The Expression Of Toll-like Receptor 2 In Rat Peritoneal Mesothelial Cells That Stimulated By Peptidoglycan,Rosiglitazone And High Glucose

ObjectivePeritoneal dialysis is the main replacement of end stsge renal disease,which has predominance that can not be replace by hemodialysis.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.In Peritoneal dialysis-related infections,the most common pathogens is bacteria,in peritonitis that caused by bacterial infection seventy percent to ninty five percent is resulted by Gram-positive bacteria.As the characteristic component of Gram-positive bacteria cell wall,peptidoglycan could lead to inflammation.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.peritoneal mesothelial cells take important part in the peritoneal dialysis-related infections,and could also be injured by the inflammation factors.It is the base of finding treatment that protect the peritoneum from the inflammation injure to study the reaction of the peritoneal mesothelial cells under the stimulation of peptidoglycan.The toll-like receptors family is an important membrane receptor family,TLRs proteins are germline-encoded receptors for various pathogen associated molecular patterns.The mammalian toll like receptors are recently recognized family of proteins implicated in direct innate immune response by components of diverse pathogens.Innate immune response mediated by toll-like receptors(TLRs)are the first line of defense against infections agents entering the organism.Until now,eleven members of the TLRs family have been reported in mammalians.Rccent in vitro studies demenstrate that TLR2,as a member of the TLRs family proteins,confers cell responsiveness to PGN,in vivo studies with mice harboring null alleles for TLR2 also provide stong evidence that TLR2 is responsible for PGN-induced signaling,The activation of TLR2 induced by PGN elicit the excessive release of proinflammatory cytokines from immune cells.these Excessive proinfiammatory cytokines(such as tumor necrosis factor-α)lead to aggregation of intraperitoneal Interleukin cells,and stimulate fibroblasts cells to generate extracelluar matrix,result in peritoneum fibrosis and ultrafiltration failure.Peroxisome proliferators activated receptors(PPARs)belong to the nuclear hormone receptor super family that regulates transcription by binding to retinoid X rece -ptor that is in turn bound to DNA in various cell types.Peroxisome proliferators activat -ed receptors have three phenotypes,Peroxisome proliferators activated receptor gamm -a is one of the three phenotypes.it take important part in the treatment of diabets.Activ -ation of Peroxisome proliferators activated receptor gamma promotes insulin-stimulate -d glucose uptake,In addition,it suppresses pro-inflammatory response,related inflamma -ation signaling pathway includes①JAK-STAT;②NF-κB;③nuclear factor ofactivated T cells(NFAT);④AP-1 and etc.We investigate the effect of peroxisome proliferators activated receptor gamma agonist rosiglitazone on the expression of TLR2 that has been stimulated by PGN to provide a base to take research in protect peritoneum from injure of inflammation.In addition,high glucose in the peritoneal dialysis fluid is an other important reason that leads to the inflammation-like changes in the peritoneum and peritoneal fibrosis,according to other study,high glucose could induce peritoneal mesothelian cells to release pro-inflammation cytokines,such as IL-1.We investigate the TLR2 expression of RPMCs that has been stimulated by high glucose to study the mechanism of peritoneum injure in peritoneal dialysis without infection. Methods1.Experimental animals:the healthy male Sprague Dawley rats were chose weighted about 120±20g,from the experimental animal center in China Medical University.2.Cells culture:RPMCs were isolated from omentum and subcutured after enzymatic digestion.They could be identified with phase contrast inverted microscope and transmission elctron microscope,scanning electron microscope and immunocytochenmistry method.the identify technology is proficiency.3.On PGN and rosiglitazone conditions,the expression of TLR2mRNA was measured with RT-PCR method,the protein expression of TLR2was measured with immunostaining via flow cytometry.the surface expression is indicated as mean fluorescence intensity.The RPMCs were treated on the follwing conditions:(1)Different concentration PGN:0,1.0,10,100μg/ml(2)10μg/ml PGN for different time:0,2,6,12hours(3)RPMCs were treated with Rosiglitazone(5μmol/L,10μmol/L,20μmol/L)for 4 hours after incubated with 10μg/ml PGNfor 2 hours to detective TLR2 protein level(4)RPMCs were treated with 10μmol/L Rosiglitazone for 4 hours after incubated with 10μg/ml PGNfor 2 hours to detective TLR2 mRNA level)(5)Different concentration high glucose:0,1.5%,2.5%,4.25%(6)2.5%high glucose for different time:0,5,10,20hoursResults1.The influences of PGN on the expression of TLR21.0μg/ml PGNcould accelerate the expression of TLR2mRNA and protein(P＜0.05),the effect of 100μg/ml PGN was the greatest(P＜0.01);on the 2^ndhour,the expression of TLR2mRNA and protein rose in the PGN group(P＜0.05);on the 6^th hour,the expression of TLR2mRNA and protein rose significantly(P＜0.01);on the 12^th hour,the expression of TLR2mRNA and protein got the highest(P＜0.01). 2.The influences of rosiglitazone on the TLR2 of RPMCs that pre-treated with PGN10μmol/L rosiglitazone inhibit the TLR2mRNA expression of RPMCs that have been pre-treated with 10μg/ml PGN for 2 hours(P＜0.05);5μmol/L rosiglitazone inhibit the TLR2 protein expression of RPMCs that have been pre-treated with 10μg/ml PGN for 2 hours(P＜0.05),the inhibition effect of 20μmol/L rosiglitazone was the greatest(P＜0.01).3.The influences of high glucose on the expression of TLR2mRNA1.5%high glucose could accelerate the expression of TLR2mRNA(P＜0.05),the effect of 4.25%high glucose was the greatest(P＜0.01);on the 5^thhour,the expression of TLR2mRNA rose in the high glucose group(P＜0.05);on the 10^thhour,the expression of TLR2mRNA a rose significantly(P＜0.01);on the 20^thhour,the expression of TLR2mRNA got the highest(P＜0.01).Conclusion1.RPMCs express TLR2.2.PGN accelerated RPMCs to express TLR2mRNA and protein in a dose- and time-dependent manner.3.Rosiglitazone inhibited the up-regulating expression of TLR2mRNA.Rosiglitazone inhibited the up-regulating expression of TLR2 protein in a dosedependent manner.4.High glucose accelerated RPMCs to express TLR2mRNA in a dose- and time-dependent manner.