| The incidence of chronic kidney diseases(CKD) is rising rapidly year by year around the world,which has become one of the most important diseases listed behind cardiovascular diseases,tumor and diabetic mellitus.In developed countries,the incidence of CKD of the common crowd is 6.5～10%.In our country,the incidence of the crowd aged over 40 years old is 8～9%,among whom most people will become uraemia according to an epidemiological investigation.Peritoneal dialysis is one of the remedies for chronic renal failure.Because it has many advantages such as less influenced glomerular filtration,more steady cardiovascular function,more handling convenience,cheaper price,and higher probability for patients to come back to the society,people have attached more and more importance to it.There are about 10 or 15 percent of ESRD patients(over 150 thousands people) using peritoneal dialysis around the world.The percentage of ESRD patients using peritoneal dialysis is different among different countries.In our country,peritoneal dialysis has been used since 1960s.With the improved living standard,the people's stronger desire for healthy,and the consummate technology,the increasing number of ESRD patients are using peritoneal dialysis.But there are still many problems such as catheter dysfunction,material histoimcompatibility,nutrition imbalance,peritonitis,and social psychic:factor slowing down the progression of peritoneal dialysis,among which peritoneum fibrosis,ultrafiltrate and convery dysfunction,even ultrafiltration failure (UFF) become the main causes to stop the remedy.A report showed that the incidence of UFF was 14%in patients who had peritoneal dialysis for 18 months and 51% patients withdrew from peritoneal dialysis after eight years owing to UFF.So it is urgent to give more emphasis on finding the pathogenesis of and developing effective remedies for UFF.The pathogenesis of peritoneal dialysis associated peritoneal fibrosis is very complicated and remains poorly understood.Most researchers think peritoneal fibrosis is relevant to the property of dialysate,peritonitis,cytokine,and so on.The histoirncompatibility factors of dialysate including low PH,high glucose,lactate and glucose degradation products damage peritoneal mesothelial cells directly,inhibit leucocytes in the abdominal cavity,cut down immune defense capability,induce glycation end products,cause angiogenesis and promote fibrosis.Peritonitis induces a series of inflammatory reaction including alexin activation,chemotatic factors release, leukocytes accumulation and inflammatory mediator release,leading to blood vessel dilatation,mesothelial cells damage,extracellular matrix accumulation and fibrosis formation.In recent years,researchers found that cytokines played important roles in UFF.UFF was related to the rising of reverse absorption by peritoneum,chronic abdominal cavity inflammation,production of glycation end products and oxygen radicals and so on.Erigeron,4-OH-scutellaria baicalensis,is the effective component of Erigeron breviscapus(Vaniot) Hand Mazz,also names fleabane.It can inhibit protein kinase C (PKC).PKC plays significant roles in cellular secretion,release and dissolve, membranous motion,receptor combination,smooth muscle constraction,cell proliferation and differentiation,and canceration.Data proved that Erigeron could block ischemia reperfusion injury,retard oxidative stress and lipid peroxidation and prevent fibrosis proliferation.Eeigeron could also prevent extracellular matrix accumulation in diabetic nephropathy,strongly inhibit monocyte chemotactic protein- 1(MCP- 1) and intercellular adhesion molecule- 1(ICAM- 1) expression and macrophage activation in renal tissues of diabetes.In a word,Erigeron can prevent tissue fibrosis,extracellular matrix accumulation and inflammation.UFF is a very serious complication during peritoneal dialysis and one of the reasons that give rise to peritoneal dialysis failure.However,the pathogenesis and remedy need to be investegated.Toll-like receptors(TLRs),which have been found on cells of human beings or rats,belong to transmembrane signal transduction receptor family,and can mediate natural immune.Up to now,at least eleven kinds have been found in human beings.The TLRs transduce identification signals into the cells via a specific motif,mediate immune and inflammation signal transduction pathways,activate fast reaction genes,produce inflammatory chemokines such as major histocompatibility complex(MHC),costimulatory molecules,chemotactic factors,etc and participate organism defense reaction.It was reported that TLR4 participated in tissue and organ damage and inflammation,but there is no report about the effects of TLR4 on UFF until now.To study TLR4 and its signal transduction will be helpful to reveal molecular mechanisms of UFF.In the above context,we investigatedthe effects of TLRs signal transduction on the development of UFF by building a peritoneal dialysis model of ureamia in rats and observing peritoneal dialysis efficacy and TLR4,MCP-1,Macrophage inflammatory protein-2(MIP-2) expression.Our study will be able to open up a new way to peritoneal dialysis investigation.Meanwhile,we researched how Erigeron affect TLR4 expression and signal transduction in order to reveal the molecular mechanisms of the protecting effects of Erigeron on peritoneal mesothelium,peritoneal dialysis efficacy and UFF.Chapterâ… To Build a Model of Peritoneal Dialysis in Uremia Rats by Self-made Peritoneal Dialysis CatheterObjective:To develop an improved model of peritoneal dialysis in rats that mimic human peritoneal dialysis.Methods:Firstly,we established the uremia rat models by feeding of amidopurine according to the dose of 300 mg·kg-1 body weight.In the following seven weeks,we detected the blood serum urea nitrogen,creatinine,patho-scopy, and determined the model built.Rats were divided into three groups:experimental group 1,experimental group 2 and control group.In experimental group 1,phlegm suction catheters were used as peritoneal dialysis catheters with tie-ins made from sterile transfuse connectors.The peritoneal dialysis catheters implanted into rats were tunneled subcutaneously to the nape.Rats with different operation mode or different operation material belonged to experimental group 2 or control group,respectively.Statistical analysis:All values were expressed as the mean±standard deviation((?)±s).Statistical analysis was performed using the statistical package SPSS for Windows Ver.13.0.Results of two groups before and after treatment were analyzed using paired t-test for comparisons.Compare to rate of sample usingχ2-test.Results:1.Peritoneal dialysis catheters in rats in experimental group 1 were intact, whereas catheters in rats in experimental group 2 were broken.2.Three rats in experimental group 1 and six rats in control group had tunnel opening infections.3.One rat in experimental group 1 and three rats in control group had peritonitis.4.In experimental group 1,the amount of exchanged solution on the first day of peritoneal dialysis was similar to that on the fifteenth day(p＞0.05),but in control group,the amount of exchanged solution on the first day was different from that on the fifteenth day(p＜0.01).5.There were no difference between experimental group 1 and control group at the amount of exchanged solution on the first day(p＞0.05),whereas there were significant difference on the fifteenth day(p＜0.05).6.Peritoneal dialysis catheters were completely wrapped by omentums in one rat in experimental group 1 and in 4 rats in control group.Catheters were partly wrapped in five rats in experimental group 1 and in 6 rats in control group.Conclusion:Correct operation modes,proper operation material and firm durable catheter tie-ins are the key for developing a successful model of peritoneal dialysis.Chapterâ…¡Experiments in VivoPartâ… Effects of Erigeron on Dialysis Efficiency and Peritoneal UItrastrueture in Uraemia RatsObjective:To study the effect of erigeron on dialysis efficiency and peritoneal ultrastructure in uraemia rats.Methods:Firstly,feeding amidopurine to make model of uraemia rats,then building model of peritoneal dialysis by made-self dialysis catheter.The death rate of rats was 13.33%after model created.4.25%glucose dialysate,Erigeron,LPS were used in peritoneal dialysis.50 male SD model rats were divided into five groups:high concentrate group,middle concentrate group,low concentrate group,control one, control two.Peritoneal Equilibration Tests was observed,and the changes of peritoneal structure as well.Statistical analysis:All values are expressed as the mean±standard deviation((?)±s).Statistical analysis was performed using the statistical package SPSS for Windows Vet.13.0.Results after treatment among multi-groups were analyzed using one-way ANOVE.Results:1.D/Pcreatinine of control two was significant higher than others(P＜0.001),its intraperitoneal volume had no significant difference than high concentrate group and middle concentrate group(P＞0.05),had significant higher than low concentrate group and control one(P＜0.001).Compare to low concentrate group and control one,high concentrate group and middle concentrate group,their D/Pcreatinine and intraperitoneal volume had no significant different(P＞0.05).2.The extent of which integrity of peritoneal mesothelial cell destroyed,base material below cells accumulated,inflammatory cells infiltrated and blood capillary hyperplasia,there are control group one,high concentrate group,middle concertrate, low concentrate group and control group two by turns.Conclusions:Erigeron could preserve peritoneal mesothelial cell,and improve peritoneal dialysis efficiency.Partâ…¡Effects of Erigeron on Toll-like Receptor 4 Gene Expression and Signal Transduetion Pathways in Rats PeritoneumObjective:The aim of this study was to bring out the effect of TLR4 gene and signal pathways on peritoneal ultrafiltration failure,also the effect of Erigeron on TLR4 gene expression.Methods:The peritoneal tissue used of this study were from the rats in partâ… . Immunohistochemical analysis was used to detect TLR4 of peritoneal tissue expression.Polymerase chain reaction assay was employed to detect TLR4 mRNA expression.ELISA was used to investigate MCP-1 and MIP-2.Statistical analysis:All values are expressed as the mean±standard deviation((?)±s).Statistical analysis was performed using the statistical package SPSS for Windows Ver.13.0.Results after treatment among multi-groups were analyzed using one-way ANOVE.If there was heterogeneity of variance,the data should be transformed first,and then analyzed.Provided that variance remained heterogeneity after data transformed,the data should be analyzed by Kruskal-Wallis H Test for multiple comparisons among multi-groups.P＜0.05 was considered to be statistically significant.Results:1.In all groups,TLR4 mRNA expression of control two was lowest,control one was highest,high concentrate group and middle concentrate group,low concentrate group and control one had same expression.Compare high concentrate group and middle concentrate group with control one had significantly higher than control one.2.TLR4 protein expression of low concentrate group and control one were most significant,high concentrate group and middle concentrate group were more significant,and control two expression was lesser.3.MCP-1 of dialysate among high concentrate group,middle concentrate group, control two were no significantly difference(P＞0.05).MCP-1 of control one was higher than low concentrate group(P＜0.05),was highest among the others.MIP-2 of control one was significantly higher than the others(P＜0.001),control two was significantly lower than all of the Erigeron group(P=0.001,0.002,0.000).Compare to high concentrate group and middle concentrate group,middle concentrate group and low concentrate group,both were no statistically difference(P=0.115,0.226). high concentrate group and low concentrate group were significantly difference(P=0. 004)Conclusion:1.TLR4 expression are positively associated with concentration of MCP-1 or MIP-2,also are positively associated with peritoneal mesothelial cell damage.2.Down-regulating TLR4 expression,prohibiting MCP-1 and MIP-2 production,may be the mechanisms of Erigeron.Chapterâ…¢Experiments in VitroPartâ… Effects of Erigeron on Peritoneal Mesothelial Cell Growth and Proliferation in RatsObjective:The purpose of the present study is to obverse effect of erigeron on peritoneal mesothelial cells growth and proliferation in vitro,to assess the mechanism of peritoneal mesothelial cells damage.Methods:SD rat's peritoneal mesothelial cells isolated from peritoneal cavity were cultured and maintained under the defined conditions in vitro.They were cultured with medium group(group 1),50ul erigeron(group 2),25ul erigeron(group 3), 12.5ul erigeron(group 4),50ul erigeron added 10ul LPS(group 5),25ul erigeron added 10ul LPS(group 6),12.5ul erigeron added 10ul LPS(group 7),10ul LPS(group 8).To investigate peritoneal mesothelial cells growth and proliferation by cell counts(After 48 hours and 72 hours) and by MTT(after 72 hours).Statistical analysis:All values are expressed as the mean±standard deviation((?)±s).Statistical analysis was performed using the statistical package SPSS for Windows Ver.13.0.Results after treatment among multi-groups were analyzed using one-way ANOVE.Results:1.Cell counting:Mesothelial cells of group 3 and group 4 grow well,both were no significantly than group 1(P＞0.05).three groups were significantly better than group 2(P＜0.05).Plus LPS groups were less than no LPS groups.Group 5 and group 6 were.,more advantage than group 8(P＜0.001),but group 7 was no significantly difference than group 8(P＞0.05).2.MTT:Mesothelial cells proliferation of plus LPS groups were worse than no LPS groups.Group 3 and group 4 were better than group 2.Group 8 was worse than group 5 and group 6,but had no significantly difference than group 7.Conclusions:1.Erigeron has the effect of protecting mesothelial cell from damage.2.Erigeron can lessen mesothelial cell damage which LPS leads to immune inflammation,and the extent has positive correlation.Partâ…¡Effects of Erigeron on Toll-like Receptor 4 Gene Expression and Signal Transduction Pathways in Peritoneal Mesothelial Cells in Vitro Objective:This study was aims at peritoneal mesothelial cells Toll-like receptor 4 gene expression and signal pathways in vitro,to assess the role of erigeron.Methods:The mesothelial cells used of this study were from mesothelial cells cultured in partâ… ,and divided group as well.After 72 hours,polymerase chain reaction assay was used to detect TLR4 mRNA expression of mesothelial cells. ELISA was used to investigate MCP-1 and MIP-2 in supernate fluid.Statistical analysis:All values are expressed as the mean±standard deviation((?)±s).Statistical analysis was performed using the statistical package SPSS for Windows Ver.13.0.Results after treatment among multi-groups were analyzed using one-way ANOVE.If there was heterogeneity of variance,the data should be transformed first,and then analyzed.Provided that variance remained heterogeneity after data transformed,the data should be analyzed by Kruskal-Wallis H Test for multiple comparisons among multi-groups.P＜0.05 was considered to be statistically significant.Results:In no LPS groups,The concentration of MCP-1 or MIP-2 in supernate fluid were no significantly difference(P＞0.05),TLR4 mRNA expression was much less.In plus LPS groups,TLR4mRNA expression and the concentration of MCP-1 or MIP-2 in supernate fluid all were remarkable higher than no LPS groups,lowest, control one was highest.High concentrate group and middle concentrate group,low concentrate group and control one had same expression.Compare high concentrate group and middle concentrate group with control one had significantly higher than control one.Group 8 was significantly difference than group 5 or group 6(P＜0.01), and no difference than group 7(P＞0.05).Conclusion:1.The concentration of MCP-1 or MIP-2 in supernate fluid depend on TLR4 mRNA expression.The results show TLR4 signal pathways is existing.2.Erigeron may down-regulate TLR4 mRNA expression and signal transduction of mesothelial cells in rats. Conclusions of the whole paper:This study was to establish a peritoneal dialysis model of uraemia rats and to culture mesothelial cells,to prove that TLR4 and its signal transduction play an important role in peritoneal fibrosis of peritoneal dialysis or ultrafiltration failure.Erigeron can improve peritoneal dialysis efficiency and inhibit mesothelial cells from damage.The mechanics may be that Erigeron can down-regulate TLR4 expression,reduce MCP-1 and MIP-2 release.And those provided overall and penetrating molecular level mechanism and laboratorial evidence for its clinic treatment on ultrafiltration failure. |
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