Curcumine Influences The Expression Of TLR2,TLR6 And TNF-α In Rat Peritoneum During Peritoneal Dialysis Associated Peritonitis

PrefaceAcute peritonitis are the most frequent and serious complication of peritoneal dialysis.Revealing the molecular mechanisms that operate in acute peritonitis is an essential goal to reduce the functional and structural changes associated with the condition.The innate immune system provides the first line of defense against invading pathogens by a number of receptors that recognize distinctive pathogenassociated molecular patterns(PAMPs).Toll-like receptors(TLRs) are types of transmembrane receptors that different members recognize distinctive PAMPs and play a key role in defense against both viral and bacterial infections.So far,at least 10 human TLRs, defined by sequence similarity,have been cloned.Among these TLRs,TLR2 and TLR6 are able to respond to Gram-positive bacterial peptidoglycan(PDG) and lipoteichoic acid(LTA),meningococcal porins,bacterial lipopeptides and lipoproteins,as well as fungal,parasitic,and viral pathogenassociated molecular patterns.The curcumin is extracted from the ginger rhizome which has the function of antioxidant, anti-inflammatory,immunomodulatory,anti-coagulation,anti-tumor,anti-mutagenic, and many other pharmacological effects.Previous studies demonstrate that curcumin has been found to exert manifold effects on cell-mediated immunity.Curcumin has been shown to be a potent immunomodulatory agent that can modulate the activation of T cells,B cells,macrophages,neutrophils,natural killer cells,and dendritic cells. Curcumin can also downregulate the expression of various proinflamrnatory cytokines including tumor necrosis factor(TNF),interleukin(IL)-1,-2,-6,-8,12,and chemokines.The purpose of this study is to examine the effect of curcumin on the expressions of TLR2,TLR6 and TNF-aduring rat Staphylococcus epidermidis peritonitis. Materials and MethodsExperimental animals72 male SD rats(250-300g,provided by the experimental animal center of China Medical University) were used in the present study.Animals in experimental subgroups(curcumin treatment group and non-curcumin treatment group) were intraperitoneally administered Staphylococcus epidermidis (10⁷/ml colony-forming units,diluted in 10%PBS).In each subgroup,control rats were administered a comparable volume of vehicle or not injected.For curcumin treatment group,rats were treated with i.p.injection of a does of curcumin(1 mg/kg body weight) in 150-200μl vehicle(1 ml DMSO in 10 ml PBS/kg body weight).After a survival time of 3,6,12,24 and 48 h after infection,rats were killed by i.p. injection of an overdose of sodium pentobarbital and peritoneum samples were processed for fixation and protein extraction as described previously. hematoxylin-eosin(H＆E) stainingPathological and biological examinations of the specimens were performed using hematoxylin-eosin(H＆E) staining.ImmunohistochemistrySamples were cut into 10μm sections in a section cutter and mounted on glass slides.The sections were rinsed twice in Tris-buffered saline(TBS,pH 7.4) and treated with 3%hydrogen peroxide(H₂O₂) in PB for 10 min to reduce endogenous peroxidase activity.After rinsing with TBS all tissue sections were preincubated for 1 h with 5% bovine serum albumin(BSA) and 3%goat serum in TBS to reduce nonspecific staining. The sections were incubated overnight at 4℃with anti-TLR2,-TLR6 and -TNF-αserum(Santa Cruz Biotechnology) diluted 1:100 in TBS containing 3%goat serum,1% BSA and 0.3%Triton-X 100.After extensive washing in TBS,sections were incubated with a 1:200 diluted biotinylated anti-rabbit IgG for 1 h at room temperature(RT).The ABC Kit was then applied for 1 h at RT in order to visualize the reaction sites.The ABC solution was diluted 1:100 in TBS.A brown colour appeared in the sections after incubation of the sections in 0.025%3,3'-diaminobenzidine with 0.0033%H₂O₂ for 10 min at RT.The sections were dehydrated in graded alcohols and coverslipped with neutral balsam for light microscopy. Western BlotSamples were homogenized 1:5(w:v) in ice-cold lysis buffer(50 mM Tris-HCl, 150 mM NaCl,1%Nonidet P-40,1 mM EDTA,0.25%sodium deoxicolate,0.1%SDS, 1 mM phenylmethylsulfonyl fluoride(PMSF),10 mg/ml leupeptin,1 mMNa3VO4 and 1 mM NaF).The resulting homogenate was centrifuged at 12,000 g for 30 min at 4℃. The supernatant was collected and total protein levels were measured by a BCA protein assay kit(Pierce Biotechnology).Sample protein(20μg) was separated on 10%SDS-polyacrylamide gels,and the proteins were transferred onto PVDF membranes(Millpore,CA) in an electronasfer device(45 V,15 h).Then the membranes were blocked in 5%nonfat milk in TBS containing 0.05%Tween 20(TTBS) for 3 h and incubated in primary antibody overnight at 4℃.The dilutions of primary antibodies were 1:500 for TLR2 and TLR6. Membranes were washed with TTBS and followed by incubation with horseradish peroxidase-conjugated secondary antibody for 2 h at RT with constant agitation.The membranes were washed and reacted with an enhanced chemiluminescence(ECL) kit (Pierce,CA),and visualized by exposure to Kodak-XAR film.RT-PCR analysisThe mRNA levels of TLR2 and TLR6 were analyzed using RT-PCR as described elsewhere.Total RNA was isolated from peritoneum according to the manufacturer's specification using an easy-BLUE RNA extraction kit(iNtRON Biotech,Korea).Total RNA(2.5 ug) was heated at 65℃for 10 min and then chilled on ice.Each sample was reverse-transcribed to cDNA for 90 min at 37℃using a cDNA synthesis kit (Amersham Pharmacia,Minneapolis,USA).Reverse-transcription polymerase chain reaction(RT-PCR) was performed with the following primers for rat(r) TLR2(5'-GGA GGT GTT GGA TGT TAG-3';5'- GCT TGA AGG GTG GGT C -3');TLR6(5'-ACT CAC CAG AGG TCC AA-3';5'- GGA TGA ATG GCG TGT C-3') and TNF-α(5' -GCCACCACGCTCTTCTG-3',5'-GCAGCCTTGTCCCTTGA-3'),which were used to verify if equal amounts of RNA were used for reverse transcription and RT-PCR amplification from different experimental conditions.The annealing temperature was 50℃for TLR2,TLR6 and TNF-α60℃forβ-Actin,respectively.Products were electrophoresed on a 1.5%agarose gel and visualized by staining with ethidium bromide.GAPDH mRNA was used as a control of mRNA loading.ResultsWBC(white blood cell) of ascitesAfter Staphylococcus epidermidis injection in 3h,WBC(white blood cell) of ascites increased.Compared with non-curcumin treatment group,the amounts of WBC of curcumin treatment group was decreased after infection in 3h,6h,12h,24h.hematoxylin-eosin(H＆E) stainingAfter Staphylococcus epidermidis injection in 24 h,retroperitoneal edema and cell morphology appeared to change.Compared with non-curcumin treatment group.The peritoneal mesothelial cell injury,cell deformation and fragmentation of curcumin treatment group was obvious;curcumin treatment group there are also a certain degree of deformation of peritoneal mesothelial cells fragmentation,but the extent of than that of non-treatment group curcumin light.TLR2,TLR6 and TNF-αImmunohistochemistryIn general,the TLR2 and TLR6 immunoreactivities were mainly located in peritoneal mesothelial cells.For control rats,no visible differences were seen in the intensity of both TLR2 and TLR6 immunoreactivity among the non- and curcumin treatment rats.TLR2 and TLR6 immunostaining progressively increased in 3,6,12,24 h after infection,whereas attenuated in 48 h after Staphylococcus epidermidis injection. Compared with non-curcumin treatment group,both TLR2 and TLR6 immunoreactivities weakened in density in 3,6,12,24h after infection and similar in control and 48 h group.In addtion,cell damage was more severe in non-curcumin treatment group in general.For control rats,no visible differences were seen in the intensity of TNF-αimmunoreactivity among the non- and curcumin treatment rats. TNF-αimmunostaining progressively increased in 3 h after infection,whereas toped in 24 h after Staphylococcus epidermidis injection.Western blot analysis for TLR2,TLR6 and TNF-αin rat peritoneumAt defined time point,the tendencies of TLR2 and TLR6 expression levels in corresponding group was surprisingly similar.For curcumin treatment group,the semiquantitative analysis of immuno-blots showed statistically significant reduction of TLR2 and TLR6 in rat peritoneum 6,12,24 h after infection as compared to non-Curcumin treatment group.In all group,statistical results revealed a clear time dependent increase in TLR2 and TLR6 from 6-24h after infection.The semiquantitative analysis of immuno-blots showed statistically significant reduction of TNF-a in rat peritoneum 6,12,24 h after infection as compared to non-Curcumin treatment group. In all group,statistical results revealed a clear time dependent increase in TNF-αfrom 6-24 h after infect.Expression of TLR2,TLR6 and TNF-αmRNAs in rat peritoneumTo analyze the mRNA expression of TLR2 and TLR6,RT-PCR were performed. At defined time point,the tendencies of TLR2 and TLR6 mRNA expression levels in corresponding group was similar.For curcumin treatment group,the semiquantitative analysis of RT-PCR showed statistically significant reduction of TLR2 and TLR6 in rat peritoneum 6,12,24h after infection as compared to non-Curcumin treatment group.In all group,statistical results revealed a clear time dependent increase in TLR2 and TLR6 from 6-24h after infection.The semiquantitative analysis of RT-PCR showed statistically significant reduction of TNF-a in rat peritoneum 6,12,24 h after infection as compared to non-Curcumin treatment group.In all group,statistical results revealed a clear time dependent increase in TLR2 and TLR6 from 6-24 h after infection.Conclusion1.There is a small amount of the expression of TLR2,TLR6 and TNF-αin normal rat peritoneum.2.TLR2 and TLR6 may contribute to the induction of immune response during acute peritonitis induced by Staphylococcus epidermidis.3.Curcumin can be served as a protective agent content during acute peritonitis induced by Staphylococcus epidermidis.