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Suppression of LPS-induced Neuroinflammation by Morin via MAPK, PI3K/Akt, and PKA/HO-1 Signaling Pathway Modulation Ji-Sun Jung, Min-Ji Choi, Yu Young Lee, Byung-In Moon, Jin-Sun Park, and Hee-Sun Kim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05147 • Publication Date (Web): 29 Dec 2016 Downloaded from http://pubs.acs.org on December 30, 2016

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Journal of Agricultural and Food Chemistry

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Suppression of LPS-induced Neuroinflammation by Morin via MAPK,

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PI3K/Akt, and PKA/HO-1 Signaling Pathway Modulation

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Ji-Sun Jung1,2, Min-Ji Choi1, Yu Young Lee1, Byung-In Moon3, Jin-Sun Park1,

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Hee-Sun Kim1,*

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Womans University Medical School, Seoul, Republic of Korea

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2

Department of Molecular Medicine and Tissue Injury Defense Research Center, Ewha

Division of Functional Food Research, Korea Food Research Institute, Gyeonggi-do,

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Republic of Korea

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3

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Korea

Department of Surgery, Ewha Womans University Medical School, Seoul, Republic of

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Running title: Anti-inflammatory Mechanism of Morin in Microglia

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*Corresponding Author: Hee-Sun Kim, Department of Molecular Medicine, Ewha

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Womans University School of Medicine, 1071, Anyangchen-ro, Yangchun-Gu, Seoul

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07985, South Korea

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Tel: 82-2-2650-5823

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Fax: 82-2-2653-8891

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Email: [email protected]

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ACS Paragon Plus Environment


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Abstract

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Morin is a flavonoid isolated from certain fruits and Chinese herbs and is known to

26

possess various medicinal properties. In this study, we investigated the anti-

27

inflammatory effects of morin on lipopolysaccharide (LPS)-induced microglial

28

activation, both in vitro and in vivo. We found that morin inhibited inducible nitric

29

oxide synthase (iNOS) and proinflammatory cytokines in LPS-stimulated BV2

30

microglial cells. Furthermore, morin suppressed the microglial activation and the

31

cytokine expression in the brains of LPS-stimulated mice. Subsequent mechanistic

32

studies revealed that morin inhibited the action of LPS-activated MAP kinases

33

(MAPK), protein kinase B (Akt) phosphorylation, nuclear factor-κB (NF-κB), and

34

activating protein-1 (AP-1). Further, the phosphorylation and the DNA binding activity

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of cAMP responsive element binding protein (CREB) was enhanced by morin.

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Moreover, morin suppressed the LPS-induced expression of nicotinamide adenine

37

dinucleotide phosphate (NADPH) oxidase subunits while it increased heme

38

oxygenase-1 (HO-1) expression and nuclear factor-E2-related factor-2 (Nrf-2)

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activation. Therefore, our data suggest that morin exerts anti-inflammatory effects in

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LPS-stimulated microglia by downregulating MAPK and PI3K/Akt signaling pathways

41

while upregulating PKA/CREB and Nrf2/HO-1 signaling pathways.

42 43

Keywords: Morin, Microglia, Neuroinflammation, Anti-inflammation, Molecular

44

mechanisms

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Introduction

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Microglia are immune cells found in the central nervous system (CNS) that

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perform homeostatic functions such as phagocytosis of apoptotic cells and debris

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throughout the CNS and support neuronal survival during brain development.1,2 There

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are two types of microglial cells, M1 and M2 cells. Activated M1 microglia initiate

51

inflammation processes in the body through the production of pro-inflammatory

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molecules such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), nitric oxide

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(NO), and reactive oxygen species (ROS), which can exacerbate brain injury.3,4

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Activated M2 microglia produce anti-inflammatory cytokines that reduce inflammation

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and repair tissue.5-7 Therefore, maintenance of the balance between the inflammatory

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M1 cells and anti-inflammatory M2 cells is crucial for homeostasis in the brain.

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However, prolonged or excessive microglial activation induces neuroinflammation,

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resulting in a homeostatic imbalance, that can cause neurodegenerative disorders.5

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Therefore, the inhibition of excessive microglial activation has been suggested as

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potential therapy for brain injury and various neurodegenerative diseases such as

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Parkinson’s disease (PD) and Alzheimer’s disease (AD).

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Morin (2′,3,4′,5,7-pentahydroxyflavone) is a well-known flavonoid that is

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naturally found in various fruits (for e.g., osage orange), almonds, red wines, and many

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Chinese medicinal herbs.8,9 Numerous studies have recently reported antioxidant, anti-

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apoptotic, and anti-inflammatory properties of morin. It has been found to be effective

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against lung injury by the suppression of the lung NLRP3 inflammasome.9 A different

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study reported the anti-inflammatory effect of morin on gastric mucosal damage via

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the modulation of the nuclear factor NF-κB signal transduction pathway in mammary

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epithelial cells.8,10 Further, recent studies have reported the protective and antioxidative

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effects of morin in mice models of cisplatin-induced kidney and acrylamide-induced

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hepatic injuries. 11,12 Morin has been shown to target multiple pathogenic mechanisms

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in AD model mice. It inhibits tau hyperphosphorylation and glial activation, promotes

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amyloid-β (Aβ) degradation, and enhances synaptic protein expression.13,14 In addition,

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morin suppresses autophagic signaling by inhibiting the production of cytokines and

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nitric oxide.15

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Although numerous studies have described the pharmacological effects of morin

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in various disease models and cell types, details of the underlying molecular

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mechanisms of its anti-inflammatory effects in activated microglia have not been

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reported so far. Therefore, we examine the underlying molecular mechanisms of the

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anti-inflammatory and antioxidant effects of morin in LPS-induced BV2 microglial

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cells in the current study. We investigate the anti-inflammatory effects of morin in vivo

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by using a sepsis model induced by the peripheral administration of LPS, based on

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previous

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neurodegeneration caused by systemic LPS.16,17

reports

that

demonstrate

neuroinflammation

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and

progressive

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Materials and Methods

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Chemicals

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Morin hydrate was purchased from Sigma-Aldrich Biotechnology (St. Louis, MO,

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USA). Reagents used for cell cultures were obtained from Gibco BRL (Grand Island,

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NY, USA). LPS (Escherichia coli serotype 055:B5) was purchased from Sigma-

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Aldrich Biotechnology (St. Louis, MO, USA). Antibodies against heme oxygenase-1

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(HO-1), TNF-α, cyclooxygenase 2 (COX-2), nuclear factor erythroid 2-related factor 2

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(Nrf2), and interleukin-6 (IL-6) were purchased from Santa Cruz Biotechnology (Santa

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Cruz, CA, USA). Antibodies against phospho-MAPK, total-MAPK, Akt, and CREB

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were purchased from Cell Signaling Technology (Beverley, MA, USA). Antibodies for

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p47phox (Ser345) were purchased from Assay Biotechnology Company, Inc. (Sunnyvale,

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CA, USA).

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Microglial cell culture and treatment

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Immortalized murine BV2 microglial cells18 were cultivated and maintained at

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37°C in Dulbecco’s modified Eagles medium supplemented with 10% heat-inactivated

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fetal bovine serum, penicillin (10 U/ml), and streptomycin (10 µg/ml). BV2 cells were

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grown to confluence and seeded into plates and incubated for 16-24 h. To examine the

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anti-inflammatory effects of morin, BV2 cells were treated with morin (100-300 µM)

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with or without LPS (0.1 µg/ml). MTT assay was performed to examine the effect of

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morin on BV2 cell viability.19 The cell viability remained unaffected by morin

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concentrations of up to 300 µM (data not shown).

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Measurement of cytokines and nitrite levels

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BV2 cells (1 × 105 cells per well on a 24-well plate) were pre-treated with morin

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(100-300 µM) for 1 h and stimulated with LPS (0.1 µg/ml). The supernatants of the

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cultured microglia were collected after 16 h of LPS stimulation and the concentrations

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of TNF-α, IL-6, and IL-1β were measured using an enzyme-linked immunosorbent

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assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA) as per the manufacturer’s

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instructions. Accumulated nitrite was also measured from the supernatants using the

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Griess Reagent System (Promega, Madison, WI, USA).

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Intracellular reactive oxygen species measurement

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Intracellular accumulation of ROS was measured using 2',7'-dichlorofluorescein

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(H2DCF-DA) and a modified version of a previously reported method.20 BV2

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microglial cells (1 × 105 cells per well in a 24-well plate) were pretreated with morin

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(100-300 µM) for 1 h, stimulated with LPS for 16 h, and stained with 20 µM H2DCF-

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DA in sodium phosphate buffer (PBS) for 1 h at 37°C. DCF fluorescence intensity was

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measured on a fluorescence plate reader (Molecular Devices, Sunnyvale, CA, USA) at

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excitation and emission wavelengths of 485 nm and 535 nm, respectively. For the

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image analysis of ROS production, BV2 cells were placed on coverslips and treated for

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1 h with morin 1 h prior to LPS stimulation. Cells were stained with H2DCF-DA and

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allowed to rest for 1 h, mounted on a clean, glass slide, and analyzed with the help of a

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confocal laser scanning microscope.

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Lipopolysaccharide-induced inflammation and morin administration

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The study involved the use of ICR mice (aged 8-9 weeks) (Orient Co., Ltd., Seoul,

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Korea). All animal experiments were approved by the Institutional Animal Care and

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Use Committee at the Ewha Womans University School of Medicine, Seoul, Republic

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of Korea. Efforts were made to minimize animal suffering, reduce the number of

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animals used in the study, and utilize alternatives to in vivo techniques when possible.

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Systemic

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mg/kg, intraperitoneal (ip) injection) to male ICR mice as described in previous

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studies.21 Morin (200 mg/kg) was dissolved in a vehicle solution (1% dimethyl

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sulfoxide (DMSO) and 0.9% sodium chloride) and administered via an intraperitoneal

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injection daily for 4 days before LPS stimulation. Samples were obtained 3 h after LPS

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stimulation.

inflammation

was

induced

by

LPS

administration

(5

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Immunohistochemistry

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Mice were anesthetized with sodium pentobarbital (120 mg/kg ip) and perfused

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transcardially with normal saline containing heparin (5 U/ml) after a 3-hour period

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involving LPS stimulation, followed by the addition of 4% paraformaldehyde (PFA) in

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0.1 M PBS (pH 7.2). The brains were removed, incubated overnight in fixatives, and

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stored in a 30% sucrose solution. Serial coronal brain sections of the cortex and the

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hippocampus (40 µm thickness, at 600 µm intervals) were collected through a freezing

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sliding microtome (Leica Biosystems Nussloch GmBH, Nussloch, Germany).

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Endogenous peroxidase was quenched with the help of 3% H2O2. Further, any

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nonspecific binding was blocked by incubating the sections in PBS containing 4%

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bovine serum albumin for 60 min at 37°C. Following an overnight incubation at 4°C

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with either the primary antibody for IBA-1 at 1:1000 dilution (Wako, Osaka, Japan) or

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CD11b at 1:500 dilution (Bio-Rad, Hercules, CA, USA), the sections were incubated

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with a biotinylated secondary antibody for 1 h at 37°C and washed with 0.1% Triton

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X-100 in PBS. The sections were incubated with avidin-biotin-HRP complex reagent

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(Vector Laboratories, Burlingame, CA, USA) for 1.5 h followed by washing with PBS,

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followed by the peroxidase reaction using diaminobenzidine tetrahydrochloride (Vector

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Laboratories, Burlingame, CA, USA). The treated sections were mounted with the help

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of a mounting medium (Thermo Fisher Scientific, Pittsburgh, PA, USA) and examined

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under a light microscope (Leica Biosystems Nussloch GmBH, Nussloch, Germany).

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Quantification of IBA-1-positive cells was conducted with the help of the ImageJ

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software (NIH, Bethesda, MD, USA). Three serial coronal brain sections were

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obtained from each mice and 1 mm2 area was quantified for each brain section for the

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lateral cortex, the hippocampus, and the dentate gyrus areas, respectively. Activated

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and resting microglia were counted according to intensity and morphological

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characteristics, as indicated in previous studies. 22,23

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Traditional and real-time reverse transcription polymerase chain reaction

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BV2 cells (7.5 × 105 cells per well on a 6-well plate) were stimulated with LPS in

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with or without morin. For the isolation of total RNA from the brain cortex, brain

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tissue was homogenized using a homogenizer (Thermo Fisher Scientific, Pittsburgh,

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PA, USA) and total RNA was extracted using the TRI reagent (Thermo Fisher

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Scientific, Pittsburgh, PA, USA). For reverse transcription polymerase chain reaction

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(RT-PCR), total RNA (1 µg) was reverse-transcribed in a reaction mixture containing 1

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U RNase inhibitor, 500 ng random primers, 3 mM magnesium chloride (MgCl2), 0.5

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mM deoxyribonucleoside triphosphate (dNTP), 1X reverse transcription (RT) buffer,

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and 10 U reverse transcriptase (Promega, Madison, WI). The synthesized cDNA was

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used as a template for the polymerase chain reaction (PCR) reaction using GoTaq

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polymerase (Promega, Madison, WI, USA) and primers for COX-2, iNOS, IL-1β, IL-6,

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TNF-α, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as described

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previously.21 GAPDH was used as an internal control for normalizing the target gene

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expression. Amplification of NADPH oxidase subunit genes was achieved through RT-

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PCR. The synthesized cDNA was amplified with SYBR Green PCR Master Mix

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(Applied Biosystems, Foster City, CA, USA), and the RT-PCR was performed on an

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ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA,

189

USA). Expression levels of the target genes were normalized against that of GADPH

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using the formula, 2(Ct test

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shown in Table 1.

gene – Ct GAPDH)

. The primers used in the PCR reaction are

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Electrophoretic mobility shift assay

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BV2 cells (7.5 × 105 cells per well on a 6 cm dish) were pretreated for 1 h with

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morin before LPS addition. Cells were harvested after LPS stimulation of the cells for

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1 h. Nuclear extracts from the stimulated microglia were prepared by a method

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described in a previous study.24 The double-stranded DNA oligonucleotides containing

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NF-κB, AP-1, antioxidant response element (ARE), and cAMP response element (CRE)

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consensus sequences (Promega, Madison, WI, USA) were end-labeled using T4

200

polynucleotide kinase (New England Biolabs, Beverly, MA, USA) in the presence of

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[γ-32P] labeled ATP. Nuclear proteins (10 µg) were incubated with a 32P-labeled probe

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on ice for 0.5 h and separated on a 5% acrylamide gel before visualization by

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autoradiography.

204 205

Western blot analysis

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Whole cell protein lysates were prepared in lysis buffer (10 mM Tris, pH 7.4, 30

207

mM NaCl, 1% Triton, 0.1% SDS, 0.1% sodium deoxycholate, and 1 mM EDTA)

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containing the protease inhibitor cocktail. The lysates were centrifuged at 13,200 rpm

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for 15 min at 4oC, following which the supernatant was collected. The protein

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concentration was determined using the Bradford protein assay. Protein samples

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ranging from 80 to 200 µg in weight were separated by SDS-PAGE, transferred to

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nitrocellulose membranes, and incubated with antibodies against the following markers:

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(a) iNOS, TNF-α, IL-1β, IL-6, COX-2 (1:1000); (b) phospho-MAPK, total-MAPK,

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Akt, and CREB (1:1000); and (c) HO-1 and Nrf2; or p-p47phox (1:1000). Following

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thorough washing with Tris-buffered saline and Tween 20 (TBST) (Bio-Rad, Hercules,

216

CA, USA), horseradish peroxidase-conjugated secondary antibody (1:2000 dilution in

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TBST) was applied. The blots developed using an enhanced chemiluminescence

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detection kit (Pierce Biotechnology, Rockford, IL, USA).

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Transient transfection and luciferase assay

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BV2 cells (2 × 105 cells per well on a 12-well plate) were transfected with 1 µg of

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plasmid DNA ([κB]3-luc, AP-1-luc, ARE-luc, and CRE-luc) using the Convoy

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Platinum transfection reagent (ACTGene, Inc., Piscataway, NJ, USA). The effect of

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morin on reporter gene activity was determined by pre-treating with morin prior to

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stimulation with LPS (100 ng/ml) followed by incubation for 6 h prior to cell harvest.

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Statistical analysis

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Unless otherwise stated, all experiments were performed using triplicated samples

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and the experiments were repeated at least thrice. The data are presented as mean and

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the standard error of the mean (SEM) and statistical comparisons between groups were

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performed using one-way analysis of variance (ANOVA), followed by the Newman-

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Keuls multiple comparison test. A p value of less than 0.05 was considered significant.

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Results

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Morin inhibits inducible nitric oxide synthase and pro-inflammatory molecules in

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LPS-stimulated BV2 microglial cells

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The anti-inflammatory effect of morin in microglia was investigated by treating

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BV2 cells with morin for 1 h prior to LPS stimulation, and examining the effects of

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morin on LPS-induced production of NO and cytokines. Pre-treated morin

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significantly suppressed the production of the proinflammatory molecules, NO, TNF-α,

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IL-6, and IL-1β (Figure 1A). Additionally, morin increased the production of the anti-

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inflammatory molecule, IL-10. We further examined the effects of morin on mRNA

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and protein expression of various pro- or anti-inflammatory molecules. RT-PCR

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(Figure 1B and Figure 1C) and Western blot analyses (Figure 1D and Figure 1E)

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showed that morin suppressed the expression of iNOS and the proinflammatory

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molecules, TNF-α, IL-1β, IL-6, and COX-2, while it upregulated the expression of IL-

247

10.

248 249

Morin suppresses lipopolysaccharide-induced microglial activation and pro-

250

inflammatory cytokine expression in brain

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The preventive and accumulative therapeutic effects of morin during

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neuroinflammation were examined by injecting morin daily into mice for 4 days before

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LPS stimulation. This was followed by determining the immunoreactivity of IBA1 (a

254

marker of microglia activation) in the cortex and hippocampus, 3 h after systemic

255

injection of LPS. The number of IBA1-positive cells with thick and densely stained

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processes, an indicator of activated microglia, increased in the cortex and hippocampus

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of LPS-stimulated mice as compared with that observed in control mice (Figure 2A).

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However, pre-treatment with morin (200 mg/kg) significantly reduced the number of

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IBA1-positive microglia in the LPS-inflamed areas of the brain. We quantified the

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reduction of IBA1-positive activated microglia by morin in the cortex and the

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hippocampus (Figure 2B). Staining with the microglial activation marker, CD11b, also

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showed inhibition of microglial activation by morin in septic mice brains

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(supplementary Figure 1). Furthermore, pre-treatment with morin significantly reduced

264

the LPS-induced expression of iNOS, TNF-α, IL-1β, IL-6, and COX-2 in the cortex

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(Figure 2C and Figure 2D).

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Morin inhibits NF-κB- and AP-1-mediated transcription and phosphorylation of

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MAPKs and Akt

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Further investigation of the anti-inflammatory mechanisms of morin involved

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examining the effect of morin on NF-κB and AP-1, known to be key modulators of

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cytokine and iNOS expression during an inflammatory response in the body.25 The

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stimulation of BV2 cells with LPS showed a strong increase in the DNA binding

273

activity of NF-κB and AP-1 (Figure 3A and Figure 3B). While morin did not affect the

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DNA binding activity of NF-κB, it inhibited the DNA binding activity of AP-1.

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However, morin suppressed the transcriptional activity of NF-κB and AP-1 (Figure 3C

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and Figure 3D). Furthermore, as shown in Figure 3E, morin inhibited the LPS-induced

277

phosphorylation of three types of MAPKs and Akt, which are important upstream

278

signaling molecules in inflammatory reactions mediated by activated microglia.

279 280 281

Morin increases the CREB signaling pathway activity The PKA/CREB signaling pathway in microglia contributes to the reduction of

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282

inflammation through anti-inflammatory gene expression regulation. In particular,

283

PKA activation and the subsequent CREB phosphorylation are upstream modulators of

284

HO-1 expression.26,27 Our group has previously reported the involvement of the

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PKA/CREB signaling pathway in reducing inflammation.21,28 The inhibitory effect of

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morin on NF-κB-mediated transcriptional activity, despite not affecting DNA binding

287

activity, might be due to the competitive binding of pCREB with CBP29, which

288

suppresses the interaction between the NF-κB subunit, p65, and the CREB-binding

289

protein (CBP). Therefore, we determined whether morin plays a role in CREB

290

phosphorylation. Our results confirmed that morin did increase the CREB

291

phosphorylation (Figure 4) and increased other CREB activities such as DNA binding,

292

transcriptional action, and nuclear translocation. Overall, the data suggests that the

293

anti-inflammatory or antioxidant mechanisms of morin involve modulation of CREB

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signaling processes.

295 296

Morin suppresses reactive oxygen species production by NADPH oxidase complex

297

modulation and heme oxygenase-1 expression upregulation

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We found that morin significantly suppressed LPS-induced ROS production in

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BV2 cells (Figure 5A and Figure 5B). To further elucidate the mechanisms underlying

300

ROS inhibition by morin, we examined the expression of NADPH oxidase subunits

301

responsible for microglial ROS production.30 RT-PCR analysis revealed that morin

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significantly inhibited the LPS-induced expression of p47phox and gp91phox but did not

303

affect the expression of p67phox and gp22phox (Figure 5C). Morin also inhibited the

304

phosphorylation of p47phox (Figure 5D), responsible for the activation of the NADPH

305

oxidase complex.

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Heme oxygenase-1 plays the role of an anti-inflammatory and antioxidant

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modulator in the microglia.31,32 Western blot and RT-PCR analyses showed that morin

308

upregulated HO-1 expression at the protein and mRNA levels (Figure 6A and Figure

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6B). To investigate the underlying mechanism of HO-1 upregulation by morin, we

310

examined its effect on Nrf2, a key transcription factor in the expression of HO-1 and

311

other phase 2 antioxidant enzymes. Morin significantly increased Nrf2 binding to the

312

antioxidant response element (ARE) (Figure 6C). Moreover, morin increased nuclear

313

translocation of Nrf2 and ARE-driven luciferase activity (Figure 6D and Figure 6E).

314

These data suggest that the upregulation of HO-1 may be partly involved in the anti-

315

inflammatory and the antioxidant mechanisms of morin in activated microglia.

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Discussion

318

The current study demonstrated the anti-inflammatory effects of morin in vitro

319

and in vivo. Morin significantly suppressed the LPS-induced production of NO and

320

proinflammatory cytokines in BV2 cells and mice brains. Further mechanistic studies

321

showed that morin inhibited the LPS-induced phosphorylation of MAPKs or Akt, and

322

downregulated the NF-κB-mediated transcriptional activity via PKA signaling. Morin

323

also suppressed intracellular ROS levels by inhibiting the NADPH oxidase subunits

324

and by upregulating the Nrf2/HO-1 axis. Our results suggest a therapeutic potential for

325

morin in the treatment of neuroinflammatory disorders.

326

Previous studies by other research groups and us have found that the PKA/CREB

327

pathway contributes to the resolution of inflammation and ROS detoxification and that

328

PKA is an upstream modulator of HO-1 expression in the microglia28,33,34 In the

329

current study, morin increased the CREB phosphorylation, which is a downstream

330

regulatory target of PKA. Furthermore, morin increased the DNA binding and

331

transcriptional activities and the nuclear translocation of CREB. Several papers have

332

demonstrated that phosphorylated CREB competes for binding to CBP with NF-

333

κB.25,29 Based on this hypothesis, morin-mediated activation of CREB may augment

334

the inhibition of NF-κB-mediated transcriptional activity. Therefore, the anti-

335

inflammatory and antioxidant activity by morin seem to be a result of the enhancement

336

of the CREB signal.

337

Oxidative stress and neuroinflammation results from an overexpression of ROS

338

through modulation of the NADPH oxidase family of enzymes.30,35 Therefore, we

339

investigated whether morin inhibits ROS production by suppressing the subunits of

340

NADPH oxidase, including p47phox, p67phox, gp91phox, and p22phox. We observed that

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morin suppressed mRNA expression of p47phox and gp91phox and inhibited the

342

phosphorylation of p47phox in LPS-stimulated microglia. Phosphorylation of p47phox

343

leads to the translocation of the p47phox-p67phox complex to the plasma membrane

344

where p47phox interacts with p22phox, and p67phox acts as the NADPH oxidase activator

345

through a direct protein-protein interaction.30 The data suggest that morin inhibits ROS

346

production by suppressing the expression and the phosphorylation of NADPH oxidase

347

subunits in microglia.

348

According to previous studies, morin is a known natural polyphenolic antioxidant.

349

In LPS-stimulated microglia, morin activates HO-1 induction to play an antioxidant

350

role,36,37 and this has been shown in an ischemia model as well.38 It is also known to

351

demonstrate neuroprotective effects in a PD model39 and contributes to the resolution

352

of inflammation in human chondrocytes40 and RAW 264.7 macrophages.41 In this study,

353

morin exerted its antioxidant activity in LPS-stimulated microglia by suppressing ROS

354

production and enhancing the HO-1 expression via Nrf2-ARE signaling. Heme

355

oxygenase-1 plays an antioxidant, anti-apoptotic, and anti-inflammatory role through

356

the modulation of the Nrf2-ARE signaling pathway.42-44 We observed that morin

357

increases the expression of HO-1 by enhancing the DNA binding activity of Nrf2 to the

358

ARE site. In addition, previous studies reported on the activation of HO-1 through

359

PKA signaling and the subsequent phosphorylation of CREB.28,45 Given this

360

perspective, morin exhibits its effects by suppressing proinflammatory and neurotoxic

361

molecules with a concomitant enhancement of anti-inflammatory and antioxidant

362

molecules such as PKA and HO-1.

363

Sepsis, which results from a severe inflammatory response to an infection, is a

364

leading cause of death and involves the dysfunction and failure of organ systems. A

17



365

previous study has reported that pro-inflammatory cytokines and other mediators are

366

important in the pathogenesis of sepsis.46 Furthermore, sepsis has been associated with

367

microglial activation.47 This study used LPS-injected mice because peripheral injection

368

with LPS is a well-known sepsis model and systemic LPS induces microglial

369

activation and progressive neurodegeneration.16,17,48 Morin was found to inhibit the

370

microglial activation and inflammatory mediator expression in LPS-injected mice

371

brains.

372

In conclusion, our study provides evidence that morin has significant potential as

373

a protective agent for LPS-induced microglial activation. This is the first study to

374

report the pharmacological effects of morin in brain microglia and the first to analyze

375

its underlying molecular mechanisms in detail. Therefore, the anti-inflammatory and

376

the antioxidant effects of morin in brain microglia may be useful as a potential

377

therapeutic agent in the treatment of various neuroinflammatory disorders related to

378

inflammation and oxidative stress.

18


Page 18 of 36

Page 19 of 36


379

Abbreviations:

380

AP-1, activator protein-1; ARE, antioxidant response element; CBP, CREB binding

381

protein; CRE, cAMP response element; COX, cyclooxygenase; CREB, cAMP

382

response element binding protein; EMSA, electrophoretic mobility shift assay; HO-1,

383

heme oxygenase-1; IL, interleukin; iNOS, inducible nitric oxide synthase; LPS,

384

lipopolysaccharide; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-

385

κB; Nrf, nuclear factor-E2-related factor; PI3K, phosphatidylinositol 3-kinase; PKA,

386

protein kinase A; ROS, reactive oxygen species; TNF, tumor necrosis factor

387 388

Funding sources

389

This research was supported by the Basic Science Research Program through the

390

National Research Foundation of Korea (NRF) and funded by the Ministry of Science,

391

ICT & Future Planning (Grant No. NRF-2010-0027945 & NRF-2015R1A2A2A0100

392

5226).

19



393

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(45) Park, S. Y.; Bae, Y. S.; Ko, M. J.; Lee, S. J.; Choi, Y. W. Comparison of antiinflammatory potential of four different dibenzocyclo-octadiene lignans in microglia; action via activation of PKA and Nrf-2 signaling and inhibition of MAPK/STAT/NF-

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574

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(47) Lemstra, A. W.; Woud, J. C.; Hoozemans, J. J.; van Haastert, E. S.; Rozemuller, A. J.; Eikelenboom, P. Microglia activation in sepsis: a case–control study. J.

578

Neuroinflammation 2007, 4, 4.

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(48) Hoogland, I. C. M.; Houbolt, C.; van Westerloo, D. J.; van Gool, W. A.; van de Beek, D. Systemic inflammation and microglial activation: systematic review of

582

animal experiements. J. Neuroinflmmation 2015, 12, 114.

25



583

Figure Legends

584

Figure 1. Inhibition of the expression of iNOS and proinflammatory molecules by

585

morin in LPS-stimulated microglia. (A) BV2 cells are pre-treated with morin for 1 h

586

followed by LPS stimulation (0.1 µg/ml) for 16 h. The supernatants are obtained and

587

the amount of NO, TNF-α, IL-1β, IL-6, and IL-10 released into the media is

588

determined. (B, D) BV2 cells are pretreated with morin for 1 h followed by LPS

589

stimulation for 6 h. RT-PCR (B) and Western blot (D) are performed to see the effects

590

of morin on mRNA and protein expression of various proinflammatory molecules. (C,

591

E) Quantification analysis of 3 independent experiments by RT-PCR (C) and Western

592

blot (E) are shown. Data are expressed as the means (± SEM). *p < 0.05, significantly

593

different from LPS-stimulated samples.

594 595

Figure 2. Reduction of inflammatory response in the brains of LPS-stimulated mice by

596

morin. (A) Morin (200 mg/kg) pre-treatment inhibits microglial activation in each

597

brain region 3 h after systemic LPS injection (5 mg/kg, ip). The images represent

598

IBA1-positive microglia in the cortex and the hippocampus. (B) Quantified data of

599

IBA1-positive microglia in the cortex and the hippocampus (Scale, 50 µm; n=6 per

600

group). (C) RT-PCR for mRNA expression levels of proinflammatory molecules in the

601

cortex. Representative gels are shown. (D) Quantified data (n = 3 per group). #p < 0.05,

602

significantly different from the control group; *p < 0.05, significantly different from

603

the LPS-stimulated group.

604 605

Figure 3. Effect of morin on the activity of NF-κB, AP-1 and the phosphorylation of

606

MAPKs and Akt in LPS-stimulated BV2 cells. EMSA for NF-κB (A) and AP-1 (B)

26


Page 26 of 36

Page 27 of 36


607

was performed using nuclear extracts isolated from the BV2 cells treated with morin in

608

the presence of LPS for 1 h. Transient transfection analysis of (C) (κB)3-luc and (D)

609

AP-1-luc reporter gene activity. (E) Cell extracts were prepared from BV2 cells

610

stimulated with LPS for 0.5 h with or without morin, followed by Western blotting

611

using antibodies against phospho-MAPK, total-MAPK, or Akt. (F) Quantified data.

612

Levels of the phosphorylated forms of MAPKs and Akt were normalized to the level of

613

each total form and expressed as relative fold changes versus the control group. Data

614

are expressed as the means ± SEM for three (A-D) or 4 (F) independent experiments.

615

*p < 0.05, significantly different from the LPS-stimulated samples.

616 617

Figure 4. Morin enhancement of the PKA-CREB signaling pathway. (A) Cell extracts

618

are prepared from BV2 cells stimulated with LPS for 1 h with or without morin and

619

subjected to Western blot analysis using antibodies against phospho- or total- CREB.

620

Levels of the phosphorylated CREB are normalized to the level of each total form.

621

Quantified data are shown in the bottom panel. (B) EMSA for CREB binding activity.

622

The data are representative of 3 independent experiments. (C) Western blot is

623

performed to detect the nuclear translocation of CREB. (D) Effect of morin on CRE-

624

luc reporter gene activity. The data are expressed as the mean ± SEM for 3 independent

625

experiments. #p < 0.05, significantly different from the control samples; *p < 0.05,

626

significantly different from the LPS-stimulated samples.

627 628

Figure 5. Morin reduction of ROS production via suppression of NADPH oxidase

629

subunits. (A) BV2 cells are pre-treated with morin 1 h prior to LPS stimulation.

630

Intracellular ROS levels are measured using the DCF-DA method after 16 h. Data are

27



631

expressed as the means ± SEM of 3 independent experiments. (B) DCF-derived

632

fluorescence in BV2 cells. (C) RT-PCR for mRNA expression levels of NADPH

633

oxidase subunits (p47phox, gp91phox, gp22phox, p67phox) in BV2 cells (n = 3). (D) Cell

634

extracts are first prepared from BV2 cells stimulated with LPS for 0.5 h in the absence

635

or presence of morin, and subsequently subjected to Western blot analysis using

636

antibodies against phospho-p47phox. Quantified data are shown in the bottom panel (n =

637

3). Levels of phosphorylated p47phox are normalized with β-actin. *p < 0.05,

638

significantly different from the LPS-stimulated group.

639 640

Figure 6. Enhancement of HO-1 expression by morin via Nrf2/ARE signaling

641

upregulation (A, B) BV2 cells are pretreated with morin for 1 h and stimulated with

642

LPS for 6 h. Western blot (A) and RT-PCR (B) for protein and mRNA expression

643

levels of HO-1 post morin treatment. (C) EMSA for Nrf2 binding activity. (D) Western

644

blot analysis to detect the nuclear translocation of Nrf2. Quantified data are shown in

645

the bottom panel (n = 3). (E) The effect of morin on ARE-luc reporter gene activity. #p

646

< 0.05, significantly different from the control samples; *p < 0.05, significantly

647

different from the LPS-stimulated samples.

28


Page 28 of 36

Page 29 of 36


Table 1. Primers used in RT-PCR Gene

Forward Primer (5’→3’)

Reverse Primer (5’→3’)

Size

p47phox

TTCACCACCATGGAGAAGGC

GGCATGGACTGTGGTCATGA

212 bp

p67phox

CTGGCTGAGGCCATCAGACT

AGGCCACTGCAGAGTGCTTG

214 bp

gp91phox

TTGGGTCAGCACTGGCTCTG

TGGCGGTGTGCAGTGCTATC

185 bp

p22phox

GTCCACCATGGAGCGATGTG

CAATGGCCAAGCAGACGGTC

164 bp

GAPDH

TTCACCACCATGGAGAAGGC

GGCATGGACTGTGGTCATGA

236 bp

29



Page 30 of 36

A

*

20 *

*

0 100 200 300 (μM) Morin: 0

80

*

600

*

400

*

200

0 0 100 200 300 (μM) Morin: 0

B 0

20

*

* *

10

0 100 200 300 (μM) Morin: 0 + LPS

+ LPS 300

30

0

+ LPS

+ LPS

Morin: 0

*

200 0

0

* *

40 20

0 0 100 200 300 (μM) Morin: 0

0 100 200 300 (μM)

+ LPS

+ LPS

100 200 300 (μM) 8

iNOS

iNOS

TNF-α

COX-2

COX-2 IL-6

Fold induction

6

TNF-α

IL-1β

*

*

4 2

*

* *

* *

0 8

IL-6

IL-1β

IL-10

6 4

*

*

*

*

*

* *

* *

2 0

IL-10

Morin: 0 300 0 100 200 300 + LPS

GAPDH

D Morin: 0

+ LPS 300

0

100 200

0 300 0 100 200 300 + LPS

0 300 0 100 200 300 (μM) + LPS

E 300 (μM) 8

iNOS TNF-α COX-2 IL-6 IL-1β

iNOS

TNF-α

6 4

COX-2

* *

2 0 8

*

* * *

IL-6

IL-1β

*

* * IL-10

* *

6 4 2

*

*

* * *

* *

0

IL-10

Morin: 0 300 0 100 200 300 + LPS

β-actin

Fig. 1 ACS Paragon Plus Environment

0 300 0 100 200 300 + LPS

*

60

C

Fold induction

Morin: 0

*

100

40

IL-10 (pg/ml)

*

400

50

800

IL-1β (pg/ml)

40

1000 IL-6 (pg/ml)

600 TNF-α (pg/ml)

NO (μM)

60

0 300 0 100 200 300 (μM) + LPS

Page 31 of 36


LPS (5 mg/kg)

A

B

cortex

morin

hippocampus

cortex

hippocampus

Iba1+ activated microglia (cells/mm2)

vehicle

Iba1+ activated microglia (cells/mm2)

saline

80

#

60

*

40 20 0

sal

LPS 60

# 40

* 20 0

sal

D

LPS (5 mg/kg) vehicle

morin

#

iNOS

4

* 2

iNOS TNF-α COX-2 IL-6 IL-1β

Fold induction

saline

6

0 6 #

TNF-α

#

IL-6

*

* 2

2 0 6

#

0 COX-2 6

*

4 2

GAPDH

6 4

4

Veh morin LPS


IL-1β

*

4

0 Sal

Fig. 2

#

2

0

veh morin LPS

Scale bar : 50 μm

C

veh morin

Sal

Veh morin LPS


A

B

+ LPS

Morin: 0

300

0

100

+ LPS

Morin:

200 300 (μM)

Page 32 of 36

300

0

0

100 200 300 (μM)

NF-κB

AP-1

C

D

► Reporter plasmid: (κB)3-luc

► Reporter plasmid: AP1-luc 4

6

*

4

Fold induction

Fold induction

8

* *

2

*

2

*

1 0

0

Morin:

*

3

0

0

100 200 300 (μM)

Morin:

0

0

+ LPS

100 200 300 (μM) p-ERK ERK p-JNK JNK p-p38

p-JNK/JNK

0

p-p38/p38

300

p-ERK/ERK

F

+ LPS

6 4

*

*

*

*

*

*

*

*

*

*

2 0 6 4 2 0 6 4

*

2 0 6

p38 p-Akt

p-Akt/Akt

Morin: 0

+ LPS

Fold induction

E

100 200 300 (μM)

4 2

*

0 Morin: 0 300 0 100 200 300 (μM)

Akt

+ LPS

Fig. 3


Page 33 of 36


A

B

+ LPS

Morin: 0

300

Morin:

100 200 300 (μM)

0

+ LPS 0

300 0 100 200 300 (μM)

pCREB CRE+ protein complex

Fold induction

CREB 8 6

#

4

*

*

*

#

2 0

Morin:

0

300

0

100 200 300 (μM)

◀F

Fold Induction:

+ LPS

C

+ LPS

Morin: 0

300

0

D

100 200 300 (μM)

► Reporter plasmid: CRE-luc

CREB

8

*

6 # 4

#

*

*

Fold induction

Fold induction

Histon H1

4 3 2

#

#

300

0

*

0

100 200 300 (μM)

0

Morin: 0

300

0

*

1

0 Morin:

2

*

+ LPS

100 200 300 (μM) + LPS

Fig. 4



B

A

ROS

(Fold induction)

4

con

LPS

Morin 300

LPS + Morin

3

*

2

* 1

*

0 Morin:

0

100 200 300 (μM)

0

+ LPS

*

Fold induction p22phox mRNA

2

*

1

0 4

*

3

Fold induction p67phox mRNA

5

*

2 1

0 Morin:

2

1

0

1.5 1.0 0.5

0 Morin:

0 300 0 100 200 300 (μM)

0 300 0 100 200 300 (μM)

+ LPS

+ LPS

D

+ LPS

Morin: 0

300

0

100 200 300 (μM) p-p47phox β-actin

Fold induction

Fold induction gp91phox mRNA

Fold induction P47phox mRNA

C

4 3

*

2

*

1 0

Morin:

0

300

0

100

*

200 300 (μM)

+ LPS

Fig. 5


Page 34 of 36


A Morin: 0

B

+ LPS 300

0

20

100 200 300 (μM) HO-1

Fold induction

β-actin 8 6 4

#

*

*

Fold induction HO-1 mRNA

Page 35 of 36

*

15

* 10

*

5

0 Morin:

*

0

#

#

300

0

#

100 200 300 (μM) + LPS

2

0 Morin: 0

300

0

D

100 200 300 (μM)

+ LPS

Morin: 0

+ LPS

300

0

100 200 300 (μM) Nrf2

C

+ LPS Histon H1 300 0

100 200 300 (μM)

ARE + protein complex

Fold induction

Morin: 0

8 6 4

*

#

#

300

0

*

*

2

0 Morin: 0

100 200 300 (μM) + LPS

E

► Reporter plasmid: ARE-luc 4

Fold induction

Fold Induction:

◀F

*

3

# #

2

*

*

1

0 Morin:

0

300

0

100 200 300 (μM) + LPS

Fig. 6



TOC Graphic

LPS

Morin

Morin PI3K/Akt, MAPKs

NF-κ κB, AP-1

iNOS, Cytokines

PKA

CREB, Nrf2

HO-1

Neuroinflammation


: activate : inhibit

Page 36 of 36

Suppression of Lipopolysaccharide-Induced ... - ACS Publications

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