| Objective : Chlamydia trachomatis is an obligate intracellular microorganism,which completes the development cycle by inhibiting apoptosis of host cells.However,the molecular mechanism of anti-apoptosis is not very clear.pORF5 protein was a secreted protein encoded by Ct plasmid,and it has been proved that pORF5 plasmid protein could activate the PI3K/AKT pathway and inhibit cell apoptosis.The purpose of this research was to examine the relationship among pORF5 plasmid protein,PI3K/AKT pathway and heme oxygenase-1(HO-1),and wether HO-1proetin was involved in anti-apoptosis induced by pORF5 plasmid protein,which would provide an experimental basis for elucidating the pathogenesis of Ct.Methods:1.The p GEX-6P/pORF5/XL1-blue E.coli was revived by LB solid medium at 37? for 24-36 h,and a single colony was inoculated into LB liquid medium and incubated for overnight at 37 ?with shaking at 200 rpm.IPTG was used to induce to express GST-pORF5 fusion protein for 4h,and the fusion protein was purified by glutathione agarose beads.Pre Scission Protease was applied to remove the GST tag of GST-pORF5 fusion protein.pORF5 plasmid protein was quantified concentration and identified by BCA method and western blotting respectively.2.He La cells were cultured and stimulated with different concentrations of pORF5 plasmid protein(3 ?g/ml,6 ?g/ml,9 ?g/ml,12 ?g/ml)for different times(4h,8h,12 h,16h),then He La cells were collected for protein extraction.Western blotting was performed to analyze HO-1 protein expression.3.He La cells were pretreated with 15?M PI3 K inhibitor LY294002 for 1h,then the optimal concentration of pORF5 plasmid protein(9 ?g/ml)was used to stimulate He La cells for another 12 h,western blotting was used to detect HO-1 expression and PI3K/AKT pathway activation,indirect immunofluorescence was applied to identify the degree of Nrf2 nuclear translocation.4.He La cells were pretreated with 50ng/ml TNF-? for 1h and divided into four groups according to the following conditions:the first group as the control;the second group: He La cells were stimulated with 9?g/ml pORF5 plasmid protein for 12h;the third group:He La cells were stimulated with 9?g/ml pORF5 plasmid protein for 12 h after pretreating with HO-1 si RNA for 6h;the fourth group: He La cells were stimulated with 9?g/ml pORF5 plasmid protein for 12 h after pretreating with control si RNA for 6h.He La cells were collected to assess the expression of HO-1,Bax and Bcl-2 by Western blotting analysis.Results:1.GST-pORF5 fusion protein with a molecular weight about 54 k Da was expressed in p GEX-6P/pORF5 transformed bacteria,then the GST tag from GST-pORF5 fusion protein was cleaved by Pre Scission protease to get pORF5 plasmid protein with a molecular weight about 28 k Da,the concentration of pORF5 protein was1.263mg/ml.2.pORF5 plasmid protein induced HO-1 protein expression in He La cells in a dose and time-dependent manner within a concentration range from 3?g/ml to 12?g/ml of pORF5 plasmid protein,when the concentration of pORF5 plasmid protein was more than 9?g/ml,the expression of HO-1 protein reduced significantly;The levels of HO-1 protein reached its peak at 12 h and reduced at 16 h.3.Western blotting showed that pORF5 plasmid protein could induce the phosphorylation of PI3K/AKT,after treatment with PI3K/AKT inhibitor LY294002 on He La cells,the expression of HO-1 protein was decreased by 83% compared with the group stimulated by pORF5 plasmid protein(P < 0.01).Indirect immunofluorescence revealed that Nrf2 translocated to the nucleus of He La cells in the presence of pORF5 obviously,but this nuclear translocation of Nrf2 was blocked by LY294002 inhibitor,the degree of Nrf2 nuclear translocation was almost not changed in LY294002 and pORF5 treatment group.4.After treatment with different factors,total protein in He La cells was collected and extracted.Western blotting showed that HO-1 si RNA effectively inhibited HO-1 protein expression,compared with the control group,the expression of HO-1 decreased by 84%(P<0.01).the expression of Bax decreased by 43%(P<0.05)and the expression of Bcl-2 increased by 112%(P<0.05)in the group teated with TNF? and pORF5 plasmid protein,in contrast to the group teated with TNF? only.The ratio of Bax/Bcl-2 was 1.26 in TNF? treatment group,and the ratio of Bax/Bcl-2 was 0.34 in the group teated TNF? and pORF5 plasmid protein,which was 73% lower than that in TNF alpha treatment group(P < 0.01).5.Bax expression increased by 31%,and Bcl-2 expression decreased by 88%,after inhibiting HO-1 protein expression with HO-1 si RNA.The ratio of Bax/Bcl-2 was 3.6451 in HO-1 si RNA treatment group,which was 10.6 times more than that of the control group(P < 0.01).There was almost not changed in the group treated with control si RNA.Conclusion:Chlamydia trachomatis pORF5 plasmid protein inhibited cell apoptosis via induction of Nrf2 nuclear translocation and HO-1 expression through activation of PI3K/AKT pathway... |
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