Li Chaohua, Sun Yanan, Yang Weifeng, Ma Shuhua, Zhang Lili, Zhao Jing, Zhao Xin, Wang Yi
Xiyuan Hospital, China Academy of Chinese Medical Sciences, No. 1 Xiyuan Playground, Haidian District, Beijing 100091, China.
Experimental Research Center, China Academy of Chinese Medical Sciences, No. 16, Nanxiao Road, Dongzhimen, Beijing 100700, China.
Evid Based Complement Alternat Med. 2019 Jan 8;2019:7276407. doi: 10.1155/2019/7276407. eCollection 2019.
Parkinson's disease (PD) is one of the most common neurodegenerative diseases worldwide. Although dopamine replacement therapy mitigates motor dysfunction in PD patients, there are no therapeutics that are currently available to reverse neuronal cell death in the substantia nigra pars compacta (SNc), which is the main region for dopamine loss in PD patients. The protein concentration of the Pilose antler extracts (PAEs) was estimated using the Bradford Protein Assay Kit. Hematoxylin and eosin (HE) staining was used to evaluate the protective effect of PAEs on 6-OHDA induced cell death in PD model rats. Immunohistochemistry (IHC) was used to detect the tyrosine hydroxylase (TH) positive neuronal cell in SNc. HPLC-MS was used to detect dopamine (DA), 3,4-Dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and glutamate (Glu) levels in the striatum and cerebrospinal fluid (CSF). The amino acid level in the striatum and CSF was measured by HPLC-FLD. Protein expression of growth associated protein-43 (GAP-43) and neurofilament heavy polypeptide (NF-H) was measured using western blotting. The components of PAEs through blood vessels were detected by HPLC/MS/MS. In this study, PAEs with proteins ranging from 10 kDa to 250 kDa molecular weight was administered to 6-OHDA-induced PD rats. We found that PAEs inhibited 6-OHDA-induced neuronal cell death and TH-positive neuronal loss in SNc. PAEs administration also increased the levels of DA, DOPAC, and 5-HT, in addition to DOPAC/DA and HVA/DA indexes in the CSF and Striatum of 6-OHDA induced rats. Conversely, PAEs decreased the levels of Glu and GABA. Treatment with PAEs and Madopar increased GAP-43 and NF-H expression in the SNc and striatum. Proteomic analysis using LC/MS/MS indicated that 11 components of PAEs may have neuropharmacological effects. These results demonstrate that PAEs protects against 6-OHDA induced toxic effects in the PD rat models. Intragastric administration of PAEs may be a novel therapeutic strategy for neurodegenerative disorders like PD.
帕金森病（PD）是全球最常见的神经退行性疾病之一。尽管多巴胺替代疗法可减轻PD患者的运动功能障碍，但目前尚无治疗方法可逆转黑质致密部（SNc）的神经元细胞死亡，而SNc是PD患者多巴胺缺失的主要区域。使用考马斯亮蓝蛋白定量试剂盒估算鹿茸提取物（PAEs）的蛋白质浓度。采用苏木精-伊红（HE）染色评估PAEs对6-羟基多巴胺（6-OHDA）诱导的PD模型大鼠细胞死亡的保护作用。免疫组织化学（IHC）用于检测SNc中酪氨酸羟化酶（TH）阳性神经元细胞。高效液相色谱-质谱联用（HPLC-MS）用于检测纹状体和脑脊液（CSF）中的多巴胺（DA）、3,4-二羟基苯乙酸（DOPAC）、高香草酸（HVA）、5-羟色胺（5-HT）、5-羟吲哚乙酸（5-HIAA）和谷氨酸（Glu）水平。通过高效液相色谱-荧光检测法（HPLC-FLD）测量纹状体和CSF中的氨基酸水平。使用蛋白质印迹法测量生长相关蛋白-43（GAP-43）和神经丝重链多肽（NF-H）的蛋白表达。通过HPLC/MS/MS检测PAEs通过血管的成分。在本研究中，将分子量范围为10 kDa至250 kDa的含蛋白质的PAEs给予6-OHDA诱导的PD大鼠。我们发现PAEs可抑制6-OHDA诱导的神经元细胞死亡以及SNc中TH阳性神经元的丢失。给予PAEs还可提高6-OHDA诱导大鼠CSF和纹状体中DA、DOPAC和5-HT的水平，以及DOPAC/DA和HVA/DA指数。相反，PAEs可降低Glu和γ-氨基丁酸（GABA）的水平。PAEs与美多芭联合治疗可增加SNc和纹状体中GAP-43和NF-H的表达。使用液相色谱-串联质谱（LC/MS/MS）进行的蛋白质组学分析表明，PAEs的11种成分可能具有神经药理学作用。这些结果表明，PAEs可保护PD大鼠模型免受6-OHDA诱导的毒性作用。胃内给予PAEs可能是一种针对PD等神经退行性疾病的新型治疗策略。
