本文選題：糖尿病腎病　切入點(diǎn)：鎘　出處：《陜西科技大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目前全球糖尿病患者達(dá)4億余人,且增長率居高不下,其中約20%患者最終將發(fā)展為糖尿病腎病。糖尿病腎病發(fā)病機(jī)制復(fù)雜,在不斷加深的器質(zhì)損傷病程中,系膜細(xì)胞和其外基質(zhì)不斷增加且基底膜不斷增厚,最終導(dǎo)致腎小球硬化。環(huán)境污染物重金屬鎘對機(jī)體產(chǎn)生氧化應(yīng)激,損傷腎臟功能,可能紊亂糖脂平衡,加劇糖尿病腎病發(fā)生,但發(fā)病機(jī)制不明。代謝組學(xué)可對體內(nèi)代謝物的變化進(jìn)行定性或定量分析,檢測方法簡單穩(wěn)定,有利于尋找潛在生物標(biāo)記物,探尋誘發(fā)生理病理變化的相關(guān)通路,對藥物篩選提供依據(jù)。本研究擬采用高脂高糖喂養(yǎng)聯(lián)合腹腔注射氯化鎘誘導(dǎo),構(gòu)建糖尿病腎病小鼠模型。通過生理生化指標(biāo)及組織形態(tài)學(xué)觀察驗(yàn)證模型,利用氣相色譜-質(zhì)譜聯(lián)用技術(shù)(GC-MS)檢測血清代謝輪廓譜,結(jié)合主成分分析等統(tǒng)計(jì)方法分析對照組、糖尿病腎病模型組及原花青素保護(hù)組代謝物,尋找與糖尿病腎病發(fā)生相關(guān)的生物標(biāo)記物。從氧化應(yīng)激水平、金屬元素水平以及相關(guān)蛋白表達(dá)水平多方面對鎘誘導(dǎo)糖尿病腎病小鼠發(fā)生機(jī)制及原花青素的保護(hù)作用進(jìn)行初步的探討,旨在為糖尿病腎病的臨床診斷及藥物開發(fā)提供實(shí)驗(yàn)依據(jù)。(1)糖尿病腎病小鼠模型的構(gòu)建及生理生化指標(biāo)檢測采用高脂高糖喂養(yǎng)聯(lián)合腹腔注射氯化鎘誘導(dǎo)的方法構(gòu)建糖尿病腎病小鼠模型,對不同實(shí)驗(yàn)組進(jìn)行生理生化指標(biāo)檢測和組織形態(tài)學(xué)觀察以確定模型構(gòu)建成功。結(jié)果表明,糖尿病腎病模型組中小鼠空腹血糖、腎臟臟器系數(shù)顯著高于對照組(*P0.05、*P0.05),小鼠體重均值極顯著低于對照組(***P0.001),甘油三酯、總膽固醇含量較對照組顯著變化(*P0.05、*P0.05),高密度脂蛋白含量顯著低于對照組(*P0.05),低密度脂蛋白含量極顯著高于對照組(**P0.01),血清中肌酐含量極顯著高于對照組(***P0.001),尿液中肌酐含量極顯著低于對照組(**P0.01),尿液微量白蛋白極顯著高于對照組(**P0.01),說明高脂高糖喂養(yǎng)聯(lián)合腹腔注射氯化鎘誘導(dǎo)的方法對小鼠造成損傷。給予原花青素治療后,體重、臟器系數(shù)、甘油三酯、總膽固醇、低密度脂蛋白、血清肌酐、尿液肌酐及微量白蛋白都有顯著改善,說明鎘能誘發(fā)糖尿病腎病,原花青素能對鎘誘發(fā)的糖尿病腎病具有一定保護(hù)作用。(2)糖尿病腎病小鼠原花青素給藥前后血清代謝物譜分析采用GC-MS檢測不同組別的血清,利用代謝組學(xué)的研究思路,對對照組、糖尿病腎病模型組、原花青素保護(hù)組的小鼠血清代謝譜進(jìn)行分析,尋找潛在的生物標(biāo)志物。結(jié)果表明,糖尿病腎病模型組血清中半乳糖、單酰甘油、α-亞麻酸含量顯著低于對照組(*P0.05、**P0.01、**P0.01),膽固醇含量顯著高于對照組(*P0.05)。經(jīng)過OPC治療四周后,半乳糖含量顯著高于模型組(#P0.05),膽固醇含量顯著低于模型組(#P0.05),單酰甘油和α-亞麻酸含量變化沒有顯著性。半乳糖和膽固醇可能與鎘誘導(dǎo)糖尿病腎病的氧化應(yīng)激有關(guān)。(3)糖尿病腎病小鼠發(fā)病機(jī)制研究對不同實(shí)驗(yàn)組小鼠腎臟的抗氧化指標(biāo)谷胱甘肽、超氧化物歧化酶(GSH、SOD)、氧化指標(biāo)蛋白羰基化、脂質(zhì)過氧化(PCO、MDA)及炎癥因子一氧化氮(NO)進(jìn)行檢測,結(jié)果表示模型組腎臟抗氧化酶GSH、SOD顯著低于對照組(*P0.05、**P0.01),氧化指標(biāo)PCO、MDA含量顯著高于對照組(**P0.01、**P0.01),炎癥因子NO含量顯著高于對照組(*P0.05);給予OPC治療后,與模型組比較抗氧化指標(biāo)活性上升(#P0.05),氧化指標(biāo)的上升被有效抑制(##P0.01)。對不同實(shí)驗(yàn)組小鼠腎臟的元素鎘、銅、鋅、鐵、鈣(Cd~(2+)、Cu~(2+)、Zn~(2+)、Fe~(2+)、Ca~(2+))進(jìn)行檢測,結(jié)果表示模型組腎臟Cd~(2+)、Fe~(2+)、Ca~(2+)含量顯著高于對照組(**P0.01、*P0.05、**P0.01),Cu~(2+)、Zn~(2+)含量顯著低于對照組(*P0.05、*P0.05);給予OPC治療后,與模型組比較Fe~(2+)、Ca~(2+)含量顯著減少(#P0.05、#P0.05),Cu~(2+)、Zn~(2+)含量顯著增加(#P0.05、#P0.05)。對不同實(shí)驗(yàn)組小鼠腎臟的MAPK信號通路(MAPK蛋白、ERK蛋白、p38蛋白)、Nrf2信號通路(Nrf2蛋白、Keap1蛋白)、Ⅱ相解毒酶(NQO1蛋白)進(jìn)行檢測,結(jié)果表示模型組腎臟MAPK信號通路中MAPK蛋白和p38蛋白表達(dá)上調(diào)(*P0.05、**P0.01),ERK蛋白表達(dá)變化不大;Nrf2信號通路中Nrf2蛋白表達(dá)下調(diào)(*P0.05),Keap1蛋白表達(dá)上調(diào)(*P0.05);Nrf2信號通路的下游蛋白NQO1表達(dá)上調(diào)(**P0.01)。高脂高糖喂養(yǎng)聯(lián)合腹腔注射氯化鎘誘導(dǎo)糖尿病腎病激活了MAPK通路中的p38蛋白,紊亂糖脂代謝物水平。原花青素可能是Nrf2通路的激動劑,調(diào)控機(jī)體氧化應(yīng)激,為新藥開發(fā)提供依據(jù)。
[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+))榪涜媯，

本文編號：1575692