中华皮肤科杂志 ›› 2019, Vol. 52 ›› Issue (8): 548-553.doi: 10.3760/cma.j.issn.0412-4030.2019.08.007

• 论著 • 上一篇    下一篇

沙眼衣原体pORF5质粒蛋白通过HMGB1抑制细胞凋亡机制初步研究

雷文波    何蓓    聂倩    文雅婷    赵钰琦    李忠玉   

  1. 南华大学衡阳医学院病原生物学研究所  特殊病原体防控湖南省重点实验室,湖南衡阳  421001
  • 收稿日期:2018-10-29 修回日期:2019-05-30 出版日期:2019-08-15 发布日期:2019-07-30
  • 通讯作者: 李忠玉 E-mail:lzhy1023@hotmail.com
  • 基金资助:
    国家自然科学基金(81772210、31470277);国家级大学生创新创业训练计划项目(201710555011);湖南省大学生研究性学习和创新性实验计划项目(湘教通[2017]205号)

Preliminary study on the anti-apoptotic mechanism of pORF5 plasmid protein of Chlamydia trachomatis through HMGB1 protein

Lei Wenbo, He Bei, Nie Qian, Wen Yating, Zhao Yuqi, Li Zhongyu   

  1. Institute of Pathogenic Biology, Hengyang Medical College, Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control, University of South China, Hengyang 421001, Hunan, China
  • Received:2018-10-29 Revised:2019-05-30 Online:2019-08-15 Published:2019-07-30
  • Contact: Li Zhongyu E-mail:lzhy1023@hotmail.com
  • Supported by:
    National Natural Science Foundation of China (81772210, 31470277); National College Students′ Innovation Entrepreneurship Training Program (201710555011); Hunan Provincial College Students Research Study and Innovative Experiment Program (Grant No. [2017]205)

摘要: 【摘要】 目的 探讨pORF5质粒蛋白抗凋亡分子机制,为进一步阐明沙眼衣原体致病机制提供实验依据。方法 将HeLa细胞分为两组,一组用凋亡诱导剂碳酰氰基-对-氯苯腙(CCCP)刺激30 min,另一组经pORF5质粒蛋白预处理18 h后,再用CCCP刺激30 min。然后,Western印迹检测细胞凋亡相关蛋白Bcl-2、Bax和胱天蛋白酶3(caspase-3)的表达水平;JC-1荧光探针检测HeLa细胞线粒体膜电位变化;间接免疫荧光观察细胞色素c释放情况。为了分析高迁移率族蛋白1(HMGB1)是否参与pORF5质粒蛋白的抗凋亡作用,分别用HMGB1 shRNA和对照RNA稳定转染HeLa细胞,再用pORF5质粒蛋白与CCCP共刺激两种细胞后,测定Bcl-2、Bax和活化的caspase-3蛋白的表达以及细胞色素c的释放情况。两组间比较采用配对样本t检验。结果 pORF5质粒蛋白能拮抗CCCP 诱导的线粒体膜电位的下降,CCCP单独处理组Hela细胞红/绿荧光强度比率为0.4 ± 0.1,显著低于pORF5与CCCP共处理组(1.7 ± 0.3,t = 6.95,P < 0.01)。与对照组相比,pORF5与CCCP共处理组HeLa细胞Bcl-2蛋白表达水平增加(5.3 ± 0.6)倍(t = 8.62,P < 0.01),而Bax和活化的caspase-3平均表达水平分别降低79% ± 10%(t = 9.23,P < 0.01)和75% ± 8%(t = 4.26,P < 0.05)。与对照RNA转染组相比,HMGB1 shRNA转染组HeLa细胞线粒体膜电位下降(t = 11.23,P < 0.01),细胞色素c释放增加,Bcl-2的表达水平降低(t = 7.19,P < 0.05),而Bax的表达水平增加(t = 13.06,P < 0.01)。结论 沙眼衣原体pORF5质粒蛋白通过HMGB1阻断线粒体凋亡途径发挥抗凋亡作用。

关键词: 沙眼衣原体, 细胞凋亡, 原癌基因蛋白质c?bcl?2, Bcl?2相关X蛋白质, 半胱氨酸天冬氨酸蛋白酶3, 高迁移率族蛋白质1, pORF5质粒蛋白

Abstract: 【Abstract】 Objective To explore the molecular mechanism underlying the anti-apoptotic activity of pORF5 plasmid protein of Chlamydia trachomatis, so as to provide an experimental basis for further clarifying the pathogenesis of Chlamydia trachomatis. Methods HeLa cells were divided into two groups: carbonyl cyanide m-chlorophenyl hydrazone (CCCP, an apoptosis inducer) group was stimulated by CCCP for 30 minutes, and pORF5 + CCCP group was pretreated with pORF5 plasmid protein for 18 hours followed by CCCP for 30 minutes. Then, Western blot analysis was performed to determine the expression of apoptosis-related proteins Bcl-2, Bax and caspase-3, JC-1 fluorescent probe was used to detect changes in the mitochondrial membrane potential in HeLa cells, and cytochrome c release from mitochondria was analyzed by indirect immunofluorescence assay. To analyze whether high-mobility group box 1 (HMGB1) protein participated in the anti-apoptotic role of pORF5 plasmid protein, HMGB1 shRNA and control RNA were separately transfected into the HeLa cells, which were then stimulated by pORF5 plasmid protein and CCCP. Then, the protein expression of Bcl-2, Bax, activated caspase-3 was determined, and cytochrome c release was analyzed. Data were compared between two groups by using paired t test. Results pORF5 plasmid protein could antagonize the CCCP-induced decrease of mitochondrial membrane potential, and the red/green fluorescence intensity ratio was significantly lower in the CCCP group (0.4 ± 0.1) than in the pORF5 + CCCP group (1.7 ± 0.3; t = 6.95, P < 0.01). The protein expression of Bcl-2 in the HeLa cells in the pORF5 + CCCP group was 5.3 ± 0.6 times more than that in the CCCP group (t = 8.62, P < 0.01), while the protein expression of Bax and activated caspase-3 in the pORF5 + CCCP group significantly decreased by 79% ± 10% (t = 9.23, P < 0.01) and 75% ± 8% (t = 4.26, P < 0.05) respectively compared with the CCCP group. Compared with the control RNA transfection group, the HMGB1 shRNA transfection group showed significantly decreased mitochondrial membrane potential in the HeLa cells (t = 11.23, P < 0.01), increased cytochrome c release, decreased Bcl-2 expresson (t = 7.19, P < 0.05) and increased Bax expression (t = 13.06, P < 0.01) after stimulation with pORF5 and CCCP. Conclusion Chlamydia trachomatis plasmid protein pORF5 plays an anti-apoptosis role by blocking the mitochondrial apoptotic pathway through HMGB1 protein.

Key words: Chlamydia trachomatis, Apoptosis, Proto?oncogene proteins c?bcl?2, Bcl?2?associated X protein, Caspase?3, High mobility group box protein 1, pORF5 plasmid protein