高糖及脂多糖对大鼠肾小球系膜细胞NOD1-RICK-NF-κB信号通路的影响

发布时间：2018-07-25 11:37

【摘要】：目的：糖尿病肾病（diabetic nephropathy,DN）是糖尿病常见且特有的微血管并发症之一，其危害巨大，然而目前具体的发病机制仍不清楚。国内外大量研究表明，炎症是糖尿病肾病发生发展的中心环节，核因子-κB（NF-κB）信号是介导糖尿病肾病（DN）炎症的重要通路。最新研究表明，作为模式识别受体的NOD样受体（NOD1）在NF-κB信号通路的调控激活中起着重要作用。细胞外刺激可经过NOD1-RICK-IκB等信号分子激活NF-κB信号通路，介导炎症反应。NOD1介导的NF-κB信号通路是否参与了糖尿病肾病的发病尚未见相关文献报道。因此，本研究采用大鼠肾小球系膜细胞(HBZY-1)进行体外培养，高糖、脂多糖（lipopolysaccharide,LPS）作为刺激因子，NOD1受体抑制剂（ML130）作为阻断剂，分别采用western-blot和RT-PCR检测NOD1、RICK、IκB、NF-κB等的蛋白及mRNA表达,旨在探讨NOD1-RICK-NF-κB信号在糖尿病肾病炎症信号中的作用，为糖尿病肾病防治提供新的理论基础。方法：1.体外培养大鼠肾小球系膜细胞，分别给予葡萄糖、LPS以及NOD1的抑制剂（ML130）干预，分成以下7组：①正常对照组(NC组)：培养基含葡萄糖浓度为5.6mmol/L；②高糖组（HG1,HG2和HG3组）：培养基所含葡萄糖浓度分别为10mmol/L、20mmol/L和30mmol/L；③渗透压对照组：培养基含葡萄糖5.6mmol/L和24.4mmol/L的甘露醇；④LPS组（LPS1组,LPS2和LPS3组）：培养基中LPS的浓度分别为1μg/L、5μg/L和10μg/L；⑤高糖+LPS组：培养基含30mmol/L葡萄糖和5μg/L LPS；⑥高糖+ML130组：含30mmol/L葡萄糖培养基中预先加入30μmol/L ML130（NOD1受体阻断剂）；⑦LPS+ML130组：含5μg/L LPS的培养基中预先加入30μmol/LML130；2.分别将各组细胞培养一定时间后，，采用免疫印迹(Western-blotting)和RT-PCR技术检测NOD1、RICK、IκB、NF-κB的蛋白及基因表达;3.统计学分析：应用SPSS17.0统计软件分析数据。所有的数据都用均数±标准差（x±s）来表示，各组之间的数据比较采用单因素方差分析，组内两两比较采用LSD t检验，检验水准α=0.05，P0.05表明有统计学意义。结果：1.与NC组比较，不同浓度的高糖作用不同时间均可上调大鼠肾小球系膜细胞的NOD1、RICK、NF-κB p65蛋白和mRNA的表达、下调IκBα表达（均P0.05），提示高糖呈时间和浓度依赖性刺激NOD1-RICK-NF-κB信号；2.与NC组比较，不同浓度的LPS作用不同时间可以上调系膜细胞NOD1、RICK、NF-κB蛋白及mRNA的表达，下调IκBα表达（均P0.05）,提示LPS能激活NOD1-RICK-NF-κB的信号；3.与高糖组和LPS组相比，高糖+LPS组可增强NOD1、RICK、NF-κB蛋白和mRNA表达，抑制IκBα表达，说明高糖和LPS能协同刺激NOD1-RICK-NF-κB信号。4.与高糖组和LPS组相比，预先加入ML130可阻断高糖和LPS诱导的NOD1、RICK、NF-κB的高表达和IκBα的低表达（均P0.05），提示高糖和LPS是通过NOD1受体激活NF-κB信号通路。结论：高糖及LPS通过NOD1受体激活系膜细胞RICK-NF-κB炎症信号通路，NOD1介导的NF-κB信号通路可能参与了糖尿病肾病发生。
[Abstract]:Objective: diabetic nephropathy (diabetic nephropathy DN) is one of the common and unique microvascular complications of diabetes mellitus. A large number of studies at home and abroad have shown that inflammation is the central link in the occurrence and development of diabetic nephropathy, and nuclear factor- 魏 B (NF- 魏 B) signal is an important pathway to mediate the inflammation of diabetic nephropathy (DN). Recent studies have shown that NOD like receptors (NOD1), as pattern recognition receptors, play an important role in the regulation and activation of NF- 魏 B signaling pathway. Extracellular stimulation can activate NF- 魏 B signaling pathway via signal molecules such as NOD1-RICK-I 魏 B, and whether NF- 魏 B signaling pathway mediated by inflammatory response. NOD1-mediated NF- 魏 B signal pathway is involved in the pathogenesis of diabetic nephropathy has not been reported. In this study, rat glomerular Mesangial cells (HBZY-1) were cultured in vitro. High glucose and lipopolysaccharide (LPS) were used as stimulator for inhibitor of NOD1 receptor (ML130). Western-blot and RT-PCR were used to detect the expression of protein and mRNA in rat glomerular Mesangial cells (HBZY-1). To explore the role of NOD1-RICK-NF- 魏 B signal in the inflammatory signal of diabetic nephropathy and to provide a new theoretical basis for the prevention and treatment of diabetic nephropathy. Method 1: 1. Rat glomerular Mesangial cells were cultured in vitro and were treated with glucose lipopolysaccharide and NOD1 inhibitor (ML130) respectively. They were divided into the following 7 groups: normal control group (NC group): the concentration of glucose was 5.6 mmol / L in culture medium; 2 High glucose group (HG1HG2 and HG3 group): the glucose concentration in the medium was 10 mmol / L, 20 mmol / L and 30 mmol / L / L, respectively. The osmotic pressure control group: mannitol 4 LPS group (LPS1 group, LPS2 and LPS3 group) containing glucose 5.6mmol/L and 24.4mmol/L: the concentration of LPS in the medium was 1 渭 g / L 5 渭 g / L and 10 渭 g / L, respectively; 5 High glucose LPS group: medium containing 30mmol/L glucose and 5 渭 g / L LPS6 high glucose ML130 group: 30mmol/L glucose medium with 30 渭 mol/L ML130 (NOD1 receptor blocker) 7 LPs ML130 group: medium containing 5 渭 g / L LPS with 30 渭 mol / L LML130 + 2. After the cells were cultured for a certain time, the protein and gene expression of I 魏 B NF- 魏 B were detected by Western blotting (Western-blotting) and RT-PCR. Statistical analysis: SPSS17.0 statistical software was used to analyze the data. All the data were represented by mean 卤standard deviation (x 卤s). The data of each group were compared with single factor ANOVA, and the intra-group comparison was performed with LSD t test. The result is 1: 1. Compared with NC group, different concentrations of high glucose could up-regulate the expression of NOD1-RICKB p65 protein and mRNA in rat glomerular Mesangial cells and down-regulate the expression of I 魏 B 伪 (P0.05), suggesting that high glucose stimulated NOD1-RICK-NF- 魏 B signal 2 in a time-and concentration-dependent manner. Compared with NC group, the expression of NOD1-RICK-NF- 魏 B protein and mRNA in Mesangial cells could be upregulated by different concentrations of LPS at different time, and I 魏 B 伪 expression was down-regulated (P0.05), suggesting that LPS could activate the signal of NOD1-RICK-NF- 魏 B. Compared with high glucose group and LPS group, high glucose LPS group could enhance the expression of NOD1-RICK- 魏 B protein and mRNA and inhibit the expression of I 魏 B 伪, indicating that high glucose and LPS could co-stimulate NOD1-RICK-NF- 魏 B signal .4. Compared with high glucose group and LPS group, the high expression of NF- 魏 B and the low expression of I 魏 B 伪 induced by high glucose and LPS were blocked by ML130, suggesting that high glucose and LPS activated NF- 魏 B signaling pathway through NOD1 receptor. Conclusion: high glucose and LPS may be involved in the pathogenesis of diabetic nephropathy through NOD1 receptor activated Mesangial cells RICK-NF- 魏 B inflammatory signaling pathway and NOD1-mediated NF- 魏 B signaling pathway.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R587.2;R692.9

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