BCG感染巨噬细胞后内质网应激对细胞焦亡的调控作用

Role of Endoplasmic Reticulum Stress on Pyroptosis of BCG Infected Macrophages

旨在探讨牛分枝杆菌疫苗株卡介苗(Bacillus Calmette-Guérin,BCG)感染人单核巨噬细胞THP-1细胞后内质网应激(endoplasmic reticulum stress,ERS)对细胞焦亡的调控作用及分子机制。在BCG单独感染THP-1细胞或与ERS抑制剂TUDCA共处理细胞后,采用qRT-PCR检测ERS标志性分子GRP78,细胞焦亡标志性分子GSDMD、Caspase1、NLRP3、IL^(-1)β和IL^(-1)8在mRNA水平的表达,采用Western blot检测GRP78、Caspase12、GSDMD、Caspase1和NLRP3在蛋白水平的表达,采用ELISA检测IL^(-1)β和IL^(-1)8的释放量,采用CCK-8检测细胞存活率。结果表明:在BCG单独感染THP-1细胞不同时间后,GRP78在mRNA水平和蛋白水平的表达均随感染时间延长而升高(P<0.01),GSDMD蛋白表达在24 h达到最高,IL^(-1)β和IL^(-1)8在mRNA水平的表达呈时间依赖性(P<0.01)。在BCG单独感染或与TUDCA共同作用细胞24 h后,与未感染对照组相比,BCG感染组GRP78、GSDMD、Caspase1、NLRP3、IL^(-1)β和IL^(-1)8 mRNA水平表达上调(P<0.05),GRP78、Caspase12、GSDMD、Caspase1和NLRP3蛋白水平表达上调(P<0.001),IL^(-1)β和IL^(-1)8的浓度增加(P<0.01),细胞存活率下降(P<0.001),而经TUDCA预处理的BCG感染组与BCG单独感染组相比,以上分子的表达下降,细胞存活率上升(P<0.01)。综上表明,在BCG感染THP-1细胞后,引起的ERS对细胞焦亡具有调控作用。

The purpose of this research is to provide the regulation and mechanism of endoplasmic reticulum stress(ERS)on pyroptosis of THP-1 cells infected by BCG.THP-1 cells were infected by BCG with or without TUDCA treatment.Then,the mRNA expression of GRP78,GSDMD,Caspase1,NLRP3,IL^(-1)β,IL^(-1)8,the protein expression of GRP78,Caspase12,GSDMD,Caspase1,NLRP3,the release of IL^(-1)β,IL^(-1)8 and the cell survival rate were detected by qRT-PCR,Western blot,ELISA and CCK-8 respectively.The results showed that both the mRNA and the protein expressions of GRP78 were gradually increased with time of BCG infection in THP-1 cells(P<0.01).The protein expression of GSDMD reached the highest level at 24 h post infection.The concentration of IL^(-1)βand IL^(-1)8 was increased in a time-dependent manner(P<0.01).Compared with the uninfected group,the mRNA expression of GRP78,GSDMD,Caspase1,NLRP3,IL^(-1)βand IL^(-1)8(P<0.05),and the protein expression of GRP78,Caspase12,GSDMD,Caspase1 and NLRP3(P<0.001)were both up-regulated,and the concentration of IL^(-1)βand IL^(-1)8 was increased(P<0.01),and cells survival rate was reduced at 24 h post infection with BCG(P<0.001).However,with the co-treatment of TUDCA and BCG,the expression of the above molecules decreased and the cell survival rate increased compared with BCG infected group(P<0.01).Our results demonstrate that ERS in THP-1 cells induced by BCG infection regulated pyroptosis.