Maslinic Acid Protected PC12 Cells Differentiated by Nerve Growth

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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

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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]

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ABSTRACT

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Beta-amyloid peptide (Abeta) was used to induce apoptosis in PC12 cells differentiated by

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nerve growth factor, and the protective activities of maslinic acid (MA) at 2-16 µM were

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examined.

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cell viability.

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increased cell viability.

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subsequent Abeta-induced release of reactive oxygen species, tumor necrosis factor-alpha,

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interleukin (IL)-1beta and IL-6.

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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,

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and nuclear factor-kappa B (NF-κB).

MA pre-treatments at 2-16 µM suppressed the

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expression of proteins including gp91phox, p47phox, p-p38 and NF-κB p65; and at 4-16 µM

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down-regulated RAGE and NF-κB p50 expression; at 8 and 16 µM reduced p-ERK1/2

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expression.

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Abeta-induced cytotoxicity.

These novel findings suggest that maslinic acid is a potent compound against

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KEYWORDS: Abeta; Maslinic acid; NADPH oxidase; RAGE

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INTRODUCTION Alzheimer’s disease (AD) is the most common progressive neurodegenerative disorder

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in many countries.

It is documented that beta-amyloid peptide (Aβ, Abeta) plays a crucial

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role in AD pathogenesis.1,2

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raised oxidative injury through promoting the production of reactive oxygen species (ROS).3

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Shao et al.4 reported that Abeta activated caspases like caspase-3 and/or caspase-8, and caused

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mitochondrial malfunctions.

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reduced the activity of Na+-K+-ATPase and perturbed potential of mitochondrial membrane

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(MMP) in astrocytes and neurons, which impaired membrane permeability and facilitated

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neuron loss.

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brain nuclear factor-kappa B (NF-κB) and/or mitogen activated protein kinase (MAPK)

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pathways, which contributed to augment the generation of several cytokines such as tumor

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necrosis factor (TNF)-alpha and interleukin (IL)-1beta.

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advanced glycation end product receptor (RAGE); furthermore, Abeta interacted with RAGE

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to induce breakage of blood-brain barrier.9

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toward neuronal cells, which consequently leads to brain disorders.

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with protective effects upon mitochondria and/or suppressive activities upon NADPH oxidase,

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RAGE and NF-κB may potentially attenuate Abeta-induced neurotoxicity and delay AD

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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

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Maslinic acid (MA) is a pentacyclic triterpenoic acid, and naturally present in some

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edible plants including brown mustard (Brassica juncea), centella (Centella asiatica L.) and

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olive (Olea europaea L.).10,11

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lipopolysaccharide induced inflammatory injury through suppressing NF-κB signal pathway.

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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

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Qian et al.14 reported that MA diminished oxygen-glucose

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scavenger and copper chelator.

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deprivation caused injury in cortical neurons. Yin et al.11 indicated that dietary MA intake

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enhanced its bioavailability in mice brain.

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anti-oxidative and anti-inflammatory agent, could protect neuronal cells against brain

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disorders.

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cells against Abeta-induced apoptosis and inflammatory stress.

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RAGE, NADPH oxidase and signaling pathways in Abeta-treated neuronal cells also remains

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unknown.

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agent against Alzhemer’s disease.

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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

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neurons.15

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investigating the protective activity and mechanism of natural compounds upon neuronal

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cells.16,17

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NGF-differentiated PC12 cell line.

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concentrations upon cell viability, MMP, Na+-K+-ATPase activity, ROS and inflammatory

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cytokines were examined.

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oxidase, NF-κB, MAPK and RAGE was evaluated.

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MATERIALS AND METHODS

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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.

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(Milwaukee, WI, USA) and Promega Chem. Co. (Madison, WI, USA), respectively.

Plates,

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medium, antibiotics and chemicals for cell culture were bought from Detroit Difco Laboratory

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(MI, USA).

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Sodium phosphate buffer (PBS, pH 7.2) at 10 mM was used to dilute this peptide to

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appropriate concentration for following experiments.

Abeta1-42 was obtained from Sigma Chemical Co. (St. Louis, MO, USA).

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Cell Culture. PC12 cells were incubated in 35-mm plates which contained Dulbecco’s

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modified Eagle’s medium (DMEM) supplied with 5% fetal bovine serum, 10%

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heat-inactivated calf serum, 100 units/mL of streptomycin and 100 units/mL of penicillin at

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37°C under 95% air and 5% CO2. Cells were further treated by 50 ng/mL NGF for 5 days to

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differentiate.

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days.

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serum-free DMEM one day before experiments, and followed by replanting in plates with 96

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wells.

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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

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further diluted by medium.

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this concentration, 0.5%, did not change PC12 cell viability (shown in supplemental figure)

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and other measurements (data not shown).

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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%

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incorporation.

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concentrations were analyzed by HPLC method form Lin et al.18

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considered as incorporated.

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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)

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was used to examine cell viability.

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Milwaukee (WI, USA).

Control or MA-treated PC12 cells were reacted with MTT, 0.25

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mg/mL at 37°C for 3 hr.

Then, MTT formazan product was quantified by using a microplate

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reader (Bio-Rad, Hercules, CA, USA) to measure the absorbance at 570 nm and 630 nm.

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Cell viability was expressed as a percentage of control without addition of MA, and without

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Abeta treatment.

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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.

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Cell culture


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supernatant at 50 µL was collected.

The activity (U/L) of LDH was determined by a

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commercial assay kit purchased from Sigma Chemical Co. (St. Louis, MO, USA).

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coefficient of variation (CV) of intra-assay and the inter-assay was 5.9% and 5.1%,

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respectively.

The

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Measurement of MMP. A fluorescent dye Rhodamine123 (Rh123) bought from Sigma

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Chemical Co. (St. Louis, MO, USA) and a Beckman-FC500 flow cytometry (Beckman

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Coulter, Fullerton, CA, USA) were used to monitor MMP.

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into to cells and followed by incubating at 37°C for 45 min. Cells were collected and further

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washed twice by PBS (pH 7.2).

Rh123 at 100 µg/L was added

Mean fluorescence intensity (MFI) of cells was measured.

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Mitochondrial Fraction Preparation. Cells were lysed by cold lysis buffer containing

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0.1 mM EDTA, 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 0.6%

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Nonidet P-40 and 1 mM PMSF for 30 min.

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xg to collect nuclei pellet.

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for 20 min to collect mitochondria pellet.

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Mitochondrial protein concentration was quantified by an assay kit purchased from Pierce

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Biotechnology Inc. (Rockford, IL, USA), in which bovine serum albumin was a standard.

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Sample was diluted by PBS to 1 mg protein/mL for following use.

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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

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determining inorganic phosphate (Pi) level released from ATP.19

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contained freshly isolated mitochondria, 2 mM ATP, 20 mM KCl, 100 mM NaCl and 30 mM

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Tris-HCl buffer at pH 7.4.

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was added to terminate assay after incubation at 37°C for 15 min.

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measured by monitoring absorbance at 640 nm. Absorbance values of MA treated cells were

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shown as a percentage of controls.

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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

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assay kits obtained from Upstate Co. (Lake Placid, NY, USA).

The intra-assay CV and the

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inter-assay CV were 3.6-4.7% and 5.2-6.3%, respectively.

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lysis buffer and further incubated in ice for ten min.

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with 5 mL reaction buffer and 2.5 mL of caspase-3 or -8 fluorogenic substrates in microplate.

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After 1 hr incubation at 37°C, caspase-3 or -8 activity was detected by a fluorophotometer at

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excitation and emission at 400 and 505 nm, respectively.

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of controls.

Briefly, cells were lysed in cold

Cell lysates at 50 µL was combined

Data were shown as a percentage

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Measurement of Glutathione (GSH), ROS and Cytokines. Cells were homogenized

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by 2 mL PBS in a Teflon glass motordriven homogenizer obtained from Glas-Col Co. (CA,

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USA).

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from

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2',7'-dichlorofluorescein diacetate was used to determine ROS level.

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50 µM dye and incubated for 30 min.

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microplate reader with excitation at 485 nm and emission at 530 nm.

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fluorescence values between time 0 and 5 min was defined as relative fluorescence unit

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(RFU).

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in supernatant were analyzed by cytoscreen immunoassay kits bought from BioSource Intl.

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(Camarillo, CA, USA).

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and for TNF-alpha was 10 pg/mL.

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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

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was measured.

Forty µg protein samples were used for 10% SDS-polyacrylamide gel

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electrophoresis; then, further transferred to nitrocellulose membranes purchased from

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Millipore (Bedford, MA, USA) for 60 min.

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60 min, membranes were followed by reacting with monoclonal antibodies against Bcl-2, Bax,

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caspase-3, caspase-8, p47phox, gp91phox (1:1000), NF-κB, RAGE and MAPK (1:2000)

After blocking with 5% skim milk solution for

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overnight at 4ºC; then, reacted with horseradish peroxidase conjugated antibody at room

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temperature for 3.5 hr.

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(GAPDH).

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blots were normalized against GAPDH, and results were expressed as arbitrary units (AU).

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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

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effect of each treatment.

Data were expressed as means ± standard deviation (SD), and used

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for analysis of variance.

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differences among means with significance defined at P0.05, data not shown).

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The influence of MA at 2, 4, 8 or 16 µM upon MTT assay for cell viability and LDH activity

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for plasma membrane integration is presented in Figure 1.

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MA did not affect either cell viability or plasma membrane integration (P>0.05).

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treatment lowered cell viability and enhanced LDH release (P

Maslinic Acid Protected PC12 Cells Differentiated by Nerve Growth

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