Influence of hydrogen peroxide on expression of calcium-independent phospholipase A2 in islet microvascular endothelial cells
AIM: To investigate the influence of hydrogen peroxide (H2O2) on the expression of calcium-independent phospholipase A2 (iPLA2) in islet microvascular endothelial cells (IMECs). METHODS: Total RNA was isolated from IMECs following induction at different concentrations of H2O2 (0, 30, 100, 300 mol/L) and for different incubation durations (0, 4, 8, 12 h). RT-PCR analysis was then performed to detect the expression level of iPLA2. Apoptotic DNA ladders were analyzed and the apoptotic cells were detected by a combination of flow cytometry with Annexin V-FTTC/PI. Mitochondrial membrane potentials were determined by flow cytometry with labeled DIOC6 ( 3) reagent. RESULTS: Electrophoretogram of RT-PCR showed that the electrophoresis strip of iPLA2 mRNA of IMECs incubated with 300 μmol/L H2O2 for 12 h was weaker than that of other groups (0, 30, 100 μmol/L). The electrophoresis strips of iPLA2 mRNA of IMECs incubated for 8 h and 12 h were weaker than those of groups incubated for 0 h and 4 h with 300 μmol/L H2O2. The apoptotic rates of IMECs were significantly different between different dosages of H2O2[(2. 0 ±0.5)%, (19. 1 ±5. 8)%, (28.4 ±4. 9)%, (40. 1 ±5. 1)% for 0, 30, 100 and 300 μmol/L respectively, P < 0. 01 ]. The apoptotic rates of IMECs between different incubation durations with 300 μmol/L H2O2 were also significantly different ( P < 0.01). The fluorescence intensity of intracellular DIOC6(3) in IMECs treated with different dosages of H2 O2 and different incubation durations was significantly different (P<0. 01). CONCLUSION: H2O2 inhibits the expression of iPLA2 of IMECs in a dose- and time-dependent manner.
hydrogen peroxidephospholipase A2islets of langerhansmicrovascular endothelial cells
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