镉致糖尿病肾病小鼠发病机制及原花青素保护作用研究
发布时间：2018-03-06 16:58
本文选题：糖尿病肾病 切入点：镉 出处：《陕西科技大学》2017年硕士论文 论文类型：学位论文
【摘要】：目前全球糖尿病患者达4亿余人,且增长率居高不下,其中约20%患者最终将发展为糖尿病肾病。糖尿病肾病发病机制复杂,在不断加深的器质损伤病程中,系膜细胞和其外基质不断增加且基底膜不断增厚,最终导致肾小球硬化。环境污染物重金属镉对机体产生氧化应激,损伤肾脏功能,可能紊乱糖脂平衡,加剧糖尿病肾病发生,但发病机制不明。代谢组学可对体内代谢物的变化进行定性或定量分析,检测方法简单稳定,有利于寻找潜在生物标记物,探寻诱发生理病理变化的相关通路,对药物筛选提供依据。本研究拟采用高脂高糖喂养联合腹腔注射氯化镉诱导,构建糖尿病肾病小鼠模型。通过生理生化指标及组织形态学观察验证模型,利用气相色谱-质谱联用技术(GC-MS)检测血清代谢轮廓谱,结合主成分分析等统计方法分析对照组、糖尿病肾病模型组及原花青素保护组代谢物,寻找与糖尿病肾病发生相关的生物标记物。从氧化应激水平、金属元素水平以及相关蛋白表达水平多方面对镉诱导糖尿病肾病小鼠发生机制及原花青素的保护作用进行初步的探讨,旨在为糖尿病肾病的临床诊断及药物开发提供实验依据。(1)糖尿病肾病小鼠模型的构建及生理生化指标检测采用高脂高糖喂养联合腹腔注射氯化镉诱导的方法构建糖尿病肾病小鼠模型,对不同实验组进行生理生化指标检测和组织形态学观察以确定模型构建成功。结果表明,糖尿病肾病模型组中小鼠空腹血糖、肾脏脏器系数显著高于对照组(*P0.05、*P0.05),小鼠体重均值极显著低于对照组(***P0.001),甘油三酯、总胆固醇含量较对照组显著变化(*P0.05、*P0.05),高密度脂蛋白含量显著低于对照组(*P0.05),低密度脂蛋白含量极显著高于对照组(**P0.01),血清中肌酐含量极显著高于对照组(***P0.001),尿液中肌酐含量极显著低于对照组(**P0.01),尿液微量白蛋白极显著高于对照组(**P0.01),说明高脂高糖喂养联合腹腔注射氯化镉诱导的方法对小鼠造成损伤。给予原花青素治疗后,体重、脏器系数、甘油三酯、总胆固醇、低密度脂蛋白、血清肌酐、尿液肌酐及微量白蛋白都有显著改善,说明镉能诱发糖尿病肾病,原花青素能对镉诱发的糖尿病肾病具有一定保护作用。(2)糖尿病肾病小鼠原花青素给药前后血清代谢物谱分析采用GC-MS检测不同组别的血清,利用代谢组学的研究思路,对对照组、糖尿病肾病模型组、原花青素保护组的小鼠血清代谢谱进行分析,寻找潜在的生物标志物。结果表明,糖尿病肾病模型组血清中半乳糖、单酰甘油、α-亚麻酸含量显著低于对照组(*P0.05、**P0.01、**P0.01),胆固醇含量显著高于对照组(*P0.05)。经过OPC治疗四周后,半乳糖含量显著高于模型组(#P0.05),胆固醇含量显著低于模型组(#P0.05),单酰甘油和α-亚麻酸含量变化没有显著性。半乳糖和胆固醇可能与镉诱导糖尿病肾病的氧化应激有关。(3)糖尿病肾病小鼠发病机制研究对不同实验组小鼠肾脏的抗氧化指标谷胱甘肽、超氧化物歧化酶(GSH、SOD)、氧化指标蛋白羰基化、脂质过氧化(PCO、MDA)及炎症因子一氧化氮(NO)进行检测,结果表示模型组肾脏抗氧化酶GSH、SOD显著低于对照组(*P0.05、**P0.01),氧化指标PCO、MDA含量显著高于对照组(**P0.01、**P0.01),炎症因子NO含量显著高于对照组(*P0.05);给予OPC治疗后,与模型组比较抗氧化指标活性上升(#P0.05),氧化指标的上升被有效抑制(##P0.01)。对不同实验组小鼠肾脏的元素镉、铜、锌、铁、钙(Cd~(2+)、Cu~(2+)、Zn~(2+)、Fe~(2+)、Ca~(2+))进行检测,结果表示模型组肾脏Cd~(2+)、Fe~(2+)、Ca~(2+)含量显著高于对照组(**P0.01、*P0.05、**P0.01),Cu~(2+)、Zn~(2+)含量显著低于对照组(*P0.05、*P0.05);给予OPC治疗后,与模型组比较Fe~(2+)、Ca~(2+)含量显著减少(#P0.05、#P0.05),Cu~(2+)、Zn~(2+)含量显著增加(#P0.05、#P0.05)。对不同实验组小鼠肾脏的MAPK信号通路(MAPK蛋白、ERK蛋白、p38蛋白)、Nrf2信号通路(Nrf2蛋白、Keap1蛋白)、Ⅱ相解毒酶(NQO1蛋白)进行检测,结果表示模型组肾脏MAPK信号通路中MAPK蛋白和p38蛋白表达上调(*P0.05、**P0.01),ERK蛋白表达变化不大;Nrf2信号通路中Nrf2蛋白表达下调(*P0.05),Keap1蛋白表达上调(*P0.05);Nrf2信号通路的下游蛋白NQO1表达上调(**P0.01)。高脂高糖喂养联合腹腔注射氯化镉诱导糖尿病肾病激活了MAPK通路中的p38蛋白,紊乱糖脂代谢物水平。原花青素可能是Nrf2通路的激动剂,调控机体氧化应激,为新药开发提供依据。
[Abstract]:The current global diabetes up to 4 more than 100 million people, and the growth rate is high, and about 20% of patients will eventually develop diabetic nephropathy. The pathogenesis of diabetic nephropathy is complex, in the course of deepening the organic injury in mesangial cells and its matrix increase and broken basement membrane thickening, resulting in glomerulosclerosis. Environmental pollutants of heavy metals cadmium causes oxidative stress on the body, the damage of kidney function, may exacerbate the disorders of glucose and lipid balance, diabetic nephropathy, but the pathogenesis is unknown. Metabonomics can change on the in vivo metabolite qualitative or quantitative analysis, the detection method is simple and stable, conducive to search for potential biomarkers, explore related pathways induced physiological and pathological changes of. Provide the basis for drug screening. The purpose of this study was feeding the high-fat diet combined with intraperitoneal injection of cadmium chloride induced diabetic nephropathy rats model, construction of small through. The validation of the model to observe the physiological and biochemical indexes and tissue morphology, using gas chromatography-mass spectrometry (GC-MS) detection of serum metabolic profiling analysis, combined with principal component analysis statistical methods such as control group, model group and diabetic nephropathy group protection of procyanidins metabolites, looking for biomarkers associated with diabetic nephropathy from oxidative stress. The level of protection of mice diabetic nephropathy and the mechanism of proanthocyanidins was studied on cadmium induced on expression level of metal elements and the related protein, to clinical diagnosis and drug development for diabetic nephropathy and provide experimental basis. (1) the construction and detection of physiological and biochemical indexes of diabetic nephropathy mouse model by feeding high fat high glucose combined with intraperitoneal injection of cadmium chloride induced by construction of diabetic nephropathy mouse model of different experimental group of physiological and biochemical indexes To observe the morphology and to determine the successful model. The results show that the model of diabetic nephropathy group were fasting blood glucose, renal index was significantly higher than the control group (*P0.05, *P0.05), the mean body weight of mice was significantly lower than the control group (***P0.001), three glycerol esters, total cholesterol content than the control group significantly (*P0.05, *P0.05), high density lipoprotein cholesterol was significantly lower than the control group (*P0.05), low density lipoprotein levels were significantly higher than the control group (**P0.01), serum creatinine levels were significantly higher than the control group (***P0.001), urine creatinine content was significantly lower than the control group (**P0.01), urine albumin was significantly higher than that of the control group (**P0.01), description method of feeding the high-fat diet combined with intraperitoneal injection of cadmium chloride induced damage on mice. Given proanthocyanidins after treatment, body weight, organ coefficient, triglyceride, total cholesterol, low density Lipoprotein, serum creatinine, urine creatinine and albumin were significantly improved, indicating that cadmium induced diabetic nephropathy, diabetic nephropathy of procyanidins induced by cadmium has certain protective effect. (2) diabetic nephropathy mice proanthocyanidins before and after administration of serum metabolite profiles were analyzed by detection of serum GC-MS in different groups, using metabolic research ideas group, the control group, diabetic nephropathy model group, procyanidins protection group serum metabolic profiling analysis, the search for potential biomarkers. The results showed that diabetic nephropathy model group serum galactose, monoacylglycerol, alpha linolenic acid content was significantly lower than the control group (*P0.05, **P0.01, **P0.01) and the content of cholesterol was significantly higher than the control group (*P0.05). After OPC after four weeks of treatment, the content of galactose was significantly higher than that of model group (#P0.05), the content of cholesterol was significantly lower than that of model group (#P0.05), mono Glycerol and alpha linolenic acid content did not significantly change. Oxidative stress and galactose cholesterol and cadmium induced diabetic nephropathy. (3) study of the pathogenesis of diabetic nephropathy mice antioxidant indexes of the different groups of mice kidney glutathione, superoxide dismutase (GSH, SOD), the oxidation index of protein carbonylation. Lipid peroxidation (PCO, MDA) and inflammatory factor nitric oxide (NO) were detected, the results show that the proposed model group renal antioxidant enzymes GSH, SOD was significantly lower than the control group (*P0.05, **P0.01), the oxidation index PCO, the content of MDA was significantly higher than the control group (**P0.01, **P0.01), NO content of inflammatory factors were significantly higher than the control group (*P0.05); after OPC treatment, compared with the model group, the antioxidant index (#P0.05) activity increased, increased oxidation index is effectively suppressed (##P0.01). The elements Cd, in different experimental groups of mice kidney copper, zinc, iron, calcium (Cd~ (2+), Cu~ (2+), Zn ~(2+),Fe~(2+),Ca~(2+))杩涜妫，
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