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

Molecules. 2020 Jan 28;25(3):553. doi: 10.3390/molecules25030553.

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-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 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-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.

Keywords: PC12 cells; epilepsy; glutamate; protective effects; protein from Bombyx batryticatus; γ-aminobutyric acid.

MeSH terms

  • Animals
  • Bombyx / metabolism*
  • Cell Survival / drug effects
  • Glutamic Acid / adverse effects*
  • Insect Proteins / isolation & purification
  • Insect Proteins / pharmacology*
  • Interleukin-1beta / metabolism
  • Interleukin-4 / metabolism
  • Nerve Growth Factor / pharmacology
  • PC12 Cells
  • Rats
  • Receptors, Glucocorticoid / metabolism
  • Serotonin / metabolism
  • Signal Transduction / drug effects*
  • Tumor Necrosis Factor-alpha / metabolism
  • gamma-Aminobutyric Acid / metabolism*

Substances

  • IL1B protein, rat
  • Insect Proteins
  • Interleukin-1beta
  • Receptors, Glucocorticoid
  • Tumor Necrosis Factor-alpha
  • glucocorticoid receptor alpha
  • Interleukin-4
  • Serotonin
  • Glutamic Acid
  • gamma-Aminobutyric Acid
  • Nerve Growth Factor