Expression Of Toll-like Receptor 4 In Alveolar Macrophages Of Diabetic Rats And Response To LPS

ObjectiveToll-like receptors(TLRs) are a group of transmembrane proteins responsible for recognizing conserved pathogen-associated molecular patterns(PAMPs).TLRs play an bridge role in the activation of innate and adaptive immunity in vertebrate animals.In view of crucial significance in immunology,molecular biology and clinical study, TLRs have been much concerned since discovery.The role of TLRs signal transduction pathway on the onset and development of diabetes and its complications becomes a hot research.Experiments confirmed that TLR2 and TLR4 expression and signaling are increased in T1DM but the specific significance of the increase are still controversial.Alveolar macrophages play an important role in the clearance of inhaled antigen and all aspects of pathogenic micro-organisms,my experiment investigate the altered expression of TLR4 in alveolar macrophages of diabetic rats after lipopolysaccharide stimulation and the effect of these changes on defending the infection.Materials and methods1.32 Male healthy wistar rats were divided into 4 groupsGroup A:the control group;group B:the diabetic group;group C:the LPS stimulated group;and groupD:the diabetic group with LPS stimulation.2.Capture and culture of alveolar macrophagesThe small fraction of cells after BAL and centificated were resuspended in RPMI-1640 meduium supplemented with 10%calf serum,the cells were plated into a 60mm petri dish with small glass slides placed in the dish,then were incubated for 2h at 37℃in 5%CO₂ with saturated humidity.Make macrophages adherent and dispose the non-adherent cells.3.Immmunocytochemical detection of TLR4TLR4 was observed in every groups referencing to two step immmunocytochemica kids.4.Detecting the expression of TLR4 by using RT-PCR tecknique5.Western blot methord to detect protein level of TLR4After extraction and qualification of protein from alveolar macrophages,the samples were electrophoresed with SDS-PAGE.The samples were cultured with primary and secondary antibody and detected.6.Statistical analysisThe data were analyzed with SPSS13.0 software.The results were represented as Mean±SD,and comparision between groups were performed using fractorial experiment,P value less than 0.05 was considered to denote significant difference.Results1.Immunocytochemical analysis for TLR4TLR4 of alveolar macrophages was expressed in the 4 groups respectively,but their strengths are different,groupB and groupC were higher than group A(P＜0.01); groupD was significantly higher than groupB and groupC(P＜0.01).2.Results of RT-PCRCompared to group A(0.113±0.017),the expression level of TLR4 mRNA in group B(0.211±0.06),group C(0.331±0.04),and D group(0.495±0.019) followed by increased significantly.3.Detecting the expression of TLR4 with Western blot methordThe expression of TLR4 in diabetic group was higher than normal group(P＜0. 01);the expression of TLR4 in diabetes+LPS group increased more significantly compared to normal+LPS group(P＜0.01). Conclusion1.The expression of TLR4 of diabetic rats was higher than that of normal rats and became more higher after LPS stimulation,which is indicated that diabetic bodies were in the proinflammatory state.2.The increase of TLR4 became more obvious after LPS stimulation.The changes of TLR4 might not only enhance the nonspecific immunity at a certain extent,but also cause damage as to the excessive immune response.