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

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.

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).

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