The Effect And Mechanism Of Human β Defensin In Dental Pulp Anti-inflammatory Response

OBJECTIVE: To express and purify human β defensin 110 in vitro prokaryotic cells,and to overexpress HBD110 in eukaryotic cells;To study the regulatory effect of HBD110 on inflammatory cytokines in human dental pulp cells and TH1 cells;To investigate the antibacterial effect of HBD110 on common pathogenic bacteria and pathogenic bacteria of pulpitis,to provide theoretical basis for elucidating the mechanism of anti-inflammatory and antibacterial effects of HBD110.MATERIALS: HBD110 polypeptide was expressed and purified from Escherichia coli by IMPACT-pTWIN1 self shearing system mediated by intein,and HBD110 was overexpressed in the supernatant of HEK293 T cells by transfection HBD110 overexpression vector;Human dental pulp cells and TH1 cells were treated with HEK293 T cells supertants containing HBD110 and then were stimulated with LPS or PGN.The expression of inflammatory factors TNF-α,IL-1α,IL-1β,IL-6,CXCL-11,TLR2 and the expression of HBD2 were detected by qRT-RCR;Dual luciferase reporter gene system was used to detect the activation of NF-kB pathway in RAW264.7 cells stimulated by LPS after HBD110 pretreatment;The CFU assay was used to analysis the antibacterial efficiency of HBD110-N short peptides against Escherichia coli,Staphylococcus aureus,Fusobacterium nucleatum and Enterococcus faecalis.RESULTS: HBD110 peptide was successfully expressed and purified from prokaryotic expression system in vitro,HBD110 peptide was also successfully overexpressed in HEK293T;HBD110 was found can regulate the expression of inflammatory cytokines in human dental pulp cells and TH1 cells stimulated by 1 μg/mL LPS or 2 μg/mL PGN,and capable of regulation the expression of HBD2 by control the expression of inflammatory factors,the regulation of HBD110 on inflammatory response may be mediated by inhibition of the NF-κB pathway;HBD110-N short peptide showed antibacterial effects on Escherichia coli,Staphylococcus aureus,Fusobacterium nucleatum and Enterococcus faecalis.CONCLUSIONS: Human β defensin 110 can inhibit the inflammatory response of human dental pulp cells and TH1 cells;Human β defensin 110 has antibacterial activity against pulpitis pathogens.