The Study Of ChTLR4 And Its Signaling Pathway In Lymphocytes Of Chicken With LPS-induced

Toll-like receptors are highly conserved pathogen pattern recognition receptor family, they are very important pathogen pattern recognition receptors in the immune response, in addition, also provide stimulating signal to the active acquired immunity. Therefore, TLR family is the important connection point of the natural immunity and acquired immunity. Toll-like receptor 4 is the earliest found and most explicit role in the TLR Familys. TLR4 is the major receptor in the natural immune system to identify pathogens, so it is very important to the body's immune response. TLR4 recognizes a variety of ligands, but the main ligand is lipopolysaccharide (LPS).A large number of experiments show that the process of TLR4 in Gram-negative bacterial infections play a very important role. Therefore, deep study the TLR4 function characteristics is effective for the immune function and control of inflammatory responses. TLR4 present on the study of mammals are more relevant reports, and for poultry and its signal transduction pathway TLR4 few studies.In this experiment, we stimulate the chicken lymphocytes in vitro with LPS. And detect the TLR4 and NF-KBp65 mRNA expression levels in the cells by Real-time PCR method at 6h,12h,24h,48h and 72h. Detect the NF-κB p65 and IL-1βexpression levels in spleen and blood cells stimulated by LPS and florfenicol by ELISA method. Then we could analyze the role of TLR4 signaling pathways in the LPS stimulate lymphocytes reaction from the level of gene and protein. We also detected the effect of the broad-spectrum antimicrobial drugs-florfenicol on LPS induction.From the Real-time PCR results, we found that in the spleen lymphocytes stimulated by LPS, the chTLR4 mRNA expression is higher than the control groups, in the experimental group it began to increase at 6h after LPS stimulation,and the highest in the 24 h, after which began to decline. Compared with the control group significant difference at 12h (p<0.05), extremely significant difference (p<0.01) at 24h. The NF-KBp65 mRNA expression levels are higher than the control group, the experimental groups began to increase at 6h after LPS stimulation, the highest in the 24 hours, and then began to decline, compared with the control group at 12h and 36h significantly different (p< 0.05), 24h extremely significant difference (p<0.01).Stimulated the lymphocytes in spleen and blood with LPS and florfenicol in 6h,12h,24h,48h and 72h. Detected the factors levels by ELISA,we found In the groups of florfenicol, IL-1βand NF-KBp65 factor levels were decreased, and significantly different (p<0.05) compared with the control groups at 24h; in the group of LPS, IL-1βand NF-KBp65 factor levels were increased, and were significant difference(p<0.05) or extremely significant difference(p<0.01) at 12h,24h,48h compared with the control groups. In groups of both LPS and florfenicol, IL-1βand NF-κBp65 factor levels were not significantly different compared with the control groups.These results show that:LPS in vitro can be recognized by lymphocytes isolated and activated chTLR4 and its signaling pathway, but the reaction is relatively weak. Florfenicol on LPS-induced reactions are inhibited, the mechanism needs further study.