| Background and objective:Ulcerative colitis(UC)is one of the most severe chronic diseases of gastrointestinal tract.Various factors,including heredity,immunity,and infection,may be involved in the pathogenesis of UC.Among these factors,immunity has been believed to play an important role.The cause of the hyperactive immune reaction in UC,either persistent stimulus of specific antigen or abnormal immune regulation,remains to be elusive.Pathologic lymphocyte homing,however,is prone to be presented in either of these situations.Immunologic derangement of UC is characterized by pathologic lymphocyte homing,which also attribute to the damage of bowel mucosa.Secondary lymphoid-tissue chemokine(SLC),a recently discovered chemokine,has been widely accepted to be involved in lymphocyte homing in vivo.However,there has been no report on its exact effect on bowel pathologic lymphocyte homing in UC.Various concentrations of 2,4-dinitrochlorobenzene(DNCB)enema were performed to establish the rat model of UC and to determinate the appropriate formula of enema. Then pathologic lymphocyte homing and the expression of SLC mRNA and associated adhesion molecule were studied in this model to elucidate their relationship.The specific antibody against SLC was used to interfere with the pathologic lymphocyte homing to determinate the role of SLC in the pathogenesis of UC.The mechanism and pathway,though which SLC exact its effect on pathologic lymphocyte homing,were studied using lymphocyte culture in vitro.Methods:1,80 Spague-Dawley rats were randomly divided into control,high-, medium- and low-dose groups,which received pure acetone,20%,10%,and 6%of DNCB enema respectively.General conditions,microscopy of colon specimen,the colon mucosa damage index(CMDI),the cytokines levels within colon tissue(IL-2, IFN-γ,and IL-6,using ELISA),were studied in various groups.2,60 Spague-Dawley rats were randomly divided into control,UC,and UC+SLC antibody intervention(intervention)group.Antibody against SLC(15μg/ml/kg)was injected via caudal vein for 5 days from the beginning of DNCB enema.The size of perye's lymph nodes within submucosum was measured microscopically.IL-2 and IL-6 levels within colon tissue were measured to reflect the inflammation of colonic mucosa.The expression of SLC mRNA and associated adhesion moleculeα4 andβ7 within colon mucosa were measured using quantitative reverse transcriptase-polymerase chain reaction(RT-PCR)and western-blot methods.The location of SLC mRNA expression was detected using histochemical method.3,Portal vein blood were aspirated in control and UC group for isolation and culture of lymphocyte,lymphocyte migration assay caused by SLC were experimented in two-compartment Boyden chamber system.The expression of adhesion moleculeα4 andβ7 covering the surface of lymphocyte were measured by flow cytometer.The expression of NF-κB-p65 within the endochylema and nucleus of lymphocyte were detected using westem-blot method.Results:part 1,Macroscopic and microscopic observations of colon mucosa in experimental rats were similar with those in patients with IBD.UC in rat model can be induced by various concentration of DNCB enema successfully.The rats receiving high or medium does of DNCB had prolonged duration of symptoms and more severe general reactions with high mortality.The opposite features were present in the low-dose group,had lowest mortality,the expression of pro-inflammation cytokines and the result of macroscopic score and microscopic score for the assessment of colonic damage show the model group and the control have statistics difference,the little-dose group have also statistics difference with big-dose and middle-dose groups. The IL-2 levels within colon tissue of high-,medium,and low-does groups were measured on 5th,14th,and 28th experimental day[D5 230±52 280±42 290±39(pg/ml);D14 210±36,270±45,280±43(pg/ml);D28 140±28,185±38, 190±32(pg/ml),respectively].The IL-2 levels in low-does group was significantly lower than those in high-does group.With respect to IFN-γ,and IL-6,the similar results were also found.part 2 Pathologic lymphocyte homing and hyperplasia of perye's lymph nodes were found in UC and intervention group.The acreage of perye's lymph nodes within slices showed significant increased in UC and intervention group as compared with the control group(9.56±0.32 and 5.84±0.62 vs 1.48±0.58,both p＜0.01)as well as significant difference between these two groups(p＜0.05).The expression of SLC mRNA within colonic tissue was increased significantly in UC and intervention group as compared with the Control group(0.846±0.07,and 0.768±0.135 vs.0.312±0.12,both p＜0.01),but there was no significant difference between these two groups. The levels of SLC within colonic tissue was increased significantly in UC and intervention group as compared with the control group(0.937±0.143,and 0.804±0.215 vs.0.532±0.201,both p＜0.01),but there was also no significant difference between these two groups.The positive expression of SLC were mainly concentrated on the surface of high endothelial venules(HEV).The SLC positive expression of HEV was increased significantly in UC and intervention group as compared with the control group(P＜0.01).The expression of adhesion moleculeα4 andβ7,which is associated with lymphocyte homing,was increased significantly in UC and intervention group as compared with the control group(0.772±0.108;0.725±0.013vs0.418±0.025,p＜0.01),but there was also no significant difference between these two groups(0.772±0.108 vs0.725±0.013,p＞0.05).The levels of IL-2 and IL-6 within colonic tissue were significantly decreased in intervention group compared with UC group(IL-2:353.6±25.7 pg/ml vs.133.1±31.2 pg/ml,p＜0.05;IL-6: 441.6±22.5 pg/ml vs.145.8±28.9 pg/ml,p＜0.01).part 3 The study in vitro showed that SLC had does-dependent effect on migration assay of lymphocyte,which was elevated by 3.7 times.The expression of adhesion moleculeα4 andβ7 covering the surface of lymphocyte was increased significantly in UC group compared with the control group(α4:64.58±9.73 vs. 16.62±7.84,p＜0.01;β7:56.76±16.13 vs.12.93±6.53,p＜0.01).Western-blot showed that NF-κB-p65 within endochylema were highly expressed in UC and the control group(0.84±0.04 vs.0.75±0.11,p＞0.05);while the expression of NF-κB-p65 within nucleus was significantly increased in UC group compared with the control group (0.94±0.34 vs.0.41±0.02,p＜0.05),as well as its activing quotiety(1.19±0.43 vs. 0.60±0.56,p＜0.01).Conclusion:part 1,UC model induced by DNCB enema(especially for the best concentration of 6%),which was a delayed-reaction immune modal type,had severe general reaction and typical local symptoms consistent with the episode of UC in human.This model can be established in batches with good controllability and long duration of symptoms.part 2,Pathologic lymphocyte homing,highly expressed SLC and adhesion moleculeα4 andβ7 were present in colon mucosa of UC rats,while SLC was mainly expressed in HEV.The intervention using antibody against SLC could reduce the inflammation effectively.part 3,SLC had strong chemotactic effect on lymphocyte,while adhesion moleculeα4 andβ7 acted as homing receptors.There is a good possibility that the activation of NF-κB within stimulated lymphocyte promote the transcription of associated gene and adjust production of the inflammatory mediators,cytokines,and adhesion molecules,which lead to the damage of mucosa in UC.
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