Antiglycation potential and mechanism of some flavonoids and their Maillard reaction adducts in endothelial cells

Abstract

Methylglyoxal (MGO), a highly reactive carbonyl species (RCS) and cytotoxic factor in mammalian cells, reacts irreversibly with the side chains of lysine, cysteine, and arginine residues of intracellular and/or extracellular proteins to form advanced glycation end products (AGEs), which might be a major pathological factor associating with diabetic complications. Thus, it is of significant importance to prevent or alleviate such diseases through inhibiting the formation of AGEs or lowering these AGEs-induced cellular damages. Firstly, in the present study, the activity of apigenin, a natural flavone in scavenging RCS and the molecular mechanism involved in its protective effect against AGEs-induced oxidative stress and inflammation were examined in vitro. Results showed that apigenin could directly trap MGO to form apigenin-MGO adducts, thus inhibiting AGEs formation. Apigenin and di-MGO apigenin adduct were further found to inhibit AGEs-induced oxidative stress and inflammation in human umbilical vein endothelial cells (HUVECs) by significantly suppressing the production of reactive oxygen species (ROS, 30% relative to control) and decreasing the protein expression of pro-inflammatory cytokines and adhesion molecules by 30-70%. Further mechanistic investigation revealed that the protective effect was likely mediated via suppression of the extracellular signal-regulated kinases (ERK)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway initiated by AGEs-acceptor for AGEs (RAGE) interaction and induction of ERK/Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway with subsequent up-regulation of antioxidant defense molecules. Secondly, based on our previous work, it was known that phloretin, an apple polyphenol and dihydrochalcone, was able to inhibit the formation of AGEs under simulated physiological conditions. In the present study, we found that phloretin prevented the formation of AGEs through trapping MGO in HUVECs. Furthermore, the presence of phloretin-MGO adducts in PBS and HUVECs were analyzed. Surprisingly, only one MGO-phloretin adduct was detected in HUVECs, which was found to form within 0.5 hr and metabolize eventually to below the detection line within 24 hrs. The specific phloretin-MGO adduct was then synthesized and identified by MS and NMR analysis. Its anti-inflammatory effect against AGEs was further investigated together with the parent compound, phloretin, which was proved to inhibit inflammation through RAGE/p38 mitogen-activated protein kinases (p38 MAPK)/NF-κB signaling pathway. Thirdly, several styryl-substituted flavonoids previously identified by our lab, were proved to be anti-inflammatory in the present study. Among them, 6-C-(E-phenylethenyl)-naringenin (6-PN) found in tomato/beef soup, was the most effective one which markedly lowered AGEs-induced oxidative stress and inflammation through RAGE/ROS/Nrf2 signaling pathway and partly through improved bioavailability in endothelial cells. Thus, our study discovered the novel health benefits of flavonoid-Maillard reaction adducts offering a hint to the beneficial effects of thermally-induced flavonoid derivatives to a certain extent while these compounds might exist in meat/vegetable soups or related food products. In summary, apigenin and phloretin were found to prevent the formation of AGEs in HUVECs through MGO-scavenging and ameliorate AGEs-induced endothelial damages. Meanwhile, a di-MGO apigenin adduct and a di-MGO phloretin adduct, also known as RCS-type flavonoid-Maillard reaction adducts, were separated and identified via chromatography, and proved to decrease AGEs-induced inflammation just as their parent compounds, indicating the possible role of flavonoids-MGO adducts in flavonoids’ protective effects against AGEs. Furthermore, 6-PN, a novel stilbene-type flavonoid-Maillard reaction adduct, showed stronger activity than its parent compound, naringenin against AGEs-evoked oxidative stress and inflammation. Therefore, this study highlights the potential of flavonoids and their Maillard reaction adducts in the prevention of diabetes-associated complication.