脑心安胶囊激活CREB/PGC-1α改善慢性脑缺血致VCI大鼠线粒体及氧化损伤的作用机制  

Naoxin’an Capsule Alleviates Mitochondrial and Oxidative Damage in Chronic Cerebral Ischemia-induced VCI in Rats via Activating CREB/PGC-1α Pathway

作　　者：冯宇娟 王乐[1] 韩广卉 余虹霓 李东岳 甄伟哲 马涛[1] FENG Yu-juan;WANG Le;HAN Guang-hui;YU Hong-ni;LI Dong-yue;ZHEN Wei-zhe;MA Tao(Dongfang Hospital,Beijing University of Chinese Medicine,Beijing 100078,China;School of Traditional Chinese Medicine,Beijing University of Chinese Medicine,Beijing 100029,China)

机构地区：[1]北京中医药大学东方医院,北京100078 [2]北京中医药大学中医学院,北京100029

出　　处：《中国实验方剂学杂志》2022年第23期19-29,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81973786,81673929)。

摘　　要：目的:以慢性脑缺血致血管性认知障碍(VCI)大鼠为模型,探究脑心安胶囊对VCI大鼠脑组织线粒体功能障碍及其导致的氧化损伤的保护作用。方法:150只大鼠采用随机数字法分为造模组(130只)和假手术组(20只),以双侧颈动脉结扎法(2-VO)造模,制成慢性脑缺血大鼠模型。经筛选,将造模组中VCI造模成功的87只大鼠,随机分为模型组、阳性药组(安理申,0.5 mg·kg^(-1)),脑心安胶囊低、中、高剂量组(0.18、0.36、0.72 g·kg^(-1)),每组17~18只大鼠。经灌胃给予脑心安胶囊8周后,采用大鼠新物体识别和Y迷宫实验测定脑心安胶囊对VCI大鼠学习记忆和工作记忆的影响。以免疫荧光染色法测定大鼠脑组织8-氧鸟嘌呤(8-OxoG)含量,分光光度法测定大鼠脑组织线粒体呼吸链复合物Ⅰ、Ⅱ、Ⅲ和Ⅳ的活性、线粒体的肿胀度、线粒体膜电位、丙酮酸脱氢酶(PDH)和α-酮戊二酸脱氢酶(KGDH)活性,电镜观察大鼠脑组织线粒体亚显微结构,蛋白免疫印迹法(Western blot)测定脑组织环磷腺苷酸反应元件结合蛋白(CREB)磷酸化水平和过氧化物酶体增殖物激活受体γ辅激活因子-1α(PGC-1α)、核呼吸因子-1(NRF-1)和线粒体转录因子A(mt-TFA)蛋白表达水平,光化学法测定大鼠线粒体状态ⅣHO生成和脑内超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性及丙二醛(MDA)水平。结果:与假手术组比较,模型组大鼠新物体辨别指数和Y迷宫自发交替反应率显著下降(P<0.01),脑组织ROS生成量明显增加(P<0.01),线粒体膜电位和肿胀度A值显著下降(P<0.01),线粒体明显肿胀,电镜观察显示脑组织线粒体出现显著的空泡、嵴断裂等亚显微结构结构损伤,CREB磷酸化水平和PGC-1α、NRF-1和mt-TFA蛋白表达水平均显著降低(P<0.01),脑组织中PDH、KGDH活性和线粒体呼吸链复合物Ⅰ、Ⅱ、Ⅲ和Ⅳ活性均明显下降(P<0.01),线粒体状态ⅣHO生成显著增加(P<0.01),MDA含量明显�Objective:To explore the effect of Naoxin’an capsule against mitochondrial dysfunction and oxidative damage in brains of rats with vascular cognitive impairment(VCI)induced by chronic cerebral ischemia.Method:A total of 150 rats were randomized into modeling group(130 rats)and sham-operated group(20 rats)with the random number table method.The two-vessel occlusion(2-VO)was employed to induce VCI in rats,and finally 87 rats developed VCI.The VCI rats were classified into model group,positive drug group(Aricept,0.5 mg·kg^(-1)),and low-,medium-and high-dose Naoxin’an capsule groups(0.18,0.36,0.72 g·kg^(-1),separately),with 17-18 rats in each group.The administration lasted 8 weeks.The learning and working memory of VCI rats were assayed by novel object recognition test and Y-maze test.The 8-oxoguanine(8-OxoG)level was measured based on immunofluorescence staining.Spectrophotometry was performed to determine the activity of mitochondrial respiratory chain complexes Ⅰ,Ⅱ,Ⅲ,and Ⅳ,mitochondrial swelling,mitochondrial membrane potential(MMP),and activity of pyruvate dehydrogenase(PDH)and α-ketoglutarate dehydrogenase(KGDH).The submicroscopic structure of mitochondria was observed with electron microscope.The phosphorylation of cAMP response element-binding protein(CREB) and the expression of peroxisome proliferator-activated receptor γ coactivator-1α(PGC-1α),nuclear respiratory factor 1(NRF-1),and mitochondrial transcription factor A(mt-TFA) were determined by Western blot.The mitochondrial status Ⅳ HOgeneration,activity of cerebral superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px),and malondialdehyde(MDA)content were measured with the photochemical method.Result:Compared with the sham-operated group,model group showed decrease in new object discrimination index(P<0.01)and spontaneous alternation rate in the Y-maze test(P<0.01),increase in ROS production in the brain(P<0.01),reduction in MMP and mitochondrial swelling A value(P<0.01),obvious mitochondrial swelling,vacuoles and cristae fractur

关 键 词：脑心安胶囊 血管性认知障碍 慢性脑缺血 氧化损伤 线粒体 环磷腺苷酸反应元件结合蛋白(CREB)/过氧化物酶体增殖物激活受体γ辅激活因子-1α(PGC-1α) 

分 类 号：R2-0[医药卫生—中医学] R22R285.5R289R33