Subscriber access provided by UNIV OF LETHBRIDGE
Article
Maslinic acid protected PC12 cells differentiated by nerve growth factor against beta-amyloid induced apoptosis Yu-wan Yang, Chia-Wen Tsai, Mei-chin Mong, and Mei-Chin Yin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04156 • Publication Date (Web): 17 Oct 2015 Downloaded from http://pubs.acs.org on October 26, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 28
Journal of Agricultural and Food Chemistry
Maslinic acid protected PC12 cells differentiated by nerve growth factor against beta-amyloid induced apoptosis
Yu-wan Yang ø,§, Chia-wen Tsai†, Mei-chin Mong⊥, Mei-chin Yin†,⊥,* ø
School of Medicine, China Medical University, Taichung City, Taiwan
§
Department of Neurology, China Medical University Hospital, Taichung City, Taiwan
†
Department of Nutrition, China Medical University, Taichung City, Taiwan
⊥
Department of Health and Nutrition Biotechnology, Asia University, Taichung City, Taiwan
Running title: Neuro-protection of MA against Abeta *To whom correspondence should be addressed: Dr. Mei-chin Yin, Professor, Department of Nutrition, China Medical University, 91, Hsueh-shih Rd., Taichung City, Taiwan TEL: 886-4-22053366 ext. 7510, FAX: 886-4-22062891 Email:
[email protected] 1
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
1
ABSTRACT
2
Beta-amyloid peptide (Abeta) was used to induce apoptosis in PC12 cells differentiated by
3
nerve growth factor, and the protective activities of maslinic acid (MA) at 2-16 µM were
4
examined.
5
cell viability.
6
increased cell viability.
7
subsequent Abeta-induced release of reactive oxygen species, tumor necrosis factor-alpha,
8
interleukin (IL)-1beta and IL-6.
9
gp91phox, mitogen-activated protein kinase, advanced glycation end product receptor (RAGE)
Abeta treatment lowered Bcl-2 expression, raised Bax expression and decreased MA pre-treatments declined Bax expression, raised Bcl-2/Bax ratio and MA pre-treatments retained glutathione content, and decreased
Abeta treatment up-regulated protein expression of p47phox,
10
and nuclear factor-kappa B (NF-κB).
MA pre-treatments at 2-16 µM suppressed the
11
expression of proteins including gp91phox, p47phox, p-p38 and NF-κB p65; and at 4-16 µM
12
down-regulated RAGE and NF-κB p50 expression; at 8 and 16 µM reduced p-ERK1/2
13
expression.
14
Abeta-induced cytotoxicity.
These novel findings suggest that maslinic acid is a potent compound against
15 16
KEYWORDS: Abeta; Maslinic acid; NADPH oxidase; RAGE
17
2
ACS Paragon Plus Environment
Page 2 of 28
Page 3 of 28
Journal of Agricultural and Food Chemistry
18 19
INTRODUCTION Alzheimer’s disease (AD) is the most common progressive neurodegenerative disorder
20
in many countries.
It is documented that beta-amyloid peptide (Aβ, Abeta) plays a crucial
21
role in AD pathogenesis.1,2
22
raised oxidative injury through promoting the production of reactive oxygen species (ROS).3
23
Shao et al.4 reported that Abeta activated caspases like caspase-3 and/or caspase-8, and caused
24
mitochondrial malfunctions.
25
reduced the activity of Na+-K+-ATPase and perturbed potential of mitochondrial membrane
26
(MMP) in astrocytes and neurons, which impaired membrane permeability and facilitated
27
neuron loss.
28
brain nuclear factor-kappa B (NF-κB) and/or mitogen activated protein kinase (MAPK)
29
pathways, which contributed to augment the generation of several cytokines such as tumor
30
necrosis factor (TNF)-alpha and interleukin (IL)-1beta.
31
advanced glycation end product receptor (RAGE); furthermore, Abeta interacted with RAGE
32
to induce breakage of blood-brain barrier.9
33
toward neuronal cells, which consequently leads to brain disorders.
34
with protective effects upon mitochondria and/or suppressive activities upon NADPH oxidase,
35
RAGE and NF-κB may potentially attenuate Abeta-induced neurotoxicity and delay AD
36
progression.
Abeta stimulated NADPH oxidase protein expression, which
Vitvitsky et al.5 and Quintanilla et al.6 indicated that Abeta
The studies of Jang and Surh7 and Kuang et al.8 revealed that Abeta activated
In addition, Abeta up-regulated the
Apparently, Abeta elicits multiple impairments Therefore, any agent
37
Maslinic acid (MA) is a pentacyclic triterpenoic acid, and naturally present in some
38
edible plants including brown mustard (Brassica juncea), centella (Centella asiatica L.) and
39
olive (Olea europaea L.).10,11
40
lipopolysaccharide induced inflammatory injury through suppressing NF-κB signal pathway.
41
Allouche et al.13 indicated that MA exhibited anti-oxidative activities via acting as free radical
Huang et al.12 reported that MA protected rat astrocyte against
3
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 4 of 28
Qian et al.14 reported that MA diminished oxygen-glucose
42
scavenger and copper chelator.
43
deprivation caused injury in cortical neurons. Yin et al.11 indicated that dietary MA intake
44
enhanced its bioavailability in mice brain.
45
anti-oxidative and anti-inflammatory agent, could protect neuronal cells against brain
46
disorders.
47
cells against Abeta-induced apoptosis and inflammatory stress.
48
RAGE, NADPH oxidase and signaling pathways in Abeta-treated neuronal cells also remains
49
unknown.
50
agent against Alzhemer’s disease.
51
These findings suggest that MA, acting as an
However, less information is available regarding the benefit of MA for neuronal The regulation of MA upon
If it could attenuate Abeta-induced neurotoxicity, it might be considered as an
PC12 cell line could be differentiated by nerve growth factor (NGF) to a sympathetic
52
neurons.15
53
investigating the protective activity and mechanism of natural compounds upon neuronal
54
cells.16,17
55
NGF-differentiated PC12 cell line.
56
concentrations upon cell viability, MMP, Na+-K+-ATPase activity, ROS and inflammatory
57
cytokines were examined.
58
oxidase, NF-κB, MAPK and RAGE was evaluated.
59 60
MATERIALS AND METHODS
61
So far, the NGF-treated PC12 cell line is widely applied as a neurons model for
In current study, Abeta was used for cytotoxicity induction in this The activities and action modes of MA at various
The impact of MA upon the protein expression of NADPH
Materials. MA (95%) and NGF (99.5%) were purchased from Aldrich Chemical Co.
62
(Milwaukee, WI, USA) and Promega Chem. Co. (Madison, WI, USA), respectively.
Plates,
63
medium, antibiotics and chemicals for cell culture were bought from Detroit Difco Laboratory
64
(MI, USA).
65
Sodium phosphate buffer (PBS, pH 7.2) at 10 mM was used to dilute this peptide to
66
appropriate concentration for following experiments.
Abeta1-42 was obtained from Sigma Chemical Co. (St. Louis, MO, USA).
4
ACS Paragon Plus Environment
Page 5 of 28
Journal of Agricultural and Food Chemistry
67
Cell Culture. PC12 cells were incubated in 35-mm plates which contained Dulbecco’s
68
modified Eagle’s medium (DMEM) supplied with 5% fetal bovine serum, 10%
69
heat-inactivated calf serum, 100 units/mL of streptomycin and 100 units/mL of penicillin at
70
37°C under 95% air and 5% CO2. Cells were further treated by 50 ng/mL NGF for 5 days to
71
differentiate.
72
days.
73
serum-free DMEM one day before experiments, and followed by replanting in plates with 96
74
wells.
75
Culture medium was renewed every 72 hr, and cells were re-cultured every 7
Then, medium was replaced by serum-deprived medium.
Cells were washed by
Experimental Design. Dimethyl sulfoxide (DMSO) was used to dissolve MA, and
76
further diluted by medium.
77
this concentration, 0.5%, did not change PC12 cell viability (shown in supplemental figure)
78
and other measurements (data not shown).
79
pre-treated by MA at 0, 1, 2, 4, 8 or 16 µM for 48 hr at 37°C, which resulted in 95.7 ± 2.0%
80
incorporation.
81
concentrations were analyzed by HPLC method form Lin et al.18
82
considered as incorporated.
83
Final DMSO concentration in medium was 0.5%.
DMSO at
NGF-treated PC12 cells, 105 cells/mL, were
MA treated cells were washed twice by PBS.
PBS was collected and MA The MA left in cells was
Then, cells were exposed by 10 µM Abeta for 24 hr at 37°C.
Viability Assay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT)
84
was used to examine cell viability.
85
Milwaukee (WI, USA).
Control or MA-treated PC12 cells were reacted with MTT, 0.25
86
mg/mL at 37°C for 3 hr.
Then, MTT formazan product was quantified by using a microplate
87
reader (Bio-Rad, Hercules, CA, USA) to measure the absorbance at 570 nm and 630 nm.
88
Cell viability was expressed as a percentage of control without addition of MA, and without
89
Abeta treatment.
90 91
MTT (99%) was obtained from Aldrich Chemical Co. in
Determination of Lactate Dehydrogenase (LDH) Activity. Plasma membrane injury of cells was detected by assaying intracellular LDH activity in the medium.
5
ACS Paragon Plus Environment
Cell culture
Journal of Agricultural and Food Chemistry
Page 6 of 28
92
supernatant at 50 µL was collected.
The activity (U/L) of LDH was determined by a
93
commercial assay kit purchased from Sigma Chemical Co. (St. Louis, MO, USA).
94
coefficient of variation (CV) of intra-assay and the inter-assay was 5.9% and 5.1%,
95
respectively.
The
96
Measurement of MMP. A fluorescent dye Rhodamine123 (Rh123) bought from Sigma
97
Chemical Co. (St. Louis, MO, USA) and a Beckman-FC500 flow cytometry (Beckman
98
Coulter, Fullerton, CA, USA) were used to monitor MMP.
99
into to cells and followed by incubating at 37°C for 45 min. Cells were collected and further
100
washed twice by PBS (pH 7.2).
Rh123 at 100 µg/L was added
Mean fluorescence intensity (MFI) of cells was measured.
101
Mitochondrial Fraction Preparation. Cells were lysed by cold lysis buffer containing
102
0.1 mM EDTA, 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 0.6%
103
Nonidet P-40 and 1 mM PMSF for 30 min.
104
xg to collect nuclei pellet.
105
for 20 min to collect mitochondria pellet.
106
Mitochondrial protein concentration was quantified by an assay kit purchased from Pierce
107
Biotechnology Inc. (Rockford, IL, USA), in which bovine serum albumin was a standard.
108
Sample was diluted by PBS to 1 mg protein/mL for following use.
109
Cells were centrifuged at 4°C for 10 min at 200
Then, the supernatant was further centrifuged at 4°C, 10,000 xg PBS was used to re-suspend this pellet.
Activity Assay of Na+-K+-ATPase. The activity of Na+-K+-ATPase was assayed by
110
determining inorganic phosphate (Pi) level released from ATP.19
111
contained freshly isolated mitochondria, 2 mM ATP, 20 mM KCl, 100 mM NaCl and 30 mM
112
Tris-HCl buffer at pH 7.4.
113
was added to terminate assay after incubation at 37°C for 15 min.
114
measured by monitoring absorbance at 640 nm. Absorbance values of MA treated cells were
115
shown as a percentage of controls.
116
The reaction mixture
ATP was added to initiate assay, and 15% trichloroacetic acid Then, released Pi was
Caspase Activity Assay. Caspase-3 and -8 activities were determined by fluorometric
6
ACS Paragon Plus Environment
Page 7 of 28
Journal of Agricultural and Food Chemistry
117
assay kits obtained from Upstate Co. (Lake Placid, NY, USA).
The intra-assay CV and the
118
inter-assay CV were 3.6-4.7% and 5.2-6.3%, respectively.
119
lysis buffer and further incubated in ice for ten min.
120
with 5 mL reaction buffer and 2.5 mL of caspase-3 or -8 fluorogenic substrates in microplate.
121
After 1 hr incubation at 37°C, caspase-3 or -8 activity was detected by a fluorophotometer at
122
excitation and emission at 400 and 505 nm, respectively.
123
of controls.
Briefly, cells were lysed in cold
Cell lysates at 50 µL was combined
Data were shown as a percentage
124
Measurement of Glutathione (GSH), ROS and Cytokines. Cells were homogenized
125
by 2 mL PBS in a Teflon glass motordriven homogenizer obtained from Glas-Col Co. (CA,
126
USA).
127
from
128
2',7'-dichlorofluorescein diacetate was used to determine ROS level.
129
50 µM dye and incubated for 30 min.
130
microplate reader with excitation at 485 nm and emission at 530 nm.
131
fluorescence values between time 0 and 5 min was defined as relative fluorescence unit
132
(RFU).
133
in supernatant were analyzed by cytoscreen immunoassay kits bought from BioSource Intl.
134
(Camarillo, CA, USA).
135
and for TNF-alpha was 10 pg/mL.
136
GSH concentration (ng/mg protein) was measured by a colorimetric kit purchased OxisResearch
(Portland,
OR,
USA).
An
oxidation
sensitive
dye,
Cells were mixed with
Fluorescence values were detected by a fluorescence
Results are presented as RFU per mg protein.
The difference in
IL-1beta, IL-6 and TNF-alpha levels
The detection limit of assays for IL-1beta and IL-6 was 5 pg/mL,
Western Blot Analyses. Cells were treated by lysis buffer, and protein concentration
137
was measured.
Forty µg protein samples were used for 10% SDS-polyacrylamide gel
138
electrophoresis; then, further transferred to nitrocellulose membranes purchased from
139
Millipore (Bedford, MA, USA) for 60 min.
140
60 min, membranes were followed by reacting with monoclonal antibodies against Bcl-2, Bax,
141
caspase-3, caspase-8, p47phox, gp91phox (1:1000), NF-κB, RAGE and MAPK (1:2000)
After blocking with 5% skim milk solution for
7
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 8 of 28
142
overnight at 4ºC; then, reacted with horseradish peroxidase conjugated antibody at room
143
temperature for 3.5 hr.
144
(GAPDH).
145
blots were normalized against GAPDH, and results were expressed as arbitrary units (AU).
146
Loading control was glyceraldehyde-3-phosphate dehydrogenase
The bands were processed by an ATTO image analyzer (Tokyo, Japan), and
Statistical Analyses. Ten different preparations (n = 10) were processed to determine the
147
effect of each treatment.
Data were expressed as means ± standard deviation (SD), and used
148
for analysis of variance.
149
differences among means with significance defined at P0.05, data not shown).
154
The influence of MA at 2, 4, 8 or 16 µM upon MTT assay for cell viability and LDH activity
155
for plasma membrane integration is presented in Figure 1.
156
MA did not affect either cell viability or plasma membrane integration (P>0.05).
157
treatment lowered cell viability and enhanced LDH release (P
