Effects Of Multiple Colon LBP Antibody On TLR4 Signaling Pathway In Alveolar Macrophage Of Acute Lung Injury Rat Induced By Lipopolysaccharide

Acute long injury (ALI) commonly caused by infection is one of emergency and critical diseases of respiratory system. ALI induced by Gram-negative bacteria infection is due to effects of cytokines from inflammatory cells activated by Lipopolysaccharide(LPS). It is helpful and important to investigate LPS signaling transduction and its blocking effects to comprehend the mechanism of ALI and search a new therapy way. Some study has shown that LPS signaling transduction of outer-membrane is finished by lipopolysaccharide binding protein (LBP) and CD14. In cytoplasma a series factors including MyD88, IL-1 receptor associated kinase (IRAK), TNF-á receptor associated kinase 6 (TRAK-6) and IêB are responsible for LPS signal transduction and finally NF-êB is translocated leading to the inflammatory cytokines such as IL-1a,TNF-á,IL-10,IL-18 et al gene transcription and translaton. Up till now, LPS signal trans-membrane transduction is not very clear. Recently TLR4 as a trans-membrane receptor in LPS target cells such as alveolar macrophage is believed to perhaps could complete this task. Its out-membrane leucine-rich repeats could recognize and bind LPS, and the domain homologuing to IL-1a in cytoplasma maybe transduce the signaling to IL-1a. So, TLR4 is the key molecular in LPS signaling pathway. TLR4 expression and its perfect function directly affect effects of LPS signaling transduction. Otherwise, depress the expression and function of TLR4 is helpful to decrease inflammatory cytokines excretion and relieve ALI induced by LPS. Different patient infected Gram-negative bacteria usually show various degree clinic syndrome. It elicit that each body has different degree reaction to LPS or has different LPS tolerance which the mechanism is still not clear and maybe relate to complex elements such as immune ability, LPS internalation and efficiency of LPS signaling transduction and so on. The structure and function of TLR4 in LPS target cell membrane and its mediated signaling pathway play a very important role in LPS tolerance. It is necessary investigate the relationship between TLR4 expression and LPS tolerance because of unclear. To decrease TLR4 expression and enhance LPS tolerance increases perhaps the resistance of body to LPS.LBP, as a "carrier" of LPS, is responsible for carrying LPS to target cell and at the same time enhance the inflammation induced by LPS in some way, therefore, LBP antibody could relieve those effects. It is worth to study the action of LBP antibody in this field which if or not relate to decrease the efficiency of LPS signaling. Because of no commercial LBP and its antibody up to now, it is very necessary to purify LBP and produce its antibody for our study.In the present studies, LBP from acute phase rat serum was purified by precipitation of ammonium sulphate, Bio-rex 70 resin and MonoQ column. Multiple colon LBP antibody (mLBPab) was produced from the anti-serum of rabbits which were stimulated with LBP for three month. Expression of TLR4mRNA and protein were detected by RT-PCR and Western Blot respectively in the alveolar macrophages from ALI rats induced by LPS and treated with mLBPab before and after LPS stimulation as well as the mRNA expression of IL-1a,IL-10, IL-18, TNF-á. Lastly, in order to investigate the role of mLBPab between LPS tolerance and the efficiency of LPS signaling mediated by TLR4, all of above expression were also determined in rats alveolar macrophages re-stimulated with different two dose LPS and treated with mLBPab.The results showed: ① 1143ug rat LBP were purified from 400ml acute phase rat serum and the purification folds reached to 1570. SDS-PAGE analysis indicated that the purified preparation of rat LBP showed homogeneity and had molecular weight of 60kD. The binding of lipopolysaccharide to abdominal cavity macrophage was enhanced by purified LBP. ② 1560ug mLBPab which could block the binding of lipopolysaccharide to abdominal cavity macrophage and be identified by Western Blot was produced from 85ml rabbit anti-serum ③ It was the...