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.
Introduction
Astragali Radix, one of the most commonly used traditional Chinese
medicine (TCM), is prepared from the roots of Astragalus membranaceus
(Fisch) Bunge and Astragalus mongholicus Bunge, and is prescribed as an
immunostimulant, hepatoprotective, antiperspirant, a diuretic or a
tonic as documented in Chinese Materia Medica [[45]1]. A brief survey
of the therapeutic functions of Astragali Radix includes
immunoregulatory, antioxidant, anti-cancer, antiviral, diuretic,
hypolipidemic, and hypoglycemic effects, as well as its protective
effects toward cardiovascular, liver, lung, and neural tissues as well
as upon renal function [[46]2–[47]5]. Total Astragalus membranaceus
extract, mainly composed of astragalosides and astragalus
polysaccharides, is often employed in different in vitro and in vivo
studies without significant toxic effects. Regarding the chemical
constituents of Astragali Radix, more than 100 compounds, such as
isoflavonoids, triterpene saponins, polysaccharides and amino acids,
have been identified so far, and various biological activities of the
compounds have been reported [[48]6]. Astragalosides are cycloartane
triterpenoid saponins which are characterized with 3-, 6- and/or 25-
conjugated glucose moieties and whose 3-glucose is acetylated [[49]7].
The basic aglycone form of these triterpenes is known as
cycloastragenol (9,10-cyclo-lanostane type; CAG) and a 3,6-glycosylated
relative without acetylation named astragaloside IV
(3-0-beta-d-xylopyranosyl-6-0-beta-d-glucopyranos-ylcycloastragenol,
AGS-IV), which is more abundant in Astragali membranaceus than in other
Astragalus species [[50]7]. AGS-IV, one of the major and active
components of the Astragalus membranaceus, has been renowned for a
series of protective effects, such as anti-oxidant [[51]8–[52]10],
anti-hypertension [[53]11], anti-diabetic [[54]12], angiogenic
[[55]13], anti-infarction [[56]14–[57]17], anti-inflammation [[58]18],
healing and anti-scar effects [[59]19].
Our recent studies have shown that AGS-IV can show cardioprotection
during myocardial ischemia in vivo and in vitro [[60]20]. AGS-IV
dilates aortic vessels from normal and spontaneously hypertensive rats
through endothelium-dependent and endothelium-independent ways
[[61]21]. Moreover, we have demonstrated that AGS-IV inhibited vessel
contraction through blocking calcium influx and intracellular calcium
release. The endothelium-dependent vessel dilation of AGS-IV is
attributed mainly to the endothelium-dependent nitric oxide (NO)-cGMP
pathway [[62]22].
Although AGS-IV can exert cardioprotection activity under
pathophysiological conditions, the targets of AGS-IV characterization
is a bottleneck and its mechanism remains to be elucidated. Recently,
we have investigated the therapeutic mechanism of AGS-IV against
cardiovascular diseases using a network-based methodology that
integrates data of drugs, targets and pathways [[63]16]. However, the
detailed molecular bases of protective effects of AGS-IV on the
proteome level remain largely unexplored.
Proteins are central to the understanding of cellular function and
disease processes. Proteomics in general deals with the large-scale
determination of gene and cellular function directly at the proteome
level, and the MS-based proteomics has established itself as an
indispensable technology to interpret the information encoded in
proteome [[64]23–[65]25]. The proteomic approach is widely applied
nowadays in the development of novel biomarker candidates for early
detection of disease and identification of new targets for
therapeutics, mainly by delineation of protein expression changes
depending on factors such as the organism’s physiological state and the
stage of development of disease [[66]26].
In the present study, we employed a high-throughput comparative
proteomic approach based on 2D-nano-LC-MS/MS to investigate the
possible signaling pathways involved in the neuroprotective effect of
AGS-IV against glutamate-induced neurotoxicity in PC12 cells. The main
purpose of the present study was to explore the detailed molecular
bases of the neuroprotective effect of AGS-IV on the proteome level
with comparative proteomics, so as to get better knowledge of
neuroprotective mechanism of AGS-IV.
Materials and Methods
Chemicals
CAG, 6-O-β-D-glucopyranosyl cycloastragenol (6-O-β-D-glu CAG),
3-O-β-D-xylopyranosyl cycloastragenol (3-O-β-D-xyl CAG), and AGS-IV
were isolated from the dried root tuber of Astragalus membranaceus and
identified by ^1H NMR, ^13C NMR, ESI-MS data and by comparing with the
published data of earlier studies [[67]27] (purity > 98% as determined
by HPLC). Their chemical structures are shown in [68]Fig 1.
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 1
[69]Open in a new tab
Reagents
Dimethyl sulfoxide (DMSO) and
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT)
was purchased from Sigma (St Louis, MO, USA). Dulbecco’s Modified
Eagle’s Medium (DMEM), fetal bovine serum (FBS) were purchased from
Gibco (Grand Island, NY). Antibodies for p-Raf, p-MEK, p-ERK1/2,
ERK1/2, p-JNK, JNK, and β-actin were obtained from Cell Signaling
Technology (Beverly, MA). Vimentin and Gap43 were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA, USA). HPLC-grade acetonitrile was
obtained from Merck KGaA (Darmstadt, German).
Cells and drug treatments
The high differentiated rat pheochromocytoma tumor cell line PC12,
obtained from the Cell Bank of the Shanghai Institute of Biochemistry &
Cell Biology, Shanghai Institute for Biological Sciences, Chinese
Academy of Sciences (Shanghai, China, [70]http://www.cellbank.org.cn),
was maintained in DMEM containing 10% FBS supplemented with 100 U/mL
penicillin and 100 μg/mL streptomycin in a humidified atmosphere
containing 5% CO[2] at 37°C as described previously [[71]28]. AGS-IV
was dissolved in DMSO and was freshly prepared each time before use
(DMSO final culture concentration < 0.1%). For cell cytotoxicity
assays, PC12 cells were inoculated at a density of 1 × 10^4 cells per
well in 96-well microplates and then maintained for a 30-h incubation
with test compounds. For neuroprotective assay, PC12 cells were treated
with test compounds for 6 h before exposure to 5 mM glutamate and then
maintained for 24 h.
MTT and LDH release assay
The effects of the test compounds on the cytotoxicity were determined
by the MTT and LDH release assay as described previously [[72]28].
Briefly, PC12 cells were seeded in 96-well plates at 1 × 10^4 cells per
well and co-incubated with the test compounds for 24 h. Cultures were
also treated with 0.1% DMSO as the untreated control. After treatment,
10 μL of MTT solution (5 mg/mL) was added to each well and the plates
were incubated for 4 h at 37°C. The supernatant was then removed from
formazan crystals and 100 μL of DMSO was added to each well. The
absorbance at 570 nm was recorded using a SYNERGY Mx microplate reader
(BioTek, Winooski, VT). The cell survival percentages were calculated
by dividing the mean OD of compound-containing wells by that of control
wells. LDH assay was performed by a commercial kit (Promega, Madison,
WI, USA) according to the manufacturer’s protocol. PC12 cells were
treated with a range of compounds concentrations for 24 h. Lysis
solution was added to 96-well plates and incubated at 37°C for 30 min.
Then supernatant of each well was moved to a new 96 well plate and
reconstituted substrate was added and the plates were kept for another
30 min in dark at room temperature. Stop solution was then added to
each well and left for 30 min. Absorbance was recorded at 490 nm. The
percentage release of LDH from the treated cells was calculated by
comparing it to the maximum release of LDH achieved by the lysis
solution used on the control cells.
Trypan blue viability assay
PC12 cells (5 × 105) were seeded in six-well plates exposed to 5 mM
glutamate and 25, 50, and 100 μM AGS-IV. After 24 h, the cells were
stained with trypan blue (Bio-Rad) and the amount of living cells was
determined using an automat Cell Counter (Bio-Rad).
Apoptosis assay
Cells in early and late stages of apoptosis were detected using an
Annexin V-FITC apoptosis detection kit from BD Biosciences. In this
procedure, 5 × 10^5 PC12 cells per well in six-well plate were exposed
to glutamate and/or test compounds and incubated for 24 h prior to
analysis. Cells were collected and washed with phosphate buffer (PBS),
then re-suspended in Annexin V binding buffer followed by addition of
Annexin V-FITC and propidium iodide (PI). The samples were incubated in
the dark for 5 min at room temperature and analyzed with a FACSCaliber
flow cytometer (BD Biosciences, San Jose, CA). Mean of % apoptosis
index was calculated by combining early apoptosis (annexin V^+/PI^+)
and late apoptosis (annexin V+/PI-) events.
LC-MS Sample preparation
Different processing PC12 cells were homogenized in lysis buffer (8 M
urea, 50 mM Tris-HCl, pH 7.5, 0.25% v/v Triton X-100, 1 mM
phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol, 1 × protease
inhibitor cocktail) and centrifuged at 12,000 rpm for 10 min at 4°C,
and the supernatant was mixed with five volumes of precipitation buffer
(ethanol: acetone: glacial acetic acid = 10: 10: 0.1). After washed
three times with cold acetone, the pellet was dissolved in denature
buffer (8 M urea, 50 mM Tris-HCl, pH 8.2). The sample was reduced by
dithiothreitol at 37°C for 2 h and alkylated by iodoacetamide in the
dark at room temperature for 30 min. Then the solution was diluted to 1
M urea with 50 mM Tris-HCl (pH 8.2). Finally, trypsin was added at an
enzyme-to-substrate of 1/25 (w/w) and incubated at 37°C overnight. The
digested mixture was loaded on a homemade C18 solid-phase cartridge (20
mg, 1 mL) conditioned with 0.5 mL × 3 acetonitrile and 0.5 mL × 3 water
(containing 0.1% trifluoroacetic acid), then the cartridge was washed
with 0.5 mL × 3 water (containing 0.1% trifluoroacetic acid) and eluted
with 0.5 mL × 2 80% acetonitrile (containing 0.1% trifluoroacetic
acid). The elution was speedvaced to dry and stored at -80°C before
LC-MS analysis.
2D-nano-LC-MS/MS analysis and database searching
The 2D-nano-LC-MS/MS system applied a quaternary surveyor pump and an
LTQ linear ion trap mass spectrometer equipped with a nanospray source
(Thermo, San Jose, CA). The tryptic samples were dissolved in 0.1%
formic acid, loaded onto a monolith strong cation exchange (SCX) column
(150 μm id × 7 cm) automatically. Then a series stepwise elution with
salt concentrations of 50, 100, 200, 300, 400, 500, and 1000 mM NH[4]AC
were used to gradually elute peptides from the phosphate monolithic
column onto the C18 analytical column. Each salt step lasts 10 min
except for the last one, which lasts for 20 min after the whole system
was re-equilibrated for 10 min with buffer A (0.1% formic acid water
solution), the binary gradient elution with buffer A and buffer B (0.1%
formic acid acetonitrile solution) (0–10 min, from 0 to 10% buffer B,
10–60 min, from 10 to 40% buffer B, 60–65 min, from 40 to 80% buffer B,
65–75 min, 80% buffer B, 75–85 min, buffer A) for reversed phase
separation was applied prior to MS detection in each cycle. The
temperature of the ion transfer capillary was set at 150°C. The spray
voltage was set at 2.2 kV. All MS and MS/MS spectra were acquired in
the data-dependent mode. The mass spectrometer was set so that one full
MS scan was followed by three MS/MS scans. The MS/MS spectra were
searched using Mascot against a composite database including both
original and reversed rat protein database of International Protein
Index (IPI_RAT RAT_v3.26). Peptides were searched using fully tryptic
cleavage constraints and up to two missed cleavages sites were allowed
for tryptic digestion. The mass tolerances are 2 Da for parent masses
and 1 Da for fragment masses. Initial searching results were filtered
with the following parameters as tasted previously: the Xcorr g 1.8 for
a singly charged peptide, 2.5 for a doubly charged peptide, and 3.5 for
a triply charged peptides; the minimum ∆Cn cutoff value of 0.08. For
semiquantitative comparisons of the proteins identified, spectral
counts for each identified protein from each experiment were extracted,
averaged, and compared as described previously [[73]29–[74]31].
Data processing and principle component analysis (PCA)
The processed data of the comparative proteomics were exported and
further processed by PCA using the SIMCA-P software package (Version
11, Umetrics, Umea, Sweden). The data were processed by unit variance
scaling and were mean-centered using the SIMCA-P software.
Network construction and Identification of pathways enriched with AGS-IV
associated proteins
Network construction was performed as previously described [[75]16].
The protein-protein interaction network was constructed based on the
Human Protein Reference Database (HPRD) data and mapped all putative
targets of AGS-IV onto this network. Then we applied the Steiner
minimal tree algorithm to identify a minimum sub-network [[76]32],
which included as many putative targets of AGS-IV and as few other
proteins as possible. Each target protein of AGS-IV was allowed to
interact with other target proteins through no more than one non-target
protein.
Differentially expressed proteins in the experiments were considered
AGS-IV associated genes. These genes were mapped onto the pathways in
the KEGG database, and then P-values were used to determine whether a
pathway was enriched with AGS-IV associated genes instead of by chance.
Assuming that the total AGS-IV associated genes (K) were mapped onto
the rat pathways in KEGG, which included N distinct genes, a
hypergeometric cumulative distribution function could model the
probability of identifying at least k genes from a pathway of size n by
chance, such that the P-value is given by:
[MATH: P=1−∑i=0k−1<
mrow>f(i)=1−∑i=0k−1<
mrow>(Ki)(N−Kn−i)(Nn) :MATH]
Given the significance level α = 0.01, a P-value smaller than α
demonstrated a low probability that the AGS-IV associated genes
appeared in the pathway by chance; i.e., the pathway can be regarded as
being significantly regulated by these gene-encoded proteins.
Western blotting study
Serial concentrations of compound were added to 1×10^6 PC12 cells in
six-well plate. After 24 h of incubation at 37°C, the cells were
collected by centrifugation at 1000 rmp for 5 min. The cell pellets
were then washed with PBS, resuspended in lysis buffer containing 150
mM NaCl, 50 mM Tris (pH 8.0), 0.02% NaN[3], 0.01% PMSF, 0.2% Aprotinin,
1% TritonX-100 supplemented with protease inhibitor cocktail (Sigma.
St. Louis, MO), and centrifuged at 12 000 rmp for 10 min. The
concentration of total proteins was determined using a BCA kit (Pierce,
Rockford, IL). 40 μg protein per lane was electrophoresed on 12% SDS
polyacrylamide gels after boiling for 5 min and transferred to
polyvinylidene difluoride (PVDF) membrane. Nonspecific reactivity was
blocked by 5% nonfat milk prepared in TBST (10 mM Tris, 150 mM NaCl,
0.05% Tween-20, pH 7.5) at room temperature for 1 h. The membranes were
incubated with antibodies diluted according to the manufacturers'
instructions. The image was captured by the Odyssey infrared imaging
system (Li-Cor Bioscience, Lincoln, NE). Protein densitometry was done
using Quantity One imaging software (Bio-Rad) and normalized against
β-actin.
Statistical analysis
Data were analyzed using GraphPad Prism software version 5 (Graph Pad
software Inc., San Diego, CA, USA). The comparison between two groups
was analyzed by unpaired Student t-test, and multiple comparisons were
compared by one-way ANOVA analysis of variance followed by Tukey post
hoc test. The quantitative data were reported as means ± SEM from at
least three independent experiments. Statistical significance was
determined as P < 0.05.
Results
AGS-IV protects against glutamate-induced neurotoxicity in PC12 cells
We evaluated whether the four cycloartane triterpenoid saponins
extracted from the root tubers of Astragalus membranaceus could protect
against oxidative glutamate cytotoxicity in vitro. As shown in [77]Fig
2A, a clear dose-dependent cell death was observed after the cells were
treated with different concentrations glutamate for 24 h. Thus, 5 mM
was the preferred concentration of choice for the rest of the
experiments. Examination of cytotoxicity of PC12 cells by the MTT
method showed that 5 mM glutamate noticeably reduced the survival of
PC12 cells with a rate of 43 ± 3.4% (P < 0.05 vs. vehicle) ([78]Fig
2B). Among the saponins examined at 50 μM, AGS-IV and 6-O-β-D-glu CAG
showed better protective effects than 3-O-β-D-xyl CAG and CAG ([79]Fig
2B). Interestingly, cytotoxicity analysis showed AGS-IV at the
concentrations exceeding 100 μM was found to be low cytotoxic to PC12
cells ([80]Fig 2C). We further characterized the effect of AGS-IV (a
major active ingredient, higher yield than that of the other three
compounds) on glutamate cytotoxicity over a wide range of
concentrations from 10 to 100 μM and found that AGS-IV could
dose-dependently mitigate glutamate-induced neurotoxicity ([81]Fig 2D).
Similar findings were also obtained with LDH release assays with the
rate of LDH release at 63 ± 3.5% in PC12 cells exposed to 5 mM
glutamate and 49 ± 3.0%, 37 ± 3.6%, and 33 ± 2.1%, respectively, in
PC12 cells exposed to 5 mM glutamate and 25, 50, and 100 μM AGS-IV (P <
0.01 vs. glumatate) ([82]Fig 2E). In addition, flow cytometric analysis
showed that glutamate induced apoptotic death of PC12 cells and AGS-IV
(25, 50 and 100 μM) attenuated these apoptotic changes ([83]Fig 2F).
Moreover, 5 mM glutamate displayed a high toxicity in the trypan blue
staining as in the MTT assay and AGS-IV could dose-dependently mitigate
glutamate-induced neurotoxicity ([84]Fig 2G).
Fig 2. AGS-IV protects against glutamate-induced neurotoxicity in PC12 cells.
Fig 2
[85]Open in a new tab
(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.
AGS-IV induces broad changes in protein expression in PC12 cells
A comprehensive shotgun proteomic profiling procedure, based on online
2D-nano-LC-MS/MS system was applied to uncover proteomic alterations
associated with 100 μM AGS-IV treated PC12 cells exposed to 5 mM
glutamate at 24 h. The numbers of total spectral counts and identified
proteins are listed in [86]Table 1. For PCA of the proteins identified
in the striatum of AGS-IV treated PC12 cells (AGS-IVs) and 5 mM
glutamate treated PC12 cells (GLUs), spectral counts for each
identified protein from each experiment were extracted, averaged,
normalized. To improve the reliability of identification of the
proteins, proteins meet the stringent filter criteria (the number of
unique peptide identified more than 2; protein identified at least four
out of six samples) were included for the further Student’s t-test
statistic. A total of 72 proteins were selected based on the filter
criteria and a Student’s t-test statistic (p < 0.05). PCA was performed
on this data set, which reveals two distinct clusters corresponding to
AGS-IVs and GLUs ([87]Fig 3). The proteins (listed in [88]Table 2) meet
the following filter criteria: (a) for the average spectral of AGS-IVs
and GLUs, the larger value more than 4; (b) the spectral count ratio
more than 3-fold and no more than 10-fold were included for the further
functional discussion [[89]33]. Two proteins, vimentin and Gap43, were
randomly selected from whose antibodies are commercially available and
their expression levels were further confirmed by Western Blots.
[90]Fig 4 shows that the altered intensity of the proteins matched well
with the differences obtained in 2D-nano-LC-MS/MS based proteomic
analysis.
Table 1. Total spectral counts and proteins identified of glutamate groups
(GLUs)[91] ^a and AGS-IV groups (AGS-IVs)[92] ^b .
GLUs AGS-IVs
GLU-1 GLU-2 GLU-3 GLU-4 GLU-5 GLU-6 AGS-IV-1 AGS-IV-2 AGS-IV-3 AGS-IV-4
AGS-IV-5 AGS-IV-6
Total spectral counts 30458 30123 36893 39785 38699 39952 40088 46583
39536 41503 41368 39329
Total protein groups 1738 1315 1612 1849 1871 1420 2602 2726 2317 2023
2711 2107
[93]Open in a new tab
a glutamate groups (GLUs): PC12 cells received 5 mM glutamate and then
maintained for 24 h.
b AGS-IV groups (AGS-IVs): PC12 cells were treated with 50 μM AGS-IV
for 6 h before exposure to 5 mM glutamate and then maintained for 24 h
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).
Fig 3
[94]Open in a new tab
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).
Table 2. Lists of the identified differentially expressed proteins.
No. Protein name Ratio [95]^a
1 Malate dehydrogenase 0.22
2 Heat shock protein HSP 90-alpha 0.33
3 14-3-3 protein epsilon 0.31
4 14-3-3 protein zeta/delta 0.24
5 Gap43 0.23
6 Neutral ceramidase 3.7
7 Heat shock 70 kDa protein 1A/1B 0.24
8 Serine/threonine-protein kinase TAO3 0.23
9 Myosin-9 0.31
10 Ribosome-binding protein 1 3.2
11 Vimentin 0.29
12 ribosomal protein S7 0.33
13 Periaxin 0.21
[96]Open in a new tab
a Ratio averaged glutamate groups (GLUs) to AGS-IV groups (AGS-IVs)
spectral counts.
Fig 4. Western blotting of vimentin and Gap43 expression in GLUs and AGS-IVs.
[97]Fig 4
[98]Open in a new tab
(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).
Protein-protein association network construction, pathways enriched and
validation with AGS-IV associated proteins
To understand the relationship among the putative targets of AGS-IV, we
mapped the differentially expressed proteins onto the protein-protein
interaction network of the human genome. We found that 27 of them can
be linked into one sub-network either through direct interactions or
with only one intermediate protein, suggesting that most of the targets
are located in the neighborhood of each other in the human protein
network ([99]Fig 5). The proximity of proteins in the interactome
indicates that they share common functions. To identify the functions
of this network between targets, the AGS-IV associated proteins are
mapped onto KEGG. The mapping pathways showed that 27 genes appeared in
a total of 8 pathways. Pathway enrichment analysis was performed to
identify pathways significantly that were regulated by AGS-IV
associated proteins, and the P-values were computed for each of the 8
pathways with AGS-IV associated proteins. The computations generated 8
pathways with P-values smaller than 0.01 ([100]Table 3), and therefore
these pathways were regarded as key pathways involved in the
neuroprotective effect of AGS-IV against glutamate-induced
neurotoxicity in PC12 cells.
Fig 5. Constructed minimum protein-protein interaction network between
targeted proteins of AGS-IV.
Fig 5
[101]Open in a new tab
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 [102]Table 2.
Table 3. Lists of KEGG pathways that are significantly regulated by AGS-IV.
Definition total genes mapped genes pathway category Fisher-P value
MAPK signaling pathway 273 7 Signal transduction 1.12×10–5
Toll-like receptor signaling pathway 101 5 Immune system 0.0081
Cell adhesion molecules (CAMs) 154 4 Signaling molecules and
interaction 0.0044
Glycerophospholipid metabolism 82 3 Energy metabolism 0.0062
Calcium signaling pathway 178 3 Signal transduction 0.0041
Glycine, serine and threonine metabolism 34 2 Energy metabolism 0.0062
Phosphatidylinositol signaling system 78 3 Signal transduction 0.0065
Metabolic pathways 1197 9 Energy metabolism 0.0081
[103]Open in a new tab
To validate the pathway enrichment result, we chosen MAPK signaling
pathway for Western blot confirmation. Interestingly, MAPK1 (ERK1) was
found as an intermediate protein in the protein-protein association
network and it is possible that the ERK MAPK activation ultimately
dictate whether glutamate induced neurotoxicity in PC12 cells. As shown
in [104]Fig 6 and 5 mM glutamate exposure significantly increased
p-Raf, p-MEK, p-ERK1/2, and p-JNK protein levels, which were inhibited
by pretreated by AGS-IV in a dose-dependent manner. Therefore,
Raf-MEK-ERK MAPK pathway was involved in the neuroprotective effect of
AGS-IV against glutamate-induced neurotoxicity in PC12 cells.
Fig 6. Involvement of Raf-MEK-ERK pathway in AGS-IV treated PC12 cells.
Fig 6
[105]Open in a new tab
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.
Discussion
Traditional Chinese medicine provides an extensive foundation for
implementing a strategically focused pharmacological research program
that aimed at the development of new drugs. Astragalus mongholicus
Bunge, known as Huangqi in China, has been used as one of the primary
Chinese tonic herbs for thousands of years. Recently there are some
reports on the neuroprotective activities of the crude extract from
Huangqi [[106]34]. Screening active ingredients is an important step in
the drug discovery of TCM. In this work, we find that AGS-IV, as a
major active ingredient, is able to significantly attenuate
neurotoxicity induced by glutamate in PC12 cells.
Glutamate is thought to be the major excitatory neurotransmitter
working at a variety of excitatory synapses in the central nervous
system [[107]35]. It plays important roles in cellular process
underlying synaptic plasticity, neuronal development and excitation via
the activation of glutamate receptors [[108]36,[109]37]. Evidence is
accumulating that high concentrations of glutamate and relative
excitatory amino acid analogs cause a specific pattern of
neurodegeneration in the brain of experiment animals, in primary
culture of brain neurons and in some cultured neuron cell lines,
including PC12 cell line, a rat pheochromocytoma cell line
[[110]38,[111]39]. In high differentiated PC12 cells, lacking
ionotropic glutamate receptors, the high concentration glutamate
inhibits cystine uptake and depletes intracellular glutathione, which
leads to the accumulation of reactive oxygen species (ROS) and
ultimately causes cell death [[112]40]. In the present study, we
investigated whether the four cycloartane triterpenoid saponins were of
neuroprotective activity in damaged PC12 induced by glutamate in vitro.
Target discovery plays an important role in clinical application of
TCM. The proteomic approach is widely applied nowadays in understanding
the molecular mechanisms of natural products and identification of new
targets for therapeutics [[113]41–[114]43]. The 2D-nano-LC-MS/MS system
applied is fully automated and displays advantages such as minimal loss
of sample, no via contamination, and no sample dilution [[115]33]. This
study implemented the proteomics scheme to search globally for the
differentially expressed proteins in PC12 cells affected by AGS-IV. In
the present study, 13 proteins whose expressions were significantly
changed under AGS-IV treatment were identified. In order to obtain an
easier and non-biased interpretation of the results and to reduce the
dimensionality of the multivariate data obtained from the LC-MS
results, we analyzed the LC-MS chromatographic data using PCA. As an
unsupervised method for pattern recognition, PCA multivariate data
analysis procedure requires preprocessing of the raw data to generate a
data matrix, the columns of which represent variables and the rows of
which contain the samples that are included for analysis. These peak
picking and peak alignment processes are important for creating a
productive data matrix. After PCA processing, the AGS-IV treatment
group (AGS-IVs) was clearly separated from the glumatate group (GLUs).
Moreover, we constructed protein-protein association network, enriched
the relevant pathways and verified that the Raf-MEK-ERK pathway was
involved in the neuroprotective effect of astragaloside IV against
glutamate-induced neurotoxicity in PC12 cells.
MAPK pathway plays a crucial role as transducers of extracellular
stimuli into a series of intracellular phosphorylation cascades, which
ultimately leads to cell differentiation, proliferation, survival or
death [[116]44,[117]45]. To date, ERK MAPK, as one of major MAPK
subfamilies, can be activated by inflammatory cytokines and
extracellular stressors such as UV light, heat, and glumatate
[[118]46,[119]47]. Activation of the Raf-MEK-ERK pathway has been shown
to be a key regulator of neuronal apoptosis [[120]48], which makes this
pathway an important molecular target of neurodegenerative diseases
therapy [[121]49]. In the present study, pretreatment with AGS-IV
dramatically inhibited the glumatate-induced increase in levels of
phosphorylated Raf, MEK, and ERK MAPKs. These results indicate that
AGS-IV prevents glumatate-induced apoptosis in PC12 cells by blocking
the phosphorylation of Raf, MEK, and ERK MAPKs. In some instances,
AGS-IV also modulates other hubs and exerts its influence on the others
by acting on their surrounding neighbors. Previous studies on the
effect of multi-target attacks in a network-based model suggested that
weak inhibition of multiple targets could be more efficient than potent
inhibition of a single hub target [[122]50,[123]51]. We can also see
that proteins grouped together tend to be correlated in functionality
or to participate in the same biological processes.
Overall, this network suggests that AGS-IV produce its beneficial
effects on the neuroprotection against glutamate-induced neurotoxicity
by several proteins associated with signal transduction, immune system,
signaling molecules and interaction, and energy metabolism. Thus, the
organization of the target protein interaction network exhibits a
modular cooperative mode and may result in synergistic therapeutic
action.
Conclusions
The present study carried out a proteomics technique to search globally
for the proteins after exposure of glutamate-treatment PC12 cells to
AGS-IV, a purified component from TCM. 2D-nano-LC-MS/MS was conducted,
and 40 differentially expressed proteins were found that might be
target-related proteins of AGS-IV. Based on the results of comparative
proteomics, we concluded that proteins associated with signal
transduction, immune system, signaling molecules and interaction, and
energy metabolism play important roles in neuroprotective effect of
AGS-IV, although the precise roles of these identified molecules in
neuroprotective effect of AGS-IV need further study. In addition,
Raf-MEK-ERK pathway was involved in the neuroprotective effect of
AGS-IV against glutamate-induced neurotoxicity in PC12 cells. The
current study improves the understanding of mechanisms of AGS-IV
neuroprotective effect, and provides prospects for the application of
the comparative proteomics based on shotgun approach in biological
mechanisms study.
In conclusion, the present results show 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.
Acknowledgments