12-脂氧化酶对糖尿病肾病肾小球P-cadherin表达的影响及其机制研究

发布时间：2018-02-02 13:49

本文关键词： 糖尿病肾病 12-脂氧化酶 P-cadherin 肾小球蛋白尿　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：背景:糖尿病肾病(Diabetic nephropathy,DN)是糖尿病最常见的并发症之一,是导致慢性肾衰竭的重要原因及死亡原因,这一趋势在发达国家更为明显。蛋白尿是DN的主要临床表现及核心发病环节,决定了病情的严重程度和发展速度,且成为一种独立的心血管危险因素。探讨DN蛋白尿的机制临床意义重大。脂氧化酶(Lipoxygenase,LO)是机体内一类重要的氧化酶,主要作用于不饱和脂肪酸。依据其作用于花生四烯酸时氧化位置分为5-、8-、12-和15-LO。12(S)-氢氧化二十碳四烯酸[12(S)-hydroxyeicosatetraenoic acid,12(S)-HETE]是12-LO氧化花生四烯酸生成的活性脂质产物。研究表明,氧化应激和炎症反应是12-LO和其脂质产物12(S)-HETE引起蛋白尿的主要机理。足细胞裂孔蛋白如nephrin、P-cadherin和NEPH1构成肾小球滤过的重要屏障,研究显示DN早期相对肥大肾小球中nephrin表达减少与蛋白尿形成相关。有研究表明P-cadherin损伤可导致非nephrin或NEPH1依赖的蛋白尿,那么其是否与nephrin一样在DN蛋白尿形成中发挥重要作用呢?目前尚未见到相关报道。之前的研究证实12-LO可通过影响nephrin的表达参与DN蛋白尿形成,那么其对同样是裂孔蛋白的P-cadherin作用如何呢?目前尚未有相关报道并因此成为本研究的目的。方法:(1)将同步化的足细胞分成四组:对照组(Ctrl);SB202190组(p38MAPK抑制剂),培养液中加入10~(-6) mol/L SB202190;12(S)-HETE组,培养液中加入10~(-7) mol/L12(S)-HETE;12(S)-HETE+SB202190组,培养液中加入10~(-7) mol/L 12(S)-HETE和10~(-6)mol/L SB202190,24小时后收集细胞,Western blot检测细胞内P-cadherin表达。(2)将微型渗透泵植入Sprague Dawley大鼠皮下,持续泵入12(S)-HETE(1 mg·Kg-1·d-1),对照组泵入乙醇胺。7天后将大鼠处死收集肾脏,应用系列过筛法分离不同大小的肾小球,应用RT-PCR和Western blot检测肾小球内P-cadherin表达。(3)将野生型和12-LO基因敲除C57/BL6小鼠分成四组:野生型对照组(WT)、野生型糖尿病组(WT+STZ)、12-LO基因敲除组(LOKO)、12-LO基因敲除糖尿病组(LOKO+STZ)。小鼠腹腔内单次大剂量注射STZ建立1型糖尿病模型,16周后处死提取肾小球,应用竞争性RT-PCR及Western blot检测肾小球内P-cadherin表达,并采用Western blot检测肾小球内p38MAPK、ERK1/2、JNK及其磷酸化水平。监测小鼠血糖,收集尿液用于测定24h尿白蛋白水平。(4)给予Sprague Dawley大鼠高脂饮食及小剂量STZ腹腔注射建立2型糖尿病模型。成模后随机分为2组:DN组和CDC(Cinnamyl-3,4-dihydroxy-α-cynanocinnamate,12-LO抑制剂)组。DN组皮下注射芝麻油,CDC组皮下注射CDC 8 mg·Kg-1,每周3次。对照组给予正常饮食。检测8周后血糖、体重、肾重,收集肾小球,Western blot、免疫荧光、免疫组化检测肾小球内P-cadherin表达,ELISA检测12(S)-HETE水平。于实验结束前留取24 h尿测尿白蛋白。结果:(1)12(S)-HETE直接刺激明显减少大鼠肾小球内P-cadherin m RNA及蛋白表达(P0.05),且大、小肾小球无明显差异。(2)12(S)-HETE可减少足细胞内P-cadherin蛋白表达(P0.05),这一作用可以部分被p38MAPK抑制剂SB202190阻断(P0.05)。(3)与对照组比较,WT+STZ组小鼠肾小球内P-cadherin表达明显减少(P0.05),而敲除12-LO基因可增加LOKO+STZ组小鼠肾小球内P-cadherin含量(P0.05)。(4)与对照组比较,WT+STZ组小鼠肾小球内p-p38MAPK、p-ERK1/2、p-JNK水平均明显增加(P0.05),敲除12-LO基因仅可以减少p-p38MAPK水平(P0.05),而对p-ERK1/2和p-JNK无明显影响(P0.05)。(5)与对照组比较,DN组大鼠血糖、肾重/体重明显增加,肾小球体积明显增大,尿白蛋白水平明显增加(P0.05);皮下注射CDC可降低糖尿病大鼠肾重/体重及尿白蛋白水平,缓解肾小球肥大(P0.05)。(6)与对照组比较,DN组大鼠肾小球内12(S)-HETE含量明显增加(P0.05),且这一趋势在相对肥大肾小球中更明显(P0.05),CDC可减少糖尿病大鼠肾小球内12(S)-HETE水平(P0.05)。(7)与对照组比较,DN组肾小球内P-cadherin m RNA和蛋白水平明显减少(P0.05),且在大、小肾小球中无明显差异(P0.05),皮下注射CDC可增加糖尿病大鼠肾小球内P-cadherin含量(P0.05)。结论:(1)12-LO作用产物12(S)-HETE直接刺激可降低足细胞及肾小球内P-cadherin表达。(2)DN肾小球内P-cadherin表达降低,抑制12-LO可上调肾小球内P-cadherin表达水平。(3)12-LO通过p38MAPK信号通路调节DN肾小球内P-cadherin的表达。
[Abstract]:Background: diabetic nephropathy (Diabetic nephropathy DN) is one of the most common complications of diabetes, is the cause of an important cause of chronic renal failure and death in developed countries, this trend is more obvious. The urine protein is the major clinical manifestations of DN and the core pathogenesis, determines the severity of the illness and the speed of development, and to become an independent cardiovascular risk factor. To investigate the mechanism and clinical significance of DN protein in the urine. The major lipoxygenase (Lipoxygenase, LO) is an important class of enzymes in the body, mainly on unsaturated fatty acids. On the basis of its role in the four arachidonic acid oxidation when the position is divided into 5-, 8-, 12- and 15-LO.12 (S twenty) - hydroxide carbon four acid [12 (S) -hydroxyeicosatetraenoic acid 12 (S) -HETE] is the activity of lipid oxidation products of 12-LO four arachidonic acid production. Research shows that oxidative stress and inflammatory reaction is 12-LO and its lipid production 12 (S) the main mechanism of proteinuria caused by -HETE. Podocyte Slit proteins such as nephrin, P-cadherin and NEPH1 constitute an important barrier of glomerular filtration, studies show that early DN relative expression of nephrin in glomerular hypertrophy associated with a decrease in proteinuria. Studies have shown that P-cadherin damage can lead to non nephrin or NEPH1 dependent protein in urine so, whether the nephrin like in the DN play an important role in the formation of proteinuria? Has yet to see the relevant reports. Previous studies confirmed that 12-LO can be formed by the expression of nephrin DN protein in urine, then the same is how the role of P-cadherin protein in hiatus? There have been no reports and thus become the the purpose of the study. Methods: (1) the synchronized podocytes were divided into four groups: control group (Ctrl); group SB202190 (p38MAPK inhibitor), was added to the culture medium 10~ (-6) mol/L SB202190 12 (S) -HE; TE was added to the culture medium 10~ (-7) mol/L12 (S) -HETE; 12 (S) -HETE+SB202190 were added to the culture medium 10~ (-7) mol/L 12 (S) -HETE and 10~ (-6) mol/L SB202190,24 hours after collection of cells, the expression of Western blot detected P-cadherin. (2) the micro penetration pump implantation of Sprague Dawley rats subcutaneously, continuous infusion of 12 (S) -HETE (1 mg - Kg-1 - D-1), the control group pump ethanolamine.7 days after the rats were sacrificed to collect the kidney, using a series of different size separation glomerular sieve method, the expression of P-cadherin and Western using RT-PCR blot detection (3 glomeruli.) and the wild type 12-LO gene knockout C57/BL6 mice were divided into four groups: wild type control group (WT), wild type diabetic group (WT+STZ), 12-LO gene knockout group (LOKO), 12-LO gene knockout diabetic group (LOKO+STZ). The establishment of type 1 diabetic mice intraperitoneal single dose injection STZ, after 16 weeks of glomerular extraction The expression of P-cadherin, RT-PCR and Western by competitive blot detection by Western and blot in glomeruli, glomerular detection within p38MAPK, ERK1/2, JNK and its phosphorylation level. Blood glucose monitoring in mice, urine was collected for determination of 24h urinary albumin levels. (4) treated with high fat diet Sprague Dawley rats and small dose of STZ intraperitoneal injection to establish type 2 the models of diabetes. The rats were randomly divided into 2 groups: group DN and CDC (Cinnamyl-3,4-dihydroxy- alpha -cynanocinnamate, 12-LO inhibitor) group.DN group subcutaneous injection of sesame oil, CDC group subcutaneous injection of CDC 8 mg - Kg-1, 3 times a week. The control group was given normal diet. After 8 weeks of testing blood glucose, body weight, kidney weight, collect Western blot, the glomeruli, immunofluorescence, immunohistochemistry to detect the expression of P-cadherin in glomeruli, ELISA was detected in 12 (S) -HETE level. At the end of the experiment before and collected 24 h urine test urine albumin. Results: (1) 12 (S) -HETE direct stimulation significantly reduce Little rat glomerular RNA and protein expression of P-cadherin m (P0.05), and big, no obvious difference. (2) 12 small glomeruli (S) -HETE can reduce the expression of P-cadherin protein in the podocyte (P0.05), this effect can be part of p38MAPK inhibitor SB202190 (P0.05) (3) and the control group. Comparison of group WT+STZ significantly decreased the expression of P-cadherin in glomeruli of mice (P0.05), and 12-LO gene knockout mice can increase the contents of P-cadherin LOKO+STZ in glomeruli (P0.05). (4) compared with the control group, WT+STZ group of mice glomeruli p-p38MAPK, p-ERK1/2, p-JNK levels were significantly increased (P0.05), 12-LO gene knockout only can reduce the level of p-p38MAPK (P0.05), but had no obvious effect on p-ERK1/2 and p-JNK (P0.05). (5) compared with the control group, the blood glucose of rats in the DN group, kidney weight / body weight increased, glomerular volume increased significantly, the level of urinary albumin was significantly increased (P0.05); subcutaneous injection of CDC can be reduced Diabetic rat kidney weight / body weight and urinary albumin levels, alleviate glomerular hypertrophy (P0.05). (6) compared with the control group, glomerulus in rats of DN group 12 (S) -HETE were significantly increased (P0.05), and this trend was more obvious in relative glomerular hypertrophy (P0.05), CDC can reduce glomerular diabetic rats in 12 (S) -HETE (P0.05). (7) compared with the control group, the level of P-cadherin m and RNA protein in DN group decreased significantly in glomeruli (P0.05), and no significant difference in large, small glomeruli (P0.05), subcutaneous injection of CDC can increase the contents of P-cadherin in glomeruli of diabetic rats disease (P0.05). Conclusion: (1) 12-LO 12 (S) -HETE product direct stimulation can decrease the expression of P-cadherin in podocytes and glomeruli. (2) decrease the expression of P-cadherin DN in glomeruli, inhibition of 12-LO can upregulate the expression of P-cadherin in glomerular level. (3) 12-LO regulation of DN P-cadhe in glomeruli through p38MAPK signaling pathway The expression of RIN.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.2;R692.9

【相似文献】