are already some subjects which studied the formula 's possible therapeutical mechanism,but there is still no research which is based on investigating the fomula's effect on neuronal synaptic remodeling based on regulating the signaling pathway of NogoA-NgR/Rho-ROCK.This subject applies the 2-VO rat vascular dementia model and hypoxia-reoxgenation cortical neuron cell,through using the methods or equipments of immunohistochemistry、RT-PCR、Western blot、electron microscope and transmission electron microscope、etc,study the Qingnao Yizhi fomula's affecting effect on the neuronal synaptic remodeling of the vascular dementia mouse's cerebral cortex and hippocampus area,and the regulating effect of cerebrospinal fluid containing Qingnao Yizhi fomula on the signaling transduction passway of NogoA-NgR/Rho-ROCK,as well as cerebrospinal fluid containing Qingnao Yizhi fomula's effect on the synapse shape and structure of the hypoxia-reoxgenation cortical neuron cell. Through studying the Qingnao Yizhi fomula's effect on synaptic remodeling and discuss its preliminary therapeutical mechanism on vascular dementia could further afford scientific evidence for the clinical application.

神经元突触重塑是目前包括血管性痴呆在内的神经退行性疾病的研究前沿，清脑益智方是基于血管性痴呆"毒损脑络"的病机理论制定的治疗血管性痴呆有效的临床经验方，前期的临床研究证明其有改善VD患者认知的作用，但它是否可通过调节NogoA-NgR/Rho-ROCK信号通路影响神经元突触重塑而起作用，目前尚无相关研究。本研究采用大鼠血管性痴呆模型和神经元细胞缺氧复氧损伤模型，运用免疫组化、RT-PCR、Western blot、电镜形态学观察及透射电镜超微结构观测等技术，研究清脑益智方对拟血管性痴呆大鼠皮层和海马区神经元突触结构的影响，及清脑益智方含药脑脊液对缺氧复氧损伤皮层神经元细胞NogoA-NgR/Rho-ROCK信号转导通路的调控作用及对神经元细胞突触重塑的影响。通过探讨清脑益智方对神经元突触重塑的作用，并阐释其治疗血管性痴呆的部分机制，为临床用药提供科学依据。

血管性痴呆（VD）属中医的“呆证”、“善忘”、“癫证”、“郁证”、“文痴”等范畴，其病位在脑，发病机理为气虚阴亏，痰饮内生，酿毒成浊，瘀血阻络，痰瘀互结而败坏脑络，殃及脑髓，阻闭清窍，蒙蔽神明。基于以上认识，张伯礼院士根据王永炎院士VD“毒损脑络”的理论并结合自己多年的临床实践经验，筛选出人参、丹参、黄连、麦冬等中药，组成清脑益智方。方中人参大补元气，增智益脑为君药；丹参活血化瘀，黄连清热解毒共为臣药；麦冬养阴生津。全方合而共奏开窍益智、清脑化毒、醒神降浊、祛痰通络的作用。. 本课题组前期研究表明，清脑益智方具有改善VD患者记忆力、提高其认知功能，并对调节脑血流状态和血液流变性有良好的作用，发挥恢复异常的行为学等作用，但其治疗VD的作用机制尚未进行系统研究。本研究首先采用双侧结扎颈总动脉法（2VO）建立SD大鼠VD模型，通过Morris水迷宫试验考察清脑益智方对VD大鼠认知功能的影响，并分别通过HE染色观察病理学变化、免疫组化实验检测清脑益智方对VD大鼠皮层、海马区组织结构及与神经元突触重塑相关指标的影响，初步探讨清脑益智方改善VD大鼠认知功能的作用机制；体外培养皮层神经元（CNC），建立缺氧/复氧（H/R）损伤模型，分别通过ELISA、Western blot及RT-PCR等分子生物学方法手段，考察清脑益智方含药脑脊液（QNYZ-CSF）对神经元突触重塑相关指标及NogoA-NgR/Rho-ROCK信号转导通路的作用；此外，还考察了QNYZ-CSF对H/R所致CNC凋亡的影响及其作用机制。. 结果表明，清脑益智方可显著缩短VD大鼠逃避潜伏期、游泳总路程，增加穿越平台次数，有效提高VD大鼠认知功能，改善其学习记忆能力；并抑制NogoA、NgR基因和蛋白表达，通过阻断NogoA-NgR/Rho-ROCK信号转导通路而增强神经元突触重塑；亦可通过PI3K/Akt-mTOR信号通路抑制H/R 诱导的CNC凋亡，为清脑益智方的临床应用提供了科学依据。