| ObjectivePeritoneal dialysis is the main replace treatment of end stage renal diseases, which has predominance that can not be vicarious by hemodialysis. But continuous peritoneal dialysis unavoidably leads to the inflammation-like changes in the peritoneum and peritoneal fibrosis, whose main pathological changes are mononuclear macrophage into peritoneum and deposition of extracellular matrix in the peritoneum, and 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 bacterium in intestinal tract into abdominal cavity through high permeable bowel walls. LPS and high glucose in peritoneal dialysis fluid are the reasons that leads to the inflammation-like changes in the peritoneum and peritoneal fibrosis.Peritoneal mesothelial cells are those on the surface of peritoneum, are the first physiologic barrier of peritoneum that directly contact peritoneal dialysis fluid, are also the first reaction of peritoneal injure. It is the base of finding the mechanism and interfering peritoneal injure to research the reaction of peritoneal mesothelial cells on LPS condition.Motocyte chemoattractant protein-1 is the important chemotatic factor and one of CC subfamily. CCR2, the receptor of MCP-1, is a kind of G protein-coupled receptor with seven transmembrane domain on different kinds of cells(monocytes, remembrance T lympholeukocyte, basophil leukocyte et al.). After binding between MCP-1 and CCR2, CCR2 changes structure and combinates with G protein to activate signal transduction pathway and make its biological effects. MCP-1 can attract monocytes or macrophage and T lympholeukocyte, and can induce monocytes and endothelial cells to express adhesion molecule to attract the inflammation cell, especially monocytes or macrophages to the disease position, start inflammatory reaction, promote the secretion of extracellular matrix and lead to fibrosis and sclerosis of tissue and organ. Recently MCP-1 was thought to play a start and aggravation role in diabetes nephropathy, peritoneal dialysis related peritonitis and peritoneal fibrosis. So far, there have not been any detailed researches about the effects of LPS on the expression of MCP-1, which was the one of focus in theNuclear factor-kappaB is the important transcription factor participating inflammation reaction, lies in many tissues and cells, and is related to happening and development of many diseases. NF-κB is nuclear factor regulating many factors to transcript. On normal condition, NF-κB and inactive regulatory subunit IκB form a dimer complex in cytoplasm, which shields its nucleic localization sequence. On many stimulus, IKK complex phosphorates IκB, p-IκB is ubiquitined by ubiquitin system and degraded by 26S proteasomes which make nucleic localization sequence of NF-κB exposed. NF-κB enters nuleus and bind withκB sequence to participate the transcription of related genes. p65: p50 dimer is largely studied and proved to activate transcription. So usually NF-κB means p65: p50. IKKβand IκBαet al. are main factors joining in the period of the activation of the dimmer. The search on gene sequence and the related researches showed there was binding sequence of NF-κB in the promoter region of MCP-1 gene. NF-κB signal pathway participated in the expression of MCP-1 in some tissues and cells. To study the expression of NF-κB signal pathway in peritoneal mesothelial cells is the foundation of research about its role participating in the expression of MCP-1. SN50, a NF-κB nucleus shift inhibitory peptide, MG-132, a proteasomes inhibitor, BAY11-7082, a IκBαphosphoration inhibitor, and SC-514, a IKKβspecific inhibitor are inhibitors of different factors in NF-κB signal pathway. Researching the expression of MCP-1 in PMC on these inhibitors and the role of NF-κB signal pathway during the period will give clue for improving ultrafiltration failure.Methods1. Tissue source: The healthy male Sprague Dawley rats were chose weighted about 120±20g, from the experimental animal center in China Medical University.2. Cells culture: PMC were isolated from omentum and subcultured after enzymatic digestion. They were identified with phase contrast inverted microscope and transmission electron microscope, scanning electron microscope and immunocytochemistry method.3. On high glucose and LPS conditions, the expression of MCP-1mRNA was measured with RT-PCR method, the protein expression of MCP-1, p-IKKβ, IκBα, p-IκBαand p65 in nucleus was measured with Western blot method. Immunocytofluorometric method was used to measure the shifting of p65 into nucleus. The PMCs were treated on the following conditions.(1) Different concentration LPS: 0, 0.1, 1.0,10, 100mg/L;(2) 1.0mg/L LPS for different time: 0, 0.5, 1, 3, 12, 24, 48 hours;(3) The combination of high glucose and LPS: normal control group, 1.5% glucose, 1.0mg/L LPS, the combination of 1.5% glucose and 1.0mg/L LPS.4. Using the inhibitors of NF-κB signal pathway, for example SN50, MG-132, BAY11-7082, SC-514, RT-PCR method was used to measure the expression of MCP-1mRNA, and Western blot method was used to measure the expression of MCP-1 protein in PMC. The PMCs were treated on the following conditions.(1) After different concentration(10,25,50mg/L) SN50 was for 3 hours, 1.0mg/L LPS was added in the media for 3 hours. Made SN50M control group(SN50 inactive control peptide).(2) After different concentration(5,10,20uM) MG-132 was for 3 hours, 1.0mg/L LPS was added in the media for 3 hours.(3) After different concentration(5,10,20uM) BAY11-7082 was for 3 hour, 1.0mg/L LPS was added in the media for 3 hours.(4) After different concentration(10,50,100uM) SC-514 was for 1 hour, 1.0mg/L LPS was added in the media for 3 hours.Results1. LPS accelerated PMC to express MCP-10.1mg/L LPS could accelerate the expression of MCP-1mRNA and protein(P<0.05), the effect of 100mg/L LPS was the greatest(P<0.01). On the 1st hour, the expression of MCP-1mRNA rose in the 1.0mg/L LPS group(P<0.05). On the 3rd hour, the expression of MCP-1 protein rose significantly(P<0.05). On the 48th hour, the expression of MCP-1mRNA and protein got the highest(P<0.01).2. The influences of high glucose and LPS on the expression of MCP-1 in PMCHigh glucose accelerated PMC to express MCP-1(P<0.01). Compared with high glucose or LPS, the combination effect of high glucose and LPS got the greatest on the expression of MCP-1 in PMC(P<0.05).3. The influences of LPS on p-IKKβ, IκBα, p-IκBα, p65 in PMC.In 0.1mg/L LPS group, p-IKKβrose(P<0.05), IκBαfell, p-IκBαrose in the cytoplasms(P<0.05), p65 rose in the nuleus(P<0.05). The effects of 100mg/L LPS were the greatest(P<0.01). On the 30th minutes, p-IKKβrose(P<0.05), IκBαfell, p-IκBαrose in the cytoplasms(P<0.05), p65 rose in the nuleus(P<0.01) in 0.1mg/L LPS group. On the 1st hour, p-IKKβrose to the peak(P<0.01). On the 3rd hour, p-IKKβfell fast, IκBαfell to the lowest, p-IκBαrose to the highest in cytoplasm(P<0.01), p65 in nucleus rose to the highest(P<0.01). Then p-IKKβfell slowly, IκBαbegan to increase, p-IκBαbegan to decrease, p65 in nucleus began to fell. On the 24th hour, p-IKKβand p-IκBαrose again, IκBαfell, p65 rose in nucleus significantly. On the 48th hour, p-IKKβand p-IκBαfell significantly, IκBαrose, p65 in nucleus decreased significantly.4. The role of NF-κB signal pathway during LPS promoted PMC to express MCP-1.Compared with LPS, 10mg/L SN50, 5uM MG-132, 5uM BAY11-7082, 10uM SC-514 all inhibited the expression of MCP-1mRNA and protein on LPS condition(P<0.05), 50mg/L SN50, 20uM MG-132, 20uM BAY11-7082, 100uM SC-514 inhibited the expression of MCP-1mRNA and protein significantly(P<0.01). SN50M had no the effects(P>0.05).Conclusion1. LPS accelerated PMC to express MCP-1mRNA and protein in a dose-and time-dependent manner.2. The effect of high glucose and LPS on the expression of MCP-1mRNA and protein in PMC was more than glucose or LPS.3. LPS could make p-IKKβincrease, IκBαfall, p-IκBαrise, p65 into nucleus in a dose-dependent manner to LPS. IKKβ-IκBα-p65 showed two-peak activities.4. LPS accelerated PMC to express MCP-1 depending on IKKβ-IκBα-p65: p50 signal pathway.
|
PDF Full Text Request