| ObjectiveContinuous ambulatory peritoneal dialysis (CAPD) is a major treatment for end-stage renal failure. The peritoneal membrane exhibits injuries that correlate with the duration of dialysis. The injuries in the peritoneum are due to its exposure to non-physiologic peritoneal dialysis solution (PDS) with a high glucose content (1), and LPS, a glycolipid that is produced and secreted by gram-negative bacteria during peritonitis, and also glucose degradation products, pH, osmolality and gram-positive bacteria and fungi related peritonitis. Here we use a HG and LPS model to stimulate the RPMCs.Peritoneal mesothelial cells have the capacity to produce a variety of cytokines. Transforming growth factor-β1 (TGF-β1) is an important profibrotic cytokine that has been suggested to affect the peritoneal membrane function during CAPD.TNF-a is another potential candidate that may play a role in progressive fibrosis of the peritoneum during CAPD. In peritoneal mesothelial cells, TGF-β1 and TNF-a is up-regulated during peritonitis and peritoneal fibrosis. Transforming growth factor (3-1 (TGF-β1) is a complex cytokine that regulates diverse biological processes, including cellular proliferation and differentiation, immune modulation, and extracellular matrix remodeling. The proinflammatory cytokine tumor necrosis factor a (TNF-a) is a pleiotropic regulator that plays an important role in the pathophysiology of numerous disorders and controls the expression of a number of inflammatory and immune regulatory genes through activation of the nuclear transcription factor kappa B (NF-κB), which may promote adhesion molecule expression.Vitamin D receptor (VDR) belongs to a nuclear receptor super-family that mediates the genomic actions of vitamin D3 and regulates gene expression by binding with vitamin D response elements (VDRE) in the promoter region of the cognate gene. It is well established that the physiological role of 1,25(OH)2D3, the active metabolite of vitamin D3, is not limited to mineral and skeletal homeostasis. In recent years, there has been increasing evidence pointing to the role of 1,25(OH)2D3 and its analogues in the regulation of cell proliferation, cell differentiation and immunomodulation. 1,25(OH)2D3 has a potent inhibitory effect on inflammation and fibrosis. We hypothesize that 1,25(OH)2D3 may constitute a regulatory mechanism that, by controlling the intensity of the inflammatory and fibrotic response of the peritoneum, will moderate tissue damage during peritonitis and peritoneal fibrosis.The present study was designed to investigate the regulatory effect of 1,25(OH)2D3 on the generation of TGF-β1 and TNF-a by RPMCs stimulated by HG and LPS. To understand the role of HG and LPS on the vitamin D system, we have investigated HG and LPS action on vitamin D system by assessing VDR expression and function in response to 1,25(OH)2D3 in RPMCs. We believe such an investigation could be of particular clinical value since in the peritoneal environment.Methods1. Experimental animals:the healthy male Sprague Dawley rats were chose weighted about.140±20g,from the experimental animal center in China Medical University.2. Cells culture:RPMCs were isolated from omentum and subcultured after enzymatic digestion. They were observed under phase contrast inverted microscope, and identified with immunocytochemistry.The expression of VDRmRNA was measured by RT-PCR,and the protein expression of VDR was measured by Western-Blot. The level of TGF-β1 and TNF-a in the supernatants of RPMCs cultures were measured by ELISA.3. The RPMCs were treated on the following conditions:(1) The Control group.(2) LPS for different concentrations:0,1,10,100μg/ml(3) lOμg/ml LPS for different times:0,2,6,12hours.(4) RPMCs were treated with 1,25(OH)2D3 (10-8mol/L,10-7mol/L,10"6mol/L)for 4 hours after preincubated with 10μg/ml LPS for 2 hours to detect the level of VDRmRNA and protein as well as TGF-β1 and TNFα.(5) High glucose for different concentrations:1.5%,2.5%,4.25%.(6) 2.5%glucose for different times:0,2,6,12hours.(7) RPMCs were treated with 1,25(OH)2D3 (10-8mol/L,10-7mol/L,10-6mol/L)for 4 hours after preincubated with 2.5%glucose for 2 hours to detect the level of VDRmRNA and protein as well as TGF-β1.Results1. The effects of LPS on the expression of VDR1μg/ml LPS could reduce the expression of VDRmRNA and protein(P<0.05),the effect of 100μg/ml LPS was the greatest<0.01);on the 2nd hour,the expression of VDRmRNA and protein descended in the LPS group(P<0.05);on the 12th hour,the expression of VDRmRNA and protein reach to the minimum(P<0.01).2. The effects of 1,25(OH)2D3 on the VDR expression that pre-treated with LPS10-8mol/L 1,25(OH)2D3 increase the VDRmRNA and protein expression of RPMCs that have been pre-treated with 10μg/ml LPS(P<0.01);the enhancement effect of 10-6mol/L 1,25(OH)2D3 was the greatest(P<0.01).3. The effects of LPS on the expression of TGF-α1 and TNF-αLPS could up-regulate the expression of TGF-β1 and TNF-αin RPMCs(P <0.01);the effect of 1 00μg/ml LPS and 12th hour was the greatest(P<0.01).4. The effects of 1,25(OH)2D3 on the expression of TGF-α1 and TNF-αthat pre-treated with LPS10-8mol/L 1,25(OH)2D3 inhibit the expression of TGF-β1 and TNF-αthat have been pre-treated with 10μg/ml LPS(P<0.01);the inhibition effect of 10-6mol/L 1,25(OH)2D3 was the greatest (P<0.01).5. The effects of high glucose on the expression of VDR1.5%glucose could reduce the expression of VDRmRNA and protein(P<0.05),the effect of 4.25%glucose was the greatest(P<0.01);on the 2nd hour,the expression of VDRmRNA and protein descended in the high glucose group(P<0.05);on the 12th hour,the expression of VDRmRNA and protein reach to the minimum(P<0.01).6. The effects of 1,25(OH)2D3 on the VDR expression that pre-treated with high glucose10-8mol/L 1,25(OH)2D3 increase the VDRmRNA and protein expression of RPMCs that have been pre-treated with 2.5%glucose(P<0.01);the enhancement effect of 10-6mol/L 1,25(OH)2D3 was the greatest (P<0.01).7. The effects of high glucose on the expression of TGF-β12.5%glucose could up-regulate the expression of TGF-β1 in RPMCs(P<0.01);the effect of 4.25%glucose and 12th hour was the greatest(P<0.01).8. The effects of 1,25(OH)2D3 on the expression of TGF-β1 that pre-treated with high glucose10-8mol/L 1,25(OH)2D3 inhibit the expression of TGF-β1 that have been pre-treated with 2.5%glucose(P<0.01);the inhibition effect of 10-6mol/L 1,25(OH)2D3 was the greatest (P<0.01).Conclusion1. RPMCs express mRNA and protein of VDR.2. LPS and high glucose down-regulate the mRNA and protein of VDR in a dose-and time-dependent manner, and increase the expression of inflammatory cytokines.3.1,25(OH)2D3 could reverse the decrease of VDRmRNA and protein in RPMCs that have been pre-treated wih LPS and high glucose, and inhibit the expression of inflammatory cytokines.4. There is a negative correlation between VDR and inflammatory cytokines, 1,25(OH)2D3 has a protect effect in peritoneal dialysis related peritonitis. |
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