Expression Of Toll-like Receptor 2 In Alveolar Macrophages Of Diabetic Rats And Response To PGN

objectiveThe lung as One of diabetes target damaged organ had been paid more attention since 70s of this century.The damages of diabetes involve pulmonary function, biochemistry metabolism and pathology of the lung and so on.The local defense function deficit of the lung leading to lower respiratory tract infect is very common.TheToll like receptors (TLRs) are molecular proteins discovered in recent years,they are syngeneic with the molecular proteins of Drosophila.And they play crucial role in the innate immune response to bacteria.Otherwise,they are firmly correlated with the diabetes.So they are now paid more and more attentions.TLRs can recognize,combinate the microorganism, cause the transduction of signals and release of inflammatory mediators.Among them, TLR2 and TLR4 are especially important.TLR2 can recognize a serious of molecular mode of microorganism,including the peptidoglycan of Gram-positive bacterial.In this experiment,we study the expression of TLR2 stimulated by peptidoglycan in the macrophages of the alveolar in normal rats and rats with diabetes.Furthermore,we hope to find the mechanism of the change of pulmonary immune function in the patientswith diabetes,reveal the relationship between the infection and diabetes.Then we can use it as a new statement to therapy and control of infection in patients with diabetes.Material and Methods1,Material(1)Experimental animals:32 male Wistar rats,level SPF,Age 12 weeks,Weight 200±20g.(2)Equipment and reagents:Univeral 32R centrifugal apparatus,Glucometer,treptozotocin(STZ), Peptidoglycan(PGN staphylococcus aureus),Rabbit-anti-Mouse TLR2 2,Methods(1)Preparation of animal model and groupingThirty two male Wistar rats,weight about 200 grams,were randomly divided into 4 groups:normal control(group A n=8),diabetes group(group B n=8),normal+PGN group(group C n=8),diabetes+PGN group(group D n=8).The rats of diabetic group received intraperitoneal injection with streptozotocin(60mg/kg body weight).The rats of normal control received intraperitoneal injection with equal volumes of sodium citrate buffer.The diabetes rats model were success if a random blood glucose＞16.67mmol/L after 72 hours,test the blood glucose each week,all together 4weeks.(2)Preparation and culture of alveolar macrophagesThe rats were Ane- sthesia with chloral hydrate,performed bronchial lavage by tracheal intubation.Then we culture the macrophages in the BALF,Group C and group D were added with the final concentration of 10ug/ml PGN.(3)Immunocytochemistry:Immunocytochemistry SABC method,The concentration of rabbit-anti-mouse TLR2 is 1:200.Membrane and cytoplasm showed brown staining are positive.(4) Test of the expression of TLR2 mRNA(5) Statistical analysis:Statistical analysis of data was conducted using SPSS 13.0 software.Experimental data were expressed as(?)±s,Statistical analysis between groups was performed using two-factor and two-level factorial analysis.P value less than 0.05 were considered to be statistically significant.Results1,The result of ImmunocytochemistryThe expressions of TLR2 are brown-yellow in cytoplasma and membrane in Normal control;The expressions of TLR2 in diabetes group are more,color is slightly deepen;The expression of TLR2 in normal+PGN group and diabetes+PGN group were dark brown-yellow in cytoplasma and membrance,part of cells necrosis. OB:Normal control(groupA):0.1146±0.002,diabetes group(groupB): 0.1731±0.011,normal+PGN group(groupC):0.2059±0.011,diabetes+ PGN group(groupD):0.2931±0.017. 2,The expression of TLR2 mRNANormal control(groupA):0.898±0.084,diabetes group(groupB): 1.202±0.07,normal+PGN group(groupC):1.628±0.11,diabetes+ PGN group(groupD): 2.307±0.13.Peptidoglycan factor and diabetic factor increase the expression of TLR2 in the macrophages of the alveolar,P＜0.001,diabetes and peptidoglycan was more visible in the main effects and interactions.Conclusion:Diabetes can promote the expression of TLR2 in the macrophages of the alveolar in rats,leading to the pro-inflammatory state in diabetic body,and the dysfunction of immune system.And this can cause the decrease of immune function in lung.