5 - Lox, Inos Expression Of Colonic Mucosa In Ulcerative Colitis Patients And Their Correlation

[Background] Ulcerative colitis (UC) is a chronic, nonspecific inflammatory bowel disease usually accompanied by recurrent attacks. Although the incidence of UC has increased gradually in recent years, its exact etiology remains unclear. Current etiological researches have proposed that the pathogenic mechanism involved in UC may be associated with immunoregulatory, genetic, environmental, infectious and psychological factors. These studies also indicate that a complex interaction of all those factors can contribute to the patients’ intestinal mucosal immune dysfunction, and give rise to excessive inflammatory cytokines and mediators, thus leading to the patiments’intestinal inflammation.5-lipoxygenase (5-LOX) is a key enzyme in the metabolism of arachidoni acid (AA) to leukotriene B4(LTB4). LTB4recruits inflammtory cells to the site of inflammation by binding to its receptors in those cells, and it has a strong chemotactic effect on neutrophils, monocytes and effective T cells. Therefore, LTB4is considered to be a potent inflammatory mediator that plays a major role in UC. Mazzon et al. have demonstrated that the symptom of diarrhea and colon inflammation is significantly alleviated in5-LOX knockout mice model. Clinical trials have also found that5-LOX inhibitors can obviously improve the patients’ clinical symptoms and their endoscopic severity of inflammtion. Although great progress has been made in figuring out the function of5-LOX, few studies have focused on the5-LOX expression in colonic tissue from patients with UC. Nitric oxide (NO) is a neurotransmitters and cell messenger molecule. It has been identified as an important inflammatory mediator involved in the incidence of UC. NO is generated by inducible nitric oxide synthase (iNOS). It is known that iNOS is not usually expressed under physiological conditions. However, in pathological conditions, iNOS is activated as a result of proinflammtory stimuli which provokes the release of excessive amounts of NO that may worsen colonic mucosa damage. Multiple studies have shown that NO can serve as an important inflammatory mediator involved in the incidence of UC, and iNOS is the key enzyme in the induction of NO production.5-LOX and iNOS play important roles in the incidence of UC since both of them can induce the production of inflammatory cytokines and mediators. However, what’s the expression pattern of5-LOX and iNOS in colonic tissue from patients with UC, and how they interact in the incidence of UC? All the issues are needed to be further investigated.[Objective] To investigate the mechanism of5-LOX and iNOS in the pathogenesis of UC by observing the expression level of5-LOX and iNOS and their interaction in intestine mucosal tissue from UC patients with different clinical classification, thus providing a theoretical basis for clinical treatment of UC. [Objects and methods]1.Case selection: we selected38patients with UC dignosed in the outpatient and inpatient of First Peopele Hospital in Jinzhong City from December2009to November2011, all cases were in line with the diagnostic criteria revised by Inflammatory Bowel Disease Collaborative Group from Branch of Chinese Medical Association in2007. The clinical classification was early-onset form and chromic recurrent form, and all cases were in active stage.2.Group:experimental group: Patients with UC were divided into3groups according to Truelove-Witts’Criteria, including mild (10cases), moderate (24cases) and severe (6cases). Patients were also divided into3groups according to Baron score in endoscope, including lev11(11cases), level2(22cases) and level3(7cases). Control group:32cases including those nornal under colonoscopy and colon polypectomy after review were collected at the same period as the control group. Age and gender difference were not statistically significant in both groups.3. sample collection and processing:4-5samples were collected from one patient in the most prominent place of a lesion, for control group, same amout of samples were collected in the sigmoid colon. All samples were fixed by10%formalin, then sequentially dehydrated, embedded in paraffin and sliced, finally subjected to hematoxylin-eosin (HE) staining or SP immunohistochemical staining.4. Methods:The immunohistochemical SP staining was used to determine the protein expression of5-LOX and iNOS in the colonic mucosa in both UC patients and normal control group. Positive staining criteria:the brown granules in the cytoplasm or nucleus were treated as positive signal for5-LOX staining, the brown granules in the cytoplasm, nucleus or cell membrane were treated as positive signal for iNOS staining. Each slice was counted200cells to calculate the percentage of positive cells under400magnification, totally five visions, and then the average of those five visions was calculated.5.Statistical analysis:statistix software SPSS13was used for statistical analysis. Data were expressed as mean±standard deviation. Difference between patients and normal control were analyzed by analysis of variance and the Wilcoxon rank sum test, differences between different clinical classification were analyzed by one-way ANOVA. The correlation between two indicators were analyzed by Pearson correlation test. Statistical significance was considered when P was less than0.05.[Results]1. With H-E staining, most of samples from patients with active UC showed intestinal mucosal congestion and edema, vascular texture disorder, part of the colonic epithelial cell loss, diffuse, acute and chronic inflammatory cells infiltration in lamina propria, neutrophils infiltration into the crypt, which leading to crypt inflammation or crypt abscess. A small number of samples showed abscess invasion into lamina propria, goblet cells decrease, mucosal surface epithelial erosion and ulceration. Whereas samples from normal control group showed normal colonic mucosa structure, tightly packed epithelial cells and intact cell structure, a large number of goblet cells, monocytes were scatterly distributed in the submucosal and there was no neutrophils and eosinophils infiltration.2. Immunohistochemical S-P staining:(1). For5-LOX staining,5-LOX was mainly expressed in the epithelial cells and inflammatory cells such as plasma cells and lymphocytes in colonic mucosa lamina propria in patients with active UC.5-LOX, shown as brown granulars, was mainly distributed in the cytoplasm of epithelial cells and inflammatory cells.5-LOX was also expressed in the nucleus, but showed dark brown and a diffuse or fine granular. Whereas in normal control group, the5-LOX was slightly expressed in the colonic mucosa epithelial cell cytoplasm with light brown and a small amount of granulars, and no positive staining of5-LOX in other cells. Statistical results:the difference of5-LOX positive staining between the experimental group and control group was statistically significant (P<0.05), the difference of5-LOX positive staining between the different clinical severity and endoscopic grading in patients with active UC was statistically significant (P <0.05).(2). Immunohistochemical S-P staining for iNOS:For iNOS staining, iNOS was mainly expressed in the epithelial cells and inflammatory cells such as plasma cells and monocytes in colonic mucosa in patients with active UC. iNOS, shown as brown or yellow granulars, was mainly distributed in the cytoplasm of epithelial cells and inflammatory cells. iNOS was also expressed in the nucleus and cell membrane. Whereas in normal control group, iNOS was slightly expressed in the colonic mucosa epithelial cell cytoplasm with light brown, only a few cases with dark brown. iNOS was occasionally expressed in alveolar epithelial cell with diffused light yellow granules in interstitial cells. Statistical results:the difference of iNOS positive staining between the experimental group and control group was statistically significant (P<0.05), the difference of iNOS positive staining between the different clinical severity and endoscopic grading in patients with active UC was statistically significant (P<0.05).3. The protein expression of5-LOX and iNOS:Pearson correlation analysis showed that the protein expression of5-LOX and iNOS was positively correlated in active UC colonic mucosa cells (r=.616, P=0.000).[Conclusion]1. Compared with normal control group, the protein expression of5-LOX and iNOS is significantly increased in colonic mucosa in experimental group.2. The protein expression of5-LOX and iNOS is correlated with the lesion severity of patients with UC and endoscopic classification.3.5-LOX expression is positively correlated with iNOS expression in colonic mucosa in patients with UC...