The Investigation On The Ability Of Enterococcus Faecalis Causing Periapical Periodontitis And The Possible Mechanism

Objectives:The purpose of this study was to investigate the ability and the mechanism of Enterococcus faecalis inducing IL-1 beta secretion in THP-1 macrophages and to investigate the ability and the mechanism of E.faecalis infection inducing apoptosis of human osteoblast-like MG63 cells.Material and methods:Part Ⅰ: THP-1 macrophages were treated with E.faecalis at a multiplicity of infection(MOI,the ratio of the number of bacteria to the number of host cells),IL-1 beta secretion were detected by Enzyme-linked immunosorbent assay.Then,Polymerase Chain Reaction and Western blot analysis were used to determine the m RNA expression level and protein expression level of IL-1β and inflammasome.Then the lactate dehydrogenase release was measured.Moreover,THP-1 macrophages were pre-treated with caspase-1 inhibitors,or NLRP3-si RNA)or ox ATP and KCl,respectively,before E.faecalis infection,then the release level of IL-1β and LDH were determined.Part Ⅱ: Human osteoblastic MG63 cells were treated with E.faecalis at a multiplicity of infection,then Cell Counting Kit was used to evaluate the effect of E.faecalis infection on the proliferation of the MG63 cells.The infected MG63 cells were subjected to flow cytometric analysis and western blot analysis to determine the amount of apoptotic cells.Cell death was measured by lactate dehy-drogenase release.Moreover,the protein expression levels of NLRP3 and caspase-1 were determined by western blot analysis,and LDH release level of MG63 cells pretreated with caspase-1 inhibitor were determined.Then small interfering RNA(si RNA)were used to silence NLRP3,and the amount of apoptotic cells and LDH release induced by E.faecalis infection were determined again.Part Ⅲ: The distribution features and expression level of NLRP3 and AIM2 inflammasome in the periapical lesions were investigated by immunohistochemistry,real-time PCR and western blot analysis.Results:Part Ⅰ: E.faecalis induced IL-1β secretion in THP-1 macrophages in dose and time dependent manner.Caspase-1 activation,pro–IL-1β up-regulation were also detected by immunoblotting,real-time reverse-transcription polymerase chain reaction,respectively.And the lactate dehy-drogenase release in infected THP-1 macrophages cells were induced by E.faecalis in MOI dose dependent manner.The caspase-1 inhibitors,NLRP3-si RNA,ox ATP and KCl significantly inhibited IL-1β secretion and LDH release in THP-1 macrophages infected with E.faecalis.Part Ⅱ: The proliferation inhibition rate of human osteoblastic MG63 cells treated with E.faecalis was increased in a MOI–dose-dependent manner.The flow cytometric and western blot analysis showed the apoptosis cells from the E.faecalis-treated samples were significantly promoted.And the activation of caspase-1 and NLRP3 were also observed in cells treated with E.faecalis.However,the LDH release decreased significantly when cells were pretreated with caspase-1 inhibitor,and the percentage of apoptotic MG63 cells and LDH release dropped significantly when NLRP3 was downregulated with NLRP3-si RNA.Part Ⅲ: The expression levels of NLRP3 and AIM2 inflammasome were increased in the periapical lesions and were mainly distributed in inflammatory cells.And the PCR and western blot analysis showed the expression levels of NLRP3 increased more than the expression levels of AIM2 in periapical lesions.Conclusions:We found that E.faecalis induced IL-1β secretion in THP-1 macrophages in dose dependent manner,and E.faecalis induces IL-1β secretion and inflammatory cell death via caspase-1 activation and NLRP3 inflammasome activation.Meanwhile,the proliferation of MG63 cells was inhibited and the cell apoptosis was induced by E.faecalis in a dose-dependent manner,and E.faecalis promotes apoptosis and inflammatory cell death(pyroptosis)of human osteoblasts at least partially through the NLRP3 inflammasome.Moreover,the inflammasome NLRP3 and AIM2 proteins play a part in the pathogenesis of periapical periodontitis.