黏膜耐受治疗对大鼠实验性结肠炎的疗效观察

目的评价口服耐受和经鼻耐受治疗对大鼠实验性结肠炎的疗效,以及分别加用佐剂对其疗效的影响。方法建立三硝基苯磺酸（TNBS）实验性结肠炎大鼠模型。以卵白蛋白（OVA）为诱导抗原,脂多糖为佐剂。将已建立的TNBS结肠炎大鼠模型根据不同的干预方法分为结肠炎组、口服耐受组、经鼻耐受组、口服加佐剂组、经鼻加佐剂组、佐剂对照组和空白对照组。各组大鼠经上述不同治疗后均于灌肠第21天处死,以肠道大体和组织病理学评分为标准评价治疗效果,以脾淋巴细胞增殖实验评价黏膜耐受诱导效果。结果肠道大体评分（平均秩次9．5VS3．5,P〈0．01）和组织病理学评分（平均秩次9．5VS3．5,P〈0．01）显示TNBS诱导的实验性结肠炎模型在无干预情况下可持续至第21天仍有肠道炎症。脾淋巴细胞增殖实验结果显示,在大鼠结肠炎状态下,口服耐受组不能诱导机体对OVA的免疫耐受,而口服加佐剂组（0．11±0．05VS0．30±0．13,P〈0．05）、经鼻耐受组（0．12±0．07,P〈0．05）和经鼻加佐剂组（0．06±0．04VS0．30±0．13,P〈0．05）均可诱导机体免疫耐受,不同途径诱导的免疫耐受效果差异无统计学意义。与结肠炎组大鼠相比,口服加佐剂组（大体评分3．0±1．3VS6．3±0．8,组织学病理评分3．0±1．1VS7．5±1．0,均P〈0．05）,经鼻加佐剂组（大体评分2．0±0．6,组织学病理评分2．7±1．0,均P〈0．05）和佐剂对照组（大体评分4．3±1．0,组织学病理评分4．6±1．6,均P〈0．05）大鼠的肠道评分均显著较低。其中经鼻加佐剂组大鼠的肠道评分下降最显著。结论TNBS实验性结肠炎干扰了口服耐受的诱导,但不影响经鼻耐受的诱导。加用佐剂的口服耐受和加用佐剂的经鼻耐受对实验性结肠炎均有一定治疗作用,单独应用佐剂也有一定疗效,其中加用佐剂的经鼻耐受疗效最好。

Therapeutic effects of mucosal tolerance on inflammatory bowel disease: experiment with rats

OBJECTIVE: To evaluate the therapeutic effect of mucosal tolerance on inflammatory bowel disease (IBD). METHODS: Forty-two SD rats underwent enema of 2, 4, 6-trinitro-benzene-sulfonic acid (TNBS) so as to establish models of experimental colitis and then divided into 7 equal groups: colitis group (without treatment), oral ovalbumin (OVA) group (undergoing gastric perfusion of OVA as inducing antigen), nasal OVA group (undergoing OVA nose dropping), oral OVA plus adjuvant group undergoing gastric perfusion OVA with lipopolysaccharide (LPS) as adjuvant], nasal OVA plus adjuvant group, adjuvant control group, and blank control group (administrated with PBS orally). Another 6 rats were used as blank control group. All of the rats were executed 21 days after enema with their spleens taken out. Splenic lymphocytes were isolated and co-cultured with OVA for 96 h. MTT method was used to calculate the proliferation index so as to detect the mucosal tolerance. Macroscopical and histolopathological scores of colon were estimated so as to evaluate the therapeutic effects. RESULTS: Typical manifestations of colitis occurred 3-4 days after enema and persisted to the day 21 after enema. The macroscopical and histolopathological scores of colon of the colitis group (mean rank were 9.5 and 9.5) were both significantly higher than those of the normal control group (mean rank were 3.5 and 3.5 both P < 0.01). The splenic lymphocyte proliferation indexes of the oral OVA plus LPS, nasal OVA, and nasal OVA plus LPS groups were 0.11 +/- 0.05, 0.12 +/- 0.07, and 0.06 +/- 0.04 respectively, all significantly lower than that of the normal control group (0.30 +/- 0.13, all P < 0.05), however, without significant differences among these 3 groups (all P > 0.05). The splenic lymphocyte proliferation indexes of the oral OVA group was 0.25 +/- 0.10, not significantly different from that of the normal control group (P > 0.05). The macroscopical scores of the oral OVA plus LPS group, nasal OVA plus LPS group, and oral LPS alone group were 3.0 +/- 1.3, 2.0 +/- 0.6, and 4.3 +/- 1.0 respectively, all significantly lower than that of the colitis group (6.3 +/- 0.8, all P < 0.05); and the histological score of the oral OVA plus LPS group, nasal OVA plus LPS group, and oral LPS alone group were 3.0 +/- 1.1, 2.7 +/- 1.0, and 4.6 +/- 1.6 respectively, all significantly lower than that of the colitis group (7.5 +/- 1.0, all P < 0.05). The colonic score of the nasal OVA plus LPS group was most significantly decreased. CONCLUSION: Colitis interferes with the induction of oral tolerance, but not the induction of nasal tolerance. Both oral tolerance combined with adjuvant and nasal tolerance combined with adjuvant have therapeutic effects on experimental colitis. Oral adjuvant alone also has therapeutic effect on colitis, and the therapeutic effect of nasal tolerance combined with adjuvant is the best.