高糖培养系膜细胞JAK_2/STAT_3信号转导通路活性的变化及对活性氧簇产生的影响
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摘要
近年来，世界范围内糖尿病(DM)的发病率明显上升。糖尿病是一组以高血糖为特征的代谢性疾病，长期高血糖状态可导致肾脏组织损伤，功能障碍和衰竭。糖尿病肾病(DN)是DM患者致残和死亡主要原因之一。据统计我国DN年发生率高达30％，而据美国肾脏资料统计，约50％的终末期肾衰(ESRD)由糖尿病所致，并呈逐年上升趋势。但目前研究尚未完全探明DN的发生发展机制，亦缺少有效的防治措施。因此，进一步探讨DN发病机制并且寻找阻止DN进展的方法已成为国内外学者研究的热点问题。
目前认为糖尿病肾病的发病过程中有多种酶和转录因子被激活，涉及多条细胞内信号转导通路(DAG／PKC、MAPK、JAK／STAT)杀伤靶器官，即高糖可以通过激活多种细胞内信号因子而导致靶器官损伤。其中Janns激酶／信号转导与转录激活子(Jams kinase／signal transducers and activators of transcription，JAK／STAT)信号传导通路在近年来越来越引起人们的重视，大量研究表明，其与系膜细胞的增殖，肥大及细胞外基质分泌有关。但此信号通路与肾脏疾病的关系的报道尚不十分清楚
目前的研究证实，氧化应激在糖尿病的发病过程中为一原发且独立的参与因素。活性氧簇(ROS)在糖尿病肾病(DN)的发生和发展过程中起着重要的作用，高糖可上调ROS，转化生长因子-β(TGF-β)在肾小球系膜细胞中的表达。ROS作为氧化应激的中心环节，参与DN的病理生理过程。还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶通过其产生的ROS，可改变肾脏血流动力学，激活细胞内信号转导等，导致糖尿病肾病(DN)的发生、发展。然而，在DN时ROS是否能够通过调控JAK_2／STAT_3通路的活性而影响DN的病理生理过程，还有待于进一步研究。
为此我们设计了以下实验：
目的
1．观察高糖培养大鼠肾小球系膜细胞(GMC)P-STAT_3的表达变化，并利用JAK_2阻断剂Ag-490阻断JAK_2／STAT_3通路，观察其对P-STAT_3表达变化的影响，探讨糖尿病状态下GMC JAK_2／STAT_3途径的活性变化。
2．观察高糖培养大鼠肾小球系膜细胞在不同时间点下活性氧簇(ROS)产生的量的变化。
3．通过NADPH氧化酶抑制剂(Apocynin)阻断ROS的产生，观察高糖培养下系膜细胞P-STAT_3的表达变化，探讨JAK_2／STAT_3通路与ROS之间的相互作用的影响。
方法
1．细胞培养：购买的大鼠肾小球系膜细胞株，选用DMEM培养基，在0.25％胰蛋白酶+0.02％EDTA消化液消化并吹打均匀后，按1：3比例进行传代，3-5天传代一次
2．分组：
(1)GMC同步化后分成：正常糖浓度组(含糖5.5mmol／L)；高糖浓度(25mmol／L)；甘露醇组(5.5mmol／L糖+19.5mmol甘露醇)；正常糖+AG-490(浓度10umol／L)组；高糖+AG-490(浓度10umol／L)组。继续观察培养24、48小时后用Western Blot及细胞免疫化学方法检测系膜细胞STAT_3、P-STAT_3表达的变化。
(2)GMC同步化后分成：正常糖浓度组(N，含糖5.5mmol／L)；高糖浓度组(H，含糖25mmol／L)；甘露醇组(M，含5.5mmol／L葡萄糖+19.5mmol／L甘露醇)；正常糖+Apocynin(N+A，Apocynin浓度为100umol／L)；高糖+Apocynin(H+A，Apocynin浓度为100umol／L)，分别于0、12h及36h收集上清液，用比色法检测系膜细胞产生ROS的量。
(3)NADPH氧化酶抑制剂Apocynin预处理，GMC同步化后分为正常糖浓度组(N，含糖5.5mmol╱L)；高糖浓度组(H，含糖25mmol／L)；甘露醇组(M，含糖5.5mmol／L，甘露醇19.5 mmol／L)：正常糖+Apocynin(N+A，Apocynin浓度为100umol／L)组；高糖+Apocynin(H+A，Apocynin浓度100umol／L)组；Apocynin提前1小时加入，与正常糖或高糖共同培养GMC48小时，培养结束后，用Western Blot方法检测系膜细胞P-STAT_3表达。
3．检测方法：
(1)用细胞免疫化学方法定性检测高糖环境下大鼠肾小球系膜细胞STAT_3、P-STAT_3表达的变化。
(2)用免疫印记法(Western Blot)半定量检测高糖状态下大鼠肾小球系膜细胞STAT_3、P-STAT_3表达的变化。
(3)用比色法检测高糖作用后培养系膜细胞上清液中ROS产生的水平。
结果
1．高糖培养大鼠肾小球系膜细胞在24小时和48小时P-STAT_3的表达较正常糖浓度组明显升高。甘露醇组与正常糖浓度组相比差异无统计学意义。而各组之间STAT_3表达差异无统计学意义。JAK_2阻断剂Ag-490可抑制高糖下P-STAT_3的表达，但对STAT_3表达无明显影响。
2．高糖条件下，ROS产生明显升高，NADPH氧化酶抑制剂Apocynin可明显降低ROS的产生水平。
3．高糖条件下，Apocynin经预处理后，在正常糖浓度和高糖浓度共同培养48小时，正常糖浓度组和正常糖+Apocynin组对比P-STAT_3的表达差异无明显差别；高糖+Apocynin组较正常糖浓度组P-STAT_3明显增加，但与高糖组相比P-STAT_3显著降低。
结论
1．高糖通过磷酸化方式激活大鼠肾小球系膜细胞(GMC)JAK_2／STAT_3信号转导通路，Ag-490可有效抑制GMC JAK_2／STAT_3信号转导通路活性的变化。
2．高糖作用下，肾小球系膜细胞ROS产生增加，并具有时间依赖性，NADPH氧化酶抑制剂Apocynin可有效抑制ROS的产生。
3．高糖激活JAK_2／STAT_3通路后，阻断ROS增加可部分抑制大鼠肾小球系膜细胞P-STAT_3的表达。
In recent years, the incidence of diabetes which is a chronic metabolic diseases characterized by hyperglycemia, in the world is significantly increased. Tissue Injury, dysfunction and failure can be found when the kidney is emerged in the long-term hyperglycemia. Today, diabetic nephropathy (DN) becomes one of most important factors causing patients disabled and death. And the incidence of DN-induced renal failure is rising annually. According to statistics, the incidence rate of DN has caught up to 47.66%, while in the United States more than 30% of end-stage renal failure (ESRD) is caused by diabetes, and the ratio is stepping upward annually.However, the present study shows neither definitely clear targets that could be blamed for the development of DN nor effective prevention and control measures. Therefore, to further exploring the pathogenesis of diabetic nephropathy and find ways to prevent DN progress is inevitable.
The activation of a variety of enzymes and transcription factors was involved in the process of diabetic nephropathy, which has been proved by many studies. That is to say, many cellular signal transduction pathways, such as DAG / PKC, MAPK, and JAK / STAT, were also involved in, which gave us a further explanation that target organs were damaged through activation of multiple intracellular signaling factors.
Among them, Janns Kinase/signal transducers and activators of transcription (JAK / STAT) signaling pathway has attracted people's attentions and numerous studies indicated that the proliferation of mesangial cells and the secretion of extracellular matrix were closely related to the pathway. But so far, few studies have focused on the relation between JAK / STAT pathway and the renal diseases, which awaits our further studies.
Oxidative stress is a primary and independent factor in the pathogenesis of diabetes.The deadly complication of diabetes - DN, has become a tremendous threat to human health. More and more evidences have proved that the reactive oxygen species (ROS) play an most important role in the start-up and development process of DN. Under high-glucose conditions, ROS can be raised, and then transforming growth factors-β(TGF-β) and extracellular matrix (ECM) express in the mesangial cells. As the central part, ROS is involved in the DN pathophysiological process. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can change the renal hemodynamics, impacting the remodeling of ECM and intracellular signal transductions, through its product - ROS. Whether can ROS regulate the signal pathway and leads to end-stage DN or not and if so what molecular mechanism it depends on is not described and proved clearly. And also, that whether ROS can affect the DN pathophysiological process by activating the JAK2/STAT3 pathway or not still stays unknown and awaits our further studies. To this end we have devised the following experiments:
1. To study the changes of JAK2/STAT3 pathway in the diabetic state by observing the changes of p-STAT_3 in the rat glomerular mesangiai cells (GMC) cultured in high glucose state.
2. To observe the levels of ROS in the GMC treated with high glucose.
3. Through the use of NADPH oxidase inhibitor (Apocynin) blocking the combination of ROS with its receptors, observe the P-STAT3 expression changes in the GMC cultured in high glucose state and study the effects of JAK_2/STAT_3 pathway on the generation of ROS and the secretion of ECM.
1. High glucose can induce the activation of JAK2/STAT3 pathway in the GMC:
Objective: To study the changes of JAK2/STAT3 signal pathway by observation the expression changes of p-STAT3 in rat GMC cultured in high-glucose state.
Methods: Synchronized GMCs are divided into following groups: the normal group (5.5mmol/L Glu), High glucose group (25mmol/L Glu), Mannitol group (5.5mmol/LGlu and 19.5mmol mannitol), the normal + AG-490 (10μmol / L), the high glucose + AG-490 (10μmol / L). After 24-48 hours' incubation, the GMCs were analyzed by Westem Blot and Immunocytochemistry to observe the changes of STAT3 and P-STAT3 expressions in mesangial cells.
Results: At 24h and 48h, the p-STAT3 expression in the GMC that cultured with high glucose is significantly higher than that in the normal group. And there is no statistical difference between the mannitol and the normal group; and also there are no significant differences among the groups on the STAT3 expressions. AG-490 inhibited P-STAT3 expression in the cells cultured with high glucose, but had no obvious effects on STAT3 expressions.
2.The levels of ROS in GMC cultured with high glucose:
Objective: To study the effects of high glucose on the generation of ROS in the rat GMC cultured under high-glucose state.
Methods: Synchronized GMCs are divided into following groups: the normal group (5.5mmol/L Glu), High glucose group (25mmol/L Glu), Mannitol group (5.5mmol/LGlu and 19.5mmol mannitol), the normal + Apocynin (100μmol/L), the high glucose + Apocynin (100μmol/L). Collect the cells at Oh, 12h and 36h respectively, and then determine the O_2~- levels with colorimetric method.
Results: At On, O_2~- level in each group is almost same and at 12h the O_2~-1evel in the High glucose group is obviously increased and higher than that in the normal (p<0.05). there is no significant difference between the Mannitol and the normal group on the O_2~- level and the O_2~- level in the high glucose + Apocynin is lower than that in the high glucose group (p<0.05). At 36h, the O_2~- levels in both the high glucose and the high glucose + Apocynin group are higher than that in the normal (p<0.01), but the O_2~- level of the high glucose + Apocynin group is significantly lower than that in the high glucose (p<0.01) and there are no statistical differences among the groups including the Mannitol, the normal, and the the normal + Apocynin.
3.Effects of high glucose on the ROS expression in GMC after activation of JAK2/STAT3 pathway
Observe: the changes of p-STAT3 expression in GMC under the high glucose condition when using the ROS blocker Apocynin to study the relation between ROS and JAK2/STAT3 pathway.
Methods: Synchronized GMCs are divided into following groups: the normal group (N, 5.5mmol/L Glu), High glucose group (H, 25mmol/L Glu), the normal + Apocynin (NA, 100μmol/L), the high glucose + Apocynin (HA, 100μmol/L). After the treatment with Apocynin for 1h, incubate the GMC of each group for 48 hours. The change of p-STAT3 expression in GMC was detected by Western Blot.
Results: The p-STAT3 expression in GMC of the H group is obviously higher than that in the N group at 48h; there is no difference between the NA and the N groups; the p-STAT3 expression in the HA group is significantly decreased but still higher than that in the N group.
Conclusion
1. Under high glucose conditions, JAK2/STAT3 signal transduction pathway can be activated in the phosphorylation manner but AG-490 can inhibit the activation. The JAK_2/STAT_3 pathway may be involved in the regulation of the secretion of ECM.
2. ROS in GMC can be increased in a time-dependent manner under high glucose condition and NADPH oxidase is the stimulator for the generation of ROS so the Apocynin ofNADPH oxidase inhibitors can inhibit ROS level effectively.
3. Under high glucose condition with the activated JAK2/STAT3 pathway, blockage of ROS can only decrease the p-STAT3 expression in the GMC partly, which means the pathway may be involved in the regulation of the secretion of ECM.
目前认为糖尿病肾病的发病过程中有多种酶和转录因子被激活，涉及多条细胞内信号转导通路(DAG／PKC、MAPK、JAK／STAT)杀伤靶器官，即高糖可以通过激活多种细胞内信号因子而导致靶器官损伤。其中Janns激酶／信号转导与转录激活子(Jams kinase／signal transducers and activators of transcription，JAK／STAT)信号传导通路在近年来越来越引起人们的重视，大量研究表明，其与系膜细胞的增殖，肥大及细胞外基质分泌有关。但此信号通路与肾脏疾病的关系的报道尚不十分清楚
目前的研究证实，氧化应激在糖尿病的发病过程中为一原发且独立的参与因素。活性氧簇(ROS)在糖尿病肾病(DN)的发生和发展过程中起着重要的作用，高糖可上调ROS，转化生长因子-β(TGF-β)在肾小球系膜细胞中的表达。ROS作为氧化应激的中心环节，参与DN的病理生理过程。还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶通过其产生的ROS，可改变肾脏血流动力学，激活细胞内信号转导等，导致糖尿病肾病(DN)的发生、发展。然而，在DN时ROS是否能够通过调控JAK_2／STAT_3通路的活性而影响DN的病理生理过程，还有待于进一步研究。
为此我们设计了以下实验：
目的
1．观察高糖培养大鼠肾小球系膜细胞(GMC)P-STAT_3的表达变化，并利用JAK_2阻断剂Ag-490阻断JAK_2／STAT_3通路，观察其对P-STAT_3表达变化的影响，探讨糖尿病状态下GMC JAK_2／STAT_3途径的活性变化。
2．观察高糖培养大鼠肾小球系膜细胞在不同时间点下活性氧簇(ROS)产生的量的变化。
3．通过NADPH氧化酶抑制剂(Apocynin)阻断ROS的产生，观察高糖培养下系膜细胞P-STAT_3的表达变化，探讨JAK_2／STAT_3通路与ROS之间的相互作用的影响。
方法
1．细胞培养：购买的大鼠肾小球系膜细胞株，选用DMEM培养基，在0.25％胰蛋白酶+0.02％EDTA消化液消化并吹打均匀后，按1：3比例进行传代，3-5天传代一次
2．分组：
(1)GMC同步化后分成：正常糖浓度组(含糖5.5mmol／L)；高糖浓度(25mmol／L)；甘露醇组(5.5mmol／L糖+19.5mmol甘露醇)；正常糖+AG-490(浓度10umol／L)组；高糖+AG-490(浓度10umol／L)组。继续观察培养24、48小时后用Western Blot及细胞免疫化学方法检测系膜细胞STAT_3、P-STAT_3表达的变化。
(2)GMC同步化后分成：正常糖浓度组(N，含糖5.5mmol／L)；高糖浓度组(H，含糖25mmol／L)；甘露醇组(M，含5.5mmol／L葡萄糖+19.5mmol／L甘露醇)；正常糖+Apocynin(N+A，Apocynin浓度为100umol／L)；高糖+Apocynin(H+A，Apocynin浓度为100umol／L)，分别于0、12h及36h收集上清液，用比色法检测系膜细胞产生ROS的量。
(3)NADPH氧化酶抑制剂Apocynin预处理，GMC同步化后分为正常糖浓度组(N，含糖5.5mmol╱L)；高糖浓度组(H，含糖25mmol／L)；甘露醇组(M，含糖5.5mmol／L，甘露醇19.5 mmol／L)：正常糖+Apocynin(N+A，Apocynin浓度为100umol／L)组；高糖+Apocynin(H+A，Apocynin浓度100umol／L)组；Apocynin提前1小时加入，与正常糖或高糖共同培养GMC48小时，培养结束后，用Western Blot方法检测系膜细胞P-STAT_3表达。
3．检测方法：
(1)用细胞免疫化学方法定性检测高糖环境下大鼠肾小球系膜细胞STAT_3、P-STAT_3表达的变化。
(2)用免疫印记法(Western Blot)半定量检测高糖状态下大鼠肾小球系膜细胞STAT_3、P-STAT_3表达的变化。
(3)用比色法检测高糖作用后培养系膜细胞上清液中ROS产生的水平。
结果
1．高糖培养大鼠肾小球系膜细胞在24小时和48小时P-STAT_3的表达较正常糖浓度组明显升高。甘露醇组与正常糖浓度组相比差异无统计学意义。而各组之间STAT_3表达差异无统计学意义。JAK_2阻断剂Ag-490可抑制高糖下P-STAT_3的表达，但对STAT_3表达无明显影响。
2．高糖条件下，ROS产生明显升高，NADPH氧化酶抑制剂Apocynin可明显降低ROS的产生水平。
3．高糖条件下，Apocynin经预处理后，在正常糖浓度和高糖浓度共同培养48小时，正常糖浓度组和正常糖+Apocynin组对比P-STAT_3的表达差异无明显差别；高糖+Apocynin组较正常糖浓度组P-STAT_3明显增加，但与高糖组相比P-STAT_3显著降低。
结论
1．高糖通过磷酸化方式激活大鼠肾小球系膜细胞(GMC)JAK_2／STAT_3信号转导通路，Ag-490可有效抑制GMC JAK_2／STAT_3信号转导通路活性的变化。
2．高糖作用下，肾小球系膜细胞ROS产生增加，并具有时间依赖性，NADPH氧化酶抑制剂Apocynin可有效抑制ROS的产生。
3．高糖激活JAK_2／STAT_3通路后，阻断ROS增加可部分抑制大鼠肾小球系膜细胞P-STAT_3的表达。
In recent years, the incidence of diabetes which is a chronic metabolic diseases characterized by hyperglycemia, in the world is significantly increased. Tissue Injury, dysfunction and failure can be found when the kidney is emerged in the long-term hyperglycemia. Today, diabetic nephropathy (DN) becomes one of most important factors causing patients disabled and death. And the incidence of DN-induced renal failure is rising annually. According to statistics, the incidence rate of DN has caught up to 47.66%, while in the United States more than 30% of end-stage renal failure (ESRD) is caused by diabetes, and the ratio is stepping upward annually.However, the present study shows neither definitely clear targets that could be blamed for the development of DN nor effective prevention and control measures. Therefore, to further exploring the pathogenesis of diabetic nephropathy and find ways to prevent DN progress is inevitable.
The activation of a variety of enzymes and transcription factors was involved in the process of diabetic nephropathy, which has been proved by many studies. That is to say, many cellular signal transduction pathways, such as DAG / PKC, MAPK, and JAK / STAT, were also involved in, which gave us a further explanation that target organs were damaged through activation of multiple intracellular signaling factors.
Among them, Janns Kinase/signal transducers and activators of transcription (JAK / STAT) signaling pathway has attracted people's attentions and numerous studies indicated that the proliferation of mesangial cells and the secretion of extracellular matrix were closely related to the pathway. But so far, few studies have focused on the relation between JAK / STAT pathway and the renal diseases, which awaits our further studies.
Oxidative stress is a primary and independent factor in the pathogenesis of diabetes.The deadly complication of diabetes - DN, has become a tremendous threat to human health. More and more evidences have proved that the reactive oxygen species (ROS) play an most important role in the start-up and development process of DN. Under high-glucose conditions, ROS can be raised, and then transforming growth factors-β(TGF-β) and extracellular matrix (ECM) express in the mesangial cells. As the central part, ROS is involved in the DN pathophysiological process. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can change the renal hemodynamics, impacting the remodeling of ECM and intracellular signal transductions, through its product - ROS. Whether can ROS regulate the signal pathway and leads to end-stage DN or not and if so what molecular mechanism it depends on is not described and proved clearly. And also, that whether ROS can affect the DN pathophysiological process by activating the JAK2/STAT3 pathway or not still stays unknown and awaits our further studies. To this end we have devised the following experiments:
1. To study the changes of JAK2/STAT3 pathway in the diabetic state by observing the changes of p-STAT_3 in the rat glomerular mesangiai cells (GMC) cultured in high glucose state.
2. To observe the levels of ROS in the GMC treated with high glucose.
3. Through the use of NADPH oxidase inhibitor (Apocynin) blocking the combination of ROS with its receptors, observe the P-STAT3 expression changes in the GMC cultured in high glucose state and study the effects of JAK_2/STAT_3 pathway on the generation of ROS and the secretion of ECM.
1. High glucose can induce the activation of JAK2/STAT3 pathway in the GMC:
Objective: To study the changes of JAK2/STAT3 signal pathway by observation the expression changes of p-STAT3 in rat GMC cultured in high-glucose state.
Methods: Synchronized GMCs are divided into following groups: the normal group (5.5mmol/L Glu), High glucose group (25mmol/L Glu), Mannitol group (5.5mmol/LGlu and 19.5mmol mannitol), the normal + AG-490 (10μmol / L), the high glucose + AG-490 (10μmol / L). After 24-48 hours' incubation, the GMCs were analyzed by Westem Blot and Immunocytochemistry to observe the changes of STAT3 and P-STAT3 expressions in mesangial cells.
Results: At 24h and 48h, the p-STAT3 expression in the GMC that cultured with high glucose is significantly higher than that in the normal group. And there is no statistical difference between the mannitol and the normal group; and also there are no significant differences among the groups on the STAT3 expressions. AG-490 inhibited P-STAT3 expression in the cells cultured with high glucose, but had no obvious effects on STAT3 expressions.
2.The levels of ROS in GMC cultured with high glucose:
Objective: To study the effects of high glucose on the generation of ROS in the rat GMC cultured under high-glucose state.
Methods: Synchronized GMCs are divided into following groups: the normal group (5.5mmol/L Glu), High glucose group (25mmol/L Glu), Mannitol group (5.5mmol/LGlu and 19.5mmol mannitol), the normal + Apocynin (100μmol/L), the high glucose + Apocynin (100μmol/L). Collect the cells at Oh, 12h and 36h respectively, and then determine the O_2~- levels with colorimetric method.
Results: At On, O_2~- level in each group is almost same and at 12h the O_2~-1evel in the High glucose group is obviously increased and higher than that in the normal (p<0.05). there is no significant difference between the Mannitol and the normal group on the O_2~- level and the O_2~- level in the high glucose + Apocynin is lower than that in the high glucose group (p<0.05). At 36h, the O_2~- levels in both the high glucose and the high glucose + Apocynin group are higher than that in the normal (p<0.01), but the O_2~- level of the high glucose + Apocynin group is significantly lower than that in the high glucose (p<0.01) and there are no statistical differences among the groups including the Mannitol, the normal, and the the normal + Apocynin.
3.Effects of high glucose on the ROS expression in GMC after activation of JAK2/STAT3 pathway
Observe: the changes of p-STAT3 expression in GMC under the high glucose condition when using the ROS blocker Apocynin to study the relation between ROS and JAK2/STAT3 pathway.
Methods: Synchronized GMCs are divided into following groups: the normal group (N, 5.5mmol/L Glu), High glucose group (H, 25mmol/L Glu), the normal + Apocynin (NA, 100μmol/L), the high glucose + Apocynin (HA, 100μmol/L). After the treatment with Apocynin for 1h, incubate the GMC of each group for 48 hours. The change of p-STAT3 expression in GMC was detected by Western Blot.
Results: The p-STAT3 expression in GMC of the H group is obviously higher than that in the N group at 48h; there is no difference between the NA and the N groups; the p-STAT3 expression in the HA group is significantly decreased but still higher than that in the N group.
Conclusion
1. Under high glucose conditions, JAK2/STAT3 signal transduction pathway can be activated in the phosphorylation manner but AG-490 can inhibit the activation. The JAK_2/STAT_3 pathway may be involved in the regulation of the secretion of ECM.
2. ROS in GMC can be increased in a time-dependent manner under high glucose condition and NADPH oxidase is the stimulator for the generation of ROS so the Apocynin ofNADPH oxidase inhibitors can inhibit ROS level effectively.
3. Under high glucose condition with the activated JAK2/STAT3 pathway, blockage of ROS can only decrease the p-STAT3 expression in the GMC partly, which means the pathway may be involved in the regulation of the secretion of ECM.
引文
1 陆菊明，潘长玉，田慧等。糖耐量减低患者尿蛋白排除率观察。中国糖尿病杂志，1995，3：210-213
2 潘长玉，陆菊明，田慧等2型糖尿病患者初诊时血管并发症患病率的调查分析。中华内分泌代谢杂志，1997，13：201-205
3 Ha H. Lee HB. Reactive Oxygen species amplify glucose signalling in renal cells cultured under high glucose and in diabetic kidney. Nephrology(carlton). 2005 oct; 10suppl: S7-10
4 Amiri F, Shaws. Wang X. et al. Angiotens: 11 activaction of the JAK/STAT pathway in messangial cells is altered by high glucose. Kidney lnt. 2002 May: 61(5): 1605-16.
5 Guh JY, Huang JS. Chen HC. et al. Advanced glycation end product-induced productproliferation in NRK-49F CEUS is dependent on the Jak_2/STATS pathway and cyclin DiAnJ Kidney Dis. 2001 Nov; 38(5): 1096-104.
6 Huang Js, Guh JY. Huang WC. Yang ML. et al. Role of the Jarms Kinase (JAK)/signal transductters and activators of transcription(STAT) Cascade in advanced glycation end-product-induced celluler mitogenesis in NRK-49F cells. Biochem J.1999 Aug 15:342.
7 Wang X, Shaw S. Amiri F, et al. Inhibition of the Jak/STAT Signaling pathway prevents the high glucose-induced increase in TGF-β eta and fibronectin synthesis in messangial cells. Diabetes. 2002 Dec; 51(12): 3505-9.
8 Sean Shaw, Xiaodan Wang, Heather Redd, et al. High Glucose Augments the Angiotensin Ⅱ-induced Activation of JAK2 in Vascular Smooth Muscle cells via the polyol pathway, J. Biol. Chem, 2003(278),Issue 33, 30634-30641.
9 Inoue H, Ogawa W, Ozaki M, et al. Role of STAT-3 in Regulation of Hepatic Gluconeogenic Genes and Carbohydrate Metabolism in vivo. Nature Medicine 2004. 10(2):168-174.
10 Kang D. Hamasaki N. Mitochondrial Oxidative Stress and mitochondrial DNA. Clin Chem Lab Med. 2003. 41(10):1281-8.
11 Ha H. Yang Y, Lee HB. Mechanisms of reactive oxygen species generation in LLC-PKI cells cultured under high glucose. J Am Soc Nephrol. 2002. 13:531A.
12 Shiose A. Kuroda J. Tsuruga K. et al. A novel Superoxide-producing NAD(P)H Oxidase in Kidney. J Biol Chem, 2001, 276:1417-1423.
13 Lai MA, Korner A, Matsuo Y, et al. Combined antioxidant and comT Inhibitor treatment reverses renal abnormalities in diabetic rats. Diabetes; 2000, 49:1381-1389.
14 Melhem MF, Craven PA, LiaChenKo J, et al. α -Lipoic acid atternantes hyperglycemia and prevents glomerular mesangial matrix expansion in diabetes. J Am Soc Nephrol, 2002, 13:108-116.
15 Studer RK, Cerubertis FR, YU HY, et al[J] Metabolism, 1997, 46:91829251.
16 HaH, YuMR, Choi YJ, et al. Activation of protein kinase C-[delta] and C-[epsilon] by oxidative stress in early diabetes rat kidney Dis , 2001, 38(S_1):S204
17 Traub 0, Monia BP, Dean NM, et al. PKC-epsilon is require for mechano sensitive activation of ERKl/2 in endothelial cells. J BioChem, 1997, 272(50):31251-31257
18 Florence Legendre, Jayesh Dudhia, Jean-Pierre. Pujol, et al. JAK/STAT but Not ERK1/ERK2 pathway Mediates Interleukin (IL)-6/Solube IL-6R Down-regulation of Type II Collagen, Aggrecan Core, And Link Protein Transcription in Articular Chondrocytes. J. Biol. Chem, Vol. 278, Issue 5, 2903-2912, January 31, 2003.
19 Yu-Ting Xuan, Yiru Guo, Hui Han, et al. An essential role of the JAK-STAT pathway in ischemic preconditioning. Biochemistry 2001, 98(16), 9050-9055.
20 Allen C, Gao. The role of Stat3 Activation in Androgen Receptor Signaling and PROState Cancer. Annual rept. 2005, 1:2004-30.
21 Eduardo Mascarenol, M、 A、 Q. Siddiqui. The role of Jak/STAT Signaling in heart tissue renin-angiotension System. Molecular and cellular Biochemistry, 2000, 212, (9); 171-175.
22 Park SK, Kim Ja, Seomum Y, et al. Hydrogen peroxide Is a Novel Inducer of Connective Tissue Growth Factor. Biochemical and Biophysical Research Communication, 2001, 284(4), 966-971.
23 EJ Lewis, LG Hunsicker, RP Bain, et al. The Effect of Angiotensin- Converting- Enzyme Inhibition on Diabetic Nephrophathy 1993, Diabetes Care, 27:1060-1065.
24 Sugimoto H, Shikita K, Matsuda M, et al. Increased expression of endothelial cell nitric oxide synthase in afferent and glomerular endothelial ceils is involved in glomerular hyperf iltration of diabetic nephropathy. Diabetologia, 1998, 41(12):1426.
25 Kern PA, Ranganathan S, Li C, Wood 2, et al. Adipose tissue tumor necROSis factor and interleukin-6 expression in human obesity and insulin resistance. Am J physiol Endocrinol Metab 2001:280:E745-51.
26 Y. Moriwaki. Elevated levdls of interleukin-18 and tumor necROSis factor-alpha in Serum of patients with type 2 diabetes mellitus: Relation ship with diabetic nephropathy. Metabolism, 2003, (52)5,605-608.
27 John De Vos, Michel Jourdan, Montpellier, et al. JAK_2tyR0Sine kinase inhibitor tyrphostin AG490 downregulates the mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription(STAT) pathways and induces apoptosis in myeloma cells. British Journal of Haematology, 2000, 109(4); 823-828.
28 Bank N, Ayni djian HS, kidney Int,2003;209-590
29 John De Vos, Michel Jourdan, Montpellier, et al. JAK2 tyeosine kinase inhibitor tyrphostin AG-490 downregulates the mitogen-activateed protein kinase (MAPK) and activator of transcription (STAT)pathways and induces apoptosis in myeloma cells. British Journal of Haematology, 2000,109(4);823-828
30 Kern PA , Ranganathan S, Li C, Wood 2, et al. Adipose tissue tumor necROSis factor and interleukin-6 expression in human obesity and insulin resistance.Am J physiol Endocrinol Metab 2001:280:E745-51.
31 Y. Moriwaki. Elevated levels of interleukin-18 and tumor nrcROSis factor-alpha in Serum of patients with type 2 diabrtrs mellitus:Relation ship with diabetic nephropathy. Metabolism, 2003, (52)5, 605-608.
32 Hannken T, Schroeder R, Stahl RA, et al. Angiotensin II-mediated expression of P~(27kipl) and induction of cellular hypertrophy in renal tubular cells depend on the generation of oxygen radicals. Kidney Int, 1998, S_4:1923-1933.
33 el Benna J. Faust Lp, Babior BM. The phosphorylation of the respiratory burst oxidase component p~(47)P~(hox) during neutrophil activation. Phosphorylation of aites recognized by protein kinase C and by proline-directed kinases. J Biol Chem, 1994,269:23431-23436.
34 Touyz RM. Chen X, Tabet F, et al. Expression of a functionally active gp91phox-containing neutrophil-tyke NADPH oxidase in smooth muscle cells from human resistance artense: regulation by angiotension II. Circ Res. 2002, 90:1205-1213.
35 Babior BM. NADPH oxidase:an update,blood, 1999,93:1464-1476.
36 Studer RK, Cerubertis FR, Yu H Y, et al. Antioxidant inhibition of protein kinase C-signaled increase in transforming growth factor-beta in mesangile cells [J]. J Metabolism, 1997,46:918.
37 Wilmer WA, Dixon CL, Herbert C, et, al. Chronic exposure of human mesangial cells to high glucose environment activates the p38MAPK pathway[J].J Kidney Int, 2001, 60:858.
1 陆菊明，潘长玉，田慧等。糖耐量减低患者尿蛋白排除率观察。中国糖尿病杂志，1995，3：210-213
2 潘长玉，陆菊明，田慧等2型糖尿病患者初诊时血管并发症患病率的调查分析。中华内分泌代谢杂志，1997，13：201-205
3 Friedman EA,, Endocrinol Metab Clin North Am, 1996;25(2):293-324
4 Centers for Disease Control and Prevention (CDC).Incidence of end-stage renal disease among persons with diabetes-United States,1990-2002. MMWR Morb Mortal Wkly Rep.2005 Nov 4;54(43):1097-100
5 Marjorie Dunlop. Aldose reductase and the role of the polyol pathway in diabetic nephrophathy. Kidney International, 2000(58),S3-S12; 1523-1755
6 Nishizuka Y. Ihtracellular Signalling by hydrolysis of phospholipids and activating of protein Kinase C. Science, 1992; 258:607
7 Traub O,Monia Bp,Dean Nm,et al.Pkc-epsilon is required for mechano sensitive activation of ERK1/2 in endothelial cells. J.Biolchem, 1997,272(50):31251-31257
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44 史永红，段惠军，王丽军等，氯沙坦对糖尿病大鼠肾小球信号转导和转录活化因子表达的影响。中国药理学通报。2005 Marj21(3)：323-326
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2 潘长玉，陆菊明，田慧等2型糖尿病患者初诊时血管并发症患病率的调查分析。中华内分泌代谢杂志，1997，13：201-205
3 Ha H. Lee HB. Reactive Oxygen species amplify glucose signalling in renal cells cultured under high glucose and in diabetic kidney. Nephrology(carlton). 2005 oct; 10suppl: S7-10
4 Amiri F, Shaws. Wang X. et al. Angiotens: 11 activaction of the JAK/STAT pathway in messangial cells is altered by high glucose. Kidney lnt. 2002 May: 61(5): 1605-16.
5 Guh JY, Huang JS. Chen HC. et al. Advanced glycation end product-induced productproliferation in NRK-49F CEUS is dependent on the Jak_2/STATS pathway and cyclin DiAnJ Kidney Dis. 2001 Nov; 38(5): 1096-104.
6 Huang Js, Guh JY. Huang WC. Yang ML. et al. Role of the Jarms Kinase (JAK)/signal transductters and activators of transcription(STAT) Cascade in advanced glycation end-product-induced celluler mitogenesis in NRK-49F cells. Biochem J.1999 Aug 15:342.
7 Wang X, Shaw S. Amiri F, et al. Inhibition of the Jak/STAT Signaling pathway prevents the high glucose-induced increase in TGF-β eta and fibronectin synthesis in messangial cells. Diabetes. 2002 Dec; 51(12): 3505-9.
8 Sean Shaw, Xiaodan Wang, Heather Redd, et al. High Glucose Augments the Angiotensin Ⅱ-induced Activation of JAK2 in Vascular Smooth Muscle cells via the polyol pathway, J. Biol. Chem, 2003(278),Issue 33, 30634-30641.
9 Inoue H, Ogawa W, Ozaki M, et al. Role of STAT-3 in Regulation of Hepatic Gluconeogenic Genes and Carbohydrate Metabolism in vivo. Nature Medicine 2004. 10(2):168-174.
10 Kang D. Hamasaki N. Mitochondrial Oxidative Stress and mitochondrial DNA. Clin Chem Lab Med. 2003. 41(10):1281-8.
11 Ha H. Yang Y, Lee HB. Mechanisms of reactive oxygen species generation in LLC-PKI cells cultured under high glucose. J Am Soc Nephrol. 2002. 13:531A.
12 Shiose A. Kuroda J. Tsuruga K. et al. A novel Superoxide-producing NAD(P)H Oxidase in Kidney. J Biol Chem, 2001, 276:1417-1423.
13 Lai MA, Korner A, Matsuo Y, et al. Combined antioxidant and comT Inhibitor treatment reverses renal abnormalities in diabetic rats. Diabetes; 2000, 49:1381-1389.
14 Melhem MF, Craven PA, LiaChenKo J, et al. α -Lipoic acid atternantes hyperglycemia and prevents glomerular mesangial matrix expansion in diabetes. J Am Soc Nephrol, 2002, 13:108-116.
15 Studer RK, Cerubertis FR, YU HY, et al[J] Metabolism, 1997, 46:91829251.
16 HaH, YuMR, Choi YJ, et al. Activation of protein kinase C-[delta] and C-[epsilon] by oxidative stress in early diabetes rat kidney Dis , 2001, 38(S_1):S204
17 Traub 0, Monia BP, Dean NM, et al. PKC-epsilon is require for mechano sensitive activation of ERKl/2 in endothelial cells. J BioChem, 1997, 272(50):31251-31257
18 Florence Legendre, Jayesh Dudhia, Jean-Pierre. Pujol, et al. JAK/STAT but Not ERK1/ERK2 pathway Mediates Interleukin (IL)-6/Solube IL-6R Down-regulation of Type II Collagen, Aggrecan Core, And Link Protein Transcription in Articular Chondrocytes. J. Biol. Chem, Vol. 278, Issue 5, 2903-2912, January 31, 2003.
19 Yu-Ting Xuan, Yiru Guo, Hui Han, et al. An essential role of the JAK-STAT pathway in ischemic preconditioning. Biochemistry 2001, 98(16), 9050-9055.
20 Allen C, Gao. The role of Stat3 Activation in Androgen Receptor Signaling and PROState Cancer. Annual rept. 2005, 1:2004-30.
21 Eduardo Mascarenol, M、 A、 Q. Siddiqui. The role of Jak/STAT Signaling in heart tissue renin-angiotension System. Molecular and cellular Biochemistry, 2000, 212, (9); 171-175.
22 Park SK, Kim Ja, Seomum Y, et al. Hydrogen peroxide Is a Novel Inducer of Connective Tissue Growth Factor. Biochemical and Biophysical Research Communication, 2001, 284(4), 966-971.
23 EJ Lewis, LG Hunsicker, RP Bain, et al. The Effect of Angiotensin- Converting- Enzyme Inhibition on Diabetic Nephrophathy 1993, Diabetes Care, 27:1060-1065.
24 Sugimoto H, Shikita K, Matsuda M, et al. Increased expression of endothelial cell nitric oxide synthase in afferent and glomerular endothelial ceils is involved in glomerular hyperf iltration of diabetic nephropathy. Diabetologia, 1998, 41(12):1426.
25 Kern PA, Ranganathan S, Li C, Wood 2, et al. Adipose tissue tumor necROSis factor and interleukin-6 expression in human obesity and insulin resistance. Am J physiol Endocrinol Metab 2001:280:E745-51.
26 Y. Moriwaki. Elevated levdls of interleukin-18 and tumor necROSis factor-alpha in Serum of patients with type 2 diabetes mellitus: Relation ship with diabetic nephropathy. Metabolism, 2003, (52)5,605-608.
27 John De Vos, Michel Jourdan, Montpellier, et al. JAK_2tyR0Sine kinase inhibitor tyrphostin AG490 downregulates the mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription(STAT) pathways and induces apoptosis in myeloma cells. British Journal of Haematology, 2000, 109(4); 823-828.
28 Bank N, Ayni djian HS, kidney Int,2003;209-590
29 John De Vos, Michel Jourdan, Montpellier, et al. JAK2 tyeosine kinase inhibitor tyrphostin AG-490 downregulates the mitogen-activateed protein kinase (MAPK) and activator of transcription (STAT)pathways and induces apoptosis in myeloma cells. British Journal of Haematology, 2000,109(4);823-828
30 Kern PA , Ranganathan S, Li C, Wood 2, et al. Adipose tissue tumor necROSis factor and interleukin-6 expression in human obesity and insulin resistance.Am J physiol Endocrinol Metab 2001:280:E745-51.
31 Y. Moriwaki. Elevated levels of interleukin-18 and tumor nrcROSis factor-alpha in Serum of patients with type 2 diabrtrs mellitus:Relation ship with diabetic nephropathy. Metabolism, 2003, (52)5, 605-608.
32 Hannken T, Schroeder R, Stahl RA, et al. Angiotensin II-mediated expression of P~(27kipl) and induction of cellular hypertrophy in renal tubular cells depend on the generation of oxygen radicals. Kidney Int, 1998, S_4:1923-1933.
33 el Benna J. Faust Lp, Babior BM. The phosphorylation of the respiratory burst oxidase component p~(47)P~(hox) during neutrophil activation. Phosphorylation of aites recognized by protein kinase C and by proline-directed kinases. J Biol Chem, 1994,269:23431-23436.
34 Touyz RM. Chen X, Tabet F, et al. Expression of a functionally active gp91phox-containing neutrophil-tyke NADPH oxidase in smooth muscle cells from human resistance artense: regulation by angiotension II. Circ Res. 2002, 90:1205-1213.
35 Babior BM. NADPH oxidase:an update,blood, 1999,93:1464-1476.
36 Studer RK, Cerubertis FR, Yu H Y, et al. Antioxidant inhibition of protein kinase C-signaled increase in transforming growth factor-beta in mesangile cells [J]. J Metabolism, 1997,46:918.
37 Wilmer WA, Dixon CL, Herbert C, et, al. Chronic exposure of human mesangial cells to high glucose environment activates the p38MAPK pathway[J].J Kidney Int, 2001, 60:858.
1 陆菊明，潘长玉，田慧等。糖耐量减低患者尿蛋白排除率观察。中国糖尿病杂志，1995，3：210-213
2 潘长玉，陆菊明，田慧等2型糖尿病患者初诊时血管并发症患病率的调查分析。中华内分泌代谢杂志，1997，13：201-205
3 Friedman EA,, Endocrinol Metab Clin North Am, 1996;25(2):293-324
4 Centers for Disease Control and Prevention (CDC).Incidence of end-stage renal disease among persons with diabetes-United States,1990-2002. MMWR Morb Mortal Wkly Rep.2005 Nov 4;54(43):1097-100
5 Marjorie Dunlop. Aldose reductase and the role of the polyol pathway in diabetic nephrophathy. Kidney International, 2000(58),S3-S12; 1523-1755
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44 史永红，段惠军，王丽军等，氯沙坦对糖尿病大鼠肾小球信号转导和转录活化因子表达的影响。中国药理学通报。2005 Marj21(3)：323-326
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