Flaxseed oil alleviates dextran sulphate sodium-induced ulcerative colitis in rats

Flaxseed oil alleviates dextran sulphate sodium-induced ulcerative colitis in rats

Journal of Functional Foods xxx (xxxx) xxxx Contents lists available at ScienceDirect Journal of Functional Foods journal homepage: www.elsevier.com...

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Journal of Functional Foods xxx (xxxx) xxxx

Contents lists available at ScienceDirect

Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff

Flaxseed oil alleviates dextran sulphate sodium-induced ulcerative colitis in rats ⁎

Qian Zhoua,b,1, Lei Maa,1, Wenyang Zhaoa, Wen Zhaoa,b, , Xue Hana, Jiahui Niua, Rongbin Lia, Changhui Zhaoc a

College of Food Science and Technology, Hebei Agricultural University, Baoding 071000, PR China Engineering Technology Research Center for Agricultural Product Processing of Hebei, Baoding 071000, PR China c College of Food Science and Engineering, Jilin University, Changchun 130062, PR China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Flaxseed oil Dextran sulfate sodium (DSS) Ulcerative colitis Inflammatory cytokine Gut microbiota

Flaxseed oil is a type of herbal oil obtained from the ripened seeds of the flax plant (Linum usitatissimum L.) that is widely utilized for baked foods. The aim of present study was to investigate the effect of flaxseed oil on ulcerative colitis. The rats were gavaged daily with flaxseed oil at doses of 400, 800 and 1600 mg/kg b.w. for six weeks, while ulcerative colitis was induced by daily administrating with 3% (w/v) dextran sulfate sodium (DSS) on the sixth week. Results showed that the rats fed with flaxseed oil had less disease activity index, colon weight, colon weight/length ratio and colon cell damage. Flaxseed oil consumption relieved the oxidative condition, reduced colon inflammation and partly restored the microbiota change by DSS. Flaxseed oil (1600 mg/kg b.w.) has a great potential in prevention of ulcerative colitis possibly by regulating the oxidative condition, inflammatory factors and cecal microbiota.

1. Introduction Ulcerative colitis is a type of chronic inflammatory bowel disease characterized by abdominal pain, tenesmus and bloody diarrhea, which can seriously reduce the quality of life (Nigg, Naumann, Käser, & Vetter, 2008). Ulcerative colitis without proper control can also increase the risk of colorectal cancer and approximately 3.7% ulcerative colitis patients will develop into colorectal cancer which substantially contributes to the morbidity and mortality associated with this disease (Eaden, Abrams, & Mayberry, 2001; Lopez, Pouillon, Beaugerie, Danese, & Peyrin-Biroulet, 2018). Conventional drugs for ulcerative colitis contain corticosteroids, salicylates, immunosuppressors and/or anti-tumor necrosis factor alpha (TNF-α) agents, which usually have serious side-effects such as headache, nausea, vomit, fever, abdominal pain, arthralgia and infection in a long-term and high-dose administration (Praengam et al., 2017; Wang, Lin, Zhao, & Li, 2015). Therefore, effective dietary supplements with no or little adverse effect are good candidates as strategies for prevention and/or treatment of ulcerative colitis. Among the dietary factors, the dietary lipids are being increasingly recognized as a kind of important modulators of the bowel disease risk (Innis & Jacobson, 2010; Wild, Drozdowski, Tartaglia, Clandinin, & Thomson, 2007).

The flaxseed is the seed of the flax plant (Linum usitatissimum L.) which is produced globally with high output (Bekhit et al., 2018). The main product of flaxseed is the flaxseed oil that is obtained from the ripened seeds of the flax plant and widely utilized for bread, cookies, cakes and other baked foods (Carter, 1993). Flaxseed oil has been recognized as one of several foods rich in α-linolenic acid (Cunnane et al., 1993) which has been reported to have anti-inflammatory and hypolipidemic effects (Lin & Tan, 2012; Zhao et al., 2004). Consumption of flaxseed oil can prevent against alcoholic hepatic steatosis by ameliorating lipid homeostasis at the adipose tissue-liver axis in mice (Wang et al., 2016), improve the lipid profile in people (Mirfatahi, Tabibi, Nasrollahi, & Hedayati, 2016), and relax both constipation and diarrhea (Palla & Gilani, 2015). However, there has some controversial results about the effect of flaxseed materials on the colitis. Palla, Iqbal, Minhas, and Gilani (2016) has reported that oral administration of flaxseed extract could exhibit mucosal protective effect in acetic acid induced colitis in mice, while Zarepoor (2013) has demonstrated that addition of flaxseed oil (3.5% of basal diet) aggravated the DSS-induced acute colitis symptoms, such as DAI and MPO activity. More importantly, the microbiota may play a critical role in etiology of ulcerative colitis, as dysbiosis of commensal microbiota is frequently reported to be associated with ulcerative colitis in humans and animals (Hartley et al.,



Corresponding author at: College of Food Science and Technology, Hebei Agricultural University, Baoding 071000, PR China. E-mail address: [email protected] (W. Zhao). 1 Qian Zhou and Lei Ma contributed equally to manuscript. https://doi.org/10.1016/j.jff.2019.103602 Received 14 May 2019; Received in revised form 27 September 2019; Accepted 1 October 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Qian Zhou, et al., Journal of Functional Foods, https://doi.org/10.1016/j.jff.2019.103602

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method (Cooper, Murthy, Shah, & Sedergran, 1993; Palla et al., 2016). The loss of body weight by 0, 1–5%, 6–10%, 11–20% and > 21% was graded as score 0, 1, 2, 3 and 4, respectively. The stool consistency at normal, soft but still formed, very soft, and diarrhea was graded as score 0, 1, 2, and 3, respectively. The stool occult blood at normal, hemooccult positive, and gross bleeding was graded as scores 0, 1 and 2, respectively. The DAI score was calculated as the sum of these parameters mentioned.

1992; Moayyedi et al., 2015; Noor et al., 2010). Pathogenic bacteria can invade the damaged epithelial barrier to aggravate the conditions of the ulcerative colitis patients and there is few detailed information on the restoration of the microbiota change in DSS induced colitis by the dietary supplement of flaxseed oil. It is noted that the α-linolenic acid was shown be able to attenuate the colonic inflammation in dextran sulfate sodium (DSS) induced colitis rats (Tyagi et al., 2012). Therefore, the present study was aimed to investigate the preventative effect of the natural herbal product flaxseed oil, which is rich in α-linolenic acid, on DSS induced ulcerative colitis mainly focusing on evaluating the oxidative stress, inflammation and microbiota related factors in Sprague Dawley rats.

2.4. Histopathological examination The colon from the ileocecal junction to the anus was quickly removed and weighted with its length measured. Representative segments (1 cm length) from the distal colon were fixed in 10% neutral buffered formalin solution, dehydrated with ethanol, clarified with chloroform, embedded with wax, and then sliced at 4–5 μm for hematoxylin and eosin staining followed by examination by the professional clerk blinded to the groups.

2. Materials and methods 2.1. Chemicals and regents The flaxseed oil with a purity of 99% was purchased from Hongjingyuan oil Co., Ltd (Inner Mongolia, China). The oil composition of flaxseed oil included linolenic acid at 56.58%, oleic acid at 18.22%, linoleic acid at 15.32%, palmitic acid at 6.28% and stearic acid at 3.53%. DSS was purchased from Shanghai Yuan Ye Co., Ltd. Commercial kits for quantifying superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA) and myeloperoxidase (MPO) were purchased form Nanjing Jiancheng Bioengineering Research Institute, and kits for interleukin 2 (IL-2), interleukin (IL-4), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 12 (IL-12), nterferon γ (IFNγ), nuclear factor κB (NF-κB) and monocyte chemoattractant protein-1 (MCP-1) were from BOSTER Biological Technology Co., Ltd, Wuhan. All other chemicals were of analytical grade.

2.5. Biochemical analysis Part of colonic tissue was completely homogenized and centrifuged at 2000g at 4 °C for 10 min and then the supernatant was collected for analysis. The SOD and MPO activities were determined by the enzymelinked immunosorbent assay (ELISA) and the levels of MDA and GSH were assayed by the colorimetric method. The inflammation indicators of IL-2, IL-4, IL-6, IL-10, IL-12, IFN-γ, NF-κB and MCP-1 were quantified by ELISA. All determination produces were performed rigorously according to the manufacturer’s instructions. 2.6. Microbial analysis of the cecal content

2.2. Animal experiment Total DNA was extracted using the TIANamp Stool DNA kit (Tiangen, Beijing, China). The V3-V4 region of the bacterial 16S rRNA gene was amplified with primers (F338 5′-barcode-ACTCCTACGGGAG GCAGCAG-3′ and R806 5′-GGACTACHVGGGTWTCTAAT-3′). The 5′ ends of the primers were tagged with specific barcodes per sample and sequencing universal primers. The program was 95 ℃ for 3 min, 27 cycles PCR (95 ℃ for 30 s, 55 ℃ for 30 s, 72 ℃ for 30 s), finally 72 ℃ for 10 min (ABI GeneAmp® 9700). The PCR products were confirmed with 2% agarose gel electrophoresis, purified by AxyPrepDNA (Axygen Scientific Inc. USA) and quantified by QuantiFluorTM-ST system (Promega, USA). The final products were sent for sequencing using IIumina MiSeq platform (IIumina SanDiego, USA) at Shanghai Majorbio Bio-pharm Technology company. The microbial analysis was carried out using UPARSE (version 7.1 http://drive5.com/uparse/). Sequences with ≥97% similarity were assigned to the same operational taxonomic units (OTUs). The sequences were trimmed with UCHIME and annotated using RDP classifier (http://rdp.cme.msu.edu/) based on Silva database (SSU123) at the threshold of 70%. Alpha diversity was applied in analyzing complexity of species diversity for a sample by Shannon index.

All experimental procedures and animal welfare were in accordance with the related ethical regulation guidelines and approved by the Research Ethics Committee of the College of Food Science and Technology in Hebei Agricultural University (2017-008). Fifty male Sprague Dawley rats (specific pathogen free, 180–220 g), from Beijing Vital River Laboratory Animal Technology Co., Ltd. were housed in an air-conditioned room at a temperature of 22 ± 2 ℃, a relative humidity of 50–60% with an illumination of 12 h light/12 h dark cycle. All rats had free access to standard chow diet and water during the experiment. After one week of acclimation to the new environment, all animals were randomly divided into 5 groups (n = 10): the normal group (NOR), the DSS colitis control group (DSS), low dose of flaxseed oil group (LDF), middle dose of flaxseed oil group (MDF) and high dose of flaxseed oil group (HDF), respectively. The assignment was according to the previous described methods (Hassan et al., 2010; Naqshbandi, Rizwan, & Khan, 2013; Naouar, Mekki, Charfi, Boubaker, & Filali, 2016; Omar, 2018; Young, Tseng, Kalaba, & Beidler, 2019). The normal and DSS groups were administered with distilled water by gavage, while LDF, MDF and HDF groups were administered with flaxseed oil at 400, 800 and 1600 mg/kg b.w. for 6 weeks. The drinking water of all groups except the normal group was replaced with 3% DSS in water (w/v) during the 6th week. Five days later, the rats were sacrificed under euthanasia with pentobarbital sodium treatment. Caecum contents were aseptically collected in sterile tubes and stored in liquid nitrogen for microbial analysis.

2.7. Statistical analysis Data were presented as mean ± SD. Statistical analysis was performed using SPSS 17.0. Significance was determined at P-value < 0.05 or P-value < 0.01 by one-way analysis of variances followed by Tukey’s test.

2.3. Evaluation of the disease activity index

3. Results

The disease activity index (DAI) was evaluated to assess the severity of ulcerative colitis during the experimental procedure. The body weight, stool consistency and stool occult blood were recorded daily by an investigator complying the protocol according to the previous

3.1. Flaxseed oil improved the colon indices of the DSS induced colitis The changes of body weight gain, DAI, colon length, colon weight and colonic weight/length ratio were presented in Table 1. No 2

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Table 1 Effects of flaxseed oil on body weight gain, DAI, colon length, colon weight and colon weight/length ratio in DSS-treated rats. Group

NOR

DSS

LDF

MDF

HDF

BWG DAI Colon length (cm) Colon weight (g) Colon weight/length ratio (mg/cm)

222 ± 6.4 0 24.05 ± 3.20 2.07 ± 0.16 86.07 ± 12.29

208 ± 11.9 7.52 ± 0.15** 20.2 ± 0.65** 2.56 ± 0.44** 126.73 ± 27.86**

210 ± 7.2 6.13 ± 0.12 21.17 ± 1.35 2.30 ± 0.42 108.64 ± 20.75

215 ± 8.8 4.29 ± 0.11# 21.29 ± 1.70 2.03 ± 0.28## 95.35 ± 16.22##

263 ± 9.8# 4.78 ± 0.17# 21.97 ± 1.87 2.17 ± 0.21# 98.77 ± 15.49##

BWG, body weight gain; DAI, disease activity index. Data were presented as the mean ± SD, n = 10. Compared with normal group, *P < 0.05, **P < 0.01; compared with DSS group, #P < 0.05, ##P < 0.01.

3.3. Flaxseed oil improved the oxidative indices in the colon

mortality of animals during the experimental period were found. No significant difference of food intake among experimental groups were found. Compared with the normal group, the DSS induced colitis rats had colitis symptom indicated by higher disease activity index (DAI) (P < 0.01), shorter colon (P < 0.01) and increased colon weight (P < 0.01) and colon weight/length ratio (P < 0.01). Compared with the DSS group, there was no significant changes of colon length in rats treated by flaxseed oil. However, rats in MDF and HDF showed significantly lower DAI (P < 0.05, P < 0.05), reduced colon weight (P < 0.01, P < 0.05) and colon weight/length ratio (P < 0.01, P < 0.01).

The results of oxidative stress in the colon were demonstrated in Table 2. The DSS induced colitis rats had significantly lower SOD activity and GSH level, higher MPO activity and MDA level (P < 0.05, P < 0.05, P < 0.01, P < 0.05), while flaxseed oil effectively improved this condition in a dose-dependent manner. After flaxseed oil administration for six weeks, SOD activities in LDF, MDF and HDF groups were increased compared with DSS group (P < 0.05, P < 0.05, P < 0.01), GSH levels in MDF and HDF groups were increased (P < 0.05, P < 0.01), MDA level in HDF was deceased (P < 0.01). MPO activity in MDF and HDF groups significantly were suppressed compared with the DSS group (P < 0.05, P < 0.01), while no difference was found between the DSS and LDF group.

3.2. Flaxseed oil ameliorated the symptoms of DSS induced colitis rats The representative results of the histopathological analysis were presented in Fig. 1. The section of NOR group showed intact and well aligned epithelial cells. Compared with NOR group, the DSS treated rats exhibited typical symptoms of colitis including sloughing off of the epithelium, rugged glands of lamina propria, reduced number of goblet cells and inflammatory cell infiltration in the mucosa. Consumption of flaxseed oil ameliorated the histopathological damage of colon induced by DSS. It was noteworthy that consumption of the middle dose of flaxseed oil had better performance for the ulcerative colitis based on epithelium integrity, goblet cell number and inflammatory infiltration compared to the other treatments of flaxseed oil.

3.4. Effect of flaxseed oil on inflammation-related factors in the colon Effect of flaxseed oil treatments on inflammatory mediators in the colon were shown in Table 3. DSS induced colitis rats had significantly lower level of IL-2 (P < 0.05) and higher level of IL-6 (P < 0.05) and MCP-1 (P < 0.05) compared with the NOR group. By contrast, IL-2 level was significantly increased in MDF group (P < 0.05) compared with DSS group. Both of MDF and HDF groups decreased IL-6 levels (P < 0.05, P < 0.01) and MCP-1 levels (P < 0.05, P < 0.01) in a dose-dependent manner compared with DSS group. IL-10 level was also enhanced in MDF group (P < 0.05). However, no significant change of

Fig. 1. Histopathological analysis of colon segment. NOR, normal group; DSS, DSS induced colitis group; LDF, low dose of flaxseed oil treated group; MDF, middle dose of flaxseed oil treated group; HDF, high dose of flaxseed oil treated group. Compared with the colon tissues of the normal rats, the rats treated with DSS showed typical symptoms of colitis. Flaxseed oil consumption improved the condition of rats. (a) represents inflammatory cells, (b) represents goblet cells depletion, (c) represents ulcerative. 3

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Table 2 Effect of flaxseed oil on oxidative status in colons. Group

NOR

SOD (U/mg protein) GSH (μmol/g protein) MDA (nmol/mg protein) MPO (U/mg protein)

3.54 0.88 0.07 0.28

DSS ± ± ± ±

2.13 0.53 0.05 0.04

2.61 0.22 0.22 0.63

LDF ± ± ± ±

0.88* 0.01* 0.07* 0.05**

4.44 0.35 0.20 0.58

MDF ± ± ± ±

1.31# 0.29 0.04 0.12

5.48 0.85 0.21 0.41

HDF ± ± ± ±

0.68# 0.37# 0.12 0.03#

8.46 1.03 0.06 0.38

± ± ± ±

4.07## 0.48## 0.13## 0.07##

SOD, superoxide dismutase; GSH, glutathione; MDA, malondialdehyde; MPO, myeloperoxidase. Data were presented as the mean ± SD, n = 10. Compared with normal group, *P < 0.05, **P < 0.01; compared with DSS group, #P < 0.05, ##P < 0.01.

IL-4, IL-12, IFN-γ or NF-κB was observed in any group.

3.5. Flaxseed oil modified the microbiota structure in the colon Compared with the normal group, the DSS treated rats had lower Shannon index (P < 0.05), indicating reduced microbiota diversity in colitis, whereas flaxseed oil increased the microbial diversity and MDF group reached significant level (P < 0.05) (Fig. 2). Different groups had their microbial composition with overlaps. Specifically, the normal group had 447 OTUs, DSS group had 386 OTUs and the flaxseed oil groups (LDF, MDF and HDF) had 376, 374 and 393 OTUs, respectively. Totally, 269 OTUs were shared by these groups. The flaxseed oil groups shared 312 OTUs. Compared with the normal group, DSS showed 47 new OTUs, part of which survived in low dose group (33 OTUs), middle dose group (29 OTUs) and high dose group (34 OTUs). Compared with the normal group, flaxseed oil treatment regained part of cecal microbial diversity. Specifically, the low dose flaxseed oil regained 24 OTUs, the middle group regained 32 OTUs and the high dose regained 29 OTUs, all of which were lost in DSS group (Fig. 3). In normal rats, we identified the main phyla of the colon including Firmicutes (73.09%), Bacteroidetes (25.01%), Proteobacteria (0.50%) and Verrucomicrobia (0.24%) (Table 4). Compared with the normal group, the abundance of Bacteroidetes significantly decreased while the Verrucomicrobia increased (P < 0.01). Consumption of flaxseed oil modified the microbiota of colon. Specifically, low dose of flaxseed oil increased the abundance of Proteobacteria (P < 0.05), the middle dose increased Proteobacteria and Verrucomicrobia (P < 0.01, P < 0.05), the high dose increased Bacteroidetes, Proteobacteria and Verrucomicrobia (P < 0.05, P < 0.05, P < 0.05). At the genus level, the cecal microbiota of DSS colitis rats changed greatly compared with the normal rats (Fig. 4). Typically, the abundance of Romboutsia and Ruminococcaceae_UCG-005 increased, while Lachnospiraceae_NK4A136_group, Lachnoclostridium, Phascolarctobacterium and Prevotellaceae_UCG-001 decreased. Flaxseed oil restored some of the microbiota change. For example, the middle dose of flaxseed oil decreased Romboutsia (P < 0.05), the middle and high dose of flaxseed oil decreased Ruminococcaceae_UCG-005 (P < 0.05, P < 0.01), the low dose of flaxseed oil increased Lactobacillus

Fig. 2. The Shannon index of cecal bacterial diversity of different groups of NOR (A), DSS (B), LDF (C), MDF (D) and HDF (E). Data were presented as the mean ± SD, n = 5. Compared with normal group, *P < 0.05, **P < 0.01; compared with DSS group, #P < 0.05, ##P < 0.01.

(P < 0.01), the low dose increased Lachnospiraceae_NK4A136_group (P < 0.05), the low dose and middle dose of flaxseed oil increased Lachnoclostridium and Phascolarctobacterium (P < 0.05), and the high dose increased Prevotellaceae_UCG-001 (P < 0.01). It needs to be noted that Alloprevotella and Eubacterium_xylanophilum group were only detected in the normal group. 4. Discussion Ulcerative colitis is one typical type of inflammatory bowel disease characterized by longstanding chronic inflammatory condition as well as an imbalance of gut microbiota or dysbiosis (Geboes et al., 2000; Goldsmith & Sartor, 2014). Studies have shown that the incidence of ulcerative colitis is increasing throughout the world and it becomes a global disease. In the present study, we demonstrated that administration of flaxseed oil alleviated the development of ulcerative colitis induced by DSS via regulation of oxidative stress, inflammatory responses and gut microbial community in a dose dependent way.

Table 3 Effect of flaxseed oil on inflammation-related factors in colons. Group

NOR

DSS

LDF

MDF

HDF

IL-2 (pg/mL) IL-4 (pg/mL) IL-6 (pg/mL) IL-10 (pg/mL) IL-12 (pg/mL) IFN-γ (pg/mL) NF-κB (pg/mL) MCP-1 (pg/mL)

364.91 ± 101.13 24.45 ± 0.97 785.2 ± 242.5 395.25 ± 16.24 14.89 ± 1.09 316.04 ± 77.38 1560 ± 688.5 366.80 ± 101.13

232.22 ± 50.57* 23.29 ± 1.69 1158 ± 117.6* 358.00 ± 32.92 14.92 ± 0.95 346.56 ± 93.60 1691 ± 497.3 451.72 ± 97.81*

309.94 ± 69.24 23.93 ± 2.91 969.4 ± 409.8 407.36 ± 74.99 14.78 ± 1.19 319.69 ± 98.22 1500 ± 253.8 336.22 ± 111.09

365.44 ± 129.97# 23.72 ± 1.57 809.5 ± 207.8# 413.52 ± 24.47# 14.08 ± 0.79 310.93 ± 80.77 1361 ± 239.6 316.33 ± 70.61#

242.11 ± 110.91 24.56 ± 2.55 623.1 ± 315.3## 393.45 ± 41.49 14.01 ± 0.92 258.00 ± 49.52 1651 ± 565.9 272.61 ± 73.76##

IL-2, interleukin 2; IL-4, interleukin 4; IL-6, interleukin 6; IL-10, interleukin 10; IL-12, interleukin 12; IFN-γ, interferon γ; NF-κB, inuclear factor κB; MCP-1, monocyte chemoattractant protein-1. Data were presented as the mean ± SD, n = 10. Compared with normal group, *P < 0.05, **P < 0.01; compared with DSS group, #P < 0.05, ##P < 0.01. 4

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ulcerative colitis (Khan et al., 2006). Be line with the previous study, DSS treatment enhanced the colonic MCP-1 level, while flaxseed oil treatment reversed the levels at MDF and HDF compared to the DSS group. The rats with ulcerative colitis showed deranged inflammation pattern, while flaxseed oil relieved some conditions. Our results were consistent with a previous report that replacement of partial corn oil with flaxseed oil in the diet prevented inflammation in the colon tissues of rats (Varma et al., 2010). Oxidative stress could occur as a result of increased level of oxidant and/or decreased antioxidant system, which is considered to be involved in the development of ulcerative colitis (Nieto et al., 2000; Seril, Liao, Yang, & Yang, 2003; Baskol, Baskol, Koçer, Ozbakir, & Yucesoy, 2008). Both of SOD and GSH play an important role in the antioxidant system. SOD as an antioxidant enzyme could convert superoxide radicals first to H2O2 which is further metabolized into molecular oxygen and water by catalase and GSH is an important intracellular free radical scavenger. In the present study, SOD activity and GSH content were significant decreased in the ulcerative colitis induced by DSS, whereas flaxseed oil treatment could reverse the reduction. Consistent with previous observations (Khan et al., 2013; Naqshbandi et al., 2013), flaxseed oil demonstrated intrinsic antioxidant properties that could ameliorate intestinal anti-oxidant status (Zeng et al., 2017). The α-linolenic acid can be transformed into eicsopentaenoic acid and docosahexaenoic acid in vivo (Simopoulos, 2002; Safarinejad, Hosseini, Dadkhah, & Asgari, 2010), which are able to increase GSH synthesis (Arab, Rossary, Soulere, & Steghens, 2006). On the other hand, under status of oxidative stress, lipid peroxidation is generated inevitably in the cellular and subcellular membranes and produces irreversible damage to structure and function of cells. Be corroborated with the previous reports, the lipid peroxidation product MDA level was increased significantly in the DSS-induced colitis (Farombi et al., 2016), while administration of flaxseed oil decreased MDA levels in colon of DSStreated rats. Additionally, MPO as an indicator of inflammation would can accelerate the oxidative tissue damage (Tong, Niu, Yue, Wu, & Ding, 2017), whereas the increase of MPO activity in DSS-treated rats colonic was significantly inhibited by the treatment of flaxseed oil. Gut microbial dysbiosis is also considered to be involved in ulcerative colitis. The alteration of microbial community in gut has been observed in patients with ulcerative colitis (Noor et al., 2010), whereas fecal transplantation can improve this condition (Moayyedi et al., 2015). Shannon index is usually used to reflect the diversity of bacterial community. There were previous reports that DSS induced colitis causes decline in the bacterial diversity of fecal samples (Nemoto et al., 2012), while some dietary intervention may increase the diversity (Wei et al., 2016). Consumption of flaxseed oil partly restored the gut microbiota diversity of the rats, along with decreased symptoms of ulcerative colitis. Two primary bacterial groups Firmicutes and Bacteroidetes make up over 90% of the gut cecal microbiota and a ratio of Firmicutes/ Bacteroidetes was always regarded as a key indicator of gut microbiota dysbiosis in many metabolic diseases (Bomhof, Saha, Reid, Paul, & Reimer, 2014; Ley, 2006). However, the deranged microbiota was frequently reported in animals and humans with no consistency. Sun et al. found DSS induced decreased Firmicutes with increased Bacteroidetes in mice (Sun et al., 2018), while decreased Firmicutes/Bacteroidetes ratio was observed in a DSS induced colitis model in rat (Tao

Fig. 3. The Venn of cecal bacteria in different groups of NOR (A), DSS (B), LDF (C), MDF (D) and HDF (E).

Flaxseed oil has been consumed for a long history, while there is few information about its effect on ulcerative colitis and the gut microbiota dysbiosis. In the present study, flaxseed oil demonstrated the protective potential for the ulcerative colitis, which was consistent with the recent report that flaxseed oil attenuated inflammatory markers, disease severity, blood pressure in an open-labeled randomized controlled trial in UC patients for 12 weeks (Morshedzadeh et al., 2019). The previous report by Zarepoor. (2013) has demonstrated that treatment of flaxseed oil (3.5% of basal diet) increased DAI and MPO activity in the DSSinduced acute colitis mice model, which may be due to the different dosage of flaxseed oil used in study. The main bioactive compound of the flaxseed oil is the α-linolenic acid (Cunnane et al., 1993), which has anti-inflammatory and hypolipidemic effects. Accumulating evidences have shown that inflammatory-related factors are closely associated with the incidence of the pathogenesis of ulcerative colitis (Baumgart & Carding, 2007). IL-6 as a pro-inflammatory cytokine is usually involved in colitis and also required to develop colitis associated cancer (Grivennikov et al., 2009; Li et al., 2009), whereas inhibition of IL-6 signaling pathway prevents development of colitis (Noguchi et al., 2007). In our study, flaxseed oil significantly decreased the over-expression levels of colonic IL-6 by DSS, which was likely due to the reduction in infiltration of activated monocytes and macrophages (primary sources of the cytokines). Interestingly, DSS treatment decreased IL-2, another pro-inflammatory factor which was previously reported to be increased in Chron’s disease but not ulcerative colitis (Mullin, Lazenby, Harris, Bayless, & James, 1992). Flaxseed oil partly restored IL-2 expression to nearly normal. The IL-10 is a well-known anti-inflammation chemokine and the deficiency can induce or aggravate the colitis in rodent animals (Devkota et al., 2012; Fuss, Boirivant, Lacy, & Strober, 2002). We showed that flaxseed oil upregulated expression of IL-10 to some degree. MCP-1 is a kind of chemokine whose elevation is always observed in colonic mucosa in models of Crohn’s disease and Table 4 Composition of the cecal microbiota at phylum level in DSS-treated rats. Phylum

NOR

DSS

LDF

MDF

HDF

Firmicutes Bacteroidetes Proteobacteria Verrucomicrobia

19369 ± 8743 6626 ± 4269 132 ± 60 62 ± 46

18040 ± 7948 1961 ± 1198* 139 ± 134 2944 ± 1670**

21639 ± 10064 1271 ± 576 315 ± 139# 3467 ± 2648

15031 ± 7377 1593 ± 1109 1080 ± 728## 4181 ± 5128#

20255 ± 10449 3276 ± 2517# 457 ± 257# 3291 ± 2009#

Data were presented as the mean ± SD, n = 5. Compared with normal group, *P < 0.05, **P < 0.01; compared with DSS group, #P < 0.05, ##P < 0.01. 5

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Fig. 4. Change of the cecal microbiota structure at genus level. NOR (A), DSS (B), LDF (C), MDF (D) and HDF (E). Data were presented as the mean ± SD, n = 5. Compared with normal group, *P < 0.05, **P < 0.01; compared with DSS group, #P < 0.05, ##P < 0.01.

5. Conclusions

et al., 2017). In the present study, the phylum Bacteroidetes was decreased and the Firmicutes was not influenced in DSS colitis group compared to those in the normal group, resulting in a decrease of the ratio of Firmicutes/Bacteroidetes, which was partly prevented by the flaxseed oil treatment. Unlike Crohn’s disease, another typical bowel disease, the bacterial flora composition of ulcerative colitis patients was not significantly altered compared with that of healthy controls (Gophna, Sommerfeld, Doolittle, & Veldhuyzen-Van-Zanten, 2006). In addition, it was noteworthy that the genus levels of Roseburia and Phascolarctobacterium were decreased in DSS group, but they were not influenced by flaxseed oil treatments, which was possibly attributable to the different degree of utilization on the polyunsaturated fatty acids. Prevotella species as the key microbia could promote the biosynthesis of bile acids, which plays an important role in the high lipid diet (Nakayama et al., 2015). The levels of Prevotella and Prevotellaceae_UCG-001 were enhanced by flaxseed oil compared to the DSS group, which was in agreement with the previous study that the Spirulina platensis 55% ethanol extract rich in polyunsaturated fatty acids increased the level of Prevotella in high-fat diet fed rats (Li et al., 2019). The abundances of Lactobacillus, Lachnoclostridium, Lachnospiraceae_NK4A136_group and Ruminococcaceae_UCG-005 were increased by flaxseed oil treatment to some extent compared to the DSS group. All of Lactobacillus, Lachnoclostridium and Ruminococcaceae produce a large amount of short-chain fatty acids including formic acid, acetic acid, lactic acid and butyric acid which can be utilized in colonocytes for sustenance. For example, more than 70% of the oxygen consumed by colonic tissues can be provided by oxidation of butyric acid (Sun et al., 2018b). These results suggested that flaxseed oil could restore the dysbiosis of microbiota induced by DSS rather than individual bacterial species in the intestinal environment, resulting in a microbiota structure that is similar to normal group.

In conclusion, the study demonstrated that flaxseed oil alleviated the development of DSS induced colitis in rats. The beneficial effect was possibly attributable to the regulation of antioxidant activity, inflammatory factors and partial recovery of intestinal flora. Flaxseed oil is a type of good food component that can be used as adjunct treatment strategy or drug carrier in protection of ulcerative colitis in future. 6. Declarations of interest None. 7. Ethics statement All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978). Acknowledgements This work was supported by the funds of the Double First-rate Subject-Food Science and Engineering Program of Hebei Province (2016SPGCA18) and Science and technology planning project of Hebei Province (16275504D). We also thanked Professor Youmin Ma of Medical College of Hebei University who carried out the histopathological analysis. Declaration of Competing Interest The authors have no conflict of interests. 6

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