Fig. 6.

Effect of CBD on HG-induced inducible nitric oxide (NO) synthase (iNOS) expression and 3-nitrotyrosine (3-NT) formation, proposed protective mechanisms of CBD. A: HCAECs were treated with either normal glucose, CBD (4 μM), HG alone for 48 h or pretreated with CBD followed by HG treatment for 48 h. Total cell lysates were prepared, and 25 μg of protein were resolved on 12% SDS-PAGE gels and probed with anti-human iNOS (monoclonal, used at 1:1,000 dilution; BD Biosciences). Shown is the representative image from 3 separate experiments with identical results. *P < 0.001 vs. 5 mM glucose; #P < 0.01 vs. HG. B: HCAECs were treated as described above, and iNOS expression was analyzed by immunofluorescene staining (mouse monoclonal anti-human iNOS at 1:150 dilution). The nuclei were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Molecular Probes, Invitrogen). Shown are representative images from 3 separate experiments. C: 3-NT formation in HG-treated cells and effects of CBD by immunofluorescence assay. Nuclei were counterstained with DAPI. Shown are images from 3 independent experiments with identical results. D: proposed protective mechanisms of CBD against HG-induced endothelial cell inflammatory response and barrier disruption. Mitochondrion is considered to be the major source of hyperglycemia-induced increased superoxide anion production; however, other sources such as xanthine and NAD(P)H oxidases, cyclooxygenase, and uncoupled NOS may also contribute to this process under certain conditions. Hyperglycemia-induced superoxide generation might also favor increased expression of iNOS through activation of NF-κB, which increases the generation of NO. Superoxide anion quenches NO, thereby reducing the efficacy of a potent endothelium-derived vasodilator system. Superoxide can also be converted to hydrogen peroxide by SOD and interact with NO to form a reactive oxidant peroxynitrite (ONOO–), which induces cell damage via lipid peroxidation, inactivation of enzymes and other proteins by oxidation and nitration, and activation of nuclear enzyme poly(ADP-ribose) polymerase (PARP-1). Red arrows/lines indicate activation, and black lines indicate inhibition.