| ObjectiveTo explore the role of thalidomide on the regulation of tight junction proteins such as occludin, claudin-1 and ZO-1 of trinitrobenzene sulfonic acid (TNBS)-induced inflammatory bowel disease rat model and further elucidate the mechanism of thalidomide’s effect on intestinal mucosa barrier function of inflammation bowel disease model rat.MethodsWe selectedSprague-Dawley rats of 4-5 weeks old with weight between 80 and 100 g. Then they were divided into 3groups. Model group (28 rats) and thalidomide group (28 rats) underwent the infusing operation of trinitrobenzene sulfonic (TNBS 150 mg/kg in ethanol) through anus with 2mm diameter rubber tube after fasting 24h. The control group (24 rats) received the same dose 0.9%NaCl solution of required TNBS and ethanol mixture. At the meanwhlile, the treatment group received treatment with thalidomide(150mg/kg).8-12 rats were sacrificed in each group on the 7th day,10th day, and specimens from blood and colon were kept back for experiments. Experiments included:(1) HE staining, electronic microscopy, generally scoring, histological injury scoring; (2)ELISA to determine the level of TNF-α in blood; (3) Western blot to evaluate the protein expressionof occludin and claudin-1; (4) Immunohistochemical to observe ZO-1 expression and location; (5) PCR to investigate the alteration of expression of mRNA of occludin, claudin-1 and ZO-1.Result1.General symptoms and scoring:After intracolonic administration of TNBS, the rat began to have diarrhea, bloating and be inactive, and colon appeared flaccid, filled with liquid stool and be adhesive with tissues around.Treatment with thalidomide significantly reduced the extent and severity of colon injury. Note that, at 4 days after intracolonic administrationof TNBS, no alteration was observed in the colon tissues collected from vehicle-treated rats.2. HE staining, histological injury scoring, electronic microscopy:After intracolonic administration of TNBS, histological scores of the model ratsincreased compared withthat of normal group. After treated with thalidomide (150mg/kg), the thalidomide group’s histological scores significantly decreased at day 7 compared with that in the model group(p= 0.0273). On 7th day and 10th day,theelectron microscope showed thatthe structure of tight junction in model group was destroyed, not straight and unclear, with desmosome decreased, whileconnection between the epithelial of treatment group seemed normal. Tight junctions werestraight and clear, and much longer. At the same timedesmosomes were increased.3. measurement of TNF-a in the blood:On 7th day, the level of TNF-awas significantly increased in the model group compared with that in the normal group(p = 0.0268). The level of TNF-a is still higher than normal group after thalidomide treatment (p= 0.0304).On 10th day, the level of TNF-a was significantly increased in model group than in normal group(p= 0.0078). The level of TNF-a in the treatment group blood was a little higher than in the normal group(p= 0.0131). The level of TNF-a in model groupwas rised to a higher level on 10th day than on 7th day, whilethe level of TNF-a in thalidomide treatment groupwas reduced.4. theexpression of occludin,claudin-1 and zo-1:(1) occludin:On 7th day,the dephosphorylated occludin protein was increased significantly in model group (p<0.001) and thalidomide group (p= 0.023) compared with that in the normal group. At the same time, the dephosphorylated occludin protein treatment group was increased significantly in the model group compared with that in thalidomide treatment group(p=0.023).On 10th day, the dephosphorylated occludin protein was increased in model group and the thalidomide group compared with thatin the normal group without statistical significance. And there was no significant difference between the model group and treatment group. On 10th day, dephosphorylated occludin protein of the model group was significantly reduced(p= 0.017) than on 7th day. The dephosphorylated occludin protein levels of thalidomide group did not change.(2)claudin-1:On 7th day, claudin-1 protein was decreased in the model group than that in the normal group (p= 0.004), and it was decreased in thalidomide group than that in the normal group without statistical significance (p=0.050). At the same time, claudin-1 protein was decreased in model group compared with that in the thalidomide group but without statistical significance. On 10th day, claudin-1 protein was decreased significantly in the model group than that in the normal group(p=0.006), while, decreased in the thalidomide group than that in the normal group but without statistical significance. At the same time, claudin-1 protein was significantly decreased in model group compared with that in the thalidomide group (p= 0.024). Claudin-1 protein was decreased in model group on 10th day compared to that on 7th day without statistical significance, while, claudin-1 protein was increased in the thalidomide group claudin-1 protein without statistical significance(p= 0.204).(3)zo-1:on 7th day, measured by immunohistochemistry, zo-1 was increased in the model group compared to the thalidomide group (p= 0.72), the normal group (p=0.31) but not without statistical significance, zo-1 was increased in the thalidomide group than in the normal group without statistical significance (p= 0.546). On 10th day, zo-1 was increased in the model group compared to the thalidomide group (p=0.426), the normal group (p=0.0503) but not without statistical significance, zo-1 was increased in the thalidomide group than in the normal group without statistical significance (p=0.212). In the normal group, zo-1 was positioned mainly at the top between the cell membrane, while, in the model group and the thalidomide group, zo-1 was distributed in the cytoplasm. And in the treatment group the situation was improved compared with in model group.5.mRNA of occludin,claudin-1 and zo-1:(1)occludin: on 7th day, level of mRNA of occludinmodel group was the highest, compared with the thalidomide group (p= 0.0006) and normal group (p= 0.0074); level of mRNA of occludin was lower in treatment group than in the normal group without statistical significance.On 10th day, the level of mRNA model group and the treatment group tends to be normal.(2)claudin-1:on 7th days, level of mRNA of claudin-1 in model group was the lowest, compared with thalidomide group (p= 0.4926) and normal group (p= 0.0033). And the level of mRNA of claudin-1 was significantly lower in thalidomide treatment group than in the normal group (p=0.0375).On 10th day, level of mRNA of claudin-1 was lower in the model group (P= 0.019) and the thalidomide group (p= 0.65) than in the normal group. And the level of mRNA of claudin-1 was higher in thalidomide group compared with in model group (p-0.0171).(3)zo-1:on 7th days, level of mRNA of ZO-1 was the highest in model group compared with the thalidomide group (p= 0.049) and normal group (p=0.0414); On 10th day, level of mRNA of ZO-1 was was significantly higher in the model group (p= 0.0016) and thalidomide group (p= 0.0073) than the normal group, in the thalidomide group, level of mRNA of ZO-lwas higher on 10th day than on 7th day (p= 0.0348).ConclusionIntracolonic administration of TNBS can cause TNF-a level to be elevated in blood, with severe inflammation in the mucosa and submucosawhere infiltrated with neutrophils. At the same time, the epithelial cells will bearranged irregularly, and the structure of tight junctions will be destroyed, with dephosphorylated occludin increased, claudin-1 protein decreased, zo-1 increased, zo-1 distributedto the cytoplasm. And with respect to mRNA, intracolonic administration of TNBS can cause expression of occludin increased, expression of zo-1 increased, whereas expression of claudin-1 decreased. Treatment with thalidomide can significantly reduce the level of blood TNF-α, and reduce the inflammatory infiltration, improve the arrangement of epithelial cells and intestinal tight junctions. Compared with the model group, dephosphorylated occludin can be reduced as zo-1,while claudin-1 protein increased. And zo-1 exist besidethe cell membrane. Maybe through inhibiting inflammation, it can inhibit the extent of change of mRNA expression. |
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