糖肾方治疗糖尿病肾病的抗氧化作用研究
发布时间：2018-07-30 07:20
【摘要】：糖尿病肾病(diabetic kidney disease, DKD)是糖尿病最主要微血管并发症,患者一旦出现大量蛋白尿,肾功能将进行性下降。由于其发病机制复杂,目前的治疗措施疗效有限。中医药治疗在控制DKD方面具有一定优势。前期研究结果显示,导师经验方糖肾方可减少2型DKD患者尿蛋白排泄、提高估算的肾小球滤过率,升高血浆谷胱甘肽水平,具有一定的保护肾功能及抗氧化作用。普遍认为,氧化应激在DKD发生发展中起到重要作用,活性氧簇(reactive oxygen species, ROS)可激活多条信号通路,引起内皮细胞、系膜细胞、足细胞、肾小管间质损伤,从而加重DKD。近年来,主要表达于肾小管上皮细胞的肌醇加氧酶(myo-inositol oxygenase, MIOX)在DKD发生发展中的作用逐渐被发现,认为其参与调控细胞内ROS生成,与氧化应激密切相关,可能成为DKD干预新靶点。为了进一步观察、验证糖肾方干预2型DKD的长期疗效,探索其可能机制,本研究选用自发性2型DKD小鼠动物模型,观察糖肾方对小鼠的干预作用。在此基础上,体外实验验证MIOX与氧化应激的关系。此外,课题组开展了糖肾方治疗DKD有效性和安全性的随机、双盲、安慰剂平行对照、多中心临床试验,将氧化应激指标纳为次要疗效指标,以期为糖肾方保护DKD的抗氧化作用提供更全面证据。目的：1.研究糖肾方对白发性2型糖尿病db/db小鼠肾脏损伤的保护作用,探索其机制是否与抗氧化应激、降低MIOX表达有关。2.探索MIOX在高糖培养的大鼠近端肾小管上皮细胞(NRK52E)中的表达变化及其在近端肾小管上皮细胞氧化应激中的作用。3. 由于糖肾方治疗DKD有效性和安全性的随机、双盲、安慰剂平行对照、多中心临床试验尚未揭盲,本研究归纳试验实施过程中影响质量控制的关键环节,为提高研究质量提供帮助。方法：1.糖肾方对db/db小鼠肾脏保护作用及机制研究采用8周龄自发性2型糖尿病模型db/db小鼠,随机分为模型组(db/db组)、糖肾方治疗组(db/db+TSF组),同周龄不发病db/m小鼠作为正常对照组(db/m组),db/db+TSF组小鼠采用糖肾方(2.4g/kg-d)连续灌胃给药12周,db/m组和db/db组小鼠给予等量蒸馏水。所有小鼠均以常规普通饲料顺应性喂养2周后,开始干预。每日观察小鼠一般状态,每周测量体重,每4周检测血糖、24h尿白蛋白。实验第12周进行动物取材,小鼠禁食12h后眼内眦取血,全自动生化仪检测肝、肾功能和血脂。之后迅速打开腹腔,摘取双侧肾脏并称重,一部分肾组织用于组织病理分析,另一部分分离出肾皮质用于分子生物学检测。肾组织进行PAS染色观察病理损伤,计算肾小球系膜基质百分比,TBA法检测血清、尿液中丙二醛含量,Wester blot.实时荧光定量PCR、免疫组化方法检测小鼠肾组织中氧化应激相关的NADPH氧化酶亚基、Nrf2通路、MIOX及足细胞标志物表达。2. MIOX在高糖诱导肾小管上皮细胞氧化应激中的作用采用5.5、10、20、30 mM浓度葡萄糖刺激大鼠近端肾小管上皮细胞(NRK52E) 48h,30 mM葡萄糖刺激NRK52E细胞12.24.36.48.60h。 Western blot方法检测细胞内MIOX、Nox4、 Nrf2表达变化,分别绘制葡萄糖对NRK52E氧化应激损伤的剂量和时间依赖曲线,确定最佳刺激剂量和时间。设计MIOX基因siRNA Oligo干扰序列,筛选出敲降效率最高者转染至NRK52E细胞,6h后更换终浓度5.5、30mM葡萄糖培养基,48h后收集细胞,DCFH-DA探针检测细胞内ROS水平,Western blot检测细胞内MIOX、Nox4、Nrf2、 NQO1蛋白表达。3. 归纳总结糖肾方治疗DKD有效性和安全性的随机、双盲、安慰剂平行对照、多中心临床试验实施过程中的质量控制要点。结果：1.糖肾方可改善自发性2型糖尿病模型db/db小鼠肾组织损伤,其机制与降脂、抗氧化应激、保护足细胞有关(1)肾脏保护作用：给药0至12周,db/db组体重明显高于db/m组(P0.001),db/db+TSF组在第6周(小鼠16周龄)出现体重下降,并持续至实验结束(P0.05)。糖肾方干预可降低db/db组升高的肾重(P0.01)。实验期间,db/m组血糖保持稳定,db/db和db/db+TSF组血糖均高于db/m组(P0.01),但两组之间无差异。与db/m组比,db/db组血清AST、ALT升高(P0.01,P0.001),糖肾方干预可有效改善其异常的肝功能(P0.05,P0.05)。db/db组血清TC、FFA、LDL-C较db/m组显著升高(P0.001,P0.05,P0.001),糖肾方可改善db/db小鼠血脂紊乱(P0.001,P0.05,P0.001)。实验过程中,db/db组24h尿白蛋白较db/m组升高(P0.001),给药第8周开始,db/db+TSF组24h尿白蛋白出现下降趋势(P0.01),至给药12周时与db/m组更接近(P0.05)。 PAS染色可见db/db组肾小球体积增大,系膜基质增生,半定量结果表明肾小球系膜基质百分比显著升高(P0.01),糖肾方治疗后肾小球系膜基质百分比降低(P0.01)。(2)抗氧化应激机制：TBA法检测MDA水平发现,db/db组小鼠血清、24h尿液MDA均较db/m组显著升高(P0.01,P0.001),糖肾方干预可使其含量显著下降(P0.05,P0.05)。实时荧光定量PCR结果显示,db/db组小鼠肾组织Nox2、Nox4、 p22phox mRNA水平较db/m组小鼠显著升高(P0.01,P0.05,P0.01),但p47phox无差异(P0.05),糖肾方干预可降低Nox2、Nox4、p22phox mRNA水平(P0.001,P0.05,P0.01)。Western blot与免疫组化结果显示,与db/m组相比,db/db组肾组织Nox2、Nox4蛋白表达升高(P0.05, P0.001; P0.05, P0.001),糖肾方可下调其表达(P0.05, P0.001; P0.05,P0.001)。实时荧光定量PCR发现,与db/m组相比,db/db组小鼠肾组织Nrf2、QO1mRNA升高(P0.05,P0.01),db/db+TSF组上述指标显著下降(P0.05,P0.05)；db/db组HO-1 mRNA水平较db/m组下降(P0.05),糖肾方干预可升高其表达(P0.05)。Western blot与免疫组化显示,糖肾方可下调db/db组肾组织内升高的Nrf2、NQO1蛋白表达(P0.05, P 0.001;P0.05,P0.001)。此外,免疫组化还显示,MIOX表达于近端肾小管,Western blot与免疫组化均证实db/db组小鼠肾小管内MIOX显著增多(P0.01,P0.001),而糖肾方可使其降低(P0.05,P0.001)。(3)保护足细胞机制：实时荧光定量PCR显示,db/db组小鼠肾组织nephrin、podocin、 WT-1 mRNA水平均较db/m组显著下降(P0.05,P0.05,P0.05),而糖肾方治疗可使其表达升高(P0.01,P0.01,P0.05)。免疫组化结果同样证实糖肾方具有升高db/db组小鼠nephrin podocin表达的作用(P0.01,P0.01)。2. MIOX介导高糖诱导的肾小管上皮细胞氢化应激发生(1)高糖刺激对MIOX表达的影响：随着葡萄糖浓度升高,NRK52E细胞MIOX、Nox4表达逐渐升高,Nrf2表达逐渐下降；30 mM葡萄糖刺激NRK52E细胞0、12、24、36、48、60h,细胞内MIOX、Nox4、Nrf2表达均先升高后降低,Nrf2在刺激12h升至高峰,48h时下降显著,MIOX、Nox4至48h时出现表达高峰。(2)敲降MIOX对细胞氧化应激的影响：筛选得到敲降效率最高(72.1%)的MIOX基因siRNA Oligo干扰序列,转染至NRK52E细胞,6h后更换终浓度5.5及30 mM葡萄糖培养基,48h后发现,细胞内ROS含量及MIOX、Nox4、NQO1蛋白表达升高,Nrf2表达下降,敲降MIOX可降低ROS水平及Nox4、NQOl表达,Nrf2表达较高糖刺激进一步下降。3.受试者招募、数据管理、多级监查、研究者和受试者依从性是试验实施过程中质量控制的重要环节。结论：1.糖肾方可降低自发性2型糖尿病模型db/db、鼠尿白蛋白排泄,改善肾组织病理损伤,具有肾脏保护作用,其机制可能与改善血脂紊乱、减轻氧化应激、降低MIOX表达、保护足细胞损伤有关。2. MIOX可促进高糖引起的肾小管上皮细胞氧化应激反应,其作用与促进Nox4表达有关,这一结果为进一步探索中药作用靶点提供了新思路。
[Abstract]:Diabetic nephropathy (diabetic kidney disease, DKD) is the most important microvascular complication of diabetes. Once a large number of albuminuria appears, the renal function will be reduced. Due to its complicated pathogenesis, the curative effect of the present treatment is limited. The treatment of traditional Chinese medicine has some advantages in the control of DKD. Fang sugar kidney can reduce the urinary protein excretion of type 2 DKD patients, improve the estimated glomerular filtration rate and increase the plasma glutathione level. It has a certain protection of renal function and antioxidant effect. It is generally believed that oxidative stress plays an important role in the development of DKD, and the active oxygen cluster (reactive oxygen species, ROS) can activate a number of signal pathways. It causes endothelial cells, mesangial cells, podocytes and tubulointerstitial damage, and thus aggravates DKD. in recent years. The role of myo-inositol oxygenase (MIOX), which is mainly expressed in renal tubular epithelial cells, is gradually discovered in the development of DKD, which is considered to be involved in the regulation of intracellular ROS formation, which is closely related to oxidative stress. To further observe the new target of DKD intervention. In order to further observe the long-term effect of sugar kidney prescription intervention on the long-term effect of type 2 DKD and explore its possible mechanism, this study selected spontaneous 2 DKD mice model and observed the intervention effect of sugar kidney prescription on mice. On this basis, the relationship between MIOX and oxygen stress was verified in vitro. In addition, the subject group carried out the sugar kidney prescription. A randomized, double-blind, placebo-controlled, multicenter clinical trial with a randomized, double-blind, placebo-controlled, multicenter clinical trial to treat DKD effectiveness and safety, and to provide more comprehensive evidence for the antioxidant effect of DKD in the sugar kidney prescription. Objective: 1. to study the protective effect of sugar kidney prescription on renal injury in db/db mice with type 2 diabetic type white hair. Whether the mechanism is associated with antioxidant stress, reducing the expression of MIOX and reducing the expression of.2. to explore the expression of MIOX in the proximal renal tubular epithelial cells (NRK52E) in high glucose cultured rats and its role in the oxidative stress of proximal renal tubular epithelial cells..3. is randomized, double blind, placebo parallel control, due to the efficacy and safety of sugar kidney prescription in the treatment of DKD. The multicenter clinical trial has not been blinded. This study summarizes the key links affecting the quality control during the implementation of the test, and provides help to improve the quality of the study. Method: 1. the protective effect and mechanism of the 1. sugar kidney prescription on the renal protection and mechanism of 8 weeks old spontaneous 2 diabetes model db/db mice were randomly divided into model group (group db/db), sugar kidney prescription In the treatment group (group db/db+TSF), the db/m mice of the same week age were used as the normal control group (group db/m), and the group db/db+TSF mice were given the sugar kidney recipe (2.4g/kg-d) for 12 weeks, and the db/m and db/db mice were given the same amount of distilled water. All the mice were fed with the routine normal feed CIS feeding for 2 weeks, and the general state of the mice was observed daily. The body weight was measured every week, blood glucose was measured every 4 weeks, 24h urine albumin was tested for Twelfth weeks, and the mice were harvested for Twelfth weeks. After fasting the mice, the blood was taken in the eye canthus. The liver, kidney function and blood lipid were detected by the automatic biochemical analyzer. Then the abdominal cavity was opened and the kidneys were extracted and weighed. Some kidney groups were used for histopathological analysis, and the other part of the renal cortex was separated. In molecular biology test, renal tissue was stained by PAS staining, calculated the percentage of glomerular mesangial matrix, TBA method to detect serum, MDA content in urine, Wester blot. real-time fluorescence quantitative PCR, and immunohistochemical method to detect the oxidative stress related NADPH oxidase subunit in mouse kidney tissue, Nrf2 pathway, MIOX and podocyte standard The effect of.2. MIOX on oxidative stress induced by high glucose in renal tubular epithelial cells was stimulated by 5.5,10,20,30 mM concentration of glucose stimulated proximal renal tubular epithelial cells (NRK52E) 48h, and 30 mM glucose stimulated NRK52E cells 12.24.36.48.60h. Western blot method to detect intracellular MIOX. The dose and time dependence curve of NRK52E oxidative stress damage were used to determine the optimal dose and time. The MIOX gene siRNA Oligo interference sequence was designed, the highest knockdown efficiency was transfected into NRK52E cells, 6h was replaced by the final concentration 5.5,30mM glucose medium, 48h was collected after the collection of fine cell, and DCFH-DA probe was used to detect the intracellular ROS level, Western blo T detection of intracellular MIOX, Nox4, Nrf2, NQO1 protein expression.3. induction and summary of the efficacy and safety of sugar kidney prescription in the treatment of DKD efficacy and safety of random, double blind, placebo parallel control, the implementation of multicenter clinical trials of quality control points. Results: 1. sugar kidney can improve the spontaneous 2 diabetes model db/db mice renal tissue damage, its mechanism and reduction Lipid, antioxidant stress, protection of podocytes (1) renal protection: 0 to 12 weeks, group db/db was significantly higher than group db/m (P0.001), group db/db+TSF had weight decline in sixth weeks (16 weeks of age of mice) and continued to the end of the experiment (P0.05). Sugar kidney intervention could reduce the renal weight of db/db group (P0.01). During the experiment, the blood sugar of db/m group was maintained. The blood glucose of group db/db and db/db+TSF was higher than that of group db/m (P0.01), but there was no difference between the two groups. Compared with the db/m group, the serum AST and ALT increased (P0.01, P0.001) in the db/db group. Rat blood lipid disorder (P0.001, P0.05, P0.001). The 24h urine albumin in the group db/db was higher than that in the db/m group (P0.001). At the beginning of the eighth week, the 24h urine albumin in the group db/db+TSF decreased (P0.01), which was closer to the db/m group (P0.05) at the 12 week of administration. The percentage of glomerular mesangial matrix increased significantly (P0.01), the percentage of glomerular mesangial matrix decreased (P0.01) after the treatment of sugar kidney. (2) the antioxidant stress mechanism: the level of MDA in the serum of db/db mice was significantly higher than that of the db/m group (P0.01, P0.001) in the serum of db/db mice (P0.01, P0.001), and the glucose and kidney intervention could significantly decrease its content (P0.05, P0.0). 5) real time fluorescence quantitative PCR results showed that the levels of Nox2, Nox4 and p22phox mRNA in the kidney tissues of db/db mice were significantly higher than those in the db/m group (P0.01, P0.05, P0.01), but there was no difference in p47phox (P0.05). The expression of Nox2, Nox4 protein increased (P0.05, P0.001; P0.05, P0.001) in the renal tissue, and the expression of P0.05, P0.001, P0.05, P0.001 was lowered in the sugar kidney. Compared with group db/m (P0.05), glucose and kidney intervention could increase its expression (P0.05).Western blot and immunohistochemistry, and sugar kidney can down regulate the Nrf2, NQO1 protein expression (P0.05, P 0.001, P0.05, P0.001) in the renal tissue of db/db group. Besides, immunohistochemistry also showed that the expression was expressed in proximal renal tubules. The MIOX in the renal tubules increased significantly (P0.01, P0.001) in the group of mice (P0.05, P0.001). (3) the protection of the podocyte mechanism: real-time fluorescent quantitative PCR showed that nephrin, podocin and WT-1 mRNA in the db/db group were significantly lower than those of the db/m group. 1, P0.05). The results of immunohistochemistry also confirmed the effect of sugar kidney on the increase of nephrin podocin expression in group db/db mice (P0.01, P0.01).2. MIOX mediated high glucose induced renal tubular epithelial cell hydrogen stress (1) the effect of high glucose stimulation on the expression of MIOX: with the increase of glucose concentration, NRK52E cell MIOX, Nox4 expression gradually increased. The expression decreased gradually; 30 mM glucose stimulated NRK52E cell 0,12,24,36,48,60h, and the expression of MIOX, Nox4 and Nrf2 in the cells all increased first and then decreased, Nrf2 in the stimulation of 12h to peak, 48h decreased significantly, MIOX, Nox4 to 48h. (2) the effect of knock down on cell oxidative stress: screening to get the highest knock efficiency (72.1%) Gene siRNA Oligo interference sequence, transfected to NRK52E cells, after 6h to replace the final concentration of 5.5 and 30 mM glucose medium. After 48h, ROS content and MIOX, Nox4, NQO1 protein expression increased, Nrf2 expression decreased. Management, multilevel monitoring, the compliance of the researchers and subjects is an important link in the quality control of the experiment. Conclusion: 1. sugar kidney can reduce the model db/db, urinary albumin excretion, improve the pathological damage of renal tissue, and have the role of renal protection. The mechanism may improve the disorder of blood lipid and reduce oxidative stress. Low MIOX expression and protective foot cell injury related to.2. MIOX can promote the oxidative stress response of renal tubular epithelial cells induced by high glucose, and their role is related to the promotion of Nox4 expression. This result provides a new idea for further exploration of the target target of traditional Chinese medicine.
【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R259
，
本文编号：2154259
[Abstract]:Diabetic nephropathy (diabetic kidney disease, DKD) is the most important microvascular complication of diabetes. Once a large number of albuminuria appears, the renal function will be reduced. Due to its complicated pathogenesis, the curative effect of the present treatment is limited. The treatment of traditional Chinese medicine has some advantages in the control of DKD. Fang sugar kidney can reduce the urinary protein excretion of type 2 DKD patients, improve the estimated glomerular filtration rate and increase the plasma glutathione level. It has a certain protection of renal function and antioxidant effect. It is generally believed that oxidative stress plays an important role in the development of DKD, and the active oxygen cluster (reactive oxygen species, ROS) can activate a number of signal pathways. It causes endothelial cells, mesangial cells, podocytes and tubulointerstitial damage, and thus aggravates DKD. in recent years. The role of myo-inositol oxygenase (MIOX), which is mainly expressed in renal tubular epithelial cells, is gradually discovered in the development of DKD, which is considered to be involved in the regulation of intracellular ROS formation, which is closely related to oxidative stress. To further observe the new target of DKD intervention. In order to further observe the long-term effect of sugar kidney prescription intervention on the long-term effect of type 2 DKD and explore its possible mechanism, this study selected spontaneous 2 DKD mice model and observed the intervention effect of sugar kidney prescription on mice. On this basis, the relationship between MIOX and oxygen stress was verified in vitro. In addition, the subject group carried out the sugar kidney prescription. A randomized, double-blind, placebo-controlled, multicenter clinical trial with a randomized, double-blind, placebo-controlled, multicenter clinical trial to treat DKD effectiveness and safety, and to provide more comprehensive evidence for the antioxidant effect of DKD in the sugar kidney prescription. Objective: 1. to study the protective effect of sugar kidney prescription on renal injury in db/db mice with type 2 diabetic type white hair. Whether the mechanism is associated with antioxidant stress, reducing the expression of MIOX and reducing the expression of.2. to explore the expression of MIOX in the proximal renal tubular epithelial cells (NRK52E) in high glucose cultured rats and its role in the oxidative stress of proximal renal tubular epithelial cells..3. is randomized, double blind, placebo parallel control, due to the efficacy and safety of sugar kidney prescription in the treatment of DKD. The multicenter clinical trial has not been blinded. This study summarizes the key links affecting the quality control during the implementation of the test, and provides help to improve the quality of the study. Method: 1. the protective effect and mechanism of the 1. sugar kidney prescription on the renal protection and mechanism of 8 weeks old spontaneous 2 diabetes model db/db mice were randomly divided into model group (group db/db), sugar kidney prescription In the treatment group (group db/db+TSF), the db/m mice of the same week age were used as the normal control group (group db/m), and the group db/db+TSF mice were given the sugar kidney recipe (2.4g/kg-d) for 12 weeks, and the db/m and db/db mice were given the same amount of distilled water. All the mice were fed with the routine normal feed CIS feeding for 2 weeks, and the general state of the mice was observed daily. The body weight was measured every week, blood glucose was measured every 4 weeks, 24h urine albumin was tested for Twelfth weeks, and the mice were harvested for Twelfth weeks. After fasting the mice, the blood was taken in the eye canthus. The liver, kidney function and blood lipid were detected by the automatic biochemical analyzer. Then the abdominal cavity was opened and the kidneys were extracted and weighed. Some kidney groups were used for histopathological analysis, and the other part of the renal cortex was separated. In molecular biology test, renal tissue was stained by PAS staining, calculated the percentage of glomerular mesangial matrix, TBA method to detect serum, MDA content in urine, Wester blot. real-time fluorescence quantitative PCR, and immunohistochemical method to detect the oxidative stress related NADPH oxidase subunit in mouse kidney tissue, Nrf2 pathway, MIOX and podocyte standard The effect of.2. MIOX on oxidative stress induced by high glucose in renal tubular epithelial cells was stimulated by 5.5,10,20,30 mM concentration of glucose stimulated proximal renal tubular epithelial cells (NRK52E) 48h, and 30 mM glucose stimulated NRK52E cells 12.24.36.48.60h. Western blot method to detect intracellular MIOX. The dose and time dependence curve of NRK52E oxidative stress damage were used to determine the optimal dose and time. The MIOX gene siRNA Oligo interference sequence was designed, the highest knockdown efficiency was transfected into NRK52E cells, 6h was replaced by the final concentration 5.5,30mM glucose medium, 48h was collected after the collection of fine cell, and DCFH-DA probe was used to detect the intracellular ROS level, Western blo T detection of intracellular MIOX, Nox4, Nrf2, NQO1 protein expression.3. induction and summary of the efficacy and safety of sugar kidney prescription in the treatment of DKD efficacy and safety of random, double blind, placebo parallel control, the implementation of multicenter clinical trials of quality control points. Results: 1. sugar kidney can improve the spontaneous 2 diabetes model db/db mice renal tissue damage, its mechanism and reduction Lipid, antioxidant stress, protection of podocytes (1) renal protection: 0 to 12 weeks, group db/db was significantly higher than group db/m (P0.001), group db/db+TSF had weight decline in sixth weeks (16 weeks of age of mice) and continued to the end of the experiment (P0.05). Sugar kidney intervention could reduce the renal weight of db/db group (P0.01). During the experiment, the blood sugar of db/m group was maintained. The blood glucose of group db/db and db/db+TSF was higher than that of group db/m (P0.01), but there was no difference between the two groups. Compared with the db/m group, the serum AST and ALT increased (P0.01, P0.001) in the db/db group. Rat blood lipid disorder (P0.001, P0.05, P0.001). The 24h urine albumin in the group db/db was higher than that in the db/m group (P0.001). At the beginning of the eighth week, the 24h urine albumin in the group db/db+TSF decreased (P0.01), which was closer to the db/m group (P0.05) at the 12 week of administration. The percentage of glomerular mesangial matrix increased significantly (P0.01), the percentage of glomerular mesangial matrix decreased (P0.01) after the treatment of sugar kidney. (2) the antioxidant stress mechanism: the level of MDA in the serum of db/db mice was significantly higher than that of the db/m group (P0.01, P0.001) in the serum of db/db mice (P0.01, P0.001), and the glucose and kidney intervention could significantly decrease its content (P0.05, P0.0). 5) real time fluorescence quantitative PCR results showed that the levels of Nox2, Nox4 and p22phox mRNA in the kidney tissues of db/db mice were significantly higher than those in the db/m group (P0.01, P0.05, P0.01), but there was no difference in p47phox (P0.05). The expression of Nox2, Nox4 protein increased (P0.05, P0.001; P0.05, P0.001) in the renal tissue, and the expression of P0.05, P0.001, P0.05, P0.001 was lowered in the sugar kidney. Compared with group db/m (P0.05), glucose and kidney intervention could increase its expression (P0.05).Western blot and immunohistochemistry, and sugar kidney can down regulate the Nrf2, NQO1 protein expression (P0.05, P 0.001, P0.05, P0.001) in the renal tissue of db/db group. Besides, immunohistochemistry also showed that the expression was expressed in proximal renal tubules. The MIOX in the renal tubules increased significantly (P0.01, P0.001) in the group of mice (P0.05, P0.001). (3) the protection of the podocyte mechanism: real-time fluorescent quantitative PCR showed that nephrin, podocin and WT-1 mRNA in the db/db group were significantly lower than those of the db/m group. 1, P0.05). The results of immunohistochemistry also confirmed the effect of sugar kidney on the increase of nephrin podocin expression in group db/db mice (P0.01, P0.01).2. MIOX mediated high glucose induced renal tubular epithelial cell hydrogen stress (1) the effect of high glucose stimulation on the expression of MIOX: with the increase of glucose concentration, NRK52E cell MIOX, Nox4 expression gradually increased. The expression decreased gradually; 30 mM glucose stimulated NRK52E cell 0,12,24,36,48,60h, and the expression of MIOX, Nox4 and Nrf2 in the cells all increased first and then decreased, Nrf2 in the stimulation of 12h to peak, 48h decreased significantly, MIOX, Nox4 to 48h. (2) the effect of knock down on cell oxidative stress: screening to get the highest knock efficiency (72.1%) Gene siRNA Oligo interference sequence, transfected to NRK52E cells, after 6h to replace the final concentration of 5.5 and 30 mM glucose medium. After 48h, ROS content and MIOX, Nox4, NQO1 protein expression increased, Nrf2 expression decreased. Management, multilevel monitoring, the compliance of the researchers and subjects is an important link in the quality control of the experiment. Conclusion: 1. sugar kidney can reduce the model db/db, urinary albumin excretion, improve the pathological damage of renal tissue, and have the role of renal protection. The mechanism may improve the disorder of blood lipid and reduce oxidative stress. Low MIOX expression and protective foot cell injury related to.2. MIOX can promote the oxidative stress response of renal tubular epithelial cells induced by high glucose, and their role is related to the promotion of Nox4 expression. This result provides a new idea for further exploration of the target target of traditional Chinese medicine.
【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R259
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本文编号：2154259
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