GLP-1抑制mTOR通路激活自噬改善糖尿病肾脏病作用机制的研究

发布时间：2018-05-26 22:34

本文选题：GLP-1 + mTOR　；参考：《南方医科大学》2017年硕士论文

【摘要】：研究背景:我国糖尿病人群超过1亿,已成为我国重大公共卫生问题,其所致的慢性并发症所引起的社会和家庭负担与日俱增。糖尿病肾脏病作为糖尿病所引起的微血管病变之一,其危害性极大,目前已成为终末期肾脏病的首要原因。终末期肾脏病的患者需要长期接受肾脏替代治疗,不仅造成沉重的家庭、社会和经济负担,且患者生活质量及长期生存率显著下降。最新研究显示,糖尿病肾脏病人群中,60.5%的患者处于糖尿病肾脏病早期阶段,为伴肾功能保留型的轻度蛋白尿增加。如何采用有效的手段早期干预肾病,阻断慢性肾脏病进展至终末期肾脏病,是目前亟需解决的问题。GLP-1作为糖尿病领域备受关注的肠道激素,可通过激活G蛋白偶联受体,在多个组织器官水平调控自噬,降低活性氧簇。既往已有报道:高糖血症发生时,肾小管上皮细胞自噬被抑制,导致细胞器及细胞内稳态受到损伤及破坏,最终造成肾功能障碍,适度激活自噬可抑制氧化应激造成的肾小管上皮细胞损伤。基于上述理论基础,我们推测机体内存在一种自噬保护机制改善糖尿病肾脏病损伤,然而GLP-1是否也通过此机制而发挥其保护作用尚未见文献报道。目的:1.探索GLP-1干预对2型糖尿病大鼠一般情况、糖脂代谢及肾功能的作用。2.探索GLP-1抑制mTOR信号通路激活自噬改善糖尿病肾脏病作用机制的研究。方法:选择8周龄的ZDF大鼠,适应性喂养2周,予高脂饲料饲养诱导为2型糖尿病大鼠模型。随后随机分为:1)非糖尿病对照组;2)糖尿病对照组;3)胰岛素治疗组;4)沙格列汀治疗组;5)利拉鲁肽治疗组。予高脂饲料喂养9周并予相对应的处理。实验期间每周监测进食量、随机血糖和体重,实验结束前测糖化血红蛋白了解近期血糖控制水平,行口服葡萄糖耐量实验以评估胰岛功能情况,并收集24h尿液检测尿微量白蛋白水平。大鼠处死后,收集血清测血脂、血尿素、血肌酐和胱抑素C等指标:肾脏组织,行HE染色检测,观察肾组织形态学改变;行PAS染色,观察肾脏组织基底膜、系膜基质形态;行Masson染色,观察肾组织纤维化情况;免疫组化检测自噬相关蛋白LC3B和P62表达情况;Western Blot法检肾脏组织mTOR通路及自噬相关蛋白表达水平;Quantitation RT-PCR法检测mTOR和Beclin-1 mRNA水平;试剂盒检测肾脏组织的自由基清除物SOD活力和GSH水平。结果:1.2型糖尿病大鼠病理结果提示,肾小球明显增生肥大、肾小球基底膜弥漫增厚、系膜细胞轻度增生及系膜基质增多,且发生明显的纤维化。2.与糖尿病对照组相比,沙格列汀治疗组和利拉鲁肽治疗组可以改善2型糖尿病大鼠的进食量、血糖、体重、血脂、肾功能及光镜下观察到的糖尿病肾脏病大鼠病理变化,且利拉鲁肽治疗组治疗效果更为明显。3.在同等降糖水平情况下,利拉鲁肽治疗组在改善ZDF大鼠肾功能指标方面并不亚于胰岛素治疗组,特别是减少24小时尿微量白蛋白要明显优于胰岛素治疗组。4.与糖尿病对照组相比,利拉鲁肽治疗组肾组织的pS6K、pS6和P62蛋白的表达水平明显减少,而肾组织Beclin-1 mRNA的表达水平明显升高。5.与糖尿病对照组相比,利拉鲁肽可增加肾组织的自由基清除物GSH水平,减少氧化应激。结论:研究表明GLP-1在降糖同时,可明显改善2型糖尿病大鼠的肾功能损伤,延缓糖尿病肾脏病的进展。这一肾功能保护作用可能与GLP-1抑制mTOR通路激活自噬,减少氧化应激有关。并发现利拉鲁肽改善2型糖尿病大鼠的内分泌代谢紊乱及对肾脏的保护作用要优于沙格列汀。
[Abstract]:Background: more than 100 million diabetic people in China have become a major public health problem in China. The social and family burdens caused by chronic complications are increasing. As one of the microvascular diseases caused by diabetes, diabetic nephropathy is very harmful, and it has become the primary cause of end-stage renal disease. Patients with renal disease need long-term renal replacement therapy, which not only causes a heavy family, social and economic burden, but also a significant decline in the quality of life and long-term survival of the patients. The latest study shows that in the diabetic group, 60.5% of the patients are in the early stage of diabetic renal disease, a mild protein with the retention of renal function. Urine increase. How to use effective means to intervene early kidney disease, block chronic renal disease progression to end-stage renal disease, it is an urgent problem to be solved.GLP-1 as an enteric hormone in the field of diabetes. By activating G protein coupling receptor, regulating autophagy at multiple tissue and organ levels and reducing active oxygen cluster. In the case of hyperglycemia, the autophagy of the renal tubular epithelial cells is inhibited, which leads to the damage and destruction of the organelle and intracellular homeostasis, eventually causing renal dysfunction. Moderate activation of autophagy can inhibit the injury of renal tubular epithelial cells caused by oxidative stress. Based on the theoretical basis, we speculate that the body is in a mechanism of autophagy protection. Improvement of diabetic renal disease damage, however, whether GLP-1 also plays its protective role has not yet been reported. Objective: 1. explore the effect of GLP-1 intervention on the general situation of type 2 diabetic rats, glycolipid metabolism and renal function.2. explore GLP-1 inhibition of mTOR signaling pathway to activate autophagy to improve the mechanism of diabetic renal disease Methods: 8 weeks old ZDF rats were selected and fed for 2 weeks, and high fat feed was given to type 2 diabetic rats. Then, they were randomly divided into 1) non diabetic control group, 2 diabetes control group, 3) insulin treatment group; 4) Shah Glenn Dean treatment group; 5) lilacin treatment group. The high fat diet was fed for 9 weeks and the corresponding treatment was given. During the period, we monitored the food intake, random blood sugar and weight. Before the end of the experiment, the glycated hemoglobin was measured to understand the recent blood glucose control level. The oral glucose tolerance test was used to assess the islet function, and the urine microalbumin level was collected by 24h urine test. After the rats were killed, the blood serum was collected to measure blood lipids, blood urea, serum creatinine and cystatin C, etc. Indexes: renal tissue, HE staining was performed to observe the morphological changes of renal tissue; PAS staining was used to observe the basal membrane of the renal tissue and the morphology of the mesangial matrix; Masson staining was used to observe the fibrosis of the renal tissue; the expression of autophagy related protein LC3B and P62 was detected by immunohistochemistry; the mTOR pathway and autophagy related protein table of renal tissue were detected by Western Blot method. The level of mTOR and Beclin-1 mRNA was detected by Quantitation RT-PCR; the kit was used to detect the SOD activity and GSH level of the free radical scavengers of the kidney tissue. Results: the pathological results of type 1.2 diabetic rats showed that the glomerular hyperplasia hypertrophy, the diffuse thickening of the glomerular basement membrane, the slight hyperplasia of mesangial cells and the increase of the mesangial matrix were observed. Compared with the diabetes control group, the Shah Glenn Dean treatment group and the llllexin treatment group could improve the diet, blood sugar, weight, blood lipid, renal function and light microscope observed in the pathological changes of diabetic rats with type 2 diabetes, and the treatment effect of lllexin treatment group was more obvious.3. at the same level of hypoglycemic control in 2 diabetic rats. In the case of the Liraru peptide treatment group, the renal function index of the ZDF rats was no less than the insulin treatment group, especially the reduction of 24 hours urine microalbuminuria was significantly better than the insulin treatment group.4. and the diabetic control group. The expression level of pS6K, pS6 and P62 protein in the renal tissue of the Liraru peptide group was significantly reduced, and the renal tissue was significantly reduced. The expression level of Beclin-1 mRNA is significantly higher than that of.5. in the diabetic control group, which can increase the level of free radical scavengers GSH in renal tissue and reduce oxidative stress. Conclusion: the study shows that GLP-1 can obviously improve renal function damage in type 2 diabetic rats and delay the progression of diabetic renal disease. The effect may be associated with GLP-1 inhibition of mTOR pathway to activate autophagy and reduce oxidative stress. It is also found that the improvement of the endocrine and metabolic disorders in type 2 diabetic rats and the protection of the kidneys is better than that of soglitetine.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R692.9

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