Abstract: 【Abstract】 Objective To explore the molecular mechanism underlying the anti-apoptotic activity of pORF5 plasmid protein of Chlamydia trachomatis, so as to provide an experimental basis for further clarifying the pathogenesis of Chlamydia trachomatis. Methods HeLa cells were divided into two groups: carbonyl cyanide m-chlorophenyl hydrazone （CCCP, an apoptosis inducer） group was stimulated by CCCP for 30 minutes, and pORF5 + CCCP group was pretreated with pORF5 plasmid protein for 18 hours followed by CCCP for 30 minutes. Then, Western blot analysis was performed to determine the expression of apoptosis-related proteins Bcl-2, Bax and caspase-3, JC-1 fluorescent probe was used to detect changes in the mitochondrial membrane potential in HeLa cells, and cytochrome c release from mitochondria was analyzed by indirect immunofluorescence assay. To analyze whether high-mobility group box 1 （HMGB1） protein participated in the anti-apoptotic role of pORF5 plasmid protein, HMGB1 shRNA and control RNA were separately transfected into the HeLa cells, which were then stimulated by pORF5 plasmid protein and CCCP. Then, the protein expression of Bcl-2, Bax, activated caspase-3 was determined, and cytochrome c release was analyzed. Data were compared between two groups by using paired t test. Results pORF5 plasmid protein could antagonize the CCCP-induced decrease of mitochondrial membrane potential, and the red/green fluorescence intensity ratio was significantly lower in the CCCP group （0.4 ± 0.1） than in the pORF5 + CCCP group （1.7 ± 0.3; t = 6.95, P < 0.01）. The protein expression of Bcl-2 in the HeLa cells in the pORF5 + CCCP group was 5.3 ± 0.6 times more than that in the CCCP group （t = 8.62, P < 0.01）, while the protein expression of Bax and activated caspase-3 in the pORF5 + CCCP group significantly decreased by 79% ± 10% （t = 9.23, P < 0.01） and 75% ± 8% （t = 4.26, P < 0.05） respectively compared with the CCCP group. Compared with the control RNA transfection group, the HMGB1 shRNA transfection group showed significantly decreased mitochondrial membrane potential in the HeLa cells （t = 11.23, P < 0.01）, increased cytochrome c release, decreased Bcl-2 expresson （t = 7.19, P < 0.05） and increased Bax expression （t = 13.06, P < 0.01） after stimulation with pORF5 and CCCP. Conclusion Chlamydia trachomatis plasmid protein pORF5 plays an anti-apoptosis role by blocking the mitochondrial apoptotic pathway through HMGB1 protein.

Key words: Chlamydia trachomatis, Apoptosis, Proto?oncogene proteins c?bcl?2, Bcl?2?associated X protein, Caspase?3, High mobility group box protein 1, pORF5 plasmid protein