CPSIT₀846 Protein Regulates Mitochondrial-mediated Apoptpsis In Chlamydia Psittaci Infected HeLa Cells Via ERKJNK Signaling Pathway

Objective: To elucidate the role of CPSIT₀846,a kind of inclusion membrane protein of Chlamydia psittaci（C.psittaci）on host cell apoptosis,and explain the function of CPSIT₀846 in the pathogenesis of C.psittaci.Methods: The recombinant CPSIT₀846 protein was expressed from pET-30a（+）-CPSIT₀846 recombinant strain induced by IPTG.After purification and concentration,the endotoxin was removed with a polymyxin B cartridge.The HeLa cells were treated with CPSIT₀846 at the concentrations of 0,2,5,8 and 10μg/mL for 24 h respectively,then the expression levels of Bcl-2 and Bax were detected by Western blot.After treated with the optimal concentration of CPSIT₀846 for 0,6,12,18 and 24 h,the expression levels of Bcl-2,Bax,p53,the activation levels of ERK1/2,SAPK/JNK,and p38 signaling pathways were examined by Western blot.After 30μM ERK1/2 inhibitor U0126 or SAPK/JNK inhibitor SP600125 treatment,HeLa cells were treated withCPSIT₀846,the expression levels of ERK1/2,p-ERK1/2,SAPK/JNK,p-SAPK/JNK,p38 and p-p38 were detected by Western blot.The treatment with STS inducer was continued,and the activation levels of Bcl-2,Bax,p53 and Caspase-3,Caspase-9 and PARP were detected.Hoechst 33258 staining and flow cytometry were used to analyze the changes of apoptosis in cells with or without U0126 or SP600125 inhibitor.After the carbonyl cyanide 3-chlorophenylhydrazone（CCCP）inducer treatment,JC-1 staining and indirect immunofluorescence technology were used to detect the changes in mitochondrial membrane potential and cytochrome c release in each group of cells with or without U0126 or SP600125 inhibitor.Results: Purified CPSIT₀846 recombinant protein was successfully obtained with the molecular weight about 24 kDa.The result of CCK-8assay showed that the recombinant CPSIT₀846 protein has no obvious toxic effect on HeLa cells.Hoechst 33258 staining results showed that,the HeLa cells with CPSIT₀846 treatment had fewer apoptotic bodies and lower apoptotic rate than that of the control group.The flow cytometry analysis results also found that the apoptotic rate（2.95%）of HeLa cells in the CPSIT₀846 treated group was significantly lower than that in the control group（5.19%）,but the apoptosis rate of the heated CPSIT₀846 protein treated group（5.62%）was similar to that of the control.After inhibiting the ERK1/2 or SAPK/JNK pathway,the resultsof Hoechst 33258 staining or flow cytometry showed that the number of apoptotic bodies and the apoptotic rate of HeLa cells in CPSIT₀846treated group were increased whether or not with STS treatment.Moreover,Western blot results showed that the expression level of Bcl-2was up-regulated while Bax was down-regulated in which cells were treated with CPSIT₀846;the phosphorylation levels of ERK1/2,SAPK/JNK and AKT were significantly increased,and this phenomenon can persist until 18 h later,but the phosphorylation level of p38 was not significantly changed.When the cells were treated with 8μg/mL CPSIT₀846 for 18 h,the expression level of Bcl-2 reached to its peak,and the expression levels of Bax and p53 were significantly down-regulated.However,after the ERK1/2 or SAPK/JNK pathway was inhibited,the ratio of Bax/Bcl-2,the expression levels of p53,Cleaved caspase-3,Cleaved caspase-9,and Cleaved PARP in the CPSIT₀846treated cells were significantly up-regulated,whether or not they were treated with STS.The results of JC-1 staining and indirect immunofluorescence detection showed that after CCCP treatment,the mitochondrial membrane potential of the cells in control group were significantly decreased,while that in the CPSIT₀846 treatment group were stabilized,and with less cytochrome c released into the cytoplasm.But after inhibiting the ERK1/2 or SAPK/JNK pathway,CPSIT₀846could not protect these cells,showed the ratio of JC-1 red-greenfluorescence signal were decreased,the MMP were downward and Cyt.c was discharged into the cytoplasm significantly.Conclusion: CPSIT₀846 proteins can up-regulate Bcl-2,down-regulate the expression levels of Bax,p53 and other pro-apoptotic proteins via ERK1/2 and SAPK/JNK signaling pathways to inhibit mitochondrial-mediated apoptosis of host cells.