PERK/ATF4/CHOP通路对BCG诱导THP-1细胞NLRP3炎性小体活化的调控作用

Regulation of PERK/ATF4/CHOP Pathway on NLRP3 Inflammasome Activation Induced by BCG in THP-1 Cells

旨在探讨牛分枝杆菌减毒株卡介苗(Bacillus Calmette-Guérin,BCG)感染人单核巨噬细胞THP-1细胞后PERK/ATF4/CHOP通路对NLRP3炎性小体的调控作用。在BCG单独感染或与PERK小干扰RNA共处理THP-1细胞后,采用qRT-PCR和Western blot方法检测NLRP3炎性小体相关分子和PERK/ATF4/CHOP通路标志性分子在mRNA、蛋白水平的表达;在BCG单独感染或与PERK抑制剂GSK2656157共处理THP-1细胞后,分别采用qRT-PCR和Western blot方法检测NLRP3炎性小体相关分子和PERK/ATF4/CHOP通路标志性分子在mRNA、蛋白水平的表达,采用ELISA方法检测白细胞介素-1β(interleukin-1β,IL-1β)和白细胞介素-18(interleukin-18,IL-18)的释放量,采用CCK-8方法检测THP-1细胞活率,采用免疫荧光检测NLRP3与ASC的共定位。结果表明:在BCG单独感染THP-1细胞不同时间后,PERK、NLRP3、ASC和Caspase-1在蛋白水平的表达均随感染时间延长而升高,且在24 h达到最高(P<0.001),IL-1β和IL-18的释放随时间递增,24 h达到最高(P<0.001)。在BCG单独感染或与PERK小干扰RNA共处理THP-1细胞24 h后,与未感染对照组相比,siNC+BCG感染组NLRP3、ASC、Caspase-1、PERK、ATF4、CHOP分子的mRNA和蛋白表达均显著(P<0.05)或极显著(P<0.001)上调,而siPERK+BCG感染组与siNC+BCG感染组相比,NLRP3等关键分子的mRNA和蛋白表达均显著(P<0.05)或极显著下调(P<0.01,P<0.001);在BCG单独感染或与GSK2656157共同作用THP-1细胞24 h后,与未感染对照组相比,BCG感染组NLRP3、ASC、Caspase-1、PERK、ATF4、CHOP分子的mRNA和蛋白表达均显著(P<0.05)或极显著(P<0.01)上调,IL-1β和IL-18的释放极显著增加(P<0.001),细胞活率极显著下调(P<0.001),而BCG+GSK2656157感染组与BCG单独感染组相比,上述分子的mRNA和蛋白表达均显著(P<0.05)或极显著下调(P<0.01),IL-1β和IL-18的释放显著(P<0.05)或极显著(P<0.01)减少,细胞活率显著上调(P<0.05),免疫荧光的结果显示NLRP3与ASC存在共定位,且GSK2656157可以极显著抑制BCG感染引起的NLRP3和ASC的表达上调(P<0.001)。以上研究结果表明,PERK/ATF4/CHOP通路对BCG感染巨噬细胞后NLRP3炎性小体的活化具有调控作用。

Our study aimed at investigating the regulatory role of the PERK/ATF4/CHOP pathway on NLRP3 inflammasome of human monocyte macrophage THP-1 cells infected with Bacillus Calmette-Guérin(BCG).THP-1 macrophages were infected with BCG alone or in the presence of small interference to PERK for 24 h,then the expressions of NLRP3 inflammasome related molecules and PERK/ATF4/CHOP pathway molecules in THP-1 cells were detected at mRNA level by qRT-PCR and protein level by Western blot;THP-1 macrophages were infected with BCG alone or in the presence of specific inhibitors to PERK for 24 h,then the expression of NLRP3 inflammasome related molecules and PERK/ATF4/CHOP pathway molecules in THP-1 cells was detected at mRNA level by qRT-PCR and protein level by Western blot,and ELISA was used to detect the release of interleukin-1β(IL-1β)and interleukin-18(IL-18),the viability of THP-1 cells was detected by CCK-8 method,the co-localization of NLRP3 and ASC was detected by immunofluorescence.The results showed that the expression of PERK,NLRP3,ASC and Caspase-1 proteins increased with the infection time,and reached the peak at 24 h(P<0.001),and the release of IL-1βand IL-18 very significantly increased with time(P<0.001).THP-1 macrophages were infected with BCG alone or in the presence of small interference to PERK for 24 h,compared with uninfected control group,the expression of NLRP3,ASC,Caspase-1,PERK,ATF4,CHOP in siNC+BCG infected group were significantly(P<0.05)or extremely significantly up-regulated(P<0.001)both at mRNA level and protein level,while compared with siNC+BCG infected group,the expression of NLRP3,ASC,Caspase-1,PERK,ATF4,CHOP in siPERK+BCG infected group were significantly(P<0.05)or extremely significantly(P<0.01,P<0.001)down-regulated both at mRNA level and protein level;THP-1 macrophages were infected with BCG alone or in the presence of inhibitor to PERK for 24 h,compared with uninfected control group,the expression of NLRP3,ASC,Caspase-1,PERK,ATF4,CHOP in BCG infected group were significantly(P<0.05)or extremely significantly up-regulated(P<0.01)both at mRNA level and protein level,and the release of IL-1βand IL-18 very significantly increased(P<0.001).Compared with BCG infection group,the expression of those molecules were significantly(P<0.05)or extremely significantly(P<0.01)down-regulated both at mRNA level and protein level,and the release of IL-1βand IL-18 significantly(P<0.05)or extremely significantly(P<0.001)decreased,the viability of THP-1 cells was significantly up-regulated(P<0.05).The results of immunofluorescence also showed that NLRP3 and ASC proteins were co-located,and GSK2656157 could significantly inhibit the expressions of NLRP3 and ASC proteins induced by BCG infection(P<0.001).The above results show that PERK/ATF4/CHOP pathway regulates the activation of NLRP3 inflammasome in macrophages infected by BCG.