| Objective: Chlamydia trachomatis can resist host cells apoptosis to evade the host immune clearance, the mechanisms of Chlamydia trachomatis resists apoptosis remains unknown. In this study, we wanted to determine the relationships among the p ORF5 plasmid protein of Chlamydia trachomatis, ERK1/2 pathway and the DJ-1 protein; and also to determine their effects on cells apoptosis. The results could provide experimental basis for futher elucidate the pathogenic mechanism of Chlamydia trachomatis.Methods: p ORF5 stable transfected He La cell line(p ORF5-He La) and control He La cell line(GFP-He La) were treated for 6h with TNF-?(20ng/ml) and cycloheximide(CHX,2?g/ml), and examined for apoptosis by Hoechst33342 staining.The m RNA and protein levels of apoptosis-related proteins Bax,Caspase-3 and Bcl-2were analyzed by Quantitative real-time PCR(q RT-PCR) and Western blot;p ORF5-He La cells were pretreated for 30 min prior to adding TNF/CHX with the ERK1/2 inhibitor PD98059(30?M/ml),and the apoptotic rate was examined by Hoechst staining,TUNEL assay and flow cytometry. q RT-PCR and Western blot were used to detected the expression of Bax,Caspase-3 and Bcl-2; To establish the DJ-1stable interfered p ORF5-He La cell line and control cell line, the sh RNA-DJ-1lentiviral particles and control sh RNA-GFP lentivirel particles were used to infect p ORF5-He La cells; The apoptotic rates of the DJ-1 stable interfered cell line and control cell line were analyzed by Hoechst33342 staining, TUNEL assay and flow cytometry, and the levels of Bax,Caspase-3 and Bcl-2 were detected by q RT-PCR andWestern blot;The p ORF5-He La cells treated with PD98059 and the control cells were harvested after 6h apoptosis induction, and the levels of DJ-1 were detected by Western blot; The DJ-1 stable interfered p ORF5-He La cells and the control cells were harvested after 6h apoptosis induction, and the levels of phosphorylated ERK1/2 was detected by Western blot.Results:1.The results of Hoechst stainning showed that the nuclears of the GFP-He La cells became pyknotic, fragmentation and hyperchromatic.Whereas, the nuclears of p ORF5- He La cells were uniform and diffuse blue-fluorescence,the apoptosis rate of p ORF5-He La cells was decreased by 32.4% compared with the control cells(P<0.05);In the p ORF5-He La cells, the levels of Bax and Caspase-3 m RNA decreased by 5.6times(P<0.01) and 18.6 times(P<0.01) respectively, when compared with the GFP-He La cells;In the p ORF5-He La cells, the expression of Bax protein decreased by 78.2% than that of the control cells(P<0.01),. However, the protein levels of Bcl-2increased by 167.8% in the p ORF5-He La cells, when compared with the control cells(P<0.01).2. The results of Hoechst and TUNEL staining showed that the apoptosis rate increased by 28.5%(P<0.05)in PD98059/p ORF5 cells compared with the control group.The percentage of TUNEL positive cells in PD98059/p ORF5 cells was(54.6±2.1)%, the percentage of TUNEL positive cells in the control grounp cells was(12.5± 3.4)% and the percentage of TUNEL positive cells in PD98059/p ORF5 cells inceased by 42.1% compared with the control cells(P<0.05); The flow cytometry analysis showed that PD98059/p ORF5 cells apoptotic rate increased by 12.57%compared with control group; In p ORF5-He La cells treated with PD98059, the levels of Bax and Caspase-3 m RNA increased 2.5 times(P<0.01)and 302.7 times(P<0.01)than the that of the control cells, the Bax protein expression level increased by264.3%(P<0.01), in contrast, Bcl-2 was downregulate by 86.6%(P<0.01) in comparison with the control cells.3. Hoechst and TUNEL assay showed that the percentage of apoptotic cells insh DJ-1-p ORF5-He La cells increased by 30.8% than that of the control cells(P<0.05);The percentage of TUNEL positive cells in sh DJ-1-p ORF5-He La cells was(45.2±2.3)%, the percentage of TUNEL positive cells in the control grounp cells was(8.3±2.0)%, and the percentage of TUNEL positive cells in sh DJ-1-p ORF5 cells inceased by 36.9% than the control cells(P<0.05); The flow cytometry analysis showed that sh DJ-1-p ORF5-He La cells apoptotic rate increased by 17.05% compared with control group; In the sh DJ-1-p ORF5-He La cells, Bax and Caspase-3 m RNA expression levels increased 187.8 times(P<0.01) and 3.6 times(P<0.01) respectively, than the control cells; and the Bax protein expression levels increased by 387.5%(P<0.01) in contrast,Bcl-2 was decreased by 91.0%(P<0.01) when compared with the control cells.4. There was no significant difference in the level of DJ-1 between the p ORF5-He La cells with PD98059 and the control cells(P>0.05).Whereas,the ERK1/2phosphorylation in sh DJ-1-p ORF5-He La cells increased 102.8% than that of the control cells(P<0.05).Conclusions:1. p ORF5 plasmid protein of Chlamydia trachomatis contributed to apoptosis resistance.2. Upregulation of DJ-1 protein confered resistance to p ORF5 plasmid protein-induced apoptosis through ERK1/2 pathways... |
PDF Full Text Request