| 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-STAT3 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 JAK2/STAT3 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 O2- levels with colorimetric method. Results: At On, O2- level in each group is almost same and at 12h the O2-1evel 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 O2- level and the O2- level in the high glucose + Apocynin is lower than that in the high glucose group (p<0.05). At 36h, the O2- levels in both the high glucose and the high glucose + Apocynin group are higher than that in the normal (p<0.01), but the O2- 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 pathwayObserve: 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 JAK2/STAT3 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.
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