Wu Dong-Mei, Wang Shan, Wen Xin, Han Xin-Rui, Wang Yong-Jian, Shen Min, Fan Shao-Hua, Zhuang Juan, Zhang Zi-Feng, Shan Qun, Li Meng-Qiu, Hu Bin, Sun Chun-Hui, Lu Jun, Zheng Yuan-Lin
Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, School of Life Science, Jiangsu Normal University, Xuzhou, China.
College of Health Sciences, Jiangsu Normal University, Xuzhou, China.
Cell Physiol Biochem. 2018;51(4):1600-1615. doi: 10.1159/000495649. Epub 2018 Nov 29.
BACKGROUND/AIMS: Parkinson's disease (PD) is a neurodegenerative movement disease with a high annual incidence. Accumulating evidence demonstrates that microRNAs play important roles in the pathogenesis of multiple neurological disorders, including PD. This study aims to investigate how microRNA-200a (miR-200a) regulates striatal dopamine receptor D2 (DRD2) to affect apoptosis of striatum in rats with PD and to explore the associated mechanism.
After successfully establishing a PD model by 6-hydroxydopamine injections, PD rats were mainly treated with miR-200a mimics, inhibitors, Forskolin or a combination of miR-200a inhibitors and Forskolin. High-performance liquid chromatography-electrochemical detection (HPLC-ECD) was employed to detect the levels of dopamine, 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and chemistry colorimetric methods were applied to detect the levels of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). A TUNEL assay and immunocytochemical staining were performed to observe apoptosis and tyrosine hydroxylase (TH)-positive cells in the striatum. The expression of miR-200a, DRD2, Bad, Bax, Bcl-2, cAMP and PKA was determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot assays.
In the cellular experiments, after transfection with the inhibitor of miR-200a, decreased levels of Bax, GSH-Px, SOD, dopamine, DOPAC and HVA but increased levels of MDA and Bcl-2 were found along with a reduced apoptosis rate and increased TH-positive cell number. In addition, downregulating miR-200a resulted in lower expression of AKT, cAMP and PKA but higher expression of DRD2 and CREB, indicating that the downregulation of miR-200a increases DRD2 expression, which blocks the cAMP/PKA signaling pathway.
This study provides evidence that the inhibition of miR-200a can repress apoptosis in the striatum via inhibition of the cAMP/PKA signaling pathway by upregulating DRD2 expression in PD rats.
背景/目的：帕金森病（PD）是一种年发病率较高的神经退行性运动疾病。越来越多的证据表明，微小RNA在包括PD在内的多种神经疾病的发病机制中发挥重要作用。本研究旨在探讨微小RNA-200a（miR-200a）如何调节纹状体多巴胺受体D2（DRD2）以影响PD大鼠纹状体的细胞凋亡，并探索相关机制。
通过注射6-羟基多巴胺成功建立PD模型后，主要用miR-200a模拟物、抑制剂、福斯高林或miR-200a抑制剂与福斯高林的组合对PD大鼠进行治疗。采用高效液相色谱-电化学检测法（HPLC-ECD）检测多巴胺、3,4-二羟基苯乙酸（DOPAC）和高香草酸（HVA）的水平，采用化学比色法检测丙二醛（MDA）、超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GSH-Px）的水平。进行TUNEL检测和免疫细胞化学染色以观察纹状体中的细胞凋亡和酪氨酸羟化酶（TH）阳性细胞。通过逆转录定量聚合酶链反应（RT-qPCR）和蛋白质免疫印迹法检测miR-200a、DRD2、Bad、Bax、Bcl-2、cAMP和PKA的表达。
在细胞实验中，用miR-200a抑制剂转染后，发现Bax、GSH-Px、SOD、多巴胺、DOPAC和HVA水平降低，但MDA和Bcl-2水平升高，同时细胞凋亡率降低，TH阳性细胞数增加。此外，下调miR-200a导致AKT、cAMP和PKA表达降低，但DRD2和CREB表达升高，表明miR-200a的下调增加了DRD2表达，从而阻断了cAMP/PKA信号通路。
本研究提供的证据表明，抑制miR-200a可通过上调PD大鼠DRD2表达抑制cAMP/PKA信号通路，从而抑制纹状体细胞凋亡。
