Protein glycation and oxidative stress in pathophysiology of diabetic complications

Diabetes mellitus is one of the most prevalent diseases found in the world population. In 2012, according to the WHO report, 1.5 million people died due to diabetic related complications. Type 2 diabetes is a progressive disease. The currently prescribed medication lacks the specificity for targeting particular pathways or molecules involved in the complications. The appropriate understanding of the mechanisms responsible for the diabetes mellitus and its related complications can pave the way to the discovery of the target specific drugs for effective treatment. The current project makes a comprehensive study to get an insight into the probable mechanisms involved and the ways to counteract them. The study specifically centers around in vitro induction of BSA glycation and its inhibition, exploration of the effect of extracellular and the intracellular AGEs on the macrophages, protein glycation analysis and inhibition in diabetic serum. The in vitro glycation model using BSA confirmed the formation of the both fluorescent and non fluorescent advanced glycation end products (AGEs) in the hyperglycaemic condition. The amount of the AGEs formed increased with the duration of exposure to the hyperglycaemic condition as reflected by increased fluorescent intensity. The oxidative stress induced by hyperglycaemic condition was validated and quantified by NBT assay and H2DCFDA assay. Moreover, the ROS generation in macrophages was increased with addition of extracellular AGEs in the medium. The reduction in the cell viability as a consequence of high ROS production and the presence of the extracellular AGEs was confirmed by MTT assay. During inhibitor study, ascorbic acid exhibited pro-oxidant activity upon adding to the diabetic serum that makes the therapeutic use of ascorbic acid alone debatable unless otherwise verified further. At the same time, GSH and EDTA act as anti-oxidants in macrophages (hence, probably reduced the AGEs formation). In silico docking study was conducted to understand the mechanism of inhibition. The binding affinity of EDTA for BSA was significantly more compared to glucose that didn’t reduce much to glycated BSA. This concludes that EDTA can prevent glycation non-competitively and may also halt the progress of glycation of the protein. The results were a step ahead in exploring the novel molecules for inhibiting the protein glycation in T2DM. For a more conclusive finding, a metacentric study involving more number of diabetic patients is needed