| Background and objective: Chlamydia trachomatis(C.trachomatis)is a kind of obligate intracellular parasitic bacteria with biphase development cycle.C.trachomatis infection can cause trachoma and is one of the potential pathogenic factors of female reproductive tract infection.To maintain its own survival,chlamydia exploit a variety of immune escape mechanisms to escape the host immune response.A large number of studies have shown that C.trachomatis infection can inhibit host cell apoptosis,but the specific mechanism remains unclear.Our previous study found that CT036 protein can inhibit apoptosis in HeLa cells by stimulating them in vitro,but the specific mechanism remains to be further clarified.Studies have reported that chlamydia infection can activate the MAPK signaling pathway and mediate the anti-apoptotic effect of host cells.Therefore,in this study,CT036 protein in vitro stimulation of HeLa cells or CT036 transfection into HeLa cells for endogenous expression as a cell model,focusing on MAPK signal,to explore the role of CT036 protein in the regulation of host cell apoptosis.Our study not only enriched the pathogenic mechanism of C.trachomatis,but also provided new information for understanding CT036 protein by preliminary exploration on anti-apoptosis of CT036 protein.Methods:1.The recombinant plasmid p ET28a-CT036 previously constructed by our research group was inoculated in LB solid medium for screening and culture.After expanded culture,the recombinant protein His-CT036 was induced and expressed by 0.5 m M IPTG.The target protein was purified by nickel column,and the refolded CT036 recombinant protein was collected and endotoxin removed.2.After stimulated with 0 μg/mL,5 μg/mL,10 μg/mL,15 μg/mL,18μg/mL,20 μg/mL of CT036 plasmid protein for 0 h,6 h,8 h,12 h,and 24 h,the protein expression levels of Bcl-2 and Bax in HeLa cells were detected by Western blot.Hoechst 33258 fluorescence staining and flow cytometry were used to detect cell apoptosis.3.After stimulating HeLa cells with 18 μg/mL CT036 recombinant protein for 0 min,5 min,15 min,30 min,60 mim,total cell proteins were collected to detect the phosphorylation levels of ERK1/2,p38 and JNK in the stimulated HeLa cells by Western blot.After HeLa cells were pretreated with 20 μM ERK1/2 inhibitor PD98059 or 1 μM p38 inhibitor SB202190 for 1 h,apoptosis inducers TNF-α(50 ng/mL)and CHX(2 ng/mL)were added for 5 h.The phosphorylation levels of ERK1/2 and p38 and the expression of apoptosis-related protein Bcl-2 Bax were detected by Western blot.Hoechst 33258 immunofluorescence staining and flow cytometry were used to detect apoptosis in treated and untreated cells.4.Transfection of CT036 into HeLa cells,inhibited host cell apoptosis by activating MAPK(ERK1/2 and p38)signaling pathways.The cells were pretreated with or without 20 μM ERK1/2 inhibitor PD98059 or1 μM p38 inhibitor SB202190 for 1 h,then treated with 50 ng/mL TNF-α and 2 μg/mL CHX for 5 h,and Flag-HeLa was used as control.Cells were collected according to different treatment factors,and the total cell proteins were extracted.The phosphorylation levels of ERK1/2,p38 and JNK,as well as the corresponding protein expression levels of Bax and Bcl-2 were detected by Western blot.Hoechst 33258 immunofluorescence staining and flow cytometry were used to detect apoptosis in treated and untreated cells.Results:1.The recombinant plasmid p ET28a-CT036 was induced by IPTG to express the recombinant protein His-CT036,with a molecular weight of about 35 KDa.The purified CT036 recombinant protein was obtained by nickel column purification and renaturation.2.The protein expression levels of Bcl-2 and Bax changed with the increase of the concentration of CT036 recombinant protein.When HeLa was stimulated by CT036 recombinant protein at 18 μg/mL,the Bcl-2/Bax ratio increased significantly.Therefore,when the concentration of CT036 recombinant protein was 18 μg/mL to stimulate HeLa cells at different time points,it was found that the expression level of Bcl-2 was significantly increased when the concentration of CT036 recombinant protein was stimulated for 24 h,the expression level of Bax was significantly decreased,and the ratio of Bcl-2 and Bax was increased.Hoechst staining showed that the apoptosis rate of CT036/TNF-α/CHX group was significantly reduced compared with TNF-α/CHX group.The results of flow cytometry were consistent with Hoechst immunofluorescence results.3.Western blot showed that the phosphorylation levels of ERK1/2 and p38 were up-regulated and differentially changed.Both ERK1/2inhibitor PD98059 and p38 inhibitor SB202190 could effectively inhibit the phosphorylation expression of ERK1/2.Compared with control group without inhibitor,PD98059/CT036 group or SB202190/CT036 group could up-regulate the expression of Bax and down-regulate the expression of Bcl-2.Hoechst staining results showed that compared with DMSO/CT036 control group,PD98059/CT036 group or SB202190/CT036 group,the cell apoptosis rate was increased,and the inhibitor group significantly increased the number of apoptotic bodies,nuclear fragmentation and nuclear pyysis were more obvious.The results of flow cytometry showed that the apoptosis rate of PD98059/CT036 group or SB202190/CT036 group was increased compared with DMSO/CT036 control group.4.The level of ERK1/2 or p38 phosphorylation of CT036-HeLa induced by TNF-α/CHX was significantly higher than that of Flag-HeLa control group(P<0.01),and the level of JNK phosphorylation was not statistically significant,which was consistent with the results of exogenous CT036 stimulation of HeLa cells.In addition,ERK1/2inhibitor PD98059 or p38 inhibitor SB202190 can effectively inhibit the phosphorylation of ERK1/2 or p38 in CT036-Hela.After TNF-α/CHX-induced apoptosis,Bcl-2 protein expression in CT036-HeLa cells was up-regulated,while Bax protein expression was down-regulated,compared with Flag-HeLa cell control group(P<0.01).However,after pretreatment with PD98059 or SB202190 inhibitors,it was found that in CT036-HeLa cells,the expression level of Bcl-2 protein was significantly decreased,while the expression level of Bax protein was up-regulated,and the Bcl-2/Bax ratio was significantly decreased.5.Hoechst staining showed that CT036-HeLa cells induced by TNF-α/CHX had significantly less apoptotic bodies than Flag-HeLa cells,and the apoptotic rate was significantly lower.However,after pretreatment with PD98059 or SB202190 inhibitors,CT036-HeLa was found to have significantly increased nuclear fragmentation and nuclear pyopysis,and significantly increased apoptosis rate.Flow cytometry detection results were consistent with Hoechst immunofluorescence results.Conclusions: C.trachomatis inclusion membrane protein CT036 inhibits host cell apoptosis by activating ERK1/2 and p38 MAPK signaling pathways... |
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