RIPK2介导自噬对高糖诱导的小鼠肾小球系膜细胞ROS-NLRP3炎症小体信号的调控研究
发布时间：2018-10-22 18:41
【摘要】：目的:糖尿病肾病(Diabetic nephropathy,DN)是一种糖尿病(Diabetes mellitus,DM)主要而严重的微血管并发症,其发病机制错综复杂,其发生发展涉及多个因素参与,近年来不少研究表明,氧化应激及免疫炎症反应可能参与糖尿病肾损害的不同病理过程并发挥核心作用。我们的前期研究表明,高糖可诱导氧化应激,激活活性氧(Reactive oxygen species,ROS)及核苷酸结合寡聚化结构域样受体蛋白3(Nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3,NLRP3)炎症小体信号通路,诱导活半胱氨酸蛋白酶1(Cysteine-aspartic acid protease1,Caspase1)及促炎细胞因子白细胞介素1-β(Interleukine-1 beta,IL-1β)的成熟与分泌。自噬(Autophagy)是一种清除损伤蛋白质或细胞器以维持细胞内稳态的降解过程,通过清除损伤线粒体减少ROS产生而实现能量回收,可能参与调节ROS及下游的NLRP3炎症小体信号通路,避免过度的免疫炎症反应。然而到目前为止,高糖刺激下肾系膜细胞(Glomerular mesangial cell,GMC)的自噬水平及调节机制研究尚少,且研究结果存在争议,既往研究表明,适当增强自噬可能减少氧化应激及免疫炎症反应,但过度自噬可能引起自噬性细胞损伤,加重细胞凋亡。在有限的自噬调节机制研究中,受体结合丝氨酸/苏氨酸激酶2(Receptor-interacting serine/threonine-protein kinase 2,RIPK2),一种Nod样受体(Nod-like receptor,NLR)家族中Nod1/Nod2的重要连接蛋白,被证明既可激活核因子-κB(Nuclear factor-κB,NF-κB)及丝裂原激活蛋白激酶(Mitogen-activated protein kinases,MAPKs)介导的炎症反应,还参与Nod1/Nod2介导的自噬反应,提示RIPK2可能参与调节自噬及ROS-NLRP3炎症小体信号通路,但目前尚缺乏研究探讨高糖刺激肾系膜细胞对RIPK2以及RIPK2与自噬、ROS-NLRP3炎症小体信号通路的影响,因此本研究通过观察小鼠肾系膜细胞中RIPK2、LC3II/I、ROS、Caspase1、IL-1β在不同时间及不同浓度高糖干预下的表达变化,同时检测细胞培养上清液中IL-1β表达,再通过细胞转染RIPK2-siRNA干扰RIPK2后再次观察自噬水平及ROS-NLRP3炎症小体关键因子的改变,旨在探讨高糖对肾系膜细胞RIPK2、自噬的影响以及自噬对ROS-NLRP3炎症小体信号通路的调控作用,为DN的防治提供新的思路。方法:(1)细胞培养及分组:体外培养小鼠肾小球系膜细胞(SV40),以高糖作为刺激因素,分为以下三组:(1)正常对照组(Normal control group,NC组):培养基含5.6 mmol/L葡萄糖;(2)渗透压对照组(Osmotic pressure group,OP组):培养基含5.6 mmol/L葡萄糖+24.4 mmol/L甘露醇。(3)不同高糖浓度干预组(High glucose group,HG组):培养基分别含10 mmol/L葡萄糖(HG1组)、20 mmol/L葡萄糖(HG2组)、30 mmol/L葡萄糖(HG3组);(2)各组细胞培养0h、2h、6h、12h、24h、48h、72h后,用Western-blot检测RIPK2、LC3II/I、Caspase1、IL-1β的蛋白表达;RT-PCR检测RIPK2、Caspase1、IL-1βmRNA表达;mRFP-GFP-LC3融合蛋白的腺病毒标记LC3以观察自噬流;各组细胞适时随机加入处理因素后装载2’,7’-二氯荧光黄双乙酸盐探针(2’,7’-Dichlorofluorescin Diacetate,DCFH—DA),并用分光光度计检测细胞内ROS水平变化;ELISA检测培养上清液IL-1β浓度。(3)RIPK2的siRNA干扰研究:筛选出最佳高糖作用浓度(30mmol/L)和时间(12h)后,细胞转染RIPK2 siRNA,再次进行分组:(1)siNC组:细胞转染control siRNA加入含5.6 mmol/L葡萄糖培养基培养12h;(2)si RIPK2组:细胞转染RIPK2siRNA加入含5.6 mmol/L葡萄糖培养基培养12h;(3)HG+siNC组:细胞转染control siRNA加入含30 mmol/L葡萄糖培养基培养12h;(4)HG+siRIPK2组细胞转染RIPK2 si RNA加入含30 mmol/L葡萄糖培养基培养12h;再次采用Western-blot、RT-PCR、mRFP-GFP-LC3标记+共聚焦显微镜、DCFH—DA标记+分光光度计及ELISA等方法检测上述指标。(4)统计学分析:采用SPSS 24.0统计软件对数据进行处理分析。符合正态分布的计量资料以均数±标准差(?)表示,采用单因素方差分析对多组间均数进行比较,组间多重比较用LSD—t法。我们定义P0.05为差异具有统计学意义。结果:(1)与NC组相比,高糖刺激可增加细胞内ROS产量、Caspase1、IL-1β蛋白及mRNA表达,差异均具有统计学意义(P值均0.05),且具有时间及浓度依赖性。(2)与NC组相比,高糖在短期作用时间内(0-12h)可诱导RIPK2、LC3II/I蛋白及RIPK2mRNA表达(P0.05),且在30 mmol/L高糖作用12h条件下表达显著增强,超过12h作用时间后RIPK2及LC3II/I表达下调(P0.05)。(3)细胞转染RIPK2 siRNA后成功抑制RIPK2表达。与siNC组相比,siRIPK2组LC3II/I表达显著下调,而细胞内ROS产量、Caspase1及IL-1β蛋白表达则显著增加(P0.05)。结论:(1)高糖可激活小鼠肾系膜细胞ROS-NLRP3炎症小体信号通路。(2)高糖对肾小球系膜细胞自噬具有双重调控作用,即高糖刺激可在短期内诱导自噬激活而在长期作用时间下内起抑制效应。(3)RIPK2通过自噬负性调节ROS-NLRP3炎症小体信号通路,可能参与糖尿病肾病的发生发展。
[Abstract]:Objective: Diabetic nephropathy (DN) is a major and serious microvascular complication of diabetes mellitus (DM). Oxidative stress and immune inflammatory responses may be involved in different pathological processes of diabetic renal impairment and play a central role. Our previous studies have shown that high sugar can induce oxidative stress, activate reactive oxygen species (ROS), and bind to oligomeric domain-like receptor protein 3 (NLRP3) inflammatory small body signal pathways, induce cysteine protease 1 (Cystine-aspinal acid protease1, The maturation and secretion of caspase-1 and interleukinine-1beta, IL-1ADH. Autophagy is a process of removing damaged proteins or organelles in order to maintain homeostasis in cells, and energy recovery is achieved by eliminating damage to mitochondria and reducing ROS generation. It is possible to participate in the regulation of the LRP3 inflammatory small signal pathways downstream of ROS and downstream, avoiding excessive immune inflammatory responses. Up to now, the autophagy level and regulation mechanism of renal membrane cells (GMC) under high sugar stimulation is less and the research results are controversial. Previous studies have shown that appropriate enhancement of autophagy may reduce oxidative stress and immune inflammatory response, However, excessive autophagy may cause autophagy cell damage and increase cell apoptosis. Receptor binding serine/ threonine kinase 2 (RIPK2), a Nod-like receptor (NLR) family, and Nod-like receptor (NLR) family, in a limited autophagy mechanism study, has been shown to be both a nuclear factor and a Nuclear factor-LacB, NF-Sepharose B and Mitogen-activated protein kinase (MAPKKs) mediate inflammatory responses, also involved in the Nod1/ No2-mediated autophagy reaction, suggesting that RIPK2 may participate in the regulation of autophagy and ROS-NLRP3 inflammatory cell signaling pathways, In this study, the expression of RIPK2, LC3II/ I, ROS, Caspas1 and IL-1 in renal membrane cells of mice was studied by observing the expression of RIPK2, LC3II/ I, ROS, Caspas1 and IL-1 in renal membrane cells of mice. meanwhile, the expression of IL-1 in the supernatant of the cell culture is detected, and the change of the key factors of the autophagy level and the inflammatory small body of the ROS-NLRP3 is again observed after the RIIPK2-siRNA interferes with RIIPK2 through the cell transfection, and aims to investigate the effect of high sugar on the RIPK2, autophagy of the renal membrane cells and the regulation effect of autophagy on the inflammatory small body signal path of the ROS-NLRP3, It provides a new idea for the prevention and cure of DN. Methods: (1) Cell culture and grouping: In vitro cultured mouse glomerular mesangial cells (NC) were cultured in three groups: (1) Normal control group (NC): medium containing 5. 6 mmol/ L glucose; (2) osmotic pressure group. OP group: The medium contains 5. 6 mmol/ L glucose + 24. 4 mmol/ L mannitol. (3) High glucose concentration intervention group (HG group): The medium contained 10 mmol/ L glucose (HG1 group), 20 mmol/ L glucose (HG2 group), 30 mmol/ L glucose (HG3 group); (2) The cells were cultured for 0h, 2h, 6h, 12h, 24h, 48h and 72h. Protein expression of Caspas1 and IL-1 protein; RT-PCR detection of RIPK2, Caspas1, IL-1, and mRNA expression; mRFP-GFP-LC3 fusion protein adenoviral marker LC3 to observe autophagy; each group of cells was randomly added to the treatment factor and loaded with 2 ', 7 The concentration of IL-1 in cultured supernatant was detected by ELISA. The concentration of IL-1 in cultured supernatant was detected by ELISA. (3) siRNA interference study of RIPK2: After screening out the optimal high sugar concentration (30mmol/ L) and time (12h), the cells were transfected with RIPK2 siRNA, and then grouped: (1) siNC group: the cell transfected control siRNA was added with 5. 6 mmol/ L glucose medium for 12h; (2) the si RIIPK2 group: Cells transfected with RIPK2 siRNA were cultured for 12h with 5. 6 mmol/ L glucose medium; (3) HG + siNC group: the control siRNA was transfected into the cells with 30 mmol/ L glucose medium for 12h; (4) the transfected RIPK2 si RNA of HG + siRIIPK2 group was added with 30 mmol/ L glucose medium for 12h; Western-blot, RT-PCR, mRFP-GFP-LC3 marker + confocal microscopy were used again. The above-mentioned indexes were tested by DCFH and DA labeling + spectrophotometer and ELISA. (4) Statistical analysis: SPSS 10.0 was used to analyze the data. The measurement data conforming to the normal distribution shall be subject to the standard deviation of the normal distribution (? The results showed that the single-factor analysis of variance was used to compare the inter-group comparisons, and the LSD-tt method was used for multiple comparisons among the groups. Results: (1) Compared with NC group, high sugar stimulation could increase ROS production, Caspas1, IL-1, and mRNA expression in cell, and the difference was statistically significant (P <0.05), and had time and concentration dependence. (2) Compared with NC group, high sugar can induce RIPK2, LC3II/ I protein and RIIPK2 mRNA expression (P0.05) in short-term action time (0-12h), and the expression of RIPK2 and LC3II/ I is down-regulated under the action of 30 mmol/ L high sugar for 12h (P0.05). (3) After transfected with RIPK2 siRNA, the expression of RIPK2 was successfully inhibited. Compared with siNC group, the expression of LC3II/ I was down-regulated in siRIIPK2 group, while in cell ROS production, the expression of caspase-1 and IL-1 was significantly increased (P0.05). Conclusion: (1) High sugar can activate the rat renal membrane cell ROS-NLRP3 inflammatory small body signal pathway. (2) High sugar has a double regulation effect on the autophagy of glomerular mesangial cells, that is, high sugar stimulation can induce autophagy in the short term and play an inhibitory effect in long-term action time. (3) RIIPK2 may be involved in the development of diabetic nephropathy by regulating ROS-NLRP3 inflammatory small body signal pathway from autophagy.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R692.9
本文编号：2287995
[Abstract]:Objective: Diabetic nephropathy (DN) is a major and serious microvascular complication of diabetes mellitus (DM). Oxidative stress and immune inflammatory responses may be involved in different pathological processes of diabetic renal impairment and play a central role. Our previous studies have shown that high sugar can induce oxidative stress, activate reactive oxygen species (ROS), and bind to oligomeric domain-like receptor protein 3 (NLRP3) inflammatory small body signal pathways, induce cysteine protease 1 (Cystine-aspinal acid protease1, The maturation and secretion of caspase-1 and interleukinine-1beta, IL-1ADH. Autophagy is a process of removing damaged proteins or organelles in order to maintain homeostasis in cells, and energy recovery is achieved by eliminating damage to mitochondria and reducing ROS generation. It is possible to participate in the regulation of the LRP3 inflammatory small signal pathways downstream of ROS and downstream, avoiding excessive immune inflammatory responses. Up to now, the autophagy level and regulation mechanism of renal membrane cells (GMC) under high sugar stimulation is less and the research results are controversial. Previous studies have shown that appropriate enhancement of autophagy may reduce oxidative stress and immune inflammatory response, However, excessive autophagy may cause autophagy cell damage and increase cell apoptosis. Receptor binding serine/ threonine kinase 2 (RIPK2), a Nod-like receptor (NLR) family, and Nod-like receptor (NLR) family, in a limited autophagy mechanism study, has been shown to be both a nuclear factor and a Nuclear factor-LacB, NF-Sepharose B and Mitogen-activated protein kinase (MAPKKs) mediate inflammatory responses, also involved in the Nod1/ No2-mediated autophagy reaction, suggesting that RIPK2 may participate in the regulation of autophagy and ROS-NLRP3 inflammatory cell signaling pathways, In this study, the expression of RIPK2, LC3II/ I, ROS, Caspas1 and IL-1 in renal membrane cells of mice was studied by observing the expression of RIPK2, LC3II/ I, ROS, Caspas1 and IL-1 in renal membrane cells of mice. meanwhile, the expression of IL-1 in the supernatant of the cell culture is detected, and the change of the key factors of the autophagy level and the inflammatory small body of the ROS-NLRP3 is again observed after the RIIPK2-siRNA interferes with RIIPK2 through the cell transfection, and aims to investigate the effect of high sugar on the RIPK2, autophagy of the renal membrane cells and the regulation effect of autophagy on the inflammatory small body signal path of the ROS-NLRP3, It provides a new idea for the prevention and cure of DN. Methods: (1) Cell culture and grouping: In vitro cultured mouse glomerular mesangial cells (NC) were cultured in three groups: (1) Normal control group (NC): medium containing 5. 6 mmol/ L glucose; (2) osmotic pressure group. OP group: The medium contains 5. 6 mmol/ L glucose + 24. 4 mmol/ L mannitol. (3) High glucose concentration intervention group (HG group): The medium contained 10 mmol/ L glucose (HG1 group), 20 mmol/ L glucose (HG2 group), 30 mmol/ L glucose (HG3 group); (2) The cells were cultured for 0h, 2h, 6h, 12h, 24h, 48h and 72h. Protein expression of Caspas1 and IL-1 protein; RT-PCR detection of RIPK2, Caspas1, IL-1, and mRNA expression; mRFP-GFP-LC3 fusion protein adenoviral marker LC3 to observe autophagy; each group of cells was randomly added to the treatment factor and loaded with 2 ', 7 The concentration of IL-1 in cultured supernatant was detected by ELISA. The concentration of IL-1 in cultured supernatant was detected by ELISA. (3) siRNA interference study of RIPK2: After screening out the optimal high sugar concentration (30mmol/ L) and time (12h), the cells were transfected with RIPK2 siRNA, and then grouped: (1) siNC group: the cell transfected control siRNA was added with 5. 6 mmol/ L glucose medium for 12h; (2) the si RIIPK2 group: Cells transfected with RIPK2 siRNA were cultured for 12h with 5. 6 mmol/ L glucose medium; (3) HG + siNC group: the control siRNA was transfected into the cells with 30 mmol/ L glucose medium for 12h; (4) the transfected RIPK2 si RNA of HG + siRIIPK2 group was added with 30 mmol/ L glucose medium for 12h; Western-blot, RT-PCR, mRFP-GFP-LC3 marker + confocal microscopy were used again. The above-mentioned indexes were tested by DCFH and DA labeling + spectrophotometer and ELISA. (4) Statistical analysis: SPSS 10.0 was used to analyze the data. The measurement data conforming to the normal distribution shall be subject to the standard deviation of the normal distribution (? The results showed that the single-factor analysis of variance was used to compare the inter-group comparisons, and the LSD-tt method was used for multiple comparisons among the groups. Results: (1) Compared with NC group, high sugar stimulation could increase ROS production, Caspas1, IL-1, and mRNA expression in cell, and the difference was statistically significant (P <0.05), and had time and concentration dependence. (2) Compared with NC group, high sugar can induce RIPK2, LC3II/ I protein and RIIPK2 mRNA expression (P0.05) in short-term action time (0-12h), and the expression of RIPK2 and LC3II/ I is down-regulated under the action of 30 mmol/ L high sugar for 12h (P0.05). (3) After transfected with RIPK2 siRNA, the expression of RIPK2 was successfully inhibited. Compared with siNC group, the expression of LC3II/ I was down-regulated in siRIIPK2 group, while in cell ROS production, the expression of caspase-1 and IL-1 was significantly increased (P0.05). Conclusion: (1) High sugar can activate the rat renal membrane cell ROS-NLRP3 inflammatory small body signal pathway. (2) High sugar has a double regulation effect on the autophagy of glomerular mesangial cells, that is, high sugar stimulation can induce autophagy in the short term and play an inhibitory effect in long-term action time. (3) RIIPK2 may be involved in the development of diabetic nephropathy by regulating ROS-NLRP3 inflammatory small body signal pathway from autophagy.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R692.9
【参考文献】
相关期刊论文 前1条
1 李锦;白雪源;崔少远;傅博;陈香美;;雷帕霉素对高糖诱导的肾系膜细胞自噬抑制、氧化损伤和衰老的影响[J];南方医科大学学报;2012年04期
，本文编号：2287995
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