College of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China.
School of Pharmacy, Chengdu Medical College, Chengdu 610500, China.
Molecules. 2020 Jan 28;25(3):553. doi: 10.3390/molecules25030553.
(BB) is a known traditional Chinese medicine (TCM) utilized to treat convulsions, epilepsy, cough, asthma, headaches, etc. in China for thousands of years. This study is aimed at investigating optimum extraction of protein-rich extracts from BB (BBPs) using response surface methodology (RSM) and exploring the protective effects of BBPs against nerve growth factor (NGF)-induced PC12 cells injured by glutamate (Glu) and their underlying mechanisms. The results indicated optimum process of extraction was as follows: extraction time 1.00 h, ratio of liquid to the raw material 3.80 mL/g and ultrasonic power 230.0 W. The cell viability of PC12 cells stimulated by Glu was determined by CCK-8 assay. The levels of γ-aminobutyric (GABA), interleukin-1β (IL-1β), interleukin-4 (IL-4), tumor necrosis factor-α (TNF-α), 5-hydroxytryptamine (5-HT) and glucocorticoid receptor alpha (GR) in PC12 cells were assayed by ELISA. Furthermore, the Ca levels in PC12 cells were determined by flow cytometry analysis. Protein and mRNA expressions of GABAA-Rα1, NMDAR1, GAD 65, GAD 67, GAT 1 and GAT 3 in PC12 cells were evaluated by real-time polymerase chain reaction (RT-PCR) and Western blotting assays. Results revealed that BBPs decreased toxic effects due to Glu treatment and decreased Ca levels in PC12 cells. After BBPs treatments, levels of GABA and 5-HT were increased and contents of TNF-α, IL-4 and IL-1β were decreased in NGF-induced PC12 cells injured by Glu. Moreover, BBPs up-regulated the expressions of GABAA-Rα1, GAD 65 and GAD 67, whereas down-regulated that of NMDAR1 GAT 1 and GAT 3. These findings suggested that BBPs possessed protective effects on NGF-induced PC12 cells injured by Glu via γ-Aminobutyric Acid (GABA) signaling pathways, which demonstrated that BBPs has potential anti-epileptic effect in vitro. These findings may be useful in the development of novel medicine for the treatment of epilepsy.
（BB）是一种在中国被用于治疗惊厥、癫痫、咳嗽、哮喘、头痛等病症的传统中药，已有数千年的应用历史。本研究旨在利用响应面法（RSM）从 BB 中提取富含蛋白质的提取物（BBPs），并探索 BBPs 对谷氨酸（Glu）诱导的神经生长因子（NGF）损伤的 PC12 细胞的保护作用及其潜在机制。结果表明，最佳提取工艺条件为：提取时间 1.00 h、液料比 3.80 mL/g、超声功率 230.0 W。CCK-8 法测定 Glu 刺激的 PC12 细胞的细胞活力。通过 ELISA 测定 PC12 细胞中γ-氨基丁酸（GABA）、白细胞介素-1β（IL-1β）、白细胞介素-4（IL-4）、肿瘤坏死因子-α（TNF-α）、5-羟色胺（5-HT）和糖皮质激素受体α（GR）的水平。此外，通过流式细胞术分析测定 PC12 细胞中的 Ca 水平。通过实时聚合酶链反应（RT-PCR）和 Western blot 测定 PC12 细胞中 GABAA-Rα1、NMDAR1、GAD65、GAD67、GAT1 和 GAT3 的蛋白和 mRNA 表达。结果表明，BBPs 降低了 Glu 处理引起的毒性作用，并降低了 PC12 细胞中的 Ca 水平。在 BBPs 处理后，Glu 诱导的 NGF 损伤的 PC12 细胞中 GABA 和 5-HT 的水平升高，TNF-α、IL-4 和 IL-1β 的含量降低。此外，BBPs 上调了 GABAA-Rα1、GAD65 和 GAD67 的表达，而下调了 NMDAR1、GAT1 和 GAT3 的表达。这些发现表明，BBPs 通过 GABA 信号通路对 Glu 诱导的 NGF 损伤的 PC12 细胞具有保护作用，这表明 BBPs 在体外具有抗癫痫作用。这些发现可能有助于开发治疗癫痫的新型药物。
