The Effect of Protein-Rich Extract from Bombyx Batryticatus against Glutamate-Damaged PC12 Cells Via Regulating γ-Aminobutyric Acid Signaling Pathway.

ABSTRACT

Bombyx Batryticatus (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-1ß (IL-1ß), 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 Ca2+ 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 Ca2+ levels in PC12 cells. After BBPs treatments, levels of GABA and 5-HT were increased and contents of TNF-α, IL-4 and IL-1ß 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.