Visceral obesity on chronic constipation, in ammation, immune function and cognitive function in patients with in ammatory bowel disease


 Objective: Obesity has gained attention among patients with inflammatory bowel disease (IBD). The impact of visceral obesity on chronic constipation, inflammation, immune function and cognition after diagnosis of IBD is still unknown.Methods: This is a cross-sectional study of 140 IBD patients. Patients’ visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) were measured by abdominal computerized tomography (CT) scans and were grouped according to visceral obesity. Baseline variables, chronic constipation status, inflammation status and immune function were compared. The implications of visceral obesity on cognitive function were evaluated using Mini-Mental State Examination (MMSE).Results: The prevalence of visceral obesity was 51% (37 out of 72) for CD patients and 26% for UC patients (18 out of 68 patients). CD patients with visceral obesity has higher incidence of chronic constipation (81% vs. 57%, P = 0.028), higher IL-6 levels (15.28 pg/ml vs. 9.429 pg/ml, P = 0.0073) and lower CD4+ T cells (32.7% vs. 44.0%, P < 0.001). For UC patients, patients with visceral obesity have the tendency of higher IL-6 levels (7.2 vs. 6.0 pg/ml, P = 0.053). VAT/SAT ratio is associated with BMI (r = 0.652, P < 0.001).Conclusions: IBD patients had high risks of visceral obesity. CD Patients with visceral obesity had higher prevalence of chronic constipation, higher inflammation levels, decreased immune function.


Introduction
In ammatory bowel disease (IBD) is gastrointestinal disorders that involve chronic in ammation of digestive tract with two types mainly diagnosed: Crohn's disease (CD) as well as ulcerative colitis (UC) 1 .
The abdominal symptoms has been well studied, however the factor that affect IBD patients' constipation, in ammation, immune function and cognition after several years of IBD disease history has not been comprehensively characterized.
Constipation is a common symptom of IBD, diagnosed by having less than 3 bowel movements per week and / or di culty in bowel movements. More than 10% IBD patients had chronic constipation 2 . Obesity has gained attention for patients with IBD. Several studies have mentioned the risk of IBD risk with obesity 3 . An observational study of 524 IBD patients proved that obesity at diagnosis was more common in CD patients versus UC patients (odds ratio 2.02, P = 0.0096) 4 . Increasing BMI was found to parallel to risk of CD, rather than UC 4 . Several studies proved that the prevalence of overweight or obesity for CD patients ranged from 40-52% in western countries 5,6 . These studies only evaluated body mass index (BMI) to de ne obesity and a threshold of obesity was de ned as BMI > 30 kg/m 2 . No published articles evaluate further into impact of visceral obesity based on computed tomography (CT) scans on clinical characteristics in the disease process of IBD patients, especially in Chinese population. Thus, we aimed to evaluate the impact of visceral obesity in the remission period of IBD.
There is growing interest that the pro-in ammatory cytokine interleukin-6 (IL-6) plays a vital role in the disease process of uncontrolled IBD 7 . It has been proved that IL-6 was increased with active CD 7 .
Moreover, IL-6 is a clinical relevant parameter for in ammatory activity of CD and well correlated with relapse of CD during disease remission 7 . Although various cytokines were studied in CD process, IL-6 gained the central pathogenetic role due to its indication of early lesions of new diagnosed patients as well as patients with long history of CD 7,8 . Actually, IL-6 has a broad effect on immune cells 9 . IL-6 de ciency could lead to impaired both the innate and adaptive immunity 10,11 . IL-6 receptor (IL-6R) has been proved to be expressed on CD4 + T cells 12 . Previous studies have demonstrated that the production of IL-6 and soluble receptors (sIL-6R) released by intestinal macrophages and CD4 + T cells in the mucosa of patients with IBD 7 . Elevated cytokines together with mood change and the chronic pain due to chronic constipation could lead to cognitive impairment of IBD patients. Although previous observational study do not support that severity of symptoms had an impact on cognitive function in IBD patients 1 . Studies did not reach consistency and more clinical research are needed in the eld of cognitive dysfunction of IBD patients. Our study aims to identify the visceral obesity of IBD patients, evaluate its impact on chronic constipation, in ammation, immune function and cognitive function in the remission period of IBD.

Study design and patients
In our observational study, all patients were aged between 22 to 53 year with at least ve years of IBD history and now in the remission period of IBD July 2016 to January 2019. Remission for UC patients mean complete cessation of rectal bleeding, urgency and increased stool frequency. Remission for CD patients mean that patients' in ammation stop causing painful damage to patients colon and rectum.
The diagnose of IBD was checked in the medical record of all recruited patients. All patients were diagnosed using a combination of endoscopy (for CD) or colonoscopy (for UC) and imaging technologies, such as magnetic resonance imaging (MRI) scans or CT scans. Stool samples were checked to guarantee that all clinical symptoms were not caused by an infection. Blood tests were run sometimes in patients' history to help con rm the IBD. Patients previous diagnosis of cognitive impairment was recorded if they had a history of treated anxiety or depression. 140 patients with IBD were recruited from department of anorectal surgery at a liated hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine between July 2016 and January 2019. Patients' abdominal CT scans, baseline clinical characteristics including chronic constipation history, patients' education status, IL-6 level, immune cells including the following cell groups: CD3 + T cells, CD4 + T cells as well as CD8 + T cells, MMSE questionnaire were taken at recruitment.

Visceral obesity evaluation
Visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) (square centimeters) were measured with SliceOmatic software (version 5.0, Tomovision, Magog, Quebec, Canada) using abdominal CT scans at recruitment. Based on previous publications, we chose the third lumbar spine (L3) as the standard delimiter because L3 levels appear to be the most relevant and recognized delimiter for whole body adipose tissue 13 . The structures of VAT and SAT were quanti ed based on pre-established thresholds of Houns eld units (HU) ranged from − 150 to -50 HU for VAT and − 190 to -30 HU for SAT according to previously published articles 14,15 . Visceral obesity was de ned using VAT/SAT ratio for males > 1.33 and for females > 0.93 as previously described 16 .

IL-6 evaluation
All serum samples were collected at the time of patients recruitment. Measurement of serum level of cytokine IL-6 was carried out using enzyme-linked immunosorbent assay (ELISA) Kit (Zhongkang Biotech, Hangzhou, China) following manufacturer's instructions.

Flow cytometry
Cells were collected after incubation time, then the cells were washed in PBS and submitted to ow cytometry to determine the proportion of T cells using antibodies from Beckman Coulter: anti-CD3, anti-CD4 and anti-CD8. Cells were incubated for 20 minutes at 4 degree Celsius in the darkness, after that cells were washed by centrifugation and acquired in ow cytometer within 24 hours. Single stained controls were used to set compensation parameters. After the acquisition, ow cytometric analyses using FlowJo v7.6.1 software to evaluate the frequencies of CD3 + T cells, CD4 + T cells as well as CD8 + T cells.

Evaluation of cognitive function analysis
For all of the recruited IBD patients, MMSE evaluation was collected at the recruitment time. The MMSE is a 30-point questionnaire that is widely used in clinical and research settings to de ne if a patients has cognitive impairment with high sensitivity of 80%-90% and also high speci city of 70%-80%. It has the advantage of being highly sensitive and easy to operate. MMSE contains 8 categories, including (1) the orientation ability to time, (2) orientation ability to place, (3) registration ability, (4) attention and calculation ability, (5) recall ability, (6) language ability, (7) repetition and (8) complex commands ability.
MMSE score is the total score of the above 8 categories. Higher scores indicate that this patient has a better cognitive function. Cognitive impairment was de ned as according the educational levels of patient, for illiterates cognitive impairment was de ned ≤ 14, for patients with elementary school cognitive impairment was de ned as ≤ 17, and for patients with over elementary school education cognitive impairment was de ned as ≤ 22 according to previous published de nitions 17 .

Statistics
Descriptive statistical analyses of baseline characteristics were conducted for all continuous variables by mean values (percentage) and categorical variables by numbers (percentage). Comparisons of IL-6 levels and immune cells between visceral obesity and non-visceral obesity subgroups were made using ANOVA. Categorical data like prevalence of chronic constipation, is presented with absolute numbers and percentages, and was analyzed using Chi-squared tests. Results of the cognitive impairment from MMSE questionnaire were initially compared between groups using ANOVA. We also draw a scatter plot to show the tendency of association of VAT/SAT ratio with BMI. A linear regression was applied to quantify the strength of the relationship between these two parameters. All analysis was performed using SPSS, version 16.0 (IBM Corporation, Armonk, NY, USA).

Patients baseline characteristics
From July 2016 to January 2019, a total of 140 IBD patients were recruited. Characteristics of the IBD patients are shown in Table 1. The prevalence of visceral obesity was 39% for IBD patients (55 out of 140 patients), with 51% (37 out of 72) for CD patients and 26% for UC patients (18 out of 68 patients) respectively. The comparison of visceral obesity with non-visceral obesity patients with little difference of BMI was illustrated in Fig. 1 using sliceOmatic. In Fig. 1, although the rst patient has lower BMI than the other patient, its VAT/SAT ratio is higher, which means that patient with lower BMI actually has more severe visceral obesity. The median age of our patients is 36 years old, with range of 22 to 53 years old.
Females take a larger portion than males (75% vs. 25%). 72 patients were diagnosed with CD and 68 patients were diagnosed with UC. Patients had an average history of IBD for 7 years. 47% of these IBD patients admitted chronic constipation at recruitment. 23% of them had a history of treated anxiety or depression (Table 1). One-way ANOVAs found signi cant differences in chronic constipation, IL-6 level, CD4 + T cells between visceral obesity group and non-visceral obesity CD group. However, there were no signi cant differences between groups for cognitive function for CD patients. The visceral obesity has no impact on chronic constipation, IL-6 level, immune function and cognitive function for UC patients. Chronic constipation status CD patients with visceral obesity has higher incidence of chronic constipation (81% vs. 57%, P = 0.028, Fig. 2). However, for UC patients, the visceral obesity has no impact on chronic constipation (50% vs. 46%, P = 0.78).
The impact of visceral obesity on UC patients' CD3 + T cells, CD4 + T cells AND CD8 + T cells did not reach statistical signi cance.

Association of VAT/SAT ratio with BMI
The Person correlation r is 0.652 between VAT/SAT ratio and BMI (Fig. 5, P < 0.001).

Cognitive function
Levels of orientation to time, orientation to place, registration, attention and calculation, recall, language ability, repetition and complex commands were measured using the MMSE questionnaire. Our observational study do not support the hypothesis that visceral obesity has statistical signi cant impact on cognitive function, neither for CD patients nor for UC patients. The prevalence of cognitive impairment for IBD in the remission period is relatively low. For 72 CD patients, only 9 patients had cognitive impairment with 5 patients with visceral obesity and 4 patients with non-visceral obesity. For UC patients, only 6 patients had cognitive impairment with 3 patients with visceral obesity and 3 patients with nonvisceral obesity.

Discussion
Obesity has been a very important issue for IBD patients, especially in the remission period. High calories diet, sedentary behavior and lack of exercise all raise patients' body weight and BMI. Cautions must be given to IBD patients in the remission period, otherwise their IBD may relapse and lead them into worse clinical outcomes. Actually previous study reported that due to their abdominal symptoms, their diet behaviors have changed a lot. 39% patients reported diet change. UC patients take higher amount of margarine, pasta and rice, and CD patients take more meat and cheese 18 . This eating behavior leads to on rising visceral obesity for IBD patients. More published articles pay attention only to BMI and reached various conclusions. A study proved that an association of increased risk of IBD patients with BMI > 40 kg/m 2 with a seven-fold higher risk of postoperative infection 19 . Several studies have shown association of obesity with increased operative times, increased blood loss, and a higher risk of conversion from laparoscopic to open surgeries 20,21 . In contrast to this, a retrospective cohort study including 391 IBD patients undergoing surgery found that 30-day postoperative complication rates including total complications, wound infection, or anastomotic leak did not vary according to BMI 22 . The inconsistency of these studies may because that they did not evaluate further into visceral obesity, which is a more representative parameter of body fat. VAT is not only a risk factor for the occurrence of gastrointestinal disorders but also can negatively impact clinical outcomes 23 . Our study suggested that VAT/SAT ratio is associated with BMI and CD patients with visceral obesity has higher chance of chronic constipation. However, constipation may due to various factors, such as a low ber diet, painful defecation with stool withholding or probably due to slow gastrointestinal transit. More clinical studies are needed to prove our ndings and interventions could be implemented for these visceral obesity CD patients.
VAT release pro in ammatory cytokines such as IL-6 24,25 and is thought to be a reason why obese patients have higher levels of in ammatory microenvironment parameters, including C-reactive protein (CRP). IL-6 can be used as an in ammatory marker for severe infection. IL-6 stimulates the in ammatory and auto-immune process in many diseases such as diabetes, cancer and Alzheimer's disease. Moreover, T-cells from adipose tissue adjacent to in amed segments of the intestine of CD patients produce more IL-6 26 . Our study proved was the rst to evaluate the association of visceral obesity with IL-6 and immune function for IBD patients in the remission period. Visceral obesity increased IL-6 and CD4 + T cells for CD patients. UC patients with visceral obesity also has the tendency to develop higher level of IL-6, indicating the role of visceral obesity on chronic in ammation and decreased immune function for IBD patients.
Previous observational studies in patients with irritable bowel syndrome and IBD proved that, IBD patients seems not to have a statistical signi cant cognitive impairment. It has been hypothesized that IBD patients with mood disorders may affect the cognitive performance of query machine speci c tasks. Our study reached the conclusion that IBD patients with visceral obesity have no impact on cognitive impairment. The IBD patients in the remission period did not develop a high prevalence of cognitive impairment.
We admit that our study has several limitations. Our study is an cross-sectional observational study. We did not record patients' diet habits or sedentary behavior, which may impact patients' visceral obesity. We did not evaluate patients' hormone levels, such as leptin and insulin, sex hormones and growth hormone that could in uence their appetite and thus have impact on their VAT/SAT ratio. However, our study is the rst to take visceral obesity into consideration for IBD patients in the remission period. It worth further studies in the eld and more interventions are needed to help patients control their visceral obesity. It is a problem that could be reverse and more interventions de nitely would lead to better control of the disease.

Conclusion
In conclusion, visceral obesity lead to chronic constipation, higher level of IL-6 and lower immune function for IBD patients in the remission period. No cognitive impairment was found to be associated with visceral obesity.

Declarations
Ethics approval and consent to participate The studies involving human subjects were reviewed and approved by the Medical Ethics Review Committee of a liated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine. All patients who participated in this study provided written informed consent during the study.

Funding
Non declared.

Competing interests
Non declared.

Consent for publication
Not applicable.

Availability of data and material
All data and material are available from the corresponding author on reasonable request.

Authors' contributions
Haihua Qian designed the study, Yemin Wan and Dan Zhang are responsible for the data collecting and visceral obesity measurement, Yunzhi Qian did the statistical analysis, Shuchen Chang drafted the manuscript. All authors read and agreed the manuscript in its nal form. Figure 1 Comparison of visceral obesity of two patients with same BMI. The two patients have the little difference in BMI (28.1 kg/m2 vs. 29.3kg/m2) but with but with different visceral obesity (VAT/SAT ratio: 1.22 vs. 2.48).  Prevalence of chronic constipation between visceral obesity and non-visceral obesity patients with