Neuroprotective Effect of Alpha-Linolenic Acid against AÎ²-Mediated

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Bioactive Constituents, Metabolites, and Functions

Neuroprotective effect of alpha-linolenic acid against A#-mediated inflammatory responses in C6 glial cell Ah Young Lee, Myoung Hee Lee, Sanghyun Lee, and Eun Ju Cho J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00836 • Publication Date (Web): 18 Apr 2018 Downloaded from http://pubs.acs.org on April 18, 2018

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Journal of Agricultural and Food Chemistry

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Neuroprotective effect of alpha-linolenic acid against Aβ-mediated

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inflammatory responses in C6 glial cell

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Ah Young Leea, Myoung Hee Leeb, Sanghyun Leec, Eun Ju Choa,*

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a

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University, Busan 46241, Republic of Korea.

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b

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Development Administration, Gyeongnam 50424, Republic of Korea.

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c

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of Korea.

Department of Food Science and Nutrition & Kimchi Research Institute, Pusan National

Department of Southern Area Crop Science, National Institute of Crop Science, Rural

Department of Integrative Plant Science, Chung-Ang University, Gyeonggi 17546, Republic

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Corresponding author: Eun Ju Cho, Department of Food Science and Nutrition, Pusan

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National University, Busan 46241, Republic of Korea. Tel: +82-51-510-2837, Fax: +82-51-

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583-3648. E-mail address: [email protected] (E. J. Cho).

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ACS Paragon Plus Environment


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ABSTRACT

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Therapeutic approaches for neurodegeneration, such as Alzheimer’s disease (AD), have been

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widely studied. One of the critical hallmarks of AD is accumulation of amyloid beta (Aβ). Aβ

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induces neurotoxicity and releases inflammatory mediators or cytokines through activation of

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glial cell, and these pathological features are observed in AD patient’s brain. The purpose of

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this study is to investigate the protective effect of alpha-linolenic acid (ALA) on Aβ25-35-

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induced neurotoxicity in C6 glial cells. Exposure of C6 glial cells to 50 µM Aβ25-35 caused

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cell death, over-production of nitric oxide (NO), and pro-inflammatory cytokines release

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[interleukin (IL)-6 and tumor necrosis factor-α], while treatment of ALA increased cell

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viability and markedly attenuated Aβ25-35-induced excessive production of NO and those

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inflammatory cytokines. Inhibitory effect of ALA on generation of NO and cytokines was

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mediated by down-regulation of inducible nitric oxide synthase and cyclooxygenase-2 protein

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and mRNA expressions. In addition, ALA treatment inhibited reactive oxygen species

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generation induced by Aβ25-35 through the enhancement of the nuclear factor-erythroid 2-

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related factor-2 (Nrf-2) protein levels and subsequent induction of heme-oxygenase-1 (HO-1)

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expression in C6 glial cells dose- and time-dependently. Furthermore, the levels of neprilysin

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and insulin-degrading enzyme protein expressions, which contribute to degradation of Aβ,

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were also increased by treatment of ALA compared to Aβ25-35–treated control group. In

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conclusion, effects of ALA on Aβ degradation were shown to be mediated trough inhibition

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of inflammatory responses and activation of antioxidative system, Nrf-2/HO-1 signaling

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pathway, in C6 glial cells. Our findings suggest that ALA might have the potential for

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therapeutics of AD.

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KEY WORDS: alpha-linolenic acid, neuroinflammation, glial cell, amyloid beta, amyloid 2


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beta degradation

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INTRODUCTION

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Alzheimer’s disease (AD) is the most common and multifactorial neurodegenerative

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diseases. One of the pathological features of AD is the deposition of amyloid beta (Aβ), a

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protein involved in oxidative stress, neuronal apoptosis and inflammatory responses. Many

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previous studies have shown that the accumulation of Aβ is accompanied by high levels of

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pro-inflammatory cytokines produced by both glial cells and neurons.1-4 Excessive

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production of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor

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necrosis factor-α (TNF-α) contributes to amyloidogenic processing of the amyloid precursor

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protein (APP). In addition, increased expression of these cytokines was found to impair Aβ

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clearance and degradation in glial cells.5,6

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Glial cells support and protect neurons from oxidative damage via modulation of synaptic

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plasticity and secretion of growth factors.7-8 Several enzymatic systems have been described

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to mediate the process of Aβ degradation in glial cells. Both neprilysin (NEP) and insulin

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degrading enzyme (IDE) are critical Aβ-degrading enzymes that reduce Aβ accumulation in

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AD brains.9-12 Moreover, glial cell dysfunction was shown to induce inhibition of Aβ

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clearance, subsequent neurotoxicity, and Aβ accumulation.13 In addition, exogenous Aβ could

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activate glial cell to generate reactive oxygen species (ROS) as well as reactive nitrogen

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species (RNS), which promote secretion of inflammatory cytokines and inflammatory

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process, thus contributing to AD progression.14-16 Therefore, suppression of inflammatory

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process in glial cells has been proposed as a therapeutic strategy for neurodegenerative

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disease.

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A number of studies demonstrated the role of omega-3 polyunsaturated fatty acids in brain

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development and function.17,18 However, most of these focused on neuroprotective effects of 4


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eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). Recently, plant-derived

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omega-3 fatty acid, rather than fish-derived products containing EPA and DHA, has attracted

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interest due to its benefits for human health.19 Alpha-linolenic acid (ALA), enriched in

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vegetable oils such as perilla oil (containing 60-70% ALA) or linseed oil (containing 30-50%

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ALA), has been shown to enhance neurological function including neurogenesis and synaptic

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function in the rodent brain.20 We previously suggested that administration of perilla oil and

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ALA improved cognitive impairment induced by Aβ25-35 in a mouse model of AD.21,22 Perilla

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oil, which has the largest proportion of ALA, suppressed the expression of inflammatory

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mediators, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in Aβ25-35-

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injected brain tissues. Furthermore, ALA supplementation significantly enhanced the

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expression of Aβ degrading enzyme, IDE. Other reports also supported that dietary ALA

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modulates oxidative or inflammatory response in vitro and in vivo.23-25 However, no studies

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have reported the neuroprotective potential of ALA in modulating glial cell function against

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Aβ. In the present study, we assessed the anti-inflammatory properties of ALA in Aβ25-35-

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treated C6 glial cell, and investigated the molecular mechanisms underlying regulation of

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inflammatory response and Aβ degradation.

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MATERIALS AND METHODS

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Sample preparation

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Perilla frutescens oil was provided by Southern Area Crop Science, Rural Development

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Administration (Miryang, Republic of Korea). ALA was obtained from perilla oil after

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treatment with urea and cooling by high-yield methods as described in a previous study.26

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ALA was freshly prepared as a stock solution in dimethyl sulfoxide (DMSO), and stored at

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4˚C with nitrogen gas for stability before use. DMSO is used for dissolving hydrophobic

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compounds including omega-3 fatty acids in vitro or cellular system.27,28 Nitrogen gas is the

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generally used to protect oil or fatty acid from air contact, and is able to avoid the

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contaminant. At low temperatures under nitrogen atmosphere, progression of lipid

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peroxidation is relatively low. 29-31

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Instruments and reagents Fetal bovine serum (FBS), Dulbecco’s modified eagle’s medium (DMEM), and

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penicillin/streptomycin

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dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dichlorofluorescin diacetate

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(DCF-DA) and DMSO were supplied by Sigma Chemical Co. (St Louis, MO, USA).

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Radioimmunoprecipitation assay (RIPA) buffer, 30% acrylamide bis solution and pre-stained

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protein size marker were obtained from Elpis Biotech (Daejeon, Korea). Polyvinylidene

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fluoride (PVDF) membrane was from Millipore (Billerica, MA, USA). Primary antibodies

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and secondary antibodies were purchased from Cell Signaling (Beverly, MA, USA).

were

supplied

by Welgene

(Daegu, Korea).

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Cell culture 6


The

3-(4,5-

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C6 glial cells were obtained from KCLB (Korean Cell Line Bank, Seoul, Korea). The cells

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were maintained in DMEM containing 1% penicillin/streptomycin and 10% FBS at 37°C in a

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5% CO2 incubator with. Cells were sub-cultured with 0.05% trypsin-EDTA in phosphate

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buffered saline (PBS).

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Cell viability

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After becoming confluent, the cells were harvested and plated at a density of 5 × 104 cells/mL

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into 96-well palate overnight. The cells were then treated with ALA (1, 2.5, 5, and 25 µg/mL)

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for 2 h, and followed by Aβ25-35 (50 µM) for 24 h. After incubation, cell viability was

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determined using the MTT assay. MTT solution was added to each 96-well plate, the plate

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was incubated for 4 h at 37°C, and the medium containing MTT was removed. The

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incorporated formazan crystals incorporated in viable cells were solubilized with DMSO and

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the absorbance of each well was read at 540 nm.

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Measurement of nitric oxide (NO) production

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NO production was investigated by quantifying nitrite accumulation in the medium treated

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with Griess reagent. To measure nitrites, cell supernatants were mixed with Griess reagent at

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1:1 volume ratio. Nitrite concentration was measured using microplate spectrophotometer at a

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wave length of 540 nm.

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Measurement of ROS production 7



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The ROS scavenging activity of ALA was measured using DCFH-DA.32 C6 glial cells were

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plated at a density of 5 × 104 cells/mL into 96-well plate overnight. The treatment with ALA

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(5 and 25 µg/mL) was performed for 2 h, after which Aβ25-35 (50 µM) was added, and the

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cells were further incubated for 24 h. The cells were then incubated with 80 µM DCFH-DA at

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37°C for 30 min. The fluorescence was read for 60 min, at 480 nm excitation and 535 nm

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emission, using a fluorescence plate reader (BMG LABTECH, Ortenberg, Germany).

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Measurement of pro-inflammatory cytokines production

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The supernatants of C6 glial cells treated with Aβ25-35 (50 µM) and ALA (5 and 25 µg/mL)

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were transferred to a separate plate. The production of TNF-α and IL-6 were was measured

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using ELISA kits (R&D system, Minneapolis, USA) according to manufacturer’s instructions.

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Reverse transcription polymerase chain reaction (RT-PCR)

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Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA) according to

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manufacturer’s instructions. Cells were lysed and transferred to microfuge tubes. RNA was

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reverse-transcribed into cDNA and used as a template for RT-PCR amplification. The primers

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and amplification conditions are listed in Table 1. PCR products were analyzed on 1%

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agarose gels, and bands were visualized using LED slider imager (Maestrogen, Las Vegas,

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NV, USA).

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Western blotting 8


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C6 glial cell extracts were prepared according to the manufacturer’s instructions using RIPA

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buffer supplemented with 1× protease inhibitor cocktail. Proteins were separated by

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electrophoresis in a precast 10-13% SDS-PAGE and blotted onto PVDF membranes. The

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membrane was blocked with 5% skim milk solution for 1 h at room temperature, and

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incubated overnight at 4°C with primary antibody [iNOS (1:200, Santa Cruz, CA, USA);

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COX-2 (1:200, Santa Cruz); IL-6 (1:1000, Santa Cruz); TNF-α (1:000, Cell Signaling);

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nuclear factor-erythroid 2-related factor-2 (Nrf-2, 1:1000, Cell Signaling, MA, USA); heme-

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oxygenase-1 (HO-1, 1:2000, Cell Signaling); IDE (1:200, Santa Cruz, CA, USA); NEP

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(1:1000, Millipore); and β-actin (1:200, Santa Cruz)]. The membrane was washed and

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incubated with the appropriate HRP-conjugated secondary antibodies. Western bands were

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visualized using a chemiluminescent imaging system (Davinci Chemi, Seoul, Korea).

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Statistical analysis

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The results are expressed as mean ± SD. Statistical significance was determined by one way

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ANOVA followed by post-hoc analysis with Tukey’s test using IBM SPSS 23 program. The

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significance was set at #P < 0.05,

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from the normal group; *significantly different from the Aβ25-35-treated control group.

##

P < 0.01, *P < 0.05, **P < 0.01. #Significantly different

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RESULTS

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Effect of ALA on cell viability in Aβ25-35-treated C6 glial cells

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The treatment with ALA at concentrations between 1 and 25 µg/mL did not show cytotoxicity

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in C6 glial cells (data not shown). C6 glial cells were treated with ALA, and the effect on

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Aβ25-35–induced cytotoxicity was determined. Compared to the normal group (100%), the 9



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exposure of cells to Aβ25-35 for 24 h induced toxicity in C6 glial cells, reducing the cell

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viability to 39.46% (Fig. 1). However, in the presence of ALA (1, 2.5, 5, and 25 µg/mL), cell

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viability was significantly increased as compared to the cells treated with Aβ25-35 alone. These

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results suggest that ALA protects C6 glial cell against Aβ25-35-induced cellular damage.

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Effect of ALA on NO production in Aβ25-35-treated C6 glial cell

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To investigate the effect of ALA on Aβ25-35-induced NO production in C6 glial cells, the cells

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were treated with various concentrations of ALA (1, 2.5, 5, and 25 µg/mL), after which Aβ

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was added for 24 h. Fig. 2 indicates significantly increased NO production by Aβ25-35

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compared to normal group. By contrast, the treatment with ALA, especially at 25 µg/mL,

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effectively attenuated Aβ25-35-induced NO production.

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Effect of ALA on protein and mRNA expression of iNOS and COX-2 in Aβ25-35-treated C6

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glial cells

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To clarify the potential effects of ALA on inflammatory mediators, iNOS and COX-2, we

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examined their protein and mRNA levels using Western blot and RT-PCR, respectively. As

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shown in Fig. 3A, the protein levels of iNOS and COX-2 were markedly increased after Aβ25-

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35 treatment.

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COX-2 protein levels, as compared to control group.

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In parallel with the inhibitory effect of ALA on iNOS and COX-2 protein over-expression, we

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further analyzed the mRNA expression. We observed that iNOS and COX-2 mRNA levels

However, the treatment with ALA leads to a significant suppression of iNOS and

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were also up-regulated following Aβ25-35 treatment, while the treatment with ALA leads to

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inhibition of iNOS and COX-2 mRNA expression (Fig. 3B). These results demonstrate the

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inhibitory effect of ALA on Aβ25-35 treatment-induced NO production associated with the

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down-regulation of iNOS and COX-2 protein and mRNA expression in C6 glial cells.

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Effect of ALA on pro-inflammatory cytokines production in Aβ25-35-treated C6 glial cells

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Fig. 4 illustrates the effect of ALA on production of TNF-α and IL-6 pro-inflammatory

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cytokines induced by Aβ25-35. The treatment with Aβ25-35 induced over-production of TNF-α

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and IL-6 in C6 glial cells, as compared to non-treated group (Fig. 4A). However, ALA

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treatment significantly decreased the over-production of both TNF-α and IL-6. In addition,

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Aβ25-35 induced up-regulation of TNF-α and IL-6 protein expression, which was markedly

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down-regulated by the treatment with ALA (Fig. 4B). These results demonstrate that

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increased production of pro-inflammatory such as TNF-α and IL-6 in C6 glial cells induced

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by Aβ25-35 was related to the inhibition of iNOS and COX-2 protein/gene expression, and that

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ALA inhibited these inflammatory responses by regulating the expression of pro-

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inflammatory cytokines and mediators.

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Effect of ALA on ROS production in Aβ25-35-treated C6 glial cells

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We examined whether ALA attenuates ROS production induced by Aβ25-35 in C6 glial cells.

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Fig. 5A shows that the treatment of C6 glial cells with Aβ25-35 significantly increased ROS

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levels in a time-dependent manner, as compared to un-treated group. Compared with Aβ25-3511



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treated control group (100%), the treatment with ALA (5 and 25 µg/mL) significantly

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inhibited the excess in ROS production, by decreasing it to 86.12% and 85.22%, respectively

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(Fig. 5B).

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Effect of ALA on Nrf-2/HO-1 signaling pathway in C6 glial cell

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Given the inhibitory effect of ALA treatment on inflammatory reaction and ROS production,

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we evaluated whether ALA regulates Nrf-2/HO-1 signaling in C6 glial cells by Western blot

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analysis. Our results indicate that ALA promotes the up-regulation of Nrf2 protein expression,

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and consequently, the increase in the expression of HO-1 protein a downstream target of Nrf-

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2, in a dose- (Fig. 6A) and time-dependent manner (Fig. 6B). These results reveal that ALA-

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dependent activation of Nrf-2/HO-1 signaling activation contributes to down-regulation of

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neuroinflammation and ROS generation.

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Effect of ALA on Aβ degradation in Aβ25-35-treated C6 glial cells

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The effect of ALA on NEP and IDE expression in C6 glial cells was also investigated.

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Western blot analysis showed that the treatment of cells with ALA significantly increased

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NEP and IDE expression dose-dependently (Fig. 7). The treatment with Aβ25-35 significantly

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reduced the levels of NEP and IDE. However, the protein levels of Aβ-degrading enzymes

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NEP and IDE were significantly increased following the treatment with ALA. Thus our

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results suggest that ALA may inhibit the accumulation of Aβ in the brain.

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DISCUSSION

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Neuroinflammation is one of the critical characteristics in the brain of AD patients.33 It has

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been reported that the initial defense against Aβ accumulation is involved in inflammatory

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responses including enzymatic degradation. Activated glial cells produce pro-inflammatory

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mediators and cytokines, resulting in promote the synthesis and APP processing. Increased

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Aβ production and deposition lead to further glial cell activation, which maintains the vicious

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cycle.34,35 The cytokines induced by Aβ in turn reduce the expression of Aβ-degrading

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enzymes, which drive the progression of AD.36,37 Since inflammatory responses have been

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implicated in neuropathological changes and the development of AD, it is conceivable that

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stimulating anti-inflammatory activity in glial cells may represent a therapeutic strategy for

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AD.

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Our study showed that the treatment of C6 glial cells with Aβ25-35 induced toxicity,

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resulting into cell death. However, ALA significantly reversed the decrease in cell viability as

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confirmed by MTT assays. Although RNS and ROS play a critical role in neurotransmission

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and synaptic function in the brain, the over-production of those are associated with

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neuroinflammation, which result in neurodegeneration.38 High levels of NO and pro-

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inflammatory cytokines in activated glial cells can be considered as a pathophysiological

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feature of AD.39 Our data revealed that Aβ25-35 elevated the levels of NO production and pro-

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inflammatory cytokines such as TNF-α and IL-6. These findings are consistent with previous

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studies showing activation of inflammatory mediators and cytokines expression by Aβ in

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glial cell lines.40,41 However, ALA prominently inhibited the production of NO, TNF-α, and

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IL-6 cytokines as well as decreased protein levels of TNF-α and IL-6 in Aβ25-35-stimulated C6

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glial cells as shown by western blotting. It has been reported that neuroprotection against Aβ 13



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can result from the inhibition of pro-inflammatory mediators through down-regulation of

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iNOS or COX-2 protein.42,43 In this study, the exposure to Aβ25-35 stimuli up-regulated iNOS

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and COX-2 protein and mRNA expression, while the ALA reduced both protein and mRNA

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levels of iNOS and COX-2. These findings suggest that the inhibition of NO and pro-

264

inflammatory cytokines production by ALA was mediated by the effect on iNOS and COX-2

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expression. Based on our results, we suggest that ALA attenuates Aβ25-35-induced glial

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activation and down-regulates inflammatory neurotoxicity.

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ROS accumulation is strongly associated with glial inflammatory responses in

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neurodegenerative disease. Increased ROS production plays an important role in the

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activation of iNOS and COX-2 by inflammatory stimuli such as Aβ.44 The inhibition of ROS

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may be an effective way to protect the cells from Aβ-induced neuroinflammation. We

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examined the intracellular ROS formation induced by Aβ25-35 in C6 glial cells. Our findings

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reveal that ALA significantly suppresses the over-production of ROS induced by Aβ25-35. A

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potential mechanism underlying the inhibition of pro-inflammatory cytokines was previously

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reported that oxidative stress and inflammation were attenuated by ALA-enriched diet

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through inactivation of nuclear factor-kappa B signaling pathway.45 Ambrozova et al. (2010)

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also demonstrated that omega-3 fatty acids such as ALA inhibited ROS and RNS generation

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in macrophage cells.46

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The up-regulation of Nrf-2 and HO-1 expression is an effective strategy to resist the

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oxidative stress. Nrf-2 has been considered as a regulator of inflammation and

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neuroprotection.47,48 The expression of Nrf-2 can decrease Aβ-induced degeneration by

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limiting cellular oxidative response and inflammatory damage.49 Activated Nrf-2 can induce

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the expression of HO-1, its downstream antioxidant target. Substantial evidence also suggests 14


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that up-regulation of HO-1 expression contributes to protection against cellular oxidative

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damage and inflammation by inhibiting iNOS activity.50,51 To investigate the molecular

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mechanism underlying the effect of ALA on inhibition of ROS generation and inflammation,

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we examined the protein levels of Nrf-2 and HO-1 in C6 glial cells. Our data shows that the

287

treatment with ALA increased Nrf-2 activation and induced HO-1 protein expression in a

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concentration- and time-dependent manner. Similar to our results, DHA treatment attenuated

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neuronal injury by activating Nrf-2 and up-regulating HO-1 expression, demonstrating

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neuroprotective effect.52 Pal and Ghosh (2012) demonstrated that ALA exerts antioxidant

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effects by reducing lipid peroxidation and modulating antioxidant enzymes such as

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superoxide dismutase, glutathione peroxidase and catalase.53 These results suggest that ALA

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protects against Aβ-induced oxidative stress and neuroinflammation through induction of

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Nrf-2/HO-1 signaling.

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We previously showed that ALA has anti-oxidant activity as a scavenger of hydroxyl

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radical, demonstrating that ALA is capable of inhibiting ROS production and lipid

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peroxidation.54 Several reports have also shown that dietary omega-3 fatty acids are able to

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modify the oxidative status by attenuating oxidative stress in vitro and in vivo.55-57

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Polyunsaturated fatty acid (PUFA) containing two or more double bonds can react with

300

oxygen, resulting in formation of hydroperoxides. This oxidation is progressed by free radical

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chain reaction through initiation, propagation, and termination at the bis-allylic positions.

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However, lipids are complex, multi-component, and heterogeneous system. Miyashita (2014)

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demonstrated that omega-3 fatty acids including ALA in micelles and liposomes can form

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tight and unique conformation, and reduce hydroxyl compounds derived from oxidation

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products, thus omega-3 fatty acids may protect against oxidative attack.58 In addition, the 15



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omage-3 fatty acids can be incorporated into membrane phospholipids or bound to membrane

307

receptors, acting as inhibitor of ROS production and regulator of biological processes.59

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Although further studies are needed to investigate the detailed physiological effects and

309

molecular mechanisms, these evidences support the antioxidant role of ALA in biological

310

system.

311

Glial-mediated oxidative stress and neuroinflammation are closely associated with the

312

degradation of Aβ in AD progression.60,61 The mechanisms underlying degradation of Aβ

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have attracted attention given their potential therapeutic application in the treatments of AD.

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This particularly applies to the roles of IDE and NEP, major Aβ-degrading enzymes in glial

315

cells.62-64 Since oxidative stress and neuroinflammation were attenuated in ALA-treated glial

316

cells, we investigated whether the activities of Aβ degrading enzymes are modified by the

317

treatment with ALA. In the present study, we found that treatment with Aβ25-35 resulted in a

318

significant decrease in the expression of IDE and NEP. However, ALA treatment significantly

319

increased protein expression levels of IDE and NEP, which are capable of degrading Aβ.

320

These effects of ALA on Aβ degradation may be affected by the inhibition of oxidative stress,

321

with a subsequent reduction of neuroinflammation induced by Aβ.

322

A number of studies demonstrated that omega-3 fatty acid consumption is associated with

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lower risk of AD and slower rate of cognitive decline.65 The protective effects of omega-3

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fatty acids, especially EPA or DHA, on AD have been well established in vitro, in vivo, and

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by several clinical studies.66,67 EPA attenuated glial-produced pro-inflammatory cytokines,

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thus subsequently inhibiting neuroinflammation.68 ALA deficiency induces abnormality of

327

nervous system, including neurons, astrocytes, oligodendrocytes, and endoplasmic

328

reticulum.69 Considering to protective effect of ALA on neurons, previous studies 16


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demonstrated that injection or administration of ALA prevented neuronal cell death with

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down-regulation of Bax protein expression in stroke animal models.70,71 Moreover, ALA

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attenuated glutamate-induced neurotoxicity and promoted neuronal survival in vitro.20,72,73

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We also confirmed that treatment of ALA reduced neuronal cell death against hydrogen

333

peroxide and Aβ by down-regulation of apoptotic signaling pathway.74,75 These results

334

suggest that ALA directly protects neuron as well as glial cells from oxidative stress. Because

335

of limited rate of conversion from ALA to EPA/DHA, the protective effect of ALA on glial

336

cells most likely does not involve bioconversion of ALA to EPA/DHA.

337

Our findings show that ALA increased cell viability and decreased ROS production in

338

Aβ25-35-treated glial cells. ALA also up-regulated the expression of Nrf-2 and HO-1,

339

demonstrated as protective against oxidative stress. The mechanisms underlying anti-

340

inflammatory effect of ALA involve interference with NO production and protein/mRNA

341

expressions of iNOS, COX-2, TNF-α, and IL-6. The induction of antioxidant activity and

342

inhibition of inflammatory cytokine release by the treatment with ALA has a potential to

343

induce degradation of Aβ as shown by promotion of IDE and NEP protein expressions. This

344

study suggests that intake of ALA should be considered as an attractive alternative to that of

345

fish oil in the treatment/prevention of neurodegenerative disorders such as AD.

346

347

ABBREVIATIONS USED

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AD, Alzheimer’s disease; ALA, alpha-linolenic acid; APP, amyloid precursor protein; COX-2,

349

cyclooxygenase-2; DCF-DA, dichlorofluorescin diacetate; DHA, docosahexaenoic acid;

350

DMEM, Dulbecco’s modified eagle’s medium; DMSO, dimethyl sulfoxide; EPA, 17



351

eicosapentaenoic acid; FBS, fetal bovine serum; HO-1, heme-oxygenase-1; IDE, insulin

352

degrading enzyme; IL, interleukin; iNOS, inducible nitric oxide synthase; MTT, 3-(4,5-

353

dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Nrf-2, nuclear factor-erythroid 2-

354

related factor-2; NEP, neprilysin; NO, nitric oxide; RIPA, radioimmunoprecipitation assay;

355

RNS, reactive nitrogen species; ROS, reactive oxygen species; PBS, phosphate buffered

356

saline; PUFA, polyunsaturated fatty acid; PVDF, polyvinylidene fluoride; TNF-alpha, tumor

357

necrosis factor-alpha

358

359

ACKNOWLEDGMENTS

360

This work was carried out with the support of the “Cooperative Research Program for

361

Agriculture Science & Technology Development (PJ01015603),” Rural Development

362

Administration, Republic of Korea.

18


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FIGURE CAPTIONS

574

Figure 1. Effect of ALA on cell viability in Aβ25-35-treated C6 glial cells. Values are mean

575

± SD (n = 6).

576

ALA; Alpha-linolenic acid.

##

P < 0.01 compared to normal group; **P < 0.01 compared to control group.

577

578

Figure 2. Effect of ALA on NO production in Aβ25-35-treated C6 glial cells. Values are

579

mean ± SD (n = 6). ##P < 0.01 compared to normal group;

580

group. ALA; Alpha-linolenic acid.

**

P < 0.01 compared to control

581

582

Figure 3. Effect of the levels of ALA on iNOS and COX-2 protein (A) and mRNA (B)

583

expressions in Aβ25-35-treated C6 glial cells. ALA; Alpha-linolenic acid. Images of band are

584

from a representative experiment and bars represent the mean ± SD (n = 3). #P < 0.05, ##P

Neuroprotective Effect of Alpha-Linolenic Acid against AÎ²-Mediated

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