Effect Of Valsartan On AGE-RAGE,Oxidative Stress And Endodermic Function In Brain Tissue Of Diabetic Rats

Background :Diabetes mellitus is one of the most frequently happened Endocrinic Metabolic diseases which is characterized by insulinopenia or insulin resistance or both of them.Pathogenesis of Diabetic complications is a constantly researched area. Among many supposed mechanisms, oxidative stress is an important one. High level active oxygen(ROS) can affect the activity and expression of antioxidases which can clear free radicals in diabetes mellitus, and hence interfere the activation of NO, induce the loss of the biological availability of NO, cause the impaired function of the endothelium, and finally evoke the damage of target organs.Moreover, advanced glycosylation end products (AGE) and its receptors (RAGE) also play an important role in the diabetic complications in pathological terms. RAGE's activation after binding with AGE, subsequent oxidative stress reaction and proinflammatory induced by RAGE downstream signal, can promote the occurring and development of Diabetic complications. The high expression of AGE in diabetes can promote the synthesis and release of angiotensin II(AngII) in tissue through activation of chymase alternative pathway. These demonstrate that AGE can promote the expression of AngII in regional tissue to cause target organs damage, while over-activation of the rennin-angiotonin system (RAS) can increase the activity of AGE-RAGE system in return.As AT1 receptor antagonist, Valsartan can slow down the development of diabetic nephropathy of type 2 diabetes mellitus due to its renal protection which is independent of lowering blood pressure. Besides, Valsartan can also reverse the myocardium interstitial fibrosis of rat diabetic cardiomyopathy. However,it is not fully clarified whether Valsartan can slow the progression to diabetic cerebropathia.In respect of: AGE-RAGE system plays an important role in the development of diabetic complications; vitro experiments have demonstrated that antioxygen treatment can decrease the experssion level of RAGE, hence implies that oxidative stress can increase the experssion of AGE and its receptor RAGE; AT1 receptor antagonist can inhibit renal experssion of AGE and its receptor RAGE of diabetic rats, and improve renal function obviously; AT1 receptor antagonist can protect blood vessel endothelium from injury, so we designed our study: we used the diabetic model of rats to observe the changes of AGE-RAGE system, oxidative stress, endothelial function in brain tissues and the influencing effect of Valsartan by Western blot, Quantitative real-time PCR, immunohistochemistry, and chromatometry. Objectives:To investigate the conditions of oxidative stress and endothelial function of brain tissue in diabetic rats, the roles of AGE-RAGE system in the diabetic cerebropathy progression and Valsartan's protection effect for diabetic cerebropathy. Methods:A total of 36 clean healthy male Wistar rats(weight 180-200g) were enrolled in this study(purchased from the experimental animal center of Shandong university). They were divided into three groups at random(12 rats in each group): NC group, normal control rats; DM group, diabetic rats; DM.V group, diabetic rats treated with Valsartan.Construction of model: rats of DM and DM.V group were given STZ (60mg/kg)by intraperitoneal injection after fasting for 10hours, and NC group was only given the isovolumic citric acid -citrate sodium buffer. Seventy-two hours later, blood was collected from tail vein and blood glucose(BG) was detected. Those rats whose BG>16.7mmol/L was consider as the successfully constructed DM model.The intervention of Valsartan: the rats of DM.V group were given Valsartan 40mg/(kgd) by the intragastric administration 24 hours after the successful construction of DM model; NC group and DM group were given the isovolumic distilled water in the same way. During the experimental session, animals were given normal diet. The blood glucose was detected once per 2 weeks.At the end of the 12th week, the weight, blood glucose, constractive blood pressure, cognition ability(assayed by Morris water maze test) were valued. The activities of superoxide dismutase (SOD), maleic dialdehyde (MDA), hydroxy radical(OH-), NO and GSH-Px were measured by chromatometry. The expression of NADPH oxidase p47phox, Endothelin-1 (ET-1) and NF-κB were detected by Quantitative real-time PCR, and the expression of AGE was detected by immunohistochemistry. Western blot was performed to detect the expression of NF-κB and RAGE. Results:1.12 weeks later, the quality ,blood glucose and contractive pressure of rats Compared with NC group, the levels of quality in DM and DM.V groups decreased significantly (both P<0.01) and the levels of blood glucose increased significantly (both P<0.01).There was no significant difference between DM and DM.V groups in terms of the levels of qualities and blood glucose. the level of contractive pressure in DM groups was significantly higher by 30mmHg than that in NC and DM.V groups(P<0.01), no significant difference betweenDM.V group and NC group.2. The experimental result of Morris water maze testMorris water maze test showed that the escape latency was longer in DM group and DM.V group than in NC group(both P<0.01),and it was short in DM.V group compared with DM group(P<0.01).3. The activities of superoxide dismutase (SOD) and GSH-Px and the level of maleic dialdehyde (MDA), hydroxy radical(OH~-) and NO .Compared with NC group, the level of OH" and MDA in DM group increased significantly, the level of NO and the activities of SOD and GSH-Px in DM group decreased significantly(all P<0.01). Compared with DM group, the level of OH~- and MDA in DM.V group decreased significantly, the level of NO and the activities of SOD and GSH-Px in DM group increased significantly(all P<0.01).4.The expression of ET-1 mRNA,NF-κB mRNA and NADPH oxidase p47phox mRNAThe expression of ET-1 mRNA,NF-κB mRNA and NADPH oxidase p47phox mRNA in DM group was significantly higher compared to those in NC group . It was 6.52,7.16 and 4.89 -fold higher than in NC group. There was significant difference between DM and NC groups ( P<0.01 ) .The expression of ET-1 mRNA,NF-κB mRNA and NADPH oxidase p47phox mRNA in DM.V group was 2.29, 1.80 and 2.67 -fold higher than that in NC group. There was significant difference between DM.V and NC groups ( P<0.05 ) .Compared with DM group ,the expressed inhibition ratio of these mRNA in DM.V group was 35%,25%and 54.5%,. There was significant difference (P<0.05) .5.The pathological measure of hippocamps in groups (HE staining) HE staining displayed that the pyramidal neuron of the hippocamp CAl in NC group were the regulated shape, affluent quantity ,clear caryotheca, conspicuous nucleolus and lined up in order; However, the pyramidal neuron of the hippocamp CA1 in DM group lined up in disorder,in loose, had the karyopycnosis,the quantity of cells were less than that in NC group; The quantity of the pyramidal neuron in DM.V group was between the two groups .The cells lined up in opposite disorder .the shape and structure of neuron approached the normal ,which can relieve the karyopyconsis obviously .6 Immunity HistochemistryThe expression of AGE was mainly on the cellular membrane and kytoplasm of the pyramidal neuron of the hippocamp CAl in NC group, the expression was fairly poor. the expression of AGE in the hippocamp CAl was strongly positive, positive cell was brown; Compared with DM group, the expression of AGE decreased significantly, the quantity of positive cell decreased.Image analysis result : The expression index of AGE in DM group were higher than that in NC group (P<0.01); compared with DM group ,the expression of AGE in DM.decreased significantly (P<0.01).7. Consequence of Wastern blotThe expression of RAGE in DM and DM.V groups was higher than that in NC group . It was 1.79-fold ( P<0.01) and 1.23-fold ( P>0.05) higher than in NC group. Compared with DM group ,the expression of RAGE in DM.V group was decreased ,it was 68.6% of that in DM group,there was significant difference (P<0.01) .The expression of NF-κB in DM and DM.V were higher than that in NC group. It was 1.76-fold (P<0.01) and 1.24 -fold (P<0.05) higher than in NC group.compared with DM group, the expression of NF-κB in DM.V group was decreased .it was 70.5% of that in DM group,there was significantdifference (P<0.01) .Conclusions:1. Diabetes mellitus can up-regulate the activity of NADPHoxidase on the brain tissue, increase the expression of ROS, decrease the activity of antioxidase, and improve the level of oxidative stress;2 . Diabetes mellitus can up-regulate the expression of AGE and RAGE, the combination of AGE and RAGE can improve the level of oxidative stress and activate NF-κB. Inversely, the high level of oxidative stress can promote the production of AGE and RAGE. Finally the relative balance between NO and ET-1 is broken and the endothelial function is damaged.These demonstrate that activation of AGE-RAGE pathway ,high level of oxidative stress and lesion of endothelial function are all important mechanisms of lesion of brain tissue in the diabetes mellitus.3. Valsartan may inhibit the activity of NADPH oxidase,decrease the level of oxidative stress in the brain tissue, reduce the expression of AGE and RAGE by blocking the combination of AngII and AT1 receptor.In the other word the lower combination of AGE and RAGE inversely decreases the level of oxidative stress, reduces inflammatory reaction,promotes production of NO, decreases the expression of ET-1, finally promotes endothelial functional recovery; the decreasion of oxidative stress may directly inhabit the expression of NF-κB,promote recovery of the relative balance between NO and ET-1, ameliorate endothelial function and brain cell damage, reverse cognition ability, delay progression of diabetic cerebropathia. Significances:The present study was performed to investigate the changes of AGE,RAGE,NF-κB,NADPH, ET-1,OH-,NO,MDA,SOD,GSH - Px and the changes of the escape latency among NC,DMand DM.V groups by using the Western blot, Quantitative real-time RT-PCR, immuno — histochemistry, chromatometry and so on. We have elucidated the roles of AGE-RAGE system in the progression of diabetic cerebropathy and changes of oxidative stress and endothelial function on brain tissue of diabetic rats.furthermore we have evaluated the role of protection effect of Valsartan and elucidated its mechanism on diabetic cerebropathy. So it is tempting to postulate that Valsartan have pharmacological effect on the prevention and cure of clinical diabetic cerebropathia.