Protein Nitration Of Diabetic Renal Tissue Damage And Alcoholic Hepatic Injury Facilitated By Iron Overload

With the change of modern dietary structure and people's attention to iron,iron deficiency and its harm has been controlled to a certain extent,and the relationship between iron overload and certain diseases has become increasingly prominent.The incidence of diabetes and alcoholic liver disease are increased in recent years.Many studies have confirmed that there is high accumulation of iron in diabetes and alcoholic liver disease,and iron overload plays a significant part in the onset and development of these diseases,but the underlying mechanism is not very clear.Reactive nitrogen-dependent protein nitration can cause changes in protein structure and function and has been recognized as a biomarker of cell and organism damage in recent years,and has been implicated in many diseases.Iron overload not only promotes the production of free radicals,but also participates in the tyrosine nitration of proteins.Therefore,diabetic nephropathy and alcoholic liver injury facilitated by iron may be related to the impact of iron on the protein nitration.The roles of protein nitration on iron promoted diabetic renal tissue damage and alcoholic hepatic injury,were studied in this paper.We also studied the effect of iron on the nitration and oxidation of an important glycolytic enzyme,and compared the difference of protein nitration between in the nitrating pathway of heme iron involved and that of SIN-1 system.The aim of this study was to elucidate the clinical and pathological features of high levels of protein nitration and iron overload in these diseases,such as alcoholic liver disease,diabetes mellitus and so on,from the viewpoint of protein nitration.The main research work are as follows:(1)Effects of iron on the promotion of renal injury and protein nitration in diabetic rats.The model of iron overload-type 2 diabetic rats was established by intraperitoneal injection of iron-dextran and streptozocin.The damage,nitration and oxidation of renal tissues in normal,iron overload,diabetic and iron overload-type 2 diabetic rats were detected by HE,immunohistochemistry and Western blotting.The effects of iron overload on diabetic renal injury were investigated by detecting blood sugar level,iron content,kidney ratio and indexes about oxidative stress.The results showed that iron supplementation on diabetic rats did not promote the rise in blood lsugar further,but iron injection increased iron content,oxidative stress and protein nitration in diabetic renal tissue and promoted kidney/body weigh ratio and kidney injury.These results suggested that iron overload aggravated renal injury by increasing oxidative stress and protein nitration in diabetic rats rather than by elevating blood sugar level.(2)Combined effects of iron overload and alcohol on protein nitration of liver injury in mice.Mouse model of liver injury was prepared by light alcohol administration in the presence of mild iron overload.Biochemical markers of liver injury,HE staining,immunoprecipitation,and Western blotting were measured to investigate the combined effects of iron and alcohol on liver injury as well as the effect of protein nitration on liver function.We also studied the nitrative modification of triosephosphate isomerase(TIM),an important glycolysis enzyme closely related to lipid metabolism.No obvious liver injury was found when mice were administrated with low doses of alcohol or iron overload alone,but severe hepatic injury was caused after co-administration of the same doses of alcohol and iron.We also found that liver damage was caused with the increased ratio of NADH/NAD+,the reduction of antioxidant ability,as well as the increasment of oxidative stress.This was followed by obvious enhancement to protein nitration.An important glycolytic enzyme,TIM,which is easy to suffer from nitration,was further studied.Tyrosine nitration of TIM was demonstrated to contribute to its inactivation.The results showed that when the mice were co-administrated with mild dose of alcohol and iron,that would cause significant liver injury,although each separate treatment seemed relatively safe.Both the decrease in the expression and the nitrative modifications of TIM resulted in enzyme inactivation.Besides,protein oxidation also played an important role in enzyme inactivation.The decrease in TIM activity and the increase of NADH/NAD+ratio would help promote the transform from dihydroxyacetone to a raw material for fat synthesis,which might lead to a metabolic block in the glycolytic pathway and disturbance of lipid metabolism associated with liver disease.(3)The study of triosephosphate isomerase tyrosine nitration induced by heme-NaNO2-H2O2 or peroxynitrite and the antagonism effects of different natural phenolic compounds.By Western blotting,circular dichroism and LC-MS,we systematically studied the difference of tyrosine nitration induced by peroxynitrite and heme-H2O2-NaNO2,respectively,and investigated the efficiencies of desferrioxamine,catechin and four natural phenolic compounds on these two ways induced TIM nitration.The results showed that SIN-1 caused both nitration and oxidation of TIM in a dose-dependent manner,it is difficult to distinguish the effects of protein oxidation and nitration on protein function.While in heme-NaNO2-H2O2 system,TIM nitration was found to be increased with the decrease of TIM oxidation,which suggested protein tyrosine nitration in this system could be controlled in a certain range.Heme-H2O2-NaNO2 induced less destructive degree of protein secondary structures and more nitrated tyrosine residues than that treated by SIN-1.While desferrioxamine or catechin could inhibite TIM nitration in both nitrating systems,protein oxidation was weakened in SIN-1 but promoted in heme-H2O2-NaNO2.We further found that in SIN-1 system the radical scavenging ability of natural phenolic compounds was consistent with their antagonistic ability of TIM nitration,while in heme-H2O2-NaNO2 system there was no similar consensus.These results indicated that protein nitration was different between in peroxynitrite and in heme-H2O2-NaNO2 system by different mechanisms and the later one could be a better model for anti-nitration compounds screening.