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中华肺部疾病杂志(电子版)

中华肺部疾病杂志(电子版) ›› 2025, Vol. 18 ›› Issue (04) : 540 -545. doi: 10.3877/cma.j.issn.1674-6902.2025.04.008

论著

衣康酸通过Nrf2抑制NLRP3激活拮抗卡介苗诱导RAW264.7细胞损伤机制研究
弓慧1, 祖力皮喀尔·阿卜杜热合曼1, 徐敬然1, 李黎1,2,†()   
  1. 1844000　喀什，喀什地区第一人民医院　新疆感染性疾病（结核病）临床医学研究中心
    2844000　喀什，喀什地区第一人民医院呼吸与危重症医学科
  • 收稿日期:2025-03-20 出版日期:2025-08-25
  • 通信作者: 李黎
  • 基金资助:
    新疆维吾尔自治区自然科学基金(2022D01C10); 新疆维吾尔自治区"天山英才"培养计划(2023TSYCJC0051)

Itaconate activates Nrf2 to inhibit NLRP3 inflammasome activation and alleviate BCG-induced inflammatory damage in RAW264.7 Cells

Hui Gong1, Abudureheman Zulipikaer1, Jingran Xu1, Li Li1,2,†()   

  1. 1The First People′s Hospital of Kashi, Clinical Research Center of Infectious Diseases (Tuberculosis) Kashi, 844000 China
    2The First People′s Hospital of Kashi, Department of Respiratory and Critical Care Medicine, XinjiangKashi, 844000 China
  • Received:2025-03-20 Published:2025-08-25
  • Corresponding author: Li Li
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弓慧, 祖力皮喀尔·阿卜杜热合曼, 徐敬然, 李黎. 衣康酸通过Nrf2抑制NLRP3激活拮抗卡介苗诱导RAW264.7细胞损伤机制研究[J/OL]. 中华肺部疾病杂志(电子版), 2025, 18(04): 540-545.

Hui Gong, Abudureheman Zulipikaer, Jingran Xu, Li Li. Itaconate activates Nrf2 to inhibit NLRP3 inflammasome activation and alleviate BCG-induced inflammatory damage in RAW264.7 Cells[J/OL]. Chinese Journal of Lung Diseases(Electronic Edition), 2025, 18(04): 540-545.

目的

分析衣康酸(Itaconate)通过核因子E2相关因子2(nuclear factor erythroid 2-related factor 2, Nrf2)抑制NLR家族含吡啶结构域蛋白3(NLR family pyrin domain-containing 3, NLRP3)激活卡介苗(Bacillus Calmette-Guérin, BCG)诱导RAW264.7细胞炎症损伤机制。

方法

采用BCG感染RAW264.7细胞，4-辛基衣康酸(OI)为Itaconate衍生物，将细胞分为对照组，BCG组、BCG＋OI组、BCG＋OI＋ML385组。BCG组采用1 200 μg/ml BCG感染4 h；BCG＋OI组分别用15、30、60 μM 4-辛基Itaconate(4-octyl-Itaconate, OI)预处理3 h＋1 200 μg/ml BCG感染4 h；BCG＋OI＋ML385组采用OI预处理＋10 μM Nrf2抑制剂ML385＋BCG处理4 h。检测细胞活力、Irg1、Nrf2、NLRP3的mRNA和蛋白表达水平及IL-1β和IL-18，通过Nrf2抑制剂ML385验证Nrf2的作用。

结果

对照组、BCG 100 μg/ml、200 μg/ml、400 μg/ml、800 μg/ml、1 200 μg/ml感染细胞活力分别为(100.00±0.00) 、(99.25±2.61)、(95.92±2.93)、(87.22±3.13)、(79.57±2.44)、(63.93±2.15)(P<0.05)。BCG组Irg1mRNA和蛋白水平分别为(2.34±0.15)、(0.74±0.06)，Nrf2为(3.16±0.51) 、(0.58±0.11)和NLRP3为(6.54±0.82)、(1.16±0.13)；对照组Irg1为(1.00±0.03)、(0.54±0.04)，Nrf2为(1.00±0.00)、(0.36±0.18)和NLRP3为(1.00±0.00)、(0.52±0.15)(P<0.05)。BCG组IL-1β(45.93±1.1035)、IL-18(1076.58±164.73)高于对照组IL-1β (37.95±1.02)、IL-18 (429.12±45.38)(P<0.05)。BCG组、BCG＋15 μM OI、BCG＋30 μM OI、BCG＋60 μM OI组细胞活力分别为(60.262±1.770)、(67.722±3.265)、(70.589±1.833)、(77.091±3.251)(P<0.05)，NLRP3 mRNA分别为(6.541±0.822)、(4.154±0.746)、(3.761±1.210)、(3.016±0.516)，NLRP3蛋白水平分别为(1.162±0.133)、(0.927±0.112)、(1.043±0.091)、(0.839±0.136)；IL-1β分别为(45.926±1.105)、(40.102±0.669)、(37.291±2.443)、(37.001±2.539)，IL-18分别为(1 076.584±164.728)、(839.663±19.818)、(748.350±97.910)、(667.800±11.420)(P<0.05)，Nrf2的mRNA和蛋白表达水平增高(P<0.05)。BCG＋OI＋ML385组Nrf2 mRNA(3.916±0.646)和蛋白表达水平(0.551±0.090)少于BCG＋OI组(5.914±0.716)、(0.753±0.060)(P<0.05)，BCG＋OI＋ML385组NLRP3 mRNA(5.164±0.512)、蛋白表达水平(1.450±0.210)、IL-1β(10.988±0.658)、IL-18(403.585±13.142)高于BCG＋OI组(3.561±0.810)、(1.010±0.310)、(6.982±0.0445)、(298.302±16.300)(P<0.05)。

结论

BCG通过NLRP3炎症小体活化加重巨噬细胞炎症损伤，Itaconate激活Nrf2抑制NLRP3发挥抗炎作用。

关键词: 结核分枝杆菌, 衣康酸, 卡介苗, 核因子E2相关因子2, NLR家族含吡啶结构域蛋白3
Objective

To analyze the mechanism by which itaconate inhibits NLRP3 activation and BCG-induced inflammatory injury in RAW264.7 cells through Nrf2.

Methods

RAW264.7 cells were infected with BCG. 4-octyl itaconate (OI) was used as an itaconate derivative. The cells were divided into the control group, BCG group, BCG+ OI group, and BCG+ OI+ ML385 group. The BCG group was infected with 1200 μg/ml BCG for 4 hours; the BCG+ OI group was pretreated with 15, 30, and 60 μM 4-octyl itaconate (OI) for 3 hours and then infected with 1 200 μg/ml BCG for 4 hours; the BCG+ OI+ ML385 group was pretreated with OI and then treated with 10 μM Nrf2 inhibitor ML385 and 1 200 μg/ml BCG for 4 hours. Cell viability, mRNA and protein expression levels of Irg1, Nrf2, and NLRP3, as well as IL-1β and IL-18, were detected. The role of Nrf2 was verified by using the Nrf2 inhibitor ML385.

Results

The cell viability of the control group, BCG 100 μg/ml, 200 μg/ml, 400 μg/ml, 800 μg/ml, and 1 200 μg/ml groups was (100.00±0.00), (99.25±2.61), (95.92±2.93), (87.22±3.13), (79.57±2.44), and (63.93±2.15), respectively (P<0.05). The mRNA and protein levels of Irg1 in the BCG group were (2.34±0.15) and (0.74±0.06), respectively; those of Nrf2 were (3.16±0.51) and (0.58±0.11), respectively; and those of NLRP3 were (6.54±0.82) and (1.16±0.13), respectively. In the control group, the mRNA and protein levels of Irg1 were (1.00±0.03) and (0.54±0.04), respectively; those of Nrf2 were (1.00±0.00) and (0.36±0.18), respectively; and those of NLRP3 were (1.00±0.00) and (0.52±0.15), respectively (P<0.05). The levels of IL-1β and IL-18 in the BCG group were (45.93±1.1035) and (1076.58±164.73), respectively, which were higher than those in the control group (IL-1β: 37.95±1.02; IL-18: 429.12±45.38) (P<0.05). The cell viability of the BCG group, BCG+ 15 μM OI group, BCG + 30 μM OI group, and BCG+ 60 μM OI group was (60.262±1.770), (67.722±3.265), (70.589±1.833), and (77.091±3.251), respectively (P<0.05). The mRNA levels of NLRP3 in these groups were (6.541±0.822), (4.154±0.746), (3.761±1.210), and (3.016±0.516), respectively; and the protein levels of NLRP3 were (1.162±0.133), (0.927±0.112), (1.043±0.091), and (0.839±0.136), respectively. The levels of IL-1β were (45.926±1.105), (40.102±0.669), (37.291±2.443), and (37.001±2.539), respectively, and the levels of IL-18 were (1 076.584±164.728), (839.663±19.818), (748.350±97.910), and (667.800±11.420) (P<0.05). The mRNA and protein expression levels of Nrf2 increased (P<0.05). The mRNA (3.916±0.646) and protein expression levels (0.551±0.090) of Nrf2 in the BCG+ OI+ ML385 group were lower than those in the BCG+ OI group (5.914±0.716) and (0.753±0.060) (P<0.05). The mRNA (5.164±0.512), protein expression levels (1.450±0.210), IL-1β (10.988±0.658), and IL-18 (403.585±13.142) in the BCG+ OI+ ML385 group were higher than those in the BCG+ OI group (3.561±0.810), (1.010±0.310), (6.982±0.0445), and (298.302±16.300) (P<0.05).

Conclusion

BCG aggravates macrophage inflammatory injury through NLRP3 inflammasome activation, while itaconate activates Nrf2 to inhibit NLRP3 and exert anti-inflammatory effects.

Key words: Mycobacterium tuberculosis, Itaconate, Bacillus Calmette-Guérin, Nuclear factor erythroid 2-related factor 2, NLR family pyrin domain-containing 3
图1 OI预处理对RAW264.7细胞活力、炎症因子及NLRP3、Nrf2表达的影响。图A为OI预处理对RAW264.7细胞活力的影响；图B、C为OI预处理对RAW264.7细胞IL-1β及IL-18浓度的影响；图D、E为OI预处理对RAW264.7细胞NLRP3、Nrf2蛋白和mRNA表达水平的影响注：BCG为卡介苗；Itaconate为衣康酸；β-actin为β-肌动蛋白(内参蛋白)；NLRP3为NLR家族含吡啶结构域蛋白3；Nrf2为核因子E2相关因子2; IL-1β为白细胞介素-1β、IL-18为白细胞介素-18；*P<0.05, **P<0.01，***P<0.001，与对照组相比。#P<0.05, ##P<0.01，###P<0.001，与BCG组相比
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