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Comparison of effects of aminosalicylic acid, glucocorticoids and immunosuppressive agents on the expression of multidrug-resistant genes in ulcerative colitis


Study subjects

A total sample of 148 UC patients (study group), 83 males and 65 females, aged 16–69 years (mean age: 42.40 ± 13.21 years), outpatients and inpatients of the First, Second and Third Affiliated Hospitals of Henan University of Science and Technology from March 2015 to December 2019 was obtained. Among them, 58 were treated with ASA drugs, 53 with glucocorticoids and 37 with immunosuppressive drugs. Forty-five patients, 25 males and 20 females, aged 18–65 years, with a mean age of (36.70 ± 11.41) years, who underwent e-colonoscopy suggestive of no intestinal symptoms, had a history of irritable bowel syndrome (IBS) or UC, and had no history of drug allergy or use of amino acid/immunosuppressive drugs or use of glucocorticoid drugs were enrolled in the control group. All patients were followed up from the admission or discharge date until December 2019. Colonoscopic biopsy and postoperative pathology were considered as the gold standard. General data were compared as shown in Table 1.

Table 1 Comparison of general data.

Classification and staging of UC

Patients with incomplete information were excluded from the follow-up trial. All enrolled patients were classified as mild and severe according to the “Chinese Consensus on the Diagnosis and Treatment of Inflammatory Bowel Disease” (hereinafter referred to as “07 Consensus”)14, which was developed by the Chinese Society of Gastroenterology in 2007, as shown in Table 2.

Table 2 Truelove-Witts Severity Index for UC.

According to the course of UC, UC can be divided into initial, chronic relapsing, chronic persistent and acute fulminant forms. In addition, according to the disease stage, UC can be divided into active and remitting stages. The diagnosis was made by reference to the Sutherland Disease Activity Index (DAI). If the index score was below 2, it was classified as remission; if it was above 2, it was classified as active, as shown in Table 3.

Table 3 Sutherland disease activity index.

Lesion site

The lesion sites can be further classified as rectum, left hemicolectum, wide colon and total colon. The left hemicolon referred to the flexure of the spleen of the colon toward the rectum. The wide colon included the splenic flexure on the proximal end of the colon. Lesions confined to the splenic flexure of the distal colon were classified as left hemicolon lesions, and those beyond the splenic flexure but not to the extent of total colon lesions were considered wide colon.

Diagnostic criteria

The diagnosis of UC can be confirmed by clinical manifestations, such as diarrhea, mucopurulent blood, with abdominal pain, defecation and various degrees of systemic symptoms; persistent, recurrent episodes; duration of UC > 1 month, colonoscopic observation of distal colon such as rectum and sigmoid colon with continuous, diffuse distribution, mucosal vascular congestion, hemorrhage and blurred texture, etc. 1 month, colonoscopic observation of rectum, sigmoid colon and other distal colon with continuous Diffuse distribution, mucosal vascular congestion, hemorrhage, edema, blurred, disorganized or absent texture, purulent secretion adhesions, barium enema examination including jagged or burr-like intestinal margins, multiple small filling defects in the intestinal wall, coarse mucosa with/ or granular changes, shortening of the intestine, disappearance of the intestinal pouch into a lead tube, pathological examination. Exclusions were those with bacillary dysentery, amebic dysentery, chronic schistosomiasis, intestinal tuberculosis and other infectious colitis, colonic Crohn’s disease (CD), ischemic colitis, and radiation colitis.

Inclusion criteria

Those who did not receive ASA medication, oral or intravenous glucocorticoids, glucocorticoids plus immunosuppressants and herbal medicine 4 weeks before the start of the study; those who received oral ASA medication after treatment: oral or intravenous glucocorticoids; oral or intravenous glucocorticoids plus immunosuppressants, ASA medication and glucocorticoids and other topical treatment medications.

Prognosis and outcome

All patients were divided into effective (complete remission and effective), ineffective and control groups according to their prognosis and efficacy. In the complete remission group, the patients’ symptoms disappeared, and the mucosa was normal on colonoscopy. In the effective treatment group, the patients’ physical symptoms of colitis largely disappeared, and diarrhea and abdominal pain decreased; colonoscopy showed mild inflammation of the mucosa or pseudo-polyps formation; pathological sections showed restoration of the mucosal layer of colonic tissue and reduction of neutrophil infiltration.

In the ineffective treatment group, patients had no relief of symptoms after treatment and frequent diarrhea and abdominal pain; colonoscopy showed severe inflammation of the mucosa and breakdown of the mucosal layer; pathological sections showed defects in the mucosal layer of colonic tissue and increased neutrophil infiltration.

Specimen preparation

Fresh colonic mucosal tissue specimens or endoscopic pathological mucosal tissues or postoperative pathological biopsies were immediately stored in a refrigerator at − 80 °C for RNA extraction. The remaining specimens were adequately fixed in 10% formalin, routinely dehydrated, and embedded in paraffin, and 4-μm tissue sections were prepared for immunohistochemical staining (P-gp).

Immunohistochemical staining

Immunohistochemical staining method

Using the SP method, antigen repair was required for the determination of P-gp. The negative control was replaced with phosphate-buffered saline (PBS) buffer, and the rest of the procedure was performed according to the manufacturer’s instructions. p-gp positive control was provided by Beijing Zhongshan Biotechnology Co. Positive controls were set up for each antibody. Routine dewaxing was hydrated using xylene twice for 15 min each, hydrated with gradient alcohol, washed three times with PBS (pH = 7.4) for 5 min each, and incubated with 3% hydrogen peroxide solution for 15 min at room temperature to eliminate endogenous peroxidase. Then, the samples were washed with distilled water for 5 min for three times. For antigen repair, 10 ml of ethylenediaminetetraacetic acid (EDTA) antigen repair solution was collected and diluted with 490 ml of triple distilled water for 3–4 min of hot repair. Finally, samples were cooled to room temperature, washed three times with PBS for 5 min each, sealed with normal goat serum working solution, and incubated at 37 °C for 40 min. The primary antibody was added to the wet box overnight at 4 °C, rewarmed at 37 °C for 45 min, and washed three times with PBS for 5 min each. The labeled secondary antibody was added to biotin drops and incubated at 37 °C for 25 min, washed three times with PBS, and horseradish peroxidase-labeled streptomycin was incubated at 37 °C for 20 min, washed three times with PBS for 5 min. The color development reaction was observed under a microscope with a chromogenic solution of 3,3-N-diaminobenzidine tetrahydrochloride (DAB). Samples were rinsed with running water, gently re-stained with hematoxylin for 10–30 s, dehydrated with a gradient of alcohol, clear xylene, and then mounted with a neutral mounting sheet.

Evaluation of immunohistochemical staining results

Using DAB immunohistochemical color development system, positive results were shown as brownish-yellow particles in the corresponding positions. p-gp positive particles were mainly distributed in the lamina propria of colonic mucosa and intestinal epithelium, and a distinct brownish-yellow color in the membrane and cytoplasm was considered positive. Ten pathological cells were randomly counted in the high magnification field. the number of P-gp treated positive cells was < 10% as negative, 10–25% as (+) and 25–75% as (+++). This refers to the criteria provided by Zhongshan Biotechnology Co.

Reverse transcription polymerase chain reaction (RT-PCR)

Primer

Primers designed by Primer Premier 5.0 were used as reference15. Primers for MDRI and β-actin were synthesized by Beijing Sebring Bioengineering Co.

Extraction of total tissue RNA

Fresh specimens collected by colonoscopic biopsy or post-surgical tissue biopsy were immediately stored at − 80 °C in a low-temperature refrigerator to detect the expression level of MDR1.

Trizol extraction16

Harvest frozen tissue samples and grind to a powder and transfer to pre-cooled 1.5 ml EP tubes before evaporation of liquid nitrogen. Add LML Trizol, insert into a microtissue homogenizer for 2–3 min, then add chloroform and shake for 15 s, allow to stand for 5 min, then centrifuge at 12,000 rpm for 15 min at 4 °C. The aqueous component of the upper layer was carefully collected into another new EP tube. Pre-chilled isopropanol at − 20 °C was added and mixed thoroughly with the supernatant phase. After 10 min of ice bath and centrifugation at 12,000 rpm for 15 min, the supernatant was discarded and dried, 1 ml of 75% ice ethanol was added and mixed well, the precipitate was washed well and centrifuged at 8000 rpm for 5 min at 4 °C, then the supernatant was discarded and the precipitate was dried. If to be used immediately, leave the sample at ambient temperature for 20 min and add 30 μl of DEPC water. If not for immediate use, add 75% ethanol LML and store at − 80 °C.

RNA quality identification

The purity of RNA was good (OD260 /OD280 ≈ 1.97) under OD260 and OD280 of ULTRAVIOLET spectrophotometer. The 5S, 18S and 28S bands were clearly visible by formaldehyde gel electrophoresis, and the extracted RNA was not degraded, as shown in Fig. 1.

Figure 1
figure 1

Total RNA integrity test.

RT-PCR

We used a two-step RT-PCR for quantification. The first step was cDNA reverse transcription. Vortex and shake the reagent solution from the melted kit for 2 s; transient centrifuge in a micro benchtop centrifuge for 5 s; transfer 4.5 µl of the bootstrap solution to a new 0.5 ml PCR tube; add 8 µl of RNA sample (5 µg of total RNA or 0.25 µg of POLY A + RNA); mix with a gun tip; incubate in a dry thermostat at 70 °C for 3 min; immediately place in an ice bath; add 8 µl of Reverse Solution (containing Invitrogen SuperScript III Reverse Transcriptase); mix well with the tip of the gun; centrifuge instantaneously for 5 s in a micro benchtop centrifuge; incubate for 60 min at 37 °C in a dry thermostat; place immediately in an ice bath; dilute with 80 µl of buffer and mix well; place in an ice bath or place in a − 20 °C refrigerator. Storage.

The second step was cDNA amplification. Transfer 30 µl of reaction solution to a new 0.5 ml PCR tube; add primers F and R (350 ng total, respectively) and Taqase; add 2 µl of the above diluted cDNA synthesis; finally add buffer to a total of 50 µl and mix well; place in a 4 °C micro benchtop centrifuge for 5 s instantaneously; add 2 drops of mineral oil using a 1000 µl gun tip. The reaction was carried out in a PCR amplifier and the products were later subjected to agarose electrophoresis for characterization.

PCR reaction conditions: pre-denaturation at 94 °C for 5 min. Cycling parameters: 94 °C for 1 min, 50 °C for 1 min, 72 °C for 1 min, 35 cycles, extension for 5 min.

Identification of amplification products

A 2% agar gel (containing ethidium bromide 0.5 μl/ml) was prepared with TAE, and 10UL amplification products were sampled by electrophoresis at 100 V for 120 min. The amplification products of MDRl and B-actin were identified under UV light at 167 bp and 301 bp, respectively, which were consistent with the designed amplification fragments of MDR1 and P-actin primers.

Quantitative analysis

Two bands of MDRl and B-actin primers were scanned with a Cs-910 chromatography scanner made by Shimadzu. The length and width were 2.1 mm and 0.1 mm. The wavelength input was 550 nm. The data were entered into a computer for relative quantitative analysis, and MDR1/B-actin was the relative amount of MDR1.

Statistical analysis

The SPSS 19.0 statistical package was used for statistical analysis (SPSS Inc., Chicago). Percentages were compared using the Chi-squire test, and t-tests were used for comparison of measurements between the two groups. p-values less than 0.05 were considered statistically significant.

Ethics approval and consent to participate

This study was approved by the ethics committee of the first affiliated hospital, and college of clinical medicine of henan university of science and technology. Informed consent for all patients (including the patients of the control group) was obtained prior to therapy and performed in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines.



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