R10, A Novel Inhibitor Of P38 Mitogen-activated Protein Kinase

p38 MAPK which has four isoforms:p38α,p38β,p38δand p38γ,was identified as a Tyr phosphorylation protein in macrophage cells which were stimulated by lipopolysaccharide (LPS) at first.As a member of mitogen-actived protein kinase(MAPK) superfamily,the p38 subgroup plays important roles in inflammation,proliferation,apoptosis etc.Especially in the production of inflammatory molecules:proinfiammatory cytokines,chemokines,degradative enzymes,adhesion molecules and others.All of them were regulated by p38 MAPK pathway. Uncontrolled inflammation is involved in many serious diseases as atherosclerosis,cancer etc. Although traditional available drugs alleviate inflammation,in most cases they are relatively non-selective and have dose-limiting side-effects.The development of new therapeutic anti-inflammatory strategies and drugs depends on a better understanding of the underlying pathogenic mechanisms of the inflammation process.p38 MAPK pathway was chosen as the target pathway for its central position in inflammatory molecules production through both transcription-dependent mechanisms and post-transcriptional regulation.So far,more than 100 kinds of p38 MAPK inhibitors have been reported in patents and literature and some of them have entered clinical trials..However,no one has been used in clinical trail because of the fail of safe test.To find a potent,safe,effective p38 MAPK inhibitor is imperative.It has been popular of life science study to base on the work of bioinformatics pro-analysis firstly.A sequence of peptide may inhibit p38 MAPK was found by statistical coupling analysis of the p38 MAPK family earlier.This project is just base on the that work of the bioinformatics to study the biology function of the peptide which is code by the sequence.And try to test the new mode of protein kinase signal pathway inhibitor screening.1.Firstly,peptide R10 was composed and the biology function was been studied by the kinases assays in vitro.The inhibition of R10 to MKK6(p38 upstream kinase) phosphorylated p38,p38 autophosphorylation and p38 phosphorylated ATF-2(one of main downstream substrates) three levels.Strong inhibition of peptide R10 to p38 phosphorylated ATF-2 was observed.However,the other two levels were not shown significant inhibition of peptide R10. In addition,the inhibition of peptide R10 increased following the concentration of peptide R10 up.But when the concentration of peptide R10 reached a level,the inhibition was balanced.No significant inhibition was observed in the phosphorylation of c-Jun by JNK.2.The function of peptide R10 in cells was been studied followed the study in vitro finished.The classical and alternative p38 activation pathways have been studied.Because our study is prepared for candidate of anti-inflammation,we dropped cell transfection to choose a new way make peptide R10 into cells directly.To deliver the peptide R10 into the cells and keep its function,TAT,classical one in cell-penetrating peptides(CPPs) was chosen as the "transport vehicle'".And an enhanced green fluorescence protein(EGFP) tag was labeled in the His-TAT-EGFP-R10 plasmid to show the position of peptide in cells.The protein was translocated into NIH/3T3 cells and its biology function was kept successfully.With the test of phosphorylation level of p38 MAPK pathway,the phosphorylation of ATF-2 by p38 but not others was inhibit significantly by His-TAT-EGFP-R10 protein.And it was identified that R10 is the functional base group in the inhibition.No significant inhibition of JNK pathway in cells was observed.3.One of important steps of inflammation amplification is the inflammatory molecules released,if the production of inflammatory molecules could be regulated at expression level, the inflammation would be controlled.Thus IL-8,one of important cytokines regulated by p38 MAPK pathway was chosen to be the test molecule.Not ELISA but LiquiChip which is based on Luminex technology was used to finish the work for its sample saving,high throughout, high sensitivity.Significant inhibition of His-TAT-EGFP-R10 protein to the production of IL-8 was observed at the concentration of 500 nmol/L,and the inhibition increased when the concentration of His-TAT-EGFP-R10 protein reached 1,000 nmol/L.R10 was identified as the functional unit of inhibition.4.There was no significant cell toxin shown in the test of XTT assays.In conclusion.the biology functions of peptide R10 in vitro,in cells and in cellular physiological levels were studied.The inhibition of peptide R10 to p38 MAPK pathway both in vitro,in vivo and the effect in physiological level were confirmed.The work makes it is possible of the study in animal model and clinical trail followed.And a primary exploration of experiment biology with bioinformatics has been made.