Leocarpinolide B attenuates LPS-induced inflammation on RAW264.7 macrophages by mediating NF-κB and Nrf2 pathways

Journal Pre-proof Leocarpinolide B attenuates LPS-induced inflammation on RAW264.7 macrophages by mediating NF-κB and Nrf2 pathways Ke-Gang Linghu, Qiu Shuo Ma, Guan Ding Zhao, Wei Xiong, Ligen Lin, Qing-Wen Zhang, Zhaoxiang Bian, Yitao Wang, Hua Yu PII:

S0014-2999(19)30806-4

DOI:

https://doi.org/10.1016/j.ejphar.2019.172854

Reference:

EJP 172854

To appear in:

European Journal of Pharmacology

Received Date: 19 May 2019 Revised Date:

4 December 2019

Accepted Date: 9 December 2019

Please cite this article as: Linghu, K.-G., Ma, Q.S., Zhao, G.D., Xiong, W., Lin, L., Zhang, Q.-W., Bian, Z., Wang, Y., Yu, H., Leocarpinolide B attenuates LPS-induced inflammation on RAW264.7 macrophages by mediating NF-κB and Nrf2 pathways, European Journal of Pharmacology (2020), doi: https://doi.org/10.1016/j.ejphar.2019.172854. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

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Leocarpinolide B attenuates LPS-induced inflammation on RAW264.7

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macrophages by mediating NF-κB and Nrf2 pathways

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Ke-Gang Linghua, Qiu Shuo Maa, Guan Ding Zhaoa, Wei Xionga, Ligen Lina,

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Qing-Wen Zhanga, Zhaoxiang Bianc, Yitao Wanga, Hua Yua,b,c*

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a

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Chinese Medicine, University of Macau 519000, Macao, China

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b

HKBU Shenzhen Research Center, Shenzhen 518000, Guangdong, China

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c

School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong 999077,

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Hong Kong, China

Institute of Chinese Medical Sciences, State Key Laboratory of Quality Research in

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*

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Hua YU

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Room 8008, Building N22,

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Institute of Chinese Medical Sciences, University of Macau,

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Avenida da Universidade, Taipa, Macao SAR, China

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Tel: +853-8822 8540

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E-mail: [email protected]

Correspondence to:

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ABSTRACT Macrophages-mediated inflammation is involved in the regulation of rheumatoid

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arthritis (RA). Sigesbeckiae Herba (SH) has been traditionally used for rheumatism.

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However, the bioactive ingredients of SH are still unclear. Recently, we isolated a

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compound (Leocarpinolide B, LB) from SH and identified its potent

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anti-inflammatory and antioxidant effects on RAW264.7 macrophages for the first

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time. LB effectively inhibited excessive production of nitric oxide (NO),

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prostaglandin E2 (PGE2), cytokines (IL-6, TNF-α and MCP-1), and the expression of

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cyclooxygenase-2 (COX-2) and inducible nitric oxide synthases (iNOS) in

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lipopolysaccharide (LPS)-induced RAW264.7 cells. LB blocked the degradation of

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inhibitor of kappa B ( IκBα) and translocation of nuclear factor kappa B (NF-κB) p65.

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Additionally, LB reduced the intracellular reactive oxygen species, and increased the

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expression of heme oxygenase-1 (HO-1) and the translocation of nuclear factor

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erythroid 2-related factor 2 (Nrf2) in the presence or absence of LPS. The results

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suggested that LB might be one of the bioactive components of SH, and be potential

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for the treatment of RA and valuable to be further investigated.

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Keywords: Rheumatoid arthritis (RA); Sigesbeckiae Herba (SH); Leocarpinolide B;

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Nuclear factor erythroid 2-related factor 2 (Nrf2) ; Nuclear transcription factor-kappa

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B (NF-κB).

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1. Introduction Rheumatoid arthritis (RA) is a kind of autoimmune disease mediated by multiple

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immune cells and their respective inflammatory mediators (Smolen et al., 2016).

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Macrophage is one of the most critical cells involved in the initiation and propagation

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of RA, activated macrophages could secret large amount of inflammatory mediators

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to drive persistent inflammation and joint destruction (Scott DL, Wolfe F, 2010).

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Therefore, a proper approach to reduce or reverse the excessive inflammatory

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mediators to maintain the normal physiological function of macrophage is especially

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significant.

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RAW264.7 cell is a kind of macrophage widely used for the research of

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inflammation (Li et al., 2018a; Ralph and Nakoinz, 1977). There are several signaling

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pathways closely related to the inflammation in RAW264.7 macrophages. Nuclear

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factor-kappa B (NF-κB) is a typical inflammation-related signaling pathway that

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regulates the expression of pro-inflammatory cytokines (Cao et al., 2019). Pathogenic

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microorganisms and pro-inflammatory mediators (such as interleukin (IL)-6, IL1β,

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tumor necrosis factor alpha) can trigger the NF-κB pathway (Lee et al., 2017). NF-κB

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is activated by phosphorylation of IκB via activating IKK in response to

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pro-inflammatory stimuli (Li et al., 2018b). Inhibition of the activation of NF-κB has

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been confirmed to ameliorate inflammation (Cao et al., 2019; Li et al., 2017).

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It is well believed that inflammation is linked with oxidative stress. Oxidative

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stress refers to elevated intracellular level of reactive oxygen species that is

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considered as the most potent inflammatory mediator (Shin et al., 2008). Antioxidants

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play a critical role in reducing inflammation (Arulselvan et al., 2016). The activation

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of nuclear factor erythroid 2-related factor 2 (Nrf2) pathway could block the

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progression of inflammation (Ahmed et al., 2017). Nrf2-mediated antioxidant gene

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expression could reduce the macrophage M1 phenotype and reactive oxygen species

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production (Jaiswal, 2004; Kobayashi et al., 2016). Nrf2 regulates the expression of

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second-stage detoxification enzymes including glutathione peroxidase, heme

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oxygenase-1 (HO-1) gene and antioxidants, which protect cells from various damages

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through their anti-inflammatory effects, thereby affecting the progression of the 3

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disease (Lee et al., 2017; Li et al., 2019).

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Sigesbeckiae Herba (SH) is a traditional anti-inflammatory herbal medicine,

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which has been used for rheumatism since Tang dynasty in China (Zhang et al., 2019).

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We have focused on the modern pharmacological research of SH in the past years, and

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identified its anti-inflammatory effects and anti-arthritis properties (Chu et al., 2018;

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Sang et al., 2018; Zhang et al., 2019; Zhong et al., 2019). However, the bioactive

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ingredients of SH are still unclear. Recently, we isolated several compounds from SH

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and identified that Leocarpinolide B (LB, C22H26O8) exhibited potent

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anti-inflammatory effects on LPS-induced RAW264.7 cells, which suggested the

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potential contribution of LB for HS on RA treatment. LB is a sesquiterpene lactones

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firstly isolated and reported in 1992 by Macías & H. Fischer from Lecocarpus

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pinnatifidus (Macías and H. Fischer, 1992). Cartagena E et al reported that LB from

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Acanthospermum hispidum exerted antibacterial action by inhibiting the production of

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biofilm (Cartagena et al., 2007). In this study, we isolated LB from Sigesbeckia and

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evaluated the anti-inflammatory effects and potential molecular mechanisms of this

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compound on RAW264.7 cells for the first time. The present study provides research

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basis and experimental data for developing new treatments on RA with LB.

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2. Materials and Methods

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2.1 Chemicals and reagents

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Acetonitrile (ACN, HPLC grade) and methanol (HPLC grade) were purchased

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from RCI Labscan Limited (Thailand). Phosphoric acid (analytical grade) was

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purchased from Sigma Chemicals Ltd. (St. Louis, MO, USA). Milli-Q water was

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prepared using a Milli-Q system (Millipore, MA, USA).

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3-[4, 5-Dimethyl-2-thiazolyl]-2, 5-diphenyltetrazolium bromide (MTT),

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dimethyl sulfoxide (DMSO) and lipopolysaccharides (LPS) from Escherichia coli

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O111:B4 were purchased from Sigma-Aldrich (St. Louis, MO, United States).

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Dulbecco's modified eagle's medium (DMEM), fetal bovine serum (FBS),

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phosphate-buffered saline (PBS), penicillin-streptomycin (10,000 U/ml, P/S), 0.25%

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Trypsin-EDTA (w/v), Nuclear and Cytoplasmic Protein Extraction Kit and Pierce 4

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LDH Cytotoxicity Assay Kit were obtained from Thermo Fisher Scientific (Waltham,

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MA, USA). Enzyme-linked immunosorbent assay (ELISA) kits were purchased from

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Neobioscience Technology Co., Ltd. (Shenzhen, China). ELISA kit for PGE2 were

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from Cayman Chemical (Cayman Chemical, Ann Arbor, MI, United States).

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Radioimmunoprecipitation assay (RIPA) lysis buffer was obtained from Beyotime

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Biotechnology (Jiangsu, China). Chemiluminescent kit was supplied by GE

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Healthcare UK Limited (Buckinghamshire, HP7 9NA, UK). Primary antibodies

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against phosphorylation (p)-IKKα/β, IKKα, IKKβ, p-IκBα, IκBα, p-NFκB p65,

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COX-2, iNOS, HO-1, Nrf2, Lamin B2 and GAPDH (glyceraldehyde-3-phosphate

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dehydrogenase), and the secondary antibody were purchased from Cell Signaling

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Technology (Danvers, MA, United States).

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2.2 Preparation of LB

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The SH samples were authenticated by the corresponding author of Dr. Hua YU.

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The voucher specimens (SG-003) were deposited at the Institute of Chinese Medical

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Sciences, University of Macau, Macao, China.

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The powdered SH herb (700g) was extracted thrice with 50% ethanol (1:10, w/v)

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for 1 h each under reflux. The combined extracts were filtered with filter paper after

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cooling and then concentrated under reduced pressure to a appreciate volume (700 ml).

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The concentrated extract was loaded onto a macroporous resin (D101) column (~700

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ml) and allowed for statically-adsorbing overnight. Subsequently, the sample was

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washed with distilled water (10-fold column volume) and then eluted with 8-fold

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column volume of 95% ethanol. The collected eluent was concentrated under reduced

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pressure to 200 ml and extracted 3 times with 200 ml of ethyl acetate. The combined

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ethyl acetate extract was dried under nitrogen at room temperature. The dried extract

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was dissolved in methanol and separated using a preparative liquid chromatographic

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system. The corresponding chromatographic peaks were collected, concentrated and

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lyophilized to obtain LB. The purity of LB was >98% (detected by HPLC analysis).

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Spectroscopy data of the prepared LB were in agreement with those reported in the

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literature (Macías and H. Fischer, 1992).

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2.3 Cell culture

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RAW264.7 cells were purchased from the American Type Culture Collection

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(ATCC; Manassas, VA, USA). The cells were cultured with 10% heat-inactivated FBS

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and 1% P/S in the atmosphere of 95% humidity and 5% CO2 at 37 °C. When the cells

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reached 80% confluence, the cells were sub-cultured after scraping from a flask (25

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cm 2 ; Thermo Fisher Scientific, MA, USA).

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2.4 Cytotoxicity assay

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RAW264.7 cells were seeded in 96-well plates (1 x 104 cells/well) and allowed

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to adhere overnight. Cells were co-treated with the indicated concentrations of LB for

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24 h in the absence or presence of LPS (1 µg/ml). Thereafter, the supernatant was

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collected to determine the release of lactate dehydrogenase (LDH) according to the

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manufacture’s protocol. The cells in the plate were incubated for an additional 3 h

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with fresh medium containing 0.5 mg/ml MTT. The supernatant was removed, and the

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absorbance of the dissolved precipitate (in 150 µl of DMSO) was measured at 490 nm

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using a microplate reader (FlexStation 3; Molecular Devices, USA).

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2.5 Measurement of NO

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RAW264.7 cells were seeded in 96-well plates (2 x 104 cells/well) and allowed

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to adhere overnight. Cells were pretreated with indicated concentrations of LB for 1 h,

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then stimulated with LPS (1µg/ml) for 12 h. Subsequently, NO production was

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determined by measuring the nitrite accumulated in the medium with Griess reagent.

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The absorbance of the final reaction solution was measured at 540 nm using a

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microplate reader.

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2.6 ELISA assay

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RAW264.7 cells were seeded in 24-well plates (1 x 105 cells/well) and allowed

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to adhere overnight. Cells were pretreated with LB (5, 10, 20 µM) for 1 h, then

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stimulated with LPS (1µg/ml) for 12 h. The cytokines (TNF-α, IL-6 and MCP-1) and

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prostaglandin E2 (PGE2) in the supernatant were detected using an ELISA kit

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according to the manufacturer's instructions.

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2.7 Determination of intracellular reactive oxygen species levels

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RAW264.7 cells were seeded in 12-well plates (2 x 105 cells/well) and allowed 6

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to adhere overnight. Cells were pretreatment with LB (5, 10, 20 µM) for 1 h, then

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stimulated with LPS (1µg/ml) for 12 h. The cells were scraped from the plates after

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washing once with PBS, then centrifugated 5 min at 200×g.

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Dichloro-dihydro-fluorescein diacetate (DCFH-DA, 10 µM in DMEM) was added to

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the cells and incubated the cells for 30 min at room temperature. After that, the cells

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were washed twice with PBS. Finally, the cells were resuspended into PBS and then

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taken photos by immunofluorescence microscope and count by flow cytometer.

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2.8 Western blot analysis

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Total protein was extracted from the harvested cells with RIPA buffer containing

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a mixture of 1 mM phenylmethylsulfonyl fluoride and protease inhibitor. Nuclear

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Protein was extracted with Nuclear and Cytoplasmic Protein Extraction Kit according

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to the manufacture’s protocol. Proteins (15-40 µg per sample) were isolated by

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SDS-PAGE (8%-12%) and then transferred to a polyvinylidene fluoride (PVDF)

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membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5%

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defatted dry milk for 2 h and then incubated overnight at 4 °C with antibodies against

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p-IKKα/β, IKKα, IKKβ, p-IκBα, IκBα, p-NFκB p65, COX-2, iNOS, HO-1, Nrf2,

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Lamin B2 and GAPDH. After washing, the membrane was incubated with the

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secondary antibody for 1.5 h at room temperature. The blot was visualized using an

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enhanced chemiluminescence kit. Digital images of the blots were generated by a

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Syngene gel imaging system (Bio-Rad) and quantified using Syngene software.

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2.9 Immunofluorescence staining

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RAW264.7 cells were seeded at 5 x 105 cells per dish into a confocal culture dish

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(NEST Biotechnology Co., Ltd; Wuxi, Jiangsu, China). Overnight, the cells were

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pre-treated with LB for 1 h then stimulated with LPS (1 µg/ml) for 1h. After treatment,

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cells were washed once with PBS, fixed with 4% paraformaldehyde for 10 min,

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washed three times with washing solution (PBS containing 0.1% Triton X-100 ), and

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blocked with 3% BSA (containing 0.1% Triton X-100 ) for 1 h at room temperature.

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Then, the cells were incubated with primary antibody against NF-κB p65 (Cell

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Signaling Technology, #8242) for 1 h at room temperature, washed three times with

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washing solution, and then incubated with Aelxa 594 Fluor (red) secondary antibody 7

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for 1 h at room temperature. Finally, cells were stained with

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4',6-diamidino-2-phenylindole (DAPI) for 4 min, then observed with a confocal laser

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microscope (Leica, Buffalo Grove, USA).

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2.10 Statistical analysis Data were analyzed using GraphPad Prism 6.0 software. All experimental data

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are presented as mean ± S.D., and each experiment was performed at least three times.

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Significant differences between groups were determined using a one-way ANOVA

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with Dunnet’s multiple comparisons test; P < 0.05 was considered difference

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significantly.

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3. Results

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3.1. Toxicity of LB on RAW264.7 cells

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The MTT results showed that LB at concentrations of 0.0625–20 µM for 24 h

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was non-toxic to RAW264.7 cells (Fig. 1B), and the LB (0.0625–20 µM) attenuated

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the LPS-induced cell injury when co-incubated with LPS (1µg/ml) for 24 h (Fig. 1C).

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Lactate dehydrogenase (LDH) in the culture medium is an index to evaluate the

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integrity of cell membrane, the Fig. 1D showed that LB reduced the LDH release

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induced by LPS (1µg/ml) dose-dependently and LB (20 µM) alone did not lead to the

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release of LDH. These results show that LB (≦20 µM) was non-toxic to RAW264.7

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cells and rescued the cells from LPS-induced injury.

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3.2. LB reduced the production of NO and pro-inflammatory proteins in

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LPS-stimulated RAW264.7 cells

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Excessive NO is a classical marker for inflammation in activated macrophages

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(Wu et al., 2018). As Fig. 2A shows, LPS induced the dramatic increase of NO, which

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could be significantly reduced by LB (1.25–20 µM) in a concentration dependent

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manner. When inflammation occurs, activated macrophages would secrete large

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amounts of pro-inflammatory proteins to aggravate inflammation (Su et al., 2017). As

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shown in Fig. 2B-E, LPS increased the expression of IL-6, TNF-a, MCP-1 and PGE2,

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which could be reduced by the pretreatment of LB.

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3.3. LB inhibited the reactive oxygen species production in LPS-stimulated RAW264.7 8

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cells It is well accepted that inflammation is linked with oxidative stress. Oxidative

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stress refers to elevated intracellular levels of reactive oxygen species that is

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considered as the most potent inflammatory mediators (Ji et al., 2018). As shown in

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Fig. 3A&B, LPS (1 µg/ml, 12 h) induced the excessive reactive oxygen species

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production, LB inhibited the LPS-induced reactive oxygen species dose-dependently.

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The Fig. 3C&D showed that LB significantly decreased the fluorescence intensity

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compared to LPS group, these data reconfirmed that LB could reverse LPS-induced

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reactive oxygen species level.

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3.4. LB suppressed the expression of iNOS and COX-2 in LPS-stimulated RAW264.7

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cells

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It has been reported that iNOS and COX-2 play an important role in the

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inflammatory process (Fu et al., 2014). Based on the significant inhibition of NO and

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PGE2 by LB, we examined the expression of iNOS and COX-2. As shown in Fig. 4,

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LPS induced the expression of iNOS and COX-2, which could be reduced by LB

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dose-dependently.

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3.5. LB blocked the activation of NF-κB in LPS-stimulated RAW264.7 cells

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NF-κB is a typical signaling pathway involved in LPS-mediated inflammation

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(Mingfeng et al., 2014). As shown in Fig. 5A&B, the expression of p-IKKα/β and

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p-IκBα in the LB group was lower than LPS group, which indicated the LB could

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reduce the phosphorylation of IKKα/β (Fig. 5A) and IκBα. Fig. 5C&D showed that

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LB decreased the expression of NF-κB p65 in nucleus and reduced the nuclear

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translocation of NF-κB p65. These data indicated that LB blocked LPS-induced

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NF-κB signaling pathway.

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3.6. LB increased the translocation of Nrf2 and the expression of HO-1 in

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LPS-stimulated or unstimulated RAW264.7 cells

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The activation of Nrf2 pathway could inhibit the progression of inflammation

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(Abd El-Twab et al., 2019). As shown in Fig. 6A, LB increased the expression of Nrf2

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in nucleus in the presence or absence of LPS, which suggested LB could activate the

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Nrf2. Nrf2 increased the transcription of its target genes, including HO-1 and NQO1 9

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(Abd El-Twab et al., 2019). Antioxidant protein HO-1 has anti-oxidative and

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anti-inflammatory effects (Pooladanda et al., 2019). In order to understand whether

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LB could exert its anti-inflammatory effects through activating Nrf2 pathway, the

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downstream protein HO-1 of Nrf2 pathway was investigated. As shown in Fig. 6B,

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the expression of HO-1 was increased significantly in the presence or absence of LPS.

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These data suggested that LB could increase the transcription of Nrf2, and further

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up-regulate the protein expression of HO-1.

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4. Discussion Sigesbeckiae Herba (SH) is a traditional Chinese medicine used for chronic

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inflammatory diseases, especially for rheumatoid arthritis (RA). Based on our

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previous research on SH since 2015, we isolated and identified an active

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sesquiterpene lactone (Lecocarpinolide B, LB) which shows stronger NO inhibitory

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effect and better solubility than kirenol, one of the main compounds widely reported

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in the literatures (Kim et al., 2014; Lu et al., 2012; Wang et al., 2011; Xiao et al.,

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2015). Further investigations showed that LB inhibited the production of TNF-α, IL-6,

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MCP-1 and PGE2 in LPS-induced RAW264.7 macrophages (Fig. 2), suggesting the

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potentiality of LB for developing an anti-inflammatory drug against RA and/or other

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chronic inflammatory diseases.

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COX-2 and iNOS are two critical proteins in the process of initiation and

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progression of inflammation, both of them are reported to be the trigger of

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pro-inflammatory mediators (NO, PGE2, TNF-α, IL-6 and MCP-1), inhibition of these

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two proteins ameliorated the inflammation (Kwon et al., 2013). COX-2 is also a key

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target for non-steroidal anti-inflammatory drugs (NSAIDs) in anti-inflammation. As

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shown in Fig. 4, LB reduced the expression of these two proteins, which was well

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consistent with the strong inhibition on the production of NO and PGE2 (Fig. 2).

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NF-κB has been confirmed to be one of the up-stream signals of inflammation

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cascade reaction, inhibition of NF-κB reduced the expression of COX-2 and iNOS

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and decreased the production of inflammatory mediators (Park et al., 2007). NF-κB

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has been widely reported to be involved in the modulation of RA (Makarov, 2001). 10

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Therefore, we examined the effects of LB on NF-κB, the results in Fig. 5 showed that

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LB blocked the NF-κB p65 activation and translocation from the cytoplasm to nucleus

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by inhibition the phosphorylation of IKKα/β and IκBα. Similarly, Helenalin, an

287

anti-inflammatory sesquiterpene lactone from Arnica, selectively inhibits transcription

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factor NF-κB (G et al., 1997). Parthenolide, a sesquiterpene lactone, has been well

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known to be an anti-inflammatory agent that inhibits NF-κB activation (Kwok et al.,

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2001; Mathema et al., 2012).

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Reactive oxygen species is also a critical mediator of oxidative stress and

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inflammation, and involved in the development and deleterious stage of several

293

inflammatory diseases (Mittal et al., 2014). Dichloro-dihydro-fluorescein diacetate

294

(DCFH-DA) is a cell-permeable fluorescent probe and has been widely used to detect

295

intracellular production of reactive oxygen species (Nitrogen, 2017), it is commonly

296

used for measuring reactive oxygen species or oxidant stress in cells or tissues in

297

combination with confocal microscopy or flow cytometry (Shen et al., 2018). As

298

shown in Fig. 3, the positive cell counting and fluorescence images all revealed that

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LB reduced the intracellular reactive oxygen species in LPS-induced RAW264.7 cells,

300

and the LB alone did not alter the level of reactive oxygen species in normal cell.

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Therefore, we further detected the effects of LB on Nrf2 which was a domain

302

mediator of oxidative stress. As shown in Fig. 6A, LB increased the expression of

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Nrf2 in nucleus in the presence or absence of LPS, LB also increased the expression

304

of HO-1 (Fig. 6B) which was a critical anti-oxidative and anti-inflammatory protein

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in the down-stream of Nrf2 signal pathway (Pooladanda et al., 2019). These data

306

suggested that the attenuation of LB on oxidative stress might be involved in the

307

activation of Nrf2/HO-1 signal pathway.

308

Sesquiterpenoids are groups of important bioactive ingredients in SH herbs.

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Based on the chemical structures, most of them are germacrane-type sesquiterpenoids

310

(including LB) (Liu et al., 2019), while only a few were cadinane-type and

311

eudesmane-type squiterpenoids (Sakuda, 1987). The reported pharmacological

312

activity of SH sesquiterpenoids including anti-cancer, anti-microbial and

313

anti-inflammation. In one recent study, Liu et al isolated 27 germacrane-type 11

314

sesquiterpenoids from SH and some of them presented anti-cancer activities against

315

human A549 and MDA-MB-231 cells in vitro (Liu et al., 2019). In addition, Jang et al

316

compared the anti-inflammatory activities of 21 compounds isolated from SH, and

317

found that highly oxygenated germacrane-type sesquiterpenoids could significantly

318

inhibit LPS-induced NO production in RAW 264.7 macrophages (Jang et al., 2018).

319

However, due to the limited amount of the obtained compounds, the potential

320

pharmacological mechanisms of these sesquiterpenoids were not further investigated

321

and reported. In this study, a germacrane-type sesquiterpenoid of LB was purified

322

from the SH extract and presented potent anti-inflammatory effect in LPS-induced

323

RAW 264.7 macrophages through regulating NF-κB and Nrf2 signaling pathways. In

324

addition, the structure-activity relationship among the SH sesquiterpenoids to their

325

anti-inflammatory activities should be further investigated and elucidated.

326

In summary, the present study isolated and identified the anti-inflammatory and

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anti-oxidative activities of natural compound (LB) from SH for the first time, and the

328

mechanisms of these actions could at least be related to the inhibition of

329

NF-κB-mediated pro-inflammatory mediators and the activation of Nrf2-mediated

330

anti-oxidant proteins.

331 332 333

Conflict of interest The authors declare that there is no conflict of interest.

334 335 336

Acknowledgments This work was supported by grants from the National Natural Science

337

Foundation of China [NSFC, No. 81470170], the Science and Technology

338

Development Fund, Macau SAR [File No. 0096/2019/A2] and the Research

339

Committee of the University of Macau [MYRG2017-00178-ICMS and

340

MYRG2018-00043-ICMS].

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Figure legends

513

Fig. 1 Toxicity of Lecocarpinolide B (LB) on RAW264.7 cells. The chemical structure

514

of LB drawn by ChemDraw software (A). RAW264.7 cells were treated with LB

515

(0.625-40 µM) in the absence (B) or presence (C) of LPS (1µg/ml) for 24 h, then the

516

cell viability was detected by MTT assay. RAW264.7 cells were treated with LB (5,

517

10, 20 µM) in the absence or presence of LPS (1µg/ml) for 24 h, then the lactate

518

dehydrogenase (LDH) in the culture medium was determined by LDH assay kit (D).

519

Data are mean ± S.D. of minimum three independent experiments. * P<0.05, **

520

P<0.01 and ***P<0.001 versus Control; ## P<0.01 and ### P<0.001 versus LPS; No

521

significance (NS).

522

Fig. 2 Lecocarpinolide B (LB) reduced the production of NO and pro-inflammatory

523

proteins in LPS-stimulated RAW264.7 cells. Cells were pretreatment indicated

524

concentrations of LB for 1 h, then stimulated with LPS (1µg/ml) for 12 h, the

525

supernatant was collected for the detection of NO with Griess reagent (A), detection

526

of IL-6 (B), MCP-1 (C), TNF-α (D), PGE2 (E) with ELISA kits. Data are mean ± S.D.

527

of minimum three independent experiments. *** P<0.001 versus Control; # P<0.05, ##

528

P<0.01 and ### P<0.001 versus LPS.

529

Fig. 3 Lecocarpinolide B (LB) inhibited the reactive oxygen species production in

530

LPS-stimulated RAW264.7 cells. Cells were pretreatment with LB (5, 10, 20 µM) for

531

1 h, then stimulated with LPS (1µg/ml) for 12 h. After incubation the cells with

532

DCFH-DA (10 µM in DMEM) for 30min at room temperature, the positive cells were

533

used to count by flow cytometer (A&B) and visualize with immunofluorescence

534

microscope (C&D). Data are mean ± S.D. of minimum three independent experiments.

535

***

536

significance (NS).

537

Fig. 4 Lecocarpinolide B (LB) suppressed the expression of iNOS and COX-2 in

538

LPS-stimulated RAW264.7 cells. Cells were pretreatment with LB (5, 10, 20 µM) for

539

1 h, then stimulated with LPS (1µg/ml) for 12 h. The expression of COX-2 and iNOS

540

were analyzed by western blotting. Data are mean ± S.D. of minimum three

P<0.001 versus Control; # P<0.05, ## P<0.01 and ### P<0.001 versus LPS; No

19

541

independent experiments. *** P<0.001 versus Control; # P<0.05 and ### P<0.001

542

versus LPS.

543

Fig. 5 Lecocarpinolide B (LB) blocked the activation of NF-κB in LPS-stimulated

544

RAW264.7 cells. Cells were pretreatment with LB (5, 10, 20 µM) for 1 h, then

545

stimulated with LPS (1µg/ml) for 1 h. The phosphorylation of IKKα/β (A),

546

phosphorylation of IκBα (B), the expression of p65 in nucleus (C), the translocation

547

of p65 to nucleus (D). Data are mean ± S.D. of minimum three independent

548

experiments. *** P<0.001 versus Control; ### P<0.001 versus LPS.

549

Fig. 6 Lecocarpinolide B (LB) increased the translocation of Nrf2 and the expression

550

of HO-1 in LPS-stimulated or unstimulated RAW264.7 cells. Cells were pretreatment

551

with LB (5, 10, 20 µM) for 1 h, then stimulation with LPS (1µg/ml) for 1 h. The

552

expression of HO-1 in the total proteins (A) and the expression of Nrf2 in nucleus (B)

553

were analyzed by western blotting. Data are mean ± S.D. of minimum three

554

independent experiments. ** P<0.01 and *** P<0.001 versus Control.

20

555 556

Fig. 1. Toxicity of Lecocarpinolide B (LB) on RAW264.7 cells

557

21

558

559 560

Fig. 2. Lecocarpinolide B (LB) reduced the production of NO and pro-inflammatory

561

proteins in LPS-stimulated RAW264.7 cells

22

562 563

Fig. 3. Lecocarpinolide B (LB) inhibited the reactive oxygen species (ROS)

564

production in LPS-stimulated RAW264.7 cells

565

23

566

567 568

Fig.4. Lecocarpinolide B (LB) suppressed the expression of iNOS and COX-2 in

569

LPS-stimulated RAW264.7 cells

570

24

571 572

Fig. 5. Lecocarpinolide B (LB) blocked the activation of NF-κB in LPS-stimulated

573

RAW264.7 cells

574

25

575

576 577

Fig. 6. Lecocarpinolide B (LB) increased the translocation of Nrf2 and the expression

578

of HO-1 in LPS-stimulated or unstimulated RAW264.7 cells

579

26