Screening The Broad-spectrum Chemotactic Inhibitors And Identification Of Its Activity

Background and purpose:Inflammation is one of the most important causes of some diseases. the target of most current clinical use of anti-inflammatory drugs (such as steroidal anti-inflammatory drugs and non-steroidal anti-inflammatory drugs)was the effect of the process of inflammatory immune response after the occurrence, these drugs are mainly organized to prevent the body injury did not prevent the further occurrence of inflammation, did not achieve the ultimate effect of the treatment of inflammatory. Research has shown that the effect of leukocyte recruitment is the most important aspect of inflammation and infection. Therefore, signaling molecules, as targets, which inhibited the excessive leukocyte recruitment played an important role in the treatment of inflammatory. In the past decade, there has been considerable progress in the research of molecular signaling network mediated leukocyte migration. In which, the most thorough research is the chemokine system. So far, the developed chemotaxis inhibitors were mainly focus on some specific inhibitors which made a single chemokine or its receptors as target, and the development of such inhibitors was relatively more mature, but the clinical trial results are not satisfactory, and the eliminate rate was too high. To Investigate the reasons, most scholars believe that the main reason was the redundancy of chemokine system. This redundancy made the chemokine system steady, and ensured the immune response occurred. Most inflammatory diseases and it's development process involved a variety of chemokines and their receptors participate in, only by inhibition of a simple particular chemokine or receptor can only obtain little effect. Therefore, the development of broad-spectrum inhibitor of chemotaxis is the best solution to overcome this bottleneck, with a very attractive prospect. Thus, the development of new molecular chemokine inhibitior with broad-spectrum will bring new prospects in inflammation treatment.In 1996, the research group of Ruoslahti, Pasqualini, Arap et al,. proved that in vivo phage display was feasible and made the technology widely used. In these years, many researchers screen some specific targeting peptide using this technology. The best advantage of in vivo phage display technology was able to obtain targeting peptide, which can combine with living tissue or organ specifically and have a relatively higher stability and specificity in vivo. In this study, an inflammation model stimulated by LPS in rats was established, a new small peptide with efficient inhibition of chemotaxis was developed by using in vivo phage display screening method, it may be as a leading peptide in the research of anti-inflammatory drug. In vivo phage display screening method was target tissues or organs in foreign, a truly targeting cytokines in vivo screening was not reported. Therefore, it has certain innovative that the broad-spectrum chemokine inhibitors in vitro screening which made a variety chemokines as targets by using phage display technology.Methods:1. An inflammation model stimulated by 5mg/kg LPS in SD rats was established, the expression of three rounds chemotactic factor IL-8, RANTES, MCP-1 representative of the chemokine CXCL class and CCL class were detected by Luminex Liquid-chip method.2. The positive phage clones broad-spectrum combined with chemokine were screened by PDT with three times 12-peptide library affinited screening in phage.3. The chemotactic activity of phage clones were further identified by transfusion experiments in vivo, in vitro experiments and Chemokine inhibition, the positive phage clones broad-spectrum inhibited with chemokine were obtained.4. Explore the mechanism of the two best positive inhibition of chemotactic clones by competitive binding experiment.5. The DNA sequence of 10 positive phage clones with better biological activity were detected, thus the inserted amino acid sequence were deduced, the obtained sequences were analyzed by bioinformatics software.Results:1. The chemokines expression of before and after the injection of LPS at different time was analyzed P<0.05, and the highest expression of chemokines was at 3h after LPS injection, we choose injection LPS 3h inflammation model as a point in time.2. After three rounds of phage display in vivo screening, the purpose phage has been effectively enriched, the amount and the recovery of the third round 19.4 times the amount and recovery of the first cycle, it also shows significantly increase compared with the negative control group.3.10 better-positive phage clones were obtained after transfusion experiments in vivo, in vitro experiments and Chemokine inhibition.4. The mechanism of the two best positive inhibition of chemotactic clones by competitive binding experiment was through blocking chemokine binding to its receptor.5. Five DNA sequences of the 10 positive phage clones with better biological activity were obtained, including the two sequences with the frequency of 30%.Conclusion:1, An animal inflammation model stimulated by LPS was successfully established by Luminex liquid chip technology. The model can simulate the abnormal expression of chemokine in the early stages of inflammatory in vivo.2, Positive phage clones with broad-spectrum chemokine inhibition was successfully obtained by using in vivo phage display screening method, proved that in vivo phage display screening method is feasible.3, Trough activity test, positive phage cloning has a relatively higher effect of in vivo-in vitro combination and chemotactic inhibition, preliminary proof that positive phage cloning play a role of chemotactic inhibition by competitive binding chemotactic factors with chemokine receptor.4, A new amino acid sequence with broad-spectrum efficient inhibition of chemotaxis was developed, it may be as a leading peptide in the research of anti-inflammatory drug.