Study design and patients
In our observational study, all patients were aged between 22 to 53 year with at least five years of IBD history and now in the remission period of IBD. 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 confirm 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 affiliated 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 quantified based on pre-established thresholds of Hounsfield units (HU) ranged from -150 to -50 HU for VAT and -190 to -30 HU for SAT according to previously published articles14, 15. Visceral obesity was defined using VAT/SAT ratio for males > 1.33 and for females > 0.93 as previously described 16.
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.
Cells were collected after incubation time, then the cells were washed in PBS and submitted to flow 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 flow cytometer within 24 hours. Single stained controls were used to set compensation parameters. After the acquisition, flow 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 define if a patients has cognitive impairment with high sensitivity of 80%-90% and also high specificity 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 defined as according the educational levels of patient, for illiterates cognitive impairment was defined ≤14, for patients with elementary school cognitive impairment was defined as ≤17, and for patients with over elementary school education cognitive impairment was defined as ≤22 according to previous published definitions 17.
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. All analysis was performed using SPSS, version 16.0 (IBM Corporation, Armonk, NY, USA).