Anti-inflammatory Activity of 8-Hydroxydaidzein in LPS-Stimulated BV2

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Bioactive Constituents, Metabolites, and Functions

Anti-inflammatory activity of 8-hydroxydaidzein in LPS-stimulated BV2 microglial cells via activation of Nrf2-antioxidant and attenuation of Akt/NF#B-inflammatory signaling pathways, as well as inhibition of COX-2 activity Pei-Shan Wu, Hsiou-Yu Ding, Jui-Hung Yen, Shu-Fen Chen, Kuan-Han Lee, and Ming-Jiuan Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00437 • Publication Date (Web): 23 May 2018 Downloaded from http://pubs.acs.org on May 23, 2018

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Journal of Agricultural and Food Chemistry

Wu Page 1 1

Anti-inflammatory activity of 8-hydroxydaidzein in LPS-stimulated BV2 microglial cells via

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activation of Nrf2-antioxidant and attenuation of Akt/NF-κB-inflammatory signaling pathways,

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as well as inhibition of COX-2 activity

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Pei-Shan Wu1†, Hsiou-Yu Ding2†, Jui-Hung Yen3, Shu-Fen Chen4, Kuan-Han Lee5, and Ming-

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Jiuan Wu1,*

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Taiwan; [email protected]

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Department of Biotechnology, Chia Nan University of Pharmacy and Science, Tainan 717,

Department of Cosmetic Science, Chia Nan University of Pharmacy and Science, Tainan 717,

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717, Taiwan; [email protected]

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[email protected]

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*Correspondence: Department of Biotechnology, Chia-Nan University of Pharmacy and Science,

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Tainan 717, Taiwan; [email protected]; Tel.: +886-6-266-4911 ext 2520; Fax: +886-6-

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266-2135

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†These authors contributed equally to this work.

Department of Molecular Biology and Human Genetics, Tzu Chi University, Hualien 970,

Department of Health and Nutrition, Chia Nan University of Pharmacy and Science, Tainan

Department of Pharmacy, Chia Nan University of Pharmacy and Science, Tainan 717, Taiwan;

ACS Paragon Plus Environment


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Abstract It was demonstrated that isoflavones can cross the blood-brain barrier, making them

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desirable candidate agents for the prevention of neurological symptoms. 8-Hydroxydaidzein (8-

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OHD, 4ʹ,7,8-trihydoxyisoflavone) is an isoflavone found only in fermented soy food. Current

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results showed that 8-OHD inhibited LPS-stimulated production of nitric oxide (NO) and

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proinflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-6, by

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inhibiting gene expression in BV2 microglial cells. Moreover, 8-OHD markedly quenched

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reactive oxygen species (ROS) and activated NF-E2-related factor 2 (Nrf2) so as to upregulate

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expression of Phase II enzymes, including heme oxygenase (HO)-1, NAD(P)H quinone

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dehydrogenase 1 (NQO1), and the modifier subunit of glutamate cysteine ligase (GCLM). 8-

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OHD also suppressed LPS-stimulated phosphorylation of Akt and NF-κB-p65. The anti-

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inflammatory activity of 8-OHD was attenuated by the HO-1 inhibitor zinc protoporphyrin

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(Znpp), but augmented by the PI3K/Akt inhibitor LY294002. 8-OHD also diminished LPS-

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induced prostaglandin E2 (PGE2) production without affecting cyclooxygenase (COX)-2

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expression. In vitro assay shows that 8-OHD displayed mixed-type inhibition of COX-2 with an

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IC50 of 8.9 ± 1.2 µM. These data suggest that the anti-inflammatory activity of 8-OHD may be

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associated with the activation of Nrf2/HO-1 and attenuation of Akt/NF-κB signaling pathways as

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well as inhibition of COX-2 enzyme activity. In conclusion, 8-OHD, a potent Nrf2 activator,

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Akt/NF-κB activation suppressor, and COX-2 enzyme inhibitor, may have health-promoting

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effects for mitigating microglia activation and preventing neuroinflammation.

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Keywords: 8-hydroxydaidzein; Nrf2; Akt; NF-κB; COX-2

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Introduction

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Microglial cells are the principle resident macrophages in the central nervous system

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(CNS)1. They are responsible for brain development, tissue maintenance and repair2. In healthy

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adult CNS, quiescent resting microglia surveil the tissue, being ready to rapidly transform to

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activated states upon real or potential danger to the CNS3. Activated microglia proliferate, exert

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phagocytic activities and produce immune-modulatory molecules, such as nitric oxide (NO),

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prostaglandin E2 (PGE2), pro-inflammatory cytokines, and reactive oxygen species (ROS)4. They

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thus play a key role in neuroinflammation, which is a fundamental process in neurodegenerative

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diseases, such as Alzheimer's disease, Parkinson's disease, prion diseases, multiple sclerosis, and

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AIDS dementia5.

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Nuclear factor E2-related factor 2 (Nrf2) and nuclear factor-κB (NF-κB) are the two key

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transcription factors that regulate cellular responses to oxidative stress and inflammation,

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respectively6. Nrf2 level is tightly regulated by binding to Keap1 (Kelch-like ECH-associated

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protein 1), which mediates Nrf2 ubiquitination and subsequent proteasomal degradation6. In the

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presence of oxidative and xenobiotic stresses, Keap1 changes conformation and prevents binding

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to Nrf2, leading to a rapid translocation and accumulation of Nrf2 in the nucleus. There, Nrf2

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forms a heterodimer with one of the small Maf proteins and binds to the antioxidant responsive

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elements (ARE) of target genes, which include a group of functionally diverse cytoprotective

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proteins, such as heme oxygenase-1 (HO-1), NAD(P)H, NAD(P)H:quinone oxidoreductase 1

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(NQO1), and glutamate-cysteine ligase (GCL)7. Small Nrf2 activators, including dietary

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polyphenolic compounds, have been known to attenuate inflammation and reduced risk of many

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chronic diseases8, 9.



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NF-κB is composed of different dimers, and a p50/p65 heterodimer is the most common

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one10. The canonical activation pathway is mediated by IκB kinase (IκKs) complex, which

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phosphorylate the inhibitors of NF-κB (IκBs) and lead to the proteasomal degradation of IκBs,

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allowing NF-κB to enter the nucleus and cause the expression of inflammatory genes. IκKs also

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phosphorylate p65 subunit at Ser536, which is involved in regulation of NF-κB nuclear

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translocation, transcriptional activity and protein stability11. Recent studies have clearly

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demonstrated that the phosphatidyl inositol 3-kinase (PI3K)/Akt signal pathway is required for

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efficient activation of NF-κB-dependent inflammatory responses in microglial cells12. There are

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functional interactions and cross-talks between the Nrf2 and NF-κB pathways to maintain the

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fine balance of cellular redox status and regulate the responses to stress and inflammation6.

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Isoflavones are a class of dietary phytoestrogens produced almost exclusively by the

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members of the Fabaceae family. Genistein (4′,5,7-trihydroxyisoflavone), daidzein (4′,7-

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dihydroxyisoflavone) and glycetein (4',7-dihydroxy-6-methoxyisoflavone) are the best known

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free isoflavones found in soy13. Due to their estrogenic activity, numerous clinical studies

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revealed the benefits of genistein and daidzein in the prevention of breast and prostate cancers,

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cardiovascular disease and osteoporosis, as well as in relieving postmenopausal symptoms14, 15.

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Genistein (Gen, SFig. 1a), the most widely studied soy isoflavone, can cross the blood-brain

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barrier (BBB), making it an especially desirable potential drug for the treatment of

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neurodegenerative diseases16. It can protect cells from oxidative stress15 and inhibit the

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production of proinflammatory mediators in lipopolysaccharide (LPS)-treated microglia cells17,

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and in TNF-α-treated human brain microvascular endothelial cells18. Animal experiments show

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that oral administration of Gen attenuated LPS-induced cognitive impairments in rat19. Daidzein


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also penetrates to the brain through the BBB20, and exhibits anti-inflammatory effects in β-

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amyloid- and LPS-activated astrocyte cells21.

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In contrast to the above-mentioned genistein and daidzein, ortho-hydroxyl isoflavones are

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usually isolated from fermented soybean products rather than from the plant per se22. 8-

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Hydroxydaidzein (8-OHD, 4',7,8-trihydroxyisoflavone, SFig. 1b) has recently attracted much

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attention due to its strong antioxidant, antitumor, anti-melanogenesis, aldose reductase

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inhibitory, and hepatoprotective activities22-25. However, the anti-inflammatory effect of 8-OHD

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has never been studied before. We thus intended to study the inhibitory effects of 8-OHD along

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with its meta-hydroxy isomer, Gen, on the production of NO, pro-inflammatory cytokines, and

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PGE2 in LPS-treated BV2 murine microglial cells. Furthermore, its effects on ROS generation

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and signal pathways including Nrf2, Akt and NF-κB were also investigated.

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Materials and Methods

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Chemicals

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Polymyxin B (PMB), lipopolysaccharide (LPS, Escherichia coli O111:B4), Griess reagent,

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3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), RIPA lysis buffer and

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other chemicals were from Sigma-Aldrich Co. (St. Louis, MO), unless otherwise indicated.

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Genistein (Purity ≥98%), Zinc Protoporphyrin-IX, and nuclear extraction kit were from Cayman

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Chemical (Ann Arbor, MI, USA). Mouse TNF-α and IL-6 ELISA Sets were from BD

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Biosciences (San Diego, CA, USA). PI3K/Akt inhibitor LY294002 was from Promega (Madison,

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WI). Prostaglandin E2 (PGE2) ELISA kit was from Abcam (Cambridge, UK). Fluorometric

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COX-2 inhibitor screening kit was purchased from Biovision (Milpitas, CA, USA).

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Isolation and Identification of 8-Hydroxydaidzein (8-OHD, 4ʹ,7,8 -Trihydoxyisoflavone)



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8-OHD was isolated from soybean fermented by Aspergillus oryzae according to a process

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described in the literature23. Soy fermented product (500 g) was refluxed with methanol (5 L) for

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3 h to give a methanol extract (102 g). The extract was suspended in water (0.1 L), and re-

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extracted with hexane and ethyl acetate. Each extraction was concentrated under vacuum to give

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hexane (54 g), ethyl acetate (5.43 g), and water (37 g) fractions. The ethyl acetate extract was

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subjected to silica gel column chromatography and eluted with hexane/ethyl acetate (3:1),

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hexane/ethyl acetate (1:1), ethyl acetate, ethyl acetate/methanol (1:1), and methanol. The ethyl

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acetate fraction was further separated on a repeated HPLC using a semi-preparative C-18 ODS2

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OBD Prep Column (Spherisorb, 80Å, 5 µm, 10 mm × 250 mm) according to previous method26.

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The mobile phase consisted of elute A (0.1% v/v acetic acid in double distilled water) and elute B

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(acetonitrile, Merck). The elution was started with 14% v/v B for 10 min (isocratically), followed

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by a linear gradient of B increased from 20% to 40% v/v over 50 min with a constant flow rate

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of 3 mL/min. The peak corresponding to 8-OHD was collected and lyopholized to white powder

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(14.7 mg). The compound was identified by mass and NMR spectrometry. 8-OHD24: The NMR spectral data were: 1H NMR (DMSO-d6) : 6.79 (2H, d, J = 8.3 Hz,

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H-3ʹ, 5ʹ), 6.94 (1H, d, J = 8.7 Hz, H-6), 7.37 (2H, d, J = 8.3 Hz, H-2ʹ, 6ʹ), 7.45 (1H, d, J = 8.7 Hz,

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H-5), 8.30 (1H, s, H-2), 9.46 (1 H, br. s, OH-7), 9.58 (1H, br. s, OH-4 ʹ), 10.37 (1H, br. s, OH-8).

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(C-8), 130.4 (C-2ʹ, 6ʹ), 123.2 (C-1ʹ), 123.0 (C-3), 117.7 (C-10), 116.0 (C-5), 115.3 (C-3ʹ, 5ʹ),

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114.5 (C-6) ; FAB MS, m/z 271 [M + H]+.

C NMR (DMSO-d6) : 175.6 (C-4), 157.4 (C-4ʹ), 153.0 (C-2), 150.2 (C-7), 147.0 (C-9), 133.2

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The purity of the isolated 8-OHD was 99% determined by the single peak with retention

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time 2.276 min on the ultra-performance liquid chromatography (UPLC) profile (SFig. 2). UPLC

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was operated through an analytic C18 reversed-phase column (Acquity UPLC BEH C18, 1.7 µm,


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2.1 mm × 100 mm, Waters, Milford, CT, USA) and a gradient elution using water (A) containing

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1% (v/v) acetic acid and methanol (B) with a linear gradient for 5 min with 15% to 35% B at a

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flow rate of 0.3 mL/min27.

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Culture of BV2 Cells

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Immortalized mouse microglial cell line BV2 was kindly provided by Professor Shun-Fen

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Tzeng (National Cheng Kung University, Tainan, Taiwan). BV2 cells were cultured in

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Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12, Invitrogen Life

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Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (HyClone, Logan, UT, USA),

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100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were maintained in a humidified

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incubator at 37°C in 5% CO2.

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Treatment of BV2 Cells and Measurement of Cell Viability, and Production of Nitrite, TNF-α,

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IL-6 and Prostaglandin E2 (PGE2)

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BV2 cells (1 × 106/mL) were pretreated with vehicle (0.1% DMSO), polymyxin B (PMB),

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8-OHD, or Gen for 30 min prior to LPS insult. Culture medium was collected 20 h later for the

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measurement of nitrite, TNF-α, IL-6, and PGE2 and cell viability was determined by MTT (3-

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(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay28.

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Nitrite (NO2−) was determined using the Griess reagent and A550 was measured against a

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sodium nitrite standard curve. TNF-α and IL-6 were measured by Mouse TNF-α and IL-6

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ELISA Sets. PGE2 was determined using PGE2 ELISA kit.

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RNA Extraction and Reverse Transcription Real-Time PCR

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Total RNA extraction was carried out using Illustra RNA Spin Mini RNA Isolation Kit (GE

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Healthcare). The reverse transcription of the RNA samples was performed using a High-

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Capacity cDNA Archive kit (Thermo Fisher Scientific). Quantitative PCR was performed using



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a Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) and ABI StepOne Real Time

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PCR System. The reactions were performed in a total volume of 20 µl that contained 0.4 µM of

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each primer (STable 1). PCR conditions were: 95 °C for 2 min, 40 cycles at 94 °C for 15 s, and

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60 °C for 60 s. The relative mRNA expression was normalized with β-actin expression and then

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calculated by the 2−∆∆CT method. The identity and purity of the amplified product was checked

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through analysis of the melting curve.

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Preparation of Cell Lysates and Nuclear Extracts and Immunoblot Analysis

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BV2 cells were washed twice with ice-cold phosphate saline buffer (pH 7.4). Total cell

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lysates and nuclear extracts were prepared by RIPA lysis buffer and nuclear extraction kit,

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respectively. The protein concentration was determined by the Bradford method (Bio-Rad

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Laboratories, Hercules, CA, USA), using bovine serum albumin as a standard reference.

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Equal amounts of protein were subjected to electrophoresis by SDS/PAGE. Proteins were

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transferred onto polyvinylidene difluoride (PVDF) membranes (Hybond-P, GE Healthcare,

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Pittsburgh, PA, USA) using CAPS buffer system (10 mM CAPS pH 10.5, 10% v/v methanol) at

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20 volts overnight at 4°C. The membranes were then incubated with freshly made blocking

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PBST buffer (5% skim milk in PBS with 0.05% v/v Tween 20, pH 7.4) for 8 h at room

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temperature, followed by overnight incubation with specific primary antibody (STable 2) at 4 °C.

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The blots were washed in PBST three times and subsequently incubated with suitable

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horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove,

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PA, USA) for 1 h before enhanced chemiluminescence detection (Amersham ECL Prime

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Western Blotting Detection Reagents, GE Healthcare). Some blots were stripped with Restore

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Western Blot stripping solution (Thermo Fisher Scientific, Rockford, IL, USA) to remove

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previous primary and secondary antibodies. After washes and blocking, the blots were re-probed


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with another primary antibody. The intensity of the band was analyzed with ImageJ software

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(National Institutes of Health, Bethesda, MD, USA).

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Cyclooxygenase (COX)-2 Inhibition Assay

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Inhibitory activities of the 8-OHD and Gen against COX-2 enzyme were determined using

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fluorometric COX-2 inhibitor screening kit following the kit protocol. To calculate the initial

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velocity of COX-2 activity (RFU/sec), the slope of the early linear range of the enzyme reaction

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progress plot was made using the observed RFU vs. time. The relative activity (Vi/Vo) was

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determined as the ratio between the initial velocity in the presence (Vi) and absence (Vo) of the

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inhibitor. The IC50 values were calculated from the regression analysis of relative activity vs.

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concentration plot, and denoted the concentration of the sample required to inhibit 50% of the

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enzyme activity.

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The enzyme kinetics of 8-OHD was further determined using various concentrations of

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substrate (arachidonic acid) either in the absence or presence of selected concentrations of 8-

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OHD. The mode of inhibition was determined by the Lineweaver–Burk double reciprocal plot

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analysis.

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Intracellular ROS Analysis

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BV2 cells were cultured in black 96-well plates (2 × 105/well) overnight. Cells were washed

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with PBS and then stained with 20 µM 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA,

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Invitrogen), a fluorogenic dye that measures hydroxyl, peroxyl and other reactive oxygen species

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(ROS) activity within the cell, at 37oC for 30 min in the dark and then washed with PBS. Cells

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were treated with indicated reagent for 30 min followed by LPS for 20 h. The signals were then

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read at EX485 nm/Em535 nm.

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Statistical Analysis



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Data were presented as means ± SD and analyzed by one-way analysis of variance

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(ANOVA) with Tukey’s post hoc test. A p value of

Anti-inflammatory Activity of 8-Hydroxydaidzein in LPS-Stimulated BV2

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