| Objective:Advanced oxidation protein products (AOPP) are protein cross-linking products isolated from the plasma of hemodialysis (HD) patients, which was firstly reported by Witko-Sarsat in 1996. AOPP as a novel markers of oxidative stress. AOPP appear to act as true inflammatory mediators and new uremic toxins with biologic activity, their role in the immune dysregulation and pathophysiology of chronic kidney disease and dialysis-related complications. Therefore, this study aim to investigate the effects of advanced oxidation protein products-human serum albumin (AOPP-HSA) on the expression of the transforming growth factor-beta 1 (TGF-β1) in human peritoneal mesothelial cells (HPMCs) cultured in vitro and the regulatory mechanism of endogenous reactive oxygen species (ROS) in this course. So that elucidating the relation between AOPP and the mechanism of CAPD-related peritoneal fibrosis which providing reliable proof for the application of antioxidant in clinic.Methods:The model of AOPP-human serum albumin (HSA) in vitro which were prepared according to the method described Witko-Sarsat et al. by combining endotoxin free HSA with hypochlorous acid at molar ration of 1/12.5,1/25,1/50,1/100 respectively. The HPMCs in vitro were separated from peritoneal dialysis effluent in chronic kidney disease patients with CAPD and primarily cultured in RPMI 1640 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 time-dependent groups, dose-dependent groups and drug groups according to different experiment required. Theprotein of TGF-β1 in the supernatant of the culture medium of cells were measured by enzyme-linked immunosorbent assay. The mRNA expressions of TGF-β1 were determined by semiquantification reverse-transcriptive polymerase chain reaction (RT-PCR). 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) and N-acetyl-cyteine (0.1mmol/L,1.0mmol/L,10mmol/L) respectively to investigate the effect of antioxidant on the expression of TGF-β1 in HPMCS and the regulatory mechanism of endogenous ROS in this course.Results:1. AOPP-HSA can induce significantly the secretion and gene expression of the TGF-β1 in cultured HPMCs in a dose-dependent manner (P<0.01). The secretion and gene expression of the TGF-β1 enhanced with the reaction time increased in the range of 0-48h (P<0.05), it achieved peak value in 48h (P<0.01) then decreased. It also correlated with the molar ratio of HSA/HOCL (P<0.01).2. AOPP-HSA can increase significantly ROS production in cultured HPMCs in a dose-dependent manner (P<0.01). The mean fluorescence intensity (MFI) in cultured HPMCs enhanced with the reaction time prolongation in the range of 0-30min when stimulated with AOPP-HSA. It achieved peak value in 30min (t=-26.073, P<0.01) and reduced gradually in 60min and 120min (t=-10.607,-7.813, P<0.05). It also correlated with the molar ratio of HSA/HOCL (P<0.01).3. Compared to the control group, the Pre-treatment of HPMCs with the different concentrations antioxidan vitamin E (12.5μmol/L,25μmol/L,50μmol/L) and N-acetyl-cyteine (0.1mmol/L,1.0mmol/L,10mmol/L) significantly reduced AOPP-induced ROS production and the secretion and gene expression of the TGF-β1 in cultured HPMCs in a dose-dependent manner (P<0.01). The 50μmol/L VitE groups and 10mmol/L NAC groups inhibited most significantly.4. Compared to the PBS control group, Non-oxidized native HSA didnot significantly increase the secretion and gene expression of the TGF-β1 and ROS production in cultured HPMCs (P>0.05).Conclusion:1. AOPP-HSA prepared in vitro significantly induced on secretion and gene expression of TGF-β1 in HPMCs, which may be partially mediated through endogenous ROS-dependent pathway. This study indicated that AOPP might be participated in the formation of CAPD-related peritoneal fibrosis by enhancing oxidative stress in vivo.2. AOPP-HSA prepared in vitro may induce HPMCs respiratory burst. The antioxidant vitamin E and N-acetyl-cyteine significantly reduced AOPP-induced ROS production.3. The antioxidant of vitamin E and N-acetyl-cyteine may significantly inhibit the secretion and gene expression of the TGF-β1 via scavenging ROS in HPMCs.4. This study demonstrate that the antioxidant may be a potential therapeutic way for prevention and treatment the peritoneal fibrosis, which providing reliable proof for the application of antioxidant in clinic.
|
PDF Full Text Request