| Objective: To investigate the effects of SINC,a type III secreted protein of C.psittaci,on apoptosis and autophagy of host cells and the mechanisms of MAPK/ERK signaling pathway on that,providing further experimental basis for the molecular pathogenic mechanisms of C.psittaci.Methods: The constructed prokaryotic expression vector pET-30a(+)/SINC was transformed into E.coli BL21,and the recombinant SINC protein was induced with 0.1mM IPTG.After purified,the concentration of SINC was determined by BCA,and endotoxin was removed by polymyxin B treatment.HeLa cells and RAW 264.7 cells were treated with SINC at different concentrations(2,4,6,8,10?g/ml)for 24 h and the expression of LC3,Belin-1,Bax and Bcl-2 was examined by western blot.Then the cells were treated with 2?g/ml SINC for different times(0,6,12,24,36h)and the expression of LC3,Belin-1,Bax and Bcl-2 was examined by western blot.The number of cells which contained more than 5 puncta of LC3 were detected by immunofluorescence analysis,and the autophagosomes and autolysosomes were detected by transmission electron microscope(TEM).Moreover,Hoechst33258 staining and Flow cytometry were applied to measure the apoptosis of HeLa cells.HeLa cells and RAW 264.7 cells were teated with SINC at the optimal concentration for different time periods(0,5,15,30,60 and 120 min or 0,6,12,18,24 and 36h)then the levels of phosphorylated ERK1/2 and total ERK1/2 were detected by western blot.After treated with specific ERK inhibitor U0126,Hoechst33258 staining and Flow cytometry were applied to measure the apoptosis of HeLa cells.The expression levels of LC3-II,Becin-1,SQSTM1,Bax,Bcl-2 and PARP were examined by western blot.The number of cells which contained more than 5 puncta of LC3 were detected by immunofluorescence analysis.Results: The prokaryotic expression vector of pET-30a(+)/SINC was successfully constructed and expressed the approximately 66 kDa target protein.The expression levels of LC3-II and Belin-1 were highest in HeLa cells after treated with 4?g/ml SINC for 6h.The immunofluorescence analysis showed that the number of cells which contained more than 5 puncta of LC3 was significantly increased and the autophagosomes and autolysosomes were found in HeLa cells.The enhanced autophagic level was revealed after 2?g/ml SINC treatment for 12 h in RAW 264.7 cells.The ratio of p-ERK1/2/ERK1/2 was maximized in HeLa cells after 4?g/ml SINC treatment for 15 min.After treated with specific ERK inhibitor U0126,the level of ERK1/2 phosphorylation was significantly decreased.Compared with SINC treated group,the expression levels of LC3-II,Becin-1 and SQSTM1 were decreased after treated with 50 uM U0126 and the immunofluorescence analysis also showed that U0126 could decreased LC3 aggregation.The ratio of p-ERK1/2/ERK1/2 was maximized in RAW 264.7 cells after 2?g/ml SINC treatment for 15 min.After treated with specific ERK inhibitor U0126,the ratio of p-ERK1/2/ERK1/2 was significantly decreased.Compared with SINC treated group,the expression levels of LC3-II and Becin-1 were decreased,while SQSTM1 was increased after treated with 50 uM U0126.And the immunofluorescence analysis also showed that U0126 could decreased LC3 aggregation.The rate of apoptotic cells in SINC treated group was significantly decreased at 10?g/ml.The expression levels of pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 showed that SINC could down-regulated Bax but up-regulated Bcl-2,and the ratio of Bax/Bcl-2 was the lowest after 10?g/ml SINC treated with 18 h.The ratio of p-ERK1/2/ERK1/2 was significantly increased after 10?g/ml SINC treated for 18 h,and U0126 could inhibit the ERK1/2 phosphorylation induced by SINC.Either the results of Hoechst33258 staining or Flow cytometry showed that SINC treatment reduced the apoptosis rate induced by STS,while the inhibition of apoptosis by SINC was significantly weakened after U0126 treatment.And the results of Flow cytometry showed that U0126 treatment could increased the apoptosis rate in the SINC treated HeLa cells in spite of STS treated or not.SINC treatment could down-regulated the ratio of Bax/Bcl-2 and the level of Cleaved PARP whether in the presence of STS or not,and U0126 could attenuate this effects.Conclusions: SINC can induce autophagy and inhibite apoptosis via the MAPK/ERK signaling Pathway. |
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