Effect Of Flavonoids From Seed Residues Of Hippophae Rhamnoides L. On Advanced Glycation End Products (AGEs) Formation And AGEs-induced Endothelial Cell Dysfunction

The accumulation of advanced glycation end products (AGEs) is considered one of the hallmarks in the pathogenesis of diabetes and various macro- or micro-vascular complications. Our previous research had confirmed that several ingredients extracted from medicinal plants, such as flavonoids, saponins, and polysaccharides are effective in controlling serum glucose and lipid levels, whereas their effects on AGEs formation and AGEs-mediated endothelial cell dysfunction are poorly known. The present study investigated these effects in order to find effective AGEs inhibitors. We also studied the effects of trace elements copper, zinc, and manganese on AGEs formation, whose content varied significantly after extraction when compared with their original plant, to clarify the functional relationship between extracted ingredients and trace elements.Fluorescence detection was used to monitor the Maillard reaction in vitro. Flavonoids exhibited excellent inhibitory effects on AGEs formation, especially the flavonoids from seed residues of Hippophae rhamnoides L. (FSH). Incubation with FSH significantly reduced levels of total AGEs and the specific pentosidines. MnCl2 also inhibited the formation of AGEs, while ZnCl2 had the opposite effect.Then we studied the effects of FSH, Zn, Mn, and the combination of FSH with Zn or Mn on AGEs induced endothelial cell dysfunction. Primary cultured bovine aortic endothelial cells (BAECs) were exposed to AGEs for 30 min. followed by FSH and trace element treatments. Cell viability of BAECs was significantly inhibited by AGEs exposure, while the addition of FSH, ZnCl2, and MnCl2 protected the cells from the injuries with cell viability greatly increased. Furthermore, AGEs exposure declined intracellular zinc levels, nitric oxide (NO) release, and endothelial NO synthase (eNOS) levels (including mRNA levels, protein expression levels, and the enzymatic activity). Simultaneously, cellular redox equilibrium was destroyed due to AGEs exposure, total anti-oxidation competence was greatly reduced, intracellular reactive oxygen species (ROS) production was obviously increased, antioxidant enzymatic ability of superoxide dismutase (SOD) was inhibited, and inflammation relevant nuclear transcription factor kappa B (NF-κB) was activated with excessive mRNA levels and protein expression. The addition of FSH improved these poor situations described above. FSH significantly increased the declined intracellular zinc levels, NO release, and eNOS expression. FSH could also restore the anti-oxidant capability, the SOD enzymatic ability, and even the abnormal cell configuration and distribution. ZnCl2 supplementations enhanced the AGEs-decreased intracellular zinc levels, NO release, eNOS levels and their enzymatic capability. ZnCl2 could also elevate the anti-oxidant capability and SOD enzymatic ability of BAECs, inhibit the activation, nuclear translocation, and overexpression of NF-κB. The MnCl2 supplementation significantly enhanced intracellular zinc levels, cleared the excessive formation of intracellular ROS, and impaired the sharpened activation and nuclear translocation of NF-κB in BAECs, as well as restored the inhibited mRNA expression and enzymatic capability of Mn-SOD.Synergistic effects of the combined treatments of FSH and ZnCl2 appeared in NO release detection, eNOS mRNA and protein expression, and eNOS enzymatic activity. Though there were no synergistic effects on cell viability and intracellular zinc uptake, the addition of FSH overcame the negative, cytotoxic effects of ZnCl2, cell viability remained high even 20μmol/L ZnCl2 solutions were supplemented. In the same manner, the presence of ZnCl2 accelerated and maintained the benefit effects of FSH on AGEs-induced endothelial cell dysfunction. With FSH supplementations intracellular zinc levels reached 150% that of the control, regardless of the increasing concentration of exogenous ZnCl2 supplementations. The combination of FSH and MnCl2 was more complicated. FSH overcame the inhibitory effects of the high leveled-MnCl2 on cell viability. With the presence of FSH, viability ratio of the AGEs-pretreated BAECs did not suppressed even when MnCl2 was supplemented at 20μmol/L, a high and toxic concentration of manganese for cells. The MnCl2 supplementations accelerated and maintained the effects of FSH just as ZnCl2 did. However, unlike the effect on increasing zinc levels, FSH hardly had any influences on intracellular manganese levels. Furthermore, the combined therapy of FSH and MnCl2 resulted in antagonistic effects on cellular zinc levels:zinc levels rapidly declined with 50μg/mL FSH and increasing amount of MnCl2. Antagonistic effects also appeared on intracellular ROS formation and NO release.In conclusion, FSH, zinc, and manganese resisted the AGEs-induced injuries through different manners. FSH and MnCl2 significantly inhibited the formation of AGEs in vitro; moderate FSH, ZnCl2, and MnCl2 supplementations improved AGEs-mediated endothelial cell dysfunction. ZnCl2 benefited the injured BAECs probably through an NO and NF-κB related mechanism, whereas MnCl2 was a potentially powerful antioxidant by refreshing Mn-SOD activity. FSH showed significant effects on eNOS activity and NO release elevation, cellular anti-oxidation ability restoration, and intracellular ROS reduction. FSH also had synergistic effect with zinc, the combination of FSH and zinc improved the general effect of FSH on AGEs inhibition, and might have advantage in further therapy for diabetic complications.