Intermedin Attenuates Hypoxia-induced-injury In Rat Glomerular Endothelial Cells

Objective:To investigate the effect of intermedin against hypoxia-induced-injury and its possible mechanism in rat glomerular endothelial cells.Methods:(1) grouping in the experiment: After subculturing r RGEC, divided them into normal group, hypoxia group and IMD groups. Cells of the normal group were cultivated in the same carbon dioxide incubator, hypoxia group and IMD groups were cultivated in the three gas incubator. IMD groups were divided into IMD1?IMD2 and IMD3 groups according to the concentrations of IMD which were 10-8mol/L?10-7mol/L and 10-6mol/L. Specimens of each group were taken after 6 hours.(2) Cell morphology changes were observed under inverted phase contrast microscope.(3) Cell apoptosis ratios were measured by flow cytometry.(4) Levels of 4-HNE, ROS and SOD in the cell supernatant were detected by Elisa method.(5) Changes of cell permeability were measured by the ratios of fluorescence intensity of endothelial cell monolayer.(6)The relative expressions of hypoxia inducing factor 1 alpha(HIF- 1?) and vascular endothelial growth factor(VEGF) m RNA were detected by quantitative-real-time-PCR.(7) The content of HIF- 1? and VEGF protein was detected by immunohistochemistry.Results:(1) Changes of morphological characteristics: morphological characteristics of cells of N group were normal, while cells of H group became swelling and there were some dead cells floating on the supernatant. Compared with H group, morphological characteristics of cells of IMD1?IMD2?IMD3 group were close to N group, and dead cells floating on the supernatant became fewer.(2) Changes of cell apoptosis ratios : Compared with N group, cell apoptosis ratios of IMD1?IMD2?IMD3 group decreased(P<0.05);Compared with H group, cell apoptosis ratios of cells of IMD1?IMD2?IMD3 group decreased(P < 0.05).(3) Levels of 4-HNE, ROS and SOD : Compared with N group, levels of 4-HNE and ROS in cell supernatant of H group elevated,SOD reduced(P < 0.05); Compared with H group, levels of 4-HNE and ROS in cell supernatant of IMD1?IMD2?IMD3 group reduced, SOD elevated(P < 0.05).(4) Changes of cell permeability : Compared with N group, cell permeability of H group increased(P < 0.05); Compared with H group, cell permeability of IMD1?IMD2?IMD3 group decreased(P < 0.05).(5) The relative expression of HIF- 1? m RNA and protein content: Compared with N group, the relative expression of HIF- 1? m RNA and protein content of H group increased(P < 0.05); Compared with H group, the relative expression of HIF- 1? m RNA and protein content of IMD2 group increased(P < 0.05).(6) The relative expression of VEGF m RNA and protein content: Compared with N group, the relative expression of VEGF m RNA and protein content of H group increased(P <0.05); Compared with H group, the relative expression of VEGF m RNA and protein content of IMD2 group increased(P <0.05).(7) HIF- 1? and VEGF m RNA were negatively correlated with the cell apoptosis ratios(r1=-0.919,r2=-0.911,P < 0.05).Conclusions: In hypoxia environment, IMD can attenuate the morphology injury and reduce the permeability of glomerular endothelial cells. When the cells were treated with IMD, the lipid peroxide reduced, the cell apoptosis ratios reduced, which indicated that IMD can attenuate hypoxia-induced-injury in glomerular endothelial cells. IMD can protect glomerular endothelial cells from hypoxia-induced-injury, the mechanism is associated with the increasing expressions of HIF-1? and VEGF.