| Peritoneal dialysis is one of the main replacements of the end stage renal disease. But continuous peritoneal dialysis unavoidably leads to peritonitis related to peritoneal dialysis,and lead to inflammatory cells into peritoneum and deposition of extracelluar matrix in the peritoneum, finally they lead to dialysis failure. Lipopolysaccharide is the main pathogenic material and only one endotoxin of Escherichia coli, whose resources are catheter related infection of continuous peritoneal dialysis and bacteriumin intestinal tract into abdominal cavity through high permeable bowel walls.LPS and high glucose in peritoneal dialysis fluid are the reasons that lead to the inflammation-like changes in the peritoneum and peritoneal fibrosis. Peritoneal mesothelial cells are the main cells of the peritoneum,and the first physiologic barrier of peritoneum that directly contact peritoneal dialysis fluid,are also the first reaction of peritoneum injure. It is the base of finding the mechanism and interfering peritoneal injure to research the reaction of peritonea lmesothelial cells on LPS and high glucose condition.Tumor necrosis factor(TNF) is the most important early proinflammatory cytokine, which may lead to some other factors like IL-1 be expressed and secreted. High mobility group box-1(HMGB-1) is recently identified as a "late" mediator of endotoxin lethality. LPS and TNF-αare its stimulus, while HMGB-1 stimulates monocytes and macrophages to secrete other proinflammatory mediators(e.g.,TNF and IL-1) in order to amplify and extend the "cytokine cascade".Meanwhile, the migration of HMGB-1 is the critical signal of the cell necrosis or apoptosis. Transforming growth factor-β1(TGF-β1) is a multifunction inflammatory active factor, which is regarded as the key factor that directly contact peritonitis with peritoneal fibrosis.We investigate the expression of TNF-a,HMGB-land TGF-β1 that has been stimulated by LPS and the relationship among them to study the mechanism of peritoneum injure and peritoneum fibrosis.Xuebijing is a Chinese crude medicine,which has a better therapeutic effect in many inflammatory and fibrous diseases. We investigate the effect of Xuebijing on the expression of TNF-a and HMGB-1 that has been stimulated by LPS to provide a base to take research in protect peritoneum from injure of inflammation.In addition, we also investigate the effect of Xuebijing on the expression of HMGB-1 and TGF-β1 that has been stimulated by high glucose to provide a base to take research in protect peritoneum from fibrosis.Methods1.Experimental animals:the healthy male Sprague Dawley rats were chose weighted about 120-160g, from the experimental animal center in China Medical University.2.Cells culture:PMCs were isolated from omentum and subcutured after enzymatic digestion.They could be identified with phase contrast inverted microscope and transmission electron microscope,scanning electron microscope and immunocytochenmistry method.The identify technology is proficiency.The expression of HMGB-lmRNA was measured with RT-PCR method,the protein expression of TNF-a,HMGB-1 and TGF-β1 were measured with enzyme linked immunosorbentassay.The PMCs were treated on the following conditions:(1) Different concentration LPS:0,1,10, 100mg/L(2) 10mg/L LPS for different time:0,2,6,12,18,21,24,36 hours(3) PMCs were treated with Xuebijing(2,10,20g/L)for 4,22 hours after incubated with lOmg/L LPS for 2 hours to detective TNF-a and HMGB-1 level(4) 2.5% high glucose for different time:0,18,24,36 hours.(5) PMCs were treated with Xuebijing(2,10,20g/L) and 2.5% high glucose for 18,24,36 hours to detective HMGB-1 and TGF-β1 levelResults 1.The influences of LPS on the expression of HMGB-1lmg/L LPS could accelerate the expression of HMGB-1mRNA and protein(P<0.05),the effect of 100mg/L LPS was the greatest(P<0.01);on the 18th hour, the expression of HMGB-1 mRNA and protein rose in the lOmg/L LPS group(P<0.05), on the 36th hour,the expression of HMGB-1 mRNA and protein got the highest(P<0.01).2.The influences of LPS on the expression of TNF-almg/L LPS could accelerate the expression of TNF-a protein(P<0.05),the effect of 100mg/L LPS was the greatest(P<0.01);In the 10mg/L LPS group, the expression of TNF-a protein rose on the 2nd hour and on the 21st(P<0.05), the expression of TNF-a appeared double hump in the 36 hour.3.The influences of LPS on the expression of TGF-β1In the 10mg/L LPS group, the expression of TGF-β1 protein rose to the peak on the 2nd hour,followingly decreased on the 6th hour and then increased in the time of 12 hour to 36 hour(P<0.05).4.The influences of Xuebijing on the HMGB-1 of PMCs that pre-treated with LPSlOg/L Xuebijing inhibit the HMGB-1mRNA expression of PMCs that have been pre-treated with 10mg/L LPS for 2 hours(P<0.05);2g/LXuebijing inhibit the HMGB-1 protein expression of PMCs that have been pre-treated with 10mg/L LPS for 2 hours(P<0.05), the inhibition effect of 20g/L Xuebijing was the greatest(P<0.01).5.The influences of Xuebijing on the TNF-a protein of PMCs that pre-treated with LPS2g/L Xuebijing inhibit the TNF-a protein expression of PMCs that have been pre-treated with 10mg/L LPS for 2 hours(P<0.05), the inhibition effect of 20g/L Xuebijing was the greatest(P<0.01).6.The influences of high glucose on the expression of HMGB-1 and TGF-β1On the 18th hour, the expression of HMGB-1 and TGF-β1 rose in the 2.5% high glucose (P<0.05),on the 36th hour, both of them got the highest(P<0.01). 7.The influences of Xuebijing on the HMGB-1 and TGF-β1 of PMCs that treated with high glucoseThe expression of HMGB-1 and TGF-β1 in groups treated with 2.5% glucose and Xuebijing was significantly lower than that in groups treated with 2.5% glucose only (P <0.05); 2g/L Xuebijing inhibit the HMGB-1 and TGF-β1 protein expression, the inhibition effect of 20g/L Xuebijing was the greatest(P<0.01).Conclusion1. HMGB-1 as a late inflammatory factor may play a role in the pathogenesis of peritonitis relating to CAPD, LPS up-regulates the expression of TNF-a,HMGB-1 and TGF-β1, leading to the continuous amplification of inflammatory processes and the aggravation of peritoneal impairment and fibrosis.2. Xuebijing might reduce peritoneal inflammatory impairment by inhibiting the up-regulation of TNF-a and HMGB-1 induced by LPS.3. Glucose of high concentration can promote the synthesis of HMGB-1 and TGF-β1 in PMC. Xuebijing can reverse these changes and it may have significance in the prevention and treating of peritoneal fibrosis.
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