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. 2022 Aug;55(8):407-412.
doi: 10.5483/BMBRep.2022.55.8.065.

NRF2 activation by 2-methoxycinnamaldehyde attenuates inflammatory responses in macrophages via enhancing autophagy flux

Bo-Sung Kim  1 , Minwook Shin  2 , Kyu-Won Kim  3 , Ki-Tae Ha  1 , Sung-Jin Bae  4
Affiliations

NRF2 activation by 2-methoxycinnamaldehyde attenuates inflammatory responses in macrophages via enhancing autophagy flux

Bo-Sung Kim et al. BMB Rep. 2022 Aug.

Abstract

A well-controlled inflammatory response is crucial for the recovery from injury and maintenance of tissue homeostasis. The anti-inflammatory response of 2-methoxycinnamaldehyde (2-MCA), a natural compound derived from cinnamon, has been studied; however, the underlying mechanism on macrophage has not been fully elucidated. In this study, LPS-stimulated production of TNF-α and NO was reduced by 2-MCA in macrophages. 2-MCA significantly activated the NRF2 pathway, and expression levels of autophagy-associated proteins in macrophages, including LC3 and P62, were enhanced via NRF2 activation regardless of LPS treatment, suggesting the occurrence of 2-MCA-mediated autophagy. Moreover, evaluation of autophagy flux using luciferase-conjugated LC3 revealed that incremental LC3 and P62 levels are coupled to enhanced autophagy flux. Finally, reduced expression levels of TNF-α and NOS2 by 2-MCA were reversed by autophagy inhibitors, such as bafilomycin A1 and NH4Cl, in LPS-stimulated macrophages. In conclusion, 2-MCA enhances autophagy flux in macrophages via NRF2 activation and consequently reduces LPS-induced inflammation. [BMB Reports 2022; 55(8): 407-412].

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors have no conflicting interests.

Figures

Fig. 1
Fig. 1
Anti-inflammatory effects of 2-MCA in LPS-stimulated macrophages. (A) RAW264.7 cells (left) and BMMs (right) were incubated with 2-MCA as indicated for 24 h. The absorbance produced by MTS was measured at 490 nm. The relative cell viability is shown as the mean ± SD (n = 3, with triplicates in each experiment). (B) RAW264.7 cells (left) and BMMs (right) were pre-incubated with 2-MCA as indicated for 4 h, followed by incubation with or without LPS (1 μg/ml) for 24 h. The secreted TNF-α in the culture media was analyzed by ELISA; *P < 0.05, **P < 0.01. (C) RAW264.7 (left) and BMMs (right) were incubated with 2-MCA as indicated for 4 h, followed by incubation with or without LPS (1 μg/ml) for 24 h. The released NO was evaluated by Griess Reagent-based analysis; *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 2
Fig. 2
Effect of 2-MCA on activation of MAPK, NF-kB, and AP-1 pathways in RAW264.7 and BMMs. (A, B) RAW264.7 cells (A) and BMMs (B) were pre-treated with or without 2-MCA (50 μM) for 4 h. After LPS (1 μg/ml) treatment within the indicated time, the phosphorylation levels p38, p44/42, and JNK were analyzed using im-munoblot assay. (C, D) RAW264.7 cells (C) and BMMs (D) were pre-treated with or without 2-MCA (50 μM) for 4 h. After LPS (1 μg/ml) treatment within the indicated time, the phosphorylation levels of IKKα, IKKβ, and p65, degradation of IκBα, and expression levels of c-Jun and c-Fos were analyzed using immunoblot assay.
Fig. 3
Fig. 3
Effect of 2-MCA on the expression of NRF2 and ATF3 in RAW264.7. (A) RAW264.7 cells were pre-treated with or without 2-MCA (50 μM) for 4 h. After LPS (1 μg/ml) treatment within the indicated time, the kinetic expression levels of NRF2 and ATF3 were analyzed using immunoblot assay. (B) RAW264.7 cells were pre-treated with or without 2-MCA (50 μM) for 4 h. After LPS (1 μg/ml) treatment within the indicated time, cells were fractionated into cyto-sol/membrane and nucleus fractions. The nucleus translocations of p65, NRF2, and ATF3 were assessed. (C) RAW264.7 cells, whether pre-treated with or without 2-MCA (50 μM) for 4 h, were stimulated with LPS (1 μg/ml) as indicated. The expression levels of NRF2, ATF3, HO-1, and NOS2 were evaluated using immunoblot assay. (D) RAW264.7 cells, whether pre-incubated with or without 2-MCA as indicated for 4 h, were stimulated with LPS (1 μg/ml) for 4 h. The expression levels of NRF2, ATF3, HO-1, and NOS2 were assessed using immunoblot assay.
Fig. 4
Fig. 4
Effects of 2-MCA on Inducing Autophagy Flux in RAW264.7 (A) RAW264.7 cells containing luciferase-LC3 were pre-treated with or without 2-MCA (50 μM) for 4 h. After LPS (1 μg/ml) or BafA1 (40 nM) treatment for 8 h, autophagy flux was measured using luciferase assay. (B, C) RAW264.7 cells were pre-treated with or without 2-MCA (50 μM) for 4 h. After LPS (1 μg/ml), BafA1 (40 nM), or NH4Cl (10 mM) treatment for 8 h, the expression levels of Tnf and Nos2 were analyzed using qRT-PCR assay. The results from three independent experiments are presented as means ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001.
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