Effect Of Vitamin E On Expression Of FN And Generation Of Reactive Oxygen Species In High Glucose Induced-human Peritoneal Mesothelial Cells

Objective : It is known that human peritoneal mesothelial cells (HPMCs) are important components of the peritoneum and play a very important role in maintaining its structural integrality and functional effectivity, which are the therapeutic foundation in continuous ambulatory peritoneal dialysis (CAPD). The chronic peritoneal dialysis will induce peritoneal mesothelial cells damage, and promote the formation of peritoneal fibrosis, result in ultrafiltration failure in the end, thereby force patient to drop out the therapy of peritoneal dialysis. accordingly, the prevention and cure of CAPD-related peritoneal fibrosis become focal point by researcher in domestic and overseas area. Therefoce, this study aim to investigate high glucose whether induce human peritoneal mesothelial cells oxidative stress injury via the generation of reactive oxygen species and effect of high glucose on expression of fibronectin. Meanwhile, we applied different concentrations antioxidant vitamin E in experiment which to observe its protection contribution under the condition of human peritoneal mesothelial cells oxidative stress injury, thus providing reliable experiment proof for the prevention and treatment of peritoneal fibrosis in clinic.Methods:The HPMCs were isolated from discarded peritoneal dialysate which came from the chronic kidney disease patients who were receiving continuous ambulatory peritoneal dialysis and primarily cultured in RPMI1640 supplemented with 10% heat inactivated fetal bovine serum. The secondarily cultured HPMCs were identified by immunohistochemistry. The thirdly cultured HPMCs were used in the experiment. HPMCs were divided into dose-dependent groups and drug groups according to experiment required. Theprotein of FN in the supernatant of the culture medium of cells were measured by enzyme-linked immunosorbent assay. The expression of FN mRNA were detected by semiquantification reverse-transcriptive polymerase chain reaction (RT-PCR). and the intracellular generation of reactive oxygen species (ROS) were assessed indirectly by measuring the fluorescent product from the oxidation of an oxidant-sensitive 2',7'-dichlorofluorescein (DCFH) fluorescence probe using flow cytometry. The Pre-treatment of HPMCS with the three different concentrations antioxidant vitamin E (12.5μmol/L,25μmol/L,50μmol/L) respectively to investigate the effect of vitamin E on the expression of FN in HPMCS and the generation of endogenous ROS in this course.Results:1. High glucose could induce significantly the secretion and gene expression of the FN in a dose-dependent manner in vitro cultured HPMCs (P<0.01). The group of 4.25%glucose showed most significantly.2. High glucose could stimulate significantly the ROS production in a dose-dependent manner in vitro cultured HPMCs (P<0.01). The group of 4.25 % glucose showed most significantly by measuring the mean fluorescence intensity in 30min.3. Compared to the blank control group, the pre-treatment of HPMCS with the three different concentrations antioxidant vitamin E could reduce significantly high glucose-induced ROS production and inhibit significantly the secretion and gene expression of the FN in vitro cultured HPMCs in a dose-dependent manner (P<0.01). The 50μmol/L VitE groups showed most obviously.Conclusion:1. High glucose may induce the secretion and gene expression of the FN in vitro cultured HPMCs and the vitamin E as an antioxidant can significantly inhibit the secretion and gene expression of the FN.2. High glucose may stimulate the generation of reactive oxygen species and result in oxidative stress injury in vitro cultured HPMCs. The vitamin E as an antioxidant can decrease ROS production.3. This study demonstrate that vitamin E may be a effective medicine for the prevention and treatment of the peritoneal fibrosis, thus providingreliable experiment proof for the application of vitamin E in clinic.