An optically active isochroman-2H-chromene conjugate potently suppresses neuronal oxidative injuries associated with the PI3K/Akt and MAPK signaling pathways

Article abstract of DOI:10.1038/s41401-020-0391-9

Increasing evidence suggests that the use of potent neuroprotective agents featured with novel pharmacological mechanism would offer a promising strategy to delay or prevent the progression of neurodegeneration. Here, we provide the first demonstration that the chiral nonracemic isochroman-2H-chromene conjugate JE-133, a novel synthetic 1,3-disubstituted isochroman derivative, possesses superior neuroprotective effect against oxidative injuries. Pretreatment with JE-133 (1–10 μM) concentration-dependently prevented H₂O₂-induced cell death in SH-SY5Y neuroblastoma cells and rat primary cortical neurons. Pretreatment with JE-133 significantly alleviated H₂O₂-induced apoptotic changes. These protective effects could not be simply attributed to the direct free radical scavenging as JE-133 had moderate activity in reducing DPPH free radical. Further study revealed that pretreatment with JE-133 (10 μM) significantly decreased the phosphorylation of MAPK pathway proteins, especially ERK and P38, in the neuronal cells. In addition, blocking PI3K/Akt pathway using LY294002 partially counteracted the cell viability-enhancing effect of JE-133. We conclude that JE-133 exerts neuroprotection associated with dual regulative mechanisms and consequently activating cell survival and inhibiting apoptotic changes, which may provide important clues for the development of effective neuroprotective drug lead/candidate.

Full text of DOI:10.1038/s41401-020-0391-9

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ARTICLE
An optically active isochroman-2H-chromene conjugate
potently suppresses neuronal oxidative injuries associated
with the PI3K/Akt and MAPK signaling pathways
Ling-xue Tao1, Sha-sha Ji1, Dóra Szalóki2, Tibor Kovács2, Attila Mándi2, Sándor Antus2, Xun Ding1, Tibor Kurtán2 and Hai-yan Zhang1
Increasing evidence suggests that the use of potent neuroprotective agents featured with novel pharmacological mechanism would
offer a promising strategy to delay or prevent the progression of neurodegeneration. Here, we provide the first demonstration that
the chiral nonracemic isochroman-2H-chromene conjugate JE-133, a novel synthetic 1,3-disubstituted isochroman derivative,
possesses superior neuroprotective effect against oxidative injuries. Pretreatment with JE-133 (1–10 μM) concentration-dependently
prevented H2O2-induced cell death in SH-SY5Y neuroblastoma cells and rat primary cortical neurons. Pretreatment with JE-133
significantly alleviated H2O2-induced apoptotic changes. These protective effects could not be simply attributed to the direct free
radical scavenging as JE-133 had moderate activity in reducing DPPH free radical. Further study revealed that pretreatment with
JE-133 (10 μM) significantly decreased the phosphorylation of MAPK pathway proteins, especially ERK and P38, in the neuronal cells.
In addition, blocking PI3K/Akt pathway using LY294002 partially counteracted the cell viability-enhancing effect of JE-133. We
conclude that JE-133 exerts neuroprotection associated with dual regulative mechanisms and consequently activating cell survival
and inhibiting apoptotic changes, which may provide important clues for the development of effective neuroprotective drug lead/
candidate.
Keywords: neurodegeneration; oxidative stress; apoptosis; MAPK pathway; PI3K/Akt pathway; SH-SY5Y neuroblastoma cells
Acta Pharmacologica Sinica (2020) 0:1–9; https://doi.org/10.1038/s41401-020-0391-9
INTRODUCTION
oxidative stress, with the mitogen-activated protein kinase (MAPK)
[18, 19] and phosphatidylinositol 3-kinase (PI3K)/Akt [16, 20, 21]
pathways being of great importance. The activation of the MAPK
signaling pathway, including the phosphorylated proteins c-Jun N-
terminal kinase (JNK), P38 and extracellular signal-regulated kinase
1/2 (ERK1/2), triggers the cleavage of apoptosis-related proteins,
such as caspase-3, and consequently leads to apoptosis [18, 22–
24]. In contrast, the PI3K/Akt pathway is critical for neuronal
survival through the promotion of cell survival and the inhibition
of apoptosis induced by deleterious stimuli [20, 21, 25–27]. The
above findings provide an alternative and promising therapeutic
strategy for resisting oxidative insults.
Our laboratory is engaged in identifying potent antioxidants
through cell-based phenotypic screening [28, 29], which is
believed to well simulate complex pathologic cellular environ-
ments [30] of interacting proteins and signaling pathways and to
possess distinct advantages over single molecular target-based
screening for drug discovery and development [30–32]. Mean-
while, this method maintains the capacity for high-throughput
screening (HTS) [30]. Employing cell-based phenotypic screening,
we discovered that the chiral nonracemic isochroman-2H-chro-
mene conjugate JE-133 (structure shown in Fig. 1a), a novel 1,3-
disubstituted isochroman derivative, shows potent antioxidative
In recent years, the utilization of antioxidants in the treatment of
Alzheimer’s disease (AD) has been a popular research topic [1–3].
Oxidative stress results from the accumulation of reactive oxygen
species (ROS) or an excess of free radicals and is involved in
multiple pathological processes of neurodegenerative diseases,
including AD [4–6]. As a consequence, a series of clinical trials have
been conducted with the aim of treating or preventing AD using
antioxidants [7, 8], such as a mixture of vitamin C and vitamin E [9],
the natural polyphenolic compound curcumin [10] and Ginkgo
biloba tree leaf extract (EGb761) [11]. Many of the abovementioned
compounds have shown promising antioxidative and cognitive
enhancing effects in animal experiments; however, there is
currently no curative therapy for AD [12, 13]. Hence, the discovery
of new lead compounds with novel antioxidative pharmacological
mechanisms is promising for achieving better therapeutic benefits.
Antioxidative interventions aimed at directly scavenging free
radicals have not yet exhibited satisfactory clinical efficacy.
Alternatively, accumulating studies have indicated that various
small molecules can significantly ameliorate neuronal oxidative
damage by activating prosurvival pathways or inhibiting the
pathogenic pathways [14–17]. Evidence has shown that many
complex signaling pathways are involved in the process of
1CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China and 2Department of Organic Chemistry,
University of Debrecen, Debrecen, P. O. Box 400, H-4002 Debrecen, Hungary
Correspondence: Tibor Kurtán (kurtan.tibor@science.unideb.hu) or Hai-yan Zhang (hzhang@simm.ac.cn)
These authors contributed equally: Ling-xue Tao, Sha-sha Ji
Received: 26 August 2019 Accepted: 23 February 2020
© CPS and SIMM 2020

JE133 regulates neuronal PI3K/Akt and MAPK pathways
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Fig. 1 Synthesis and stereochemical analysis of the compound JE-133. a Synthesis of the compound JE-133. b Twenty computed low-energy
overlapping solution conformers (level of DFT optimization: CAM-B3LYP/TZVP PCM/MeCN) of (1R,3R,2’S)-JE-133 with an overall Boltzmann
population of 87.4%. c Experimental ECD spectrum of JE-133 (black curve) compared with the Boltzmann-weighted PBE0/TZVP (PCM/MeCN)
ECD spectrum (red curve) of (1R,3R,2’S)-JE-133 computed for the B97D/TZVP PCM/MeCN conformers. The bars represent the rotational
strengths of the lowest-energy conformer
effects on SH-SY5Y neuroblastoma cells. To better understand the
protective potential of JE-133, the present study was designed to
further verify the antioxidant effects of JE-133 in SH-SY5Y cells and
rat primary cortical neurons, and to elucidate whether the MAPK
and PI3K/Akt signaling pathways are involved in the above-
mentioned neuroprotective effects.
Briefly, the cortices were rapidly dissected and minced on ice
and then dissociated in 0.125% trypsin with 0.2 mg/mL DNase I at
37 °C for 15 min. Next, the tissues were triturated with a pipette in
HG-DMEM medium with 10% FBS and filtered through sieves
(300/400 mesh). The cells were plated in plates coated with poly-L-
lysine (Sangon Biotech, Shanghai, China). The culture medium was
replaced with neurobasal medium (Invitrogen, Carlsbad, CA, USA)
containing 0.5 mM L-glutamine, 2% B27 supplement, penicillin
(60 mg/L) and streptomycin (50 mg/L) after 4 h. Half of the culture
medium was refreshed every 3 days. The neurons were allowed to
grow for 9 days before further treatment.
MATERIALS AND METHODS
Preparation of compounds
JE-133 and N-acetylcysteine (NAC, Sigma-Aldrich, St. Louis, MO,
USA) were dissolved in dimethyl sulfoxide (DMSO) at
concentration of 10 mM to generate stock solutions, stored at
a
Cell viability measurement
−20 °C and diluted with culture medium before use.
Morphological changes in SH-SY5Y cells and primary cortical
neurons were observed with an inverted optical microscope
(Nikon TE2000, Melville, NY, USA), and cell viability was evaluated
by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium
bromide (MTT, Sigma-Aldrich) assay. Briefly, SH-SY5Y cells or
primary cortical neurons (3 × 104 cells/well in 96-well plates) were
incubated with 100 μM or 300 μM H2O2 at 37 °C for 24 h after
being pretreated with or without JE-133 (or NAC) and then
incubated with MTT at a final concentration of 0.5 mg/mL for 3 h.
The medium was removed, and 100 μL DMSO was added. The cell
plates were shaken for 5 min, and the absorbance of each well was
read at 490 nm using a microplate reader (DTX 800 Multimode
Detector, Beckman Coulter, Fullerton, CA). The optical density (OD)
values of all groups were normalized to the values of the control
SH-SY5Y cell culture
Human SH-SY5Y neuroblastoma cells were purchased from
American Type Culture Collection (ATCC, Manassas, VA, USA).
SH-SY5Y cells were cultured in MEM/F12 medium (Gibco, Carlsbad,
CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco),
60 mg/L penicillin and 50 mg/L streptomycin in a humid atmo-
sphere of 5% CO2 at 37 °C. SH-SY5Y cells were seeded into 96- or
6-well plates at a density of 3 × 105 cells/mL.
Primary cortical neuron culture
Primary cortical neuron cultures were prepared from 17-day-old
embryos of Sprague-Dawley rats as previously described [33].
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group, and cell viability is presented as the percentage of
surviving cells relative to that in the control group.
RESULTS
Synthesis and stereochemical analysis of JE-133
JE-133 was obtained by the diastereoselective oxa-Pictet-Spengler
cyclization of 4-[(R)-2-hydroxypropyl]-benzene-1,2-diol (3) with ethyl
(2S)-3-formyl-6-methoxy-2H-chromene-2-carboxylate (4) (Fig. 1a).
Optically active 3 [34] was prepared in two steps; 1,2-bis-
(benzyloxy)-4-bromobenzene (1) was lithiated with n-butyllithium
and reacted with (R)-propylene oxide in the presence of BF3·OEt2,
and then the benzyl protective groups were removed by catalytic
hydrogenation (2 → 3). Optically active 4 was obtained from
2-hydroxy-5-methoxybenzaldehyde through an enantioselective
organocatalytic oxa-Michael/aldol condensation domino reaction
according to the procedure of Córdova et al. [35] with 93%
enantiomeric excess. The oxa-Pictet-Spengler cyclization of 3 with 4
retained the (R) and (S) absolute configuration of the reactants, and
a new chirality center was established at C-1 of the isochroman unit
diastereoselectively with (R) absolute configuration. The (1R,3R)
absolute configuration of the isochroman moiety afforded the low-
energy equatorial arrangement of the C-1 and C-3 substituents,
which was confirmed by the NOE correlation between the axial 1-H
and 3-H oriented toward the same face of the molecule.
Apoptosis assay
Apoptosis was detected by Annexin V-FITC and PI staining
following the procedures described in the apoptosis detection
kit (Rainbio, Shanghai, China). Annexin V is used to detect early
apoptotic cells, and propidium iodide (PI) can penetrate into the
nuclei of cells undergoing late-stage apoptosis, while intact cells
cannot be stained by Annexin V or PI. SH-SY5Y cells (6 × 105 cells/
well in 6-well plates) were incubated with 100 μM H2O2 for 24 h
after being pretreated with or without JE-133 (or NAC). The cells
were then collected, suspended in 500 μL binding buffer, and
stained with 5 μL Annexin V-FITC and 5 μL PI. After 15 min of
reaction in the dark, the apoptosis rates of the different groups
(10,000 cells for each group) were analyzed by flow cytometry (BD
Biosciences, San Jose, CA, USA). The FL-1 and FL-3 channels were
chosen for the detection of Annexin V-FITC- and PI-labeled cells,
respectively.
DPPH free radical scavenging assay
The free radical scavenging ability of JE-133 was analyzed using
stable 1,1-diphenyl-2-picrylhydrazyl (DPPH, Sigma-Aldrich) dis-
solved in 95% ethanol. Briefly, various concentrations of JE-133
(or NAC) in 20 μL solution were added to 180 μL DPPH radical
solution (80 μM) and incubated for 60 min in the dark at room
temperature. The absorbance of the mixture was measured with a
microplate reader (DTX 800 Multimode Detector, Beckman Coulter,
Fullerton, CA) at 517 nm. The absorbance value was then converted
to the DPPH free radical scavenging rate according to the following
equation: DPPH scavenging rate (IP%) = [1−(Ai−Aj)/Ac] × 100%,
with Ai representing the absorbance value of the compounds upon
reaction with DPPH; Aj representing the absorbance value of the
compounds upon reaction with 95% ethanol; and Ac representing
the absorbance value of DPPH solution.
Since the three-dimensional shape of chiral nonracemic JE-133
is believed to be an important factor for its activity, the solution
conformation and absolute configuration of JE-133 were studied
by electronic circular dichroism (ECD) measurements supported
by the solution time-dependent density functional theory-
electronic circular dichroism (TDDFT-ECD) calculation protocol
[36, 37]. In all the computed low-energy conformers, the isochro-
man and 2H-chromene subunits had the same well-defined
relative arrangement, even though they were attached by a
rotatable single bond (Fig. 1b). The C-1 and C-3 substituents
adopted equatorial orientation, while the C-2′ ethoxycarbonyl
group had axial orientation. The Boltzmann-weighted computed
ECD spectra gave good agreement with the experimental ECD
spectrum of (1R,3R,2′S)-JE-133, which confirmed the (1R,3R,2’S)
absolute configuration and determined the preferred solution
conformation.
Western blot analysis
After exposure to 100 μM (SH-SY5Y cells) or 300 μM (primary
neurons) H2O2 for 15 min (6 h or 24 h) following pretreatment with
or without JE-133 (or LY294002, purchased from Selleck, Shanghai,
China), the cells were lysed in RIPA buffer [50 mM Tris 50, 150 mM
NaCl, 0.5% (v/v) sodium-deoxycholate, 1% (v/v) Triton X-100, 0.1%
(v/v) SDS 0.1%, and 2 mM EDTA, pH 7.4] plus protease inhibitor
cocktail (Sigma-Aldrich) for 30 min on ice and then centrifuged at
12,000 × g for 15 min at 4 °C. The supernatants were collected, and
the protein concentrations were determined using a BCA assay kit
(Pierce, Rockford, IL, USA). The proteins were separated by 10% or
12% SDS-PAGE and transferred to 0.2-μm nitrocellulose mem-
branes. After blocking with 5% nonfat milk for 1 h at room
temperature, the membranes were incubated with the following
primary antibodies at 4 °C overnight: anti-cleaved caspase-3 (1:500,
Cell Signaling, Beverly, MA, USA); anti-caspase-3 (1:2000, Cell
Signaling); anti-p-Akt (1:1000, Cell Signaling); anti-Akt (1:3000, Cell
Signaling); anti-p-JNK (1:800, Cell Signaling); anti-JNK (1:1000, Cell
Signaling); anti-p-P38 (1:1000, Cell Signaling); anti-P38 (1:2000, Cell
Signaling); anti-p-ERK1/2 (1:1000, Cell Signaling); anti-ERK1/2
(1:2000, Cell Signaling); and anti-β-Actin (1:10,0000, Sigma-Aldrich).
Then, they were incubated with HRP-conjugated secondary
antibodies (1:5,000; Kangcheng, Shanghai, China) for 2 h at room
temperature. The bands were visualized by autoradiography after
development by an ECL plus kit (Millipore, Billerica, MA, USA), and
the intensity of each band was quantified with ImageJ software.
The best agreement was produced by the PBE0/TZVP PCM/
MeCN ECD computed for the CAM-B3LYP/TZVP PCM/MeCN
conformers, which is shown in Fig. 1c.
The spectroscopic characterization and additional stereochemi-
cal results are shown in supplementary Figs. S1 –S7.
JE-133 attenuated H2O2-induced neurotoxicity
SH-SY5Y cells exposed to H2O2 for 24 h exhibited morphological
cell damage characterized by dendrite fragmentation, body
swelling and decreased cell number (Fig. 2a). In contrast, the cell
morphological damage induced by H2O2 was markedly attenu-
ated by pretreatment with 10 μM JE-133 (Fig. 2a). Consistent with
the morphological observations, H2O2-induced cytotoxicity was
also confirmed by a significant reduction in values in the MTT
assay (50.62% 3.23%, P < 0.001) (Fig. 2b) and excessive release of
LDH (34.28% 1.84%, P < 0.001) (supplementary Fig. S8) com-
pared to those of the control group. However, pretreatment with
JE-133 markedly attenuated cytotoxicity induced by H2O2 in a
concentration-dependent manner, with the maximum protective
effect at a concentration of 10 μM (cell viability restored to 95.88%
3.06%, P < 0.001 vs the H2O2 group) (Fig. 2b). Similarly, in cells
pretreated with 3 and 10 μM JE-133, intracellular LDH release was
significantly inhibited (25.25% 2.37%, P < 0.05; 12.97% 1.44%,
P < 0.001, respectively) (Supplementary Fig. S8). The antioxidant
NAC, the positive control, significantly attenuated the decline in
cell viability in the MTT and LDH assays (78.25% 2.14%, P < 0.001;
16.21% 2.48%, P < 0.001, respectively) (Fig. 2b and supplemen-
tary Fig. S8). To verify whether the neuroprotective effects of JE-
133 against H2O2 are limited to SH-SY5Y cells, we also evaluated
its protective effects on primary cultured cortical neurons. Similar
as in SH-SY5Y cells, JE-133 markedly attenuated H2O2-induced cell
Statistical analysis
All data are presented as the mean SEM and were analyzed by
Student’s t-test for single comparisons or by one-way ANOVA
followed by Dunnett’s test for multiple comparisons. P-values
below 0.05 were considered statistically significant.
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Fig. 2 Effects of JE-133 on H2O2-induced cytotoxicity in neuronal cells. Cells were pretreated with different concentrations of JE-133 (1, 3 or
10 μM) or NAC (100 μM) for 2 h and then exposed to H2O2 for 24 h. The morphology of SH-SY5Y cells (a) and primary cortical neurons
(c) observed under a microscope at 100× magnification after different treatments. Scale bar = 100 μm. The viability of SH-SY5Y cells (b) and
primary cortical neurons (d) was determined using the MTT assay, n = 4. The data are presented as the mean SEM. ###P < 0.001 vs the control
group; *P < 0.05, **P < 0.01, ***P < 0.001 vs the H2O2 group
morphological damage (Fig. 2c) and alleviated the H2O2-induced
reduction in cell viability in a concentration-dependent manner in
primary cultured cortical neurons (Fig. 2d).
98.67%. In contrast, the clearance ratio of DPPH free radicals by
10 μM JE-133, the working concentration against H2O2-induced
oxidative cytotoxicity, was 15.00% (Fig. 3d).
JE-133 pretreatment protected SH-SY5Y cells against H2O2-
induced apoptosis
JE-133 prevented the upregulation of MAPK pathway protein
phosphorylation induced by H2O2 in neuronal cells
To explore whether the neuroprotective effects of JE-133 in SH-
SY5Y cells are associated with the amelioration of apoptotic
changes, flow cytometry with Annexin V-FITC and PI double
staining was performed. SH-SY5Y cells in the upper and lower
right quadrants represented cells that underwent the late and
early stages of apoptosis, respectively. As shown in Fig. 3a, b, after
exposure to H2O2 for 24 h, the percentage of SH-SY5Y cells in the
early and late stages of apoptosis was greatly increased from
24.45% 3.89% to 49.25% 1.60% (P < 0.01 vs the control group).
However, pretreatment with 3 and 10 μM JE-133 suppressed the
apoptosis ratio to 30.83% 4.35% (P < 0.05 vs the H2O2 group)
and 25.79% 5.13% (P < 0.01 vs the H2O2 group), respectively.
Similarly, 100 μM NAC significantly decreased the rate of apoptosis
to 29.12% 5.46% (P < 0.05 vs the H2O2 group). The cleavage of
caspase-3 was examined by Western blot analysis. As shown in
Fig. 3c, H2O2 caused a robust increase in the level of cleaved
caspase-3 compared to that in the control group (9.86 1.09-fold
of the control group, P < 0.01). In contrast, the activation of
caspase-3 was markedly alleviated in SH-SY5Y cells upon
preincubation with JE-133 (10 μM) for 2 h followed by exposure
to H2O2 for 24 h, as shown by a significant reduction in cleaved
caspase-3 levels (1.60 0.26-fold of the control group, P < 0.01 vs
the H2O2 group) (Fig. 3c).
To reveal the potential molecular mechanism involved in the
antioxidative effect of JE-133, its influence on ERK1/2, P38 and JNK
phosphorylation was measured. The exposure of SH-SY5Y cells to
H2O2 for 6 h significantly increased the levels of phosphorylated
ERK1/2, P38 and JNK (3.14 0.34-fold of the control group, P <
0.01; 12.19 1.76-fold of the control group, P < 0.01; and 2.28
0.06-fold of the control group, P < 0.001 vs the control group,
respectively) (Fig. 4a–c). In contrast, upon pretreatment with
10 μM JE-133, the H2O2-induced increases in ERK1/2, P38 and JNK
phosphorylation were markedly decreased (1.48 0.09-, 4.96
1.10-, and 1.40 0.18-fold of the control group, respectively, P <
0.05 for all vs the H2O2 group) (Fig. 4a–c). We also evaluated
changes in the MAPK pathway in primary cortical neurons
exposed to H2O2 after pretreatment with or without JE-133. As
shown in Fig. 4d–f, the exposure of primary cortical neurons to
H2O2 for 6 h significantly increased the levels of phosphorylated
ERK1/2 and P38 (1.64 0.08-fold of control group, P < 0.001;
1.80 0.11-fold of the control group, P < 0.01, respectively). Upon
pretreatment with 10 μM JE-133, the H2O2-induced increases in
ERK1/2 and P38 phosphorylation were markedly decreased
(1.27 0.11- and 1.38 0.12-fold of the control group, respec-
tively, P < 0.05 for both vs the H2O2 group) (Fig. 4d, e). H2O2
treatment with or without JE-133 had no effect on JNK
phosphorylation (Fig. 4f).
JE-133 (at its working concentration) had a weak ability to
scavenge DPPH free radicals against H2O2-induced oxidative
cytotoxicity
We next examined the DPPH radical scavenging capacity of JE-133
to further understand its antioxidative mechanism using the
antioxidant NAC as a positive control. The scavenging ratio of
100 μM NAC, the working concentration that was effective against
H2O2-induced oxidative cytotoxicity, on DPPH free radicals was
PI3K/Akt pathway partially mediated the neuroprotective effects
of JE-133
As the PI3K/Akt signaling pathway plays a key role in cell survival,
we further evaluated whether the neuroprotective effect of JE-133
against H2O2-induced neurotoxicity is related to the PI3K/Akt
pathway. The exposure of SH-SY5Y cells to H2O2 for 5–60 min
increased the phosphorylation of Akt, and the maximal
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Fig. 3 Effects of JE-133 on H2O2-induced apoptosis in SH-SY5Y cells and on free radical scavenging. Cells were pretreated with various
concentrations of JE-133 or 100 μM NAC for 2 h and then exposed to H2O2 for 24 h. a Representative FACS analyses of apoptosis. Q1: necrotic
cells; Q2: late apoptotic cells; Q3: early apoptotic cells; Q4: live cells. The apoptosis rate is equal to the sum of Q2 and Q3. b A histogram of the
percentage of apoptotic cells, n = 4. c Statistical results of cleaved caspase-3. The protein levels of caspase-3 and cleaved caspase-3 were
detected by Western blotting using β-actin as a loading control, n = 3. d The direct radical scavenging capacity of FAN was determined using
the DPPH free radical scavenging assay, n = 3. The data are presented as the mean SEM. ##P < 0.01 vs the control group; *P < 0.05, **P < 0.01
vs the H2O2 group
phosphorylation level of Akt was reached 15 min after H2O2
exposure (data not shown). In contrast, pretreatment with JE-133
(10 μM) for 2 h significantly enhanced the phosphorylation of Akt
(P < 0.05 vs the H2O2 group) in H2O2-exposed SH-SY5Y cells
(Fig. 5a). We then used LY294002 (an inhibitor of PI3K/Akt
activation) to further evaluate how the PI3K/Akt signaling pathway
is involved in the antioxidative neuroprotective effects of JE-133 in
SH-SY5Y cells. As shown in Fig. 5b, the neuroprotective effects of
JE-133 against H2O2-induced SH-SY5Y cell injury (95.58% 0.43%,
P < 0.001 vs the H2O2 group) were effectively antagonized by 5 μM
LY294002 (80.24% 0.30%, P < 0.05 vs the H2O2 + JE-133 group).
In contrast, pretreatment with 5 μM LY294002 for 2 h exerted no
influence on the viability of SH-SY5Y cells incubated with or
without H2O2. As in SH-SY5Y cells, the protective effect of JE-133
against H2O2-induced reduction in cell viability was inhibited by
LY294002 in primary cortical neurons (Fig. 5c). JE-133 pretreat-
ment had no effect on the phosphorylation of Akt in H2O2-
exposed primary neurons (supplementary Fig. S9).
Blocking the PI3K/Akt pathway influenced the suppressive effects
of JE-133 on the phosphorylation of MAPK pathway proteins in
H2O2-exposed SH-SY5Y cells
We further investigated the relationship between the activation of
PI3K/AKT signaling and the inhibition of MAPK regulation induced
by JE-133 treatment. The cells were pretreated with LY294002
and/or JE-133 (10 μM) for 2 h and then exposed to H2O2 for 6 h,
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Fig. 4 Effects of JE-133 on the phosphorylation of MAPK pathway proteins in H2O2-exposed neuronal cells. Cells were pretreated with JE-133
(10 μM) for 2 h and then exposed to H2O2 for 6 h. The protein levels of p-ERK1/2 (a), p-P38 (b), and p-JNK (c) in SH-SY5Y cells, n = 3. The protein
levels of p-ERK1/2 (d), p-P38 (e), and p-JNK (f) in primary cortical neurons, n = 4. The above protein levels were measured by Western blotting
using β-actin as a loading control. The data are presented as the mean SEM. ##P < 0.01, ###P < 0.001 vs the control group; *P < 0.05 vs the
H2O2 group
and the phosphorylation of MAPK pathway proteins was then
measured. Pretreatment with LY294002 significantly reversed the
inhibitory effects of JE-133 on P38 phosphorylation (P < 0.01 vs the
H2O2 + JE-133 group, Fig. 6a) and ERK1/2 phosphorylation (P <
0.05 vs the H2O2 + JE-133 group, Fig. 6b) but did not reverse the
inhibitory effects of JE-133 on JNK phosphorylation (Fig. 6c) in
H2O2-exposed SH-SY5Y cells. In addition, pretreatment with
LY294002 had no influence on P38 and ERK1/2 phosphorylation
in cells not exposed to H2O2 (Fig. 6a, b).
Hydrogen peroxide (H2O2), a major form of ROS produced
during oxidative stress, plays an important role in the develop-
ment and progression of neurodegenerative diseases, including
AD. Exogenous H2O2 can penetrate the cell membrane, generate
various free radicals and cause toxicity to neurons in the AD brain
[38]. H2O2 is widely used as an inducer of oxidative stress to
establish cellular oxidative damage models [39–42]. In our study,
an H2O2-stimulated SH-SY5Y neuronal injury model was used for
cell-based phenotypic screening to identify effective lead
compounds that protect against oxidative neuronal injury.
Consistent with previous studies [40, 43–45], the addition of
H2O2 to SH-SY5Y cells induced obvious neuronal damage, as
indicated by cell morphology observations and MTT and LDH
measurements (Fig. 2a, b and Supplementary Fig. S8). These
aberrant changes were significantly ameliorated by JE-133 in a
concentration-dependent manner to a greater extent than the
positive control (Fig. 2a, b and Supplementary Fig. S8). Moreover,
similar neuroprotective effects of JE-133 were also verified in
DISCUSSION
In the current study, we demonstrated for the first time that (i) JE-
133, a novel optically active isochroman-2H-chromene conjugate,
exhibited potent neuroprotective effects against H2O2-induced
aberrant changes in SH-SY5Y cells and primary cortical neurons
and (ii) the MAPK and PI3K/Akt pathways were both closely
associated with the neuroprotective effects mediated by JE-133.
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JE133 regulates neuronal PI3K/Akt and MAPK pathways
LX Tao et al.
7
Fig. 5 The PI3K/Akt pathway mediated the neuroprotective effect of JE-133 in H2O2-exposed neuronal cells. a The level of p-AKT detected by
Western blotting, n = 3. b The effect of LY294002 on the H2O2-induced reduction in viability of SH-SY5Y cells, n = 3. c The effect of LY294002
on the H2O2-induced reduction in viability of primary cortical neurons, n = 4. Cell viability was detected by the MTT assay. The data are
presented as the mean SEM. #P < 0.05, ###P < 0.001 vs the control group; *P < 0.05, ***P <0.001 vs the H2O2 group; &P < 0.05 vs the H2O2 + JE-
133 group
Fig. 6 Effect of LY294002 on suppressive effect of JE-133 on the phosphorylation of MAPK pathway proteins in H2O2-exposed SH-SY5Y cells.
a The protein levels of p-P38 and P38. b The protein levels of p-ERK1/2 and ERK1/2. c The protein levels of p-JNK and JNK. The above protein
levels were analyzed by Western blotting using β-actin as a loading control. A histogram of the protein expression ratio of p-P38 (p-ERK1/2, p-
JNK)/β-actin based on the results of six independent experiments. The data are presented as the mean SEM. #P < 0.05, ##P < 0.01 vs the
control group; *P < 0.05, ***P < 0.001 vs the H2O2 group; &P < 0.05, &&P < 0.01 vs the H2O2 + JE-133 group
H2O2-stimulated primary cortical neurons (Fig. 2c, d). The above
results prove that JE-133 is a novel small molecule with potent
antioxidant effects and the capacity to attenuate oxidative stress-
induced neuronal injury.
oxidative stress. Free radicals damage the plasma membrane,
impair mitochondrial function, result in insufficient cell energy
supply, and give rise to DNA fragmentation [49]. Antioxidants such
as NAC [50], a classic antioxidant used as a positive control in our
study, directly react with free radicals. The antioxidative capacity
of JE-133 was estimated using DPPH free radicals, which have
excellent stability and are widely used for testing free radical
scavenging activity [51]. The results showed that JE-133 had
moderate free radical scavenging activity (Fig. 3d), indicating that
the neuroprotective effect of JE-133 is not mainly attributed to its
free radical scavenging ability.
Numerous studies have indicated that the MAPK signaling
pathway plays a critical role in oxidative stress-induced cell
damage [52, 53]. MAPK signaling initiates the mitochondrial
apoptotic pathway through enhanced pro-apoptotic protein
activation [54, 55]. Consistent with previous reports [24, 56],
H2O2 stimulated all three MAPK signaling proteins in SH-SY5Y cells
in our study. Similar stimulation of the ERK and P38 pathways but
A large number of studies have shown that oxidative stress
injury eventually leads to apoptosis. Under oxidative stress insult,
excessive ROS accumulation can activate caspase-3 in various
cells, including neurons [22, 41, 46–48]. By double staining SH-
SY5Y cells with Annexin V-FITC and PI, we confirmed that the
apoptosis rate of the H2O2-exposed group was significantly
increased and that this increase was effectively suppressed by
JE-133 treatment (Fig. 3a, b). Taken together, these results suggest
that the ability of JE-133 to downregulate caspase-3 cleavage in
H2O2-exposed SH-SY5Y cells (Fig. 3c) and the neuroprotective
effects afforded by JE-133 may be partially attributed to its anti-
apoptotic ability.
The imbalance between the generation and scavenging of
intracellular free radicals is considered the key factor in triggering
Acta Pharmacologica Sinica (2020) 0:1 – 9

JE133 regulates neuronal PI3K/Akt and MAPK pathways
LX Tao et al.
8
on P38 and ERK phosphorylation may be a downstream effect of
its ability to trigger Akt signaling. In contrast, our results indicate
that the inhibitory effect of JE-133 on JNK phosphorylation is
independent of its regulation of Akt signaling, which is in line with
the incomplete inhibitory effect of LY294002 on the JE-133-
induced enhancement of cell survival. This dual regulatory effect
of JE-133 on the MAPK and PI3K/Akt pathways may provide the
first insight into the molecular mechanisms underlying the
neuroprotective effect of this novel small molecule. Overall, as a
novel synthetic 1,3-disubstituted isochroman derivative obtained
by diastereoselective oxa-Pictet-Spengler cyclization, JE-133
affords superior neuroprotective efficacy against H2O2-induced
oxidative neuronal injuries associated with the regulation of the
MAPK and PI3K/Akt signaling pathways (Fig. 7). Our study might
provide important clues for the development of effective drug
leads/candidates against oxidative stress-associated neurodegen-
eration. However, further animal studies are needed to determine
the in vivo efficacy of JE-133.
ACKNOWLEDGEMENTS
This work was supported by the National Natural Science Foundation of China (grant
numbers. 81703507, 81872859, 81522045). The research of the Hungarian authors
was supported by the EU and cofinanced by the European Regional Development
Fund under the project GINOP-2.3.2-15-2016-00008 and the National Research
Development and Innovation Office (grant number: K-120181).
AUTHOR CONTRIBUTIONS
LXT and HYZ designed the pharmacological experiments; LXT, SSJ, and XD performed
the pharmacological assays and analyzed the data; DS, TK, AM, and SA synthesized
JE-133; TK instructed the chemical synthesis and wrote the part of the manuscript
involving the chemical synthesis; LXT, SSJ, and HYZ wrote the manuscript with input
from all of the authors.
Fig. 7 Schematic diagram of the beneficial effects of JE-133 against
H2O2-induced neurotoxicity and the potential mechanisms. H2O2
exposure induces intracellular oxidative stress, which triggers the
overphosphorylation of the MAPK pathway proteins ERK1/2, P38
and JNK, and induces the upregulation of Akt phosphorylation,
consequently activating neuronal apoptosis and ultimately leading
to neuronal damage. JE-133 prevents H2O2-induced neuronal
damage, which might be mediated by the downregulation of
ERK1/2 and P38 phosphorylation and associated with the PI3K/Akt
pathway. The dual regulatory effects on the PI3K/Akt and MAPK
pathways might synergistically contribute to the neuroprotective
effects of JE-133
ADDITIONAL INFORMATION
The online version of this article (https://doi.org/10.1038/s41401-020-0391-9)
contains supplementary material, which is available to authorized users.
Competing interests: The authors declare no competing interests.
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