Nauclea officinalis inhibits inflammation in LPS-mediated RAW 264.7 macrophages by suppressing the NF-κB signaling pathway

Author’s Accepted Manuscript Nauclea officinalis inhibits inflammation in LPSmediated RAW 264.7 macrophages by suppressing the NF-κB signaling pathway Xiao-Ting Zhai, Zhi-Yuan Zhang, Cui-Hua Jiang, Jia-Quan Chen, Ji-Qing Ye, Xiao-Bin Jia, Yi Yang, Qian Ni, Shu-Xia Wang, Jie Song, Fen-Xia Zhu www.elsevier.com/locate/jep

PII: DOI: Reference:

S0378-8741(16)30019-8 http://dx.doi.org/10.1016/j.jep.2016.01.018 JEP9929

To appear in: Journal of Ethnopharmacology Received date: 10 July 2015 Revised date: 14 January 2016 Accepted date: 18 January 2016 Cite this article as: Xiao-Ting Zhai, Zhi-Yuan Zhang, Cui-Hua Jiang, Jia-Quan Chen, Ji-Qing Ye, Xiao-Bin Jia, Yi Yang, Qian Ni, Shu-Xia Wang, Jie Song and Fen-Xia Zhu, Nauclea officinalis inhibits inflammation in LPS-mediated RAW 264.7 macrophages by suppressing the NF-κB signaling pathway, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2016.01.018 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 galley proof before it is published in its final citable 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.

Nauclea officinalis inhibits inflammation in LPS-mediated RAW 264.7 macrophages by suppressing the NF-κB signaling pathway Xiao-Ting Zhai a,b,c, Zhi-Yuan Zhangd, Cui-Hua Jiang a,b, Jia-Quan Chenc, Ji-Qing Yee, Xiao-Bin Jia a,b, Yi Yang a,b, Qian Ni a,b, Shu-Xia Wang a,b, Jie Song a,b, Fen-Xia Zhu a,b* a Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210028, P.R.China b Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing 210028, P.R. China c Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, P.R.China

d College of Pharmacy, Hainan Medical University, Haikou 570102, P.R.China eJiangsu Key Laboratory of Drug Design & Optimization, Department of Medicinal Chemistry, China Pha rmaceutical University, Nanjing 210009, P.R.China  *Authors for correspondence: Dr. Fen-xia Zhu Key Laboratory of New Drug Delivery System of Chinese Meteria Medica, Jiangsu Provincial Academy of Chinese Medicine, No.100 Shizi Road, Nanjing 210028, Jiangsu Province, P.R.China Tel: +86-25-52362107, Fax: +86-25-85637817 E-mail: [email protected]

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Abstract Ethnopharmacological relevance: Nauclea officinalis has been traditionally used in China for the treatment of fever, pneumonia and enteritidis etc. This study aims to investigate effects of Nauclea officinalis on the inflammatory response as well as the possible molecular mechanism in LPS-stimulated RAW 264.7 murine macrophage cells. Materials and methods: Anti-inflammatory activity of Nauclea officinalis (10, 20, 50, 100 µg/mL) was investigated by using LPS-induced RAW 264.7 macrophages. The NO production was determined by assaying nitrite in culture supernatants with the Griess reagent. The levels of TNF-α, IL-6 and IL-1β in culture media were measured with ELISA kits. Real time fluorescence quantitative PCR was detected for mRNA expression of iNOS, TNF-α, IL-6 and IL-1β. Western blot assay was performed to illustrate the inhibitory effects of Nauclea officinalis on phosphorylation of IκB-α and NF-κB p65. Results:Treatment with Nauclea officinalis (10-100 µg/mL) dose-dependently inhibited the production as well as mRNA expression of NO, TNF-α, IL-6 and IL-1β in RAW 264.7 macrophages. Western blot assay suggested that the mechanism of the anti-inflammatory effect was associated with the inhibition of phosphorylation of IκB-α and NF-κB p65. Conclusions: The results indicated that Nauclea officinalis potentially inhibited the activation of upstream mediator NF-κB signaling pathway via suppressing phosphorylation of IκB-α and NF-κB p65 to inhibit LPS-stimulated inflammation.

Key words: Nauclea officinalis; Anti-inflammation; Pro-inflammatory cytokines; NF-κB. 2

Chemical compounds studied in this article: Pumiloside (PubChem CID: 10346314); 3-epi-pumiloside (CAS No: 126722-26-7); Strictosamide (PubChem CID: 11969629); Vincosamide (PubChem CID: 44567197); DMSO (PubChem CID: 679); NaNO2 (PubChem CID: 23668193); EtOH (PubChem CID: 702); Acetonitrile (PubChem CID: 6342); Methanoic acid (PubChem CID: 284); 3˄4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide.

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1. Introduction Inflammation is an important part of the complex biological interactions that arise in any tissue in the response to bacterial, chemicals or physical injury (Song et al., 2014). It is well known that many systemic diseases generate and develop along with serious inflammatory reaction including inflammatory bowel disease, atherosclerosis multiple sclerosis, hyperlipidemia and bacterial pneumonia (Cho et al., 2014). The inflammatory process is normally elicited by numerous stimuli such as physical, noxious chemical stimuli or microbiological toxins (Shin et al., 2010), which leads to production of various molecules Among them, the nitric oxide (NO) and various pro-inflammatory cytokines including tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) plays a crucial role (Xu et al., 2014; Nguyen et al., 2015). Excessive production of these inflammatory mediators and cytokines cause inflammatory activities, tissue necrosis, even inflammatory diseases. Nuclear factor-kappa B (NF-κB) represents an essential family of eukaryotic transcription factors which are involved in regulating the expression of numerous genes in immune response and cell growth (Xu et al., 2003). Extenssive studies have shown that phosphorylation and degradation of IκB-α can activate the downstream NF-κB signaling pathway via p65 translocation into the nucleus to change the expression of related genes in response to LPS stimulation (Kwon et al., 2014; Fan et al., 2015). Overproduction of TNF-α, IL-1β and IL-6, which are regulated by NF-κB signaling pathway, aggravates the early immune response and inflammatory reaction after NF-κB activation. Therefore, restraining NF-κB activation has potential application in reducing inflammatory status in organism (Bocchini et al., 1992). 4

Nauclea officinalis, one of the commonly used traditional medicine in China (Wang et al., 2012), is the only species of genus Nauclea in China (Sun et al., 2007). It is widely used for the treatment of cold, fever, throat swelling, pink eyes, etc (Chen et al., 2014). It is reported that Nauclea officinalis exhibits various biological properties such as antimalarial (Kahunu et al., 2010), antibacterial (Hu et al., 2009) and anti-inflammatory effect (Fu et al., 2002). The in vivo anti-inflammatory activity indicates that Nauclea officinalis can inhibit early symptoms of acute inflammation such as seepage and swelling (Fu et al., 2002). However, up to now no more detail regarding the inhibitory effects of Nauclea officinalis on the inflammatory response has been reported and the molecular mechanism by which Nauclea officinalis enhances pro-inflammatory cytokine production remains unclear. In this paper, the production and relative mRNA expression of NO, iNOS, TNF-α, IL-6 and IL-1β in cell supernatant were determined. Besides, the phosphorylation of IκBα and p65 was also determined to explore the possible anti-inflammatory molecular mechanism.

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Materials and methods 2.1. Chemicals and reagents Nauclea officinalis (110101) was provided by Hainan Pharmaceutical Factory Co., Ltd. (Wuzhishan, China). Pumiloside, 3-epi-pumiloside, strictosamide and vincosamide (HPLC ≥ 98%) were separated from Nauclea officinalis and their chemical structures were analyzed by MS, 1H-,

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C-NMR (Zhu et al., 2013)Lipopolysaccharide (LPS), Griess reagent and

dimethyl sulfoxide (DMSO) were obtained from Sigma (California, USA). MTT and NaNO2 were obtained from Nanjing Naoao Science and Technology Co., Ltd. (Nanjing, China). Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS) and trypsin were obtained from Nanjing KeyGEN Biotech.Co., Ltd. (Nanjing, China) and the Trizol reagent was purchased from Invitrogen (California, USA). Mouse TNF-α, IL-1β and IL-6 ELISA kits were acquried from MultiSciences Biotech Co. Ltd. (Hangzhou, China). Mouse monoclonal TNF-α antibody, IL-1β antibody, IL-6 antibody, β-acitn antibody, mouse monoclonal phospho-NF-κB p65 antibody and mouse polyclonal IκBα antibody were obtained from Cell Signaling Technology (Massachusetts, USA). RAW 264.7 mouse macrophage cells were purchased from Shanghai cell bank, Chinese academy of sciences (Shanghai, China). All other chemicals were of reagent grade. The sample of Nauclea officinalis (50 g) was extracted under reflux in boiling 80 % EtOH (1000 mL) for 2 h and the extraction was repeated three times. After solvent removal, 80 % EtOH crude extract (2.92 g) was obtained.

2.2. UPLC-PDA analysis

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UPLC-PDA (Massachusetts, USA) was used to analyze the Nauclea officinalis extract and a standard solution including pumiloside, 3-epi-pumiloside, strictosamide and vincosamide. Waters Acquity BEH C18 column (2.1 mm × 100 mm, 1.7 µm) was eluted with the mobile phases of acetonitrile (A) and methanoic acid/water (1:1000, v/v) (B) in gradient mode. The flow rate was 0.4 mL/min with the following gradient program: 0-0.5 min, 2 % A; 0.5-6 min, 2 %~10 % A; 6-20 min, 10 %~33 % A. The column temperature was 40°C. The injection volume was 0.3 µL. The detected wavelength was set at 245 nm for pumiloside and 3-epi-pumiloside, 226 nm for strictosamide and vincosamide. The concentrations of four compounds in the exact were calculated with reference to standard curve of the corresponding compound.

2.3. Cell culture RAW 264.7 cells were cultured in DMEM which was supplemented with 10 % FBS, 100 EU/mL penicillin and 100 µg/mL streptomycin at 37°C in a humidified atmosphere of 5 % CO2. Experiments performed in Nauclea officinalis were repeated three times independently.

2.4. MTT assay for cell viability RAW 264.7 cells were seeded in 96-well plates at a density of 4×106 cells/mL and incubated for 24 h. The cells were then treated with 0.05 % DMSO, or Nauclea officinalis (10, 20, 50, 100, 200, 500 µg/mL) in the absence or presence of 1 µg/mL LPS for 24 h. MTT reagent (5 mg/mL) which was dissolved in PBS was added to each well. After incubation at 37°C for 4 h, the culture medium was discarded and then 150 µL of DMSO was added to 7

dissolve the crystals. Optical density was measured at 570 nm using a microplate reader.

2.5. NO assay RAW 264.7 cells (4×106 cells/mL) were plated in 96-well plates and subsequently treated with or without LPS (1 µg/mL) in the presence of different concentrations of Nauclea officinalis (0, 10, 20, 50, 100 µg/mL) for 24 h. Each culture supernatant (100 µL) was mixed with Griess reagent (50 µL) for 10 min at room temperature. The absorbance values were detected at 550 nm. The NO production was determined with reference to standard curve of sodium nitrite.

2.6. Enzyme-linked immunosorbent assay RAW 264.7 cells were divided into six groups randomly and plated at a density of 4×106 cells/mL in 96-well plates. The control group was only treated with DMEM which contained 0.05 % DMSO and the other five groups were subsequently treated with LPS (1 µg/mL) in the presence of various concentrations of Nauclea officinalis (0, 10, 20, 50, 100 µg/mL) for 24 h. The assay was performed according to the manufacturer’s instruction. The OD of the microplate was read at 570 nm.

2.7. Real time fluorescence quantitative PCR RAW 264.7 cells (4×106 cells/mL) were treated with 0.05% DMSO (control), or Nauclea officinalis (0, 10, 20, 50, 100 µg/mL) in the absence or presence of 1 µg/mL LPS for 24 h. Total RNA was isolated using Trizol. The quantity of total RNA were determined at 260 nm and 280 nm. cDNA was generated using the 1st strand cDNA synthesis Kit. cDNA was used as a template for real-time PCR in triplicates with Fast SYBR Green Master Mix 8

(TOYOBO, Japan) and gene-specific primers. PCR was performed for 40 cycles in 20 µL reaction volumes by real-time qPCR using the DA7600 Real-Time PCR System. GAPDH was used as the internal control for normalization. The upstream and downstream primer sequences were as follows: for iNOS: sense primer: 5-AGCAACTACTGCTGGTGGTG-3, antisense primer: 5-TCTTCAGAGTCTGCCCATTG-3; for TNF-α: sense primer: 5-ATGAG AAGTTCCCAAATGGC-3, antisenseprimer: 5-CTCCACTTGGTGGTTTGCTA-3; for IL-1β: sense primer: 5-GAAGAAGAGCCCATCCTCTG-3, antisenseprimer: CCTGTAGTG-3;

for

IL-6:

sense

primer:

5-TCATCTCGGAG

5-AGTCCGGAGAGGAGACTTCA-3,

antisenseprimer: 5-ATTTCCACGATTTCCCAGAG-3; for GAPDH: senseprimer: 5-GGCCT TCCGTGTTCCTACC-3, antisenseprimer: 5-TGCCTGCTTCACCACCTTC-3. Cycling was initiated at 95°C for 5 min, followed by 40 cycles of 95°C for 15s and 60°C for 30 s.

2.8. Western blot analysis The treated RAW 264.7 macrophages (4×106 cells/mL) were mearsured for the relative ratio of p-IκBα/β-actin and p-p65/β-actin. The cellular proteins, extracted by pancreatic enzyme (containing 0.25 % EDTA), ice-cold PBS and cell lysis buffer, were measured with Brandford assay in order to calculate the quantity of proteins. Equal amounts of protein samples were electrophoresed by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane. The PVDF membranes, blocking with 5 % nonfat milk overnight, were subsequently incubated with the primary antibody (1:1000) at 4°C overnight and then incubated with peroxidase conjugated second antibody(1:5000) at room temperature for 2 h. The protein bands were detected using the KeyECL Plus Kit and exposed to X-ray film.

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2.9. Statistical analysis All data were analyzed using the statistical program SPSS software. Multiple group comparisons were performed using one-way ANOVA and Tukey's test. The values were presented as the means ± SD; Values of *P < 0.05, **P < 0.01 and ***P < 0.001 were considered statistically significant.

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3. Results 3.1. Measurement of major compounds of Nauclea officinalis As shown in Fig.1 and Table 1, strictosamide and pumiloside were the major compounds of Nauclea officinalis extract, which were 11.53 % and 4.68 % of the total extract, respectively. It was no doubt that the proportion of strictosamide was highest in Nauclea officinalis extract.

Fig.1 UPLC-PDA analysis of the exacted Nauclea officinalis (B) and standard mixtures (A) including pumiloside (1), 3-epi-pumiloside (2), strictosamide (3) and vincosamide (4) at 245 nm.

Tab 1 Proportions of individual polyphenols in Nauclea officinalis extract Peak

Retention time /min

Tentative identity

Concentration /%

1

10.942

pumiloside

4.68

2

12.024

3-epi-pumiloside

0.64

3

16.570

strictosamide

11.53

4

18.959

vincosamide

0.86

3.2. Effect of Nauclea officinalis on cell viability of RAW 264.7 murine macrophage cells 11

When RAW 264.7 macrophages were treated with Nauclea officinalis at a concentration of 0, 10, 20, 50, 100, 200, 500 µg/mL and 1 µg/mL LPS, the viabilities of RAW 264.7 cells were 100.06 ± 4.73, 98.98 ± 1.61, 95.99 ± 1.77, 97.21 ± 5.08, 90.33 ± 3.00, 63.81 ± 2.99, 42.78 ± 5.16 %, respectively. The results of statistical analysis showed treatment with Nauclea officinalis (10, 20, 50, 100 µg/mL) had no obvious toxic effect on cell growth compared to 0.05 % DMSO group (100.00 ± 6.17 %) (P > 0.05). While the viability of cells exposed to 200, 500 µg/mL Nauclea officinalis was significant different from 0.05 % DMSO group (P < 0.05) (Fig.2). These results indicated that no significant cytotoxicity was observed at concentrations ranging from 10 to 100 µg/mL. Therefore, the cells were incubated with Nauclea officinalis at concentrations from 10 to 100 µg/mL in the following experiments.

Fig.2 Effect of Nauclea officinalis on the viability of RAW 264.7 macrophages. Cell viability was determined by MTT assay. Cells were treated with LPS (1 µg/mL) without or with Nauclea officinalis extract for 24 h. Error bars represent the mean ± SD (n = 5 per group). Values of * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. LPS were considered statistically significant.

3.3. Effect of Nauclea officinalis on NO production Examination of the inhibitory effect of Nauclea officinalis showed significant difference between the control group (11.44 ± 0.30 µM) (P < 0.01) and the LPS-alone stimulated (19.52 ± 0.78 µM) cells. As can be seen in Fig.3, no obvious inhibitory effect on NO production was observed at a concentration of 10 µg/mL (18.63 ± 0.50 µM) (P > 0.05), compared with the 12

LPS-stimulated cells. When the tested drug was at 20 µg/mL (17.92 ± 0.09 µM) (P < 0.01), 50 µg/mL (14.08 ± 0.28 µM) (P < 0.001), 100 µg/mL (11.33 ± 0.44 µM) (P < 0.001), there was significant inhibition in a concentration-dependent manner (P < 0.01) (Fig.3). These results suggested that Nauclea officinalis significantly reduced the NO production at a range of 10-100 µg/mL and the secretion of NO was at the basal level of the control group when the cells were treated with 100 µg/mL Nauclea officinalis.

Fig.3 Effect of Nauclea officinalis on NO production in LPS-induced RAW 264.7 macrophages. RAW 264.7 cells were incubated with LPS (1 μg/mL) and then treated with Nauclea officinalis (10, 20, 50, 100 µg/mL) for 24 h. The culture supernatant was subjected to griess reagent. Error bars represent the mean ± SD (n = 5 per group). Values of *P < 0.05,**P < 0.01 and ***P < 0.001 vs. LPS were considered statistically significant.

3.4. Effects of Nauclea officinalis on levels of TNF-α, IL-6 and IL-1β ELISA assay was used to invesatigate the effects of Nauclea officinalis on levels of TNF-α, IL-6 and IL-1β in LPS-attenuated RAW 264.7 cells. After LPS-stimulated for 24 h, the RAW 264.7 cell secretion of three pro-inflammatory cytokines was strongly increased compared with the untreated control cells. Fortunetaly, Nauclea officinalis (10-100 µg/mL) significantly reduced the production of TNF-α, IL-6 and IL-1β in dose-dependent manner (Fig.4) (P < 0.05). The inhibition following treatment with 100 µg/mL Nauclea officinalis 13

treatment was 38.98 % for TNF-α, 54.66% for IL-6 and 56.05% for IL-1β, respectively. However, none of these cytokine levles returned to the basal level of the untreated group.

Fig.4 Effect of Nauclea officinalis on TNF-α, IL-6 and IL-1β production in LPS-induced RAW 264.7 maerophages. RAW 264.7 cells were incubated with LPS (1 μg/mL) and then treated with Nauclea officinalis (10, 20, 50, 100 µg/mL) for 24 h. The culture supernatant was subjected to ELISA kits. Error bars represent the mean ± SD (n = 6 per group). Values of *P < 0.05, **P < 0.01 and ***P < 0.001 vs. LPS were considered statistically significant.

3.5. Effect of Nauclea officinalis on iNOS mRNA expression 14

The relative mRNA expression of iNOS was quantitated with real time fluorescence quantitative PCR. Nauclea officinalis showed mild inhibitory effect in LPS-stimulated RAW 264.7 murine macrophage cells at the concentrations of 10 µg/mL (8.58 ± 0.67 %) (P < 0.05) and 20 µg/mL (5.95 ± 0.33 %) ( P < 0.01), compared to LPS-stimulated cells (10.43 ± 0.70 %). Significant inhibitory effect was obtained when the concentrations of the tested drug are 50 µg/mL (3.07 ± 0.24 %) (P < 0.001) and 100 µg/mL (1.48 ± 0.11 %) (P < 0.001) (Fig.5). The results suggested that Nauclea officinalis significantly reduced the iNOS expression at dose-dependently manner, which was in line with NO production.

Fig.5 Effect of Nauclea officinalis on iNOS expression in LPS-induced RAW 264.7 maerophages. RAW 264.7 cells were incubated with LPS (1 μg/mL) and then treated with Nauclea officinalis (10, 20, 50, 100 µg/ml) for 24 h. The mRNA expression levels of iNOS were quantitated with real time fluorescence quantitative PCR. Error bars represent the mean ± SD (n = 3 per group). Values of *P < 0.05, **P < 0.01 and ***P < 0.001 vs. LPS were considered statistically significant.

3.6. Effect of Nauclea officinalis on TNF-α, IL-6 and IL-1β mRNA expression According to Fig.6, Nauclea officinalis (10, 20, 50, 100 µg/mL) had strong and concentration-dependent effects on reducing relative expression of TNF-α, IL-6 and IL-1β mRNA in LPS-induced RAW 264.7 cells (P < 0.05). The high dosage of Nauclea officinalis (100 µg/mL) showed the best inhibitory effect, compared with that of other groups. The 15

percent inhibition of 100 µg/mL Nauclea officinalis was 80.83 % for TNF-α, 80.00 % for IL-6 and 86.51 % for IL-1β, respectively. Although there were high levels of inhibition of cytokine production, none of them returned to their basal level, compared to the control group.

Fig.6 Effect of Nauclea officinalis on TNF-α, IL-6 and IL-1β mRNA expression in LPS-induced RAW 264.7 maerophages. RAW 264.7 cells were incubated with LPS (1 μg/mL) and then treated with Nauclea officinalis (10, 20, 50, 100 µg/mL) for 24 h. The mRNA expression level of TNF-α (A), IL-6 (B) and IL-1β (C) were quantitated with real time fluorescence quantitative PCR. Error bars represent the mean ± SD (n=3 per group). Values of *P < 0.05, **P < 0.01 and ***P < 0.001 vs. LPS were considered statistically 16

significant.

3.7. Effects of Nauclea officinalis on phosphorylation of IκB-α and p65 As shown in Fig.7, 10-100 µg/mL of Nauclea officinalis obviously suppressed the phosphorylation of IκB-α and phosphorylated protein expression of p65 in RAW 264.7 cells (P < 0.05). Nauclea officinalis showed substantial inhibitory effect on IκBα phosphorylation with following dose-dependently reducing relative ratio: 47.32 ± 3.10 %, 41.07 ± 2.68 %, 33.37 ± 2.19 %, 11.69 ± 0.83 %. That of control group and LPS-alone attenuated group were 0.59 ± 0.19% and 76.22 ± 3.45%. The relative ratio of p-p65/β-actin in Nauclea officinalis treatment group (control group, 10, 20, 50, 100 µg/mL) were 4.29 ± 0.47%, 54.47 ± 4.59%, 46.65 ± 1.44%, 29.76 ± 1.51%, 10.90 ± 1.21%, respectively, while that of LPS-alone induced group was 70.33 ± 7.31 %. The results suggested the signifigant inhibitory effect of Nauclea officinalis on LPS-induced phosphorylation of IκBα and p65.

Fig.7 Effect of Nauclea officinalis on the LPS-induced activation of the NF-κB pathway in RAW 264.7 cells. The cells were treated with Nauclea officinalis (10, 20, 50, 100 µg/mL) in the presence of 1 μg/mL

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LPS for 24 h. Protein samples were analyzed by Western blot using phospho-specific anti-IκBα and phospho-specific anti-p65 antibodies (A). GAPDH was used as the internal control for normalization. The bar chart shows the quantitative evaluation of p-IκB-α and p-p65 bands by densitometry. The data represent the mean ± SD (n=3 per group) (B and C). The bars not sharing the same letter are significantly different at *P < 0.05,**P < 0.01 and ***P < 0.001 vs. LPS were considered statistically significant.

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4. Discussion In this research, Nauclea officinalis extract reduced production of NO, TNF-α, IL-1β and IL-6, and significantly suppressed phosphorylation of IκB-α and p65 subunit, which suggested Nauclea officinalis had effective anti-inflammatory effects on LPS-induced inflammatory responses. NO is a short-lived free radical that participates in kinds of physiological processes such as regulating neuro transmission, inflammation, mitochondrial functions and apoptosis, and directly damaging normal cells (Kim et al., 2014). Its production is associated with iNOS via activated NF-κB signaling pathways (Chen et al., 2014). In this research, the results indicated Nauclea officinalis extract dose-depedently suppressed NO production as well as iNOS expression in the presence of LPS in RAW 264.7 macrophages and restored expression to the basal level of the control group. This suggested that the surpressing effect on iNOS expression is one of the anti-inflammatory mechanisms of Nauclea officinalis. LPS-activated macrophages results in the secretion of pro-inflammatory cytokines including TNF-α, IL-1β and IL-6, all of which play key roles in anti-inflammatory response (Sandro et al., 2005). TNF-α is a crucial mediator that activates the expression of inflammatory molecules to improve the inflammatory response (Fan et al., 2015). IL-1β and IL-6 also have pro-inflammatory properties and are considered as important players in the immune response (Yang et al., 2013). In general, inhibition of TNF-α, IL-1β and IL-6 is regarded as an effective treatment strategy for inflammatory diseases and tissues damage (Chang et al., 2012; Eli et al., 2009). The present study suggested that Nauclea officinalis suppresses the production and mRNA level of TNF-α, IL-1β and IL-6 in a dose-dependent 19

manner in LPS-activated RAW 264.7 macrophages. However, the production of these cytokines as well as their mRNA expression did not return to the basal level of the untreated cells. The unsatisified results may be associated with the incompleted inhibition of the phosphorylation of IκB-α and p65 subunit which regulate the secretion of these pro-inflammatory cytokines. NF-κB signaling pathway plays an important role in inflammatory reaction as a transcription factor for pro-inflammatory mediators (Lee et al., 2012). TNF and/or IKKβ overexpression appear to suppress NF-κB gene expression by augmenting the transcriptional activity of NF-κB p65 subunit (Xu et al., 2003). IκB-α degradation is also a key step in NF-κB activation and most stimuli activates NF-κB by IKK-mediated IκBα phosphorylation on N-terminal serine residues (Wu et al., 2003). Phosphorylated IκB-α stimulates the degradation of IκB-α, which leads to p65 subunit translocates to the nucleus (Fan et al., 2015). The results in present study indicated that Nauclea officinalis significantly and concentration-dependently restrained phosphorylation of IκB-α and p65 in LPS-induced RAW 264.7 cells, which resulted in less expression of inflammation-associated proteins, including iNOS, TNF-α, IL-6 and IL-1β. Hence, suppressing LPS-induced NF-κB activation may be a pivotal strategy to reduce inflammatory status in organism. Furthermore, the reason of the incompleted inhibition of phosphorylation of IκB-α and NF-κB p65 in the furture research will be investigated to study the anti-inflammatory mechanism of Nauclea officinalis in more detail. Many researches have reported that alkaloids are the major active compounds in Nauclea officinalisˈand they possess good anti-inflammatory activities (Wang et al., 2012; 20

Sun et al., 2007; Sun et al., 2008; Zhu et al., 2013). In our previous studies, pumiloside, 3-epi-pumiloside, strictosamide and vincosamide were found to be the major chemical compounds in the EtOH extract of Nauclea officinalis (Chen et al., 2014; Zhu et al., 2014). In this experiment, we discovered that alkaloids were possibly the main contributors on anti-inflammatory effects of Nauclea officinalis, and strictosamide assumed to be the active ingredient of anti-inflammatory activities. However, the assumption has not been confirmed with present experiments and it will be studied in the further study.

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5. Conclusions In this study, Nauclea officinalis significantly inhibited the pro-inflammatory mediator such as NO and cytokines production including TNF-α, IL-1β and IL-6 in LPS-mediated RAW 264.7 macrophages. In addition, Nauclea officinalis suppressed phosphorylation of IκBα and NF-κB p65 in the NF-κB signaling pathway (Viatour et al., 2005; Fan et al., 2015; Xu et al., 2003). The research showed that Nauclea officinalis had a good anti-inflammatory activity. Since strictosamide is the highest content in the extract of Nauclea officinalis, it maybe be the effective anti-inflammatory chemical compound, and the possible anti-inflammatory mechanism of strictosamide as well as Nauclea officinalis in more detail will be demonstrated in our future research.

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Acknowledgements This work was supported by the grants of the National Natural Science Foundation of China (No. 81203001 and No. 81160558).

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