Astragaloside IV Attenuates Glutamate-Induced Neurotoxicity in PC12 Cells through Raf-MEK-ERK Pathway

Abstract

Astragaloside IV (AGS-IV) is a main active ingredient of Astragalus membranaceus Bunge, a medicinal herb prescribed as an immunostimulant, hepatoprotective, antiperspirant, a diuretic or a tonic as documented in Chinese Materia Medica. In the present study, we employed a high-throughput comparative proteomic approach based on 2D-nano-LC-MS/MS to investigate the possible mechanism of action involved in the neuroprotective effect of AGS-IV against glutamate-induced neurotoxicity in PC12 cells. Differential proteins were identified, among which 13 proteins survived the stringent filter criteria and were further included for functional discussion. Two proteins (vimentin and Gap43) were randomly selected, and their expression levels were further confirmed by western blots analysis. The results matched well with those of proteomics. Furthermore, network analysis of protein-protein interactions (PPI) and pathways enrichment with AGS-IV associated proteins were carried out to illustrate its underlying molecular mechanism. Proteins associated with signal transduction, immune system, signaling molecules and interaction, and energy metabolism play important roles in neuroprotective effect of AGS-IV and Raf-MEK-ERK pathway was involved in the neuroprotective effect of AGS-IV against glutamate-induced neurotoxicity in PC12 cells. This study demonstrates that comparative proteomics based on shotgun approach is a valuable tool for molecular mechanism studies, since it allows the simultaneously evaluate the global proteins alterations.

Fig 1. Chemical structures of the four compounds, astragaloside IV (AGS-IV), 6-O-β-D-glucopyranosyl cycloastragenol (6-O-β-D-glu CAG), 3-O-β-D-xylopyranosyl cycloastragenol (3-O-β-D-xyl CAG), cycloastragenol (CAG).

Fig 2. AGS-IV protects against glutamate-induced neurotoxicity in PC12 cells.

(A) Dose-dependent cell death was observed after the cells were treated with different concentrations glutamate for 24 h. (B) Effects of 50 μM AGS-IV, 6-O-β-D-glu CAG, 3-O-β-D-xyl CAG, and CAG on glutamate-induced PC12 cell neurotoxicity. (C) Survival of PC12 cells after exposure to 0.1% DMSO or various concentrations of AGS-IV for 30 h. (D, E) Effects of AGS-IV on glutamate-induced PC12 cell injury. PC12 cells were treated with 10, 25, 50 and 100 μM AGS-IV and then co-incubated with or without 5 mM glutamate for 24 h, and cell cytotoxicity was determined by MTT assay and LDH activity. (F) Apoptosis assay of PC12 cells exposed to glutamate and/or AGS-IV were examined by flow cytometry. PC12 cells were gated for an annexin V⁺ (x-axis) versus PI⁺ (y-axis) contour plot. The numbers on dot plots represent the percentages of annexin V⁺/PI⁺ cells and annexin V⁺/PI^- cells. (G) The percentage of living cells was tested by trypan blue exclusion for PC12 cells exposed to glutamate and/or AGS-IV. All the data are presented as mean ± SEM of three independent experiments. ## < 0.01 versus vehicle and **P < 0.01 versus glutamate by one-way ANOVA analysis of variance with Tukey’s HSD post hoc test.

Fig 3. Score plot of PCA performed on the spectral count data of glutamate-treated groups (GLUs) and AGS-IV-treated groups (AGS-IVs) (Pentagram, GLUs; Triangle, AGS-IVs).

Proteins meet the following filter criteria were included for PCA: (1) the number of unique peptide identified more than 2; (2) protein identified at least four out of six samples; (3) statistical significance (P < 0.05) were obtained by Student’s t-test (GLUs and AGS-IVs).

Fig 4. Western blotting of vimentin and Gap43 expression in GLUs and AGS-IVs.

(A) Modulation of vimentin and Gap43 by AGS-IV on glutamate-induced PC12 cell injury. Data shown are the results of three different experiments and are represented as the relative densities of vimentin (B) and Gap43 (C) protein bands normalized to β-actin. Results are presented as means ± SEM of three assays. ** Significant difference compared with GLUs by Student’s t-test (P < 0.01).

Fig 5. Constructed minimum protein-protein interaction network between targeted proteins of AGS-IV.

Square nodes denote identified differentially expressed proteins of AGS-IV. Circular nodes are proteins that link the identified proteins together. The network was decomposed into topological compact modules by a simulated annealing algorithm. Symbols and full names of the intermediate partners in the network are shown in Table 2.

Fig 6. Involvement of Raf-MEK-ERK pathway in AGS-IV treated PC12 cells.

PC12 cells were treated with different concentrations (25, 50 and 100 μM) of AGS-IV and then co-incubated with or without 5 mM glutamate (Glu) for 24 h. Data shown are the results of three different experiments and are represented as the relative densities of protein bands normalized to β-actin. Results are presented as means ± SEM of three assays. ** Significant difference compared with GLUs by one-way ANOVA analysis of variance with Tukey’s HSD post hoc test.