丁氏溃结灌肠液对DSS诱导的溃疡性结肠炎大鼠肠道屏障作用的相关研究
发布时间：2018-08-03 07:13
【摘要】：目的:研究丁氏溃结灌肠液对葡聚糖硫酸钠(Dextran Sulfate Sodium,DSS)诱导的溃疡性结肠炎(Ulcerative Colitis,UC)大鼠的结肠组织中紧密连接蛋白(Occludin)、粘蛋白2(MUC2)和诱导型一氧化氮合酶(iNOS)、血清免疫球蛋白A(IgA)的影响,探讨其可能的作用机制。方法:随机挑选10只大鼠,归入到空白对照组,其余的50只通过饮用DSS水溶液的方法建立大鼠UC模型。模型复制成功后,将参加造模的大鼠随机的分为5组,即模型组、丁氏溃结灌肠液低剂量组、丁氏溃结灌肠液中剂量组、丁氏溃结灌肠液高剂量组、美沙拉秦灌肠液组。除空白对照组外,其余各组给予生理盐水和相应药物连续灌肠14d。治疗14d后采用腹主动脉取血的方法,将全部大鼠处死,并剖取结肠标本。观察大鼠的体重、大便性状、便血情况及结肠组织的损伤变化并评分,评估疾病活动指数(Disease Activity Index,DAI),肉眼观察结肠组织大体情况并评分,光镜下观察结肠组织病理损伤并评分,酶联免疫吸附(ELISA)法测定血清IgA含量、蛋白质印迹(Western Blot)法检测结肠组织Occludin蛋白水平、实时荧光定量PCR(Real-Time PCR)检测结肠组织MUC2mRNA、iNOS mRNA表达水平。结果:1、造模后大鼠精神萎靡、体重下降、大便溏稀伴有黏液脓血,DAI评分与空白对照组比较增高(P0.05)。灌肠治疗后,丁氏溃结灌肠液组和美沙拉秦灌肠液组大鼠上述症状好转,DAI评分与模型组比较降低(P0.05)。其中丁氏溃结灌肠液低剂量组情况不及其他用药祖,但也好于模型组。2、肉眼观察,模型组大鼠结肠粘膜出现不完整损伤,明显充血、水肿,并伴有溃疡形成,与空白对照组比较大体评分增高(P0.05)。丁氏溃结灌肠液各组及美沙拉秦灌肠液组大鼠结肠粘膜损伤后得以修复,仅出现了充血、水肿、轻度糜烂,未见到明显的溃疡形成,与模型组比较大体评分下降(P0.05)。3、光镜下观察,模型组大鼠结肠粘膜损伤后出现坏死脱落,伴发不同程度的溃疡灶,大量炎症性细胞浸润,粘膜腺体被破坏,排列稀疏紊乱,甚至缺如,并有程度不一的扩张,与空白对照组大鼠比较病理评分显著增加(P0.01)。丁氏溃结灌肠液各组和美沙拉秦灌肠液组大鼠结肠粘膜未有明显的溃疡形成,粘膜水肿充血和炎症细胞浸润减少,与模型组比较病理评分明显的降低(P0.01)。4、模型组大鼠血清IgA含量、结肠组织Occludin蛋白表达水平,与空白对照组相比显著下降(P0.05),经丁氏溃结灌肠液及美沙拉秦灌肠液治疗后,各组血清IgA含量、结肠组织Occludin蛋白表达水平升高,与模型组比较有统计学差异(P0.05)。5、模型组大鼠结肠组织iNOSmRNA的表达水平,与空白对照组比较明显的升高(P0.01),经丁氏溃结灌肠液和美沙拉秦灌肠液治疗后,各组iNOSmRNA的表达呈下降趋势,与模型组比较有统计学意义(P0.05)。模型组大鼠结肠组织MUC2 mRNA的表达水平,与空白对照组比较明显的减低(P0.01),经丁氏溃结灌肠液和美沙拉秦灌肠液治疗后,MUC2mRNA的表达水平显示升高,与模型组比较有统计学差异(P0.05)。在mRNA表达水平上的比较,丁氏溃结灌肠液中高剂量组与美沙拉秦灌肠液组之间无明显差异。结论:1、丁氏溃结灌肠液对DSS诱导的UC有一定治疗作用,可有效减轻其大便不成形、便血、体重下降等症状,使肠粘膜组织炎症好转,降低DAI评分和大体、病理评分。2、对于DSS诱导的UC肠道损伤,丁氏溃结灌肠液可显著升高Occludin蛋白、MUC2 mRNA表达水平,增高血清IgA含量,降低iNOSmRNA表达水平,从肠道机械屏障、免疫屏障及化学屏障方面,减轻大鼠肠粘膜炎症损伤。
[Abstract]:Objective: To study the effects of Ding's ulceration enema on the colonic tissue, mucin 2 (MUC2) and inducible nitric oxide synthase (iNOS) and serum immunoglobulin A (IgA) in the colonic tissue of rats with Dextran Sulfate Sodium (DSS) induced ulcerative colitis (UC). Mechanism. Methods: 10 rats were randomly selected and returned to the blank control group. The other 50 rats were established by drinking DSS water solution. After the model replication was successful, the model rats were randomly divided into 5 groups, that is, the model group, the low dose group of Ding's ulceration enema, the middle dose group of the Ding's ulcerative enema solution, and the Ding's ulceration enema. In the high dose group, the Mesalazine Enemas group. Except for the blank control group, the other groups were given the normal saline and the corresponding drug continuous enema 14D. for the treatment of 14d after the treatment of the abdominal aorta. All rats were killed and the colon specimens were dissected. The body weight, stool, blood condition and colonic tissue injury of the rats were observed and evaluated. The disease activity index (Disease Activity Index, DAI) was evaluated and the colonic tissue was observed and graded by naked eyes. The pathological damage of colon tissue was observed under light microscope and the serum IgA content was measured by enzyme linked immunosorbent assay (ELISA). The level of Occludin protein in colon tissue was detected by the method of Western blot (Western Blot), and real time fluorescent quantitative PCR (Real) -Time PCR) detected the expression level of MUC2mRNA and iNOS mRNA in colonic tissue. Results: 1, the rats were mentally depressed, weight loss, loose stool with mucous blood, DAI score and blank control group increased (P0.05). After enema treatment, the symptoms of DINK and clyster group and mesalazine enema group were improved, DAI score and model Group comparison decreased (P0.05). Among them, the low dose group of Ding's ulcerative enema was inferior to that of other drugs, but it was also better than.2 in the model group. The colonic mucosa of the model group had incomplete injury, obvious congestion, edema, and the formation of ulceration, and the gross score was higher in the blank control group (P0.05). The colonic mucosa of the Mesalazine Enemas group was repaired. Only the hyperemia, edema, mild erosion, no obvious ulcers were found, and the gross scores of the model group decreased (P0.05).3. Under the light microscope, the colonic mucosa of the model rats showed necrosis and shedding, accompanied by varying degrees of ulcer, and a large number of inflammatory lesions. Cell infiltration, mucous glands were destroyed, the arrangement of a sparse disorder, or even the absence of a degree of expansion, compared with the blank control group, the pathological score of the rats was significantly increased (P0.01). There was no obvious ulcer formation in the colonic mucosa of the group of Ding's ulcerative enema and the mesalazine enema group, and the congestion of mucous edema and the infiltration of inflammatory cells. Less, compared with the model group, the pathological score was significantly lower (P0.01).4, the serum IgA content of the model group and the expression level of Occludin protein in the colon tissue were significantly lower than that in the blank control group (P0.05). The serum IgA content and the expression of Occludin protein in the colon tissues were increased after the treatment of the Ding ulceration enema and Mesalazine Enemas. Compared with the model group, there was a statistically significant difference (P0.05).5. The expression level of iNOSmRNA in the colon tissue of the model group was significantly higher than that in the blank control group (P0.01). After the treatment of the Ding ulceration enema and the mesalazine enema, the expression of iNOSmRNA decreased in each group, and was statistically significant (P0.05) compared with the model group (P0.05). The model group was larger than the model group. The expression level of MUC2 mRNA in the rat colonic tissue was significantly lower than that in the blank control group (P0.01). The expression level of MUC2mRNA increased after the treatment of Ding's ulcerative enema and mesalazine enema, compared with the model group (P0.05). In the mRNA expression level, the high dose group and the beauty in the Ding's ulcerative enema solution were compared. There was no obvious difference between the Shalin enema group. Conclusion: 1, Ding's ulcerative enema has a certain therapeutic effect on DSS induced UC. It can effectively alleviate the symptoms of unformed bowel, blood, weight loss and so on, improve the inflammation of the intestinal mucosa, reduce the DAI score and gross, the pathological score of.2, DSS induced UC intestinal injury, and Ding's ulceration enema The solution could significantly increase the expression of Occludin protein, MUC2 mRNA, increase the content of serum IgA, reduce the level of iNOSmRNA expression, and reduce the injury of intestinal mucositis in rats, from the intestinal mechanical barrier, immune barrier and chemical barrier.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5
[Abstract]:Objective: To study the effects of Ding's ulceration enema on the colonic tissue, mucin 2 (MUC2) and inducible nitric oxide synthase (iNOS) and serum immunoglobulin A (IgA) in the colonic tissue of rats with Dextran Sulfate Sodium (DSS) induced ulcerative colitis (UC). Mechanism. Methods: 10 rats were randomly selected and returned to the blank control group. The other 50 rats were established by drinking DSS water solution. After the model replication was successful, the model rats were randomly divided into 5 groups, that is, the model group, the low dose group of Ding's ulceration enema, the middle dose group of the Ding's ulcerative enema solution, and the Ding's ulceration enema. In the high dose group, the Mesalazine Enemas group. Except for the blank control group, the other groups were given the normal saline and the corresponding drug continuous enema 14D. for the treatment of 14d after the treatment of the abdominal aorta. All rats were killed and the colon specimens were dissected. The body weight, stool, blood condition and colonic tissue injury of the rats were observed and evaluated. The disease activity index (Disease Activity Index, DAI) was evaluated and the colonic tissue was observed and graded by naked eyes. The pathological damage of colon tissue was observed under light microscope and the serum IgA content was measured by enzyme linked immunosorbent assay (ELISA). The level of Occludin protein in colon tissue was detected by the method of Western blot (Western Blot), and real time fluorescent quantitative PCR (Real) -Time PCR) detected the expression level of MUC2mRNA and iNOS mRNA in colonic tissue. Results: 1, the rats were mentally depressed, weight loss, loose stool with mucous blood, DAI score and blank control group increased (P0.05). After enema treatment, the symptoms of DINK and clyster group and mesalazine enema group were improved, DAI score and model Group comparison decreased (P0.05). Among them, the low dose group of Ding's ulcerative enema was inferior to that of other drugs, but it was also better than.2 in the model group. The colonic mucosa of the model group had incomplete injury, obvious congestion, edema, and the formation of ulceration, and the gross score was higher in the blank control group (P0.05). The colonic mucosa of the Mesalazine Enemas group was repaired. Only the hyperemia, edema, mild erosion, no obvious ulcers were found, and the gross scores of the model group decreased (P0.05).3. Under the light microscope, the colonic mucosa of the model rats showed necrosis and shedding, accompanied by varying degrees of ulcer, and a large number of inflammatory lesions. Cell infiltration, mucous glands were destroyed, the arrangement of a sparse disorder, or even the absence of a degree of expansion, compared with the blank control group, the pathological score of the rats was significantly increased (P0.01). There was no obvious ulcer formation in the colonic mucosa of the group of Ding's ulcerative enema and the mesalazine enema group, and the congestion of mucous edema and the infiltration of inflammatory cells. Less, compared with the model group, the pathological score was significantly lower (P0.01).4, the serum IgA content of the model group and the expression level of Occludin protein in the colon tissue were significantly lower than that in the blank control group (P0.05). The serum IgA content and the expression of Occludin protein in the colon tissues were increased after the treatment of the Ding ulceration enema and Mesalazine Enemas. Compared with the model group, there was a statistically significant difference (P0.05).5. The expression level of iNOSmRNA in the colon tissue of the model group was significantly higher than that in the blank control group (P0.01). After the treatment of the Ding ulceration enema and the mesalazine enema, the expression of iNOSmRNA decreased in each group, and was statistically significant (P0.05) compared with the model group (P0.05). The model group was larger than the model group. The expression level of MUC2 mRNA in the rat colonic tissue was significantly lower than that in the blank control group (P0.01). The expression level of MUC2mRNA increased after the treatment of Ding's ulcerative enema and mesalazine enema, compared with the model group (P0.05). In the mRNA expression level, the high dose group and the beauty in the Ding's ulcerative enema solution were compared. There was no obvious difference between the Shalin enema group. Conclusion: 1, Ding's ulcerative enema has a certain therapeutic effect on DSS induced UC. It can effectively alleviate the symptoms of unformed bowel, blood, weight loss and so on, improve the inflammation of the intestinal mucosa, reduce the DAI score and gross, the pathological score of.2, DSS induced UC intestinal injury, and Ding's ulceration enema The solution could significantly increase the expression of Occludin protein, MUC2 mRNA, increase the content of serum IgA, reduce the level of iNOSmRNA expression, and reduce the injury of intestinal mucositis in rats, from the intestinal mechanical barrier, immune barrier and chemical barrier.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5
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