目的 探讨鹦鹉热衣原体SINC蛋白对宿主细胞凋亡的影响，及丝裂原活化蛋白激酶/细胞外信号调节激酶(mitogen-activated protein kinase/extracellular signal-regulated kinase，MAPK/ERK)信号通路在其中的调控作用。 方法 用重组SINC蛋白刺激HeLa细胞，Western blot检测凋亡相关蛋白Bax和Bcl-2的蛋白质表达水平及ERK1/2的磷酸化程度，Hoechst 33258染色检测SINC蛋白刺激对HeLa细胞凋亡的影响。用50 μmol/L MEK1/2抑制剂U0126预处理HeLa细胞，流式细胞术检测不同浓度SINC蛋白刺激后细胞凋亡率的变化，Western blot检测Bax和Bcl-2的蛋白质表达水平。 结果 Western blot检测结果显示，用10 μg/ml SINC蛋白刺激HeLa细胞18 h后，Bax表达下调，Bcl-2表达上调，Bax/Bcl-2比值最低；p-ERK1/2与ERK1/2的比值明显上调；Hoechst 33258染色检测发现5、10、15 μg/ml SINC蛋白刺激后，HeLa细胞内凋亡小体数量明显减少。加入抑制剂U0126后，Bcl-2表达下调，cleaved PARP表达量显著增加；流式细胞术显示细胞凋亡率明显增高。 结论 SINC蛋白通过激活MAPK/ERK信号通路抑制HeLa细胞发生凋亡。 Objective To investigate the effects of SINC, a novel secreted protein of Chlamydia psittaci, on the apoptosis of host cells and the regulatory role of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway in it. Methods HeLa cells were treated with recombinant SINC. The expression of Bax and Bcl-2 at protein level and the phosphorylation of ERK1/2 were analyzed by Western blot. Hoechst 33258 staining was used to detect the apoptosis of HeLa cells after SINC stimulation. Moreover, HeLa cells were pretreated with MEK1/2 inhibitor U0126 (50 μmol/L), and then stimulated with different concentrations of SINC for different time. Flow cytometry was used to detect the changes in cell apoptosis rates and Western blot was performed to detect the expression of Bax and Bcl-2 at protein level. Results Treating HeLa cells with 10 μg/ml of SINC for 18 h resulted in down-regulated Bax and up-regulated Bcl-2 at protein level. Besides, the ratio of Bax/Bcl-2 was the lowest and a significant increase in the ratio of phosphorylated ERK1/2 (p-ERK1/2) to ERK1/2 was observed. Hoechst 33258 staining showed that the number of apoptotic bodies decreased significantly after stimulating HeLa cells with 5, 10 and 15 μg/ml of SINC. In the presence of MEK1/2 inhibitor U0126, the expression of Bcl-2 at protein level was down-regulated, while the expression of cleaved PARP was significantly up-regulated. Flow cytometry showed a significantly enhanced apoptosis of HeLa cells. Conclusions SINC can inhibit the apoptosis of HeLa cells through activating the MAPK/ERK signaling pathway.
Chlamydia psittaci SINC protein inhibits host cell apoptosis through activating MAPK/ERK signaling pathway
Objective To investigate the effects of SINC, a novel secreted protein of Chlamydia psittaci, on the apoptosis of host cells and the regulatory role of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway in it. Methods HeLa cells were treated with recombinant SINC. The expression of Bax and Bcl-2 at protein level and the phosphorylation of ERK1/2 were analyzed by Western blot. Hoechst 33258 staining was used to detect the apoptosis of HeLa cells after SINC stimulation. Moreover, HeLa cells were pretreated with MEK1/2 inhibitor U0126 (50 μmol/L), and then stimulated with different concentrations of SINC for different time. Flow cytometry was used to detect the changes in cell apoptosis rates and Western blot was performed to detect the expression of Bax and Bcl-2 at protein level. Results Treating HeLa cells with 10 μg/ml of SINC for 18 h resulted in down-regulated Bax and up-regulated Bcl-2 at protein level. Besides, the ratio of Bax/Bcl-2 was the lowest and a significant increase in the ratio of phosphorylated ERK1/2 (p-ERK1/2) to ERK1/2 was observed. Hoechst 33258 staining showed that the number of apoptotic bodies decreased significantly after stimulating HeLa cells with 5, 10 and 15 μg/ml of SINC. In the presence of MEK1/2 inhibitor U0126, the expression of Bcl-2 at protein level was down-regulated, while the expression of cleaved PARP was significantly up-regulated. Flow cytometry showed a significantly enhanced apoptosis of HeLa cells. Conclusions SINC can inhibit the apoptosis of HeLa cells through activating the MAPK/ERK signaling pathway.
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