Protein Nitration Determination In Heart And Aorta Of Diabetic Rats

Diabetes mellitus is a serious and growing health problem. Diabetes complications are major injury to diabetics, and cardiovascular complications cause the majority of diabetes-related deaths. There is emerging evidence that RNS/ROS make a significant contribution to the progression of diabetes cardiovascular complications. Many studies found a positive correlation between protein nitration and cardiovascular disease progression. This means that protein nitration may be as a biological marker of diabetes cardiovascular complications.In this thesis, protein nitration in different course of diabetes rat heart and aorta homogenates had been detected by SDS-PAGE and Western blotting. At the same time, the correlation between protein nitration and diabetes cardiovascular was discussed. The main results obtained were as follows:(1) In order to obtain the optimal experiment conditions of Western blotting, protein nitration in protein-liposome mixtures by different nitrating systems, including Hemin/NO2-/H2O2, Hemoglobin/NO2-/H2O2 and Fe3+/NO2-/H2O2, were detected. Meanwhile, the effects of pH and liposome on protein nitration were also preliminarily studied. The optimal experiment conditions of Western blotting were determined, which were as follows:â‘ the optimal time of semi-dry transfer was 1 h 40min,â‘¡the optimal dilution ratio of anti-nitrotyrosine antibody was 1:600,â‘¢the optimal dilution ratio of secondary antibody conjugated to HRP was 1:3000.(2) On the basis of the optimal experiment conditions of Western blotting obtained in vitro, protein nitration, oxidation and iNOS expression in different course of diabetes rat heart homogenates were detected. The results showed that protein nitration, oxidation and iNOS expression were increased in diabetes groups at 0 day and 60 days, but were decreased at 30days. In addition, nitrated and oxided proteins were not the same ones.(3) Protein nitration in different course of diabetes rat aorta homogenates was detected. The results showed that the kinds of nitrated proteins in aorta were similar to the ones in heart homogenates. However, the kinds of nitrated proteins were needed to be identified by 2D-MS.