The Effects of Flaxseed Supplementation On Circulating Adipokines Concentration in Patients With Ulcerative Colitis

Introduction: Inammatory bowel disease (IBD) is one of the most common gastrointestinal diseases that can affect people of all ages. Adipokines secreted from adipose tissue have been shown to play an important role in the pathogenesis of ulcerative colitis (UC). The aim of this study was to evaluate the effect of supplementation with your seed on the concentrations of adiponectin, resistin and visfatin in patients with UC. Methods: This trial is an open-labeled randomized controlled trial which conducted among 70 patients with UC. Patients were randomly divided into two groups: axseed and control. Patients in the intervention were received 30 g/day axseed powder for 12 weeks. Anthropometric, nutritional and biochemical factors of patients were evaluated at the beginning and end of the intervention period. Results: Totally, 64 patients (36 men and 28 women) with mean age of 31.12 ± 9.67 included in the nal analysis. There wasn’t any signicant difference between two groups in term of baseline weight and height (P>0.05). After the 12 weeks' intervention, axseed supplementation led to a signicant reduction in the resistin (-4.85 ± 1.89 vs. -1.10 ± 2.25, P<0.001) and visfatin concentration (-1.33± 1.14 vs. -0.53 ± 1.63, P=0.018). Moreover, we found a signicant increase in the adiponectin levels after the axseed supplementation (3.49 ± 1.29 vs. -0.35 ± 0.96, P<0.001). Conclusion: It has been reported in this study that axseed supplementation could exert benecial effects on adipokine levels in patients with UC.


Introduction
Crohn's disease (CD) and ulcerative colitis (UC) are in ammatory conditions and include a wide range of in ammatory conditions called in ammatory bowel disease (IBD). In fact, IBD has not been shown to be a simple disease and manifests itself in the form of a systemic in ammation with a variety of clinical manifestations [1,2]. Chronic in ammation in the intestinal tract of patients with UC may penetrate the surrounding adipose tissue and cause mesenteric hypertrophy of adipose tissue. There has also been evidence of mesenteric adipose tissue in imaging techniques of patients with IBD [3].
Adipose tissue acts as an active component in the secretion of various hormones and in the regulation of the immune system via the secretion of peptides detectable in relevant levels in the systemic circulation, the so-called "adipo(cyto)kines" [4]. These adipocytokines such as leptin, adipontin, resistin, and visfatin in addition to their role in adipose tissue metabolism, also play an important role in the pathogenesis of various diseases such as UC. Various clinical studies have evaluated the association between adipose tissue changes and its role in the pathophysiology of IBD, especially UC [5,6]. Resistin is one of the adipokines secreted by adipose tissue and immune cells (mainly produced by PBMC (peripheral blood mononuclear cells) and macrophages) and as a mediator plays an important role in adipose tissue response to in ammatory factors. Also, expression and serum levels of visfatin are affected by the amount of visceral fat and mesenteric adipose tissue and is involved in the pathogenesis of UC by stimulating leukocytes to produce in ammatory cytokines such as interleukin-6 [7,8].
Flaxseed as a owering plant containing various active compounds, has been widely used in traditional and modern medicine of different countries. Flaxseed contains active compounds that include a high concentration of alpha linolenic acid (ALA), composing approximately 55% of the total fatty acid content; lignans, a class of phytoestrogen; and dietary ber (28% by weight), a third of which is soluble ber [9,10].
In recent years, various studies have shown that axseed has the ability to exert antioxidant and antiin ammatory effects due to its active ingredients [11,12]. Rahimlou et al. in a meta-analysis study in 2019 were reported that axseed consumption reduces the serum concentration of some in ammatory factors [13].
In addition to these anti-in ammatory and antioxidant effects, various studies have shown that supplementation with axseed can regulate the concentration of some adipocytokines. Fukumitsu et al. reported that oral administration of axseed active components in animal models caused a signi cant improvement in the adiponection gene expression [14]. Moreover, some animal studies have shown that the active compounds in axseed such as ALA can act as a ligand for peroxisome proliferator-activated receptor gamma (PPAR-γ), which in turn induces the expression of some anti-in ammatory adipocytokines [15].
Considering the role of adipose tissue and adipokines in the pathogenesis of IBD, we suggest that dietary axseed may improve adipokine levels, as an additional mechanism to protect against UC. The purpose of the present study, therefore, is to examine the effects of 12-week dietary supplementation with axseed, the richest dietary source of ALA and other active components, on circulating levels of adiponectin, resistin and visfatin.

Subjects
Participants in this project were selected from patients referred to the gastroenterology and liver diseases clinic in Shahid Beheshti University of Medical Sciences, Tehran, Iran. Potential Participants were screened for UC by a gastroenterologist, and those who met the criteria were included in the study. The criteria for diagnosing UC by gastroenterologist was based on histopathological results in the last three months. As shown in Fig. 1, out of 84 volunteers to participate in this trial, 64 patients met the inclusion criteria. The age range of patients participating in this trial was between 18 and 55 years old and BMI > 20. Patients were excluded from the study if there was evidence of other intestinal diseases, in ammatory diseases, and autoimmune diseases. Other criteria for not including regular consumption of omega-3, axseed or any supplements with antioxidant and anti-in ammatory properties during the past month, pregnancy and lactation, sensitivity to axseed compounds and the use of anti-in ammatory drugs using anti-in ammatory drugs (corticosteroids, immune-modulators (such asAzathioprine, 6mercaptopurine, Methotrexate and Cyclosporine A),and anti-TNF-αmedications(such as Adalimumab, Certolizumab pegoland In iximab)) in the baseline or during the study, and unwilling to participate.

Study design
This trial is an open-labeled randomized controlled trial which carried out among the 64 UC patients.
Patients participating in this study were randomly divided into two groups receiving axseed and the control group. The duration of intervention in this trial was 12 weeks and the protocol of the project was approved by the ethics Committee of the Shahid Beheshti University of Medical Sciences and carried out in accordance with the Helsinki Declaration (IRCT registration no. IRCT20180311039043N1). Informed consent taken from the participants before the start of the study.
At the beginning of the study, after recording the demographic information of all patients, the patients in the intervention group were asked to consume 30 grams of axseed powder daily and the control group was advised to follow their routine medication regimen. The axseed was provided from a farm in Khoy, West Azerbaijan province of Iran. Flaxseed powder was analyzed by the School of Pharmacy and the composition of macronutrients and micronutrients per 100 g of powder was as follows: energy: 450 kcal; fat:41 g; ALA:21.5; protein: 20 g; carbohydrate: 29 g and ber:28 g. The rounded axseed (GF) was cleaned, milled and packed (250 g each pack) with a 15 g measure. Subjects in the GF group were asked to use one serving (15 g) of grounded axseed mixed in a glass of cold water after breakfast and one serving at the evening with an hour interval of taking medications. Packages were given to the participants at the start, 4th and 8th weeks of the study. Patients were asked not to change their diet or routine medication during the intervention period and to avoid consuming axseed containing products. To assess patient compliance, they were asked to return empty packages boxes each time to receive a new package. Patients who did not consume more than 15% of the given axseed were excluded from the study.

Biochemical assessment
To assess the serum concentration of adipokines, at the beginning and end of the study, 10 cc of blood was taken from all patients after 12 hours of fasting. To separate the serum, 10 cc of the blood samples were centrifuged at room temperature with 3000 rpm for 10 min and the isolated serum was stored at -80°C until the biochemical tests were carried out. Serum concentrations of adiponectin, resistin and visfatin were measured using ELISA kits (EASTBIOPHARM, PRC, and DBC, Canada) Dietary intake and physical activity assessment Energy, macronutrients and micronutrients intake in all patients at the beginning and end of the study were assessed using three (two consecutive days and a day-off) 24 -h food recalls. Then, each food item entered to Nutritionist IV software (1997, First DataBank Inc., San Bruno, CA) and mean intake of energy, micronutrients, and macronutrients were calculated at the baseline and after 12 weeks of the study.
Physical activity was assessed using the MET questionnaire.

Anthropometric measurements
At the beginning and end of 12 weeks, the weight and height of patients participating in both groups were assessed using standard equipment. Patients 'weight was assessed using Seca device with 100 g precision and patients' height was assessed using Seca stadiometer in a standing position next to the wall and without shoes, with a precision of 0.1 cm. The standard formula was used to calculate the BMI.

Statistical methods
Quantitative and qualitative data were reported as mean (standard deviation) and frequency (%), respectively. Kolmogorov-Smirnov test was used to evaluate the normality of the data. Qualitative variables were compared using the chi-square test. Also, One-way analysis of variance and LSD post-hoc test were used to compare groups in terms of quantitative variables. Also, analysis of covariance (ANCOVA) was used to adjust the effect of confounding variables (dietary intake of energy, protein, fat, polyunsaturated fatty acids, omega 3 polyunsaturated fatty acids, and omega 6 polyunsaturated fatty acids). Moreover, paired sample t-test was used to compare the change of variables over the study period in each group. The P-value < 0.05 was considered as statistically signi cant. All statistical analyses were performed using SPSS software version 24 (IBM Corp. IBM SPSS Statistics for Windows, Armonk, NY).

Study baseline characteristics
Preliminary information of patients participating in this trial is reported in Table 1. Totally, 64 patients (36 men and 28 women) with mean age of 31.12 ± 9.67 included in the nal analysis. The mean diagnosis duration in the intervention group was 5.21 ± 2.45 years vs. 5.00 ± 3.05 years in the control group which wasn't signi cant differences between two groups (P = 0.73). There wasn't any signi cant difference between two groups in term of baseline weight and height (P > 0.05).

Compare dietary intakes of the participants
The caloric intake, macronutrients and micronutrients of the axseed and control group are shown in Table 2. At the beginning of the study, the mean caloric intake in the intervention group was 2295 ± 281.02 kcal and in the control group was 2226.56 ± 268.27 kcal, but no signi cant difference was observed between the two groups (P = 0.24). Also, we not found any signi cant difference between two groups in term of calorie, protein, carbohydrate, total fat, PUFA, omega-3 and omega-6 at the beginning and end of the study (P > 0.05).
Effect of axseed on the adipokine parameters Table 3 compares the mean levels of adiponectin, resistin and visfatin between GF and control groups at the baseline and following 12 weeks of the study. At the beginning of the study, there was no difference between the two groups in terms of mean serum concentrations of resistin, adiponectin and visfatin (P > 0.05). After the 12 weeks' intervention, axseed supplementation led to a signi cant reduction in the resistin (-4.85 ± 1.89 vs. -1.10 ± 2.25, P < 0.001) and visfatin concentration (-1.33 ± 1.14 vs. -0.53 ± 1.63, P = 0.018). Moreover, we found a signi cant increase in the adiponectin levels after the axseed supplementation (3.49 ± 1.29 vs. -0.35 ± 0.96, P < 0.001). After adjusting the results for confounding variables, there was no difference in the signi cance of the results.

Discussion
The results of present study showed that axseed supplementation in patients with UC led to a signi cant reduction in the resistin and visfatin concentration. Also, we found a signi cant improvement in the adiponectin level in the intervention group than control. We found that adiponectin concentration increased signi cantly after the axseed supplementation. Weigert et al. in a cross sectional study were showed that circulating levels of chemerin and adiponectin are higher in ulcerative colitis [19]. Also, Karmiris et al. [20]described signi cantly elevated adiponectin levels in UC, whereas Valentini et al.
[6] identi ed reduced adiponectin in the serum of CD and UC patients. Waluga et al., in a study examining the effect of corticosteroid therapy on adipokine concentrations in forty patients with UC, showed that no signi cant differences in adiponectin concentrations were observed at baseline and end of the study between the two groups [5].
One of the reasons for the difference in the results observed in different studies is the higher concentration of adiponectin in women compared to men and the lack of proper distribution of participants in different studies in terms of sex [21]. Various studies have shown that adiponectin has the ability to exert anti-in ammatory effects by increasing the synthesis of interleukin receptor antagonist and decreasing the dendritic cell release of interferon gamma. Adiponectin also has the ability to induce macrophages to perform more phagocytosis [22,23].
On the other hand, in line with the results of our study, some ndings have indicated an increase in the concentration of adiponectin following supplementation with axseed. Haidari et al. were reported an increase in the adiponectin levels after the axseed supplementation in women with polycystic ovary syndrome [24]. Also, Sekine et al. showed that ALA-rich axseed oil (FSO) oral administration in rats led to a signi cant increase in the adiponectin levels [25].
However, the results of some studies are contradictory and no signi cant change in adiponectin concentration was observed following axseed supplementation [26,27]. ALA, the main component of axseed products, acts as a ligand for PPAR-γ, which can increase expression and circulating level of adiponectin [28]. Some researchers have also reported that one of the reasons for the increase in adiponectin concentration after the axseed supplementation is weight loss. It has been reported that adiponectin levels and its gene expression increased following weight loss [29]. On the other hand, axseed can cause weight loss due to their high ber content and active ingredients [30]. However, in our study, no signi cant change was observed in the weight of patients in the intervention group compared to the control group.
Resistin expression in human monocytes was markedly increased by treatment with endotoxin and proin ammatory cytokines [31,32]. Based on the ndings of various studies, the serum level of resistin is strongly associated with the concentration of some in ammatory factors [33,34]. Konrad et al. showed that patients with UC had signi cantly higher concentration of resistin [35]. Also, Abedimanesh et al. observed that resistin levels was higher in patients with UC compared healthy subjects and correlated with disease activity scores, hs-CRP levels and fat mas [36]. Resistant plays an important role in exacerbating chronic in ammation by inducing nuclear factor-kappa B in ammatory pathways [37].
Bokarewa et al. reported that higher levels of resistin can upregulate IL-6 and TNF-a related genes [38]. Another study revealed that human resistin signi cantly increase the production and secretion of TNF-a and IL-12 by activation of NF-kB transcription factor [39]. In the present study, we found a signi cant reduction in the resistin concentration after the axseed supplementation. According to our search, no previous study has evaluated the effect of axseed on resistin concentration. However, it seems that these positive effects may be due to the high content of ALA in axseed.
Another result of the present study was the signi cant effect of axseed supplementation on the concentration of visfatin in patients with UC. Moschen et al. reported that visfatin can increase production and secretion of in ammatory cytokines and may be considered a new pro in ammatory adipocytokine [40]. Also, it has been reported that visfatin plasma levels and its mRNA expression signi cantly increased in the patients with UC[6]. Researchers have suggested that visfatin exacerbates IBD through a variety of mechanisms. These mechanisms include effects on peripheral blood mononuclear cells, direct stimulation of pro-in ammatory cytokine production and suppression of neutrophil apoptosis [41,42]. Moreover, it has been reported that visfatin has also been shown to increase the expression of some genes involved in in ammatory pathways, such as NF-κB p65(RelA) DNA-binding activity in human leukocytes by p38 and MEK-1 [43,44].
Prior to our study, no study evaluated the effect of axseed supplementation on visfatin concentration. However, some studies have shown that omega-3 supplementation signi cantly reduces serum visfatin concentrations [45,46].
Our study was the rst clinical trial to evaluate the effect of axseed supplementation on adipokine levels in patients with UC. However, there were some limitations in the present study that should be considered in analyzing the results. In this study, serum leptin concentration was not assessed. Due to the strong correlation between leptin concentration and the severity of in ammation, evaluation of this factor could increase the accuracy of the results. On the other hand, measuring the expression of genes related to some in ammatory factors could be helpful. On the other hand, one of the most important limitations of this study was the type of study design, which due to the lack of a suitable placebo, the possibility of double blinding in the study was not provided.

Conclusion
In conclusion, our study showed that axseed supplementation led to a signi cant reduction in the visfatin and resistin concentration and a signi cant improvement in the adiponectin levels. However, to con rm the present results, further studies with higher sample size and measurement of more adipokines are needed.   Flow chart of participant's enrollment in the study