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

doi:10.11843/j.issn.0366-6964.2022.07.023

摘要/Abstract

摘要： 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。

关键词: 卡介苗, PERK/ATF4/CHOP通路, NLRP3炎性小体, THP-1细胞

Abstract: 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-1β and 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-1β and 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-1β and 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.

Key words: BCG, PERK/ATF4/CHOP pathway, NLRP3 inflammasome, THP-1 cell

中图分类号:

Q939.91

马伯利, 聂雪伊, 刘悦阳, 苗申奥, 陈通, 杨易, 徐金瑞. 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[J]. 畜牧兽医学报, 2022, 53(7): 2268-2281.

MA Boli, NIE Xueyi, LIU Yueyang, MIAO Shenao, CHEN Tong, YANG Yi, XU Jinrui. Regulation of PERK/ATF4/CHOP Pathway on NLRP3 Inflammasome Activation Induced by BCG in THP-1 Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7): 2268-2281.

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