艾塞那肽对高果糖诱导胰岛素抵抗大鼠肾脏纤维化的影响

发布时间：2018-05-25 08:48

本文选题：胰岛素抵抗 + 艾塞那肽　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:当今人民生活水平不断提高,伴随而来的是食品生产和饮食结构的变化,致使果糖的摄入量逐年增加,高果糖饮食与代谢综合征的发生密切相关,胰岛素抵抗和高胰岛素血症是其共同的病理基础,是心、脑血管病变以及糖尿病发生的危险因素并和慢性肾脏病(Chronic kidney disease,CKD)高度相关。胰高血糖素样肽1(Glucagon like peptide-1,GLP-1)类似物是一种新型的降糖药物,可促进胰岛素的生物合成和分泌,保护胰岛β细胞,改善胰岛素抵抗,抑制胰高血糖素的分泌,抑制食欲及摄食,延缓胃内容物排空,从而降低血糖并使之维持在恒定水平。但GLP-1对胰岛素抵抗大鼠肾脏疾病的作用尚不明确。本研究首先建立了高果糖诱导的胰岛素抵抗大鼠模型,其后探讨了艾塞那肽对高果糖诱导胰岛素抵抗大鼠肾脏组织的作用及其可能机制,为临床用药提供理论依据。方法:雄性Wistar大鼠30只(体重180g-200g),适应性喂养1周后,随机分为2组:正常对照组(Con)12只和高果糖饮食组(HF)18只。正常对照组予普通饲料进行喂养,高果糖组予等热量的高果糖饲料进行喂养。喂养8周后每组随机抽取6只大鼠,进行高胰岛素-正葡萄糖钳夹实验,通过计算葡萄糖输注率(Glucose infusion rate,GIR)来评估其胰岛素敏感性,随后处死大鼠并留取血液及肾脏组织标本。8周高果糖诱导胰岛素抵抗大鼠造模成功后,将剩余高果糖组大鼠随机分为2组:高果糖模型组(HF)、艾塞那肽治疗组(HFG),继续进行高果糖喂养并给予相应药物干预4周。药物干预4周后,行高胰岛素-正葡萄糖钳夹试验,计算GIR后处死大鼠,留取血清及肾脏标本,测空腹血糖(Fasting blood glucose,FBG)、空腹血胰岛素(Fasting insulin,FINS)、总胆固醇(Total cholesterol,TC)、甘油三酯(Triglyceride,TG)、肌酐(Creatinine,Cr)、尿素氮(Blood urea nitrogen,BUN)、尿酸(Uric acid,UA),应用Western Blot检测肾脏组织中转化生长因子β1(Transforming growth factor beta 1,TGF-β1)、骨形态发生蛋白7(Bone morphogenetic protein 7,BMP-7)、Smad4、结缔组织生长因子(Connective tissue growth factor,CTGF)、Toll样受体4(Toll-like receptor4,TLR-4)、核转录因子(Nuclear factor kappa B,NF-κB)及p38丝裂原激活蛋白激酶(p38 Mitogen activated protein kinase,p38 MAPK)的蛋白表达水平,ELISA试剂盒测肾组织骨形态发生蛋白7(Bone morphogenetic protein 7,BMP-7)蛋白水平,HE染色、电镜观察肾脏形态变化。结果:1高果糖喂养8周后,各项指标变化:1.1体重两组大鼠试验初体重无明显差异,喂养8周后,高果糖组大鼠的体重稍高于正常对照组,两组间无统计学差异(P0.05)。1.2血清生化指标与正常对照组相比,高果糖组大鼠FINS、BUN、Cr、TC、TG、UA均显著提高,差异均有统计学意义(P0.05),但两组间FBG无统计学差异(P0.05)。1.3高胰岛素-正葡萄糖钳夹实验高果糖组大鼠的葡萄糖输注率明显低于正常对照组,差异有统计学意义(P0.05)。2药物干预4周后,3组大鼠各项指标变化:2.1体重药物干预4周后,高果糖组大鼠体重稍高于正常对照组与艾塞那肽组,各组间无统计学差异(P0.05)。2.2血清生化指标与正常对照组相比,高果糖组大鼠FINS、BUN、Cr、TC、TG、UA均显著增高,差异均有统计学意义(P0.05)。艾塞那肽组各项指标较正常组高,差异有统计学意义(P0.05),与高果糖组相比明显降低,差异有统计学意义(P0.05),但三组间FBG无统计学差异(P0.05)。2.3血清骨形态蛋白-7与正常对照组相比,高果糖组大鼠血清中骨形态蛋白-7含量显著降低,差异有统计学意义(P0.05)。艾塞那肽干预组较正常组低,差异有统计学意义(P0.05),较高果糖组明显增高,差异有统计学意义(P0.05)。2.4肾脏HE染色经HE染色后在光镜高倍镜(×400)下观察两组大鼠肾组织切片可见,正常对照组肾小球及肾小管结构清晰可见,未见明显病理改变。高果糖组肾小管上皮细胞肿胀增生,肾小球体积缩小,基底膜增厚,系膜区增宽,系膜细胞增生,硬化明显。艾塞那肽组的上述异常明显改善。2.5透射电镜观察肾组织经过4周药物干预后,通过透射电子显微镜(×15000)观察各组大鼠肾组织,发现正常对照组肾小球毛细血管基底膜均匀一致,足细胞足突排列整齐有序。高果糖组可见肾小球毛细血管基底膜不均匀性增厚,足细胞足突广泛融合甚至消失,系膜细胞明显增生,系膜区明显增宽。艾塞那肽组肾小球结构异常较高果糖组明显改善。2.6肾脏纤维化指标与正常对照组相比,高果糖组大鼠肾脏组织TGF-β1、Smad4、CTGF的蛋白表达明显升高,差异有统计学意义(P0.05),BMP-7的蛋白表达明显降低,差异有统计学意义(P0.05)。艾塞那肽干预组较高果糖组TGF-β1、Smad4、CTGF的蛋白表达下降,而BMP-7的表达上调,差异有统计学意义(P0.05)。2.7 TLR-4信号通路与正常对照组相比,高果糖组大鼠肾脏组织TLR-4、NF-κB、p38MAPK的蛋白表达显著增高,差异有统计学意义(P0.05)。艾塞那肽干预组较高果糖组TLR-4、NF-κB、p38MAPK的蛋白表达明显下降,差异有统计学意义(P0.05)。2.8高胰岛素-正葡萄糖钳夹实验高果糖组大鼠的葡萄糖输注率明显低于正常对照组,差异有统计学意义(P0.05)。艾塞那肽组的葡萄糖输注率较高果糖组明显升高,差异有统计学意义(P0.05)。结论:1高果糖饮食可诱导大鼠发生胰岛素抵抗,表现为高胰岛素血症,同时伴有高血脂、高尿酸血症等代谢紊乱。2艾塞那肽可以改善高果糖饮食诱导的胰岛素抵抗,并有降脂、保护肾功能作用。3艾塞那肽可能是通过下调TGF-β1、Smad4、CTGF上调BMP-7来减轻肾脏纤维化。4艾塞那肽可抑制肾脏TLR-4信号通路,通过抑制NF-κB、p38MAPK的蛋白表达来达到肾脏保护作用。
[Abstract]:Objective: the living standard of the people is constantly improving, accompanied by the changes in food production and dietary structure, resulting in increased intake of fructose year by year. High fructose diet is closely related to the occurrence of metabolic syndrome. Insulin resistance and hyperinsulinemia are the common basis of disease, heart, cerebrovascular disease and diabetes. The risk factors are highly related to Chronic kidney disease (CKD). The glucagon like peptide 1 (Glucagon like peptide-1, GLP-1) analogue is a new type of hypoglycemic agent that promotes the biosynthesis and secretion of insulin, protects islet beta cells, improves insulin resistance, inhibits the secretion of glucagon and inhibits appetite. And feeding, delaying gastric content emptying, reducing blood sugar and keeping it at a constant level. But the effect of GLP-1 on insulin resistance in rats is not clear. First, the rat model of insulin resistance induced by high fructose was first established, and then the effect of alcisin on the renal tissue of insulin resistant rats induced by high fructose was discussed. Methods: 30 male Wistar rats (180g-200g) and 1 weeks after adaptive feeding were randomly divided into 2 groups: normal control group (Con) 12 and high fructose diet group (HF) 18. Normal control group was fed with ordinary feed, high fructose group was given equal calorie high fructose diet. 8 weeks after feeding, 6 rats in each group were randomly selected to carry out the high insulin positive glucose clamp experiment. The insulin sensitivity was evaluated by calculating the glucose infusion rate (Glucose infusion rate, GIR). Then the rats were killed and the blood and kidney tissue specimens were left after.8 week high fructose induced insulin resistance in rats. The rats of residual fructose group were randomly divided into 2 groups: high fructose model group (HF), altna peptide group (HFG), continuous high fructose feeding and corresponding drug intervention for 4 weeks. After 4 weeks of drug intervention, high insulin positive glucose clamp test was performed, rats were sacrificed after GIR, serum and kidney specimens were left, and fasting blood glucose (Fasting blood glucose) was measured. FBG, Fasting insulin (FINS), total cholesterol (Total cholesterol, TC), triglyceride (Triglyceride, TG), creatinine (Creatinine, Cr), urea nitrogen (Blood), uric acid (FINS), bone shape Protein 7 (Bone morphogenetic protein 7, BMP-7), Smad4, connective tissue growth factor (Connective tissue growth factor, CTGF), Toll like receptor 4, nuclear transcription factor and fibrinogen activator protein kinase Expression level, ELISA kit was used to measure the protein level of bone morphogenetic protein 7 (Bone morphogenetic protein 7, BMP-7), HE staining, and electron microscope observation of renal morphological changes. Results: after 8 weeks of 1 high fructose feeding, various indexes were changed: there was no significant difference in the initial weight of the two groups of 1.1 body weight rats. After feeding for 8 weeks, the weight of the high fructose group was slightly higher. In the normal control group, there was no statistical difference between the two groups (P0.05).1.2 serum biochemical indexes compared with the normal control group, the FINS, BUN, Cr, TC, TG, UA in the high fructose group were significantly increased, the difference was statistically significant (P0.05), but there was no statistical difference between the two groups (P0.05).1.3 high insulin positive glucose clamp experiment of high fructose group rats The rate of glucose infusion was significantly lower than that of the normal control group. The difference was statistically significant (P0.05) after 4 weeks of.2 intervention. After 4 weeks of 2.1 weight drug intervention, the body weight of the high fructose group was slightly higher than that of the normal control group and the alaninin group. There was no statistical difference between the groups (P0.05).2.2 serum biochemical indexes compared with the normal control group. The FINS, BUN, Cr, TC, TG, UA in the high fructose group were all significantly higher, the difference was statistically significant (P0.05). The indexes of the altna peptide group were higher than those of the normal group, the difference was statistically significant (P0.05), compared with the high fructose group, the difference was statistically significant (P0.05), but there was no statistical difference in the FBG between the three groups (P0.05).2.3 serum bone Morphin protein Compared with the normal control group, the serum bone morphogenetic protein -7 content in the serum of high fructose group was significantly lower than that of the normal group (P0.05). The difference was statistically significant (P0.05), the higher fructose group was significantly higher than that of the normal group (P0.05), and the difference was statistically significant (P0.05).2.4 kidney HE staining after HE staining in the optical mirror high magnification ( The renal tissue sections of two groups of rats were observed. The glomerulus and renal tubule structure of the normal control group were clearly visible and no obvious pathological changes were seen. The renal tubular epithelial cells in the high fructose group were swelling, the glomerular volume narrowed, the basement membrane thickened, the mesangial region broadened, the mesangial cells proliferated, and the sclerosis was obvious. The abnormality of the alscanine group was obvious. The renal tissue after 4 weeks was observed by.2.5 transmission electron microscopy. The renal tissue of rats in each group was observed through transmission electron microscope (x 15000). It was found that the glomerular capillary basement membrane was uniform and orderly in the normal control group, and the foot cells were arranged orderly and orderly. The glomerular capillary basement membrane was not uniform thickening and the foot thin was seen in the high fructose group. The mesangial cells proliferated and even disappeared, the mesangial cells were obviously proliferated and the mesangial area was broadened obviously. The expression of TGF- beta 1, Smad4, CTGF in the kidney tissue of the high fructose group was significantly higher than that in the normal control group. The difference was statistically significant (P0.0 5) the protein expression of BMP-7 was significantly decreased, and the difference was statistically significant (P0.05). The expression of TGF- beta 1, Smad4, CTGF in the high fructose group decreased, and the expression of BMP-7 was up, and the difference was statistically significant (P0.05).2.7 TLR-4 signaling pathway was compared with the normal group, and TLR-4, NF- kappa B, and NF- B in the rats of high fructose group. The expression of protein was significantly higher (P0.05). The protein expression of TLR-4, NF- kappa B and p38MAPK decreased significantly in the high fructose group of the group of alnannin intervention, and the difference was statistically significant (P0.05).2.8 hyperinsulinemia positive glucose clamp experiment with high fructose group, the glucose infusion rate was significantly lower than that of the normal control group, the difference was statistically significant Significance (P0.05). The glucose infusion rate of the alsna peptide group was higher than that of the high fructose group. The difference was statistically significant (P0.05). Conclusion: 1 high fructose diet can induce insulin resistance in rats, showing hyperinsulinemia, accompanied by hyperlipidemia, hyperuricemia and other metabolic disorders such as.2 alaninin can improve the induction of high fructose diet. Insulin resistance, and lipid lowering, protection of renal function.3 alnthe peptide may be by reducing TGF- beta 1, Smad4, CTGF up regulation of BMP-7 to reduce renal fibrosis.4 alnin inhibits renal TLR-4 signaling pathway, by inhibiting the protein expression of NF- kappa B, p38MAPK to achieve renal protection.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R965

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