Application of interleukin 1β and 8 mRNA expression and other indicators to monitor the activity of ulcerative colitis

Abstract Objective To study the role of IL-1β and IL-8 mRNA expression, myeloperoxidase (MPO) and superoxide dismutase (SOD) activity in monitoring the activity of ulcerative colitis (UC). Methods 20 patients with ulcerative colitis (UC) active period (AUC group), 23 patients with UC inactive period (IUC group), 14 patients with non-UC intestinal inflammation (NUC group) and 12 patients with control group were observed. Colonoscopy mucosal biopsy specimens MPO and SOD activity and IL-1β and IL-8 mRNA expression. In the 14 patients in the AUC group, after 2 months of treatment, the condition was stable and the measurement of the above indicators was repeated. Results The MPO activities of colon mucosa in the above four groups were (19.37 ± 0.54), (11.59 ± 1.41), (12.97 ± 0.49) and (9.49 ± 0.51) U / g tissue, and the SOD activities were (5.03 ± 1.07) and ( 7.66 ± 0.79), (6.98 ± 0.61) and (8.82 ± 0.58) U / mg protein, the first 3 groups were significantly different from the control group; the AUC group was also significantly different from the IUC and NUC groups (P < 0.01). In 14 patients in the AUC group, the MPO and SOD activities before and after treatment were (12.61 ± 0.74) U / g tissue vs (19.31 ± 0.44) U / g tissue and (7.44 ± 0.55) U / mg protein vs (5.11 ± 1.05) U / mg protein, the former activity decreased significantly after treatment, the latter increased (P <0.05). In situ hybridization showed that all AUC specimens, 9 IUC specimens, and 7 NUC specimens detected the expression of IL-1β mRNA in colonic mucosal glandular epithelial cells and inflammatory cells of the lamina propria, but 14 AUC patients were not detected after treatment IL-1β mRNA expression; IL-8 mRNA expression can also be detected in the inflammatory cells of the colonic lamina propria and submucosa of the AUC group, but IL-8 mRNA expression was not detected in IUC, NUC, and 14 AUC patients after treatment . The mRNA of the two interleukins was not expressed in the colonic mucosa of the control group. Conclusion MPO, SOD, IL-1β and IL-8 mRNA of intestinal mucosa can be used as indicators to monitor UC disease activity and evaluate clinical efficacy. IL-1β mRNA can also monitor early or potential activities of UC.

Keywords: ulcerative colitis interleukin 1β interleukin 8 myeloperoxidase superoxide dismutase

Ulcerative colitis (ulcerative colitis, UC) is a chronic relapsing disease, its clinical features are alternating relapse and remission. Early detection of signs of UC recurrence and corresponding preventive and therapeutic measures are important for controlling the development of the disease. However, there are currently no indicators for monitoring UC activity. In this study, based on previous animal experiments, the activity of myeloperoxidase (MPO) and superoxide dismutase (SOD) and interleukin-1β (interleukin-1β, IL-1β) mRNA were detected And interleukin-8 (IL-8) mRNA expression, used to monitor the disease activity of UC patients.

1 Materials and methods

The main reagent is used for digoxin labeling and detection kit for mRNA in situ hybridization (Boehringer Mannheim). Human IL-1β cDNA probe, 210 bp, kindly donated by Glaxo Kawashima. Human IL-8 cDNA probe, 229 bp, kindly donated by Yoko Matsushima from UCSF University.

Subjects selected 62 patients with reference to the standard of literature [1], and all were diagnosed as UC by colonoscopy and histopathological examination. The activity of the disease is comprehensively judged by clinical symptoms, erythrocyte sedimentation rate, C-reactive protein, α2 globulin, and colonoscopy results (including microscopic findings and histopathological examination). All patients were divided into 5 groups: (1) Control group: 12 patients, who underwent colonoscopy due to abdominal distension, abdominal pain, constipation and diarrhea to exclude inflammation or tumor. (2) AUC group: 20 patients, except 6 newly diagnosed patients who have not taken medicine, the other patients are all treated with glucocorticoids (such as prednisone, hydrocortisone succinate, etc., the dose is equivalent to prednisone 30-40mg / d), and taking sulfasalazine salicylate (SASP) 3 ~ 4 g / d. Medication 1 to 4 days before inspection. No immunosuppressants were used. (3) IUC group: 23 patients. All patients received glucocorticoid equivalent to prednisone at a dose of 10 to 30 mg / d and SASP 2 to 4 g / d for 3 weeks to 6 months. No immunosuppressants were used. (4) NUC group: 14 cases, including 2 cases of appendicitis, 3 cases of anastomotic stomatitis (intestinal anastomosis due to resection of intestinal tumors), 4 cases of colon cancer, and 5 cases of colon polyps. In addition, 14 patients in the AUC group who had been in the inactive phase of UC after 2 months of treatment with prednisone and SASP voluntarily received colonoscopy, and were listed as the post-treatment group.

Methods Colonoscopy biopsy was used to obtain materials. The patients with AUC, IUC and NUC were all taken at the obvious site of inflammation, and the patients in the control group were randomly selected. Determine the MPO and SOD of biopsy specimens [1], and use in situ hybridization to detect the expression of IL-1β and IL-8 mRNA in specimens [2].

Statistical methods Experimental data is expressed as mean ± standard deviation (x ± s), and t-test is used for comparison between groups. Before and after treatment, the difference t test was used.

2 results

Colonic mucosal MPO and SOD activity AUC, IUC, and NUC groups had higher colonic mucosal MPO activity than the control group, and AUC group MPO activity was also higher than IUC and NUC groups; while SOD activity was opposite to MPO, and SOD activity was higher in all three groups The control group was low (schedule). U / g tissue of 14 patients in AUC group after MPO activity treatment (12.61 ± 0.74) was lower than before treatment (19.31 ± 0.44) U / g tissue, P <0.01; and after SOD activity treatment (7.44 ± 0.55) U / mg protein Compared with (5.10 ± 1.07) before treatment, U / mg protein increased, P <0.01.

The activities of myeloperoxidase and superoxide dismutase in colon mucosa of patients in different groups

Table MPO and SOD activity in different group patients (X ± S)

Control

AUC

IUC

NUC

(n = 12)

(n = 20)

(n = 23)

(n = 14)

MPO (U / g tissue)

9.49 ± 0.51

19.37 ± 0.54 ** #

11.59 ± 1.41 *

12.97 ± 0.49 **

SOD (U / mg protein)

8.82 ± 0.58

5.03 ± 1.07 ** #

7.66 ± 0.79 *

6.98 ± 0.61 *

* P ＜ 0.05, ** P ＜ 0.01 compared with control group; # P ＜ 0.01 compared with IUC and NUC group

AUC, IUC: active and inactive ulcerative colitis respectively; NUC: nonulcerative colitis inflammation; MPO: myeloperoxidase; SOD: superoxide dismutase

The expression of IL-1β and IL-8 mRNA in the colonic mucosa was measured using antisense IL-1β cRNA. The expression of IL-1β mRNA was detected (Figure 1). However, the expression of IL-1β mRNA was not detected in 14 AUC specimens after treatment. No sense IL-1 β cRNA probe was detected in the intestinal mucosa of patients in the above groups.

Figure 1 Anti-intentional IL-1β cRNA probe detected the expression of IL-1 β mRNA in the gut epithelium of intestinal mucosa in patients with active UC

Fig 1 Anti-sense IL-1 β cRNA probe demonstrated IL-1 β mRNA expression in epithelial cells in active ulcerative colitis patients (arrow) × 20

Figure 2 IL-8 cRNA probe detected IL-8 mRNA expression in intestinal mucosal lamina propria of active UC patients

Fig 2 Anti-sense IL-8 cRNA probe demonstrated IL-8 mRNA expression in inflammatory cells of lamina propria in active ulcerative colitis patients (arrow) × 40

Using antisense IL-8 cRNA as a probe in the inflammatory cells of the mucosal lamina propria and submucosa of AUC group specimens can detect the expression of IL-8 mRNA. However, the expression of IL-8 mRNA was not detected in the colonic mucosa of IUC, NUC, and 14 AUC groups after treatment. No sense IL-8 cRNA probe was detected in the intestinal mucosa of patients in the above groups.

The sense and antisense cRNA probes of IL-1 and IL-8 were not detected in the corresponding colonic mucosa in the control group.

3 Discussion

At present, the clinical evaluation of UC activity is mainly based on the comprehensive judgment of the recurrence of clinical symptoms, the increase of erythrocyte sedimentation rate, C-reactive protein, α2 globulin, and the performance under endoscopy. The determination of most UC activities is relatively accurate. However, the statistics of 365 outpatient and inpatient UC medical records of Peking Union Medical College Hospital in the past 20 years have found that there are a small number of patients and some patients with early active stage, the blood biochemical performance is not obvious, and endoscopic examination has not found obvious signs of acute UC activity. Clinical Symptoms suggest that the disease recurs [3]. Given that the onset of UC is due to the local immune response induced by the contact of external antigens with the intestine, only when the local immune response develops to a certain stage and reaches a certain severity will the systemic systemic manifestations appear, so patients with UC early active stage are normal Blood biochemical indicators may not accurately reflect the inflammation of their intestines. Endoscopy, as a morphological observation method, can not detect early internal active lesions. As a result, the treatment of some patients' condition was delayed due to untimely judgment, which caused the disease to be difficult to control and relieve, and even complications. Therefore, it is very meaningful for clinical work to establish more sensitive laboratory indicators for monitoring UC activity. The author has confirmed from animal experiments that MPO and SOD in intestinal tissue are positively correlated with the severity of intestinal inflammation [1,2], suggesting that the detection of tissue MPO and SOD activity can be used to monitor the activity of intestinal inflammation.

The MPO activity of intestinal mucosa biopsy specimens in this study showed that the MPO activity of patients in the AUC, IUC, and NUC groups was higher than that of the control group, indicating that MPO activity was not disease-specific in monitoring intestinal inflammation activity. However, the MPO activity of the AUC group was significantly higher than that of the IUC group, and after 14 patients with AUC were stabilized by clinical treatment, the MPO activity decreased significantly, indicating that for patients who have been diagnosed with UC, MPO can be used to monitor the activity of the disease and evaluate An indicator of clinical efficacy. Although the MPO activity of IUC patients is lower than that of the active phase, it is higher than that of the control group, indicating that some tissue enzyme activities that are clinically judged as "inactive UC" still show potential activity. The results of this experiment also showed that the reduction of SOD activity in the intestinal mucosa of patients can also reflect changes in intestinal inflammation and disease, and can also be used as a non-specific indicator for monitoring UC activity. However, because the detection method of SOD is more complicated than MPO and the cost is higher, it is not as practical as an index for clinical monitoring of UC activity.

Although the detection of MPO and SOD activity in intestinal mucosa tissue is two sensitive indicators reflecting the activity of UC, it is not as easy as blood test because it requires specimens to be obtained through endoscopic examination. As an auxiliary method for clinical monitoring of UC activity, it is mainly suitable for UC patients who have unclear judgment of the disease activity by conventional means. From the perspective of the specificity of diagnosis, although they cannot distinguish the type of intestinal inflammation, they can reflect the acute degree of intestinal inflammation, especially for patients with confirmed UC, which can be used to monitor the activity of the disease.