Screening Broad-spectrum Correlated Inflammatory Chemokine Inhibitor Leader

Inflammation, including acute inflammation and chronic inflammation,an important feature of them is that the leukocyte are over accumulation in the local inflammatory area. Almost any stimulation which can hange cell stabilizing factor could induce the secretion of chemokines, raising the specific white blood cells to the inflammatory area, the over accumulative white blood cells can induce produce excessive cytokines,intensify cell-cytotoxicity reaction, then induce inflammation. Early in inflammation, viral could infect, stimulate and activate inflammation effector cells, resulting in a large number of chemokines and causing substantial accumulation of inflammatory cells. However, the continued high level of chemokine expression is not only inappropriate, but also it will raise a significant increase in white blood cells and the formation of destructive damage, resulting in occurrence of autoimmune disease.Meanwhile,the white blood cell mediated injury also caused high expression of chemokines and the other types cytokines,which resulting in further tissue damage, destoryed the white blood cells of the body's protective effect and caused diseases. Thus, antagonists of the chemokines and chemokine receptor of inflammatory response may become a new target for therapy way.This subject proposed to use phage display technology from phage peptide library to screen the phage clones which could bind early inflammatory chemokine production to block inflammation.In recent years, with the scope of application of phage display technology expanding and deepening in the study, it plays an increasing role in the inflammatory reaction, inflammation chemokines are as targets,screened the short peptide with high affinity which can inhibit the infection of early inflammtory chemokines production,thereby blocking the body's inflammtory response,and overcome the disadvantages of the conventional macromolecular drugs,such as,the molecular weight, the weak of the tissue permeability, unstable, vivo immunogenicity and poor transport capacity,it can provide a new way for the development of inflammatory drugs.Then,by DNA sequencing analysising, study the inflammatory response inhibition theory, the targets of it is to find a new anti-inflammatory factors, and a series of efficacy and toxicology experiments have shown that broad-spectrum inhibitor of chemokine function is not only a powerful anti-inflammatory effects, and compared with steroidal anti-inflammatory drugs significantly,it reduced the role of toxic side effects,so it has a very good development prospects.Object:By screening the early inflammatory chemokine antagonist lead compound,which is to inhibit the production of cytokines and to block the body's inflammatory response for exploring a new way for infection prevention and control of inflammation,.Methods:1. Conventional method to obtain and develop in peripheral blood mononuclear cells (PBMC), using lipopolysaccharide to stimulate PBMC to set up vitro inflammation model, then collected supernatant and analyzed the production of chemokines.2. Using phage random peptide library to obtain positive phage clones which are screened by four rounds of affinity and initial validation.3. Sequenced positive phage clones, used a variety of bioinformatics software to analysis peptide sequence and found the broad spectrum of inflammatory chemokine binding peptide.4. Selected the high inhibition rate of clones and researched by chemokine inhibition test;5. On the synthesis of short peptides of MTT testing in vitro activity and detection of its inhibition of inflammatory effects of chemokines;6. Use of calcium flow test study the relationship the peptide with the chemokine receptor;7. According to broad-spectrum chemokine inhibitors molecular mechanism, using RT-PCR test to detect the level of chemokine mRNA expression, to study peptide mechanism of action of reducing the expression of chemokines.Results:1. Using phage display screening method by 4 rounds of panning, phage clones recovery rate from 5.51×10"8 to 5.06×10-6, the fourth round phage clones recovery is about the first round of 91.83 times,it showed that the target phage clones were effectively enriched.2. Titer determination experiments found that 16 clones had a higher titer, agarose gel study found that 10 clones of them were in the same position, relatively clear bands, so sequenced these peptides. 3. The sequencing results showed that 5 clones have the same insertion sequence, named P1 peptide, another five were poor homologous,then use of PROTPARAM bioinformatics software to analyze various physical and chemical nature of peptides.4. Chemotactic inhibition was found that P1 peptides could significantly inhibit the expression of various types of inflammatory chemokine on acute inflammation model,, other short peptides inhibition is not ideal, it may be that the P1 is the basic framework or lead compounds of the inflammatory chemokine inhibitors.5. The MTT test results indicated that the P1 peptide did not affect the activity of PBMC cells.6. LUMINEX chip test results showed that the P1 peptide could significantly reduce the various types of inflammatory chemokine expression.7. Calcium flow test results show that the P1 peptide can inhibit the inflammatory chemokine receptor binding probability, and the P1 peptide itself does not cause changes in intracellular calcium, indicating the P1 has no significant relationship with the chemokine receptors.8.RT-PCR results showed that all kinds of mRNA of chemokines expression were significantly reduced under the action of the P1 peptide, but it did not affect by the expression of TTP, indicating the reduced mRNA expression is not achieved by the TTP, may involve the expression of other genes, which are to be further experimental studies.Conclusion:1 screened a short peptide successfully by 4 rounds of panning;2 activity experimental studies found that the peptide does not produce toxic side effects on the cells;3 Mechanism of P1 peptide was found that it could inhibit the amount of chemokines by inhibiting various inflammatory chemokine mRNA expressions but the specific inhibition mechanism needs further study to prove.