7-Deoxy-trans-dihydronarciclasine Isolated from Lycoris chejuensis

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Bioactive Constituents, Metabolites, and Functions

7-Deoxy-trans-dihydronarciclasine Isolated from Lycoris chejuensis Inhibits neuroinflammation in Experimental Models Dong Zhao, Ming-Yao Gu, Li Jun Zhang, Hyo Jin Jeon, Yong-Baik Cho, and Hyun Ok Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03307 • Publication Date (Web): 08 Aug 2019 Downloaded from pubs.acs.org on August 9, 2019

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Journal of Agricultural and Food Chemistry

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7-Deoxy-trans-dihydronarciclasine Isolated from Lycoris chejuensis Inhibits

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neuroinflammation in Experimental Models

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Dong Zhao,†,‡ Ming-Yao Gu,§ Li Jun Zhang, †,‡ Hyo Jin Jeon,┴ Yong-Baik Cho,┴ Hyun

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Ok Yang *,†,‡

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†Natural

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Gangneung 25451, Gangwon-do, Republic of Korea

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‡Division

Product Research Center, Korea Institute of Science and Technology,

of Bio-Medical Science & Technology, KIST School, Korea University of

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Science and Technology, Seoul 02792, Republic of Korea

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§Department

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Shenzhen University Health Science Center, Shenzhen 51801, China

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┴Pharmaceutical

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*Corresponding author

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Tel.: +82 33 650 3501; Fax: +82 33 650 3529.

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E-mail address: [email protected]

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ORCID: 0000-0003-1604-0843

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Running title: CJ extract and its active compound E144 inhibit neuroinflammation

of Cell Biology and Medical Genetics, School of Basic Medical Sciences,

R&D Center, Kolmar Korea, Sejong 30003, Republic of Korea

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ACS Paragon Plus Environment


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ABSTRACT: Over-activated microglia and persistent neuroinflammation hold

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important role in the pathophysiology of neurodegenerative diseases. The extract of

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Lycoris chejuensis (CJ) and its active compound, 7-deoxy-trans-dihydronarciclasine

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(named E144) attenuated expressions of pro-inflammatory factors, including nitric

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oxide, prostaglandin E2, inducible nitric oxide synthase, cyclooxygenase (COX)-2,

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tumor necrosis factor (TNF)-α, and interleukin (IL)-6, secreted by lipopolysaccharide-

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activated BV-2 microglial cells, as measured by enzyme-linked immunosorbent assay

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or western blotting. In contrast, CJ extract and E144 promoted the secretion of the anti-

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inflammatory cytokine, IL-10. Moreover, we found that E144 attenuated the expression

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of TNF-α and COX-2 in the cerebral cortex of lipopolysaccharide-treated mice and/or

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T2576 transgenic mice, as well as reducing the reactive immune cells visualized by

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ionized calcium binding adaptor molecule-1. Our results suggest the possibility of E144

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to serve as a potential anti-neuroinflammatory agent by preventing excess production

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of pro-inflammatory factors.

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KEYWORDS:

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Lycoris chejuensis, 7-Deoxy-trans-dihydronarciclasine, Anti-inflammation, Pro-

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inflammatory factors, Neuroinflammation

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Introduction

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Natural foods and herbs are an important source for the development of health-

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beneficial ingredients, such as Gingko biloba and cinnamon to prevent dementia and

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ginseng and Lycoris radiata to treat brain diseases. (1-4) Lycoris radiata is well-known

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not only for ornamental use but also for its medicinal value; for example, the bulb has

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been used as a traditional Chinese medicine to treat emesis, rheumatoid arthritis, and

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pyogenic infections, as recorded by the Compendium of Materia Medica. In addition,

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Lycoris bulb is rich in starch, and has thus been traditionally used as a hunger crop after

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boiling; Lycoris chejuensis K. Tae et S. Ko (CJ) has also been used for this purpose. (5)

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CJ is another Lycoris species and originates from Jeju Island of Korea. (6,7) The Lycoris

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genus was found to have a wide range of biological activities because its active

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compounds generally contain 7 representative alkaloids, including lycorine-,

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homolycorine-,

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narciclasine-compounds.

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acetylcholinesterase (AchE) inhibitor, has been approved by the FDA to treat

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Alzheimer’s disease (AD) in the USA and Europe. Interestingly, in addition to its anti-

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AD effect, galanthamine also significantly inhibited neuroinflammation by attenuating

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tumor necrosis factor (TNF)-α and nitric oxide (NO) production in murine microglia.

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(12)

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inhibitor but also because it contributes to the inhibition of pro-inflammatory factors,

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such as TNF-α.

haemanthamine-, (8-11)

tazettine-,

montanine-,

galanthamine-,

and

Among of these alkaloids, galanthamine, as an

Thus, galanthamine was approved as an anti-AD drug not only because it is an AchE

(13)

Recently, we found that a CJ ethanol extract inhibited the



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pathological hallmarks of AD, including amyloid-beta (Aβ) production and Aβ plaque

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formation in Aβ protein precursor (APP)-transfected HeLa cells, and improved

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cognitive function in PS1/APP transgenic mice. (5) More recently, an active compound

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isolated from CJ, 7-deoxy-trans-dihydronarciclasine (named E144), was shown to be a

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potential drug candidate because it reduces the pathological hallmarks and promotes

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the memory index of APP-transgenic mice. (14) E144 may be a potential anti-AD drug,

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but whether it inhibits neuroinflammation is still unclear.

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Neurodegenerative diseases impact millions of patients’ lives worldwide and cause

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various socioeconomic problems. These diseases, including AD, Parkinson’s disease

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(PD), and multiple sclerosis (MS), are characterized by progressive loss of neurons that

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results in functional disorders such as mobility, memory, and learning impairments

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related to dysfunction in the central nervous system (CNS). (15-17) Although the process

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of neurodegenerative diseases is not fully understood, the greatest risk factors for

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neurodegenerative diseases normally occur with aging, depression, obesity, and

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infection, all of which cause the activation of glial cells, particularly microglia. (17,18)

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Microglia are the major CNS innate immune cells and responsible for immune defense

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and tissue repair with a strict self-limiting mechanism, but sustained activation and

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over-activation of microglia have been identified as hallmarks of neurodegenerative

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diseases because excessive phagocytosis occurs together with production of neurotoxic

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mediators, such as interleukin (IL)-6, TNF-α, prostaglandin (PG)E2, and NO.

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However, there are some feedback mechanisms that serve to attenuate pro-


(19-21)

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inflammatory mediators via induction of the immunosuppressive cytokines IL-10 and

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transforming growth factor (TGF)-β to inactivate microglial cells.

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evidence has suggested that selective inhibition of neurotoxic factors or promotion of

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neuroprotective factors produced by excessive glial activation might be an approach to

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altering neurodegenerative disease progression. Furthermore, targeting microglial cells

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might have beneficial effects on neurodegenerative diseases, and could be considered

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an important therapeutic strategy.

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Our current study evaluated the anti-inflammatory effect of CJ extract and E144 on

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microglial cells by assessing the pro- and anti-inflammatory factors secretion, including

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NO, PGE2, TNF-α, iNOS, COX-2, IL-6, and IL-10, in lipopolysaccharide (LPS)-

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activated BV-2 microglial cells. In addition, we also demonstrated that E144 inhibits

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the pro-inflammatory factors, COX-2 and TNF-α, in LPS-treated mice. Moreover,

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E144 inhibited the expression of COX-2 in AD transgenic mice. Finally, we found that

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E144 inhibits the activation of immune cells in the brain visualized using Iba-1, which

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is a marker up-regulated in reactive microglia of both LPS-injected mice and APP-

(22,23)

Increasing

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transgenic AD mouse models.

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MATERIALS AND METHODS

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Chemicals. Antibodies (primary anti-bodies: anti-iNOS, anti-COX-2, anti-GAPDH,

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and HRP-linked anti-rabbit IgG secondary antibody) were purchased from Cell

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Signaling Technology (Boston, MA, USA). The primary anti-Iba-1 and anti-TNF-α

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antibodies were obtained from Abcam (Cambridge, UK). The secondary anti-rabbit



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Alexa Fluor 488 antibody was purchased from Invitrogen (Carlsbad, CA, USA). Fetal

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bovine serum (FBS) was purchased from ATCC (Manassas, VA, USA).

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Penicillin/streptomycin (P/S), Dulbecco’s Modified Eagle’s Medium (DMEM), and

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trypsin-EDTA were purchased from Gibco (Grand Island, NY, USA). Highly pure

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solvents, including ethanol, n-hexane, CH2Cl2, and n-BuOH were purchased from

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BUKSAN (Gyeonggi-do, Republic of Korea). Analytical grade ACN, water, 4ʹ-6-

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diamidino-2-phenylindole (DAPI), and LPS originated from Escherichia coli 055:B5

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were obtained from Sigma-Aldrich (St. Louis, MO, USA).

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Preparation of CJ extract and 7-deoxy-trans-dihydronarciclasine (E144). CJ bulbs

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were obtained on August 2017 from the Garden of Gangneung National Forest

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(Gangwon-do, Republic of Korea), and were identified by Ms. Jung Hwa Kang who

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works in Hantaek Botanical Garden, and compared to a specimen (KNB00065) from

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the Korea Institute of Science and Technology (KIST) herbarium. For CJ extract and

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E144 preparation, we used the same method described in our previous study. (5) Briefly,

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CJ bulbs were cut into little piece, and air dried (10 kg), then refluxed three times using

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ethanol (50% v/v) and evaporated under reduced pressure at 40°C, yielding 3.3 kg of

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CJ extract (yield of 33.1%). Subsequently, CJ extract (3 kg) suspended in water was

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successively subjected to liquid partitioning with equivalent volumes of n-hexane (CJ-

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1), CH2Cl2 (CJ-2), and n-BuOH (CJ-3). Then, 272.4 g of CJ-3 was fractionated using a

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Diaion-HP20 (SUPELCO, MO, USA) column with stepwise elution of aqueous ACN,

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resulting in eight fractions (CJ-3-F1-8) was obtained. CJ-3-F4 was further isolated


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using preparative HPLC with a C18 reversed-phase column for 40 min with detection

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at 223 nm, a flow rate of 10 mL/min, and a gradient elution of 10-20% ACN adding

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0.02% trifluoroacetic acid. Finally, 46.5 mg of E144 (purity > 97%) was isolated, and

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its chemical structure was identified using LC-MS (Agilent, CA, USA) and an NMR

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spectrometer (Bruker, MA, USA).

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Cell culture and viability. BV-2 microglial cells were maintained in DMEM/F12

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(Gibco) supplemented with 5% heat-inactivated FBS and 1% P/S under 5% CO2

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humidified incubator at 37°C. BV-2 cells were seeded (1×104 cells/well) for 24 h into

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96-well plates, and treated for 12 h with indicated concentrations of CJ extract and E144

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in the presence or absence of 100 ng/mL LPS. Cell viability was measured with the

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protocol described previously, (24, 25) by using EZ-Cytox reagent (DAEILLAB Co. Ltd.,

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Seoul, Republic of Korea). After 30 min of incubation, the optical density was

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measured using a microplate reader at the wavelength of 450 nm.

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Animals and drug administration. All animal experiments were conducted following

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guidelines of the KIST Animal Care and Ethics Committee. Both 7-week-old

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C57BL/6N male mice (Orient Bio Inc., Seongnam, Republic of Korea) and 3-month-

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old Tg2576 Swedish mutant APP transgenic male mice (Taconic, Germantown, NY,

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USA) were randomly grouped (8 mice/group), maintained in cages, and placed in a

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humidity- and temperature- (22 ± 3°C) controlled room under a 12 h light/dark cycle

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with food and water available ad libitum. The LPS-treated mouse model was designated

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according to our preliminary experiments and previous study,


(26)

Seven-week-old


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C57BL/6N mice were administered with designated concentrations (0.5, 1, and 1.5

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mg/kg/day) of E144 and a positive control (genipin, 25 mg/kg/day) via oral gavage for

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8 days, and intraperitoneally injected after 30 min E144 treatment with LPS of 2.5

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mg/kg/day started on the 6th day. In the APPsw transgenic mouse model, as described

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previously, (14) drug administration started at the age of 9 months with PBS as a control

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or E144 at 1.5 mg/kg/day for 3 months.

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Measurement of LPS-induced NO production. To assess the effect of E144 on NO

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production as described previously,

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plates for 24 h, and E144 was pre-treated for 1 h with the indicated concentrations, and

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then co-treated the cells with LPS of 100 ng/mL for 12 h. The collected medium was

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centrifuged at 13000 rpm for dead cell removal. The medium samples (50 μL) and equal

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volume of Griess reagents [1% sulfanilamide, and N-(1-naphthyl)-ethylenediamine

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dihydrochloride in 2.5% H3PO4] were reacted for 10 min. Standard curve was prepared

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using sodium nitrite, and the optical density was measured at 540 nm using a microplate

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reader.

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Measurement of LPS-induced IL-6, TNF-α, and PGE2 production. To evaluate pro-

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inflammatory mediators described previously, (24,25) conditioned medium was prepared

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according to the same protocol used for the NO assay. Three specific enzyme-link

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immunosorbent assay (ELISA) kits, IL-6 (M6000B), TNF-α (MTA00B), and PGE2

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(KGE004B, R&D Systems Inc., MN, USA) were used according to the manufacturer’s

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protocols.

(24, 25)

we seeded BV-2 cells (5×105) into 6-well


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Microglia in mice measured by immunofluorescence assay. To observe microglia in

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the mouse brain, we performed an immunofluorescence assay according to our

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previously described method. (24,25) Briefly, mice were anesthetized with pentobarbital

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sodium (60 mg/kg), perfused through the left ventricle with 10 mL of ice-cold 0.1 M

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(pH 7.4) PBS, and fixed with 10 mL of ice-cold 4% formaldehyde solution (pH 6.9).

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Then, the mouse brains were carefully isolated, stored in 4% formaldehyde at 4°C for

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1 day, and then kept in 20% sucrose dissolved in 0.1 M PBS at 4°C. Mouse brain slices

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of 25 μm were sectioned using cryo-sectioning and preserved in a storage solution at -

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20°C. The brain slices were immersed into the solution (0.5% Triton X-100 in PBS) for

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30 min, and blocked using 5% bovine serum albumin (BSA) at room temperature for 1

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h. Then, the slices were incubated overnight with an anti-Iba-1 antibody (1:100) in 5%

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BSA, and the secondary Alexa Fluor 488-labeled IgG (1:200) in 5% BSA was added

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and protected from light at room temperature for 1 h. Finally, cell nuclei were stained

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with DAPI in PBST at 37°C for 30 min under dark. After adding fluorescence mounting

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medium (Agilent & Dako), the slides were sealed with coverslips. Images were

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captured using a confocal fluorescence microscope (Leica, Solms, Germany) with

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excitation/emission wavelengths of 493/519 nm and 358/461 nm for Alexa Flour 488

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and DAPI, respectively.

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Total protein isolation from BV-2 cells. Total protein was isolated according to a

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previous study. (24,25) Briefly, the cells were washed three times with ice-cold PBS and

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lysed with lysis buffer containing 1× protease inhibitor cocktail (PIC, Roche, Penzberg,



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Germany) and 1× phenylmethylsulfonyl fluoride (PMSF). The lysates were centrifuged

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(13000 rpm) for 20 min. The quantification of protein was conducted with Bradford

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reagent (Bio-Rad, Hercules, CA, USA), and standard was prepared with BSA (0-20

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μg/mL). Western blot samples were prepared with lysate and an equal volume of 2X

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NuPAGE LDS sample buffer (Thermo Fisher Scientific Inc., Lafayette, CA, USA) with

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10% 2-mercaptoethanol.

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Total protein isolation from the mouse brain and western blot analysis. For the

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preparation of mouse brain samples as described previously,

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cortex was collected, stored in liquid nitrogen, homogenized with PRO-PREP protein

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extraction buffer (iNtRON, Gyeonggi, Republic of Korea) and 1× PIC set І (Sigma-

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Aldrich, MO, USA), and then centrifuged (13000 rpm) for 20 min. Protein

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quantification assays were performed as described for cell sample preparation. Western

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blotting was performed according to previous method. (24,25) The total protein samples

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(20 μg) were separated using sodium dodecyl sulfate-polyacrylamide gel

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electrophoresis (SDS-PAGE) with 8%, 10%, or 12% Acrylamide/Bis gels and

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electrotransferred to polyvinylidene difluoride (PVDF, Millipore, USA) membranes.

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Then, the membranes were blocked with 5% BSA in TBST and incubated overnight

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under 4°C with specific primary antibodies (1:1000). After the membranes were

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washed with TBST, they were incubated with secondary horseradish peroxidase

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(HRP)-conjugated IgG (1:2000) at room temperature for 1 h, and visualized using ECL

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reagents (Thermo Fisher Scientific). Densitometry analysis of the bands was performed


(14)

briefly, the mouse

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with the LAS4000 system (Fujifilm, Tokyo, Japan).

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Statistical analysis. Statistical analysis was preformed using GraphPad Prism 7

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software (GraphPad Software Inc., San Diego, CA, USA). After the one-way ANOVA

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with Tukey multiple comparison test, the values with p < 0.05 were considered

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significant. All data are expressed as the mean ± standard error of the mean (SEM).

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Results and Discussion

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Effect of CJ extract on the cell viability of BV-2 microglial cells, and the

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inflammatory factors. CJ extract was prepared using 50% ethanol according to our

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previous study.

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measure the cell viability of BV-2 cells, and 20 μg/mL CJ extract did not show

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significant cytotoxicity in the MTT assay (Fig. 1A). Subsequently, CJ extract at

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concentrations of 5, 10, and 20 μg/mL were used to test whether CJ extract affects the

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production of pro-inflammatory cytokines, such as IL-6 and TNF-α, as measured using

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specific ELISA kits. As shown in Fig. 1B and C, treatment with LPS significantly

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activated microglial cells to secrete the pro-inflammatory cytokines TNF-α (52.2 ± 1.2

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fold of control) and IL-6 (20.7 ± 2.9 fold of control) in culture medium, but the levels

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of TNF-α (7.0 ± 5.6%, 35.2 ± 8.4%, and 76.7 ± 8.9%) and IL-6 (30.3 ± 13.6%, 67.6 ±

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7.7%, and 89.7 ± 5.9%) were dose-dependently decreased when co-treated with the

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indicated concentrations of CJ extract. Furthermore, NO production is a key player in

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the pathogenesis of inflammation.

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treatment, LPS significantly induced NO production (35.0 ± 2.4 fold of control)

(5)

Various concentrations of CJ extract (0-50 μg/mL) were used to

(27)

In the present study, compared to the control



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measured using Griess reagents, and treatment with CJ extract significantly inhibited

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production by 24.7 ± 1.8%, 46.8 ± 2.2%, and 79.4 ± 1.3% in a dose-dependent manner

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(Fig. 1D). In addition, we tried to assess whether CJ extract can affect the production

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of anti-inflammatory cytokines such as IL-10 that can directly regulate innate and

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adaptive immune cell activation and differentiation by suppressing the production of

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pro-inflammatory mediators such as TNF-α, iNOS, and IL-1β.

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could be considered as an important anti-inflammatory modulator of glial activation in

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the CNS.

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inflammatory cytokine IL-10. These results indicated that CJ extract may contain some

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constituents that can inhibit the production of pro-inflammatory factors, and promote

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the secretion of the anti-inflammatory cytokine IL-10.

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Effect of E144 on cell viability and inflammatory factors in BV-2 microglial cells.

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To identify the active compound in the CJ extract, E144 (> 97%) was isolated from

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bulbs of CJ (CJ-3-F4), its purity was quantified and identified by LC-MS and NMR, as

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shown in the Supporting Information (Fig. S1, and S2), as well as by comparison with

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previously reported data.

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Before further study, the cytotoxicity of E144 was determined by treating cells with

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varying concentrations of E144 in the presence or absence of LPS for 12 h. As shown

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in Fig. 2B, E144 at a concentration of 0.5 μM did not significantly affect the cell

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viability of BV-2 cells, thus, E144 were used at concentrations of 0.1, 0.3, and 0.5 μM

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for subsequent experiments. We assessed whether the CJ compound, E144 reduced the

(29)

(28)

Therefore, IL-10

As shown in Fig. 1E, CJ extract promoted the expression of the anti-

(30, 31)

The chemical structure of E144 is shown in Fig. 2A.


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production of pro-inflammatory cytokines, including TNF-α and IL-6, secreted by LPS-

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stimulated BV-2 microglial cells. BV-2 cells were pre-incubated with designated

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concentrations of E144 for 1 h, and then co-treated with 100 ng/mL LPS for 12 h.

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Specific ELISA kits were used to measure the production of TNF-α and IL-6 in the

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medium, as shown in Fig. 2C and D. TNF-α and IL-6 levels were increased by 47.6 ±

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4.7 and 17.9 ± 1.3 times compared to those in the control group, respectively, but these

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excessive pro-inflammatory cytokines were significantly attenuated by 18.3 ± 5.6%,

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46.2 ± 6.8%, and 76.4 ± 6.7% and 5.5 ± 10.9%, 53.2 ± 7.2%, and 87.6 ± 1.2% after

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treatment with 0.1, 0.3, and 0.5 μM E144, respectively. In addition, similar to the CJ

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extract, E144 enhanced the IL-10 level in LPS-activated BV-2 cells as shown in Fig.

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2E. Interestingly, treatment with E144 alone does not significantly affect the production

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of IL-6, TNF-α, and IL-10 compared with the control group. These data were consistent

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with those obtained after treatment with the CJ extract, indicating that E144 may be an

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active compound in CJ, and both of these preparations significantly decreased LPS-

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induced pro-inflammatory cytokines in a dose-dependent manner.

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Effect of E144 on the production of NO and PGE2, as well as the regulators iNOS

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and COX-2. Excessive NO production has been identified as a cause of the

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pathogenesis of neurodegenerative diseases, such as NO-induced dopaminergic neuron

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degeneration and nitrated tau in AD patients. (32,33) Thus, we investigated whether E144

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inhibits the production of NO by reactive microglial cells. The amount of NO secreted

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by LPS-activated BV-2 microglial cells was dramatically increased by 27.9 ± 2.5 times



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compared with the control group, but NO production was inhibited by 5.6 ± 5.6%, 37.6

275

± 6.7%, and 65.9 ± 3.7% after treatment with the indicated concentrations of E144 in a

276

dose-dependent manner (Fig. 3A). Pathological NO production is mainly regulated by

277

the inducible enzyme iNOS.

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significantly induced the expression of the iNOS enzyme in BV-2 microglial cells,

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however, it was significantly attenuated by the treatment of E144 (Fig. 3B). Moreover,

280

another important pro-inflammatory mediator, PGE2, is also involved in the

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pathogenesis of inflammatory neurodegeneration by potentiating toxic and oxidative

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stress, and PGE2 was initially significantly elevated in AD patients at an early stage. (35-

283

38)

284

of PGE2 by 4.9 ± 1.6 fold of control group, but it was inhibited by 24.0 ± 2.0%, 50.1 ±

285

3.8%, and 65.1 ± 5.4% after treatment with 0.1, 0.3, and 0.5 μM E144 (Fig. 3C),

286

respectively. Pathological PGE2 was upregulated by the inducible enzyme COX-2,

287

which is normally not present in microglial cells, but can be dramatically induced by

288

neuroinflammation.

289

valuable strategy for slowing the progression of the neurodegenerative process. In this

290

study, LPS-induced COX-2 was significantly attenuated by treatment with E144, as

291

shown in Fig. 3D. These data indicated that E144 may be a potential anti-

292

neuroinflammatory candidate by blocking the production of NO and PGE2 and their

293

respective regulators, iNOS and COX-2.

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Effect of E144 on the expression of TNF-α and COX-2 in LPS-treated mouse

(34)

Consistent with the NO production data, LPS

In this study, we found that treatment with LPS significantly increased the secretion

(39,40)

These data suggest that inhibition of COX-2 may be a


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brains. To further determine whether E144 can inhibit pro-inflammatory factors in

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mouse brain, we used a mouse model treated with LPS at 2.5 mg/kg/day, and we found

297

that mice intraperitoneally injected with LPS showed significant over-expression of

298

TNF-α and COX-2 in the mouse cortex using western blot analysis, as shown in Fig.

299

4A and B. In this study, we used a positive control, genipin, which showed anti-

300

neuroinflammatory activity in vitro and in vivo. (41,42) Genipin at 25 mg/mL significantly

301

inhibited the expression of TNF-α and COX-2. Mice treated orally with E144 also

302

showed significantly reduced expression of pro-inflammatory factors, including TNF-

303

α and COX-2, in the mouse brain. These data suggest that E144 may prevent

304

neuroinflammation by inhibiting pro-inflammatory factors such as TNF-α and COX-2.

305

Effect of E144 on the neuroinflammation in LPS-treated mouse brains. To assess

306

whether E144 inhibits neuroinflammation, we used the immune cells marker, Iba-1,

307

which is upregulated in reactive microglia, and is often used to visualize these cells. (43)

308

Thus, we determined the content of the expression of Iba-1 by western blot analysis.

309

As shown in Fig. 5A, Iba-1 was significantly enhanced in LPS-treated mice brain, but

310

it was significantly reduced by treatment with a positive control (genipin), (41,44) and

311

E144. To confirm that the Iba-1 was affected by treatment with E144, we performed an

312

immunofluorescence assay to observe the Iba-1 positive cells in mouse brains.

313

Consistent with the western blot data, histological Iba-1 immunoreactivity was detected,

314

as shown in Fig. 5B, which demonstrated that LPS obviously induced higher levels of

315

Iba-1 than the control treatment. In contrast to the LPS treated group, co-treatment with



316

E144 obviously attenuated the expression of Iba-1. This finding indicated that E144

317

may inhibit the neuroinflammation in an LPS-treated mouse model.

318

Effect of E144 on the neuroinflammation including COX-2 enzyme and Iba-1 in

319

the APPsw transgenic (TG) mouse model. Emerging evidence suggests that Aβ fibrils

320

trigger microglial responses characterized by the release of inflammatory mediators,

321

which contribute to the progression and severity of AD. (45) Thus, we evaluated whether

322

E144 inhibits neuroinflammatory responses during the development of AD in the

323

APPsw transgenic mouse model. We measured the expression of COX-2 in the cerebral

324

cortex of TG mice by western blot analysis and found that COX-2 levels were

325

significantly induced compared to normal mice with the same age background group.

326

Treatment with E144 at 1.5 mg/kg/day significantly reduced the over-expression of

327

COX-2 in AD transgenic mice (Fig. 6A). Furthermore, Iba-1 level were determined

328

using western blotting in Fig. 6B and confocal microscopy (Leica) in Fig. 6C, and Iba-

329

1-positive cells were quantified in Fig. 6D by using ImageJ (National Institutes of

330

Health, NIH Image in the public domain, USA). As shown in Fig. 6B, C, and D, there

331

were more Iba-1 positive cells in the TG mouse brains than background mouse brains,

332

and the number of Iba-1-marked cell was attenuated by treatment with E144. These

333

data indicated that E144 may inhibit the neuroinflammation in AD-TG mouse model.

334

In conclusion, CJ extract and its active compound E144 both attenuated the production

335

of pro-inflammatory mediators, including TNF-α, IL-6, NO, PGE2, iNOS, and COX-2.

336

Both of CJ extract and E144 increased the production of IL-10 in LPS-activated


Page 16 of 33

Page 17 of 33


337

microglia. An in vivo study revealed that E144 reduced the expression of TNF-α or

338

COX-2, and Iba-1 in both LPS-treated mice and AD-TG mice. This study supports our

339

previous finding that E144 may have neuroprotective effect on cognitive function and

340

Aβ production, possibly by reducing pro-inflammatory mediators and promoting anti-

341

inflammatory cytokines.

342

Supporting Information

343

Supporting Information presenting HPLC analysis of CJ extract, HPLC chromatograms

344

and NMR data for E144.

345

Funding

346

This work was funded by the National Research Foundation of the Ministry of Science,

347

ICT & Future Planning of Republic of Korea (NRF-2015M3A9A5030735) and the Bio-

348

Synergy Research Project (NRF-2012M3A9C4048793).

349

Notes

350

All authors declare no conflicts of interest.

351

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Figure legends

487

Fig. 1. Effect of CJ extract on cell viability and inflammatory mediators in BV-2 cells.

488

Cells were treated with the indicated concentrations of CJ extract for 12 h in the

489

presence or absence of LPS. (A) Cell viability measured using EZ-Cytox reagent (n =

490

6), and culture medium was measured using with specific ELISA kits for the

491

measurement of the pro-inflammatory factors (B) TNF-α (n = 4), (C) IL-6 (n = 4), (D)

492

NO production (n = 6), and the anti-inflammatory cytokine (E) IL-10 (n = 4). All data

493

are expressed as the mean ± SEM, **p < 0.01, ***p < 0.001 significantly different from

494

the control group, #p < 0.05, ##p < 0.01, and ###p < 0.001 significantly different from

495

the LPS group.

496

Fig. 2. Effect of E144 on cell viability and inflammatory factors in BV-2 cells. Cells

497

were treated with the indicated concentrations of E144 for 12 h in the presence or

498

absence of LPS. (A) Chemical structure and (B) Cell viability of E144 measured using

499

EZ-Cytox reagent (n = 6), and culture medium was measured with specific ELISA kits

500

for the measurement of the pro-inflammatory factors (C) TNF-α (n = 5), (D) IL-6 (n =

501

5), and anti-inflammatory cytokine (E) IL-10 (n = 5). All data are expressed as the mean

502

± SEM, **p < 0.01, ***p < 0.001 significantly different from the control group, #p

7-Deoxy-trans-dihydronarciclasine Isolated from Lycoris chejuensis

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