Propofol Protects Against High Glucose-Induced Endothelial Dysfunction In Human Umbilical Vein Endothelial Cells

BACKGROUND:Hyperglycemia, via peroxynitrite-mediated endothelial nitric oxide synthase (eNOS) enzymatic uncoupling, induced endothelial dysfunction. Propofol has been reported to improve high glucose-induced endothelial dysfunction. However, its mechanisms of action remain unclear. We hypothesized that propofol could improve hyperglycemia-induced endothelial dysfunction by decreasing the peroxynitrite level and thus restoring eNOS coupling.METHODS:At the end of3days of incubation in medium with30mM glucose, human umbilical vein endothelial cells were treated with different concentrations (0.2,1,5, and25uM) of propofol for different times (0.5,1,2, and4h). In parallel experiments, cells were cultured in5mM glucose for3days as a control. Nitric oxide (NO) production was measured with a nitrate reductase assay. Superoxide anion (O2’-) accumulation was measured with the reduction of ferricytochrome c and dihydroethidine fluorescence assay. The treatment that had maximal effect on30mM glucose-induced NO production and02’-accumulation was applied in the following studies to examine the underlying signaling pathways. eNOS total protein, eNOS dimer and monomer expression, eNOS phosphorylation at Ser1177, inducible NO synthase total protein, inducible NO synthase dimer and monomer expression, peroxynitrite, and guanosine triphosphate cyclohydrolase I expression were measured by Western blot. Tetrahydrobiopterin (BH4) level was measured with liquid chromatography-mass spectrometry.RESULTS:Compared with5mM glucose treatment,30mM glucose significantly decreased NO production by60%(p<0.001) and increased02’-accumulation by175%(p=0.0026), which were both attenuated by propofol in a concentration-and time-dependent manner. Compared with5mM glucose treatment, total eNOS protein expression was increased by30mM glucose (p<0.001), whereas the ratio of eNOS dimer/monomer (p<0.0001) and eNOS phosphorylation (p<0.001) were decreased by30mM glucose. Propofol did not affect30mM glucose-induced total eNOS protein expression, but restored the ratio of eNOS dimer/monomer (p=0.0005) and increased eNOS phosphorylation (p=0.001).30mM glucose-induced O2’-accumulation was inhibited by the eNOS inhibitor hydrochloride. Furthermore, compared with5mM glucose treatment,30mM glucose decreased the BH4level (p=0.0001) and guanosine triphosphate cyclohydrolase I expression (p<0.001), whereas it increased peroxynitrite level (p=0.0003), which could all be reversed by propofol (p=0.0045, p<0.001, p=0.0001vs30mM glucose treatment, respectively).CONCLUSIONS:Propofol has beneficial effects on30mM glucose-induced NO reduction and O2’-accumulation in human umbilical vein endothelial cells. This may be mediated through inhibiting peroxynitrite-mediated BH4reduction, and restoring eNOS coupling.