The Inhibition And Mechanism Of Protein N~ε-carboxymethyllysine Formation By Catechin

Advanced glycation end products (AGEs), the end products of protein non-enzymaticglycation, is a key risk factor of chronic persistent inflammation, obesity, fatty liver andmetabolic syndrome in animal and human. Glyoxal (GO) and methylglyoxal (MGO),dicarbonyl compounds, are reactive carbonyl species (RCS) and precursors of carboxymethyllysine (N-carboxymethyllysine, CML), can start the protein non-enzymatic glycation andform AGEs. Therefore, trapping dicarbonyl compounds and the inhibiting of AGEs and CML,become an important way for the prevention of diabetes and fatty liver. This study willinvestigate if natural products catechins can inhibit MGO or glyceraldehyde/Fenton inducedcalf thymus histone H1and BSA non-enzymatic glycation and CML formation. Theexperimental results are as follows:To detect the MGO trapping ability of catechin by Girard’s reagent T method, MGO andcatechin was incubated in37℃. The results show that catechin significantly remove80.73%MGO at a concentration equimolar to that of MGO, much better than the traditionaldicarbonyl trapping agent aminoguanidine (AG). Early glycation products Schiff baseformation was measured by DNPH method and result shows that Schiff base formation can beinhibited significantly by catechin at a1:6molar concentration to that of MGO. Histone H1generation of fluorescent substances was significantly increased by MGO and this fluorescentsubstances can be inhibited by catechin. MGO induced histone H1non-enzymatic glycationmodel was primarily established by SDS-PAGE electrophoresis, and successfully confirmedby histone H1expression and CML increased through western blot.MGO induced histone H1glycation was almost completely inhibited by catechins at themolar ratio of2:1to MGO. The MGO induced histone H1CML formation can be inhibited bylow concentration catechin (12.5-100μM) but was increased by high concentration catechin(200-800μM). H2O2formation was significantly increased by catechin when incubated withMGO and histone H1at37℃, and this increased H2O2formation is relative to concentrationof catechin but not MGO. In the Fenton reaction conditions, GO was formed byglyceraldehyde in37℃. Glyceraldehyde/EDTA/Fe2+/H2O2induced GO formation can be trapped by N-Acetyl-L-cysteine (NAC) and catechin. Glyceraldehyde/EDTA/Fe2+/H2O2induced BSA Schiff base formation can be inhibited by NAC;Glyceraldehyde/EDTA/Fe2+/H2O2induced BSA CML formation can be inhibited by NAC andcatechin.Conclusion: Histone H1non-enzymatic glycation model and CML model wassuccessfully established in this study. Dicarbonyl induced histone H1non-enzymaticglycation can be inhibited by catechin probably through the mechanisms relative to trappingMGO, inhibition of total AGEs and Schiff base formation. Low concentrations of catechinscan effectively inhibit the MGO induced histone H1CML formation, and its mechanism maybe associated with trapping MGO not the ability of free radical scavenging. Highconcentration catechin cannot suppress the amount of CML formed because of a large numberof H2O2formation. Catechins may inhibit glyceraldehyde induced CML formation probablythrough the mechanisms relative to trapping GO.