愈溃方对TNBS诱导UC大鼠结肠屏障功能障碍的影响
发布时间：2018-07-31 10:02
【摘要】：目的:本实验旨在通过愈溃方(YKR)对三硝基苯磺酸(TNBS)诱导的溃疡性结肠炎大鼠结肠黏膜屏障功能调节的作用,通过对紧密连接蛋白Claudin-2和Claudin-7及免疫蛋白MBL和MASP-2蛋白表达情况的研究,探讨愈溃方在溃疡性结肠炎大鼠的肠道粘膜修复功能以及对肠道免疫功能调节作用。材料与方法:成年SD级雄性大鼠60只,随机分配6组,分为A.空白组、B.模型组、C.YKRH剂量组、D.YKRI剂量组、E.YKRL剂量组,F.美沙拉嗪组。除正常对照组外,B、C、D、E、F组大鼠均应用TNBS诱导SD大鼠UC发病,造模前禁食不禁水24h,灌肠前抚摸大鼠背部刺激远端大便排空,腹腔注射水合氯醛麻醉后,倒悬位,将直径0.2cm的聚乙烯管插入结肠8cm处,缓慢灌入TNBS溶液(TNBS为5%水溶液,与无水乙醇按1:1体积配制,乙醇终浓度50%),剂量为60mg/kg,灌肠后倒悬位持续1min,建模第2天发现各组模型大鼠均出现稀便及血便情况,开始灌胃给予中药及美沙拉嗪,A、B组大鼠灌胃给予0.5%羧甲基纤维素钠,给药剂量根据人体转换公式进行转换计算后C、D、E组大鼠灌胃给予不同剂量中药,中药高剂量组14mg/g/d,中药正常剂量组7mg/g/d,中药低剂量组4mg/g/d给药剂量,F组大鼠灌胃给予0.2g/kg/d的美沙拉嗪混悬液,分别溶于5ml纯净水中给予灌胃,连续给药14天,处死大鼠,对大鼠DAI、CDMI、TDI进行评分,并取结肠标本,采用免疫组化技术,对Claudin-2和Claudin-7及MBL和MASP-2蛋白表达进行检测,通过统计学软件进行分析。结果:1.YKR可以有效改善UC大鼠症状,在改善大鼠体重、隐血、大便效果上,YKR高剂量组美沙拉嗪组YKR中剂量组YKR低剂量组。TNBS诱导UC大鼠在使用YKR中药高中剂量后,大鼠DAI、CMDI、TDI评分,效果与美沙拉秦组无统计学意义,均有效降低评分。而YKR低剂量组,大鼠DAI、CMDI、TDI评分与模型组无差别,故无效;2.TNBS诱导SD级大鼠结肠粘膜损伤,相关紧密连接蛋白Claudin-2升高,Claudin-7降低进而影响肠黏膜重建,通透性增加。在使用YKR中高剂量组可使Claudin-2降低,Claudin-7升高,有效改善肠黏膜恢复,低剂量组不明显,故无效;3.TNBS诱导SD级大鼠结肠可使免疫蛋白MBL蛋白与MASP-2蛋白下降。TNBS诱导SD级大鼠在使用YKR中高剂量组可使MBL蛋白与MASP-2蛋白回升,低剂量组不明显。结论:1、高中YKR可有效改善UC大鼠病理状态,低YKR影响较低。2、高中YKR可以调整Claudin-2与Claudin-7趋正常恢复,有效恢复肠道粘膜功能低剂量无效。3、YKR高中剂量组可以有效调整肠道免疫功能,促使MBL和MASP-2趋于正常状态发展,低剂量无效。
[Abstract]:Aim: to investigate the effects of Yukui recipe (YKR) on the regulation of colonic mucosal barrier function induced by trinitrobenzene sulfonic acid (TNBS) in rats with ulcerative colitis, and to investigate the expression of tight junction protein (Claudin-2) and Claudin-7 and immunoglobulin MBL and MASP-2 in rats with ulcerative colitis. To investigate the function of Yukui recipe in repairing intestinal mucosa and regulating intestinal immune function in rats with ulcerative colitis. Materials and methods: 60 adult SD male rats were randomly assigned into 6 groups. B. C. YKRH dose group, D. YKRI dose group, E. YKRL dose group. Mesalazine group. Except for the normal control group, the rats in the control group were treated with TNBS to induce the onset of UC in SD rats. The rats fasted for 24 hours before the model was made. The distal defecation was stimulated by caressing rats before enema. After intraperitoneal injection of chloral hydrate anesthesia, the rats were suspended upside down. The polyethylene tube with diameter 0.2cm was inserted into the colon 8cm, and the TNBS solution (TNBS was 5% aqueous solution) was infused slowly into the colon, and the TNBS solution was prepared with anhydrous ethanol at 1:1 volume. The final concentration of ethanol was 50%), the dose was 60 mg / kg, the suspension lasted 1 min after enema. On the second day of modeling, it was found that the rats in each group had dilute stool and blood stool, and the rats in group B were given 0.5% sodium carboxymethyl cellulose. After the dose was calculated according to the conversion formula of human body, the rats in group C were given different doses of traditional Chinese medicine by intragastric perfusion. 14 mg / g / d in high dose group, 7 mg / g / d in normal dose group, and intragastric administration of mesalazine suspension of 0.2g/kg/d in low dose 4mg/g/d group and F group, respectively. The rats were given intragastric administration of mesalazine in purified water of 5ml for 14 days, and the rats were killed. The expression of Claudin-2, Claudin-7, MBL and MASP-2 were detected by immunohistochemical technique. Results 1. YKR could effectively improve the symptoms of UC rats. In improving the weight, occult blood and stool of UC rats, YKR high dose group, mesalazine group, middle dose group, YKR low dose group. TNBS induced UC rats to score CMDIDI score after the high dose of YKR. There was no significant difference between the effect and the mesaladine group, and the scores were significantly decreased. However, in the low dose group of YKR, there was no difference between the model group and the model group. 2. TNBS-induced colonic mucosal injury in SD rats, and the increase of Claudin-2 related to tight junction protein decreased Claudin-7, which affected the intestinal mucosal remodeling and permeability. 2. In the middle and high dose group of YKR, the increase of Claudin-7 in Claudin-2 was decreased, and the recovery of intestinal mucosa was improved effectively, but the low dose group was not obvious. MBL protein and MASP-2 protein were decreased in SD rats colon induced by TNBS. MBL protein and MASP-2 protein were increased in middle and high dose group of SD rats induced by TNBS, but not in low dose group. Conclusion: high school YKR can effectively improve the pathological state of UC rats, and the effect of low YKR is low. High school YKR can adjust the normal recovery of Claudin-2 and Claudin-7, and effectively restore intestinal mucosal function. 3YKR high dose group can effectively adjust the intestinal immune function. Promote the development of MBL and MASP-2 in normal state, low dose is not effective.
【学位授予单位】：辽宁中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5
本文编号：2155235
[Abstract]:Aim: to investigate the effects of Yukui recipe (YKR) on the regulation of colonic mucosal barrier function induced by trinitrobenzene sulfonic acid (TNBS) in rats with ulcerative colitis, and to investigate the expression of tight junction protein (Claudin-2) and Claudin-7 and immunoglobulin MBL and MASP-2 in rats with ulcerative colitis. To investigate the function of Yukui recipe in repairing intestinal mucosa and regulating intestinal immune function in rats with ulcerative colitis. Materials and methods: 60 adult SD male rats were randomly assigned into 6 groups. B. C. YKRH dose group, D. YKRI dose group, E. YKRL dose group. Mesalazine group. Except for the normal control group, the rats in the control group were treated with TNBS to induce the onset of UC in SD rats. The rats fasted for 24 hours before the model was made. The distal defecation was stimulated by caressing rats before enema. After intraperitoneal injection of chloral hydrate anesthesia, the rats were suspended upside down. The polyethylene tube with diameter 0.2cm was inserted into the colon 8cm, and the TNBS solution (TNBS was 5% aqueous solution) was infused slowly into the colon, and the TNBS solution was prepared with anhydrous ethanol at 1:1 volume. The final concentration of ethanol was 50%), the dose was 60 mg / kg, the suspension lasted 1 min after enema. On the second day of modeling, it was found that the rats in each group had dilute stool and blood stool, and the rats in group B were given 0.5% sodium carboxymethyl cellulose. After the dose was calculated according to the conversion formula of human body, the rats in group C were given different doses of traditional Chinese medicine by intragastric perfusion. 14 mg / g / d in high dose group, 7 mg / g / d in normal dose group, and intragastric administration of mesalazine suspension of 0.2g/kg/d in low dose 4mg/g/d group and F group, respectively. The rats were given intragastric administration of mesalazine in purified water of 5ml for 14 days, and the rats were killed. The expression of Claudin-2, Claudin-7, MBL and MASP-2 were detected by immunohistochemical technique. Results 1. YKR could effectively improve the symptoms of UC rats. In improving the weight, occult blood and stool of UC rats, YKR high dose group, mesalazine group, middle dose group, YKR low dose group. TNBS induced UC rats to score CMDIDI score after the high dose of YKR. There was no significant difference between the effect and the mesaladine group, and the scores were significantly decreased. However, in the low dose group of YKR, there was no difference between the model group and the model group. 2. TNBS-induced colonic mucosal injury in SD rats, and the increase of Claudin-2 related to tight junction protein decreased Claudin-7, which affected the intestinal mucosal remodeling and permeability. 2. In the middle and high dose group of YKR, the increase of Claudin-7 in Claudin-2 was decreased, and the recovery of intestinal mucosa was improved effectively, but the low dose group was not obvious. MBL protein and MASP-2 protein were decreased in SD rats colon induced by TNBS. MBL protein and MASP-2 protein were increased in middle and high dose group of SD rats induced by TNBS, but not in low dose group. Conclusion: high school YKR can effectively improve the pathological state of UC rats, and the effect of low YKR is low. High school YKR can adjust the normal recovery of Claudin-2 and Claudin-7, and effectively restore intestinal mucosal function. 3YKR high dose group can effectively adjust the intestinal immune function. Promote the development of MBL and MASP-2 in normal state, low dose is not effective.
【学位授予单位】：辽宁中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5
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