The Effects Of Hydroxysafflor Yellow A On The Formation Of Advanced Glycation End Products And On The MGO-induced Injury In The Cultured HBMEC

Advanced glycation end products (AGEs) are a group of complex and heterogeneous compounds. They are the final products of the nonenzymatic reaction between reducing sugars and amino groups in proteins, lipids, and nucleic acids. Recently, AGEs accumulation in vivo has been implicated as a key pathogenic process in diabetic complications, including neuropathy, nephropathy, retinopathy, cataract and other health disorders such as atherosclerosis, Alzheimer’s disease, and normal aging. In the development process of these diseases, AGEs play an important role. On the one hand, AGEs directly involved in the diseases, affecting cell and tissue functions. On the other hand, it also mediate a series of pathological reactions through specific receptors.Therefore, the discovery and investigation of AGEs inhibitors would offer a potential therapeutic approach for the prevention and treatment of diabetic or other pathogenic complications.Objective:To investigate the inhibitory effects of hydroxysafflor yellow A (HSYA) on the protein glycation in vitro. Using bovine serum albumin (BSA)-glucose assay, BSA-methylglyoxal (MGO) assay, N-acetylglycyl-lysine methyl ester (G.K.) peptide-ribose assay, and the cultured human brain microvascular endothelial cell (HBMEC) injured by MGO, inhibitory effects of HSYA were investigated.Methods:In reagent:BSA-glucose assay:BSA and glucose in phosphate buffer were incubated under sterile, dark conditions at37℃for7d. Fluorescence of samples was measured at the excitation and emission maxima of330nm and410nm, respectively. In certain experiments, the indicated HSYA was added to the model system in the concentration range of0.01-1mmol/1. Aminoguanidine (AG,50mmol/1) was used as a positive control.BSA-MGO assay:The mixture of BSA and MGO was incubated under sterile, dark conditions at37℃for24h. Fluorescence of samples was measured at the excitation and emission maxima of330nm and410nm, respectively. In certain experiments, the indicated HSYA was added to the model system in the concentration range of0.01-1mmol/1. AG (50mmol/1) was used as a positive control. The effects of MGO modification on the cross-linking and aggregation of BSA were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).G.K. peptide-ribose assay:The mixture of G.K. peptide and ribose was incubated under sterile, dark conditions at37℃for24h. At the end of the incubation, samples were analyzed for specific fluorescence (excitation340nm; emission420nm). The HSYA was added to the model system in the concentration range of0.01-1mmol/1, respectively. AG (50mmol/1) was used as a positive control.Cellular level:HBMEC were incubated with different concentration of MGO (10μmol/1-10mmol/1) for24h. Cell viability was determined by MTT assay. We used2mmol/1MGO to injure cells in the follow-up study, at which, the cell viability was around50%. To investigate the protective effect of HSYA on MGO-induced injury in the cultured HBMEC, cells were treated with different concentrations of HSYA20min prior to MGO treatment. Cell viability was determined by the MTT assay. The amount of lactate dehydrogenase (LDH) released by cells was determined by a LDH assay kit. And then, we investigated the cultured HBMEC apoptosis via AnnexinV/PI staining and expression of caspase-3.Finally, we determined the levels of AGEs in the cultured HBMEC.AG (1mmol/1) was adopted as a positive control.Results:In all these models, HSYA suppressed fluorescence in a concentration-dependent manner. At the concentration1mmol/l, the inhibitory effects of HSYA was86.61%in BSA-glucose assay,55.25%in BSA-MGO assay,53.68%in G.K. peptide-ribose assay. The result of SDS-PAGE showed that, when BSA incubated with MGO, there was a decrease in the detectable amount of BSA in its usual position together with the less resolution and spreading of bands compared to untreated protein. However, when HSYA or AG was added in the incubation mixture, both loss of BSA and formation of the high molecular weight protein were inhibited.Cell viability decreased obviously in the MGO (2mmol/1) group (52.05%). In contrast, HSYA mediated high up-regulation of cell viability at100μmol/1(68.99%). This response to HSYA was similar to that of LDH release, the cell death percentage declined significantly in HSYA treated group:MGO (2mmol/1) group (60.12%), HSYA (100μmol/1) group (38.93%). HSYA inhibited MGO-induced cell apoptosis via AnnexinV/PI staining and expression of caspase-3. Cell apoptosis increased after24h MGO treatment (48.50%) than those treated with HSYA (100μmol/1)(35.23%). The expression of casepase-3shared a similar trend:MGO group3.27, HSYA group2.64. AGEs accumulation was detectable in control and increased to a higher level after24h MGO treatment (1.906) than those treated with HSYA (100μmol/1)(1.557). Conclusions:Our research revealed that HSYA can inhibit all stage glycation and also supported a specific protective effect of HSYA on MGO-induced injury in the cultured HBMEC. The results of present study indicate that HSYA has excellent antiglycation effects in vitro.