Construction Of A Dual Functional Polypeptide With Antibacterial And Anti-inflammatory And Its Efficacy In Periodontitis Microenvironment

Objective:Bacteria and lipopolysaccharides are powerful activators of different immune cells including monocytes and macrophages,which are closely related to the pathogenesis of periodontitis.The objectives of this study were to design and fabricate a dual functional polypeptide,and to evaluate its antimicrobial and anti-inflammatory efficacy in periodontitis microenvironment.Materials and Methods: A novel polypeptide was developed by connecting the human antimicrobial peptide LL-37 to the anti-inflammatory peptide C15.The polypeptide was synthesized,purified by high performance liquid chromatography,and identified by mass spectrometer.The antibacterial activity of LL37-C15 against Porphyromonas gingivalis was measured by the double dilution method,and its antibacterial mechanism was explored by laser confocal microscope and scanning electron microscope.Flow cytometry was used to detect the macrophage typing ratio.Real-time quantitative PCR and western blot were used to detect the gene and protein expression levels of cytokines.Cell Counting Kit-8 was used to detect the biocompatibility of LL37-C15.Molecular dynamics simulations were performed to analyze the mode of LL37-C15 action on animal and bacterial cell membranes.Sprague-Dawley rats were used to establish a periodontitis model and evaluate the anti-inflammatory efficacy in vivo.Results: The constructed dual functional polypeptide LL37-C15 can inhibit the growth,adhesion,and biofilm formation of bacteria.Compared with the control group,the proinflammatory cytokines in experimental group,such as interleukin and tumor necrosis factor,were significantly reduced.The proportion of macrophages M1 phenotype induced by LPS was decreased.The Cell Counting Kit-8 results show that the dual functional polypeptide has no obvious inhibitory effect on cell proliferation with good biocompatibility.Molecular dynamics simulations showed that LL37-C15 improved the antibacterial properties and biocompatibility of LL-37.In vivo results showed that LL37-C15 reduced alveolar bone loss,gingival inflammation,and the number of osteoclasts in the periodontitis model.Conclusion: LL37-C15 has the advantages of high activity and low toxicity,and it expresses good antibacterial and anti-inflammatory properties in the periodontitis microenvironment.It can provide a potential method for the treatment of periodontitis.What’s more,the excellent function of dual functional polypeptide develops a molecular biology platform for bacterial infectious disease,providing a new therapeutic approach for diseases with complex etiology like periodontitis.