Study On The Regulation Mechanism Of Staphylococcus Aureus Protein A On Autophagy Signal Of Osteoblasts In Chronic Osteomyelitis

Objective:To explore the regulatory mechanism of Staphylococcus aureus protein A(SpA)on autophagy signal of osteoblasts in chronic osteomyelitis,and to study the effect of SpA on proliferation and differentiation of MC3T3-E1 osteoblasts in vitro,as well as the regulatory mechanism of apoptosis and autophagy signal.In vivo,we used S.aureus wild-type Newman strain and S.aureus ?SpANewman strain to construct SD rat chronic osteomyelitis model to study the effect on autophagy signal of osteoblasts,and to explore the regulatory mechanism of SpA on apoptosis and autophagy signal of osteoblasts.Methods:1.TNFR1-/-MC3T3-E1 cells were cultured and identified by CRISPR/cas9 gene editing technique.Firstly,MC3T3-E1 cells were infected with HBLV-cas9-puro chronic virus with MOI value of 0,5,10,20 and 40,respectively.Monoclonal cell lines were screened and identified by 10 ?g/mLpuromycin,and stable Cas9-PURO MC3T3-E1 cells were successfully constructed.Secondly,MC3T3-E1 cells were infected with HBLV-BSD-RFP NC,HBLV-m-Tnfrsf1a-gRNA1-BSD-RFP,HBLV-m-Tnfrsfla-gRNA2-BSD-RFP and HBLV-m-Tnfrsfla-gRNA3-BSD-RFP chronic virus,and 10 ?g/mL was used to kill blasticidin 72 hours after infection.The transfection efficiency of each group was observed and calculated under fluorescence microscope.The TNFR1-null mutants of cell lines were selected and identified,and the stable TNFR1-/-MC3T3-E1 cell line was successfully constructed.2.WT and TNFR1-/-MC3T3-E1 cells were induced by different concentrations of SpA at different time in vitro.The cell vitality was detected by MTS,the apoptotic rate of MC3T3-E1 cellswas detected by flow cytometry,the number of calcified nodules were observed by alizalin red staining,Expression of apoptosis and autophagy-related proteins was detected by Western blottingand immunofluorescence.The expression levels of autophagy related proteins in MC3T3-E1 cells were detected under different conditions.SD rats were inoculated with 5%sodium morrhuate solution and 1 × 108 CFU/mL of S.aureus newman wild-type strain and S.aureus newman?SpA strain in 50 ?L of PBS.After 2,3 and 4 weeks of infection,Ratswere sacrificed,fixed,decalcified,paraffin embedded and sectioned and evaluated by HE staining.Expression of apoptosis related proteinsin each group were detected by Western blotting and immunocytofluorescence.Results:1.Lentivirus-mediated HBLV-cas9-PURO modified MC3T3-E1 cells.Cas9+/+MC3T3-E1 cells were successfully constructed by identification and selection of monoclonal culture.Lentivirus-mediated HBLV-m-Tnfrsfla-gRNA3-BSD-RFP Modified MC3T3-E1 cells.The stable red fluorescence transfer efficiency of the cells was the highest under fluorescence microscope.The stable TNFR1-/-MC3T3-E1 cells were successfully constructed by the culture and identification of monoclonal cell lines.2.WT and TNFR1-/-MC3T3-E1 cells were induced with 10,30,60,90,120?g/mL SpA for 1 and 7 days,and the cell activity was detected by MTS in a doseand timedependent manner.WT and TNFR1-/-MC3T3-E1 cells were induced by 120?g/mL SpA for 3 days,and apoptosis was detected by flow cytometry in a doseand time dependent manner.Compared with TNFR1-/-group,the number of calcified nodules in TNFR1-/-group induced by 120 ?g/mL SpAdecreased in a dosedependent manner on the 14th and 28th day.The expression of pro-caspase-3 was downregulated,while the expression of cleaved-caspase-3 and BCL-2 was up-regulated,the expression of Beclin-1,ATG5,ATG7,and LC3-? in autophagy was downregulated,while the expression of LAMP-2 and SQSTM1/p62 was up-regulated.The chronic osteomyelitis model of SD rats infected with S.aureus Newman wildtype strains and S.aureus Newman ?SpA strains was established and verified.SD rat chronic osteomyelitis model was fed for 2 weeks,3 weeks and 4 weeks after successful establishment of chronic osteomyelitis model.Materials,fixation,decalcification,paraffin embedding and slicing.Routine he staining was performed in S.aureus newman wildtype strains group and S.aureus newman group.Conclusion:In vitro,it was confirmed that SpA could induce apoptosis of MC3T3-E1 cells by up regulating the expression of apoptosis related proteins such as cleaved-caspase-3 and Bcl-2 mediated by TNFR1.In vitro and in vivo studies showed that SpA couldinhibit autophagy by TNFR1 mediated inhibition of autophagy related proteins Beclin-1,ATG5,ATG7 and LC3-?,and directly or indirectly inhibit the formation of autophagosomeand inhibit the autophagy of osteoblasts.In addition,up regulation of the expression of LAMP-2 and SQSTM1/p62 showed that SpA did not affect the fusion of autophagosomeand lysosomes to form autolysosome,and the subsequent degradation of autolysosome.Autophagy flux was not inhibited.The results of this study indicate that SpA is a major virulence factor of S.aureusstrains in inhibiting autophagy signal of osteoblasts in chronic osteomyelitis may lay a foundation for more effective treatment of the disease,and regulating autophagy signal of osteoblasts may be an alternative treatment target.