Lan Yu-Long, Zhou Jun-Jun, Liu Jing, Huo Xiao-Kui, Wang Ya-Li, Liang Jia-Hao, Zhao Jian-Chao, Sun Cheng-Peng, Yu Zhen-Long, Fang Lin-Lin, Tian Xiang-Ge, Feng Lei, Ning Jing, Zhang Bao-Jing, Wang Chao, Zhao Xin-Yu, Ma Xiao-Chi
College of Pharmacy, College (Institute) of Integrative Medicine, College of Medical Laboratory, Department of Neurosurgery, The Second Affiliated Hospital of Dalian Medical University, Dalian Medical University, Dalian, China.
Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Department of Natural Products Chemistry, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, China.
Cell Physiol Biochem. 2018;47(4):1453-1464. doi: 10.1159/000490837. Epub 2018 Jun 21.
BACKGROUND/AIMS: Uncaria rhynchophylla, known as "Gou-teng", is a traditional Chinese medicine (TCM) used to extinguish wind, clear heat, arrest convulsions, and pacify the liver. Although U. rhynchophylla has a long history of being often used to treat central nervous system (CNS) diseases, its efficacy and potential mechanism are still uncertain. This study investigated neuroprotective effect and the underlying mechanism of U. rhynchophylla extract (URE) in MPP+-induced SH-SY5Y cells and MPTP-induced mice.
MPP+-induced SH-SY5Y cells and MPTP-induced mice were used to established Parkinson's disease (PD) models. Quantitative proteomics and bioinformatics were used to uncover proteomics changes of URE. Western blotting was used to validate main differentially expressed proteins and test HSP90 client proteins (apoptosis-related, autophagy-related, MAPKs, PI3K, and AKT proteins). Flow cytometry and JC-1 staining assay were further used to confirm the effect of URE on MPP+-induced apoptosis in SH-SY5Y cells. Gait analysis was used to detect the behavioral changes in MPTP-induced mice. The levels of dopamine (DA) and their metabolites were examined in striatum (STR) by HPLC-EC. The positive expression of tyrosine hydroxylase (TH) was detected by immunohischemical staining and Western blotting.
URE dose-dependently increased the cell viability in MPP+-induced SH-SY5Y cells. Quantitative proteomics and bioinformatics results confirmed that HSP90 was an important differentially expressed protein of URE. URE inhibited the expression of HSP90, which further reversed MPP+-induced cell apoptosis and autophagy by increasing the expressions of Bcl-2, Cyclin D1, p-ERK, p-PI3K p85, PI3K p110α, p-AKT, and LC3-I and decreasing cleaved caspase 3, Bax, p-JNK, p-p38, and LC3-II. URE also markedly decreased the apoptotic ratio and elevated mitochondrial transmembrane potential (DΨm). Furthermore, URE treatment ameliorated behavioral impairments, increased the contents of DA and its metabolites and elevated the positive expressions of TH in SN and STR as well as the TH protein.
URE possessed the neuroprotective effect in vivo and in vitro, regulated MAPK and PI3K-AKT signal pathways, and inhibited the expression of HSP90. U. rhynchophylla has potentials as therapeutic agent in PD treatment.
背景/目的：钩藤，又称“钩藤”，是一种传统中药，用于熄风、清热、止痉、平肝。尽管钩藤长期以来常用于治疗中枢神经系统（CNS）疾病，但其疗效和潜在机制仍不明确。本研究探讨了钩藤提取物（URE）对MPP⁺诱导的SH-SY5Y细胞和MPTP诱导的小鼠的神经保护作用及其潜在机制。
使用MPP⁺诱导的SH-SY5Y细胞和MPTP诱导的小鼠建立帕金森病（PD）模型。采用定量蛋白质组学和生物信息学方法揭示URE的蛋白质组学变化。Western印迹法用于验证主要差异表达蛋白并检测HSP90客户蛋白（凋亡相关、自噬相关、MAPKs、PI3K和AKT蛋白）。流式细胞术和JC-1染色试验进一步用于确认URE对MPP⁺诱导的SH-SY5Y细胞凋亡的影响。步态分析用于检测MPTP诱导的小鼠的行为变化。通过高效液相色谱-电化学法检测纹状体（STR）中多巴胺（DA）及其代谢产物的水平。通过免疫组织化学染色和Western印迹法检测酪氨酸羟化酶（TH）的阳性表达。
URE剂量依赖性地增加了MPP⁺诱导的SH-SY5Y细胞的活力。定量蛋白质组学和生物信息学结果证实HSP90是URE的一种重要差异表达蛋白。URE抑制HSP90的表达，通过增加Bcl-2、Cyclin D1、p-ERK、p-PI3K p85、PI3K p110α、p-AKT和LC3-I的表达以及降低裂解的caspase 3、Bax、p-JNK、p-p38和LC3-II的表达，进一步逆转MPP⁺诱导的细胞凋亡和自噬。URE还显著降低了凋亡率并提高了线粒体跨膜电位（ΔΨm）。此外，URE治疗改善了行为障碍，增加了DA及其代谢产物的含量，并提高了黑质（SN）和STR中TH的阳性表达以及TH蛋白水平。
URE在体内和体外均具有神经保护作用，调节MAPK和PI3K-AKT信号通路，并抑制HSP90的表达。钩藤具有作为PD治疗药物的潜力。
