ATF6/CHOP信号通路对减毒牛结核分枝杆菌感染的THP-1细胞焦亡的调控作用

ATF6/CHOP signaling pathway regulates pyroptosis of THP-1 cells infected with Bacillus Calmette-Guérin

目的:探讨转录激活因子6(ATF6)/CCAAT/增强子结合蛋白同源蛋白(CHOP)信号通路对牛分枝杆菌减毒株(卡介苗,BCG)感染的THP-1细胞焦亡的调控作用。方法:在感染复数为10∶1的BCG感染THP-1细胞2、6、12、24和48 h基础上,设置si-NC组、si-NC+BCG组、si-ATF6+BCG组及si-CHOP+BCG组。si-NC组转染干扰阴性对照24 h后正常培养;si-NC+BCG组先转染阴性对照24 h后以感染复数为10∶1的BCG感染24 h;si-ATF6+BCG组和si-CHOP+BCG组分别转染si-ATF6和si-CHOP 24 h后再用感染复数为10∶1的BCG感染24 h。采用Western blot检测cleaved ATF6、CHOP、核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、pro-caspase-1、caspase-1 p20、含caspase募集结构域的凋亡相关斑点样蛋白(ASC)、IL-1βp17、IL-18 p22及消皮素D-N端片段(GSDMD-N)蛋白表达;采用免疫荧光检测GSDMD蛋白表达;采用CCK-8法检测细胞活力。结果:Western blot结果显示,BCG感染THP-1细胞后,cleaved ATF6和CHOP蛋白的表达随感染时间延长逐渐升高,48 h达到最高,而GSDMD-N蛋白在24 h表达最高(P<0.01)。与si-NC组相比,si-NC+BCG组的cleaved ATF6、CHOP、NLRP3、pro-caspase-1、caspase-1 p20、ASC、IL-1βp17、IL-18 p22及GSDMD-N蛋白表达显著上调(P<0.01),细胞内GSDMD含量显著增加(P<0.01),细胞活力显著下降(P<0.01);与si-NC+BCG组相比,si-ATF6+BCG组上述蛋白表达显著下调(P<0.01),细胞内GSDMD含量显著下降(P<0.01),细胞活力显著上升(P<0.01);与si-NC+BCG组相比,si-CHOP+BCG组上述蛋白表达显著下调(P<0.01),细胞内GSDMD含量显著下降(P<0.01),细胞活力显著上升(P<0.01)。结论:ATF6/CHOP信号通路对BCG感染的THP-1细胞焦亡具有调控作用。

AIM:To investigate the role of activating transcription factor 6(ATF6)/CCAAT/enhancer-binding protein homologous protein(CHOP)signaling pathway in the regulation of pyroptosis in human monocyte/macrophage THP-1 cells infected with Bacillus Calmette-Guérin(BCG).METHODS:On the basis of BCG infection of THP-1 cells at multiplicity of infection(MOI)of 10∶1 for 2,6,12,24 and 48 h,THP-1 cells were divided into si-NC group,si-NC+BCG group,si-ATF6+BCG group and si-CHOP+BCG group.The cells in si-NC group were transfected with negative control for 24 h and cultured normally.In si-NC+BCG group,the cells were transfected with negative control for 24 h and then infected with BCG at MOI of 10∶1 for 24 h.The cells in si-ATF6+BCG and si-CHOP+BCG groups were transfected with siATF6 and si-CHOP for 24 h,respectively,and then infected with BCG at MOI of 10∶1 for 24 h.Western blot was used to detect the protein levels of cleaved ATF6,CHOP,nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),pro-caspase-1,caspase p20,apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC),IL-1 p17,IL-18 p22 and N-terminal fragment of gasdermin D(GSDMD-N).The GSDMD protein level was determined by using immunofluorescence,and cell viability was determined by CCK-8 assay.RESULTS:After BCG infection,cleaved ATF6 and CHOP were increased along with the infection time,reaching the greatest level at 48 h,while GSDMD-N peaked at 24 h(P<0.01).Compared with si-NC group,the protein levels of cleaved ATF6,CHOP,NLRP3,pro-caspase-1,caspase-1 p20,ASC,IL-1βp17,IL-18 p22,GSDMD-N and GSDMD in si-NC+BCG group were significantly upregulated(P<0.01),while the cell viability was significantly decreased(P<0.01).Compared with si-NC+BCG group,the above protein levels in si-ATF6+BCG group were significantly down-regulated(P<0.01),and the cell viability was significantly increased(P<0.01).Compared with si-NC+BCG group,the above proteins in si-CHOP+BCG group were significantly decreased(P<0.01),and the cell viability was significantly increased(P<0.01).CONCLUSION:The ATF6/CHOP signaling pathway regulates the pyroptosis of THP-1 cells infected with BCG.