| BackgroundThe original damage caused by diabetes mellitus is due to all kinds of diabetic complications. So it is very important for us to find out the mechanisms of these diabetic complications. The traditional ideas of the mechanisms of diabetic complications include : increased polyol pathway flux, increased intracellular formation of AGEs, activation of protein kinase C, increased flux through the hexosamine pathway. Recently, a new theory — unifying mechanism, was put forword by professor Michael A. Brownlee,which means that the four traditional pathyways are the result of the overproduct of superoxide radical produced in the cell mitochondrial,in other words, the result of oxidative stress.Oxidative stress is considered as the imbalance between the production and clearance of all kinds of radicals.This is usually affected by some factors and results in the tissue damage. Some radicals,including O2-, H2O2, OH- and so on ,are produced during the normal metabolism in different tissues.These radicals are called ROS(reactive oxygen species).On the other hand ,there are some different systems to clear these radicals so as to inhibit the damage caused by them and keep the normal metabolism . These systems are very complicated.The main part of these protective systems are the antioxidantive enzymes,including SOD, CAT, GSH-Px et al.The main reason of the oxidative stress in the diabetic patients is that hyperglycemiaresults in the overproduction of electron donors from the TCA (tricarboxylic acid) cycle and generate a high mitochondrial membrane potential by pumping protons across the mitochondrial inner membrane. This inhibits electron transport at complex III, increasing the half-life of free-radical intermediates of coenzyme Q, which reduce O2 to O2". Inhibition by MnSOD or UCP-1 of hyperglycaemia-induced overproduction of mitochondrial superoxide completely prevented an increase in polyol pathway flux, increased intracellular AGE formation, increased PKC activation and an increase in hexosamine pathway activity in endothelial cells. From above , we can conclude that oxidative stress is the main reason of the diabetic complications ,it is a new area for research about diabetes. There is just a few researches about this in China. So,based on the diabetic rats made by the injection of STZ and utilyzing the RT-PCR -. immunochemihistryN chromatometry and other methods,we observered the damaged to the kidney^ heart and liver tissues in the diabetic rats caused by oxidative stress in diabetic rats.Objective: This research was based on the model of diabetic rats by the injection of streptozotocin (STZ). 12 weeks later,the products of oxidative stress,the activities and gene expression of different antioxidant enzymes and the expression of nitrotyrosine in different tissues were observed and evaluated,at the same time , the pathologic changes in different tissues were observed too.Methods: Diabetic rats were induced by the injection of STZ.36 rats were divided into three groups randomly:( 1) NC group,normal control rats,(2) D I group, diabetic rats received PZI 2U -4U/2days; (3) DII group,diabetic rats received PZI 9-12U/kg body weigh/day. 12 weeks later,rats were killed ,blood glucose,blood cholesterol,serum creatinine,blood urea nitrogen,HbAlc,urinary creatinine,and urinary protein for 24 hours were measured.The activities of antioxidant enzymes in different tissues including TSOD^ Cu-Zn SOD ? Mn SOD^ CAT\ GSH-Px,and MDA were measured by chromatometry. RT-PCR was performed to detect the expression of different antioxidant enzymes mRNA in the kidney tissue . The pathologic changes of different rat tissue were studied by HE and PAS staining.Results:1. General data: 12 weeks later, rats in NC group or DII group were more heavy than they were in the 0 week,however,rats in D I group weighed less in the 12th week than in 0 week.Some items,including blood glucose, HbAl^ TCU TG, increased significantly in D I group than in NC group or DII group.There was no significant difference between NC group and DII group.2. Kidney tissue: Of the rats in D I group/the renal function evaluated by the urinary protein and creatine clearance rate,was impaired compared with that of NC group or DII group.The level of urinary protein was higher ,while the creatine clearance rate was lower than the other two groups; the level of MDA was significantly higher,and the total antioxidant capacity decreased significantly; the activities of TSODn Cu-Zn SODn CAT decareased ,while the activeity of GSH-Px increased significantly; the expression of NT was more strong than that in NC group or DII group. The gene expression levels of GSH-Px and Cu-Zn SOD mRNA in D I group were more strong than in the other two groups, while the expression level of CAT decreased. MnSOD mRNA was expressed without significant difference in all three groups.By HE and PAS staining,we found that there was much more PAS positive material and mesangial cells in the glomeruli in D I group than in NC or DII group.3. Liver tissue:The activities of ALT and AST increased significantly in serum of DI group than those of NC group or DII group. Pathologic changings were found by HE and PAS staining,there was evident hepatic steatosis in D I group compared with the other groups, Less PAS positive material was also found in the livers of D I group,and it was disorderly distributed.The above pathologic changings were improved in the DII group.In D I group,the level of MDA in livers increased ,so did the total antioxidant capacity;the activeities of TSOD^ CAT\ GSH-Px increased significantly ,and the expression of NT were more strong in livers of D I group than those of NC group or DII group.4. Heart tissue: The cardial myocyte hypertrophy, myocyte steatosis and PAS positive material could be found in the hearts of rats of D I group bye HE and PAS staining.Sometimes even necrosis myocytes could also be found.These changings wereseldom seen in NC or DII group.The level of MDA was elevated obviously in the hearts of D I group, at the same time,the activeities of TSODs GSH-Px decreased significantly, while that of CAT increased obviously;the total antioxidant capacity decreased significantly;The expression of NT was strong in D I group,and NT was less expressed in DII group, and was almost not expressed in NC group. Conclusions:1. Oxidative stress could be caused by diabetes in the kidney,liver and heart tissues in diabetic rats;2. Oxidative stress is an important mechanism for high blood glucose to damage different tissues and results in different diabetic complications;3. High blood glucose has effect on the activities and gene expression of different antioxidant enzymes and this may be an important way to increase oxidative stress in different tissue in diabetic rats;4. Better control of blood glucose with insulin could lower the level of oxidative stress and lessen damage in different tissues in diabetic rats.Significance:This research foud out that based on the model of diabetic rats,hyperglycemia could result in the onset of oxidative stress,and this was related to the damage of different tissues.The main reason of the oxidative stress is the overproduction of super anion in mitochondrial. We also found that the activities and gene expression of antioxidant enzymes were affected by hyperglycemia,the ability to clear radical was inhibited, so the damage to different tissues was doubled.The use of insulin lowered the level of blood glucose and the negative effect of oxidative stress to tissues was alleviated.On the other hand,it was proved that oxidative stress could be caused by diabetes mellituss and was the main reason for diabetic complications.This provide a new method for us to prevent and treat diabetic complications.New antioxidant drugs are very promising.
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