| Summary: | 碩士 === 台北醫學院 === 藥學研究所 === 87 === (1) Reducing sugars react with amino groups of proteins to form a variety of fluorescent advanced glycosylation end products (AGEs). Serum low molecular weight-AGEs (LMW-AGEs) have been linked to the development of diabetic-associated cardiac vascular complications. In this report, we raised antibodies specifically against AGEs and developed a competitive enzyme-linked immunosorbent assay (ELISA) to determine the serum LMW-AGEs in two age groups of non-diabetics and one group of diabetics. By defining 1 AGEs unit (AU) as the inhibition that results from 1:5 diluted pooled serum in competitive ELISA, We found that the circulatory AGEs levels in the young group as well as those in the elderly group of non-diabetics fit normal distributions (P<0.05) and their reference ranges were 3.120.52(N=30) and 4.411.2 AU (N=36), respectively. The circulatory AGEs levels in diabetic patients were 8.962.13 AU (N=32) which is significantly higher than that in both age groups of non-diabetics (P<0.01). The circulation AGEs data correlated well with the HbA1c values obtained from patients with diabetes (r=0.86). In conclusion, these data reveal that circulation AGEs are higher in the elderly as compared to those of the young group and may serve as a circulation marker reflecting the severity of the diabetic sequel.
(2) AGEs are the reactive derivatives of non-enzymatic glucose-macromolecules condensation products. These compounds have been implicated in the structural and functional alterations of proteins that occur during aging and long-term diabetes. In the present studies, rat C6 glioma cells were incubated with the BSA-AGEs, the Cox-2 protein expression and PGE2 production were examined. Since the MAPKK inhibitor, PD98059,the p38 MAP kinase inhibitor, SB203580 and the NF-kB inhibitor, PDTC abolished the BSA-AGEs-induced COX-2 expression and PGE2 production, MAP kinase and NF-kB appear to be involved in the signaling pathway. Our data suggest that the AGEs-induced COX-2 expression and PGE2 production are mediated through MAP kinase/ NF-kB pathways which may play a role in the progression of neurological disorders implicated with diabetic mellitus.
(3)AGEs stimulated a dose dependent NO production from a variety of cell lines. In the present study, we demonstrated that AGEs stimulated inducible NO synthase (iNOS) expression as well as NO production in C6 glioma cell line. The AGEs-stimulated NO production was blocked by anti-AGEs Ab in C6 glioma cells. The AGEs—stimulated NO production from C6 glioma cell was inhibited by PD98059, the MAPKK inhibitor, SB203580 (10uM), the p38 MAPK inhibitor and PDTC (50uM), the NF-kB inhibitor. Thus, AGEs-induced iNOS expression may mediate through the MAPK/NF-kB pathway. The NO synthase non-selective inhibitor, l-NAME inhibited the AGEs-stimulated NO accumulation suggesting that the NO accumulation may be due to iNOS expression. Since the AGEs-stimulated p21ras activation is mediated by increasing intracellular oxidative stress, we investigate the role of antioxidant, l-NAC on AGEs-stimulated NO release from C6 glioma cells. Consistently, the endogenous glutathione inhibitor, l-Buthionine-(S,R)-Sulfoximine, enhanced AGEs-stimulated NO production in C6 glioma cells. Pretreatment of C6 glioma cells with PKC inhibitor, Go6976, specifically inhibited the subseqeunt AGEs-stimulated NO release from C6 glioma cells, suggesting that PKC may also involve in AGEs-stimulated NO production. In conclusion, although the detail signaling mechanism is not totally understood, the AGEs-stimulated effect is clearly dependent on MAPK/NF-kB pathway mediated by oxidative stress. Possibly AGEs also trigger the PKC pathway in iNOS expression.
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