The Effect Of Enterococcus Faecalis On The Osteoclastogenesis Of Bone Marrow-derived Macrophages Under Hypoxia

Objective:Enterococcus faecalis（E.faecalis）is considered the main pathogen causing refractory periapical inflammation,and hypoxia can inhibit osteoclast differentiation.Refractory periapical inflammation is often associated with infection and periapical hypoxic microenvironment,but the mechanism of bone destruction remains unclear.This study was aimed to investigate the effect of Enterococcus faecalis on osteoclast formation under hypoxia.Methods:Firstly,bone marrow-derived macrophages（BMMs）were isolated and identified.The receptor activator of NF-κB ligand（RANKL）and macrophage colony stimulating factor（M-CSF）were added to induce the formation of osteoclasts in BMMs and tartrate-resistant acid phosphatase（TRAP）staining techniques were used to identify the osteoclasts-derived from BMMs.Secondly,cobalt chloride（Co Cl₂）was used to simulate hypoxia and western blot was used to detect hypoxia inducible factor-1α（HIF-1α）.Thus,an effective cell culture model in hypoxic environment was established.Enzyme-linked immunosorbent assay（ELISA）was used to detect the expression of HIF-1αand tumor necrosis factor-α（TNF-α）.The hypoxic inflammatory models were established and evaluated in vitro.In addition,CCK-8 was used to detect the effects of hypoxia alone and Enterococcus faecalis on BMMs proliferation under hypoxia.The effect of Enterococcus faecalis on cell apoptosis under hypoxia was studied using flow cytometry and western blot,respectively.Finally,flow cytometry and western blot were used to study the expressions of m RNA and protein related to osteoclast differentiation in BMMs,including Blimp,c-Fos,NFATc1 and RBP-J.TRAP staining and immunofluorescence detection were used to measure the effects of hypoxia alone and Enterococcus faecalis on osteoclast formation under hypoxia.SPSS26.0 was used to conduct independent sample t test and one-way analysis of variance,respectively,to compare the differences between two groups of data and the differences between multiple groups of data.Results:RANKL-treated BMMs showed the ability of osteoclast formation,which was characterized by TRAP staining.In this study,50 ng/m L RANKL was more effective than20 ng/m L RANKL-induced osteoclast formation of BMMs.In addition,western blot results showed that the expression level of HIF-1αwas significantly increased after 1 day of Co Cl₂treatment compared with the control group.The result of ELISA assay indicated that,the expression of TNF-αwas significantly increased after 72 h treatment of 1000 MOI Enterococcus faecalis stimulated BMMs under hypoxia conditions,thus establishing an in vitro model of infection under hypoxia.The proliferation of cells stimulated by 100μM Co Cl₂ was significantly increased within 5 days.When Enterococcus faecalis at 10,100and 1000 MOI was added under hypoxia,cell proliferation decreased significantly.western blot analysis showed that the expression of apoptosis-related protein Bcl-2 was increased in 100μM Co Cl₂-treated cells,while the expression of Caspase-3 was decreased.After1000 MOI Enterococcus faecalis was added for 1 days under hypoxia,the expression of Caspase-3 was significantly increased,while the expression of Bcl-2 was significantly decreased.TRAP staining and immunofluorescence detection showed that the RANKL-induced osteoclastogenesis of BMMs was significantly decreased under the condition of Co Cl₂ mimicking hypoxia.When the stimulation of heat-killed Enterococcus faecalis was added under hypoxia,the osteoclast formation ability of BMMs was significantly enhanced.The results of RT-PCR and western blot showed that the m RNA and protein expression levels of Blimp,c-Fos and NFATc1 were significantly increased,while the expression level of RBP-J was significantly decreased.Conclusion:In conclusion,Enterococcus faecalis promotes RANKL-induced osteoclast formation under hypoxia independent of cell proliferation and apoptosis in vitro.This study illustrates that thorough root canal disinfection is crucial,in addition to impenetrable root canal obturation.