复方地黄颗粒通过泛素-蛋白酶体系统治疗PD阴虚动风证大鼠的分子机制研究  

Molecular Mechanism Study of Compound Dihuang Granule in Treating PD Rats with Yin-Deficiency and Dynamic Wind Syndrome by Ubiquitin-Proteasome System

作　　者：吕子微 梁建庆 王行玲 孙光洁 何建成[4] 康永强 LYU Ziwei;LIANG Jianqing;WANG Xingling;SUN Guangjie;HE Jiancheng;KANG Yongqiang(School of Basic Medical Sciences,Gansu University of Chinese Medicine,Lanzhou 730050,China;Key Laboratory of TCM Prescription and Drug Exploration and Innovation Transformation,Lanzhou 730050,China;The Key Laboratory of the Ministry of Education of Dunhuang Medicine and Transformation,Lanzhou 730050,China;School of Traditional Chinese Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Department of Neurology,The Second People’s Hospital of Lanzhou City,Lanzhou 730050,China)

机构地区：[1]甘肃中医药大学基础医学院,甘肃兰州730050 [2]甘肃省中医方药挖掘与创新转化重点实验室,甘肃兰州730050 [3]敦煌医学与转化教育部重点实验室,甘肃兰州730050 [4]上海中医药大学中医学院,上海201203 [5]兰州市第二人民医院神经内科,甘肃兰州730050

出　　处：《南京中医药大学学报》2024年第6期608-617,共10页Journal of Nanjing University of Traditional Chinese Medicine

基　　金：国家自然科学基金地区项目(82160929);2021年度甘肃省科技厅第十一批省级科技计划自然科学基金(自筹经费)项目(21JR11RA143);敦煌医学与转化教育部重点实验室开放课题(DHYX22-06);2022年度中医学一级学科“岐黄英才”导师专项基金硕导项目(ZYXKSD-202211);甘肃中医药大学“中医老年病学”学科建设项目(2022-3)。

摘　　要：目的探讨复方地黄颗粒通过调节泛素-蛋白酶体系统相关蛋白变化治疗帕金森病(PD)阴虚动风证的分子机制。方法随机选取26只SPF级雄性SD大鼠作为正常对照组、假手术组,每组13只,其余大鼠采用6-羟基多巴胺(6-OHDA)脑部偏侧注射损毁黑质法制备PD阴虚动风证大鼠模型,将成模大鼠随机分为模型组,美多芭组(150 mg·kg^(-1)),复方地黄颗粒低、中、高剂量组(1.75、3.5、7 g·kg^(-1)),每组13只。各组连续灌胃给药28 d。观察各组大鼠一般情况及神经行为学表现;免疫组化法、Western blot及qPCR法检测各组大鼠脑部损毁侧纹状体组织中泛素(UB)、泛素激活酶(UBE1)、泛素结合酶(UBE2A)、泛素羧基末端水解酶(UCH-L1)阳性细胞表达、蛋白及mRNA表达水平;ELISA法检测各组大鼠脑部损毁侧纹状体中多巴胺(DA)含量变化。结果正常对照组与假手术组一般情况无明显差异,旋转圈数、悬挂时间及移动格数差异无统计学意义,与假手术组比较,模型组大鼠精神差,易激惹,肢体震颤,行动迟缓,毛色黯黄,饮食少,体质量减轻,旋转圈数增加、悬挂时间及移动格数减少(P<0.01),损毁侧纹状体中UB、UBE1、UBE2A、UCH-L1阳性细胞表达、mRNA及蛋白表达水平明显降低(P<0.01),DA含量明显减少(P<0.01);与模型组比较,美多芭组及复方地黄颗粒各剂量组大鼠一般情况有所改善,旋转圈数减少、悬挂时间及移动格数增加(P<0.05,P<0.01),UB、UBE1、UBE2A、UCH-L1阳性细胞表达、mRNA及蛋白表达水平提高(P<0.05,P<0.01),DA含量增加(P<0.01);行为学检测及各指标表达水平以美多芭组和复方高剂量组改善最明显(P<0.05,P<0.01),此二者无统计学意义(P>0.05)。结论复方地黄颗粒可能通过调控泛素-蛋白酶体系统相关蛋白的表达,减少异常蛋白聚集,从而减轻DA含量缺失,发挥治疗PD阴虚动风证的作用,并以复方地黄颗粒高剂量效果最佳。OBJECTIVE To explore the molecular mechanism of Compound Dihuang Granule in treating Parkinson’s disease(PD)with yin-deficiency and dynamic wind syndrome by regulating the changes of ubiquitin-proteasome system-related proteins.METHODS SPF male SD rats were randomly selected as normal control group and sham operation group,13 rats in each group.The remaining rats were treated with 6-hydroxydopamine lateral brain injection to damage the substantia nigra to establish PD yin-deficiency and dynamic wind syndrome rat model.The established rats were randomly divided into model group,madopar group(150 mg·kg^(-1)),low,medium and high dose groups of Compound Dihuang Granule(1.75,3.5,7 g·kg^(-1)),13 rats in each group.Each group was given drugs by intragastric gavage for 28 d.The general conditions and neurobehavioral manifestations of the rats were observed;the expression of ubiquitin(UB),ubiquitin activating enzyme(UBE1),ubiquitin conjugating enzyme(UBE2A)and ubiquitin carboxyl terminal hydrolase(UCH-L1)positive cells,protein and mRNA expression levels in the damaged lateral striatum of the brain of rats were detected by immunohistochemistry,Western blot and qPCR methods;changes of dopamine(DA)content in the damaged lateral striatum of the brain of rats were detected by ELISA.RESULTS There was no significant difference in general conditions between the normal control group and the sham operation group,and there was no significant difference in the number of rotations,suspension time and number of moving grids.Compared to the sham-op group,the model group rats showed poor spirit,irritability,limb tremor,slow movement,dark yellow fur,less food intake,reduced body weight;the modeling increased number of rotations,decreased hanging time and number of moving grids(P<0.01),significantly decreased expression of UB,UBE1,UBE2A,and UCH-L1 positive cells,mRNA and protein expression levels in the damaged lateral striatum(P<0.01),and significantly decreased DA content(P<0.01).Compared with the model group,the general condition of

关 键 词：帕金森病 阴虚动风证 复方地黄颗粒 泛素-蛋白酶体系统 分子机制 

分 类 号：R285.5[医药卫生—中药学]