基于血清外泌体的芎麻药对对血管性认知障碍大鼠胆碱能突触信号通路的干预研究

Exploration of Intervention in Xiongma Dropping Pills on Cholinergic Synaptic Signal Pathway in Vascular Cognitive Impairment Rats Based on Serum ExosomesLiu Lin, QI Xin, Yang Yang, ZHANG Jiaojiao, WANG Dongxue (College of Pharmacy, Harbin University of Commerce, Harbin 150076)

关键词:  血管性认知障碍  血清外泌体  芎麻药对  胆碱能突触信号通路

Key words:  Vascular cognitive impairment  Serum exosome  Xiongma dropping pills  Cholinergic synaptic signaling pathway

摘要:

目的 探究芎麻药对对血管性认知障碍大鼠脑和血清中外泌体表达的影响及其干预作用机制。方法 选用SPF级8-10周龄雄性Wistar大鼠50只,利用2-VO法复制大鼠VCI模型,依据脑血流量结果将2-VO组大鼠随机分为VCI模型组(生理盐水10mL/kg),阳性药组(盐酸多奈哌齐0.03g/kg),芎麻药对高剂量组(相当于生药3g/kg)、芎麻药对低剂量组(相当于生药1.5g/kg)；假手术组(生理盐水10mL/kg),于模型建立后三天开始灌胃给药,每天两次。观察芎麻药对对大鼠脑血流量的干预作用；通过Morris水迷宫实验检验大鼠的学习记忆能力,社交实验考察大鼠社交能力；对Ach含量、AChE活性检测,检测脑内、血清、血清外泌体中的AKT1、AChE和PKCβ1的表达水平及含量。结果 Morris水迷宫实验和社交实验结果显示,模型组大鼠在28d开始到56d持续发生学习记忆障碍以及社交障碍,给药组能够有效改善VCI大鼠学习记忆能力和社交行为障碍。根据各组大鼠脑内ACh含量与AChE活力值测试结果表明,与模型组相比,芎麻高剂量组及阳性药组大鼠脑内ACh含量显著升高,各给药组AChE活力值均显著降低。各组大鼠脑内相关蛋白表达结果显示,与模型组相比,各给药组大鼠脑内AKT1和AChE的表达显著上调；芎麻高剂量组及阳性药组大鼠脑内PKCβ1表达显著上调。各组大鼠血清外泌体中胆碱能突触信号通路中关键蛋白表达结果显示,与模型组相比,各给药组大鼠血清外泌体中PKCβ1及AChE表达水平显著上调。差异均具有统计学意义(p均<0.05)。结论 芎麻药对通过抑制VCI大鼠脑内AChE活性并增加ACh的含量来达到治疗VCI, 胆碱能突触通路中的关键蛋白AChE与PKCβ1在脑中和血清外泌体中的变化基本一致,显示血清外泌体可能携带AChE和PKCβ1,在外周血液与脑组织之间传递疾病信息。

Abstract:

OBJECTIVE To investigate the effect of Xiongma dropping pills on the expression of exosomes in brain and serum of rats with vascular cognitive impairment and its intervention mechanism. METHODS a A total of 50 male Wistar rats aged 8-10 weeks in SPF grade were selected to replicate the VCI model using 2-VO method. According to the results of cerebral blood flow, the rats in 2-VO group were randomly divided into VCI model group (10mL/kg normal saline) and positive drug group (Donepezil hydrochloride 0.03g/kg). High dose of Xiongma dropping pill group (equivalent to 3g/kg of crude drug), low dose of Xiongma dropping pill group (equivalent to 1.5g/kg of crude drug); The sham operation group (10mL/kg normal saline) was given intragastric administration twice a day three days after the establishment of the model. To observe the intervention effect of Xiongma dropping pills on cerebral blood flow in rats; The learning and memory ability of rats was tested by Morris water maze experiment, and the social ability of rats was tested by social experiment. Ach content and AChE activity were detected. AKT1, AChE and PKCβ1 expression levels in brain, serum and serum exosomes were detected. RESULTS The results of Morris water maze experiment and social interaction experiment showed that the model group of rats continued to develop learning and memory disorders and social interaction disorders from day 28 to day 56, and the drug administration group could effectively improve the learning and memory ability and social behavior disorders of VCI rats. According to the test results of ACh content and AChE activity value in brain of rats in each group, ACh content in brain of rats in high dose group and positive drug group was significantly increased, and AChE activity value in each drug administration group was significantly decreased compared with the model group. The expression of related proteins in the brain of rats in each group showed that compared with the model group, the expressions of AKT1 and AChE in the brain of rats in each administration group were significantly up-regulated; The expression of PKCβ1 in the brain of rats in the high dose group and the positive group was significantly up-regulated. The expression of key proteins in cholinergic synaptic signaling pathway in serum exosomes of rats in all groups showed that PKCβ1 and AChE expression levels in serum exosomes of rats in all groups were significantly up-regulated compared with that in model group. The differences were statistically significant (p <0.05).CONCLUSION Xiong Ma dropping pills can treat VCI by inhibiting AChE activity in the brain of VCI rats and increasing ACh content. The changes of AChE, a key protein in cholinergic synaptic pathway, and PKCβ1 in brain and serum exosomes are basically consistent, indicating that serum exosomes may carry AChE and PKCβ1. Transmitting disease information between peripheral blood and brain tissue.