The Therapeutic Effect Of Thalidomide On Trinitrobenzene Sulfonate (TNBS)-induced Colitis In Young Rats And Studies On Its Mechanism

PartⅠEstablishment of model of TNBS-induced colitis in young Sprague-Dawley (SD) ratsBackground:Inflammatory bowel disease (IBD) is a group of chronic inflammatory disease of the gastrointestinal tract which comprises mainly ulcerative colitis (UC) and Crohn's disease (CD). The prevalence is highest in North America and Europe, but its incidence appears to be on the rise in Asian and other regions. The exact etiology and pathogenesis are unknown, and there is no single highly effective therapeutic modality. Therefore it is important to develop an animal model of (IBD) to study the effectiveness and therapeutic mechanism of new medications because such results would be important when applied to human patients.Aims:To establish a model of TNBS-induced colitis in young rats.Material & Method:25 rats of age 4-5 weeks were randomly separated to TNBS treatment group and controls. TNBS 150mg/kg was introduced rectally through a catheter for the treatment group and for the control group, normal saline. The daily eating behavior, fur condition and physical activities were monitored, and the body weights, characteristics of and presence or absence of blood in the stool were recorded. On days 4,7 and 14, four rats of each group were sacrificed. The colons were examined morphologically and the severity of macroscopic damage graded according to the scoring system proposed by Wallace and Keenan. Histopathologically, the colonic damage was graded according to the scoring system proposed by Cooper.Results:In the control group, the fur appeared normal, and the eating behavior and physical activities were also normal. The diarrhea and per rectal bleeding lasted for 2.08±0.67 and 1.83±0.72 days. No rat in the control group died during the study period. In the TNBS treatment group, the fur lacked luster, the appetite decreased, and the rats appeared apathetic. Abdominal distension, diarrhea and rectal bleeding appeared rapidly after TNBS treatment, and the diarrhea and bleeding persisted for 6.00±1.71 and 6.08±1.68 days respectively, being statistically different from the controls (P< 0.01). The mortality rate of the treatment group was 7.69%. The body weights did not increase but rather reduce in the first four days. They resumed the original weight by day 7 and increased gradually but lacked behind the control group (P<0.01).In the treatment group, the colon appeared dilated, there was mucosal erythema and edema, the colonic wall appeared thinner, and ulcerative lesions were seen in the colonic mucosa. The serosal surface appeared eroded and fluid was seen in the peritoneal cavity. On day-14, there is some improvement in the inflammatory changes, scarring is noted in the ulcerated areas, the thinned regions became thickened, and adhesion to adjacent organs became apparent. The morphological scores of the colon on days 4,7 and 14 were:4.25±0.50,4.25±0.96,2.75±0.5 respectively, while those of the control group were 0,0 and 0; and there is marked statistical difference (P< 0.01) between groups.In the treatment group, under light microscopy, numerous ulcers were seen in the colonic mucosa with depletion of goblet cells. The majority of the crypts and colonic glands were destroyed, only a small amount of the epithelial cells remained. The normal architecture of the colonic glands was lost, the arrangement became erratic and the glandular lumens became obliterated by infiltration of inflammatory cells. The inflammation was transmural, affecting all layers of the colon. On day-14, the small ulcers became more circumscribed, crypts were seen on the edges and goblet cells reappeared. There was regeneration of the crypts, but inflammatory infiltrates were still seen, mostly in the mucosal and submucosal layers, occasionally involving the muscularis. An increase in fibrosis was present. On days 4,7 and 14 the histopathological scores were:3.75±0.50,3.75±0.50,2.50±0.58 respectively, while those of the control group were 0,0, and 0. There is significant statistical difference between groups (P< 0.01).Conclusion:The rat TNBS-induced colitis model is characterized by:long periods of bloody diarrhea and poor weight gain. Morphologically, mucosal erythema, edema, ulceration and increase in thickness of colonic wall were present. Histologically, massive infiltration of inflammatory cells and destruction of crypts and cryptal glands were the salient changes. To a certain extent these may reflect the changes in IBD in humans hence the model may be a valid one for the studies on related issues connected with IBD in children. PartⅡThe therapeutic effect of thalidomide on trinitrobenzene sulfonate (TNBS)-induced colitis in young rats and studies on its mechanismBackground: We and others have previously reported the successful use of thalidomide in a group of patients with inflammatory bowel disease who failed traditional treatment. This drug can be effective in inducing remission, improvement in symptoms and closing of fistulas. The specific effects of thalidomide in treating patients with intractable inflammatory bowel disease have aroused interest in the medical community.Aims:To observe the therapeutic effect of thalidomide by using the established animal model of IBD (model of TNBS-induced colitis in Sprague-Dawley rats) and to investigate the possible mechanism of action, thereby postulate the basis of therapeutic action of thalidomide in the management of IBD in children.Material and Method:Eighty-two Spraque-Dawley rats, about 4-5 weeks old, were randomized into three groups. The first group (25 rats) was the normal controls, the second group (29 rats), the TNBS treated group, and the third group (28 rats), the thalidomide treatment group. The first group was treated with normal saline through a catheter inserted through the anus, the second group was treated with 150 mg/kg of TNBS intrarectally, and the third group the same treatment as the second group plus oral administration of thalidomide 150 mg/kg/day. Daily activities were recorded as described in the previous section. At least eight rats from each group were sacrificed on days 4,7 and 14. The morphological and histological changes in the colon were individually assessed as described in the first part. Serum was collected and the levels of TNF-a and interleukins (IL-13, IL-6, IL-10 and IL-12) were essayed by the ELISA method. The colonic mucosal nuclear factor NF-κB expression was essayed with the immunohistochemical method.Results:In the control group, the diarrhea and bleeding recovered rapidly (2.04±0.68 days and 1.96±0.84 days) and no death was recorded. In the TNBS treated group, the diarrhea and per rectal bleeding persisted for a longer period of time (5.77±1.70 days and 5.92±1.74 days) and statistically different from that of the control group (P< 0.01). There was a mortality rate of 10.34% during the observation period, the weight gain of this group was significantly lower than that of the control group. In the thalidomide treatment group, the diarrhea and per rectal bleeding persisted for 3.78±0.58 days and 3.89±1.15 days which were significantly shorter than the TNBS only group(P< 0.01). This group of rats also exhibited faster weight gain after the seventh day compared with the TNBS only group but still lower than that of the control group. The mortality rate of the thalidomide treatment group was 3.57%.Macroscopic morphological scores of colon:TNBS model group:4.30±0.48,4.25±0.71and 2.88±0.64 on days 4,7 and 14 respectively and significantly different from control group (P< 0.01). Microscopic histopathological scores:TNBS model group: 3.80±0.42,3.88±0.35 and 2.38±0.52 (significantly different from controls, P< 0.01). In the thalidomide-treated group, the day-4 morphological score was 4.00±0.71 and the histopathological score was 3.78±0.44, both not statistically different from those of the TNBS only group(P>0.05). On day-7, there was improvement in the colonic distension, the ulcers became smaller and more circumscribed as observed under light microscopy and there was regeneration of the crypts, colonic glands and goblet cells. Adhesion to the surrounding structures was also observed. The morphological and histopathological scores were 3.55±0.73 and 3.00±0.50 respectively, and compared with the scores of day-4, had demonstrated statistically significant improvement(P< 0.01). On day-14, there was no obvious distension of the colon, scarring of the ulcers became evident, and under light microscopy the structures became better defined-the mucosa appeared more organized and less inflammatory cellular infiltration was observed. Macroscopic and microscopic scores were 1.11±0.60 and 1.11±0.60 respectively, and were significantly lower than those of the same group on day-7(P< 0.01), and those of the TNBS model group on the same day(P< 0.01). These findings are suggestive of better colonic recovery in the thalidomide treated group.NF-κB expression is lower in the colonic mucosa of the control group, mainly distributed in the cytoplasm. A large amount of intranuclear and cytoplasmic staining was observed (more prominent intranuclearly) in the TNBS model group (59.80%±9.30%) and the thalidomide treatment group (56.78%±6.72%); both being significantly higher than that in the control group (6.75%±1.39%, P< 0.01). On days 7 and 14, intranuclear NF-κB containing cells are still significantly lower in the thalidomide treatment group (36.00%±9.97%,13.22%±2.91%) than those in the TNBS model group (52.63%±5.13%,31.13%±6.66%, P< 0.01).In the control group, the cellular inflammatory factors (TNF-α, IL-1β, IL-6, IL-10and IL-12) were expressed at a low level while in the other two groups they were already expressed at a significantly higher level on day-4 (Pcontrol vs model,Pcontrol vs thalidomide <0.01). On day-7 the same inflammatory markers expression was lower in the thalidomide treatment group compared with the TNBS model group (Pcontrolvs model, Pcontrol vs thalidomide<0.01). On day-14, the inflammatory markers were significantly lower in the thalidomide treatment group than in the TNBS model group (Pcontrol vs thalidomide<0.05). On day-4, the IL-10 level of the thalidomide treatment group became significantly elevated (44.90±21.66 pg/ml), compared with control (12.41±4.03 pg/ml) and TNBS model groups (18.81±7.07 pg/ml) (Pconrol vs thalidomide, Pmodel vs thalidomide< 0.01). The levels gradually decreased but still remained at a higher level. On day-7 and day-14 IL-10 levels were 32.11±10.10 pg/ml and 31.14±7.46 pg/ml respectively. In the TNBS model group, the IL-10 expression peaked later than in the thalidomide treatment group. On day-4, compared with that of the control group, there was no statistically significant difference but on days-7 and-14 the levels were 25.84±10.07 pg/ml and 24.91±8.44 pg/ml respectively, and significantly different from those of the control group (11.98±4.09 pg/ml and 12.28±3.10 pg/ml, respectively; Pcontrol vs model< 0.01). These levels were significantly lower than those of the thalidomide group.Conclusion:Thalidomide was effective in the management of TNBS-induced colitis in young rats. It shortened the duration of diarrhea and passage of blood in the stool, enhanced the recovery of intestinal mucosal damage and modified the intestinal inflammatory reaction. Thalidomide in our study demonstrated good anti-inflammatory and immune-modulatory effects. This may be due to the suppression and down-regulation of nuclear factor NF-κB and the expression of the downstream inflammatory mediators (TNF-α,IL-1β,IL-6 and IL-12). There is also indication that expression of the anti-inflammatory cytokine (IL-10) is concomitantly up-regulated as well.