痛泻二草方对肝郁脾虚型溃疡性结肠炎模型大鼠NF-κB通路相关分子表达影响的研究

发布时间：2018-06-01 07:20

本文选题：痛泻二草方 + 溃疡性结肠炎　；参考：《甘肃中医学院》2014年硕士论文

【摘要】：目的：本课题采用束缚刺激加三硝基苯磺酸（TNBS）和乙醇混合灌肠的病证结合方式建立肝郁脾虚型溃疡性结肠炎（Ulcerative colitis，UC）大鼠模型，观察痛泻二草方（TXECF）对肝郁脾虚型UC大鼠结肠组织病理学的影响及NF-κB信号通路相关基因和蛋白的影响，从而阐明TXECF治疗肝郁脾虚型UC的作用机理和治疗靶点。方法：将90只SPF级Wistar大鼠，根据体重及雌雄随机分为空白组、模型组、TXECF高剂量组、TXECF中剂量组、TXECF低剂量组、阳性组（SASP组），每组15只。空白组给予蒸馏水等体积灌肠，其余各组懫用束缚刺激加TNBS和乙醇混合灌肠的病证结合法建立肝郁脾虚型UC大鼠模型，动物模型制备成功后，中药各治疗组给予相应浓度的TXECF灌胃，阳性组给予柳氮磺胺吡啶（SASP）灌胃，空白组及模型组给予等体积的蒸馏水灌胃，每日1次，连续21d。各组大鼠末次灌胃结束后禁食不禁水24h，水合氯醛麻醉，剖开整个结肠组织，先用肉眼仔细观察各组大鼠结肠黏膜的充血、水肿、溃疡和糜烂数目及面积病理改变情况，然后取少量结肠组织石蜡包埋，进行HE染色，光镜下观察各组结肠组织病理形态变化。剩余的结肠组织标本用于PCR-Array及RT-PCR和蛋白免疫印迹(Western blot)等技术检测。结果：1.与空白组比较，模型组及各治疗组的结肠黏膜在肉眼观察下存在一定程度的充血、水肿、溃疡及糜烂；光镜下观察也存在不同程度的溃疡、炎症及并发的肉芽和纤维化增生。给予痛泻二草方治疗后与模型组比较，各组大鼠的结肠黏膜损伤评分和病理切片评分均降低，差异显著（P0.01或P0.05）。 2.与空白组比较，模型组结肠组织中Nfkb1、Nfrkb、Ikbkb基因表达量均升高，差异有统计学意义（P0.01）。给予痛泻二草方治疗后Nfkb1、Nfrkb、Ikbkb基因表达量与模型组比较均有所降低（P0.01或P0.05），其中高剂量组各基因的表达量差异显著（P0.01）。痛泻二草方高剂量组与SASP治疗组比较，Nfkb1、Nfrkb表达差异无统计学意义，Ikbkb表达差异有统计学意义（P0.05）。 3.与空白组比较，模型组结肠组织中NF-κB-P65、IκB-α、IκB-β蛋白相对表达水平增高，差异显著（P0.01）。给予痛泻二草方治疗后NF-κB-P65、IκB-α、IκB-β蛋白表达水平降低（P0.01或P0.05），且高剂量组各蛋白表达量降低差异显著（P0.01）。痛泻二草方高剂量组与SASP治疗组比较各蛋白表达差异均有统计学意义（P0.01或P0.05）。结论：1.痛泻二草方可以抑制肝郁脾虚型UC病程中炎症细胞浸润，减轻炎症反应，对肠黏膜的损伤修复有促进作用，，能够使肠黏膜分泌功能恢复正常。说明本研究肝郁脾虚型UC模型复建成功，且痛泻二草方可以通过减轻炎症反应，修复肠黏膜，进而起到治疗肝郁脾虚型UC的作用。 2.肝郁脾虚型UC结肠黏膜Nfkb1、Nfrkb、Ikbkb的表达水平能够反映结肠黏膜的损伤程度，对病情的评估及治疗效果的判断可能具有潜在指导意义。 3.痛泻二草方的治疗作用可能与下调Nfkb1、Nfrkb、Ikbkb基因的表达水平，进而减轻黏膜炎症反应，起到修复黏膜的作用有关。提示Nfkb1、Nfrkb、Ikbkb基因位点可能是痛泻二草方治疗肝郁脾虚型UC的关键靶位。 4.痛泻二草方对肝郁脾虚型UC大鼠结肠黏膜NF-κB-P65、IκB-α、IκB-β蛋白的表达具有下调作用，进而可以抑制炎症的发生发展，促进修复损伤的黏膜。提示痛泻二草方对肝郁脾虚型UC的治疗作用与下调NF-κB-P65、IκB-α及IκB-β蛋白表达水平有关。
[Abstract]:Objective: to establish a rat model of Ulcerative colitis (UC) with liver depression and spleen deficiency type ulcerative colitis (UC) by combination of binding stimulation plus three nitrobenzene sulfonic acid (TNBS) and ethanol mixed enema, and observe the effect of TXECF on the histopathology of intestinal histology in UC rats with liver depression and spleen deficiency and the genes related to NF- kappa B signaling pathway Thus, we can elucidate the mechanism and therapeutic targets of TXECF in treating UC of liver depression and spleen deficiency.
Methods: 90 SPF grade Wistar rats were divided into blank group, model group, TXECF high dose group, middle dose group, TXECF low dose group, TXECF low dose group, positive group (SASP group), 15 rats in each group. The blank group was treated with distilled water and equal volume enema. The rest groups were combined with the combination of TNBS and ethanol mixed enema with the combination of TNBS and ethanol enema. After the success of the model of UC rats with spleen deficiency and spleen deficiency, the treatment group was given the corresponding concentration of TXECF. The positive group was given the gavage of sulfasalazine (SASP). The blank group and the model group were given equal volume of distilled water to the stomach, 1 times a day. After the last time of the end of the last perfusion of the 21d. rats, the fasting was 24h, and chloral hydrate anaesthetized with hydrated. The whole colonic tissue was opened, and the colonic mucosa in each group was carefully observed with the naked eye. The number and area of the colonic mucosa were carefully observed. Then a small amount of colonic tissue was embedded in paraffin and stained with HE. The pathological changes of the colon tissue were observed under light microscope. The remaining colonic tissue specimens were used for PCR-Array and RT-PCR. And protein immunoblot (Western blot) technology detection.
Results: 1. compared with the blank group, there was a certain degree of congestion, edema, ulcers and erosions in the colonic mucosa of the model group and the treatment groups. Under the light microscope, there were different degrees of ulcers, inflammation and complicated granuloma and fibrotic hyperplasia. After the treatment of the two herbs of pain and diarrhea, the colon was compared with the model group, and the colon of the rats in each group Mucosal injury score and pathological section score decreased significantly (P0.01 or P0.05).
2. compared with the blank group, the expression of Nfkb1, Nfrkb and Ikbkb in the colon tissue of the model group increased, and the difference was statistically significant (P0.01). The expression of Nfkb1, Nfrkb, Ikbkb in the group of Nfkb1, Nfrkb, Ikbkb decreased (P0.01 or P0.05), and the expression of each gene in the middle high dose group was significantly different (P0.01). Two of the pain diarrhea. There was no significant difference in the expression of Nfkb1 and Nfrkb between the high dose group and the SASP group. The difference of Ikbkb expression was statistically significant (P0.05).
3. compared with the blank group, the relative expression level of NF- kappa B-P65, I kappa B- alpha and I kappa B- beta protein in the colon tissue of the model group was increased significantly (P0.01). The expression level of NF- kappa B-P65, I kappa B- alpha, I kappa protein expression level decreased after the treatment of pain diarrhea two grass prescription, and the expression of protein expression in the high dose group decreased significantly. Compared with the SASP treatment group, the difference of protein expression was statistically significant (P0.01 or P0.05).
Conclusion: 1. pain and diarrhea two grass can inhibit the infiltration of inflammatory cells in the course of liver depression and spleen deficiency of UC, alleviate the inflammatory reaction, promote the repair of intestinal mucosa damage and restore the secretion function of the intestinal mucosa to normal. It shows that the liver depression and spleen deficiency type UC model is successfully rebuilt, and the pain and diarrhea two grass can repair the intestine by alleviating the inflammatory reaction and repairing intestines. Mucosa plays a role in the treatment of UC of liver depression and spleen deficiency.
2. the expression level of Nfkb1, Nfrkb, Ikbkb in the UC colon mucosa of the liver depression and spleen deficiency can reflect the damage degree of the colonic mucosa, and the evaluation of the disease and the judgment of the therapeutic effect may be of potential guiding significance.
3. the therapeutic effect of Tong Xie two grass recipe may be related to the reduction of the expression level of Nfkb1, Nfrkb, Ikbkb gene and the mucosal inflammation, which may play a role in the repair of mucous membrane. It is suggested that the Nfkb1, Nfrkb, and Ikbkb gene loci may be the key target for the treatment of the liver depression and spleen deficiency type UC.
4. pain and diarrhea two grass recipe has a down-regulation effect on the expression of NF- kappa B-P65, I kappa B- alpha and I kappa B- beta protein in the colon mucosa of UC rats with liver depression and spleen deficiency, which can inhibit the occurrence and development of inflammation and promote the repair of damaged mucosa. It suggests that the expression level of NF- kappa B-P65, I kappa B- alpha and kappa beta protein in the treatment of the liver depression and spleen deficiency type UC by the pain diarrhea two herb prescription. Close.
【学位授予单位】：甘肃中医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R259;R285.5

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