Expression of Tight Junction Proteins According to Functional Dyspepsia Subtype and Sex (original) (raw)

Subjects

From March 2013 to May 2016, we prospectively enrolled study subjects. Korean subjects who received upper GI endoscopy were enrolled. Questionnaires about FD, emotional state, and quality of life (QoL) were obtained from subjects under the guidance of a well-trained interviewer. Subjects who had a GI surgery history, a recent peptic ulcer history, or any malignancy history were excluded. Non-steroid anti-inflammatory drug or anticoagulant users and patients who take medications due to chronic disorders were also excluded.

According to the Rome III criteria,17,18 the control or FD group were assigned. FD group were classified into the PDS, EPS, and mixed subtype based on the Rome III criteria.19 The control group was defined as individuals who had no GI symptoms and showed normal endoscopic findings. This study was approved by the Institutional Review Board (B-1101/119-010).

Severity of Dyspepsia Symptoms, Assessment of Anxiety, Depression, and Quality of Life

The scores of epigastric pain/burning, postprandial fullness, early satiation, and overall abdominal pain (not restricted to the epigastric area) were ranked by a 5-point scale (0, none; 1, mild; 2, moderate; 3, severe; 4, very severe) using a validated Korean version of Talley’s bowel disease questionnaire.20 The Bristol Stool Form Scale21 was used to evaluate stool consistency, and the number of bowel movements was also estimated. The anxiety and depression of the study subjects was evaluated using the hospital anxiety and depression scale (HADS).22 It is subclassified into anxiety and depression scales, both of them contains 7 items. Each response is ranked on a 4-point (0-3) scale. As higher scores by HADS demonstrating more depressive or anxious subjects, with a score more than 7 for each scale revealing potential anxiety or depression.23 To evaluate the QoL, the World Health Organization quality of life scale field trial version (WHOQOL-BREF) was used.24 WHOQOL-BREF contains questionnaires about overall QoL and general health with 4 domains, including physical and psychological, social, and environmental domain (overall: range 0-100, domain: range 0-20, higher scores mean a higher QoL). All these 3 questionnaires have been validated in Korea.20,25,26

Upper Endoscopy, Histology, and Helicobacter pylori Evaluation

Gastric biopsy specimens were obtained from the antrum and body for histology during the upper endoscopy. These biopsy specimens were used to measure the mRNA expression of CLDN1, CLDN2, CLDN4, OCLN, and ZO-1. The H. pylori infection status was evaluated by histology by using the updated Sydney system,27 CLOtest (Delta West, Bentley, Australia), and culture. If one of these 3 invasive H. pylori tests was positive, the patient was diagnosed with current H. pylori infection. When these tests were all negative then serum anti-H. pylori IgG antibody test (Genedia ELISA; Green Cross Medical Science Corp, Eumsung, Korea) was performed. If the H. pylori serology was positive, but no bacteria were found in the invasive studies, it was defined as a past H. pylori infection. Similarly, if the patient has H. pylori eradication history then this case was also regarded as past H. pylori infection. The absence of both current and past H. pylori infections was defined as the H. pylori uninfected group.

Quantitative Reverse Transcription Polymerase Chain Reaction

To stabilize and protect RNA from degradation, gastric biopsy specimens were stored in RNAlater Solution (Ambion, Austin, TX, USA) at 4℃. According to manufacturer’s recommendations, total RNAs were extracted from the gastric mucosal biopsy specimen using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and RNA purification was done by using RNeasy mini kits (Qiagen, Valencia, CA, USA). Synthesis of cDNA was performed using 1 µg of total RNA with a High Capacity cDNA kit (Applied Biosystems, Foster City, CA, USA). The thermal cycling parameters for the reverse transcription were as follows: 10 minutes at 25℃, 120 minutes at 37℃, and 5 minutes at 85℃. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed in triplicate using a StepOnePlus Real-time PCR system (Applied Biosystems) with SYBR Premix Ex TaqTM (Takara Bio, Shiga, Japan). Detail information about the primers of each TJPs were described in Supplementary Table. The thermal cycling conditions were as follows: initial denaturation at 95℃ for 10 seconds, followed by 40 cycles of 95℃ for 5 seconds and 60-65℃ for 33 seconds. Relative expression levels of mRNA from the target genes were compared with that of the endogenous control β-actin using the 2−ΔΔCt method.

Helicobacter pylori Infection of HFE-145 Cells

HFE-145 normal male human gastric epithelial cells were kindly provided by Dr Hassan Ashktorab and Duane T Smoot (Howard University, Washington, DC, USA). Cells were seeded onto 6-well slide chambers or 6-cm diameter petri dishes (Thermo Fisher Scientific Nunc, Inc, Waltham, MA, USA). Cells were grown in medium with 2.5% fetal bovine serum and without antibiotics for 20 hours at 37℃ prior to H. pylori infection. Cells were washed once with sterile phosphate buffered saline (PBS), and then H. pylori ATCC 43504 (O4 strain; cagA+ and vacA+) that was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) were added at a multiplicity of infection of 100:1 for different time points, followed by washing with PBS 6 times to remove nonadherent bacteria. Bacteria were cultured under microaerophillic conditions (5% O2, 10% CO2, and 85% N2) at 37℃. The effect of H. pylori infection on expression of ZO-1, OCLN, and CLDN2 in human gastric cell lines was investigated.

Western Blot Analysis

After 24 hours and 48 hours of co-culture, cells were lysed with RIPA buffer (Cell Signaling Technology, Beverly, MA, USA) after washing twice with PBS, and BCA protein assay was performed for determining protein concentration (Pierce, Rockford, IL, USA). Cell extracts (20 µg protein) were subjected to 10% SDS-PAGE and the separated proteins were transferred to PVDF membranes. After blocking the non-specific binding sites with non-fat dry milk, the membranes were incubated with anti-Claudin-2 antibodies (1:500; 51-6100; Thermo Fisher Scientific, Inc, Waltham, MA, USA), anti-ZO-1 antibodies (1:1000, 61-7300; Thermo Fisher Scientific, Inc), and anti-Occludin antibodies (1:1000; 33-1500; Thermo Fisher Scientific, Inc) at 4℃ overnight, then blots were incubated with secondary antibody (goat-anti rabbit antibody, 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA). Detection was achieved with an enhanced chemiluminescence agent (Amersham BioSciences, Buckinghamshire, UK).

Statistical Methods

Data are presented as the median (interquartile range) or mean ± SD. Categorical variables are presented as numbers and percentages and were analyzed by the Chi-square test or Fisher’s exact test. Continuous variables were analyzed using the Mann-Whitney U test between 2 groups or the Kruskal-Wallis test among 3 and more groups. Post hoc analysis was performed by the Mann-Whitney U test. SPSS software (version 20.0; IBM Corp, Armonk, NY, USA) was used for Statistical analyses. Two-sided P-values of < 0.05 were considered statistically significant.