N-乙酰氨基葡萄糖对5-FU所致大鼠肠粘膜屏障损伤的保护及机制初探

发布时间：2018-06-28 21:58

本文选题：化疗 + 5-FU　；参考：《第三军医大学》2012年硕士论文

【摘要】：目的：本研究旨在探索肠粘膜屏障的保护方法。肠粘膜屏障是机体内外联系的重要界面，包括化疗在内的多种应激因素可致肠粘膜屏障损坏。化疗药物在杀灭肿瘤细胞的同时，对消化道粘膜上皮组织细胞产生毒性。导致肠粘膜萎缩、绒毛变短肠通透性增加、肠局部免疫功能受损以及肠菌群失调等。化疗对肠粘膜屏障的这些损伤可导致患者恶心、呕吐、腹泻，严重则会出现粘膜坏死、脱落、便血及全身不良反应，甚至导致病情恶化，危及生命。因此，在化疗时保护肠粘膜屏障结构完整和功能正常是防治化疗不良反应的重要课题。氨基葡萄糖(glucosamine, GlcN)是在2位碳上氨基取代的葡萄糖，N-乙酰氨基葡萄糖(N-Acetyl-D-glucosamine, GlcNAc)则是GlcN的氨基上发生乙酰化的衍生物，分子量小，水溶性好，具有抗氧化、免疫调节作用；能有效改善肠粘膜局部生态环境、提高上皮细胞活性、能进入细胞发挥蛋白的糖基化修饰等作用，为此，本研究观察GlcNAc对5-FU引起的肠粘膜结构与功能损伤的保护作用，并探索其作用机理，为化疗期间肠粘膜屏障受损新的防治方法研究奠定实验基础。材料与方法： 1．动物的分组与模型建立：SPF级SD雄性大鼠50只，体重190~210g，随机分为空白对照组、5-FU模型组、Gln组、GlcN组和GlcNAc组(n=10)。采用5-FU腹腔注射方法制备大鼠肠粘膜屏障损伤模型，按分组分别给药，连续6d； 2．实验过程中，密切检测大鼠的体重变化；所有实验动物每天称量体重，并以此为依据调整给药剂量； 3．采用HE染色法，，光镜下观察大鼠小肠粘膜组织病理形态学改变；通过透射电镜，观察小肠粘膜上皮细胞和细胞连接超微结构改变； 4．采用分光光度法检测大鼠血浆中D-乳酸的含量；采用ELISA法检测大鼠血浆中DAO的含量； 5．Western blot法检测大鼠小肠组织中O-GlcNAc蛋白的表达。结果： 1.GlcNAc对大鼠体重变化的影响及解剖时大鼠小肠外观观察: 与实验前相比，空白对照组、Gln组和GlcNAc组大鼠体重增加，而5-FU模型组和GlcN组大鼠体重减轻。解剖可见，空白对照组和GlcNAc组大鼠小肠无明显变化，5-FU组、GlcN和Gln组不同程度存在肠胀气、肠粘膜充血以及腹泻。 2.GlcNAc对大鼠小肠组织病理形态学改变的影响: HE染色高倍镜观察显示：空白对照组大鼠小肠肠壁结构清晰完整，粘膜绒毛较细长，发达，排列规则紧密；5-FU模型组和GlcN组损伤明显；GlcNAc组和Gln组大鼠小肠粘膜有轻度损伤表现。 3.GlcNAc对大鼠小肠组织超微结构变化的影响透射电镜观察显示：5-FU细胞连接结构局部出现异常；GlcNAc组大鼠小肠上皮细胞紧密连接结构与空白对照组相似。 4.GlcNAc对大鼠血浆中D-乳酸含量的影响 GlcNAc组大鼠D-乳酸的含量较5-FU模型组和GlcN组显著降低(p0.01)，分别降低了31.5%和33.7%。而GlcN组与5-FU模型组相比较没有统计学差异(p0.05)。 5.GlcNAc对大鼠血浆中DAO含量的影响与模型组和GlcN组相比较，Gln组和GlcNAc组大鼠DAO的含量明显降低(p0.01)；而GlcN组与5-FU模型组相比较没有统计学差异(p0.05)。 6.GlcNAc对大鼠小肠组织中O-GlcNAc蛋白表达的影响与空白对照组相比，5-FU模型组和GlcN组大鼠小肠组织中O-G1cNAc蛋白水平明显降低(p0.01)；与模型组和GlcN组相比较，Gln组和GlcNAc组大鼠O-G1cNAc蛋白水平明显增高(p0.01)；而GlcN组与5-FU模型组相比较没有统计学差异(p0.05)。结论： 1．N-乙酰氨基葡萄糖能减轻化疗所致大鼠肠粘膜损伤，降低肠粘膜通透性，保护小肠粘膜及其屏障功能； 2． N-乙酰氨基葡萄糖保护小肠粘膜及其屏障功能可能与上调小肠组织中O-GlcNAc蛋白表达有关。
[Abstract]:Objective:
The purpose of this study is to explore the protection of intestinal mucosal barrier. The intestinal mucosal barrier is an important interface between the internal and external contact of the body, and a variety of stress factors, including chemotherapy, can cause damage to the intestinal mucosal barrier. The chemotherapeutic drugs are toxic to the epithelial tissue cells of the digestive tract mucosa while killing the tumor cells. The intestinal mucosa atrophy and the villi become shorter. Intestinal mucosal permeability increases, intestinal immune function is damaged, and intestinal flora imbalance. These injuries to the intestinal mucosal barrier can cause nausea, vomiting, diarrhea, and severe mucosal necrosis, exfoliation, blood and systemic adverse reactions, even exacerbation of life. Therefore, protective intestinal mucosal barrier structure is protected during chemotherapy. The normal function of integrin is an important topic in the prevention and treatment of adverse reactions of chemotherapy.
Glucosamine (glucosamine, GlcN) is an amino - substituted glucose in 2 bits of carbon, and N- acetyl glucosamine (N-Acetyl-D-glucosamine, GlcNAc) is a derivative of acetylation on the amino group of GlcN, with small molecular weight, good water solubility, antioxidant and immune modulation; it can effectively improve the local ecological environment of the intestinal mucosa and improve the epithelium. In this study, the protective effect of GlcNAc on the structural and functional damage of intestinal mucosa caused by 5-FU was observed, and the mechanism of its action was explored, which lay the foundation for the study of new methods for preventing and controlling intestinal mucosal barrier during chemotherapy.
Materials and methods:
1. group and model of animals: 50 SPF male SD male rats, weight 190~210g, were randomly divided into blank control group, 5-FU model group, Gln group, GlcN group and GlcNAc group (n=10). The intestinal mucosal barrier damage model of rats was prepared by 5-FU intraperitoneal injection.
2. during the experiment, the weight changes of rats were closely monitored; all the experimental animals weighed daily and adjusted the dosage according to this.
3. HE staining was used to observe the pathological changes of the intestinal mucosa of the rats under light microscope, and the ultrastructural changes of the epithelial cells and cells of the small intestinal mucosa were observed by transmission electron microscopy.
4. the content of D- lactate in rat plasma was detected by spectrophotometry and the content of DAO in plasma was detected by ELISA.
5.Western blot was used to detect the expression of O-GlcNAc protein in the small intestine of rats.
Result:
Effects of 1.GlcNAc on body weight and the appearance of small intestine in rats during dissection:
Compared with before the experiment, the weight of the blank control group, the Gln group and the GlcNAc group increased, while the weight of the 5-FU model group and the group GlcN decreased, and the small intestine of the blank control group and the GlcNAc group had no obvious changes. In group 5-FU, there were flatulence, intestinal mucous membrane congestion and diarrhea in the GlcN and Gln groups.
Effects of 2.GlcNAc on the pathomorphological changes of small intestine in rats:
The observation of HE staining with high magnification showed that the intestinal wall structure of the small intestine in the blank control group was clear and complete, the mucous villi were long, developed, and the arrangement of the intestinal mucosa was close, the 5-FU model group and the GlcN group were injured obviously, and the small intestinal mucosa of the rats of group GlcNAc and Gln group had mild damage.
Effect of 3.GlcNAc on ultrastructural changes of small intestine in rats
Transmission electron microscopy showed that 5-FU cells were abnormal in connection structure. The tight junction structure of intestinal epithelial cells in GlcNAc group was similar to that in blank control group.
Effect of 4.GlcNAc on the content of D- lactate in rat plasma
The content of D- lactate in group GlcNAc rats was significantly lower than that in 5-FU model group and GlcN group (P0.01), which decreased by 31.5% and 33.7%. respectively, while there was no statistical difference between the GlcN group and the 5-FU model group (P0.05).
The effect of 5.GlcNAc on the content of DAO in rat plasma
Compared with the model group and the GlcN group, the content of DAO in the Gln group and the GlcNAc group decreased significantly (P0.01), but there was no statistical difference between the GlcN group and the 5-FU model group (P0.05).
Effect of 6.GlcNAc on O-GlcNAc protein expression in rat small intestine
Compared with the blank control group, the O-G1cNAc protein level in the small intestine of the 5-FU model group and the GlcN group was significantly lower (P0.01). Compared with the model group and the GlcN group, the level of O-G1cNAc protein in the Gln and GlcNAc groups increased significantly (P0.01), but there was no statistical difference between the GlcN group and the 5-FU model group (P0.05).
Conclusion:
1.N- acetylglucosamine can alleviate intestinal mucosal injury induced by chemotherapy, reduce intestinal permeability and protect intestinal mucosa and barrier function.
The protective effect of 2.N- acetylglucosamine on intestinal mucosal and barrier function may be related to the up regulation of O-GlcNAc protein in the small intestine.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

【参考文献】