Subjects and samples
We identified endoscopic biopsy samples from 83 patients with CD and 372 controls by searching pathological report files at Chonnam National University Hospital between Jan 2018 and June 2019. All 372 control group subjects had no significant pathological findings and no clinical history of inflammatory bowel disease or neoplasia. Among 83 CD patients and 372 controls, 43 of each group were selected through propensity score matching to reduce the effects of age and sex.
Immunohistochemistry of Cyr61
Cyr61 protein expression in colon mucosal tissues was evaluated by immunohistochemistry (IHC). Briefly, formalin-fixed paraffin-embedded (FFPE) blocks were cut at 3-μm thickness and immunostained with a specific antibody against Cyr61 protein (1:1500 dilution; catalog no. ab10760; Abcam, Cambridge, UK) using an automated immunostainer (Bond-maX DC2002; Leica Biosystems, Bannockburn, IL, USA). For antigen retrieval, programmed heat-induced epitope retrieval was carried out using bond epitope retrieval solution 1 (containing citrate buffer at pH 6.0) for 15 min. All immunostained slides were evaluated twice by an experienced pathologist (LKH) with no knowledge of the clinical data. The staining intensity and the stained area proportion were measured. The intensity of cytoplasmic immunoreactivity was initially classified into four grades: no staining, weak positivity, moderate positivity, and strong positivity. No cases were completely immunonegative for Cyr61. Cases with weak staining intensity were categorized as ‘low-expression’ (weighed as intensity grade scale 1, Figure 1A and 1C), and those with moderate or strong staining intensity were considered as ‘high-expression’ (weighed as intensity grade scale 2, Figure 1B and Figure 1D). The proportion of the stained area was estimated by the ratio of positively stained area over the whole area and expressed as a percentage. IHC scores for Cyr61 expression were calculated by multiplying the intensity grade scale by the stained area percentage.
RNA isolation and real-time polymerase chain reaction of pro-inflammatory genes and Cyr61
To evaluate the degree of inflammation and Cyr61 expression between inflamed and noninflamed lesions, we used colonic mucosal biopsy specimens from 11 patients with CD, which were obtained from our previous study. Total RNA was extracted using Trizol (Takara, Tokyo, Japan). Briefly, 1 mL of Trizol solution was added into each well, and then the suspension was collected into a 1.5 mL tube. After adding 200 μL of chloroform (Sigma-Aldrich, St. Louis, MO, USA) and vortexing for 15 secs, the mixture was centrifuged at 20,000 x g for 20 min. The supernatant was then collected and mixed with equal amounts of isopropyl alcohol (MERCK, Kenilworth, NJ, USA) followed by centrifugation at 20,000 x g rpm for 20 min. The pellet was washed with 1 mL of 70% ethyl alcohol (MERCK) and centrifuged at 20,000 x g for 5 min. After removing the remaining ethyl alcohol, the RNA pellet was air dried at room temperature and then suspended in 50 μL of diethyl pyrocarbonate water.
The clinical disease activities of 83 patients with CD were evaluated around the time of colonoscopy by experienced gastroenterologists (KDH and KHS) using the Crohn’s disease activity index (CDAI), simple endoscopic score of Crohn’s disease (SES-CD) scale, and Crohn’s disease endoscopic index of severity (CDEIS).
We also reviewed the clinical course of patients in medical records after acquiring biopsy specimens. We defined clinical recurrence as a change in prescription, bowel resection, fistulotomy, strictureplasty, stoma formation, CD-related hospitalization, or flare during the follow-up period. CD-related hospitalization was defined as hospitalization because of complications including the following: CD-related surgery, hospitalization for nonsurgical CD-related events such as CD-related flares, hospitalization related to complications/extraintestinal manifestation of CD, and disease flare.
Results are expressed as mean (standard deviation, SD) or median value (range). Continuous variables in 2 groups were compared using Student’s t-test or the Mann-Whitney test. IHC scores for Cyr61 expression in patients with CD were divided into tertiles: 1st, <60 (n=27); 2nd, 60–80 (n=30) and 3rd, ≥80 (n=26). We compared the clinical disease activities such as CDAI, SES-CD and CD-EIS according to tertiles of IHC score for Cyr 61 expression using Kruskall Wallis test. The association between Cyr61 expression and clinical recurrence was assessed using univariate and multivariable binary logistic regression. Odd ratio (OR) and P value are presented. Tests for trend were performed using the Cyr61 expression tertiles as ordinal variables in the corresponding logistic regression models. All statistical analyses were performed using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). Two-sided P values<0.05 were considered statistically significant.
The study protocol was approved by the Ethics Committee of Chonnam National University Hospital (IRB No. CNUH-2020-121) and was conducted according to the Declaration of Helsinki and Good Clinical Practice guidelines.