Inhibitory effects of geraniin on LPS-induced inflammation via regulating NF-κB and Nrf2 pathways in RAW 264.7 cells

Accepted Manuscript Inhibitory effects of geraniin on LPS-induced inflammation via regulating NF-κB and Nrf2 pathways in RAW 264.7 cells Peng Wang, Qi Qiao, Ji Li, Wei Wang, Li-Ping Yao, Yu-Jie Fu PII:

S0009-2797(16)30184-3

DOI:

10.1016/j.cbi.2016.05.014

Reference:

CBI 7693

To appear in:

Chemico-Biological Interactions

Received Date: 8 January 2016 Revised Date:

25 March 2016

Accepted Date: 8 May 2016

Please cite this article as: P. Wang, Q. Qiao, J. Li, W. Wang, L.-P. Yao, Y.-J. Fu, Inhibitory effects of geraniin on LPS-induced inflammation via regulating NF-κB and Nrf2 pathways in RAW 264.7 cells, Chemico-Biological Interactions (2016), doi: 10.1016/j.cbi.2016.05.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

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GRAPHICAL ABSTRACT

ACCEPTED MANUSCRIPT Inhibitory effects of geraniin on LPS-induced inflammation via regulating NF-κB and Nrf2 pathways in RAW 264.7 cells Peng Wang 1,†, Qi Qiao 1, Ji Li 2,3, Wei Wang 1, Li-Ping Yao 1, Yu-Jie Fu 1,*

Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry

University, Harbin 150040, P. R. China,

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Department of Cardiology, The 2nd

Affiliated Hospital of Harbin Medical University, Harbin 150001, P. R. China and

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the Key Laboratory of Myocardial Ischemia, Ministry of Education, Harbin Medical

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University, Harbin, 150001, P. R. China.

* Corresponding author. Phone/fax: +86-451-82190535.

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E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract Geraniin, a major polyphenolic compound of Geranium sibiricum L, has long been used as an important Chinese herbal medicine for the treatment of a variety of

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inflammatory pathologies. However, the underlying anti-inflammatory molecular mechanisms of this compound are not clear. The aim of the present study was to investigate the anti-inflammatory activities of geraniin and elucidate the underlying

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mechanisms. The anti-inflammatory effects of geraniin were studied by using

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lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Geraniin suppressed the inducible nitric oxide synthase (iNOS) expression, and inhibited reactive oxygen species (ROS) production. Subsequent studies demonstrated that geraniin effectively reduced production of NO and pro- inflammatory cytokines. These effects were

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mediated by impaired translocation of nuclear factor (NF)-κB and inhibition of the phosphorylation of Akt in LPS-stimulated RAW 264.7 cells. Furthermore, geraniin induced heme oxygenase-1 (HO-1) expression via activation of transcription factor

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Nrf2. This study gives scientific evidence that geraniin inhibits the LPS-induced

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expression of inflammatory mediators via suppression of Akt-mediated NF-κB pathway as well as up-regulation of Nrf2/HO-1 pathway, indicating that geraniin has a potential application in inflammatory conditions. Keyword: NF-κB; Anti-inflammation; RAW 264.7 cells; Geraniin; Nrf2; ROS

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ACCEPTED MANUSCRIPT 1. Introduction Inflammation has been recognized as a complex physiological defense process that removes the injurious stimuli and initiates the healing process regulated by the

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immune system. However, excessive inflammation causes chronic inflammatory conditions including arthritis, asthma, multiple sclerosis and atherosclerosis. [1-4]. The activation of pro-inflammatory cells plays a critical role in the inflammatory

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response, mainly macrophages and monocytes. Bacterial lipopolysaccharide (LPS), a

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major component of the cell walls of Gram-negative bacteria [5], is one of the factors that activates cytokine networks via inducing the release of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and inflammatory mediators including nitric oxide (NO) and

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prostaglandin E2 (PGE2) [6]. NO can be synthetized from L-arginine by a family of nitric oxide synthases. An inducible isoform of NOS (iNOS or NOS2) is expressed only after exposure to pro-inflammatory conditions. Once expressed, iNOS generates

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large amounts of NO, and this excessive NO plays an important role in acute and

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chronic inflammation [7, 8].

It has been known that LPS initially promotes the production of reactive oxygen

species (ROS) [9]. Reactive oxygen species (ROS), as modulators of redox-sensitive signaling molecules, contributes to the innate immune response against multiple pathogens [10]. Nuclear transcription factor kappa-B (NF-κB), a ROS-sensitive transcription factor, plays a pivotal role in the regulation of genes in response to inflammatory stimuli [11]. Under quiescent conditions, NF-κB is bound to inhibitory

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ACCEPTED MANUSCRIPT proteins in the cytoplasm. When stimulated with LPS, IκB was phosphorylated by IKK, thereby, releasing NF-κB into the nucleus to drive the expression of target genes [12]. It has been demonstrated that LPS stimulation contributes to activation

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of the PI3K/Akt pathway. Moreover, pharmacologic PI3K inhibitors prevent LPS-induced NF-κB translocation [13]. Several studies have reported that HO-1 plays a critical role in inhibiting of the excessive production of pro-inflammatory

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cytokines as well as reactive oxygen species (ROS) in LPS-stimulated RAW 264.7

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cells [14]. HO-1 expression is tightly regulated by nuclear transcription factor erythroid 2-related factor (Nrf2). Thus, Nrf2-mediated HO-1 expression has shown a beneficial effect against the inflammatory response and NF-κB activation [15]. Herbs have been extensively used in foods and traditional medicines in oriental

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countries for millennia. Geranium sibiricum L, a widely spread herb, has been used for healing of diarrhea and intestinal inflammation in traditional folk medicine of China. Recent studies have showed that a number of polyphenolic compounds

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isolated from various Geranium species have antinociceptive and anti-inflammatory

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effects in inflammatory animal model systems [16]. Among phenolic compounds, geraniin, the main polyphenolic compound of Geranium sibiricum L, has long been used as an important Chinese herbal medicine possessing considerable pharmacological activities, including radioprotective [17], antihypertensive [18] and antioxidant effects [19]. However, detailed molecular mechanisms underlying the anti-inflammatory effects of geraniin have not been clarified. In this study, we found that geraniin attenuated the expression levels of iNOS protein, as well as their

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proinflammatory

mediators

NO

in

lipopolysaccharide

(LPS)-stimulated RAW 264.7cells by inhibiting NF-κB activation. Furthermore, our results showed that geraniin suppressed LPS-induced NO production by inducing

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Nrf2-dependent HO-1 expression.

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ACCEPTED MANUSCRIPT 2. Materials and methods 2.1. Reagents and antibodies Geraniin (purity ≥ 98%) was purchased from Delta Co. Ltd. (Anhui province, China). A 10 mM stock solution of geraniin was prepared in dimethyl sulfoxide

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(DMSO) and stored at − 80 °C.

Polyclonal antibodies against phospho-Akt (Ser473), Akt, phosphor-IKKα/β (Ser176/177), NF-κB p65, IκKα, β-Actin and LY294002 (PI3K inhibitor) were

purchased

from

dimethylsulfoximine

Santa

(DMSO),

Cruz

Biotechnology

(Santa

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were

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purchased from Beyotime Institute of Biotechnology (Beijing, China). HO-1, Nrf-2,

LPS

(from

Escherichia

Cruz,

coli

CA).

055:B5 ),

N-acetyl-L-cysteine (NAC), and MTT were obtained from Sigma (St. Louis, MO, USA). Zn-protoporphyrin (ZnPP) was from Merck (Darmstadt, Germany). Other reagents and chemicals were purchased from Beijing Chemical Reagents Co. (Beijing, Secondary antibodies

were obtained

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China).

from

Beyotime

Institute of

Biotechnology (Beijing, China). Deionized water was purified by a Milli Q Water Purification system from Millipore (Millipore Corp., Bedford, MA). Polyvinylidene

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MA).

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fluoride (PVDF) membrane was purchased from Millipore (Millipore Corp., Bedford,

2.2. Cell culture

RAW264.7 macrophage cells, purchased from the CBCAS (Cell Bank of the

Chinese Academic of Sciences, Shanghai, PR China), were cultured in DMEM (Invitrogen) containing 10% (v/v) fetal bovine serum (Hyclone) and antibiotics (100 U/ml penicillin and 100 µg/ml strepto-mycin) (Hyclone) at 37 °C in a humidified 5% CO2 incubator. 6

ACCEPTED MANUSCRIPT 2.3. Cytokine measurement RAW264.7 cells (5 × 105 cells/well) were plated into 24-well plates 24 h before pretreated with or without different concentrations of geraniin for 2 h, then were

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stimulated with LPS (1 µg/mL) for 24 h. After stimulation, the culture media were collected. The amounts of cytokines in the supernatants for IL-6, IL-1β and TNF-α, respectively, were determined by using enzyme-linked immunosorbent assay kits

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according to the manufacturer's instructions. Three replicates were carried out for

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each of the different treatments. 2.4. Nitrite analysis

RAW 264.7 cells (5 × 105 cells/well) were seeded in 24-well cell culture plates and allowed to adhere for 24h. After being treated with geraniin and LPS, the

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supernatant was collected from each well. NO production in culture supernatant was spectrophotometrically evaluated by measuring nitrite, an oxidative product of NO. Nitrite was determined with the Griess reaction by mixing 100 µl of culture

in

5%

(w/v)

phosphoric

acid

and

0.1%

(w/v)

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sulfanilamide

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supernatant with 100 µl of Griess reagent containing equal volumes of 1% (w/v)

of N-(1-naphthyl)ethylenediamine solution. Absorbance was measured at 540 nm and nitrite concentration was determined using sodium nitrite as a standard. Three replicates were carried out for each of the different treatments. 2.5 iNOS Enzyme Activity Assay RAW 264.7 (1 × 106 cells/well) cells were plated in 6-well plates and allowed to adhere for 24h. After being treated with geraniin and LPS, RAW 264.7 cells were

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ACCEPTED MANUSCRIPT were lysed by three freeze-thaw cycles, and centrifuged 20 min at the speed of 2000-3000 rpm. The supernatants were used for iNOS activity. The iNOS enzyme activity in lysate was determined spectrophotometrically at 450 nm. The

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concentration of iNOS in the samples is then determined by comparing the OD of the samples to the standard curve. Each experiment was performed in triplicate. 2.6. Cell viability assay

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Cell viability was determined by MTT assay. Briefly, RAW264.7 cells were

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seeded into 96-well plates at a density of 1 × 104 cells per well 24 h before treatment. Cells were treated with various concentrations of geraniin in the presence or absence of LPS (1 µg /ml) for 24 h followed by incubating with 5 mg/ml of MTT working solution for 4 h at 37 °C. After added 100 µl of DMSO to dissolve the crystals, the

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absorbance of each well at 570 nm was measured. Three replicates were carried out for each of the different treatments.

2.7. Measurement of GSH/GSSG ratio

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Total glutathione and oxidized glutathione (GSSG) were measured using a

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GSH/GSSH Assay kit (Beyotime, China) according to the manufacturer’s protocol. After treatment, RAW264.7 cells collected and centrifuged at 10,000 × g for 10 min at 4°C. The cells were mixed with 30 µl protein removal reagent M, then frozen and thawed twice using liquid nitrogen and 37 °C water. The samples were centrifuged and the supernatant was used for GSH and GSSG assays. The total GSH level was measured by the DTNB-GSSG recycling assay. The GSSG level was quantified by the same method as for total GSH after the supernatant was treated with 1 mol/L

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ACCEPTED MANUSCRIPT 2-vinylpyridine solution to remove the reduced GSH. The amount of reduced GSH was obtained by subtracting the amount of GSSG from that of total GSH. 2.8. Western blotting

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RAW 264.7 cells (1 × 106 cells/well) were seeded in 6-well cell culture plates and allowed to adhere for 24h. After being treated with geraniin and LPS, the cells were washed twice with cold PBS. For isolation of total protein fractions, media were

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removed and cells were washed twice with ice-cold PBS and then lysed using cell

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lysis buffer [20 mM Tris pH 7.5, 150 mM NaCl, 1%Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM EDTA, 1%Na3CO4 , 0.5 µ g/mL leupeptin, 1 mM phenylmethanesulfonyl fluoride(PMSF)]. The lysates were collected by scraping from the plates and then centrifuged at 10 000 rpm at 4 °C for 5 min.

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Total protein samples (20 µg) were loaded on 12% SDS polyacrylamide gels for electrophoresis, and then transferred onto PVDF transfer membranes (Millipore, Billerica, MA) at 0.8 mA/cm2 for 2 h. Membranes were blocked at room temperature

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for 2 h with blocking solution (1% BSA in PBS plus 0.05% Tween-20). Membranes

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were then incubated overnight at 4 ° C with primary antibodies at a dilution of 1: 500 in blocking solution. After thrice washings in TBST (Tris buffered saline with Tween 20) for 5 min each, membranes were incubated for 1 h at room temperature with alkaline phosphatase peroxidase-conjugated secondary antibody (1:500 dilution) in blocking solution. Detection was performed by the BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime Institute of Biotechnology, Beijing, China) according to the manufacturer ’ s instructions. Bands were then recorded by a

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ACCEPTED MANUSCRIPT digital camera (Canon, EOS 350D, Tokyo, Japan). 2.9. Preparation of cytoplasmic and nuclear extracts Nuclear and cytoplasmic extractions were performed using the Nuclear and

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Cytoplasmic Extraction kit (Biosynthesis Biotechnology Company, Beijing, China)

assayed by Western blotting, as described above. 2.10. Detection of reactive oxygen species

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following manufacturer ’ s instructions. Then, nuclear and cytoplasmic proteins were

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The intracellular accumulation of ROS was monitored using the fluorescent probe DCFH-DA. At the end of the treatment, cells were collected and washed twice with PBS, then PBS were removed and 10 µM of DCFH-DA was added and incubated for 30 min at 37 °C in the dark. Fluorescence generation due to the hydrolysis of

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dichlorodihydrofluorescein-diacetate(DCFH-DA) to DCFH by non-specific cellular esterases and subsequent oxidation of DCFH by peroxides. The fluorescence was analyzed by a Partec PAS flow cytometer (Partec GmbH, Münster, Germany).

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2.11. Immunofluorescence and confocal microscopy

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The cells were immunofluorescence-labeled according to the manufacturer’s instruction using a Cellular NF-κB Translocation Kit (Beyotime Biotech). Briefly, after washing and fixing, cells were incubated with a blocking buffer for 1 h to suppress non-specific binding. Next, cells were incubated with the primary NF-κB p65 antibody for 1 h, followed by incubation with a Cy3-conjugated secondary antibody for 1 h, then with DAPI for 5 min before observation. p65 protein and nuclei fluoresce red and blue, respectively, and can be simultaneously viewed by

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ACCEPTED MANUSCRIPT laser confocal microscope at an excitation wavelength of 350 nm for DAPI and 540 nm for Cy3. To create a two-color image, the red and blue images were overlaid, producing purple fluorescence in areas of co-localization.

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2.12. Plasmids, transfections and luciferase assay The reporter plasmid pGL6- NF-κB -Luc for testing NF-κB transcriptional activity was purchased from Beyotime Institute Biotechnology, China. RAW264.7 cells (5 ×

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105 cells/well) were seeded in 24-well plates before transfection and grew to about

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90 % confluence. Cells were then transiently co-transfected with 1µg of NF-κB -Luc plasmid and 0.1µg of pRL-TK plasmid (Promega) as control using Lipofectamine 3000 (Invitrogen). After 24 h, the cells were washed with fresh medium, pretreated with different concentrations of of geraniin for 2 h and then stimulated with LPS

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(1 µg/mL). After 12 h of stimulation, cells were lysed and the luciferase activity was determined using the Promega luciferase assay system (Promega) and luminometer (GloMax®20/20, Promega, CA, USA).

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2.13. Statistical analysis

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All results were expressed as mean values ± standard deviation (n = 3). Differences between groups were calculated by one-way ANOVA. An analysis of ANOVA variance with a Tukey post hoc test was used for multiple comparisons. All statistics were calculated using the STATISTICA program (StatSoft, Tulsa, OK). Correlations were calculated using the ReglinP function and inverted Student’s t test. p < 0.05 was considered as statistically significant.

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ACCEPTED MANUSCRIPT 3. Results 3.1. Effect of geraniin on cell viability In this study, MTT assay was used to determine cell viability. Cells were treated

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with different concentrations of geraniin in the presence or absence of LPS (1 µg /ml) for 24 h, and the percentage of cell viability in RAW 264.7 macrophages were shown in Fig. 1. The result showed that non-cytotoxic level of geraniin suppressed

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LPS-induced inflammatory responses in RAW 264.7 cells. Therefore, we excluded

geraniin on RAW 264.7 cells.

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the possibility that anti-inflammatory effect was caused by the cellular toxicity of

3.2. Effects of geraniin on production of IL-6, IL-1β,TNF-α, NO and expression of iNOS protein in LPS-stimulated RAW264.7 cells

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To test the inhibitory effects of geraniin on the production of the inflammatory cytokines (TNF-α, IL-6 and IL-1β) and mediators (NO) from LPS-stimulated RAW 264.7 cells, RAW264.7 cells were pretreated with various concentrations of geraniin

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for 2 h and then were incubated with or without 1 µg/ml of LPS for another 24 h for

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TNF-α, IL-1β, IL-6 and NO, respectively. As shown in Figs. 2A-D, it was obvious that LPS stimulation led to an increase in the production of TNF-α, IL-6, IL-1β and NO, while pretreatment with geraniin (15 and 30 µM) showed a significant inhibitory effect.

Since geraniin was found to inhibit NO production, we used western blotting to determine whether inhibitory effect of geraniin was related to inducible nitric oxide synthase (iNOS) expression. Our data showed that geraniin significantly inhibited

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ACCEPTED MANUSCRIPT iNOS compared with LPS-induced macrophages (Fig. 2E). In addition, geraniin reduced LPS- stimulated iNOS enzyme activity in Raw264.7 cells (Fig. 2F). 3.3. Geraniin suppressed LPS-induced NF-κB activation in RAW264.7 cells

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The NF-κB pathway plays a critical role in regulating the expression of several cytokines through activation of IKKs, which is an important upstream kinase for phosphorylation of IκB and subsequent IκB degradation in macrophages. Hence, the

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protein expression of phosphorylated IKKα/β and IκBα were detected. The results

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showed that geraniin inhibited the phosphorylation of IKKα/β and degradation of IκBα (Fig. 3A and 3B). To investigate the inhibitory effect of geraniin on suppression of LPS-induced translocation of NF-κB into the nucleus, western blotting was performed to analyze the transfer of NF-κB p65 to the nucleus. Results

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suggested that LPS treatment decreased the cytosolic p65 subunit level, resulting in an increase in nuclear p65 level, and treatment with geraniin reversed this effect (Fig. 3C). Additionally, we also evaluated NF-κB activity using its luciferase gene

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transcriptional activity, and found LPS significantly enhanced NF-κB activity up to

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4.8-fold over the basal level, while pretreatment of the cells with geraniin resulted in significant reduction of the luciferase activity in Fig. 3D. We also confirmed this result with immuno- fluorescence. Upon LPS stimulation, the p65 subunit of NF-κB was translocated into the nucleus. However, the maximum dose of geraniin dramatically influenced this translocation in Fig. 3E. 3.4. Geraniin affected the phosphorylation of PI3K/Akt pathway in LPS-induced RAW 264.7 cells

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ACCEPTED MANUSCRIPT PI3K/Akt has been shown to be associated with the induction of proinflammatory genes in activated macrophages [20]. First, we determined the effect of geraniin on the activation of Akt in LPS-stimulated RAW264.7 cells. LPS-induced

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phosphorylation continued from 15 min to 60 min and reached a peak at 30 min. Application of geraniin attenuated the LPS-induced phophorylation of AKT (Fig. 4A and B). To further confirm the PI3K/Akt was involved in the inhibitory effect of

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geraniin on LPS-induced NO production, specific inhibitor LY294002 (for AKT) was

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used in our system. As shown in Fig. 4C, pretreatment with LY294002 abolished NO production.

3.5. Effect of geraniin on the regulation of HO-1 and Nrf2

It is well known that activated macrophages release a massive amount of ROS

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during inflammation [21], which acts as a second messenger for regulating Nrf2 activation [22]. We assessed the effects of the geraniin on intracellular ROS level in LPS-stimulated RAW 264.7 cells. As expected, the DCFH-DA assay demonstrated

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that pretreatment with geraniin significantly attenuates the LPS-induced ROS

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generation. N-acetyl-cysteine（NAC）has been known to act as an antioxidant/free radical scavenger or reducing agent. As shown in Fig. 5A, NAC also down-regulates LPS-induced intracellular ROS production, similar to the activity of geraniin. Meanwhile, our results also showed that geraniin and NAC (10 mM) inhibited LPS-induced decrease in the intracellular GSH/GSSG ratio (Fig. 5B). Heme oxygenase (HO)-1, which is an enzyme for heme degradation, plays an important role in cellular protection against oxidative stress. Therefore, our results suggest that

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ACCEPTED MANUSCRIPT the anti-inflammatory activity of geraniin could be linked to HO-1. As shown in Fig. 5C and D, geraniin treatment led to a significant increase in HO-1 protein and Nrf2 nuclear translocation. To confirm that HO-1 protein induced by geraniin inhibited

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LPS-induced NO production. As shown in Fig. 5E, ZnPP, an inhibitor of HO-1, significantly reversed the regulatory effect of geraniin on NO production in LPS-stimulated RAW 264.7 macrophages, indicating that HO-1 induced by geraniin

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inhibited NO production.

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ACCEPTED MANUSCRIPT 4. Discussion Geranium sibiricum L has been used as a traditional folk medicine in eastern Asia. Many classic texts of oriental medicine showed Geranium sibiricum Linne had a

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wide array of pharmacological actions for healing of bacteria, intestinal inflammation, dermatitis and diarrhea [23, 24]. Here, we identified geraniin, a major polyphenolic compounds in G. sibiricum, has anti-inflammatory activity. In this study,

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we found that geraniin could reduce the release of inflammatory mediators and

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proinflammatory cytokines, further underlying mechanism study revealed that the anti-inflammatory properties of geraniin were due to free radical scavenging, leading to the suppression of LPS-stimulated AKT- mediated NF-κB signaling as well as up-regulating Nrf2/HO-1 signaling in LPS-stimulated RAW264.7 macrophages.

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Inflammation is a host response to foreign pathogens or tissue injury by the organism to eliminate harmful stimuli as well as to initiate the healing and repair process of the damaged tissue [25]. LPS is a major constituent of the cell wall of

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gram-negative bacteria, which can bind with the TLR 4 receptor of macrophages and

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induce the inflammation [26]. In response to LPS, macrophages synthesize and release inflammatory mediators. One of the major inflammatory mediators NO is markedly up-regulated by inducible NOS (iNOS). Apart from NO, macrophages also produce pro-inflammatory cytokines, such as TNF-α, IL-1 β and IL-6. These cytokines not only are involved in the inflammatory response, but also deepen the development

of inflammatory conditions. Thus,

blocking the

effects

of

pro-inflammatory mediators offers an attractive therapeutic strategy. Our study

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ACCEPTED MANUSCRIPT showed that geraniin significantly reduced production of these pro-inflammatory cytokines and inflammatory mediators in LPS-induced RAW264.7 cells (Fig. 2). Furthermore, our results revealed geraniin suppressed the expression of iNOS and

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enzyme activity, which supported the conclusion that production of NO was decreased.

Several recent studies indicate that triggering the production of LPS-induced

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inflammatory cytokine is associated with the activation of NF-κB [27]. NF-κB,

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known as a major transcription factor, plays a key role in regulating the expression of inflammation-induced enzymes and cytokines such as TNF-α, IL-1β and IL-6. Under normal conditions, NF-κB is constitutively localized in the cytoplasm, which is bound to its distinct inhibitory factor, IκB. Upon stimulation with LPS, IκB is

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rapidly phosphorylated by IKKα/β, and NF-κB dissociating from IκBs, translocates to the nucleus, where it binds to the promoter of target genes and activates transcription [28]. So we evaluated the effect of geraniin on NF-κB activation.

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Reporter gene assay for determining the transcription activity of NF-κB revealed that

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geraniin inhibited NF-κB activation. Western blot results revealed that geraniin significantly attenuated LPS-induced IKKα/β phosphorylation and inhibited NF-κB p65 subunit, which was also confirmed by immunofluorescence analysis by attenuating LPS-induced NF-κB p65 (Fig. 3). These collective findings indicated that geraniin suppressed the LPS-induced activation of NF-κB signaling probably resulting in the reduced productions of pro-inflammatory cytokines. The PI3K/AKT pathway has been shown to control a variety of cellular processes, including cell

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ACCEPTED MANUSCRIPT survival and proliferation [29]. Recently, numerous studies have shown that the PI3K/Akt signaling pathway, as the upstream molecules of NF-κB, plays an important role in NF-κB activation in LPS-activated macrophages [30, 31]. We

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found that treatment with geraniin significantly decreased phosphorylation of Akt. To reconfirm that geraniin could inhibit AKT activity and improved the inflammatory response, macrophages were treated with PI3K/AKT inhibitors, which led to

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decreased NO production in LPS-stimulated macrophages. Therefore, the inhibition

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of NF-κB activation by geraniin may be due to inhibition of Akt phosphorylation. HO-1 is a protein that can protect against cell injury from oxidative stress and inflammatory responses [32], which catalyzes degradation of heme, producing iron, carbon monoxide, and biliverdin. These products have anti-inflammatory,

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anti-apoptotic, and anti-proliferative effects [33]. Accumulating evidence indicates that HO-1 induction blocks LPS-induced inflammation by activation of Nrf2 signaling [34]. In this study, we found that geraniin induced the expression of HO-1

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and translocation of Nrf2 into nucleus (Fig. 5). Geraniin-induced HO-1 expression

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appears to be concomitant with the inhibition of ROS in LPS-stimulated cells. Our findings showed that geraniin induced HO-1 expression, meanwhile, HO-1 inhibitor, ZnPP, significantly reversed geraniin -mediated suppression of NO production. In summary, the present study suggests that geraniin diminishes LPS-induced pro-

inflammatory cytokines and NO production, most likely by affecting iNOS activation by suppressing LPS-induced ROS/PI3K/Akt-dependent NF-κB activation and up regulating Nrf2-dependent HO-1 expression.

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ACCEPTED MANUSCRIPT Conflict of Interest statement The authors declare that there are no conflicts of interest.

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Acknowledgements

The authors gratefully acknowledge the financial supports by the Application Technology Research and Development Program of Harbin (2013AA3BS014),

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Special Fund of National Natural Science Foundation of China (31270618)

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values for each geraniin concentration tested represent the average (mean ± S.D.) from three replicate wells and are representative of three separate experiments.

Fig. 2. Effects of geraniin on NO, TNF-α, IL-1β, IL-6 production and expression of

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iNOS in LPS-induced RAW264.7 cells

RAW264.7 cells were pretreated with geraniin (5, 15 and 30 µM) or not for 2 h

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followed by co-treatment with LPS for 24 h or 18 h in RAW264.7 cells. (A–D) The levels of TNF-α, IL-1β, IL-6 and NO were measured in the culture medium by ELISA kits or Griess reagents. (E) The protein level of iNOS was determined by Western blot. β-actin was used as control. (F) iNOS activity was analyzed with enzyme

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immunoassay kit. Each bar represents the mean ± SD of three independent P < 0.01 compared to vehicle control, * P<0.05, **P<0.01 and

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Fig. 3. Effects of geraniin on NF-κB activation in LPS-induced RAW264.7 cells.

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RAW264.7 cells were pretreated indicated concentrations of geraniin (5, 15 and 30 µM) for 2 h, and followed by LPS stimulation (1 µg/ml) for 15 min, 30 min or 1 h. IKK (A), IκKα (B) and the nuclear translocation of NF-κB (C) were determined by Western blot. Histone H3 was used as the nuclear marker and β-actin as the cytosol protein marker. (D) RAW264.7 cells were transiently transfected with a NF-κB-dependent reporter gene construct. Twenty-four hours after transfection, the cells were pretreated for 2 h with the indicated concentrations of geraniin, and followed by LPS stimulation (1 µg/ml) for 12 h. The cells were lysed and luciferase

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Fig. 4. Effects of geraniin on LPS-induced phosphorylation of PI3K/AKT.

(A) RAW264.7 cells were pretreated for 2 h with geraniin (30 µM), and followed by LPS stimulation (1 µg/ml) for the indicated time points. (B) RAW264.7 cells were

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pretreated for 2 h with the indicated concentrations of geraniin, and followed by LPS stimulation (1 µg/ml) for 30 min. The total lysates were blotted with the indicated

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antibodies. (C) Nitrite was determined in RAW264.7 cells treated with LPS for 24 h in the presence or absence of 25 µM LY294002 by Griess reagents. The results were expressed as mean ± SD of three independent experiments. ##P < 0.01 compared to vehicle control, * P<0.05, **P<0.01 and ***P<0.01 versus LPS-stimulated groups

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Fig. 5. Effect of geraniin on the regulation of HO-1 and Nrf2

RAW264.7 cells were stimulated with LPS (1 µg/ml) for 24 h in the presence of indicated concentrations of geraniin or NAC. Intracellular ROS (A) and the

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GSH/GSSG ratio (B) were measured. Cells were pretreated with indicated

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concentrations of geraniin for 2 h and stimulated with LPS (1 µg/ml) for 6 h and 12 h, The cytoplasmic and nuclear Nrf-2 (C), HO-1 (D) and β-actin were detected by Western blot using corresponding antibodies. (E) Cells were cultured with 1µg/ml LPS under geraniin treatment for 24 h in the presence or absence of 10 µM of ZnPP. Culture media were harvested for measurement of NO. The results were expressed as mean ± SD of three independent experiments. ##P < 0.01 compared to vehicle control, * P<0.05, **P<0.01 and ***P<0.01 versus LPS-stimulated groups.

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Highlights (1) Geraniin inhibited LPS-induced the release of the pro-inflammatory cytokines. (2) Geraniin suppressed LPS-induced iNOS expression and enzyme activity.

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(3) Geraniin regulates NF-κB activation and phosphorylation of AKT.

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(4) Geraniin reduced reactive oxygen species (ROS) accumulation via Nrf2 pathways.