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Nrf2 activation by 2-methoxycinnamaldehyde attenuates macrophage-
proinflammatory responses via enhancing autophagy flux

Minwook Shin 1,# , Bo-Sung Kim 2,3,# , Jin-Yeong Kim 2,3 , Ki-Tae Ha 2,3 , Sung-Jin Bae 3,*
1 RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
2 Department of Korean Medical Science, School of Korean Medicine, Pusan National University, Yangsan, Gyeongnam 50612, Korea
3 Korean Medical Research Center for Healthy Aging, Pusan National University, Yangsan, Gyeongnam 50612, Korea

ABSTRACT RESULTS

Inflammation is the body's response to infection, tissue Fig. 1. Anti-inflammatory effects of 2-MCA in LPS- Fig. 2. Effect of 2-MCA on activation of MAPK,
damage, and cellular stress. A well-controlled stimulated macrophages. NF-kB, and AP-1 pathways in RAW264.7 and
inflammatory response has a beneficial effect for BMMs.
recovery and maintainance of tissue homeostasis. 2-
methoxycinnamaldehyde (2-MCA) is a natural
compound found in cinnamon, the most common
spice. The anti-inflammatory response of 2-MCA has
been already known; however, the mechanism
underlying anti-inflammatory action was not fully
elucidated. It was confirmed that the LPS-stimulated
production of TNF-α and NO was reduced by 2-MCA
treatment in murine bone marrow macrophages
(BMM) and macrophagic RAW264.7 cells. To
investigate the mechanism by which 2-MCA has anti-
inflammatory effects, MAPK, NF-κB, and AP-1
signaling pathways activated by LPS were examined.
2-MCA treatment did not affect the MAPK, NF-κB,
and AP-1 signaling pathways. However, 2-MCA
increased the expression and nuclear localization of
Nrf2 in macrophages. Previous studies have suggested
that Nrf2 is associated with regulation of autophagy.
Our result showed that 2-MCA increased the mRNA
and protein levels of p62 and Lc3. 2-MCA increased
the luciferase activity in RAW264.7 cells expressing (A) RAW264.7 (left) and BMMs (right) were incubated with 2-MCA
Lc3-Luciferase fusion protein which was designed for
investigating autophagic flux. In addition, LPS- as indicated for 24 hours and the absorbance induced by MTS was (A, B) RAW264.7 cells and BMMs were pre-treated with or without 2-
stimulated expression of Tnf-α and Nos2 was reduced measured at 492 nm, and the relative cell viability is shown as the MCA (50 μM) for 4 hours. After the treatment of LPS (1 μg/mL) for the
by 2-MCA treatment and reversed by autophagy mean ± SD (n = 3, with triplicates in each experiment). (B) RAW264.7 indicated time, the phosphorylations of p38, p44/42, and JNK were
inhibitors such as Bafilomycin A1 and NH 4 Cl. In (left) and BMMs (right) were pre-incubated with 2-MCA as indicated
conclusion, 2-MCA treatment activates Nrf2 in analyzed by immunoblot assay. (C, D) RAW264.7 cells and BMMs (D)
macrophages, induces autophagy, and consequently for 4 hours followed by incubation with or without LPS (1 μg/mL) for were pre-treated with or without 2-MCA (50 μM) for 4 hours. After the
reduces LPS-induced inflammatory responses. Thus, 24 hours. The secreted TNF-α in culture media was analyzed by treatment of LPS (1 μg/mL) for the indicated time, the phosphorylations of
here we suggest that anti-inflammatory effect of 2- ELISA; *P < 0.05, **P < 0.01. (C) RAW264.7 (left) and BMMs (right)
MCA might be regulated by Nrf2-mediated autophagy. IKKα, IKKβ, and p65, degradation of IκBα, and expression of c-Jun and c-
were incubated with 2-MCA for 4 hours as indicated followed by Fos were analyzed by immunoblot assay.
incubation with or without LPS (1 μg/mL) for 24 hours. The released
METHODS NO was evaluated by Griess Reagent-based analysis; *P < 0.05, **P
< 0.01, ***P < 0.001.
Fig. 4. Effects of 2-MCA on Inducing Autophagy
Flux in RAW264.7
Cell viability assay
Cell viability was measured by reduction of MTS. Fig. 3. Effect of 2-MCA on expression of Nrf2 and
The cultured medium was changed to MTS ATF3 in RAW264.7.
containing medium and then incubated 1 h for MTS
reduction by viable cell dehydrogenases. The
soluble formazan product of MTS was measure at
490 nm.
Nitric oxide (NO) assay
The cultured supernatant was collected and
removed cells by centrifugation at 500 g for 3 min.
100 μl of the cultured supernatant was mixed with
100 μl of Griess reagent (1:1 mixture of 1 %
sulfanilamide in 30 % acetate and 0.1 % N-1-
naphthyl ethylenediamine dihydrochloride in 60 %
acetate) at RT for 10 min. The absorbance of the
incubated samples was measured using a microplate
reader at 540 nm. A standard curve drawn with (A) RAW264.7 cells were pre-treated with or without 2-MCA (50 μM)
known concentrations of sodium nitrite was applied
to calculate the concentration of nitrite, the stable for 4 hours. After the treatment of LPS (1 μg/mL) for the indicated time,
end product of NO. the kinetic expressions of Nrf2 and ATF3 were analyzed by immunoblot
assay. (B) RAW264.7 cells were pre-treated with or without 2-MCA (50
Measurement of TNF-α secretion μM) for 4 hours. After the treatment of LPS (1 μg/mL) for the indicated
The cultured supernatant was collected. The amount
of secreted TNF-α was measured using Mouse TNF- time, cells were fractionated into cytosol/membrane and nucleus fractions. (A) RAW264.7 cells containing luciferase vector were pre-treated with or
α ELISA MAX kit (BioLegend, San Diego, CA) as The nucleus translocations of p65, Nrf2, and ATF3 were assessed. (C) without 2-MCA (50 μM) for 4 hours. After the treatment of LPS (1 μg/mL),
the manufacturer’s instructions. RAW264.7 cells, pre-treated with or without 2-MCA (50 μM) for 4 hours, Bafilomycin A1(40nM) for 8 hours, autophagy flux was measured by
were stimulated with LPS (1 μg/mL) as indicated and the expressions of luciferase assay. (B, C) RAW264.7 cells were pre-treated with or without
Immunoblot analysis Nrf2, ATF3, HO-1, and NOS2 were evaluated by immunoblot assay. (D) 2-MCA (50 μM) for 4 hours. After the treatment of LPS (1 μg/mL),
Proteins were extracted in cell lysis buffer
containing 50 mM Tris-Cl (pH 7.4), 300 mM NaCl, RAW264.7 cells, pre-incubated with or without 2-MCA as indicated for 4 Bafilomycin A1 (40 nM), NH 4 Cl (10 mM) for 8 hours, the expressions of
5 mM EDTA, 0.02 % (w/v) sodium azide, 1 % hours, were stimulated with LPS (1 μg/mL) for 4 hours, and the Tnf-α and Nos2 were analyzed by qPCR assay. The results from 3
(w/v) Triton X-100, 10 mM iodoacetamide, 1 mM expressions of Nrf2, ATF3, HO-1, and NOS2 were assessed by independent experiments are presented as means ± SD. *p<0.05, **p<0.01
phenylmethanesulfonyl fluoride, 2 μg/ml leupeptin, immunoblot assay. and ***p<0.001 comparisons with each group.
and protease inhibitor cocktail (Calbiochem,
Billerica, MA). Lysates were separated with SDS-
PAGE and transferred to nitrocellulose membrane CONCLUSION
(GE Healthcare Life Sciences, Pittsburgh, PA). The
transferred membrane was probed with the specific
antibodies. Image was acquired using LAS3000
machine (GE Healthcare Life Sciences). 2-MCA treatment activates Nrf2 in macrophages, induces autophagy, and consequently
reduces LPS-induced inflammatory responses. Thus, here we suggest that anti-
inflammatory effect of 2-MCA might be regulated by Nrf2-mediated autophagy.