Structure based modification of chalcone analogue activates Nrf2 in the human retinal pigment epithelial cell line ARPE-19

Article abstract of DOI:10.1016/j.freeradbiomed.2019.12.033

Oxidative stress-induced degeneration of retinal pigment epithelial (RPE) cells is known to be a key contributor to the development of age-related macular degeneration (AMD). Activation of the nuclear factor-(erythroid-derived 2)-related factor-2 (Nrf2)-mediated cellular defense system is believed to be a valid therapeutic approach. In the present study, we designed and synthesized a novel chalcone analogue, 1-(2,3,4-trimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-acrylketone (Tak), as a Nrf2 activator. The potency of Tak was measured in RPE cells by the induction of the Nrf2-dependent antioxidant genes HO-1, NQO-1, GCLc, and GCLm, which were regulated through the Erk pathway. We also showed that Tak could protect RPE cells against oxidative stress-induced cell death and mitochondrial dysfunction. Furthermore, by modifying the α, β unsaturated carbonyl entity in Tak, we showed that the induction of antioxidant genes was abolished, indicating that this unique feature in Tak was responsible for the Nrf2 activation. These results suggest that Tak is a potential candidate for clinical application against AMD.

Full text of DOI:10.1016/j.freeradbiomed.2019.12.033

Journal Pre-proof
Structure based modification of chalcone analogue activates Nrf2 in the human retinal
pigment epithelial cell line ARPE-19
Yuting Cui, Yuan Li, Na Huang, Yue Xiong, Ruijun Cao, Lingjie Meng, Jiankang Liu,
Zhihui Feng
PII:
S0891-5849(19)31631-4
DOI:
https://doi.org/10.1016/j.freeradbiomed.2019.12.033
FRB 14542
Reference:
To appear in: Free Radical Biology and Medicine
Received Date: 30 September 2019
Revised Date: 19 December 2019
Accepted Date: 23 December 2019
Please cite this article as: Y. Cui, Y. Li, N. Huang, Y. Xiong, R. Cao, L. Meng, J. Liu, Z. Feng,
Structure based modification of chalcone analogue activates Nrf2 in the human retinal pigment
epithelial cell line ARPE-19, Free Radical Biology and Medicine (2020), doi: https://doi.org/10.1016/
j.freeradbiomed.2019.12.033.
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Structure based modification of chalcone analogue activates Nrf2 in the human
retinal pigment epithelial cell line ARPE-19
Yuting Cui1a, Yuan Li2a, Na Huang3, Yue Xiong1, Ruijun Cao3, Lingjie Meng3,
Jiankang Liu1,4*, Zhihui Feng1,4*
1Center for Mitochondrial Biology and Medicine, The Key Laboratory of Biomedical
Information Engineering of Ministry of Education, School of Life Science and
Technology, Xi’an Jiaotong University, Xi’an, 710049, China.
2Institute of Basic Medical Science, Xi'an Medical University, Xi'an, 710021, PR
China.
3MOE Key Laboratory for Nonequilibrium Synthesis and Modulation of Condensed
Matter, School of Science, Xi'an Jiaotong University, Xi'an, China.
4Frontier Institute of Science and Technology, Xi’an Jiaotong University, Xi’an,
710049, China.
aThese authors contribute equally
*Corresponding authors:
Correspondence and requests for materials should be addressed to J. L.
(j.liu@mail.xjtu.edu.cn) or Z. F. (email: zhfeng@mail.xjtu.edu.cn).

Abstract
Oxidative stress-induced degeneration of retinal pigment epithelial (RPE) cells is
known to be a key contributor to the development of age-related macular degeneration
(AMD). Activation of the nuclear factor-(erythroid-derived 2)-related factor-2
(Nrf2)-mediated cellular defense system is believed to be a valid therapeutic approach.
In the present study, we designed and synthesized a novel chalcone analogue,
1-(2,3,4-trimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-acrylketone (Tak), as a Nrf2
activator. The potency of Tak was measured in RPE cells by the induction of the
Nrf2-dependent antioxidant genes HO-1, NQO-1, GCLc, and GCLm, which were
regulated through the Erk pathway. We also showed that Tak could protect RPE cells
against oxidative stress-induced cell death and mitochondrial dysfunction.
Furthermore, by modifying the α, β unsaturated carbonyl entity in Tak, we showed
that the induction of antioxidant genes was abolished, indicating that this unique
feature in Tak was responsible for the Nrf2 activation. These results suggest that Tak
is a potential candidate for clinical application against AMD.
Key words: Nrf2, oxidative stress, mitochondrial dysfunction, acrolein, macular
degeneration

Introduction
Age-related macular degeneration (AMD) is an eye disease characterized by the
presence of extracellular depositions, known as drusen, accumulating between
Bruch’s membrane and the neural retina [1]. AMD affects the macula, a highly
specialized region of the central retina responsible for fine and color vision, and has
been recognized as a leading cause of vision loss that affects millions of individuals
over the age of 65 years, especially in the United States and Europe [2]. The current
incidence of AMD has reached 8.7% worldwide and is projected to be 196 million by
2020 and 288 million by 2040 [3]. In the past few decades, dramatic progress has
been made in understanding the mechanisms of AMD progression, and multiple risk
factors have been identified that are useful to the public for knowledge of AMD
prevention. However, there is neither definitive treatment for the disease nor
intervention to slow the progression of AMD, and targeted drug and nutrient
screening remains of popular interest in the study of AMD [4].
It is commonly accepted that oxidative stress plays a pivotal role in AMD
pathogenesis, and many risk factors, such as aging, smoking, UV and blue light
exposure, chronic inflammation, and improper diet, have been identified as related to
oxidative stress [5-9]. Retinal pigment epithelial (RPE) cells are highly specialized
epithelial cells with an apical side in contact with photoreceptor segments. RPE cells
maintain the health and regular activity of these photoreceptors [10] and function in
the outer blood-retinal barrier to provide selective entry and removal of metabolites
and molecules [11]. Given the remarkable and diverse functions of RPE cells, it has
been well demonstrated that impairing RPE cell function is a major contributor to
AMD progression [12], and it has been suggested that oxidative stress is the major
factor for the protein damage and aggregation that ultimately leads to the degeneration
of the RPE cells [13].
Previous studies have shown that the endogenous antioxidant defense system is
activated in response to oxidative stress, which is regulated by nuclear

factor-(erythroid-derived 2)-related factor-2, also known as Nrf2. Nrf2 is a major
regulator that binds to antioxidant response elements (AREs) to induce the expression
of antioxidant enzymes, also known as phase II enzymes, which has been suggested to
play vital roles in protecting RPE cells [14-18]. Under resting conditions, Nrf2 is
trapped in the cytoplasm by Keap1 protein, thereby promoting Nrf2 ubiquitination
and subsequent degradation by the proteasome [19, 20]; however, under stress
conditions, Keap1 serves as the primary sensor of the stress signals and undergoes
conformational changes due to protein modification, thereby inhibiting Nrf2
ubiquitination [21]. Nrf2 can translocate to the nucleus, where it binds to a small Maf
protein and activates the transcription of target genes, such as heme oxygenase-1
(HO-1) [22], NAD (P) H:quinone oxidoreductase 1 (NQO-1) [23] and
γ-glutamyl-cysteine ligase (GCL) [24]. GCL is the rate-limiting enzyme for the
synthesis of glutathione (GSH), which is one of the major antioxidants produced in
vivo [25], and Nrf2 can regulate GSH levels by modulating the expression of the GCL
catalytic (GCLc) and modifier (GCLm) subunits. Consistently, Nrf2 mRNA and
protein levels have been shown to decline with aging, which renders aging RPE cells
vulnerable to oxidative damage [18, 26]. Therefore, it has been suggested that Nrf2
signaling is a promising target for novel pharmacological and genetic therapeutic
strategies [27].
Many reports have demonstrated that dietary polyphenols beneficially contribute
to enhanced human health, such as by improving vision. In recent years, several
polyphenolic compounds that target Nrf2, such as quercetin, resveratrol, curcumin,
and chlorogenic acid, have been identified as protectors of RPE cells [4]. Chalcones,
which constitute a subclass of the flavonoid family, are the precursors of the flavones
that participate in the biosynthesis of flavonoids and have been reported to exhibit a
broad spectrum of pharmacological properties such that they have continuously
attracted increased attention [28]. Chalcones often exhibit low bioavailability and
poor absorption, leading to limited efficacy in vivo [29]. Studies have been dedicated
to improving the chalcone derivatives that can induce Nrf2-mediated HO-1 expression
and GSH synthesis [30]. A recent report suggested that the ability of chalcone

derivatives to increase intracellular GSH levels was drastically improved with the
addition of three methoxyl and/or hydroxyl groups on phenyl ring A [31]. Here, we
synthesized
a
new
chalcone
analogue,
1-(2,3,4-trimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-acrylketone (Tak), which has
three methoxyl groups on both phenyl ring A and phenyl ring B. It is tempting to
speculate that Tak activates the phase II detoxifying enzyme system to create
pronounced antioxidant capacity. We also designed another two compounds with
modified carbonyl entities between the phenyl rings to compare to Tak and thus
confirm the key structure responsible for Nrf2-induced activity.
Material and Method
Chemicals and reagents. LY294002, SB203580, SP600125, U0126, JC-1, and ATP
mixes were obtained from Sigma (St. Louis, MO, USA). Antibodies against p-Akt,
Akt, p-Erk1/2, Erk1/2, p-p38, p38, JNK, p-JNK and β-actin were purchased from Cell
Signaling Technology (Danvers, MA, USA). Antibodies against PGC-1, TFAM,
OPA1, Mfn1, Mfn2, Drp1, Fis1, HO-1 and NQO-1 were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). Antibodies against complexes I, II, and III
were purchased from Life Technologies (San Diego, CA, USA). The cell culture
medium, Mito-Tracker Green/Red and MitoSOX Red were purchased from Life
Technologies (San Diego, CA, USA). The synthesis scheme and compound structure
are provided in Figure 1. The reactions were monitored by thin-layer chromatography
1
(TLC) and visualized under UV light. The H NMR and 13C NMR spectra were
measured on a Bruker Advance III 400 MHz instrument (Supplementary method).
The purity (≥95%) of each compound was verified by HPLC (Fig. S1-3).
Cell culture. The human ARPE-19 cell line was obtained from the ATCC (Manassas,
VA, USA) and was cultured in DMEM-F12 supplemented with 10% fetal bovine
serum, 0.348% sodium bicarbonate, 2 mM L-glutamine, 100 U/ml penicillin and 100
µg/ml streptomycin. The cell cultures were maintained at 37 °C in a humidified
atmosphere of 95% air and 5% CO2. The cell medium was changed every two days.

ARPE-19 cells were harvested within the first 10 passages.
Cell viability assay. Cell viability was analyzed using the MTT
(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay. The cells
were seeded at a density of 3.5×104 cells/ml in 96-well plates. After incubation, 200
µL of MTT- medium solution (1:9) was added to each well. The plates were further
incubated for 4 h and then 200 µL of DMSO was added to each well. The absorbance
was measured at 490 nm using a microplate fluorometer (Fluoroskan Ascent; Thermo
Fisher Scientific, Inc.). The bar graph was generated by the GraphPad Prism. Three
independent experiments with six repeats in each group were performed for the assay.
JC-1 assay for the MMP. Cells were cultured at a density of 3.5×104 cells/ml in
96-well plates. After treatment, the mitochondrial membrane potential (MMP) was
measured
using
the
lipophilic
cationic
probe
5,5ʹ,6,6ʹ-tetrachloro-1,1ʹ,3,3ʹ-tetraethyl-imidacarbocyanine iodide (JC-1). The cells
were washed with PBS once and scanned with a microplate fluorometer (Fluoroskan
Ascent; Thermo Fisher Scientific, Inc.) at 488 nm excitation and 538 and 590 nm
emission wavelengths to measure green and red JC-1 fluorescence, respectively. The
red/green fluorescence intensity ratio reflects the MMP. The bar graph was generated
by the GraphPad Prism. Three independent experiments with six repeats in each
group were performed for the assay.
Intracellular ATP levels. Cells were cultured in six-well plates. After treatment, the
cells were lysed using 0.5% Triton X-100 in 100 mM glycine buffer at pH 7.4. To
determine intracellular ATP levels, an ATP bioluminescent assay kit (Sigma, St. Louis,
MO, USA) was used. The assay was performed following manufacture instruction.
The ATP was consumed, and light was emitted when the firefly luciferase catalyzed
the oxidation of D-luciferin [32]. The bar graph was generated by the GraphPad Prism.
Three independent experiments with three repeats in each group were performed for
the assay.

Mitochondrial superoxide measurement. Cells were cultured at a density of 5×104
cells/ml in 24-well plates. After treatment, the generation of mitochondrial
superoxides was observed by an MitoSOXTM Red Mitochondrial Superoxide
Indicator, and the mitochondria were assessed with Mito-Tracker Green FM. Briefly,
cells were stained with Mito-Tracker Green FM at 500 nM in serum-free medium for
30 min, followed by 10 µM MitoSOXTM Red incubation in serum-free medium for
10 min. After washing with PBS, the cells were visualized by laser scanning confocal
microscopy (Zeiss, Jena, Germany). Similar procedure was used to observe
mitochondria distribution with Mitotracker Red.
Real-time PCR. Total RNA was extracted using TRIzol (Invitrogen, San Diego, CA,
USA) following the manufacturer's protocol. Reverse transcription was performed
using a PrimeScript RT-PCR kit (Otsu, Shiga, Japan) and was followed by
semiquantitative real-time PCR using specific primers. Data were normalized to the
mRNA levels of beta-actin, which was used as a housekeeping gene and were
analyzed by the 2-∆∆CT method. The final results are presented as percentages of the
control. The primer sequences are presented in detail in Table S1. The bar graph was
generated by the GraphPad Prism. Three independent experiments with three repeats
in each group were performed for the assay.
Western blot analysis. Cells were lysed with western and IP lysis buffers (Beyotime,
Nanjing, China). The lysates were centrifuged at 13,000 g for 6 min at 4° C. The
supernatants were collected, and the protein concentration was determined with a
BCA protein assay kit (Pierce, San Diego, CA, USA). Equal aliquots of protein
samples were loaded onto 10% SDS-PAGE gels and transferred to nitrocellulose
membranes. After blocking with 5% nonfat milk for 1 h, the membranes were
incubated with primary antibodies overnight at 4° C, and then, the membranes were
incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at
room temperature. Western blots were developed using an enhanced

chemiluminescence (ECL) western blot detection kit (Pierce, Rockford, IL) and
quantified by scanning densitometry.
Statistical analysis. Data are presented as the mean ± standard error of the mean
(SEM) from at least three independent experiments and were analyzed using
GraphPad Prism-5 software. The statistical significance was evaluated using one-way
analysis of variance (ANOVA) followed by post hoc comparisons with Tukey’s HSD
test. The differences between two groups were analyzed using Student’s t-test, and the
level of significance was set at a value of P<0.05.
Results
Effects of Tak, Tap, and Tbp on cell viability and MMP. The chalcone analogue
Tak was synthesized from 1,2,3-trimethoxybenzene with three methoxyl groups in
both phenyl ring A and phenyl ring B and an α, β unsaturated carbonyl entity between
the two phenyl rings. The compounds Tap and Tbp have the same phenyl ring
structure as Tak, although Tab has two carbonyl units and Tbp has one carbonyl group
between the phenyl rings (Fig. 1). The three compounds were tested in ARPE-19 cells
with an acrolein challenge, which is a well-established cellular model that mimics
smoking-promoted RPE cell death during AMD progression [33]. As shown in Fig.
2A, Tak at 5 and 10 µM could sufficiently inhibit acrolein-induced loss of cell
viability, while neither Tap nor Tbp had a protective effect (Fig. 2B, C). The
mitochondrial membrane potential (MMP) is a key factor in maintaining normal
mitochondrial function and cellular activity. Following a cell viability assay, we
further tested the protective effects of three compounds on the MMP. Consistently,
Tak presented significant protection against acrolein-induced MMP loss, while Tap
and Tbp had no effect (Fig. 2E, F). In addition, we also applied Tak to a
t-BHP-challenged cellular model. As expected, Tak at 10 µM significantly protected
both cell viability and MMP against t-BHP-induced damage (Fig. 3A, B).
Effects of Tak on mitochondrial superoxide. It is estimated that 1-3% of the

mitochondrial oxygen consumed during the process of oxidative phosphorylation is
incompletely reduced, which results in the formation of superoxide anions, a
predominant form of ROS produced in mitochondria, and its accumulation could
induce oxidative stress to suppress mitochondria activity [34]. Since MMP was
significantly decreased after acrolein treatment, we analyzed the extent of
mitochondrial superoxide with confocal microscopy and found that the acrolein
challenge dramatically increased superoxide levels in the RPE cells and that this
increase was significantly prevented by Tak pretreatment (Fig. 3C).
Effects of Tak on mitochondrial dynamic markers. To explore the mechanism by
which Tak confers its protective effect, we first analyzed mitochondrial dynamic
markers, including mitochondrial biogenesis, fusion and fission related proteins. After
treating the RPE cells with 1 µM and 10 µM Tak for 24 h, we analyzed the protein
levels of PGC-1, TFAM, complexes I, II, and III and found no significant changes
(Fig. 4A). Consistently, the mRNA levels of the mitochondrial complex subunits were
not affected (Fig. 4B). Considering these results, together with the unchanged
mitochondrial copy number (Fig. 4E), we speculated that Tak had no effect on
mitochondrial biogenesis. Furthermore, fusion- and fission-related proteins were also
tested, and no significant changes were observed (Fig. 4C). Meanwhile, Mitotracker
Red staining of mitochondria showed no significant changes on general morphology
and cellular localization (Fig.4D). In addition, the cellular ATP level was not affected
by Tak treatment (Fig. 4F). Taken together, these data suggested that Tak had no
significant effect on mitochondrial number, cellular localization, and general
fusion/fission related proteins.
Effects of Tak on Nrf2-regulated phase II enzymes. Since the Nrf2/Keap1 complex
is the regulator of endogenous antioxidative enzymes, also known as phase II
enzymes, we found that the mRNA levels of both Nrf2 and Keap1 were not changed
after treatment with each of the three compounds (Fig. 5A, B). Interestingly, further
analysis of phase II enzymes revealed that Tak could dramatically induce the

expression of HO-1, NQO-1, GCLc and GCLm, indicating that Tak could activate
Nrf2 for the induction of phase II enzymes (Fig. 5C-F). Consistent with the
observations from the MTT and MMP assays, neither Tap nor Tbp had significant
effects on phase II enzyme levels (Fig. 5C-F). In addition, the changes in mRNA
levels were further confirmed by measuring the protein levels of Nrf2, HO-1 and
NQO-1, and the findings showed unique induction of phase II enzymes only with the
Tak treatment (Fig. 6). Since total Nrf2 protein level was not affected by Tak, we
further analyzed nuclear level of Nrf2 and found that Tak treatment at 6 h could
sufficiently promote Nrf2 nucleus translocation (Fig. 6D, E). Taken together, these
data suggested that Tak could activate Nrf2 through promoting its nuclear
translocation for the induction of phase II enzymes, which was the key factor
responsible for the protection conferred by Tak.
Tak activates phase II enzymes through Erk signaling. To further explore the
upstream pathway that regulates phase II enzymes by Tak, we treated RPE cells in a
time-dependent manner and found that Tak could efficiently increase the levels of
p-Akt and p-Erk without affecting other MAP kinases (Fig. 7A). Employment of
pathway inhibitors showed that only inhibition of the Erk pathway by U0126[35]
could significantly abolish the induction of phase II enzymes, which included HO-1,
NQO-1, GCLc and GCLm (Fig. 7B-E), while inhibition of Akt by LY294002[36],
inhibition of JNK by SP600125[37], and inhibition of p38 by SB203580 [38] had no
significant effects on Tak-induced phase II enzyme expression (Fig. 7B-E). These data
suggested that Tak could induce antioxidant enzyme expression through activation of
the Erk pathway, which is consistent with previous reports showing that Erk
activation is required for Nrf2 nuclear localization in HepG2 cells [39, 40].
Discussion
AMD is a multifactorial disease with no definitive treatment. Nutritional and
chemical interventions targeting Nrf2-mediated phase II enzymes are acknowledged

and popular strategies for disease prevention. Even though chalcones have reportedly
exhibited multiple benefits, their bioactivities in RPE cells have not been evaluated.
Since the low bioavailability and poor absorption of chalcone limit its efficacy in vivo,
recent studies have been dedicated to developing functional chalcone derivatives. In
the present study, we designed and synthesized a series of chalcone analogues and
showed that the chalcone analogue Tak could dramatically induce Nrf2-mediated
phase II enzymes through activation of the Erk pathway and thus protect RPE cells
from oxidative stress-induced mitochondrial dysfunction and cell death. We also
confirmed that the α, β unsaturated carbonyl entity in Tak is the key structural feature
that mediates Nrf2 activation.
Accumulating evidence has now been consolidated to show a role for oxidative
stress in aging and many diseases, and retinal diseases are no exception to this pattern.
As a result, targeting oxidative stress has been extensively studied in disease
pathogenesis and prevention, and increasing data have suggested a role for Nrf2 in
these processes [41]. A decrease in Nrf2 DNA-binding activity was reported in the
retina of patients with diabetic retinopathy, and a role for Nrf2 in the onset of AMD
has been well documented [42, 43]. Data from Nrf2-knockout mice contribute to the
rationale for the suggested Nrf2-mediated cellular protection. Even though
Nrf2-knockout mice develop without critical deficits, the abnormal ocular phenotypes
observed in these mice were exacerbated under oxidative stress, and age-dependent
deterioration in RPE cells was also observed in the Nrf2-knockout mice [44, 45]. An
increasing number of studies have proposed that activation of Nrf2 would be an ideal
therapy for treating retinal diseases. We have previously reported that natural
compounds such as hydroxytyrosol [46], zeaxanthin [17], and alpha-tocopherol [47]
are all specific Nrf2 activators and each offer RPE cell protection. Through structural
modification, we also designed curcumin analogues that had improved efficiency
compared with curcumin in activating Nrf2 [48]. Therefore, targeting Nrf2 by natural
compounds and synthetic chemicals can protect RPE cell function against oxidative
damage. Since oxidative stress is the major risk factor for the early progression of
AMD, also named “dry AMD”, there’s another aggressive type of AMD, named “wet

AMD”, which presents unique angiogenesis. It’s been proposed that chalcones may
work as an inhibitor of angiogenesis in macular degeneration, which has not been
extensively studied [49]. In the present study, we found that Tak had no significant
effect on angiogenesis related gene expression in ARPE-19 cells (Fig. S4), future
studies shall be dedicated to exploring whether Tak could prevent stress induced
angiogenesis both in vitro and in vivo.
Previous studies have reported that chalcone derivatives induce Nrf2-mediated
HO-1 expression and GSH synthesis [30], and this inducement was drastically
enhanced with the addition of up to three methoxyl and/or hydroxyl groups on phenyl
ring A [31]. Here, we synthesized a new chalcone analogue with three methoxyl
groups on both phenyl ring A and phenyl ring B. We speculated that Tak activates the
phase II detoxifying enzyme system and has pronounced antioxidant capacity. The
results of the present study, obtained by measuring phase II enzyme expression,
confirmed these roles of Tak. In addition, to further identify the key structure that
modulates this Nrf2 activation, we synthesized another two analogues, Tap and Tbp,
which had the same phenyl rings as Tak but different linkages between the rings.
Interestingly, we found that removal of the α, β unsaturated ketone completely
abolished the effect of Tak on Nrf2 activation, indicating the vital role of the α, β
unsaturated carbonyl entity in regulating Nrf2 activity.
Nrf2 is the ubiquitously expressed transcription factor that regulates the
expression of over 1000 genes [50]. Nrf2 can be regulated at the transcriptional level,
posttranscriptional level or by other transcription factors [51]. Even though, the most
efficient way of activating Nrf2 is to disrupt the binding between Nrf2 and Keap1,
which locates in cytoplasm and target Nrf2 for ubiquitination and degradation. Known
Nrf2 inducers such as ROS, sulforophane, tert-butyl hydroquinone can oxidize the
active cysteine residues in Keap1 to induce conformational change and prevent Nrf2
ubiquitination [52]. Meanwhile, several kinases including the PI3K/Akt and the
MAPK, including JNK, Erk, and p38 were also reported to regulate Nrf2 through
phosphorylation of Keap1 [53-55]. We have previously found that natural compound
hydroxytyrosol could promote Nrf2 nuclear translocation through JNK activation,

while zeaxanthin mediates Nrf2 activation through PI3/Akt pathway [17, 46]. Here
we found that Tak could activate Nrf2 through Erk1/2 activation, further
investigations should be explored for the mechanisms of Tak structure feature in
Erk1/2 activation.
In conclusion, our study demonstrated that the newly synthesized chalcone
analogue could induce Nrf2-mediated phase II enzyme expression to improve the
cellular redox status and enhance mitochondrial function through activation of the Erk
pathway. Such beneficial bioactivity could further protect RPE cells against oxidative
stress-induced cell damage. We also confirmed that the α, β unsaturated carbonyl
entity of the chalcone is the key structural feature that mediates Nrf2 activation. All
together, these findings suggest that Tak may be an attractive agent for the prevention
and treatment of retinal diseases.
Acknowledgment
This work was supported by the National Natural Science Foundation of China
(81571050; 31770917; 31570777; 91649106; and 31701025), the General Financial
Grant from the China Postdoctoral Science Foundation (2017M613108) and the
Fundamental Research Funds for the Central Universities (Z201806073).
Author Contributions
Z.F. and J.L. conceived the study, analyzed the data. N.H., R.C., and L.M. synthesized
the compounds. Y.X., Y.L., and Y.C. performed the biological experiments and
analyzed the data. Y.L., Y.C., and Z.F. drafted the manuscript.
Y.X., Y.L., and Y.C. contributed equally to the study.
Competing interests
The authors declare that they have no competing interests.
Supporting information

List of primers for real-time PCR; HPLC analysis of compounds Tak, Tap, and Tbp
was presented in supporting information.
References
[1] K.M. Gehrs, D.H. Anderson, L.V. Johnson, G.S. Hageman, Age-related macular
degeneration--emerging pathogenetic and therapeutic concepts, Annals of medicine 38(7) (2006)
450-71.
[2] H. Kokotas, M. Grigoriadou, M.B. Petersen, Age-related macular degeneration: genetic and clinical
findings, Clinical chemistry and laboratory medicine : CCLM / FESCC 49(4) (2011) 601-16.
[3] W.L. Wong, X. Su, X. Li, C.M. Cheung, R. Klein, C.Y. Cheng, T.Y. Wong, Global prevalence of
age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic
review and meta-analysis, Lancet Glob Health 2(2) (2014) e106-16.
[4] E. Pawlowska, J. Szczepanska, A. Koskela, K. Kaarniranta, J. Blasiak, Dietary Polyphenols in
Age-Related Macular Degeneration: Protection against Oxidative Stress and Beyond, Oxid Med Cell
Longev 2019 (2019) 9682318.
[5] O.S. Kozhevnikova, E.E. Korbolina, N.I. Ershov, N.G. Kolosova, Rat retinal transcriptome: effects of
aging and AMD-like retinopathy, Cell Cycle 12(11) (2013) 1745-61.
[6] S.S. Ni Dhubhghaill, M.T. Cahill, M. Campbell, L. Cassidy, M.M. Humphries, P. Humphries, The
pathophysiology of cigarette smoking and age-related macular degeneration, Adv Exp Med Biol 664
(2010) 437-46.
[7] S. Kraljevic Pavelic, M. Klobucar, M. Sedic, V. Micek, P. Gehrig, J. Grossman, K. Pavelic, B. Vojnikovic,
UV-induced retinal proteome changes in the rat model of age-related macular degeneration, Biochim
Biophys Acta 1852(9) (2015) 1833-45.
[8] N. Piippo, E. Korhonen, M. Hytti, K. Kinnunen, K. Kaarniranta, A. Kauppinen, Oxidative Stress is the
Principal Contributor to Inflammasome Activation in Retinal Pigment Epithelium Cells with Defunct
Proteasomes and Autophagy, Cell Physiol Biochem 49(1) (2018) 359-367.
[9] D.L. Bhoiwala, Y. Song, A. Cwanger, E. Clark, L.L. Zhao, C. Wang, Y. Li, D. Song, J.L. Dunaief, CD1
Mouse Retina Is Shielded From Iron Overload Caused by a High Iron Diet, Invest Ophthalmol Vis Sci
56(9) (2015) 5344-52.
[10] M. Saint-Geniez, T. Kurihara, E. Sekiyama, A.E. Maldonado, P.A. D'Amore, An essential role for
RPE-derived soluble VEGF in the maintenance of the choriocapillaris, Proceedings of the National
Academy of Sciences of the United States of America 106(44) (2009) 18751-6.
[11] O. Strauss, The retinal pigment epithelium in visual function, Physiological reviews 85(3) (2005)
845-81.
[12] S. Datta, M. Cano, K. Ebrahimi, L. Wang, J.T. Handa, The impact of oxidative stress and
inflammation on RPE degeneration in non-neovascular AMD, Prog Retin Eye Res 60 (2017) 201-218.
[13] K. Kinnunen, G. Petrovski, M.C. Moe, A. Berta, K. Kaarniranta, Molecular mechanisms of retinal
pigment epithelium damage and development of age-related macular degeneration, Acta Ophthalmol
(2011).
[14] W. Li, A.N. Kong, Molecular mechanisms of Nrf2-mediated antioxidant response, Molecular
carcinogenesis 48(2) (2009) 91-104.
[15] T. Nguyen, P. Nioi, C.B. Pickett, The Nrf2-antioxidant response element signaling pathway and its
activation by oxidative stress, The Journal of biological chemistry 284(20) (2009) 13291-5.

[16] X. Gao, P. Talalay, Induction of phase 2 genes by sulforaphane protects retinal pigment epithelial
cells against photooxidative damage, Proceedings of the National Academy of Sciences of the United
States of America 101(28) (2004) 10446-51.
[17] X. Zou, J. Gao, Y. Zheng, X. Wang, C. Chen, K. Cao, J. Xu, Y. Li, W. Lu, J. Liu, Z. Feng, Zeaxanthin
induces Nrf2-mediated phase II enzymes in protection of cell death, Cell Death Dis 5 (2014) e1218.
[18] M.M. Sachdeva, M. Cano, J.T. Handa, Nrf2 signaling is impaired in the aging RPE given an oxidative
insult, Experimental eye research 119 (2014) 111-4.
[19] K. Itoh, N. Wakabayashi, Y. Katoh, T. Ishii, K. Igarashi, J.D. Engel, M. Yamamoto, Keap1 represses
nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal
Neh2 domain, Genes & development 13(1) (1999) 76-86.
[20] M. Furukawa, Y. Xiong, BTB protein Keap1 targets antioxidant transcription factor Nrf2 for
ubiquitination by the Cullin 3-Roc1 ligase, Molecular and cellular biology 25(1) (2005) 162-71.
[21] T. Suzuki, H. Motohashi, M. Yamamoto, Toward clinical application of the Keap1-Nrf2 pathway,
Trends Pharmacol Sci 34(6) (2013) 340-6.
[22] J. Alam, D. Stewart, C. Touchard, S. Boinapally, A.M. Choi, J.L. Cook, Nrf2, a Cap'n'Collar
transcription factor, regulates induction of the heme oxygenase-1 gene, The Journal of biological
chemistry 274(37) (1999) 26071-8.
[23] R. Venugopal, A.K. Jaiswal, Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the
human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductase1
gene, Proceedings of the National Academy of Sciences of the United States of America 93(25) (1996)
14960-5.
[24] J.H. Suh, S.V. Shenvi, B.M. Dixon, H. Liu, A.K. Jaiswal, R.M. Liu, T.M. Hagen, Decline in
transcriptional activity of Nrf2 causes age-related loss of glutathione synthesis, which is reversible
with lipoic acid, Proceedings of the National Academy of Sciences of the United States of America
101(10) (2004) 3381-6.
[25] D. Botta, C.C. White, P. Vliet-Gregg, I. Mohar, S. Shi, M.B. McGrath, L.A. McConnachie, T.J.
Kavanagh, Modulating GSH synthesis using glutamate cysteine ligase transgenic and gene-targeted
mice, Drug metabolism reviews 40(3) (2008) 465-77.
[26] M. Suzuki, T. Betsuyaku, Y. Ito, K. Nagai, Y. Nasuhara, K. Kaga, S. Kondo, M. Nishimura,
Down-regulated NF-E2-related factor 2 in pulmonary macrophages of aged smokers and patients with
chronic obstructive pulmonary disease, American journal of respiratory cell and molecular biology
39(6) (2008) 673-82.
[27] I. Bellezza, Oxidative Stress in Age-Related Macular Degeneration: Nrf2 as Therapeutic Target,
Front Pharmacol 9 (2018) 1280.
[28] P. Singh, A. Anand, V. Kumar, Recent developments in biological activities of chalcones: A mini
review, European Journal of Medicinal Chemistry 85 (2014) 758-777.
[29] C.E. Gutteridge, D.S. Thota, S.M. Curtis, M.P. Kozar, Q. Li, L. Xie, J. Zhang, V. Melendez, C.O. Asher,
T.T. Luong, L. Gerena, D.A. Nichols, G. Montip, In vitro biotransformation, in vivo efficacy and
pharmacokinetics of antimalarial chalcones, Pharmacology 87(1-2) (2011) 96-104.
[30] J.A. Lee, H.J. Son, J.W. Choi, J. Kim, S.H. Han, N. Shin, J.H. Kim, S.J. Kim, J.Y. Heo, D.J. Kim, K.D. Park,
O. Hwang, Activation of the Nrf2 signaling pathway and neuroprotection of nigral dopaminergic
neurons by a novel synthetic compound KMS99220, Neurochem Int 112 (2018) 96-107.
[31] R. Kachadourian, B.J. Day, S. Pugazhenti, C.C. Franklin, E. Genoux-Bastide, G. Mahaffey, C.
Gauthier, A. Di Pietro, A. Boumendjel, A Synthetic Chalcone as a Potent Inducer of Glutathione

Biosynthesis, Journal of Medicinal Chemistry 55(3) (2012) 1382-1388.
[32] X. Li, Z. Liu, C. Luo, H. Jia, L. Sun, B. Hou, W. Shen, L. Packer, C.W. Cotman, J. Liu, Lipoamide
protects retinal pigment epithelial cells from oxidative stress and mitochondrial dysfunction, Free
radical biology & medicine 44(7) (2008) 1465-74.
[33] L. Jia, Z. Liu, L. Sun, S.S. Miller, B.N. Ames, C.W. Cotman, J. Liu, Acrolein, a toxicant in cigarette
smoke, causes oxidative damage and mitochondrial dysfunction in RPE cells: protection by
(R)-alpha-lipoic acid, Invest Ophthalmol Vis Sci 48(1) (2007) 339-48.
[34] M. Fukui, H.J. Choi, B.T. Zhu, Mechanism for the protective effect of resveratrol against oxidative
stress-induced neuronal death, Free Radical Biology and Medicine 49(5) (2010) 800-813.
[35] J.I. Chuang, J.Y. Huang, S.J. Tsai, H.S. Sun, S.H. Yang, P.C. Chuang, B.M. Huang, C.H. Ching,
FGF9-induced changes in cellular redox status and HO-1 upregulation are FGFR-dependent and
proceed through both ERK and AKT to induce CREB and Nrf2 activation, Free Radic Biol Med 89 (2015)
274-86.
[36] C.J. Vlahos, W.F. Matter, K.Y. Hui, R.F. Brown, A specific inhibitor of phosphatidylinositol 3-kinase,
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), J Biol Chem 269(7) (1994) 5241-8.
[37] B.L. Bennett, D.T. Sasaki, B.W. Murray, E.C. O'Leary, S.T. Sakata, W. Xu, J.C. Leisten, A. Motiwala, S.
Pierce, Y. Satoh, S.S. Bhagwat, A.M. Manning, D.W. Anderson, SP600125, an anthrapyrazolone inhibitor
of Jun N-terminal kinase, Proc Natl Acad Sci U S A 98(24) (2001) 13681-6.
[38] S.H. Yang, A. Galanis, A.D. Sharrocks, Targeting of p38 mitogen-activated protein kinases to MEF2
transcription factors, Mol Cell Biol 19(6) (1999) 4028-38.
[39] S.Y. Yang, M.C. Pyo, M.H. Nam, K.W. Lee, ERK/Nrf2 pathway activation by caffeic acid in HepG2
cells alleviates its hepatocellular damage caused by t-butylhydroperoxide-induced oxidative stress,
BMC Complement Altern Med 19(1) (2019) 139.
[40] L.M. Zipper, R.T. Mulcahy, Erk activation is required for Nrf2 nuclear localization during pyrrolidine
dithiocarbamate induction of glutamate cysteine ligase modulatory gene expression in HepG2 cells,
Toxicol Sci 73(1) (2003) 124-34.
[41] Y. Nakagami, Nrf2 Is an Attractive Therapeutic Target for Retinal Diseases, Oxid Med Cell Longev
2016 (2016) 7469326.
[42] Q. Zhong, M. Mishra, R.A. Kowluru, Transcription factor Nrf2-mediated antioxidant defense
system in the development of diabetic retinopathy, Invest Ophthalmol Vis Sci 54(6) (2013) 3941-8.
[43] M.L. Lambros, S.M. Plafker, Oxidative Stress and the Nrf2 Anti-Oxidant Transcription Factor in
Age-Related Macular Degeneration, Adv Exp Med Biol 854 (2016) 67-72.
[44] Z. Zhao, Y. Chen, J. Wang, P. Sternberg, M.L. Freeman, H.E. Grossniklaus, J. Cai, Age-related
retinopathy in NRF2-deficient mice, PLoS One 6(4) (2011) e19456.
[45] M. Cano, R. Thimmalappula, M. Fujihara, N. Nagai, M. Sporn, A.L. Wang, A.H. Neufeld, S. Biswal,
J.T. Handa, Cigarette smoking, oxidative stress, the anti-oxidant response through Nrf2 signaling, and
Age-related Macular Degeneration, Vision Res 50(7) (2010) 652-64.
[46] X. Zou, Z. Feng, Y. Li, Y. Wang, K. Wertz, P. Weber, Y. Fu, J. Liu, Stimulation of GSH synthesis to
prevent oxidative stress-induced apoptosis by hydroxytyrosol in human retinal pigment epithelial cells:
activation of Nrf2 and JNK-p62/SQSTM1 pathways, J Nutr Biochem 23(8) (2012) 994-1006.
[47] Z. Feng, Z. Liu, X. Li, H. Jia, L. Sun, C. Tian, L. Jia, J. Liu, alpha-Tocopherol is an effective Phase II
enzyme inducer: protective effects on acrolein-induced oxidative stress and mitochondrial dysfunction
in human retinal pigment epithelial cells, J Nutr Biochem 21(12) (2010) 1222-31.
[48] Y. Li, X. Zou, K. Cao, J. Xu, T. Yue, F. Dai, B. Zhou, W. Lu, Z. Feng, J. Liu, Curcumin analog 1, 5-bis

(2-trifluoromethylphenyl)-1, 4-pentadien-3-one exhibits enhanced ability on Nrf2 activation and
protection against acrolein-induced ARPE-19 cell toxicity, Toxicol Appl Pharmacol 272(3) (2013)
726-35.
[49] R.S. Sulaiman, H.D. Basavarajappa, T.W. Corson, Natural product inhibitors of ocular angiogenesis,
Exp Eye Res 129 (2014) 161-71.
[50] D. Malhotra, E. Portales-Casamar, A. Singh, S. Srivastava, D. Arenillas, C. Happel, C. Shyr, N.
Wakabayashi, T.W. Kensler, W.W. Wasserman, S. Biswal, Global mapping of binding sites for Nrf2
identifies novel targets in cell survival response through ChIP-Seq profiling and network analysis,
Nucleic Acids Res 38(17) (2010) 5718-34.
[51] C.A. Silva-Islas, P.D. Maldonado, Canonical and non-canonical mechanisms of Nrf2 activation,
Pharmacol Res 134 (2018) 92-99.
[52] D.D. Zhang, M. Hannink, Distinct cysteine residues in Keap1 are required for Keap1-dependent
ubiquitination of Nrf2 and for stabilization of Nrf2 by chemopreventive agents and oxidative stress,
Mol Cell Biol 23(22) (2003) 8137-51.
[53] K. Nakaso, H. Yano, Y. Fukuhara, T. Takeshima, K. Wada-Isoe, K. Nakashima, PI3K is a key molecule
in the Nrf2-mediated regulation of antioxidative proteins by hemin in human neuroblastoma cells,
FEBS Lett 546(2-3) (2003) 181-4.
[54] G. Filomeni, S. Piccirillo, G. Rotilio, M.R. Ciriolo, p38(MAPK) and ERK1/2 dictate cell death/survival
response to different pro-oxidant stimuli via p53 and Nrf2 in neuroblastoma cells SH-SY5Y, Biochem
Pharmacol 83(10) (2012) 1349-57.
[55] R. Vari, M. D'Archivio, C. Filesi, S. Carotenuto, B. Scazzocchio, C. Santangelo, C. Giovannini, R.
Masella, Protocatechuic acid induces antioxidant/detoxifying enzyme expression through
JNK-mediated Nrf2 activation in murine macrophages, J Nutr Biochem 22(5) (2011) 409-17.
Figure Legends
Figure 1. Synthesis scheme and compounds structure. (A) Synthesis of
1-(2,3,4-trimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-acrylketone
Synthesis of 1-(2,3,4-trimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-acetophenone
(Tap). (C) Synthesis of (2,3,4-trimethoxyphenyl)
(3,4,5-trimethoxyphenyl)-benzophenone (Tbp).
(Tak).
(B)
-
Figure 2. Effects of Tak, Tap, Tbp on cell viability and MMP. ARPE-19 cells were
treated with compounds at indicated doses for 24 h, followed by 75 µM acrolein for
another 24 h, cell viability was assessed by MTT assay, MMP was measured with

JC-1 staining. (A) Effect of Tak on cell viability. (B) Effect of Tap on cell viability. (C)
Effect of Tbp on cell viability. (D) Effect of Tak on MMP. (E) Effect of Tap on MMP.
(F) Effect of Tbp on MMP. Values are means ± S.E.M. from at least three independent
experiments. *P < 0.05, **P < 0.01 vs. relative control.
Figure 3. Effect of Tak on mitochondrial superoxide. ARPE-19 cells were treated
with Tak at indicated doses for 24 h, followed by 300 µM t-BHP for another 6 h, cell
viability (A) and MMP (B) was analyzed. (C) ARPE-19 cells were treated with 10 µM
Tak for 24 h, followed by 75 µM acrolein for another 24 h, mitochondrial superoxide
level was assessed by fluorescent confocal microscope. Values are means ± S.E.M.
from at least three independent experiments. *P < 0.05, **P < 0.01 vs. relative
control.
Figure 4. Effect of Tak on mitochondrial dynamics. ARPE-19 cells were treated
with Tak at indicated doses for 24 h, cells were collected for (A) mitochondrial
biogenesis related proteins, (B) mitochondrial complex subunits mRNA level, (C)
mitochondrial fusion and fission related proteins, (D) Mitotracker Red staining by
fluorescent confocal microscope, (E) mtDNA copy number, (F) cellular ATP level.
Values are means ± S.E.M. from at least three independent experiments. *P < 0.05,
**P < 0.01 vs. relative control.
Figure 5. Effect of compounds on phase II enzymes mRNA. ARPE-19 cells were
treated with compounds at indicated doses for 6 h, mRNA levels of phase II enzymes
were analyzed by qPCR. (A) mRNA level of Nrf2. (B) mRNA level of Keap1. (C)
mRNA level of HO-1. (D) mRNA level of NQO-1. (E) mRNA level of GCLc. (F)
mRNA level of GCLm. Values are means ± S.E.M. from at least three independent
experiments. *P < 0.05, **P < 0.01 vs. relative control.
Figure 6. Effects of Tak on phase II proteins. ARPE-19 cells were treated with Tak
at indicated doses for 24 h, protein levels of HO-1 and NQO-1 were analyzed by
Western blot. (A) Western blot image. (B) Statistical analysis of HO-1. (C) Statistical
analysis of NQO-1. Cells were treated with Tak at indicated doses for 6 h, cytosolic
and nuclear fractions were prepared for analyzing Nrf2 protein level. (D) Western blot
image. (E) Statistical analysis of nuclear Nrf2. Values are means ± S.E.M. from at
least three independent experiments. *P < 0.05, **P < 0.01 vs. relative control.
Figure 7. Tak activates phase II enzymes through Erk pathway. ARPE-19 cells
were treated with Tak at 5 and 10 µM for indicated time, Akt and MAPK pathway
activation was analyzed by western blot (A). ARPE-19 cells were treated with Akt and

MAPK pathway inhibitors for 1 h, followed by Tak treatment for another 6 h, mRNA
levels of (B) HO-1, (C) NQO-1, (D) GCLc, and (E) GCLm were analyzed by qPCR.
Values are means ± S.E.M. from at least three independent experiments. *P < 0.05,
**P < 0.01 vs. relative control.
Table of Contents Graphic

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Highlights
ò Modified chalcone analogue activates Nrf2 through Erk1/2 kinase
ò The compound eliminates acrolein induced mitochondrial ROS overproduction
ò The compound protects ARPE-19 cells against oxidative stress induced cell death
ò α, β unsaturated carbonyl entity of the compound is responsible for Nrf2
activation