| Objective:Ulcerative colitis, an idiopathic chronic inflammatory disease in intestine, mainly involves colonic and rectal mucosa, as well as submucosa, as its pathogenesis is inadequately understood, which is considered to be the result of the interaction of multiple factors mostly including hereditary, infective, environmental and immunological factors. It is demonstrated in researches that lack of methyl donor provides much of the impulsion for the increasing expression of some certain inflammatory cytokines and chemical factors in the rat model of ulcerative colitis, and even induces aggravation of colonic mucosal inflammation in connection with cell apoptosis and oxidative stress. In addition, it is indicated that the rectal mucosa interferon y expression significantly increases in patients of ulcerative colitis, and hypomethylation is tested in the promoter region of interferon gene, which might make a critical difference in the pathogenesis of ulcerative colitis. Through testing the serum folic acid level in both normal and colitis rat model, the expression level of rectal mucosa interferon y together with the methylation level of interferon gene in colonic structure, and analyzing the relativity between the level of folic acid and the expression level of interferon y, as well as the methylation level of interferon gene, this study explores the impact of level of folic acid on severity of inflammatory colonic mucosal inflammation, and the relativity between the abnormal expression of interferon y and methylation level of interferon gene. Moreover, it will further determine the influence of folic acid and methylation of interferon gene on the pathogenesis of ulcerative colitis, which is able to provide the basis for ulcerative colitis diagnosis and treatment.Materials and Methods:1. The establishment of animal models:24female BALB/C rats which met the experiment requirements were chosen and divided randomly into4groups within6rats in each group. Two groups were randomly selected and fed with no folic acid diet, while the other two groups were provided standard diet of equal quantity. There was no limitation of water intake for all groups. Since the6th week, one group of both no folic acid and standard diet were selected to be offered dextran sodium sulfate solution as drinking water, while the other two groups remained unchanged for7days, which was meant to build rat models of acute colitis. The groups were categorized as follows:Group A:deficiency of folic acid (D) and the models were set up (DDS+); Group B:Standard diet (C) and the models were set up (DDS+); Group C:Standard diet (C) and the models were not set up (DDS-); Group D: deficiency of folic acid (D) and the models were not set up (DDS-).2. The rats’ weight should be respectively measured before the experiment, at the5th and6th weekend. Vital signs should also be observed everyday such as rats’diet, water intake, active state, and so on. The rats’characteristics of feces, crissum inflamed and the degree of erosion and hematochezia were supposed to be observed and recorded from the6th week till the end of the experiment. After the experiment, the caudal venous blood and fresh colonic mucosa specimens of4groups of rats needed to be collected.3. The biotin double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was used to test the rats’of serum folic acid level in peripheral vein.4. Evaluate disease activity index of colitis:according to the degree of weight loss in mice, stool, blood in the stool to score,"normal" is0points, and the rest were scored1,2,3,4points according to different levels, record the total score, based on "disease activity index"=total score/3(0-4),0is healthy,4represents the greatest degree of inflammatory activity.5. The immunohistochemical staining method was used to test the interferon γ expression in colonic mucosa of rats.6. The bioinformatics technology was used to predict CpG Island in the promoter region of interferon gene and design the primer of methylation specific PCR (MSP). DNA of colonic structure was extracted and then used to test the methylation level of CpG Island in the promoter region of interferon gene through methylation specific PCR (MSP).Results:1. Serum folic acid level in peripheral vein The folic acid level of Group A and D obviously decreased comparing to Group B and C, and there was significant difference between the two sets of groups (P<0.05); There was no significant difference between Group B and C and between Group A and D (P>0.05).2.Interferon y expression in colonic mucosaThe interferon y expression of Group A and B obviously increased comparing to Group C and D, and there was significant difference between the two sets of groups (P<0.05); The interferon γ expression of Group A obviously increased comparing to Group B, and there was significant difference between the two groups (P<0.05) There was no significant difference between Group C and D (P>0.05).3.Correlation analysis of interferon y expression and colitis activity index In both Group A and B, the interferon y expression was positively correlated with colitis activity index, and the correlation coefficient r was0.796(P<0.01).4.Correlation analysis of folic acid level and interferon y expression as well as colitis activity indexIn Group A, the folic acid level was negatively correlated with the interferon γ expression and colitis activity index, and the correlation coefficients r were respectively-0.880、-0.997(P<0.05); In Group B, the folic acid level was not correlated with the interferon y expression and colitis activity index (P>0.05); In Group C, the folic acid level was not correlated with the interferon y expression (P>0.05); In Group D, the folic acid level was negatively correlated with the interferon y expression, and the correlation coefficient r was-0.867(P<0.05).5.There was no methylation tested in CpG Island of the promoter region in MSP test for every group of rats’interferon gene in colonic mucosa.The methylation of IFNG were detected with MSP products by2.5%agarose gel electrophoresis, the methylated bands were not detected in MSP products of A,B,C,D groups which were amplified by the methylated specific primers, and unmethylated bands were dected in products of four groups which were amplified by the unmethylated specific primers. The size of MSP products is about70bp(50bp100bp); but amplified band does not appear in the control group.Conclusion:1. In the rat model of ulcerative colitis, the deficiency of folic acid promoted the expression of inflammatory factor interferon y which aggravated the degree of colonic mucosal inflammation; but it did not make impact on interferon y expression in colonic mucosa.2. In the rat model of ulcerative colitis, the methylation in CpG Island of the promoter region of interferon gene may not be the precipitating factor for leading to increased expression of interferon y of colonic mucosa. It was indicated that there probably was other mechanism causing the unusually high expression of inflammatory factor interferon y.3. the deficiency of folic acid didn’t result in the methylation in CpG Island of the promoter region of interferon gene, and caused the abnormal expression of interferon γ in UC models of mice. |
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