Patients and samples
The study was performed on outpatients who were referred to the endoscopic ward of a general hospital in Tehran, Iran from January to August of 2019. This study was approved by the ethical committee of the Research Center in Tehran University of Medical Science (accepted Number, IR.TUMS.SPH.REC.1398.167 1398/7/3) and an informed consent form was obtained from all the patients. Demographic and clinical information of patients recorded in a questionnaire form. Patients who had received non-steroidal anti-inflammatory drugs or antibiotics within the last six weeks were excluded from this study. Three biopsy specimens of patients with gastric complaints were obtained during endoscopy from the antrum (the distal region of the stomach), and were used for histological examination, H. pylori culture and rapid urease test, and RNA extraction.
Histological examination
Biopsy specimens in the pathology department were histologically examined by hematoxylin-eosin staining method and the grade of gastritis was described based on histological parameters and updated Sydney System. Patients samples with chronic gastritis were selected for further analysis.
Isolation and identification
Each biopsy specimen for culture were kept in a transport medium consisting of thioglycollate with 1.3 g/L Agar (Merck, Germany) with 3% yeast extract (Oxoid, UK) and were transferred to the laboratory in less than 2 hours, then were homogenized and cultured on Brucella Agar supplemented with 7% sheep blood, Campylobacter selective supplement (vancomycin 2.0 mg, polymyxin 0.05 mg, trimethoprim 1.0 mg), 10% fetal calf serum, and amphotericin B (2.5 mg/L). Incubation was performed in microaerophilic conditions at 37 °C for 5-7 days. Identification of H. pylori isolates was performed by analyzing colony morphology, Gram staining, positive reactions of oxidase and catalase, and urease activities. Confirmation of the identity was done using specific primers for H. pylori (glmM) by polymerase chain reaction (PCR) as described before (13). The isolates were preserved in BHI broth containing 20% glycerol and 10% fetal calf serum and stored at -70 °C.
Genotyping of H. pylori isolates
DNA extraction
Genomic DNA of harvested colonies was extracted as described by Douraghi M (14). Briefly, harvested colonies of the H. pylori isolates were suspended in 1ml PBS. After centrifugation (6,000 rpm, 5 min), the pellets were resuspended in 50 mM/L NaOH and heated at 100° C for 20 minutes. After a quick spin and addition of 1 M Tris-HCl, pH 7.5, centrifuged for 5 min at 3,000 rpm. The supernatants containing genomic DNA samples were stored at -20° C until used for molecular studies.
Genotyping
ollowed by a 10-min incuba-
tion in 1 mol ⁄ L Tris ⁄ HCl (pH 7.5)
ollowed by a 10-min incuba-
tion in 1 mol ⁄ L Tris ⁄ HCl (pH 7.5)
In this study, genotyping of the isolates was done by PCR. Primers for detection of cagA, cagL, cagY, vacA (s and m alleles), and iceA1 and iceA2 genes were used in separate reactions as described before (13, 15, 16). Each genotype was defined according to a distinct pattern of the virulence genes detected as follows, cagA+/-/cagL+/-/cagY+/-/vacA (s1/2/m1/2)/iceA1+/-/A2+/-.
Gene expression analysis
RNA extraction and cDNA synthesis
To analyze the extent of alteration in the transcription of inflammatory genes in patients with chronic gastritis, RNA extraction of the gastric biopsy samples was done using TRIzol reagent (BlueZol, Iran) following the manufacturer’s guidelines with some modifications. Briefly, the tissue samples were cut into small pieces and mixed thoroughly with 600 μl of the extraction solution (BlueZol, Iran). After the addition of 150 μl chloroform and vigorous vortex for 15 seconds, the resulting mixture was centrifuged at 12000 rpm for 15 min at 4 ° C. The colorless upper aqueous phase was transferred to a new clean tube and mixed with 400 μl of isopropanol. The mixture was frozen at -70 ° C for 30 minutes, and the obtained RNA pellet (12,000 rpm, 15min at 4° C) was mixed with 1 ml 80% ethanol. A gentle vortex was applied to suspended the white plate at the bottom of the microtube. The extracted RNA in the pellet of the mixture was obtained after centrifuged at 7500 rpm for 5 min at 4° C. Each pellet was resuspended in 20-30 µl DEPC treated water, and stored at − 70 ° C after heat treatment in 60 °C for 5 minutes. The extracted RNA concentration was measured by quantitative method (Nano Drop™ One Microvolume UV-Vis Spectrophotometers). cDNA synthesis was done after adjustment of RNA concentrations using the easy cDNA synthesis kit (Parstous, Iran), according to the manufacturer’s instruction.
Detection of primer efficiency
The efficiency of primers targeting TNF-α, IL-8, Noxo1, MMP-7, Atp4A, B2M, and ACTB genes was measured before each analysis. The nucleotide sequence of these primers is shown in Table 1.
Relative quantitative real-time PCR
The expression level of IL-8, TNF-α, Noxo1, MMP-7, ATP4A genes in the H. pylori-infected compared with H. pylori non-infected patients with chronic gastritis was measured using SYBR green quantitative real-time PCR. B2M and ACTB genes were used as endogenous genes as described before (17). The reaction mixture consisted of 0.5 µL of each primer, 12.5 µL RealQ Plus Master Mix Green (Ampliqon, Denmark), 2 µL of cDNA, and distilled water up to the final volume of 25 µL. The thermal cycling conditions in Rotor Gene 6000 Corbett Sequence Detection System have comprised an initial denaturation step at 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 30 seconds, annealing at 60 °C for 60 seconds, elongation at 72 °C for 60 seconds, and a final extension step at 72 °C for 3 min. All the reactions were tested in duplicate. To show the accuracy of the amplification for each gene, primer efficiency, melting curve analysis, and gel electrophoresis were done. Relative gene expressions for all samples were determined from the obtained Ct (Crossing Threshold) values and using the 2-∆∆ct (2^- (Sample ∆ct- Average control group ∆ct) method. Up- and down-regulation were defined based on RQ values ≥2 and ≤ 0.5, respectively (18).
Statistical analysis
Statistical analyses and graphical representation of results were performed using SPSS (25 version) and GraphPad Prism7 softwares. The correlation between the relative expression values of IL-8, TNF-α, Noxo1, MMP-7, ATP4A genes in the infected group was evaluated by Spearman's correlation nonparametric test. As well, the correlation between H. pylori genotypes and relative expression values of these genes in the H. pylori-infected patients with chronic gastritis was evaluated by Kruskal-Wallis nonparametric test. p-value ≤ 0.05 was considered statistically significant.