Characterization of the Metrnl Serum Levels in Patients with Inammatory Bowel Disease and its Association with Inammatory Cytokines

Background Meteorin-like (Metrnl) is a newly discovered adipokine with the anti-inammatory and insulin sensitizing properties. Accordingly, Metrnl regulates glucose tolerance, enhances metabolism in body tissues, and induces thermogenesis in white adipose tissue. However, the correlation among Metrnl, IBD, and obesity still remains unexplored. The present study was conducted on 54 healthy control, 42 Ulcerative Colitis (UC), and 43 Crohn's disease (CD) patients who were diagnosed by pathological examination. In these patients, serum levels of adiponectin, Metrnl, IL-6, and TNF-α were measured using ELISA kits. serum Metrnl and IBD as the this the pro-inammatory cytokines. In this we examined the serum levels of Metrnl in the patients with inammatory bowel disease and also its association with the hall-markers of inammatory cytokines (TNF-α and IL-6) in IBD. insulin


Introduction
In ammatory Bowel Disease (IBD) is known as one of the causes of mortality in modern societies, with the characteristics of gastrointestinal chronic in ammation (1,2). Moreover, IBD includes two pathogenic form Crohn's disease (CD) and ulcerative colitis (UC). In this regard, the exact etiology of IBD is not wellunderstood yet, which resulted in identifying no-de nitive cure (3). Although underweight and malnutrition are historically associated with IBD, new studies have revealed a rising prevalence of the IBD obese patients (15-40% are obese, and 20-40% are overweight) in general population (4). Recent epidemiological studies have also shown that, the increasing rate of IBD is parallel with the obesity prevalence (4,5). Moreover, hospitalization and surgery are more frequent among the obese IBD patients (6). Although it is not fully understood yet, scientist studies have suggested that, genetics, the gut microbiome, and the immune system may play critical roles in IBD (7). IBD (UC and CD disease) also displays the characteristics of chronic in ammation and metabolic syndrome, which is affectedly altered in metabolism (8,9). Adipocytokines are secreted by white adipose tissue affecting the gut microbiome, in ammation, and metabolism pathways (10,11). Conversely, IBD can be considered as a risk factor for obesity by changes in the intestinal microbial metabolism (9,12).
On the other hand, the in ammatory condition of colon tissue can be affected by the impaired white adipose tissue (WAT) function such as abnormal adipocytokine secretion from adipocytes [12]. For example, leptin has decreased in the serum sample obtained from the IBD patients (with or without overweight). While the levels of resistin, adiponectin, and active ghrelin have remarkably increased (13).
Meteorin-like (Metrnl, known as Subfatin) is a novel adipo-myokine that is mainly expressed in WAT. In addition, some studies have shown that, Metrnl is expressed in the colon epithelium. Recently, it has been revealed that, Metrnl exerts an anti-in ammatory activity (14). Moreover, it can enhance lipid metabolism, decrease adipose in ammation, and ameliorate obesity-mediated insulin resistance. Notably, in this study, Metrnl expression was higher in mesenteric WAT of the CD patients in comparison to the controls (15). Studies reported that, the expression of Metrnl is high in intestinal cells, white adipose tissue, and skin. In addition, Metrnl express in various other tissues including muscle, liver, heart, spleen, and central nervous system (CNS). Moreover, Metrnl is produced by the activated macrophages, thus it can be associated with the in ammatory disorders such as IBD (16). A few studies have been done on tissue Metrnl and IBD disease. However, up to now, no studies have been performed on investigating the association between serum Metrnl and IBD disease as well as the association among this protein, obesity, and the pro-in ammatory cytokines. In this study, we examined the serum levels of Metrnl in the patients with in ammatory bowel disease and also its association with the hall-markers of in ammatory cytokines (TNF-α and IL-6) in IBD.

Study population
This case-control study was conducted on 54 control subjects and 85 IBD patients including 42 CD and 43 UC who were recruited from endoscopy unit of Valiasr Hospital, Birjand, Iran. The patient and control groups were selected by clinical examination, radiologic, endoscopic, and pathologic criteria. All the individuals aged between 35 and 60 years old. Moreover, the subjects with any history of cancer, diabetes, autoimmune diseases or active infectious disease were excluded from this study.

Anthropometric data and laboratory measurements
All the participants lled out a questionnaire to report their demographic data and medical history. Furthermore, weight and height were measured and body mass index (BMI) was also calculated using the following formula: [height (m2)/weight (kg)]. Afterward, systolic and diastolic blood pressures of all the participants were evaluated using a standard sphygmomanometer after 15 min resting in sitting posture.
After 12 hours of fasting, 5 ml of blood was obtained from all the participants and the serum was then separated by centrifugation. Subsequently, blood glucose serum levels (FBS) and lipid pro les including triglycerides (TG), cholesterol (Chol), HDL, and LDL were measured using auto-analyzer and the commercially available kits (Pars Azmoon, Iran).

Statistical analysis
Statistical analysis was performed using SPSS version 18. Categorical data were presented by frequency and percentage, which were tested by chi-square test. Continuous variables were also examined by student t-test and one-way ANOVA, and were then presented by mean and standard deviation (SD). Correlation analysis was performed by Pearson correlation test. Furthermore, multinomial logistic regression was conducted to estimate the risk of diseases status in terms of the Metrnl levels.

Anthropometric and biochemical measurement
The details of anthropometric and biochemical variables of the studied population are given in Table 1. In this regard, the studied groups showed no signi cant difference in terms of age, sex, and BMI. Similarly, SBP and DPB indicated no considerable difference among these 3 groups. Although FBG illustrated no signi cant difference between the controls and patients with UC and CD, insulin and HOMA-IR signi cantly increased in the patients with CD compared to the controls. It should be noted that, higher levels of insulin and HOMA-IR in the UC patients compared to the controls did not reach the signi cant threshold. Furthermore, lipids pro le including TG, TC, HDL-C, and LDL-C demonstrated no considerable variation between the patients and controls.

Serum levels of adipokines and cytokines
The ELISA results (Fig. 1) showed that, adiponectin concentration signi cantly decreased in the patients with UC and CD compared to the controls. Furthermore, TNF-α considerably elevated in the patients with UC and CD in comparison to the controls. Moreover, the patients with CD showed higher IL-6 levels compared to the controls, while the serum levels of IL-6 did not reach the signi cant threshold in the patients with UC compared to the controls. Furthermore, Metrnl serum concentration considerably diminished in the patients with UC and CD compared to the controls.
Regarding the crucial role of adipose tissue on Metrnl levels, analysis was performed according to the BMI cutoff (Over Wight, BMI ≥ 25 and normal weight, BMI < 25). In this regard, Metrnl serum levels were found to be lower in all the overweight subgroups (Table 2).

Correlation analysis
Correlation analyses were performed in 2 subgroups, as controls and patients. In the control group, Metrnl was found to be inversely correlated with BMI. Also, in the patient groups, Metrnl had an inverse correlation with BMI, IL-6, and TNF-α (Table 4).

Discussion
Adipokines have several effects on the immune system through regulating the expression and secretion of various cytokines. Therefore, they can play a crucial role in in ammatory diseases like IBD, which also have a metabolic background (17). Several studies have shown that, adipokines such as leptin, resistin, visfatin, retinol-binding protein-4, adiponectin, glucose, and insulin are deregulated in the IBD patients (13). Metrnl is a novel adipokin, which plays a key role in in ammation and insulin resistance improvement (18). Accordingly, this adipokine has been investigated in several metabolic and Rheumatoid Arthritis, Psoriatic Arthritis, and Osteoarthritis. As a result, they found the elevated level of Metrnl in Psoriatic Arthritis (22). To the best of knowledge, this is the rst report on the serum levels of Metrnl in the IBD patients. In addition, our results show the lower serum levels of Metrnl in the IBD patients compared to the controls. However, Metrnl was not different between the patients with UC and CD. LI et al. demonstrated that, Metrnl is highly expressed in the gastrointestinal tract of normal donors as well as mice. On the other hand, they produced intestinal epithelial cell-speci c knockout mice, which showed no signi cant serum reduction, despite the reduction in the GUT expression of Metrnl (16). A recent study performed by Zuo et al. reported that Metrnl expression is higher in mesenteric adipose tissue (MAT) of the CD patients compared to the controls. They also showed that, systemic treatment of Metrnl can improve the adipocyte function, and reduce the macrophage in ltration and in ammation by acting on the PPARγ pathway in mice. Therefore, they suggested that, Metrnl upregulation in the MAT of these patients may be caused by a compensatory response ( (14). Regarding the inconvenient results, it seems likely that, Metrnl expression can have an organ dependent pattern; however, establishing this concept needs more studies. When we strati ed the population based on obesity, the serum level of Metrnl was signi cantly lower in obese subjects than in non-obese ones. Consistently, AlKhairi et al. reported that, Metrnl is signi cantly higher in the T2D obese patients, in a way that this elevation can be considered as a compensatory response (23). However, Zhi-Yong Li et al. showed no correlation between serum Metrnl level and BMI (24). As the adipose tissue is the main source of Metrnl secretion, it is expected that, BMI can affect the levels of this adipokine, and adipose tissue in ammation and dysfunction might be considered as the causes for the decrease in Metrnl levels.

Conclusion
In conclusion, the current study is the rst to show that serum level of Metrnl has decreased in the IBD patients. Moreover, it was found that, serum level of Metrnl has a negative correlation with serum levels of TNF-α, IL-6 and BMI in the patients with IBD. Altogether, the present study demonstrated a relationship among Metrnl, in ammation, and obesity that suggested a possible role of Metrnl in the pathogenesis of IBD. However, the cross-sectional design of the study limited us in concluding a cause and effect relationship, so further studies are needed to dissect the possible mechanism for the reported relationship.

Declarations
Availability of data and material Additional data are available from the corresponding authors for reasonable requesting.
Ethics approval and consent to participate The written informed consent was signed by all the participants and the research was con rmed by the Ethics Committee of Birjand University of Medical Sciences.

Consent for publication
No applicable

Competing interest
The authors declare no con ict of interest.

Funding
The present study was supported by research council of Birjand University of Medical Sciences.
Author's contribution