Octacosanol Attenuates Inflammation in Both RAW264.7

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Octacosanol attenuates inflammation in both RAW264.7 macrophages and a mouse model of colitis Tianyi Guo, Qinlu Lin, Xinhua Li, Ying Nie, Long Wang, Limin Shi, Wei Xu, Tao Hu, Ting Guo, and Feijun Luo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05465 • Publication Date (Web): 26 Jan 2017 Downloaded from http://pubs.acs.org on January 28, 2017

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Journal of Agricultural and Food Chemistry

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Octacosanol Attenuates Inflammation in Both RAW264.7

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Macrophages and a Mouse Model of Colitis

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Tianyi Guo, †,§ Qinlu Lin, †,§ Xinhua Li, ‡ Ying Nie, † Long Wang, † Limin Shi, † Wei Xu, † Tao Hu, † Ting Guo, † and Feijun Luo†,#

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†

Laboratory of Molecular Nutrition, National Engineering Laboratory for Rice and

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Byproducts, College of Food Science and Engineering, Central South University of

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Forestry and Technology, Changsha, Hunan 41004, China

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‡

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Changsha, Hunan 410008, China

Department of Gastroenterology, Xiangya Hospital, Central South University,

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Contributed equally to this work.

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#

Corresponding author, Dr. Feijun Luo, Laboratory of Molecular Nutrition, College

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of Food Science and Technology, Central South University of Forestry and

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Technology, 498, Shaoshan Road, Changsha, 410004, China.

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Tel: +86-731-85623240, Feijun Luo Email: [email protected];

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[email protected].

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ACS Paragon Plus Environment


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ABSTRACT: Octacosanol has multiple biological functions. In this study, the

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anti-inflammatory effect and molecular mechanism of octacosanol were evaluated by

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using dextran sulfate sodium (DSS)-induced ulcerative colitis model in mice and

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lipopolysaccharide (LPS)-stimulated mouse macrophage RAW264.7 cells. The colitis

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mouse model was induced by 3.0% DSS in 8-week ICR mice and octacosanol oral

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administered with 100mg/kg/day. The results showed that octacosanol significantly

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improved the health status of mice and reduced DSS-induced pathological damage in

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the colonic tissues. Octacosanol obviously inhibited the mRNA and protein expression

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levels of pro-inflammatory factors of colonic tissues. In vitro study, octacosanol

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administration significantly reduced the expression of mRNA or protein of

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pro-inflammatory cytokines and the phosphorylation of c-Jun N-terminal kinase and

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p38, and it also partly prevented LPS-induced the translocations of NF-κB and AP-1.

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Octacosanol has anti-inflammatory effect and its molecular mechanism may be

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involved in down-regulating the expression of inflammatory factors and blocking of

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MAPK/ NF-κB/AP-1 signaling pathway.

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KEYWORDS: octacosanol, colitis, inflammation factor, MAPK, NF-κB, AP-1

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INTRODUCTION

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With the improvement of living standards and the diversification of diets, the

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incidence of digestive disease including inflammatory bowel disease (IBD) has

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increased considerably over the past decades.1 The ulcerative colitis disease (UC) is

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induced by multi factors and is one of the common non-specific inflammatory bowel

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diseases. UC has a long disease course and is a great risk factor leading to colorectal

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cancer. 2 Blood stool and pus, diarrhea and abdominal pain are the main symptoms of

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UC patients. Its ulcer lesions are mainly located in the mucosal layer of the colon,

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involving the rectum, distal colon even extended to the proximal or entire colon. 3 UC

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was listed as one of the modern refractory diseases by the WHO.4,5 So far, the etiology

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of UC has not yet completely been understood. Anti-inflammation and regulating the

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immune response are the main measures for UC treatment, including medication of

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5-ASA drugs, corticosteroids and immunosuppressors, cell therapy or surgical

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treatments. However, these therapies have high risks of adverse effects (such as sleep

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and mood disturbances, dyspepsia, and glucose intolerance), high recurrence rate and

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poor tolerance, they are not suitable as long term therapies, and new measures for

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adjunct therapies are needed. 6,7

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Octacosanol, the main component of policosanol, is a kind of high-level saturated

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fatty alcohol which exists in natural plants such as rice, sugar cane in the form of wax

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ester.8 Many studies have reported that octacosanol plays a positive role in many

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physiological functions, including reducing blood sugar and lipid,9,10 resisting fatigue,

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regulating cardiovascular function13,14 and so on.15 In addition, toxicity

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experiments have demonstrated that it has wide safety margin when administered

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chronically in rats,16 which is safer compared to the same dose of salt. Benefited from

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its high security, octacosanol could be used as a kind of drug or food additives without

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any

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anti-inflammatory effects. For example, it can increase neutrophil influx of

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carcinogen-induced mouse and inhibit the expression of tumor necrosis factor-α

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(TNF-α).17 A long chain fatty alcohol mixture extracted from evening primrose seed

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oil (octacosanol content is 7.64%) reduces nitric oxide (NO) content and release of

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interleukin-1β (IL-1β) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells.18 It

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suggests that the octacosanol may have a potential anti-inflammatory effect.

side

effects.

Some

evidences

suggest

that

octacosanol

may

have

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To simulate the clinical symptoms of UC, a classic model of the colitis induced

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by dextran sulfate sodium (DSS) was used in this study. The model has the advantages

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of low cost, simple preparation, good repeatability and easy operation. Experimental

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animals induced by DSS exhibits the characteristics resembling human UC, for

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instance severe diarrhea, weight loss, ulceration and leukocyte infiltration.19,20 As a

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physiological response to infection or injury, the macrophage plays a critical role in

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inflammation.21,22 It produces different cytokines or releasing of lysosomal enzymes,

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macrophages mediate directly or indirectly the process of inflammatory reaction.23

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LPS has widely used to induce the inflammatory response of macrophages for the

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study of anti-inflammatory mechanism. LPS could stimulate a series of expression of

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cytokines proteins caused by activating cell signaling pathway, including

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mitogen-activated protein kinases (MAPK) and nuclear factor kappa-B (NF-κB),

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which are the major kinase families associated with the inflammation process. 24,25

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So far, there is no report provides direct evidences about octacosanol inhibiting

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inflammation in classical inflammatory models. In this study, we examined the

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anti-inflammatory effects of octacosanol by using both LPS-stimulated RAW264.7

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cell and DSS-induced mouse model. The study also evaluated the effects of

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octacosanol on NF-κB and MAPK signaling pathways and revealed the possible

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anti-inflammation mechanism of octacosanol.

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MATERIALS AND METHODS

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Materials and Reagents. DSS (purity: 100% MP Biomedicals, Illkirch, France.

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Molecular weight 36-50 kDa, Lot number M2066); Octacosanol (flake-like white

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powder, extracted from rice bran, purity 99%, Sigma Aldrich Co., St. Louis, Mo.,

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USA); Stock solutions of octacosanol were dissolved in ethanol and octacosanol

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solutions were performed by ultrasonic concussion for 5 min. NO assay kit, MDA

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assay kit and MPO assay kit were purchased from Nanjing Jiancheng, Nanjing, China.

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LPS from Escherichia coli O127:B8 was purchased from Sigma Aldrich Co. (St.

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Louis, Mo., USA). RPMI 1640 and fetal bovine serum were purchased from

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Gibco-BRL (Gibco-BRL, Carlsbad, CA, USA). Nuclear and Cytoplasmic Protein

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Extraction Kit (P0028) and Enhanced BCA protein kit (P0009) were purchased from

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Beyotime Biotechnology (Shanghai, China). Polyclonal antibody TNF-α (Cat#11948),

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IL-1β (Cat#12507), inducible nitric oxide synthase (iNOS) (Cat#13120), interleukin-6

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(IL-6) (Cat#12912), β-Actin (Cat#12620), phospho-MAPK Sampler Kit (Cat#9910),

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MAPK Family Antibody Sampler Kit (Cat#9926), SP600125, U0126 and SB203580

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were purchased from Cell Signaling Technology Company (Beverly, MA, USA).

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Anti-Mouse IgG HRP Conjugate (W4021) and anti-rabbit IgG HRP Conjugate

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(V7951) were purchased from Promega Corporation (Madison, Wl, USA). All other

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reagents of this study were the analytical grade.

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Experimental Animals. Total 60 8-week-old，weight between 30-35 g, half of

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each sex ICR mice were purchased from Hunan SJA Laboratory Animal Co., Ltd

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(SLAC, Changsha, Hunan, China) and were fed with standard laboratory chow in the

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animal room with 12h dark/light cycles and constant temperature of 23±2℃. Before

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treatment, animals were fed with a laboratory diet (SLAC, Changsha, Hunan, China)

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and water ad libitum. After one week of adaptation period, these mice were randomly

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divided into 3 groups (20 mice per group). Mice in control group were given drinking

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water ad libitum for 13 days. The DSS group and the DSS+Oct group were given

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drinking water with 3% (w/v) DSS only from the third day to the end of the

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experiment (DSS treatment period were 11 days). And the mice of octacosanol group

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were additionally administrated octacosanol suspension with water (100mg/kg/day)

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from the third day of DSS treating to the end of 13 days experimental period. The use

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of the animals and experimental protocols were approved by the Guidelines for the

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Care and Use of Experimental Animals, the Hunan Normal University School of

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Medicine (SYXK(Xiang) 2014-0006), and the study was approved by the office of

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Animal Experiment Ethnics, Central South University of Forestry and Technology.

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Evaluation of Disease Activity Index (DAI). During the experimental period,

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the body weight, food and water intake, mental state, stool consistency and gross

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bleeding were recorded at the same time every day. DAI score was determined by

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combining scores of body weight loss, stool consistency and gross bleeding26 and it

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was described in Table 1. Determination of fecal occult blood: small amount of feces

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was smeared evenly on the glass slide with sterilized cotton swab. After adding a few

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drops of orthotolidine (o-TD), add a few more drops of 3% H2O2. Judgment standard

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of fecal occult blood: the fecal color has no significant change in 2 minute should be

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determined as negative; the color changes blue and becomes gradually (blue brown)in

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10s should be determined as weak positive; the color changes from light blue brown

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to deep (blue brown) should be determined as positive; the color shows the blue

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brown immediately should be strong positive. At the end of the experiment, body

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weight and spleen weight were measured immediately，and the colon tissues of

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sacrificed mice were separated from the proximal rectum. The colon length between

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the ileo-cecal junction and the proximal rectum was subsequently measured.

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MPO and MDA Assay. The middle segments of the colon tissues were

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intercepted and myeloperoxidase (MPO) and malonic dialdehyde (MDA) contents

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were detected. The tissues (50-100mg) were washed and homogenized in pre-cold

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phosphate buffered saline (homogenate concentration is 10% w/v). According to the

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protocols specification, MPO and MDA assays were carried strictly. The MPO

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activity was expressed in terms of units/g tissue (IU/g), and the content of MDA was

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in terms of nmol MDA/mg tissue protein (nmol/mg).

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NO Assay. NO is instable in vivo and easy to be converted to nitrite (NO2-) or

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nitrate (NO3-), so the NO production was indirectly calculated by measuring the nitrite

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and nitrate levels. Centrifuged the mice blood at 4000 rpm for 20 min at 4°C, and the

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supernatants were analyzed the content of nitrite and nitrate content according to the

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protocol of NO assay kits.

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Histopathology Analysis. The middle segments of the colon tissues were

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intercepted and fixed in 10% (100 g/L) formalin solution for 24h, then dehydrated

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with increasing concentrations of ethanol, transparented the sections using xylene and

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embedded in molten paraffin. Sections of colon tissue were cut into 5µm slices on a

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microtome and then put on clean glass slides and dried at 37°C overnight. Tissues

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were dewaxed by xylene and dehydrated by different concentrations of ethanol. These

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sections were stained with hematoxylin and eosin (H&E), and the morphometry of

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intestinal epithelia cell, loss of crypt glands and density of neutrophil and lymphocyte

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infiltration would be observed under the microscope.

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Cell culture. The mouse monocyte-macrophage cell line RAW264.7 was

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purchased from BioHermes Co. (Wuxi, China) and the cell line are originated from

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ATCC (American Type Culture Collection, USA). RAW264.7 cells were incubated in

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Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat inactivated fetal

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bovine serum (FBS), 100 U/ml streptomycin and 100 U/ml penicillin at 37℃ and 5%

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CO2. The medium was replenished three times a week and cells were subcultured

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using 0.25% trypsin timely.

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Total RNA extraction and quantitative RT-PCR. Tissues (100mg) of the

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middle segment of the colon were intercepted and were ground in liquid nitrogen

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pre-cooling mortar. Raw264.7 cells were seeded at 1×106 cells per mL in 6-wells

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plates and incubated overnight. Cells were treated with octacosanol (0, 10, 30 and

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100µg/mL) for 2 hours followed by adding LPS (1µg/mL) for 6 hours. Total RNA

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from cells or colon tissues was extracted by using Transzol-Up reagent according to

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the manufacturer's instructions. The quality, purity and concentration of RNA sample

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were analyzed using Nanodrop ultramicro spectrophotometer instrument. The

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resulting RNA (2µg) was used as a template for reverse-transcribing to first-strand

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cDNA synthesis by using High-Capacity cDNA Reverse Transcription Kits in a total

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volume of 20 µL. Real-time quantitative polymerase chain reaction (RT-qPCR) was

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performed by the CFX96 Real Time PCR system (Applied Bio systems) using

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SYBR® Select Master Mix (Applied Bio systems) referring to the protocol. The

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sequences of primers (pro-inflammatory cytokines TNF-α, IL-1β, iNOS, IL-6 and

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β-actin, control for qPCR) used for RT-qPCR analysis. The amplification conditions:

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94℃，initial denaturation for 3 min；94℃，denaturation for 30 s；60℃，annealing for

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40 s；72℃，extension for 1 min, 40 cycles. Relative expression levels of the target

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genes were calculated based on 2-△△Ct（RQ）method. The PCR primers were used as

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following: IL-1β forward: 5’ GAG CAC CTT CTT TTC CTT CAT CTT 3’, reverse: 5’

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TCA CAC ACC AGC AGG TTA TCA TC 3’; IL-6 forward: 5’ ATG GAT GCT ACC

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AAA CTG GAT 3’, reverse: 5’ TGA AGG ACT CTG GCT TTG TCT 3’; iNOS

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forward: 5’CAG CTG GGC TGT ACA AAC CTT-3’; reverse: 5’CAT TGG AAG

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TGA AGC GTT TCG3’；TNF-α forward: 5’ CAA AAT TCG AGT GAC AAG CCT G

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3’, reverse: 5’ GAG ATC CAT GCC GTT GGC 3’; β-actin forward: 5’ ATC ATG TTT

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GAG ACC TTC AAC ACC3’, reverse: 5’ TAG AGC AAC ATA GCA CAG CTT CTC

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TT 3’.

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Preparation of Total/Nuclear Lysates. Raw264.7 cells were seeded at a

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concentration of 1×106 cells/mL in 10cm petri dish and incubated overnight. Cells

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were treated with octacosanol (0, 10, 30 and 100µg/mL) for 2 hours followed by

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exposure to LPS (1µg/mL) for 1 hours. Before the extracting of protein, stimulated

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RAW264.7 cells were harvested and washed twice with ice-cold PBS. Tissues (100mg)

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removed from colon were ground in liquid nitrogen pre-cooling mortar. Total protein

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from cells or colon tissues was extracted with RIPA buffer (0.1% deoxycholate, 1%

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Triton X-100, 0.5% SDS, 2mM PMSF, 2mM EDTA and 2mM orthovanadate)

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supplemented with a cocktail of protease and phosphatase inhibitors. The cell nuclear

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and cytoplasmic proteins were extracted using cytoplasm/nucleus protein extraction

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reagents referring to manufacturer's instructions. The concentration of protein samples

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were determined using bicinchoninic acid (BCA) protein assay kit with Nanodrop

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ultramicro spectrophotometer instrument.

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Western Blot Analysis. The prepared protein samples were mixed in equal

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amounts (10-20µg) with 2×SDS loading buffer (125mM Tris-HCl, pH 6.8, 10%

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2-mercaptoethanol, 4% SDS, 20% glycerol, and 0.3% bromophenol blue), boiled for 5

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min at 95℃. Then the mixture was loaded to electrophoresis on 10%-15% SDS-PAGE

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gel followed by transferring onto a polyvinylidene difluoride (PVDF) membrane. The

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membranes combined with protein of target gene were blocked with 5% bovine serum

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albumin (BSA) in Tris-buffered saline with Tween20 (TBST) for about 1h at room

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temperature. After being blocked, the membrane was incubated with a 1:1000 dilution

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of primary antibody in 5% BSA with TBST at 4℃ for overnight. Then the membrane

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was washed three times for 30min with TBST at room temperature and then incubated

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with a 1:5000-10000 dilution of anti-mouse/ anti-rabbit IgG secondary antibody for

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1h at 4℃. After washed three times for 30min with TBST, the immunoreactive

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proteins were detected using the ECL Plus™ Western blot detection system (Pierce,

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Rockford, USA) and was imaged in the Gel Imaging System (ChemiDoc™ XRS+,

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BIO-RAD). The relative expression quantity of the target protein compared to the

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control protein was determined through calculating the integral light density value of

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each band using the imaging system.

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Luciferase Reporter Activity Assay. RAW264.7 cells were seeded at a density

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of 5×105/mL in 24-well plates for overnight incubation. After 24h incubation, the

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culture medium was removed and replaced with fresh medium without serum,

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antibiotics. After achieving 80% confluence of cells, culture medium was replaced

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with fresh serum free medium again at 300 µL/well containing transfection mixture

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(2µL of pNF-κB/AP-1-luc in 2µL of lipofectamine 2000 reagents) and cells were

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transferred for 4h. Then the medium mixed with transfection mixture was removed

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and cells were cultivated in complete medium for 5h. Cells were pre-treated with

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octacosanol (0, 10, 30 and 100 µg/mL) for 2 hours before stimulation with LPS (1

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µg/mL). 12 hours after stimulation, the cell were then obtained with 1× reporter lysis

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buffer, luciferase activity from cell lysates was determined using the luciferase assay

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system and reporter lysis buffer from Promega. Results of luciferase expression were

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expressed as a relative light units (RLU)/mg protein.

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Statistical Analysis. Each experiment repeated three times, and the data were

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analyzed by SPSS 17.0 software (SPSS, Chicago, USA). Results are expressed as the

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mean±SD and were analyzed by means of one-way ANOVA test analysis followed by

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Tukey’s test when comparisons proceed among groups. Percentage (ratio)

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comparisons were used Chi-square test. The value of p less than 0.05 was considered

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statistically significant.

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RESULTS

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Octacosanol ameliorates inflammatory symptoms of DSS-induced colitis in

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mice. In order to investigate the effect of octacosanol on colitis, DSS-induced ICR

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mice inflammation model was established in the experiments. During the

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experimental course, phenotypic changes of mice were observed and recorded. After

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DSS administration, mice show body weight loss, reduced food intake, diarrhea,

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reduced movements and even hematochezia. DSS-induced mice gavaged with

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octacosanol (100mg/kg/day) significantly mitigate those symptoms of colitis. DSS

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treatment resulted in a visible hematochezia at day 7, loss of body weight from

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35.67±2.13g to 34.05±1.44g. The body weight loss of protection group (DSS+Oct)

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mice were improved at end of the experiment (Fig. 1A). Contrasted with control

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group, the score of DAI increased significantly after DSS intake, however, markedly

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attenuated in octacosanol-treated group. The scores of the two groups were

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11.05±0.94 and 8.15±1.03 (p

Octacosanol Attenuates Inflammation in Both RAW264.7

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