The Application Study On A Novel Anti-inflammatory Peptide

Purpose:NLRC5is a has strong anti-inflammatory effect.In this paper, by means ofbioinformatics, we try to filter out NLRC5-specificity polypeptide, and to study itsanti-inflammatory effect in cell and animal level.Methods:1.Screening peptide LS22through bioinformatics methodsBy using the principle of the bioinformatics, we will first screen sequence usingSMART software to carry on the multiple sequence alignment in order to find out theconserved sequence of NLRC5protein.We intend to use clustalx software to dohomologous NLRCx Protein sequence alignment. The CLC Protein Work Benchsoftware is adopted to analysis predominate hydrophilic, antigenicity of NLRC5Protein and other physical properties.Finally, from the LS22peptide is selected.2. In vitro study of anti-inflammatory effect of LS22peptidesThe MTS experiments was used to test the ability of LS22peptides in cell vitality.Bystimulating mouse macrophage cell line Raw264.7,the epithelial cells of the humanumbilical vein HUVEC and human mononuclear cells THP-1with lipopolysaccharide(LPS) respectively to establish a in vitro model of inflammation.ELISA and Q-PCRwere employed to detect the effect of different concentrations of LS22polypeptide inthe inflammatory response induced by LPS.Chemotaxis and adhesion assay wereintroduced to test whether LS22peptide also affects the cell chemotactic response and adhesive ability.3. In vivo study of anti-inflammatory effect of LS22peptidesEIU was induced by a single footpad injection of100ml sterile pyrogen free salinecontaining200mg LPS. A separate group received the same volume of sterilizedsaline in one foot pad for comparison.The rats were treated with PBS, LPS, controlpeptide,LS22peptide or dexamethasone (10mg/eye)1h before LPS (200mg)injection, and were evaluated24h after LPS challenge.We assessed microscopicexamination of the rat eyeball living tissue, tissue section, EIU disease rating, proteinleakage and cell infiltration into the aqueous humor and inflammatory cytokines.4. anti-inflammatory mechanism of LS22peptideLuciferase report assay was used to test the effect of LS22peptides on NF-KBsignaling activation induced by LPS.The effect of LS22peptides on thephosphorylation and translocation of transcription factor p65were tested by confocaland western blot.Results:1.Screening peptide LS22through bioinformatics methodsWe selected polypeptide LS22.The amino acid sequence isLDLSHNSISQESALYLLETLPS.2. In vitro study of anti-inflammatory effect of LS22peptidesResults show that the LS22polypeptide showed no influence on the cell vitality.Nomatter from the level of protein or mRNA level, for the detection of three kinds of celllines,50uM,100uM LS22polypeptide displayed significant anti-inflammatoryeffects compared with positive control and control polypeptide group, with adose-dependent manner.1uM polypeptide had no effect.LS22polypeptide alsoinhibited the LPS-induced chemotaxis reactions, while showed no influence on theTNF-a-induced cell adhesion ability. 3. In vivo study of anti-inflammatory effect of LS22peptidesResults show that the LS22peptide significantly attenuated clinical manifestation ofEIU rats, reduced protein leakage and cell infiltration into the aqueous humor (AqH),suppressed TNF-a and IL-6production.4. anti-inflammatory mechanism of LS22peptideLS22polypeptide inhibited the LPS-induced NF-KB luciferase reporter geneactivation.Confocal experiment and immune experiment showed LS22peptidesinhibited p65phosphorylation and translocation to nucleus, but had no effect on totalp65.Conclusion:1. By means of bioinformatics, we predict and select LS22polypeptide2. LS22peptide inhibits inflammation in a dose-dependent manner in vitro3. LS22peptide inhibits rat uveitis and inflammation in vivo4. LS22peptide can inhibit p65phosphorylation and translocation into the nucleus...