The Screening Of Mimic Peptide Of Lipopolysaccharide Binding Protein Binding To CD14 And Its Protective Effects On Endotoxin-induced Acute Lung Injury

Background and ObjectiveAcute lung injury (ALI) is an acute progressive respiratory failure induced by various reasons except cardiogenic factors with high mortality. Lipopolysaccharide (LPS), a major component of Gram-negative bacterial endotoxin, is the leading cause of ALI. So far, there are still no effective antiendotoxin drugs. Physiologically, the LPS concentration is very low in human body. Even in pyemia induced by gram-negative bacilli, the plasma concentration of LPS is only 0.01-1ng/ml. The increasing sensitivity system of LPS-binding protein (LBP)/CD14 plays a pivotal role in LPS-mediated inflammatory responses. The previous studies have shown that LBP enhances the effect of LPS on TNF-αreleases in membrane CD14 (mCD14)-positive cells such as mononuclear macrophages to 1000 folds. The binding site for LPS is situated in the N-terminal part of the LBP molecule, while the C-terminal part of the LBP mediates the binding of LBP to CD14. LBP/CD14 is at the upstream of LPS signal transduction. So, inflammatory response may be attenuated by LBP/CD14 pathway intervention before cell activation and inflammatory media release in endotoxin-induced ALI. In this study we will screen the mimic peptide of LBP binding to CD14 with phage display peptide library, then detect the antiendotoxin activity of mimic peptide in vitro and observe its protective effects on ALI rat model induced by endotoxin.Methods1. CD14 was coated and the four rounds of bio-pannings were carried out using LBP as eluant with a phage display peptide library. One hundred bacteriophage clones were selected randomly and used in binding test, competition test and inhibition test of cytokine production by enzyme linked immunosorbent assay (ELISA). Totally, eight positive clones with high affinity to CD14 and powerful competition with LBP were sequenced. These 12-mer peptides were compared with primary amino acid sequence of LBP.2. A mimic peptide was synthesized by Fmoc-based solid phase peptide synthesis. The affinity binding to CD14 and the activity competing against LBP were determined by ELISA. U937 cells were treated with LPS and incubated with mimic peptides at a high (10ug/ml), middle (1.0ug/ml) and low (0.1ug/ml) dose, respectively. The TNF-αexpression in U937cells were determined at the mRNA and the protein level, respectively. And the effects of MP12 on cell TNF-αrelease induced by LPS were detected by ELISA at different time point.3. A rat model of ALI was established by jugular injection of LPS. Rats were randomly divided into three groups: N, LPS, MP12. Group N was untreated as control. Group LPS was injured by LPS injection as ALI model. Group MP12 was treated with LPS and MP12. After 2 hours treatment, the artery blood and lung tissue were collected. The level of PaO2 in artery blood and the wet/dry ratio of lung tissue were measured. The pulmonary microvascular permeability was detected. The histological changes of lung tissue were observed under microscope. The Alveolar macrophages were isolated from bronchoalveolar lavage fluid and the effects of mimic peptides on LPS conjugation with cells were observed.Results1. Phages were found to be enriched effectively by pannings. The binding test showed that 73 phages clones exhibited high affinity with CD14. The competition test showed that 26 clones among them competed powerfully against LBP for binding to CD14. The inhibition test of cytokine showed that LPS-induced TNF-αwas suppressed by 8 clones significantly.2. Three 12-mer sequences of FHRWPTWPLPSP, AAFHRAHHLTSP and MHRHPPPITLPL were obtained from eight positive phage clones, which core sequence was FHRXPXXXXPXX. Blast showed that there were no homoplastic sequences between mimic peptide and primary structure of LBP.3. The mimic peptide of AC-FHRWPTWPLPSP-NH2 was synthesized successfully by Fmoc-based solid phase peptide synthesis. The purity was above 95% and the molecular weight was 1562.00. The binding test and competition test showed that this mimic peptide exhibited higher affinity with CD14 and more powerful competition against LBP for binding to CD14 at high and middle dose. 4. TNF-αexpression in U937 cells stimulated with LPS increased significantly both at the mRNA and the protein level. The mimic peptide suppressed the expression of TNF-αinduced by LPS at the mRNA and the protein level, especially at earlier intervension stage..5. Compared with group N, PaO2 decreased and W/D ratio, pulmonary microvascular permeability, mean fluorescence intensity of alveolar macrophage in group LPS increased significantly. However, compared with group LPS, the above indexes in group MP12 also decreased significantly, except PaO2 increased.Conclusion1. Positive phage clones are screened successfully from a phage display peptide library, which exhibit high affinity with CD14, powerful competition against LBP and inhibits the production of TNF-αinduced by LPS.2. Three 12-mer sequences of FHRWPTWPLPSP, AAFHRAHHLTSP and MHRHPPPITLPL are screened from positive phage clones. The core sequence is FHRXPXXXXPXX, and there are no homoplastic sequences between mimic peptide and primary structure of LBP.3. The mimic peptide of AC-FHRWPTWPLPSP-NH2 exhibits high affinity with CD14 and inhibits LBP binding to CD14.4. The mimic peptide suppresses the TNF-αexpression in mononuclear macrophage induced by LPS at the mRNA and the protein level, especially at the earlier intervention stage.5. The mimic peptide improves oxygenation and attenuates lung injury in rats. It inhibits the conjugation between LPS and alveolar macrophages, suggesting that the mimic peptide could protect rats from endotoxin-induced ALI.