HO-1 signaling pathways

Journal Pre-proof Anti-inflammatory effects of Aureusidin in LPS-stimulated RAW264.7 macrophages via suppressing NF-κB and activating ROS- and MAPKs-dependent Nrf2/HO-1 signaling pathways

Jie Ren, Dan Su, Lixia Li, Heng Cai, Meiju Zhang, Jingchen Zhai, Minyue Li, Xinyue Wu, Kun Hu PII:

S0041-008X(19)30454-5

DOI:

https://doi.org/10.1016/j.taap.2019.114846

Reference:

YTAAP 114846

To appear in:

Toxicology and Applied Pharmacology

Received date:

4 July 2019

Revised date:

17 November 2019

Accepted date:

28 November 2019

Please cite this article as: J. Ren, D. Su, L. Li, et al., Anti-inflammatory effects of Aureusidin in LPS-stimulated RAW264.7 macrophages via suppressing NF-κB and activating ROS- and MAPKs-dependent Nrf2/HO-1 signaling pathways, Toxicology and Applied Pharmacology (2019), https://doi.org/10.1016/j.taap.2019.114846

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© 2019 Published by Elsevier.

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Anti-inflammatory effects of Aureusidin in LPS-stimulated RAW264.7 macrophages via suppressing NF-κB and activating ROS- and MAPKs-dependent Nrf2/HO-1 signaling pathways Jie Ren a†*, Dan Su b† , Lixia Li a, Heng Cai a, Meiju Zhang a, Jingchen Zhai a

, Minyue Li a, Xinyue Wu a, and Kun Hu a

a

School of Pharmaceutical Engineering & Life Science, Changzhou University,

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Changzhou, Jiangsu, 213164, People’s Republic of China. b

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Changzhou No.2 People's Hospital，Changzhou, Jiangsu，213164，P. R. China.

†

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J. R. and D. S. contributed equally to this work.

Abbreviations: Akt, protein kinase B; CO, carbon monoxide; COX-2, cyclooxygenase-2; DCFH-DA, 2',7'-dichlorodihydrofluorescein diacetate; DM EM , Dulbecco’s modified Eagle’s medium; DM SO, dimethyl sulfoxide; DS, Discover Studio; ELISA, enzyme linked immunosorbent assay; ERK, extracellular signal-regulated kinase; FBS, Fetal bovine serum; FITC, fluoresceine isothiocyanate; HO-1, heme oxygenase; HRP, horseradish peroxidase; IκBα, inhibitor α of NF-κB; iNOS, inducible nitric oxide synthase; IL-1β, Interleukin-1β; IL-6, interleukin-6; JNK, Jun N-terminal kinase; Keap1, Kelch like ECH associated protein1; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; M TT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NAC, N-acetylcysteine; NF-κB, nuclear factor-κB; Nrf2, nuclear factor E2-related factor 2; NO, nitric oxide; NQO1, NAD(P)H:quinone oxidoreductase 1; NSAIDs, Non-steroidal anti-inflammatory drugs; PGE2, prostaglandin E2; PVDF, polyvinylidenefluoride; qRT-RCR, quantitative real time polymerase chain reaction; RIPA, Radio-Immunoprecipitation Assay; ROS, reactive oxygen species; SD, standard deviation; SD S-PAGE, sodium dodecylsulphate polyacrylamide gel electrophoresis; TNF-α, tumor necrosis factor-α.  Correspondence to: Dr. Jie Ren, School of Pharmaceutical Engineering & Life Science, Changzhou University, 1 Gehu Road, Wujin district, Changzhou, Jiangsu 213164, P. R. China. Email addresses: [email protected]. Ms. Dan Su, Changzhou No.2 People's Hospital, 68 Gehu Road, Wujin district, Changzhou, Jiangsu 213164, P. R. China. Email addresses: [email protected].

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ABSTRACT Aureusidin, a naturally-occurring flavonoid, is found in various plants of Cyperaceae such as Heleocharis dulcis (Burm. f.) Trin., but its pharmacological effect and active mechanism are rarely reported. This study aimed to investigate the anti- inflammatory effect and action mechanism of Aureusidin in LPS-induced mouse

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macrophage RAW264.7 cells. The results suggested that lipopolysaccharide

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(LPS)- induced nitric oxide (NO), tumor necrosis factor-α (TNF-α) and prostaglandin

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E2 (PGE2) production were obviously inhibited by Aureusidin. Moreover, Aureusidin

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also significantly decreased the mRNA expression of various inflammatory factors in

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LPS-stimulated RAW264.7 cells. Furthermore, mechanistic studies showed that Aureusidin significantly inhibited nuclear transfer of nuclear factor-κB (NF-κB),

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while increasing the nuclear translocation of nuclear factor E2-related factor 2 (Nrf2)

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as well as expression of Nrf2 target genes such as heme oxygenase (HO-1) and

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NAD(P)H:quinone oxidoreductase 1 (NQO1), but the addition of the HO-1 inhibitor Sn-protoporphyrin (Snpp) significantly abolished the anti- inflammatory effect of Aureusidin in LPS-stimulated RAW264.7 cells, confirming the view that HO-1 was involved in the anti- inflammatory effect. In addition, Aureusidin increased the levels of reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) phosphorylation in RAW264.7 cells. Antioxidant N-acetylcysteine (NAC) or three MAPK inhibitors blocked the nuclear translocation of Nrf2 and HO-1 expression induced by Aureusidin, indicating that Aureusidin activated the Nrf2/HO-1 signaling pathway through ROS and MAPKs pathways. At the same time, co-treatment with the

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NAC blocked the phosphorylation of MAPKs. Results from molecular docking indicated that Aureusidin inhibited the NF-κB pathway by covalently binding to NF-κB. Thus, Aureusidin exerted the anti- inflammatory activity through blocking the NF-κB signaling pathways and activating the MAPKs and Nrf2/HO-1 signaling pathways. Based on the above results, Aureusidin may be an attractive therapeutic

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candidate for the inflammation-related diseases.

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Keywords

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Aureusidin; Inflammation; NF-κB; MAPKs; Nrf2; HO-1

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1 Introduction Inflammation is a series of complex defense-related reactions caused by various damage factors. Any inflammation begins with metamorphism, after exudation defense, and finally proliferative repair (Zhang et al., 2017). Chronic inflammation is mainly caused by hyperplasia, usually with lymphocytes and plasma. Cell infiltration

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is the main pathological manifestation (Shieh et al., 2015). In general, inflammation is

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potentially harmful, and the inflammatory response is the basis of some diseases, such

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as severe hypersensitivity, when the inflammation is too severe, it can threaten the

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patient's life (Shen et al., 2019).

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The characteristics of inflammatory diseases are complex and difficult to cure, so the establishment of an inflammatory model has practical significance for screening

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the inflammatory drugs and treating the inflammatory diseases (Jang et al., 2014).

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LPS is recognized and combined by receptors distributed on the membrane of

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macrophages, dendritic cells and B cells to trigger innate immunity, stimulate immune cells to release inflammatory factors, and cause local inflammation (Volk et al., 2014). In addition, it can cause sepsis and multiple organ dysfunction. NO is an important signal molecule in the body, which is produced by inducible nitric oxide synthase (iNOS) catalyzing L-arginine (Karan and Dubey, 2016). iNOS is a carrier of gas-to-cell intercellular information transmission, which is activated in the inflammatory response, which increases iNOS expression and catalyzes NO production. Excessive NO can induce the development and development of inflammatory diseases (Wang et al., 2017). Therefore, the excessive secretion of NO

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is effectively inhibited as one of the important measures to control the inflammatory response. TNF-α is mainly secreted by mononuclear macrophages, which activates the cytokine cascade in inflammatory responses and induces macrophage to produce Interleukin-1β (IL-1β) and interleukin-6 (IL-6) (Park et al., 2010). PGE2 is a kind of prostaglandin mainly produced by cyclooxygenase-2 (COX-2), which is both an

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inflammatory mediator and an immune regulator, and participates in a series of

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physiological and pathological processes. COX-2 can also regulate the activity of

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NF-κB and other transcription factors, and synergistic inflammation (Kang et al.,

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2018).

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NF-κB, as a multi-directional, pleiotropic regulator, is at the core of inflammation-anti- inflammatory (Han et al., 2017). More and more studies have

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shown that the activation of NF-κB induced by LPS exerts an important effect in the

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occurrence and development of many critical illnesses. After LPS interacts with the

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corresponding receptors on the macrophage membrane, it initiates intracellular signal transduction and activates NF-κB, which initiates transcription of various inflammatory factor genes, leading to the releasing in large quantities of inflammation factors such as TNF- and IL-1, enhancing and amplifying the inflammatory response (Jin et al., 2014). Nrf2 is a key and important transcription factor, which regulates the expressions of the phase II detoxification enzymes and a series of antioxidant enzymes (Chen et al., 2015). HO-1, as a phase II detoxification enzyme regulated by Nrf2, plays an important role not only in anti-oxidation but also in suppressing immune response and

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inducing immune tolerance (Suh et al., 2006). In addition, the metabolite carbon monoxide (CO) produced by HO-1 by catalyzing the heme reaction also has anti- inflammatory, anti-apoptotic, and diastolic blood vessels and other tissue protection effects. ROS is a small highly reactive molecule. Various defense mechanisms may be activated in intracellular processes with the accumulation of ROS.

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Furthermore, ROS are products of normal cellular metabolism. The balance between

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anti-oxidative and pro-oxidative system can be broken due to excessive ROS (Bao et

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al., 2018). MAPKs including extracellular signal- regulated kinase (ERK), Jun

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N-terminal kinase (JNK), and p38 which were regulated via phosphorylation cascade

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are associated with inflammation-related disorders (Koo et al., 2014). Non-steroidal anti- inflammatory drugs (NSAIDs) regulate inflammation- induced

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diseases by inhibiting cyclooxygenase expression, and these drugs are also used for

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cardiovascular disease and tumor prevention (Wu et al., 2004). There are many kinds

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of inflammation, various inducing factors and complicated pathogenesis. Therefore, anti- inflammatory drugs are widely used in clinical practice and are one of the most widely used drugs in the world. Natural products play an important role in the discovery of novel lead compounds and new chemical entities, and much effort has been directed toward the search for compounds or herbs that treat all kinds of diseases. As a kind of flavone, Aurones have a wide range of biological activities, such as anti-tumor, anti-oxidation, anti- microbial and other activities. However, Aureusidin, a naturally-occurring representative aurones, is found in various plants of Cyperaceae such as Heleocharis dulcis (Burm. f.) Trin., and has potential anti-antioxidant activity

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(Roussaki et al., 2014), but its anti- inflammatory effect and action mechanism have not been investigated. The purpose of this study is mainly to explore the potentially signaling pathway for the anti-inflammatory activity of Aureusidin. 2 Materials and Methods 2.1 Chemicals and reagents Aureusidin (Fig. 1A) was chemically synthesized by Dr. Kun Hu and the purity

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of compound was > 95% (Hu et al., 2011). Dulbecco’s modified Eagle’s medium

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(DMEM) was obtained from Gibco (Carlsbad, CA, USA). Fetal bovine serum (FBS)

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and trypsin were from PAA Laboratories (Pasching, Austria). ELISA Kit was

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purchased from Shuangying Biological Technology Co., Ltd. (Shanghai, China). Griess reagent and all the antibodies except for iNOS, COX-2, and NF-κB from

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Solarbio Life Science (Beijing, China), were purchased from Beyotime (Shanghai,

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China). SB203980, PD98059 and SP600125 were purchased from Spectrum

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Vibration Biotechnology (Shanghai, China). Quantitative real time polymerase chain reaction (qRT-PCR) reagents were obtained from TransGen Biotech Co., Ltd. (Beijng, China). All the primers were synthesized by General Biosystems, Inc. (Anhui, China). LPS and other reagents not referred were obtained from Sigma (St. Louis, MO, USA). 2.2 Cell culture and cell viability assay RAW264.7 cells (obtained from Cell Source Center, Chinese Academy of Science) were cultured in DMEM culture medium (containing 8% FBS, 100 U/mL penicillin and 100 μg/ml streptomycin) at 37 °C incubator with 5% CO 2 . Cells in 96-well plates were co-treated with Aureusidin (1,10, 20, 40, 60, 80 and 100 μM) and

Journal Pre-proof LPS (250 ng/mL) for 24 h (Ren et al, 2019). Then, each well was added 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution (5 mg/mL) and cultured for another 4 h. After discarding the medium in the well, each well was added 100 μL of dimethyl sulfoxide (DMSO) and placed in the incubator for 10 min, the absorbance of the well at 570 nm was measured, and the reference

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wavelength was 630 nm.

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2.3 Nitrite assay

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Cell supernatants were aspirated and the nitrite content in the supernatant was

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detected by Griess reagent. Firstly, remove the Griess Reagent I and II from a 4 °C

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freezer and allow it to equilibrate for 20 min at 25 °C. Then take a 96-well plate and take 50 μL of the cell supernatant into the corresponding well. Set triple replicates for

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each set of experiments. Then add 25 μL of Griess I reagent to each well. After

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incubation for 10 min, each well was added 25 μL of Griess II reagent and incubated

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in the dark for another 10 min. Finally, the absorbance at 540 nm was measured in a microplate reader.

2.4 Enzyme linked immunosorbent assay (ELISA) for TNF-α and PGE2 detection The macrophage cells culture supernatant was collected and diluted 5 times with DMEM medium. The levels of TNF-α and PGE2 released from RAW264.7 cells were measured according to the manufacturer's protocol by a commercial mouse ELISA kit. 2.5 ROS level detection RAW264.7 cells were treated with Aureusidin for 24 h, the upper medium in

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each well of 96 plates was discarded, and the ROS content of each experimental group was determined by ROS detection kit. First, 50 μL of Rosup reaction was added to the positive control group for 30 min, then 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was diluted at 1:1000 with fresh serum- free medium, and each well was added 50 μL of diluted DCFH-DA. Incubate in a 37 °C cell incubator for 20 min, then

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wash the cells in each well 2-3 times with 100 μL of cold serum- free fresh medium.

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Finally, the intensity of fluorescence before and after stimulation was detected in

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real-time or time-by-time using a microplate reader at the emission wavelengths of

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488 nm and 525 nm. The fluorescence intensity indicates the content of ROS.

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2.6 RNA extraction and qRT-PCR analysis

Total RNA was extracted from RAW264.7 cells using Trizol reagent (Invitrogen,

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USA) and the extracted RNA was assayed for concentration and quality by a

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spectrophotometer. RNA having an absorption ratio (OD260 nm/OD280 nm) of about

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2.0 was selected and converted into cDNA using TransScript II All- in-One First-Strand cDNA Synthesis SuperMix for qPCR (TransGen Biotech Co., Ltd., Beijng, China). At last, 1 μL of cDNA template, 0.2 μL of two primers and 5 μL of SYBR-Green PCR mixture (Thermo, USA) were used for PCR amplification. Specific primer sequences for all genes are shown in (Table 1). Table 1 2.7 Immunofluorescence assay RAW264.7 cells were fixed with 4% paraformaldehyde at room temperature. Then, block the cells with 2% of BSA for 30 min, wash twice with pre-chilled PBS,

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then incubate overnight with NF-κB or Nrf2 primary antibody at 4 °C. Subsequently, add the fluoresceine isothiocyanate (FITC)-conjugated secondary antibody and incubate for 1 h in the dark. After rinsing 3 times with PBS, the nuclei were stained with Hoechst 33258 fluorescent dye for 30 min. Finally, a fluorescence microscope (OLYMPUS, FSX100, Japan) was used to analyze the stained cells.

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2.8 Western blotting

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RAW264.7 cells were harvested and lysed with Radio-Immunoprecipitation

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Assay (RIPA) buffer for 20 min to extract total protein. Nuclear and cytoplasmic

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proteins were isolated by nuclear extraction kits (Beyotime Shanghai, China). After

separated

by

sodium

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quantifying the protein concentration, proteins of different molecular weights were dodecylsulphate

polyacrylamide

gel

electrophoresis

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(SDS-PAGE) and transferred onto polyvinylidenefluoride (PVDF) membrane. The

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blotted PVDF membrane was incubated with a horseradish peroxidase (HRP)

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conjugated secondary antibody after reaction with the primary antibody. Band intensity was measured using chemiluminescence western blot detection system. 2.9 Molecular docking

The crystal structure of NF-κB (PDB ID: 1VKX) has been obtained from the RCSB protein data bank. For protein preparation, the natural ligand was extracted and the resulting crystal structure was then freed of the water molecules and added the polar hydrogen atoms. Subsequently, the receptor was anticipated by Protein Preparation module in Discover Studio software (Version 2017 R2). For ligand preparation, 3D structure of Aureusidin was copied from ChemBioDraw and

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anticipated by Ligand Preparation module. Then the prepared ligand was docked into the prepared protein through the CDOCKER module. After completion of docking, the model with the lowest value was selected and analyzed to investigate the type of interaction. 2.10 Data analysis

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All data are presented as mean ± SD of independent experiments. Statistical

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evaluation of the results was performed by one-way ANOVA and P value of 0.05 or

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less being considered statistically significant.

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3 Results

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3.1 Cytotoxicity of Aureusidin on RAW264.7 macrophages 1-100 μM of Aureusidin and 250 ng/mL of LPS were used to co-treated the

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RAW264.7 cells for 24 h, MTT assay was carried out to calculate the cell survive rate.

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We found that Aureusidin did not inhibit the proliferation of mouse macrophage

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RAW264.7 cells till the concentration of 60 μM. However, 80 and 100 μM of Aureusidin significantly inhibited the survival rate of RAW264.7 cells (Fig. 1B). Therefore, 60 μM of Aureusidin was used as the maximum dose in the following study. 3.2 Effect of Aureusidin on NO release NO has been shown to be an effective molecule for determining the extent of inflammation (Lai et al., 2017). To initially explore the anti- inflammatory activity of Aureusidin, different concentrations of Aureusidin (1, 10, 20, 40 and 60 μM) and LPS (250 ng/mL) were administrated in the RAW264.7 cells. Griess reagent was used to

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measure the content of NO in the supernatant. As can be seen, compared to the blank control group, LPS treatment led to an obvious increase of NO release in RAW264.7 cells, and the increase in LPS- induced NO release was significantly decreased by Aureusidin with a dose-dependent manner (Fig. 1C). 3.3 Effect of Aureusidin on PGE2 and TNF-α release

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PGE2 and TNF-α are two important inflammatory cytokines involved in

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mediating inflammatory responses (Ryu et al., 2015). PGE2 and TNF-α ELISA kit

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were used to detect their content in RAW264.7 cells culture supernatant. As shown,

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LPS significantly induced the production of PGE2 and TNF-α. However, Aureusidin

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could obviously inhibit PGE2 and TNF-α release induced by LPS, and the inhibition of PGE2 and TNF-α by Aureusidin was dose-dependent (Fig.1D and E).

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3.4 Effects of Aureusidin on iNOS and COX-2 expressions

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Previous studies have shown that iNOS and COX-2 are two important

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inflammatory factors involved in the inflammatory response (Raish et al., 2018). When an inflammatory reaction occurs, the body produces a large amount of these two inflammatory factors. Therefore, the reduction of the expression of these two inflammatory factors exerts a vital effect in slowing inflammation. Here, we investigated the effect of Aureusidin on LPS-induced inflammatory factors iNOS and COX-2 expressions in RAW264.7 cells. From the western blot results, we found that LPS significantly promoted the expression of iNOS and COX-2 proteins, while Aureusidin obviously inhibited this effect induced by LPS (Fig. 1F and 1G). Figure. 1

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3.5 Effect of Aureusidin on the expression of pro-inflammatory cytokine mRNAs In order to verify whether the regulation of inflammatory factors by Aureusidin is based on the level of genes, we further examined the expression of various inflammatory factors by qRT-PCR. As shown, the gene expressions of inflammatory factors iNOS, TNF-α, COX-2, IL-1β and IL-6 were detected in RAW264.7 cells after

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co-treatment with LPS and aureusidin for 4 h. Their mRNA expression levels were

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significantly inhibited by aureusidin, and the degree of inhibition was dose-dependent

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(Fig. 2A-2E). It is indicated that the Aureusidin can exert anti- inflammatory activity

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by inhibiting the expression of various inflammatory factors at the level of gene

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transcription. Figure. 2

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3.6 Effect of Aureusidin on NF-κB signaling pathway

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LPS is known to activate NF-κB, which regulates the inflammatory response by

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modulating multiple pro- inflammatory cytokines in macrophages (Liu et al., 2014). To further investigate whether the anti- inflammatory effects of Aureusidin are mediated by the NF-κB signaling pathway, the nuclear localization of nuclear factor NF-κB was examined in RAW264.7 cells by immunofluorescence. We found that LPS (250 ng/mL) treatment made NF-κB gradually transfer from the cytoplasm to the nucleus. However, after treatment with Aureusidin, NF-κB is mainly concentrated in the cytoplasm (Fig. 3A). These experimental results indicated that Aureusidin could inhibit LPS- induced NF-κB nucleus transfer. Furthermore, we determined the protein expression of NF-κB and the phosphorylation level of its inhibitory protein inhibitor α

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of NF-κB (IκBα) by Western blot. As can be seen from the results, Aureusidin prevented the expression of p-IκBα induced by LPS and significantly inhibited the expression of NF-κB in the nucleus (Fig. 3B and 3C). Overall, these findings indicated that Aureusidin limited the ability of NF-κB nuclear transfer by inhibiting the expression of p-IκBα.

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Figure. 3

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3.7 Effect of Aureusidin on Nrf2/HO-1 signaling pathway

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Nrf2 is recognized as a key transcription factor in cellular homeostasis, and it is

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confirmed that Nrf2 has antioxidant and anti- inflammatory effects by interacting with

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a variety of signaling pathways (Dolunay et al., 2016). The localization of nuclear factor Nrf2 in RAW264.7 cells was examined by immunofluorescence. As shown,

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Nrf2 was significantly transferred to the nucleus after treatment with Aureusidin, and

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the greater the concentration of Aureusidin, the more obvious the nuclear transfer of

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Nrf2 (Fig. 4A). At the same time, Western blot was performed to further explore the effect of Aureusidin on the Nrf2 signaling pathway. As we expected, Aureusidin treatment significantly decreased the expression of Kelch like ECH associated protein1 (Keap1) in the cytosol and increased the expression of Nrf2 protein in the nucleus (Fig. 4B and 4C). Furthermore, this study also detected the effect of Aureusidin on Nrf2 signaling pathway-related gene expressions by qRT-PCR. From the results, we found that Aureusidin treatment significantly increased Nrf2, HO-1 and NQO1 mRNA expressions while obviously inhibited the mRNA expression of Keap1 in RAW264.7 cells (Fig. 4D).

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To test whether HO-1 is involved in the anti- inflammatory effects of Aureusidin, Western blot was carried out to test the expression of HO-1 protein in RAW264.7 cells. The results showed that Aureusidin dose-dependently increased HO-1 protein expression. (Fig. 4E). In addition, we test the effect of Aureusidin on the release of LPS-induced inflammatory factor NO in RAW264.7 cells pretreated with the HO-1

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specific inhibitor Snpp. As shown, the treatment by Aureusidin alone significantly

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inhibited LPS- induced NO production, while the inhibitory effect of Aureusidin on

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NO release obviously decreased after the addition of Snpp (Fig. 4F). These two results

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indicated that Aureusidin had an anti- inflammatory effect by inducing HO-1

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expression. Figure. 4

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3.8 Aureusidin activates Nrf2/HO-1 via MAPKs signaling pathway

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MAPKs pathway is thought to be the primary mechanism of inflammation and

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participate in the regulation of HO-1 and Nrf2 signaling pathways (Nah et al., 2007). We treated the cells with Aureusidin for 15, 30, and 60 min, and detected the phosphorylated proteins expression of protein kinase B (Akt), ERK, JNK, and p38 in RAW264.7 cells. As shown, Western blot results suggested that Aureusidin could obviously increase phosphorylation of Akt, ERK, p38 and JNK at different indicated time and peaked at 15 min (Fig. 5A and 5B). So Aureusidin could promote the phosphorylation of Akt and activate the MAPKs signaling pathway. Furthermore, we treated RAW264.7 cells with p38 specific inhibitor (SB203580), JNK specific inhibitor (SP600125) and ERK specific inhibitor (PD98059), and the expression of

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Nrf2 and HO-1 proteins was evaluated by Western blot. As shown, all three MAPKs specific inhibitors significantly decreased the Nrf2 and HO-1 protein expressions induced by Aureusidin. Furthermore, we found that SB203580 has the most significant inhibition of Nrf2 nuclear translocation and HO-1 expression (Fig. 5C and 5D). Therefore, we could infer that the Aureusidin induced the expression of Nrf2 and

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HO-1 mainly by activation of p38. In short, these results indicate that Aureusidin exert

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activate Nrf2 to up-regulate HO-1 expression.

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an anti- inflammatory effect by activating MAPKs signaling pathway to further

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Figure. 5

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3.9 Aureusidin induces the activation of Nrf2/HO-1 signaling pathway via ROS Cumulative evidence suggested that ROS have an important influence on the

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activation of Nrf2 (Hou et al., 2015). Our results showed that Aureusidin could

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significant increase intracellular ROS levels in RAW264.7 cells (Fig. 6A). To verify

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whether ROS is involved in the activation of Nrf2/HO-1 by Aureusidin, the protein expression of Nrf2 and HO-1 in RAW264.7 cells co-treated with Aureusidin and NAC were examined. Western blot results showed that Aureusidin alone significantly increased the protein expression of Nrf2 and HO-1. However, NAC significantly inhibited the nuclear translocation of Nrf2 and the protein expression of HO-1 in RAW264.7 cells induced by Aureusidin (Fig. 6B and 6C). This result indicated that ROS was involved in the activation of the Nrf2/HO-1 signaling pathway mediated by Aureusidin. 3.10 Aureusidin activates MAPKs signaling pathway through ROS.

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To further explore the link between the MAPKs signaling pathways and ROS generation, we treated RAW264.7 cells with specific inhibitors of ROS (NAC), and detected phosphorylation levels of MAPKs by Western blot. The results showed that the ROS inhibitor NAC significantly inhibited the phosphorylation of ERK, JNK, and p38 proteins induced by Aureusidin (Fig. 6D and 6E). Therefore, this result revealed

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that Aureusidin activated the MAPKs signaling pathway by up-regulating ROS.

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Thence, Comprehensive results illustrated that Aureusidin induced the activation of

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Nrf2/HO-1 via ROS-dependent MAPKs signaling pathway.

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Figure. 6

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3.11 Docking analysis

In order to gain better understanding of the correlation between Aureusidin and

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NF-κB domain, CDOCKER was running by fitting aureusidin into the active site of

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NF-κB. Hydrophobic effects play an important role in molecular recognition,

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aureusidin penetrated well into the hydrophobic pocket of the defined site obviously. As shown in Fig. 7, Aureusidin mainly relied on hydrogen bonds to interact with amino acids. First, 15’-OH group in Aureusidin formed a hydrogen bond (2.81 Å) with -NH group of the residue ARG35. Second, 16’-OH group in Aureusidin formed two hydrogen bonds (2.40 Å and 2.95 Å) with –CO group of the ALA43 residue and SER42 residue, respectively. The 9’-O atom of Aureusidin also formed a hydrogen bond (2.10 Å) with -NH group of the residues SER42. In addition, 1’-O group in Aureusidin formed Salt Bridge (3.00Å) with –NH group of the residue ARG41. Pi-alkyl, Van der Waals and carbon hydrogen bond interactions between Aureusidin

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and NF-κB protein further stabilized the interaction (Fig. 7A and 7B). Figure. 7 4 Discussion Cytokines are micro- molecule proteins that mediate cell-to-cell communication. Inflammatory cytokines are various cytokines involved in the inflammatory response

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and directly damage the vascular endothelium, resulting in increased vascular

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permeability. In addition, it can cause fever, pain, vasodilation, increased

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permeability, leukocyte exudation and other inflammatory reactions (Thiyagarajan et

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al., 2016). Macrophages are central cells involved in the inflammatory response,

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which have the functions of phagocytosis, secretion and antigen presentation. As a central cell that initiates the production of inflammatory mediators in vivo,

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macrophages play a leading role in regulating the inflammatory response, and can

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produce a large number of inflammatory cytokines when activated by LPS (Elia et al.,

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2007). Experimental evidence showed that Aureusidin significantly inhibited NO, TNF-α and PGE2 production as well as reduced the mRNA expressions of pro-inflammatory factors including iNOS, TNF-α, COX-2, IL-1β and IL-6. This suggested that Aureusidin had good anti- inflammatory activity against LPS-induced RAW264.7 cells. NF-κB is a nuclear factor that regulates gene transcription during inflammation and immunity, and its activation is mainly determined by the phosphorylation of IκB (Scherle et al., 1998). Ubiquitination and proteasomal degradation of IκB allow NF-κB to be released from the cytosol into the nucleus to initiate the expression of

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related genes (Su et al., 2011). Activation of NF-κB initiates the expression of various inflammatory and adhesion factors, which directly leads to inflammation (Feng et al., 2017). Here, results indicated that NF-κB was involved in the anti- inflammatory effects of Aureusidin, and Aureusidin regulated nuclear transfer of NF-κB by inhibiting phosphorylation of IκBα. In addition, the results of molecular docking

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showed that Aureusidin was firmly bonded to NF-κB by interaction with each other.

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This interaction was of significance for rapid translocation of NF-κB to the nucleus.

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Therefore, we could conclude that Aureusidin was likely to be an inhibitor of NF-κB.

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Nrf2/HO-1 is an anti- injury mechanism for the formation of defense against

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external stimuli in the evolution of organisms, and it also is the main signaling pathway for antioxidative stress. Previous studies have shown that the Nrf2/HO-1

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signaling pathway acts as the primary cellular sensor for oxidative stress (Li et al.,

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2018). Further researches on Nrf2 and its related pathways are still of great

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significance for the clinical treatment of inflammation-related diseases (Xu et al., 2019). Here, the results showed that Aureusidin could inhibit Keap1 expression to promote the transfer of Nrf2 into the nucleus. Furthermore, qRT-PCR results showed that Aureusidin could up-regulate the expressions of Nrf2, HO-1 and NQO-1 mRNA and inhibit the Keap1 mRNA expression. Therefore, we believed that Aureusidin exerted the anti- inflammatory effect by promoting the nuclear transfer of Nrf2. As the main antioxidant molecule, HO-1 has an important effect on maintaining the redox balance of cells, and its activity is regulated by Nrf2 (Wang et al., 2019). To determine whether Aureusidin also affects HO-1 expression, Western blot was carried out to

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examine the expression of HO-1 protein. The results showed that Aureusidin significantly promoted HO-1 protein expression. And

we found that the

anti- inflammatory action of Aureusidin was related to HO-1 in RAW264.7 cells. The experimental results revealed that Aureusidin exerted an anti- inflammatory effect in RAW264.7 cells by up-regulating the expression of HO-1.

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ROS, functioned as an endogenous signaling molecule, may regulate multiple

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signaling pathways in various intracellular processes, which is of importance for the

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activation of Nrf2 (Intayoung et al., 2015). To further understand the potential

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molecular mechanisms underlying the anti- inflammatory activity of Aureusidin, we

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examined the effects of Aureusidin on ROS release and MAPK phosphorylation. The results showed that Aureusidin could promote the release of ROS in RAW264.7 cells,

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thereby activating the nuclear transfer of Nrf2 to up-regulate the expression of HO-1

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to exert anti- inflammatory effects. Activation of MAPKs is known to induce

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Nrf2- mediated HO-1 expression (Kyriakis et al., 2001). Furthermore, ROS is reported to mediate the activation of various kinases, such as MAPKs (Cheng et al., 2017). Here, the results showed that treatment with specific inhibitors of ERK1/2, JNK1/2 and p38 significantly inhibited the nuclear transfer of Nrf2 and the expression of HO-1 induced by Aureusidin in RAW264.7 cells. ROS inhibitor NAC significantly inhibited the phosphorylation of ERK, JNK, and p38 proteins induced by Aureusidin. These results indicated that Aureusidin may mediate Nrf2 activation via a ROS-dependent MAPKs pathway. 5 Conclusions

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In summary, our results clearly indicated that Aureusidin significantly attenuated the expression of pro- inflammatory cytokines induced by LPS. Mechanism studies further elucidated that Aureusidin exerted anti- inflammatory effects by inhibiting NF-κB nuclear translocation and activating MAPKs-and ROS-dependent Nrf2/HO-1 signaling pathways (Fig. 8). The results exhibited that Aureusidin might be a

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promising candidate for novel inflammatory inhibitors.

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Figure. 8

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Conflict of interest statement

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We declare that we do not have any commercial or associative interest that

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represents a conflict of interest in connection with the work submitted. Transparency document

version.

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Acknowledgments

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The transparency document associated with this article can be found, in online

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Captions Figure. 1 Cell viability and effect of Aureusidin on NO, PGE2 and TNF-α production and the protein expressions of iNOS and COX-2 in LPS- induced RAW264.7 macrophages. (A) Chemical structure of Aureusidin. (B and C) Cell viability and NO assay. RAW264.7 cells were pretreated with Aureusidin (1, 10, 20, 40, 60, 80, and 100

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μM) for 1 h and then co-treated with LPS (250 ng/mL) for 24 h. Cell viability was

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measured by MTT assay and the content of NO was measured using Griess reagent.

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(D and E) Effect of Aureusidin on level of PGE2 and TNF-α production in

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LPS-induced RAW264.7 macrophages. RAW264.7 cells were pretreated with

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Aureusidin (20, 40, and 60 μM) for 1 h and then co-treated with LPS (250 ng/mL) for 24 h. The culture supernatant was subjected to ELISA kits to determine PGE2 and

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TNF-α production. (F and G) Effect of Aureusidin on iNOS and COX-2 protein

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expressions. RAW264.7 cells were pretreated with various concentrations of

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Aureusidin (20, 40 and 60 μM) for 1 h and then co-treated with LPS (250 ng/mL) for 24 h. The cells were harvested and the lysates were subjected to Western blot analysis for (E) iNOS and (F) COX-2. β-Actin was used as the internal control for normalization. Data shown are the means ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs LPS-treated group and

###

p < 0.001vs control.

Figure. 2 Effects of Aureusidin on pro- inflammatory cytokines-related gene expressions in LPS-stimulated RAW264.7 cells. RAW264.7 macrophages were pretreated with various concentrations of Aureusidin (20, 40 and 60 μM) for 1 h and then co-treated with LPS (250 ng/mL) for 4 h. (A-E) Pro- inflammatory cytokines

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mRNA expressions. The mRNA expression levels of iNOS (A), COX-2 (B), TNF-α (C), IL-6 (D) and IL-1β (E) were quantitated by qRT-PCR in RAW264.7 macrophages. Data shown are the means ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs LPS-treated group and ###p < 0.001vs control. Figure. 3 Effects of Aureusidin on LPS- induced NF-κB activation in RAW264.7

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cells. (A) Effect of Aureusidin on immunofluorescence subcellular localization of p65

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protein in LPS-stimulated RAW264.7 cells. RAW264.7 cells were pretreated with

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Aureusidin (60 μM) for 1 h and then co-treated with LPS (250 ng/mL) for 1 h, p65

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protein localization was immunochemically detected using anti-p65 antibody. The

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same fields were stained with Hoechst 33258 for the location of nuclei. (B) Effects of Aureusidin on LPS-induced IκBα phosphorylation. RAW264.7 cells were pretreated

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with Aureusidin (20, 40 and 60 μM) for 1 h and then co-treated with LPS (250 ng/mL)

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for 1 h. The effect of Aureusidin on IκBα phosphorylation was immunochemically

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assessed using phospho-specific anti-IκBα for cytosol extract of RAW264.7 cells. β-Actin was used as the internal control for normalization. (C) Effect of Aureusidin on LPS-induced NF-κB activation. Protein samples for nuclear and cytosol extract of RAW264.7 cells were analyzed by Western blot using anti-p65 antibody. ERK and β-actin were used as the internal control for normalization. The data represent the means ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs LPS-treated group and

##

p < 0.01, ### p < 0.001vs control.

Figure. 4 Effects of Aureusidin on Nrf2/HO-1 pathway in RAW264.7 cells. (A) Effect of Aureusidin on immunofluorescence subcellular localization of Nrf2 protein in

Journal Pre-proof RAW264.7 cells. RAW264.7 cells were treated with 60 μM Aureusidin for 1 h. Fixed cells were incubated with anti-Nrf2 antibody and FITC-conjugated anti- rabbit IgG antibody, then nuclei were stained with Hoechst 33258 and observed by fluorescence microscopy. (B) Effects of Aureusidin on Nrf2 protein expression in RAW264.7 cells. RAW264.7 cells were treated with Aureusidin (20, 40 and 60 μM) for 1 h. Protein samples for nuclear and cytosol extract of RAW264.7 cells were analyzed by Western

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blot using anti-Nrf2 antibody. ERK and β-actin were used as the internal control for

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normalization. (C) Effect of Aureusidin on Keap1 protein expression in RAW264.7

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cells. RAW264.7 cells were treated with Aureusidin (20, 40 and 60 μM) for 1 h.

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Keap1 protein expression was immunochemically assessed using anti-Keap1 antibody for cytosol extract of RAW264.7 cells. β-Actin was used as the internal control for

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normalization. (D) Effects of Aureusidin on Nrf2, Keap1, HO-1 and NQO1 gene

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expressions in RAW264.7 cells. The cells were treated with Aureusidin (20, 40 and 60

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μM) for 4 h. (E) Effect of Aureusidin on HO-1 protein expression in RAW264.7 cells. The cells were treated with Aureusidin (20, 40 and 60 μM) for 6 h. HO-1 protein expression was immunochemically assessed using anti-HO-1 antibody. β-Actin was used as the internal control for normalization. (F) Effect of Aureusidin on NO production in presence of Snpp. Cells were pretreated with or without Snpp (1 μM) for 1 h in presence of 60 μM Aureusidin, then treated with LPS (250 ng/mL) for another 24 h. The culture medium was collected to detect the concentration of NO by Griess reagent. Data shown are the means ± SD from three independent experiments, *p<0.05, **p<0.01 and ***p<0.001 vs control group, $$$ p<0.001 vs Aureusidin plus

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LPS-treated group. Figure. 5 Aureusidin activates Nrf2/HO-1 via MAPKs signaling pathway. (A and B) Effect of Aureusidin on phosphorylation of Akt/MAPKs. RAW264.7 cells were treated with Aureusidin (60 μM) for the indicated time. The whole-cell lysates were analyzed by Western blot using various antibodies against specific antibodies. (C and

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D) Effect of Aureusidin on Nrf2 and HO-1 expression in the presence of MAPKs

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inhibitors. Cells were treated with SB203580 (p38 inhibitor), PD98059 (ERK

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inhibitor), or SP600125 (JNK inhibitor) for 1 h and then treated with Aureusidin for

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another 1 h (Nrf2) or 6 h (HO-1). Nuclear fraction for Nrf2 and the total proteins for

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HO-1 were subjected to Western blot analysis. ERK and β-actin were used as the internal control for normalization. The data represent the means ± SD from three

p < 0.01, &&&p < 0.001 vs Aureusidin-treated group.

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&&

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independent experiments. *p<0.05, **p < 0.01, ***p < 0.001 vs control and &p < 0.05,

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Figure. 6 Aureusidin induces the activation of Nrf2/HO-1 via ROS-dependent MAPK signaling pathway. (A) Effect of Aureusidin on the intracellular ROS generation. RAW264.7 cells were treated with the indicated concentrations of Aureusidin for 24 h. DCF-DA was used to assess intracellular ROS generation. (B and C) RAW264.7 cells were treated for 1 h (Nrf2) or 6 h (HO-1) with Aureusidin (60 μM) alone, or in the presence of NAC (100 μM). Nuclear extracts and total proteins were subjected to Western blot analysis to determine Nrf2 and HO-1 levels. LaminB and β-actin were used as the internal control for normalization. (D and E) RAW264.7 cells were treated for the indicated time with Aureusidin (60 μM) alone, or in the presence of NAC (100

Journal Pre-proof μM), and subsequently whole cell lysates were blotted with the indicated antibodies. *p < 0.05, **p < 0.01, ***p < 0.001 vs control and & p < 0.05,

&&

p < 0.01,

&&&

p<

0.001 vs Aureusidin-treated group. Figure. 7 Molecular docking. Predicted docking pose of Aureusidin in complex with NF-κB (PDB: 1VKX). (A) Overall structure of NF-κB/Aureusidin complex. The hydrophilic surface is coloured in blue. (B) Local view of residues interacting with

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NF-κB and Aureusidin. Green dashed lines indicate hydrogen bonds. Purple dashed

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lines indicate Pi-Alkyl interactions. Orange dashed lines indicate salt bridge and

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attractive charge interaction.

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Figure. 8 Proposed mechanism for anti-inflammatory action of Aureusidin.

Journal Pre-proof Table. 1 Primer sequences used in qRT-PCR. β-actin

F: 5’-ATGTGGATCAGCAAGCAGGA-3’ R: 5’-AAGGGTGTAAAACGCAGCTCA-3’

iNOS

F: 5’-AGCCAAGCCCTCACCTACTT-3’ R: 5’-GCCTCCAATCTCTGCCTATC-3’

COX-2

F: 5’-CCAGCACTTCACCCATCAGT-3’ R: 5’-GGGATACACCTCTCCACCAA-3’ F: 5’-TATGGCTCAGGGTCCAACTC-3’

f

TNF-α

IL-6

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R: 5’-GGAAAGCCCATTTGAGTCCT-3’ F: 5’-CACGGCCTTCCCTACTTCAC-3’

F: 5’-GTTGACGGACCCCAAAAGAT-3’

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IL-1β

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R: 5’-TGCAAGTGCATCATCGTTGT-3’

NQO1

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R: 5’-CCTCATCCTGGAAGGTCCAC-3’ F: 5’-AGTGGCATCCTGCGTTTCT-3’

HO-1

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R: 5’-TCTCCTCCCAGACGGTTTC-3’ F: 5’-TCTGGATGGAGGGAGATACC-3’

F: 5’-TCAGCGACAGAAGGACTAAG-3’

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Nrf2

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R: 5’-CAGCAGTCGTGGTCAGTCAA-3’

R: 5’-AGGCATCTTGTTTGGGAATG-3

Keap1

F: 5’-AGTGGCATCCTGCGT TTCT-3’ R: 5’-CAACACCACACCAACATTA-3

Note: F: forward; R: reverse.

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Journal Pre-proof Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Highlights: •Aureusidin might be a good candidate against inflammatory-related diseases. •Aureusidin exerts anti-inflammatory activity by inhibiting NF-κB signaling pathway. •Aureusidin activates the Nrf2/HO-1 signaling pathway via ROS-dependent MAPK

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pathway.

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Author's Contributions J. R. and D. S. conceived and designed the experiments. L.-X. L., H. C. and M.-J. Z. performed the experiments. J.-C. Z., M.-Y. L. and X.-Y. W. analyzed the data. L.-X. L.,

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J. R. and K. H. wrote the manuscript.