Expression And Significance Of Gastric Toll-like Receptor 2/5 In Patients With Hp Infection

Background and ObjectivesHelicobacter pylori is a spiral,microaerophilic,Gram-negative bacteria that colonizes gastric epithelial cells and the gastric mucosal layer.Approximately 50%of the world's population is colonized by Helicobacter pylori,and,although the majority of these infected individuals are asymptomatic,long-term interactions between Helicobacter pylori and humans significantly increase the risk for peptic ulcer disease and distal gastric adenocarcinoma.Helicobacter pylori is a causative agent of chronic gastritis,peptic ulcer disease,gastric adenocarcinoma,and gastric mucosa-associated lymphoid tissue(MALT) lymphoma.Approaching the mechanisms by which Helicobacter pylori and/or host factors cause disease means more significant.Helicobacter pylori activates the transcription factor NF-kB,leading to proinflammatory cytokine production such as TNF-a,IL-8,IFN-γsecretion by gastric epithelial cells,which is much more robust than people without infection.However, the receptors for the initial bacterial interaction with host cells which activate downstream signaling events have not been completely defined.Toll-like receptors(TLRs) are an evolutionarily conserved family of eukaryotic receptors that function in innate immunity via recognition of somewhat invariant regions in bacterial molecules,which are called pathogen-associated molecular patterns or PAMPs.TLRs are recently found to play an essential role in the first line of host defense by recognition of microbial components.Transcription factor NF-kB and activator protein-1 are activated by TLRs recognition PAMP.Responses to extracellular PAMPs are mediated through transmembrane receptors such as the Toll-like receptors(TLRs),which induce the expression of proinflammatory cytokines,chemokines,and other antimicrobial defense molecules.In addition to directly controlling the microbial infection,the innate immune response is also instructive to the adaptive immune response.TLR2 and TLR5 are required for Helicobacter pylori-induced NF-κB activation and chemokine expression by gastric epithelial cell lines.Studies from numerous laboratories have now demonstrated that the specific clinical outcome is determined by different expression of TLRs gene.TLRs was always taken as investigated object in AGS,MKN cell lines in the past,however,there was rare report about expression and biological function of TLR2 and TLR5 in gastric mucosa in vivo so far.The mRNA level of TLR2 and TLR5 was measured by SYBR GreenⅠReal-time-polymerase chain reaction,and the expression levels of each sample were corrected by glyceraldehyde-3-phosphate dehydrogenase (GAPDH)mRNA levels.The S-P immunohistochemical technique was used to detect the expression of TLR5.The aim is to observe the expression of TLR2 and TLR5 in gastric mucosa,and to analyze their relationship with clinicopathologic significance.Materials and methodsGastric biopsy specimens were obtained from 68 patients who underwent electronic gastroscope examination in the first affiliated hospital of Zhengzhou university,department of gastroenterology from August 2008 to October 2008.¹⁴C -Urea Breath Test and Rapid Urease Test were employed to diagnose Helicobacter pylori infection.Tissues samples were snap-frozen immediately in liquid nitrogen immediately for using.The total RNA were extracted using RNAprep pure Tissue Kit(TIANGEN), according to the manufacture's recommended protocol.And then the intensity ratio of 28S to 18S rRNA in total RNA samples was examined by agarose gel electrophoresis to estimate the total RNA integrity.The absorptan was examined at 260/280nm by spectrophotometer.And then SYBR GreenⅠreal-time quantitative PCR was performed according to the protocol provided by TIANGEN.Amplication product was tested by 2%agarose gel electrophoresis.Immunohistochemistry were performed in SP method.The pathological section was scanned and photoed by Scan Scope Vitural Microscopy System.The 2(-Delta Delta C(T)) method was used to analysis of relative gene expression data.The data was treated statitically with SPSS10.0 revolved in t-test,x² test,and the test criteria is 0.05(a=0.05).Results1.TLR5mRNA was expressed in both groups.The mRNA level of TLR5 in experimental group was significantly lower than that in control group(P＜0.05).2.TLR2mRNA was expressed in both groups.There was no difference in expression of TLR2 between control group and experimental group(P＞0.05).3.The expression level of TLR5 was 60%(21/35)in experimental group detected by Immunohistochemistry SP method,while the expression level in control group was 84.8%(28/33).It had significant differences between control group and experimental group(P＜0.05)Conclusions1.Gastric epithelial cells express TLR2 and TLR5 mRNA,so the TLRs on the gastric epithelial cells may interact with Helicobacter pylori.They cause inflammation to gastric mucosa and damage to target organ.2.Helicobacter pylori infection can down regulate TLR5 through mRNA and protein expression,which maybe the reason for Helicobacter pylori infection lasted.3.TLR2 might not participate in the pathogenesis of Helicobacter pylori infection.