Abstract: Chen Wentao, Xue Yaohua, Huang Jinmei, Yang Jieyi, Zhao Yunhu, Lan Yinyuan, Fang Mingheng, Zheng Biying, Zheng Heping Clinical Laboratory, Dermatology Hospital of Southern Medical University, Guangdong Provincial Dermatology Hospital, Guangzhou 510000, China（Chen WT, Xue YH, Huang JM, Zhao YH, Lan YY, Fang MH, Zheng HP）; Graduate College, Guangdong Medical University, Dongguan 523808, Guangdong, China（Yang JY, Zheng BY） Corresponding authors: Zheng Heping, Email: zhhpf@hotmail.com; Zheng Biying, Email: 469069691@qq.com 【Abstract】 Objective To evaluate the regulatory role of azithromycin-induced persistent Chlamydia trachomatis （Ct） infection in the apoptosis of Hela229 cells. Methods Hela229 cells were firstly co-cultured with Ct for 22 hours, and then cultured with Dulbecco′s modified Eagle′s medium （DMEM） containing 0.08 mg/L azithromycin for 26 hours to establish a cell model of persistent Ct infection （persistent infection group）. These infected Hela229 cells cultured with azithromycin-free DMEM served as a cell model of acute Ct infection （acute infection group）. After 48-hour infection with Ct, azithromycin was removed, and infected Hela229 cells in the above 2 groups were successively cultured with DMEM for the resurgence of Ct. Immunofluorescence assay and electron microscopy were performed to verify the persistent Ct infection model. The Hela229 cells in the persistent infection group and acute infection group as well as uninfected Hela229 cells （control group） were treated with staurosporine （STS） for 4 hours to induce the apoptosis, and then cell apoptosis was detected by Hoechst 33258 staining, annexin V/propidium iodide staining and flow cytometry. Results After the treatment with azithromycin, atypical inclusions with aberrant reticulate bodies appeared in the Ct-infected cells. After removing azithromycin, cells were cultured until 96 hours after infection, and infectious elementary bodies reappeared in the Ct inclusions. After the treatment with STS, Hoechst staining showed that there was loose chromatin in the persistently infected cells, while chromatin condensation was observed in the uninfected cells. After 24-hour infection with Ct and 4-hour induction with STS, the apoptosis rate was significantly higher in the persistent infection group （45.567% ± 2.631%） than in the acute infection group （38.567% ± 1.701%, t = 2.686, P = 0.028）, but significantly lower in the persistent infection group than in the uninfected group（69.800% ± 2.835%, t = 8.187, P < 0.001）. After 48-hour infection with Ct and 4-hour induction with STS, there was a significant difference in the apoptosis rate between the persistent infection group （46.700% ± 5.257%） and acute infection group （61.767% ± 1.815%, t = 5.781, P < 0.001）, as well as between the persistent infection group and the uninfected group （68.667% ± 3.156%, t = 7.421, P < 0.001）. Conclusion This study showed that azithromycin-induced persistent Ct infection regulated the apoptosis of host cells, and this effect lasted 48 hours.

Key words: Chlamydia trachomatis, Azithromycin, Treatment failure, Apoptosis, Persistent infection