何首乌苷对大脑中动脉栓塞再灌注模型大鼠皮层线粒体自噬的调控作用及神经保护机制  

Regulation and neuroprotective mechanism of 2,3,5,4'-tetrahydroxyl diphenylethylene-2-O-glucoside on mitochondrial autophagy in cortex of rats with middle cerebral artery occlusion/reperfusion model

作　　者：向韵[1,2] 黄丹[1,2] 雷昌[1,2] 周梓洋 邱峰 刘桐赫 施佳仪 谭开媚 曾红雨 易韬 伍大华[3] 张秀丽[1,2] XIANG Yun;HUANG Dan;LEI Chang;ZHOU Ziyang;QIU Feng;LIU Tonghe;SHI Jiayi;TAN Kaimei;ZENG Hongyu;YI Tao;WU Dahua;ZHANG Xiuli(Hunan University of Chinese Medicine,Changsha 410208,China;Science and Technology Innovation Center,Hunan University of Chinese Medicine,Changsha 410208,China;Affiliated Hospital of Hunan Academy of Traditional Chinese Medicine,Changsha 410006,China)

机构地区：[1]湖南中医药大学,湖南长沙410208 [2]湖南中医药大学科技创新中心,湖南长沙410208 [3]湖南省中医药研究院附属医院,湖南长沙410006

出　　处：《中草药》2024年第14期4771-4781,共11页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金面上项目(82374441);湖南省自然科学基金资助项目(2023JJ30465,2023JJ40483);长沙市自然科学基金资助项目(kq2208186);湖南省教育厅青年项目(22B0351);湖南中医药大学校级科研项目(2022XJZKC013,2021XJJJ026);湖南中医药大学中药粉体与创新药物省部共建国家重点实验室培育基地开放基金资助项目(21PTKF1013,21PTKF1022,2022FTKFJJ08)。

摘　　要：目的研究何首乌苷(2,3,5,4'-tetrahydroxyldiphenylethylene-2-O-glucoside,THSG)在脑缺血再灌注(cerebral ischemia/reperfusion,CI/R)中对大鼠皮层神经元的保护作用。方法采用线栓法建立大鼠短暂性大脑中动脉栓塞再灌注(transient middle cerebral artery occlusion/reperfusion,tMCAO/R)模型。雄性SD大鼠随机分为假手术组、模型组和THSG低、中、高剂量(10、20、40 mg/kg)组,每组10只。采用Longa 5分法评估神经功能缺损情况;TTC染色检测大鼠脑梗死体积百分比;苏木素-伊红(HE)染色检测神经元结构;Nissl染色检测Nissl小体数量;Western blotting检测半胱氨酸天冬氨酸蛋白酶-3(cystein-asparate protease-3,Caspase-3)、B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax)、PTEN诱导的激酶1(PTEN induced putative kinase 1,PINK1)、Parkin、微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)、p62的蛋白表达;透射电镜观察皮层神经元线粒体自噬情况;免疫荧光检测神经元Parkin、LC3的表达。结果THSG显著降低了MCAO/R大鼠脑梗死体积百分比和神经功能缺损评分(P<0.05、0.001),降低了MCAO/R大鼠皮层神经元结构损伤和Nissl小体丢失的现象,下调了皮层区cleaved Caspase-3、Bax、p62蛋白表达(P<0.05、0.01、0.001),上调了皮层区Bcl-2、LC3-Ⅱ/LC3-Ⅰ蛋白表达(P<0.01),促进皮层神经元中自噬体的数量增加以及PINK1、Parkin蛋白表达(P<0.05、0.001)。结论THSG能显著改善MCAO/R大鼠皮层神经元损伤,其神经保护机制与促进PINK1/Parkin依赖性线粒体自噬途径有关。Objective To investigate the protective effect of 2,3,5,4'-tetrahydroxyl diphenylethylene-2-O-glucoside(THSG) on cortical neurons in rats during cerebral ischemia/reperfusion(CI/R).Methods The rat transient middle cerebral artery occlusion/reperfusion(tMCAO/R) model was established by the wire bolt method.Male SD rats were randomly divided into sham group,model group and THSG low-,medium-,high-dose(10,20,40 mg/kg) groups,with 10 rats in each group.Longa quintile was used to assess neurological deficits;TTC staining was used to detect the volume percentage of cerebral infarction in rats.Hematoxylineosin(HE) staining was used to detect neuronal structure.Nissl staining was used to detect the number of Nissl bodies;Western blotting was used to detect the protein expressions of cystein-asparate protease-3(Caspase-3),B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),PTEN induced putative kinase 1(PINK1),Parkin,microtubule-associated protein 1 light chain 3(LC3) and p62;Mitochondrial autophagy of cortical neurons was observed by transmission electron microscopy;Immunofluorescence assay was used to detect the expressions of Parkin and LC3 in neurons.Results THSG significantly reduced the percentage of cerebral infarction volume and neurological deficit score in MCAO/R rats(P < 0.05,0.001),reduced the structural damage and Nissl body loss of cortical neurons in MCAO/R rats,down-regulated the expressions of cleaved Caspase-3,Bax,and p62 proteins in cortical area(P < 0.05,0.01,0.001),up-regulated the expressions of Bcl-2,LC3-Ⅱ/LC3-Ⅰ proteins in cortical area(P < 0.01),promoted the increase of autophagosomes in cortical neuron and expressions of PINK1 and Parkin proteins(P < 0.05,0.001).Conclusion THSG can significantly improve cortical neuronal damage in MCAO/R rats,and its neuroprotective mechanism is related to promoting PINK1/Parkin dependent mitochondrial autophagy pathway.

关 键 词：何首乌苷 脑缺血再灌注 皮层 线粒体自噬 PINK1/Parkin通路 

分 类 号：R285.5[医药卫生—中药学]