Primary Research On The Role Of Anti-oxidative Stress And Inhibition Of IL-6 And IL-12 Of Chlamydia Trachomatis PORF5 Plasmid Protein

Objective:To investigate whether Ct pORF5 plasmid protein could protect host cells from oxidative stress and inhibit inflammation factors such as IL-6 and IL-12 via up-regulation of NQO1,which could provide theoretical foundation for further clarify the role of pORF5 plasmid protein in the Ct pathogenesis.Methods:The E.coli XL 1-blue strain which has transformed pGEX-6p-1/pORF5 recombinant plasmid was inoculate to LB solid medium containing Amp,after enlarge cultivation,IPTG was used to induce the expression of GST-pORF5 fusion protein.GST-pORF5 fusion protein was purified by Glutathione Sepharose 4B and GST tag was excised by PreScission Protease.The fusion protein and pORF5 plasmid protein were both analyzed and identified by SDS-PAGE and Western blotting.BCA was used to test the concentration of pORF5 plasmid protein.And polymyxin B was applied to eliminate endotoxin.RT-qPCR was used to detect the transcription mRNA level of NQOl after HeLa cells were stimulated with pORF5 plasmid protein at different concentrations(0,5,10,15 ?g/mL)for 24 h.HeLa cells were stimulated with pORF5 plasmid protein at different concentrations and different times(0,6,12,18,24 h),Western blotting was applied to evaluate the protein expression of NQO1 in HeLa cells.The contents of MDA,T-SOD and T-AOC level in HeLa cells were semi-quantificationally detected by spectrophotometer after stimulated with pORF5 plasmid protein at different concentrations for 18 h.HeLa cells were infected with Ct and stimulated with 10 ?g/mL pORF5 plasmid protein for different times,then spectrophotometer was used to detect the contents of MDA,T-SOD and T-AOC level,IFA was used to detect the formation of inclusions.THP-1 cells which pretreated with PMA were stimulated with pORF5 plasmid protein at different concentrations for 24 h and 15 ?g/mL pORF5 plasmid protein for different times,ELISA was applied to detect the levels of IL-6 and IL-12.HeLa cells were interfered with siRNA-NQO1 and stimulated with 10 ?g/mL pORF5 plasmid protein for 18 h,MDA,T-SOD and T-AOC contents were detected.THP-1 cells were interfered with siRNA-NQO1 and stimulated with 15 ?g/mL pORF5 plasmid protein for 18 h,the levels of IL-6 and IL-12 were detected by ELISA.Results:1.GST-pORF5 fusion protein whose molecular weight is about 54 kDa was successfully expressed and pORF5 plasmid protein whose molecular weight is about 28 kDa was successfully obtained after purification and removal of GST-tag.2.HeLa cells were stimulated with pORF5 plasmid protein at different concentrations for 24 h,RT-qPCR and Western blotting results showed that the mRNA and protein expression levels of NQO1 presented dose-dependent up-regulation and the peak value of NQO1 mRNA and protein levels were appeared when the concentration reached to 10 ?g/mL.And HeLa cells were stimulated with 10 ?g/mL pORF5 plasmid protein for different times,Western blotting result showed that the protein expression levels of NQO1 presented time-dependent up-regulation and the peak value of NQO1 protein level was appeared when the time was 18 h.3.HeLa cells were stimulated with pORF5 plasmid protein at different concentrations for 18 h,MDA test result showed that compared with LPS positive control group,the MDA content presented dose-dependent down-regulation and the minimum value 0.21+0.02 nmol/mg(P<0.01)appeared at 10 ?g/mL.T-SOD and T-AOC test results showed that the T-SOD and T-AOC contents presented dose-dependent up-regulation and the peak value 48.79±1.83 U/mL(P<0.01)and 50.76+0.94 U/mL(P<0.01)appeared at 10 ug/mL.4.HeLa cells which infected with Ct were stimulated with 10 ?g/mL pORF5 plasmid protein for different times,compared with PBS stimulation group,MDA test result showed that the most obvious reduction degree 1.35±0.04 nmol/mg(P<0.01)of MDA content appeared at 18 h;T-SOD and T-AOC test results showed that the most obvious increasing extent 26.61±2.21 U/mL(P<0.01)and 34.40±2.50 U/mL(P<0.01)of T-SOD and T-AOC contents appeared at 18 h.IFA showed that the IFU in control group was 7.13×105/mL,in pORF5 stimulation group was 9.03×105/mL,more than control group by 26.7%(P?0.05).5.THP-1 cells pretreated with PMA were stimulated with pORF5 plasmid protein at different concentrations for 24 h,ELISA results showed that the IL-6 and IL-12 levels presented dose-dependent down-regulation and the values 3.83±0.37 pg/mL(P<0.01)and 2.24±0.01 pg/mL(P<0.01)appeared at 15 ?g/mL;THP-1 cells pretreated with PMA were stimulated with 15 ?g/mL pORF5 plasmid protein for different times,ELISA results showed that the IL-6 and IL-12 levels presented time-dependent down-regulation and the values 2.07±0.55 pg/mL(P<0.05)and 2.31±0.14 pg/mL(P<0.01)appeared at 18 h.6.HeLa cells were stimulated with pORF5 plasmid protein after interfered by siRNA-NQO1,m contrast to the control group,the MDA content increased by 0.92±0.27 pg/mL(P<0.05),the T-SOD and T-AOC contents decreased by 21.90±3.36 U/mL(P<0.05)and 22.51+2.82 U/mL(P<0.01);THP-1 cells were stimulated with pORF5 plasmid protein after interfered by siRNA-NQO1,ELISA results showed that IL-6 and IL-12 levels increased by 11.16±3.13 pg/mL(P<0.05)and 6.91+0.32 pg/mL(P<0.01)compared with the control groupsConclusions:Chlamydia trachomatis plasmid-encoded protein pORF5 played antioxidant stress effect and inhibited the production of IL-6 and IL-12 through up-regulation of NQO1 of host cells.