N,N-disubstituted azines attenuate LPS-mediated neuroinflammation in microglia and neuronal apoptosis via inhibiting MAPK signaling pathways

Article abstract of DOI:10.1186/s12868-017-0399-3

Background: Activated microglia interact with astrocytes and neuronal cells to induce neuroinflammation, which can contribute to the pathogenesis and progression of Alzheimer's and Parkinson's disease. To identify the most effective anti-neuroinflammatory agent, we designed and synthesized a family of 13 new azine derivatives and investigated their anti-neuroinflammatory activities in LPS-activated BV-2 microglial cells. Results: Out of 13 derivatives, compound 3 [4,4'-(1E,1'E,3E,3'E)-3,3'-(hydrazine-1,2-diylidene) bis-(prop-1-ene-1-yl-3-ylidene) bis-(2-methoxyphenol)] exhibited excellent anti-neuroinflammatory activities (IC₅₀ = 12.47 μM), which protected neurons from microglia-mediated neurotoxicity. Specifically, the anti-neuroinflammatory effects of compound 3 inhibited MAPK signaling pathways through the inhibition of p38 and JNK mediated signaling and the production of pro-inflammatory cytokines, and inflammatory mediators. Additionally, compound 3 strongly exhibited neuroprotective effect by inhibiting LPS-mediated necrosis and apoptosis. Preliminary SAR analysis suggests that the presence of methoxyphenol and the substitution pattern within hydrazine may influence the anti-neuroinflammatory activity. FACS analysis also strongly supports the neuroprotective effect of compound 3. Conclusions: Based on our results, the compound 3 exhibited excellent anti-neuroinflammatory activity against LPS-activated microglia, which resulted in the inhibition of neuronal apoptosis and neuronal degeneration.

Full text of DOI:10.1186/s12868-017-0399-3

et al. BMC Neurosci (2017) 18:82
Subedi
BMC Neuroscience
https://doi.org/10.1186/s12868-017-0399-3
Open Access
RESEARCH ARTICLE
N,N-disubstituted azines attenuate
LPS-mediated neuroinfammation in microglia
and neuronal apoptosis via inhibiting MAPK
signaling pathways
Lalita Subedi1†, Oh Wook Kwon2†, Chaeho Pak1, Goeun Lee3, Kangwoo Lee3, Hakwon Kim3*‡
and Sun Yeou Kim1*‡
Abstract
Background: Activated microglia interact with astrocytes and neuronal cells to induce neuroinfammation, which
can contribute to the pathogenesis and progression of Alzheimer’s and Parkinson’s disease. To identify the most efec-
tive anti-neuroinfammatory agent, we designed and synthesized a family of 13 new azine derivatives and investi-
gated their anti-neuroinfammatory activities in LPS-activated BV-2 microglial cells.
Results: Out of 13 derivatives, compound 3 [4,4′-(1E,1′E,3E,3′E)-3,3′-(hydrazine-1,2-diylidene) bis-(prop-1-ene-1-yl-3-
ylidene) bis-(2-methoxyphenol)] exhibited excellent anti-neuroinfammatory activities (IC50 = 12.47 µM), which pro-
tected neurons from microglia-mediated neurotoxicity. Specifcally, the anti-neuroinfammatory efects of compound
3 inhibited MAPK signaling pathways through the inhibition of p38 and JNK mediated signaling and the production
of pro-infammatory cytokines, and infammatory mediators. Additionally, compound 3 strongly exhibited neuropro-
tective efect by inhibiting LPS-mediated necrosis and apoptosis. Preliminary SAR analysis suggests that the presence
of methoxyphenol and the substitution pattern within hydrazine may infuence the anti-neuroinfammatory activity.
FACS analysis also strongly supports the neuroprotective efect of compound 3.
Conclusions: Based on our results, the compound 3 exhibited excellent anti-neuroinfammatory activity against
LPS-activated microglia, which resulted in the inhibition of neuronal apoptosis and neuronal degeneration.
Keywords: Azine, Lipopolysaccharide, Neuroinfammation, MAPK, Apoptosis
major cause of neurodegenerative diseases like Alzhei-
mer’s disease (AD) and Parkinson’s disease (PD) [1–3].
Determining the regulators of neuroinfammation is very
important for the treatment of neurodegenerative dis-
eases. Extracellular stimuli such as lipopolysaccharide
(LPS), and interferon-γ (IFN-γ) can activate microglia
and secrete pro-infammatory mediators, which will initi-
ate several major cellular responses that contribute to the
pathogenesis of neuroinfammation [4–7]. Pro-infamma-
tory mediators such as nitric oxide (NO), prostaglandin
E2 (PGE2), interleukin-6 (IL-6), interleukin-1β (IL-1β),
and tumor necrosis factor-α (TNF-α) are released by acti-
vated microglia and will further activate astrocytes, and
vice versa. Although the normal activation of microglia
Background
Microglia are the resident immune cells that play a crucial
role in the innate immune response in the normal brain
and central nervous system (CNS). However, chronic
neuroinfammation induced by activated microglia is the
*Correspondence: hwkim@khu.ac.kr; sunnykim@gachon.ac.kr
†Lalita Subedi and Oh Wook Kwon contributed equally as frst author
‡Hakwon Kim and Sun Yeou Kim contributed equally to this work
1 College of Pharmacy, Gachon University, #191, Hambakmoero,
Yeonsu-gu, Incheon 21936, Republic of Korea3 Department of Applied
Chemistry and Institute of Natural Sciences, Kyung Hee University, Global
Campus, #1732 Deogyeong-daero, Giheung-gu, Yongin, Gyenggi-do
446-701, Republic of Korea
Full list of author information is available at the end of the article
© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

et al. BMC Neurosci (2017) 18:82
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and astrocytes is critical for the health of the CNS, address the regulation of activated microglia. In this
chronic activation of microglia and astrocytes can induce work, we report the synthesis of N,N-di-substituted azine
neuronal toxicity and cell death. Upon activation, micro- derivatives and their anti-neuroinfammatory and neuro-
glial cells grow large, assume an amoeboid shape, and ini- protective efects in vitro.
tiate the infammatory cascades in the brain [8–10].
Chronic injury and toxins with chronic infammation Methods
can eventually lead to neurodegeneration. LPS, as TLR4 Chemicals and reagents
agonist, can activate the receptor followed by an increase Chemicals used in cell culture experiments includ-
in the phosphorylation of MAPK signaling proteins, and ing Dulbecco’s modifed Eagle medium (DMEM), fetal
can further activate nuclear factor κB (NF-κB) medi- bovine serum (FBS), and penicillin–streptomycin were
ated transcription of various infammatory factors like obtained from Invitrogen (Carlsbad, CA, USA). LPS,
inducible nitric oxide synthase (iNOS), NO, cyclooxy- N-monomethyl-l-arginine (NMMA). Enzyme link
genase 2 (COX-2), PGE2, arachidonic acid, eicosanoids, immune sorbent assay (ELISA) development kit, TNF-α
and reactive oxygen species (ROS). In addition, chronic (DY410), IL-6 (DY406), and PGE-2 (514010). Primary
infammation can increase the levels of proinfammatory and secondary antibodies for iNOS (610333), COX-2
cytokines such as TNF-α, IL-6, and IL-1β. NO, biosyn- (Sc1745), pERK (9101s), ERK (9102s), pJNK (9251s), JNK
thesized from l-arginine by inducible nitric oxide syn- (4671s), pp38 (9211s), p38 (9212s), and tubulin (T5168)
thase (iNOS) plays an important role in the modulation were purchased from Santa Cruz (Capitola Rd, Santa
of infammatory responses [11]. Similarly, iNOS is also Cruz, CA, USA), and Cell Signaling (Beverly, MA, USA)
an important enzyme regulator of neuroinfammation respectively. All other chemicals and reagents were pur-
[12–14]. PGE2 production is catalyzed by COX-2, which chased from Sigma Chemical (St. Louis, MO) unless oth-
is a key neuroinfammatory enzyme that controls the erwise stated.
immune system and ascending pain pathway in the brain
[15]. Furthermore, LPS-induced infammatory cytokines General procedure for azines
play a major role in the brain immune system. LPS- A vial containing aldehyde (1 mM) and solid hydrazin-
activated microglia, infammatory mediators, and pro- ium carboxylate (0.5 mM) was heated at 60–80 °C in an
infammatory cytokines, such as activated astrocytes, can oven until the reaction was complete. Subsequently, the
further increase infammatory cascades by inducing the generated water was removed by evaporation. A high
activation of ramifed microglia. Tis negative crosslink- yield of the corresponding azine was produced. Te azine
ing of microglia and astrocytes induce the degeneration was used in the next reaction without further purifcation
and death of neurons. AD and PD are characterized by (Scheme 1).
such conditions [16].
Murine BV-2 microglia cells exhibit morphological, 1,2‑Bis((E)‑3‑(4‑methoxyphenyl)allylidene)hydrazine (1)
functional, and phenotypical properties similar to pri- Yield: 77.5%; 1H NMR: (300 MHz, CDCl3) δ 8.35 (d,
mary microglia. Terefore, BV-2 microglial cells are the J = 8.8 Hz, 2H), 7.47 (d, J = 8.8 Hz, 4H), 6.90–7.01 (m,
perfect alternative to primary microglia in which to study 8H), 3.84 (s, 6H); 13C NMR: (75 MHz, CDCl3) δ 163.6,
the various microglial responses and interactions in vitro. 160.7, 142.8, 128.9, 128.7, 123.2, 114.3, 55.3; LC–MS
Several of our previous studies demonstrated the anti- (ESI): 321.1 (M + 1).
infammatory or protective efects of natural derivatives
in LPS-stimulated BV-2 cells. For example, 6-shogaol 1,2‑Bis((E)‑2‑1methyl‑3‑phenylallylidene)hydrazine (2)
modulated neuroinfammation [17], and wogonin, a fa- Yield: 98%; H NMR: (300 MHz, CDCl3) δ 8.35 (s, 2H),
vone from the root of Scutellaria baicalensis, protected 7.39–7.53 (m, 10H), 6.91 (s, 2H), 2.27 (s, 6H); 13C NMR:
the brain from oxygen and glucose deprivation [18].
(75 MHz, CDCl3) δ 166.3, 140.3, 136.5, 134.9, 129.5,
Azine have been reported to have biological actions 128.3, 127.9, 13.3; LC–MS (ESI): 289.1 (M + 1).
such as antiviral efects, anticonvulsant activity [19, 20],
and histamine H4 receptor antagonistic activity [21, 4,4′‑(1E,1′E,3E,3′E)‑3,3′‑(hydrazine‑1,2‑diylidene)
22]. Several pharmacological studies have suggested the bis(prop‑1‑ene‑1‑yl‑3‑ylidene)bis(2‑methoxyphenol) (3)
potential utility of histamine H4R antagonists/inverse Yield: 41.1%; 1H NMR: (300 MHz, CDCl3) δ 8.35 (d,
agonists in the treatment of infammatory diseases such J = 8.8 Hz, 2H), 6.89–7.06 (m, 10H), 5.82 (br s, 2H), 3.93
as allergic rhinitis, asthma, atopic dermatitis, colitis, and (s, 6H); 13C NMR: (75 MHz, CDCl3) δ 163.5, 147.3, 146.8,
pruritus. However, no anti-neuroinfammatory studies on 143.2, 128.5, 123.1, 122.3, 114.7, 108.5, 55.8; LC–MS
azine derivatives have been performed that specifcally (ESI): 353.2 (M + 1).

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Scheme 1 Synthetic schemes for azine derivatives
1,2‑Bis(quinolin‑3‑ylmethylene)hydrazine (4)
1,2‑Bis(3,3‑diphenylallylidene)hydrazine (8)
Yield: 96%; H NMR: (300 MHz, CDCl3) δ 9.51 (s, 2H), Yield: 97%; 1H NMR: (300 MHz, CDCl3) δ 8.25 (d,
8.93 (s, 2H), 8.53 (s, 2H), 8.18 (d, J = 8.4 Hz, 2H), 7.95 J = 10.2 Hz, 2H), 7.23–7.43 (m, 20H), 6.97 (d, J = 10.3 Hz,
(d, J = 8.1 Hz, 2H), 7.82 (t, J = 7.7 Hz, 2H), 7.64 (t, 2H); 13C NMR: (75 MHz, CDCl3) δ 162.4, 154.1, 140.8, 138.2,
J = 7.3 Hz, 2H); 13C NMR: (75 MHz, CDCl3) δ 160.4, 130.3, 129.1, 128.4, 128.1, 124.1; LC–MS (ESI): 413.2 (M + 1).
149.5, 149.2, 136.9, 131.0, 129.6, 128.6, 127.5, 127.4,
1
126.9; LC–MS (ESI): 311.1 (M + 1).
4,4′‑Hydrazine‑1,2‑diylidenebis(methan‑1‑yl‑1‑ylidene)
bis(N,N‑diphenylaniline) (9)
1,2‑Dibenzylidenehydrazine (5)
Yield: 96%; 1H NMR: (300 MHz, CDCl3) δ 8.59 (s, 2H), 7.66
Yield: 99.8%; 1H NMR: (300 MHz, CDCl3) δ 8.68 (s, (d, J = 8.8 Hz, 4H), 7.27–7.33 (m, 8H), 7.05–7.16 (m, 16H);
2H), 7.83–7.86 (m, 4H), 7.45–7.47 (m, 6H); 13C NMR: 13C NMR: (75 MHz, CDCl3) δ 160.9, 150.4, 147.0, 129.5,
(75 MHz, CDCl3) δ 162.0, 134.0, 131.1, 128.7, 128.5; LC– 129.4, 127.3, 125.4, 123.9, 121.7; LC–MS (ESI): 543.2 (M + 1).
MS (ESI): 209.1 (M + 1).
We designed and synthesized several derivatives of
azine using the solvent-free reaction of aldehyde with
solid hydrazine to produce the corresponding azine [23–
1,2‑Bis((E)‑3‑(2‑nitrophenyl)allylidene)hydrazine (6)
Yield: 93.8%; 1H NMR: (300 MHz, CDCl3) δ 8.40 25] (Fig. 1).
(d, J = 8.8 Hz, 2H), 8.04 (d, J = 8.1 Hz, 2H), 7.77 (d,
J = 7.7 Hz, 2H), 7.70 (d, J = 7.32 Hz, 2H), 7.63 (d,
J = 15.4 Hz, 2H), 7.52 (t, J = 7.7 Hz, 2H), 7.02–7.11 (m,
2H); 13C NMR: (75 MHz, CDCl3) δ 163.1, 148.0, 137.8,
133.4, 131.2, 130.1, 129.7, 128.4, 125.0; LC–MS (ESI):
350.1 (M + 1).
1,2‑Bis((E)‑3‑(4‑chlorophenyl)allylidene)hydrazine (7)
Yield: 85%; 1H NMR: (300 MHz, CDCl3) δ 8.36 (t,
J = 4.4 Hz, 2H), 7.46 (d, J = 8.8 Hz, 4H), 7.36 (d,
J = 8.8 Hz, 4H), 7.05 (d, J = 4.1 Hz, 4H); 13C NMR:
(75 MHz, CDCl3)
δ 163.5, 141.9, 135.3, 134.2,
129.2, 128.5, 125.9; LC–MS (ESI): 329.0 (M+), 331.0
Fig. 1 General structure of azine
(M + 2).

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Cell culture
using a microplate reader. Sodium nitrite was used to
create a standard curve from which the NO2 concentra-
tion was calculated. Te cell viability was evaluated by
spectrophotometrically measuring the reduction of yel-
low 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT) to purple formazan crystals. MTT was
dissolved in DMEM, added to the culture plate contain-
ing cells at a fnal concentration of 0.5 mg/mL, and incu-
bated for 1 h. Te medium containing the MTT solution
was carefully removed, and 200 µL DMSO was added to
each well. After solubilizing the stained cells to produce
a formazan-colored solution, the optical density (OD)
was measured on a microplate reader at 570 nm, and the
results were expressed as a percentage of the LPS-treated
group. Te same plate was used for the NO and MTT
assays. Te cell supernatant was used for the NO assay,
and the plated cells were used for the MTT assay.
Te BV-2 microglial cell line was obtained as a gift from
by Dr. E. Choi at Korea University (Seoul, Korea) and
the murine neuroblastoma cell line (N2a) was origi-
nally obtained from American Type Culture Collection
(Manassas, VA, USA). BV-2 and N2a cells were grown in
uncoated tissue culture plates and incubated at 37 °C in a
humidifed atmosphere of 5% CO2 and 95% air. Te cells
were maintained in Dulbecco’s modifed Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum,
100 U/mL penicillin, and 100 U/mL streptomycin. Te
culture medium was changed every 24 h [26]. Te over-
all treatment strategy for this experiment is shown in
Scheme 2.
Nitrite oxide and MTT assay
Nitrite oxide or MTT assay was performed as described
by our previous publication with slight modifcation
[27]. In order to measure MTT and NO production,
BV-2 cells were plated into a 96-well plate at a density of
4 × 104 cells/well. Seeded cells were pretreated with 5,
10, and 20 µM compound 3 and incubated for 30 min.
After the incubation, 100 ng/mL LPS was applied to all
the wells with compound 3, except the untreated con-
trol group, and incubated for a further 24 h. Ten, nitrite
(a soluble oxidation product of NO) was detected in the
culture media by using the Griess reaction. Te superna-
tant (50 µL) was collected and mixed with an equal vol-
ume of Griess reagent (1% sulfanilamide in phosphoric
acid and 0.1% N-1-napthylethylenediamine dihydro-
chloride). Te absorbance was measured at 570 nm by
ELISA
To measure TNF-α, IL-6, and PGE2, BV-2 cells were
seeded in a 24-well plate at a density of 3 × 105 cells/well.
Te cells were treated with LPS (100 ng/mL) in the pres-
ence or absence of compound 3 for 24 h, and the media
was collected and centrifuged [26]. IL-6, TNF-α, and
PGE2 produced in microglial culture supernatants (inter-
cellular) were measured by competitive enzyme immu-
noassay kits (R&D systems, Minneapolis, MN, USA) in
accordance with the manufacturer’s protocol.
Western blot analysis
BV-2 cells were seeded in a 6-well plate at a density of
6 × 105 cells/well and exposed to LPS (100 ng/mL) in
the presence or absence of compound 3 for required
time [26]. After treatment, cells were collected and
lysed in cell lysis bufer (50 mM Tris–HCl, pH 8.0, 0.1%
SDS, 150 mM NaCl, 1% NP-40, 0.02% sodium azide,
0.5% sodium deoxycholate, 100 µg/mL PMSF, and 1 g/
mL aprotinin) for 24 h at 4 °C. Te protein content was
measured using a Bradford assay. Equal amounts of pro-
tein (30 µg) were separated using SDS-PAGE and trans-
ferred to a nitrocellulose membrane. Te membrane was
blocked for 1 h with 5% skim milk in tris-bufered saline
with Tween-20 (TBST) and then incubated for 12 h with
primary antibodies against α-tubulin (Sigma-Aldrich Cat.
no: T5168), iNOS (Bioscience Cat. no: 610333, COX-2
(Santa Cruz Cat. no: sc-1745), ERK (Cell Signaling Cat.
no: 9107s), pERK (Cell Signaling Cat. no: 5013s), JNK
(Cell Signaling Cat. no: 9252s), pJNK (Cell Signaling Cat.
no: 4671s), p38 (Cell Signaling Cat. no: 8690s), and pP38
(Cell Signaling Cat. no: 9211s), followed by incubation for
1 h with horseradish peroxidase-conjugated secondary
Scheme 2 Treatment strategy for various parts of this experiment

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antibodies (Cell Signaling) at 15 °C. Te blots were devel- reproducibility of the results, each experiment was per-
oped using ECL Western Blotting Detection Reagents formed in triplicate.
(Amersham Pharmacia Biotech, Buckinghamshire, UK).
Densitometri™c analysis of the bands was performed using Results
ImageMaster 2D Elite software (version 3.1, Amersham Screening of efects of azine derivatives on LPS‑induced
Pharmacia Biotech) [26].
NO production in BV‑2 cells
We investigated whether azine derivatives had an inhibi-
tory efect on the LPS-induced infammatory response in
Annexin V/PI staining
An annexin V/PI apoptosis kit was used in accordance BV-2 cells. Te NO production and cell viability of LPS-
with the manufacturer’s instructions to quantify the activated BV-2 cells were measured after treatment with
percentage of cells undergoing apoptosis. First, BV-2 azine derivatives using Griess reagent and MTT assay,
cells were incubated for 24 h with 5, 10, and 20 µM of respectively [29]. Azine derivatives exhibited inhibitory
compound 3 or LPS (100 ng/mL). Ten, N2a cells were efects on LPS-induced NO production and cell viabil-
treated with the supernatant of the treated BV-2 cells and ity in BV-2 cells. As shown in Table 1, the azine deriva-
incubated. After 24 h, the cells were collected, washed tives suppressed LPS-induced NO production in BV-2
twice with cold PBS, and re-suspended in binding bufer cells, except for compound 4. Among these derivatives,
at a concentration of 1 × 106 cells/µL. Annexin V-FITC compounds 6, 7, 8, and 9 were cytotoxic to BV-2 cells at
(5 µL) and PI (10 µL) were added, and the cells were incu- 20 µM. Since compound 3 displayed an inhibitory efect
bated for 15 min at room temperature in the dark. Fol- on NO production in BV-2 cells without cytotoxicity or
lowing incubation, 200 µL of binding bufer was added, cell death, it was selected for the in vitro anti-neuroin-
and the cells were immediately analyzed by fow cytom- fammatory study.
etry (FACSAriaIII; BD Biosciences, Franklin Lakes, NJ,
USA). Te fow cytometric analysis was performed using Efect of compound 3 on the LPS‑induced
the Cell Quest software (BD Biosciences). Te annexin neuroinfammatory response in BV‑2 cells
V+/PI− cells and annexin V−/PI+ cells were used to We investigated the inhibitory efect of compound 3 on
identify apoptotic cells and necrotic cells, respectively. LPS-induced iNOS and COX-2 expression in BV-2 cells.
Te procedure was repeated three times for each sample.
As shown in Fig. 2a, pretreatment of BV2 cells with com-
pound 3 signifcantly reduced the expression of iNOS
and COX-2 at all tested concentrations (5, 10, and 20 µM)
Neuronal cell viability
N2a cells were used as a representative neuronal cell line (Fig. 2b, c). Tis efect was more potential even at the 24 h
for the examination of apoptotic neuron viability using of chronic LPS activation. Compound 3 dramatically res-
direct treatment of compound 3 with TNF-α activation cued BV2 cells against LPS induced iNOS and COX-2
as well as conditioned media (CM) from LPS-stimulated
BV-2 cells treated with compound 3 [28]. Tis assay was
Table 1 Efects of azine derivatives on NO production
performed following the methods described in our pre-
in LPS-activated BV-2 cells
vious study, with slight modifcations [26]. To measure
Compound
ICa50(µM)
Cell viabilityb(%)
neuron viability, BV-2 cells were frst seeded and treated
with 100 ng/mL LPS in the presence or absence of com-
pound 3. After 24 h, the CM was collected and trans-
ferred to N2a cells for a further 24 h. Te cell viability of
N2a cells was measured using the MTT assay. Well dif-
ferentiated neuronal cells were used for the experimental
purpose as shown in Additional fle 1: Fig. S1.
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
Compound 6
Compound 7
Compound 8
Compound 9
L-NMMAc
58.52
50.12
12.47
> 500
59.91
47.36
39.94
47.89
26.27
18.32
90.33 9.37
96.58 1.75
100.73 2.53
91.03 3.39
97.55 10.59
87.90 19.53
77.24 5.67
69.45 3.25
83.54 7.78
100.40 3.41
Statistical analysis
Te data were analyzed using Statistical Analysis Sys-
tem (SAS) software (PRISM). All data were expressed as
the mean standard error of the mean. Statistical com-
parisons between diferent treatments were performed
using one-way ANOVA followed by the Newman-Keuls
post hoc test, using GraphPad Prism 5 (GraphPad Soft-
ware Inc., La Jolla, CA, USA). Values of P < 0.05 were
considered statistically signifcant. To confrm the
a
The IC50 value of each compound was defned as the concentration (in µM)
that caused 50% inhibition of NO production in LPS-activated BV-2 cells
b
The cell viability was measured at 20 µM of compound treatment and it was
expressed as a percentage (%) of the LPS-only treatment group. The results are
the average of three independent experiments and the data are expressed as
mean standard deviation
c
L-NMMA was used as a positive control

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Fig. 2 Efect of compound 3 on LPS-induced iNOS and COX2 expression in BV-2 cells. BV-2 cells were pretreated with various concentrations (5, 10,
and 20 µM) of compound 3 for 30 min and activated with 100 ng/mL LPS. After 6 h, the cells were collected for analysis of iNOS and COX-2 expres-
sion using western blot analysis (a–c). Similarly, LPS activation was made for 24 h as chronic BV2 activation and the iNOS and COX-2 expression was
measured (d). α-Tubulin was used as the loading control. The densitometric analysis of bands was performed as described in the methods section.
After 24 h, the culture medium was subsequently collected to measure the quantity of PGE2 (e), IL-6 (f), and TNF-α (g) using ELISA analysis. All data
are presented as the mean standard error of the mean of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 indicates signifcant
diferences compared with treatment with LPS alone while ###P < 0.001 indicates the signifcant diferences compared with untreated control
group. Here Ctl indicates untreated control and LPS indicates lipopolysaccharide
Efect of compound 3 on the MAPK pathway
in LPS‑induced BV‑2 cells
activation as shown in Fig. 2d. Protein expression was nor-
malized to the expression of α-tubulin, and the quantitative
Te mitogen-activated protein kinase (MAPK) sign-
aling pathway plays an important role in controlling
the synthesis and release of pro-infammatory factors
in LPS-stimulated BV-2 cells [30, 31]. Terefore, we
investigated the efects of compound 3 on MAPK acti-
vation in BV-2 cells. Compound 3 signifcantly inhib-
ited the LPS-induced phosphorylation of p38 as well as
c-Jun N-terminal kinase (JNK) however, pretreatments
of compound 3 did not alter the phosphorylation of
extracellular signal-regulated kinase (ERK) (Fig. 3).
Te pattern of activity is similar at short term LPS
activation i.e. 30 min and chronic activation i.e. 24 h
suggesting that, compound 3-mediated anti-infam-
matory efect is through the inhibition of p38 and JNK
phosphorylation.
band intensity was calculated.
To investigate whether compound 3 infuenced the
secretion of pro-infammatory factors such as TNF-α,
IL-6, and PGE2 in activated microglia cells, BV-2 cells were
treated with compound 3 for 30 min before LPS was added
for 24 h. LPS-activated PGE2 production was reduced
by compound 3 at concentrations of 5, 10, and 20 µM in
BV-2 cells (Fig. 2e). Similar results were obtained from
the measurement of IL-6 and TNF-α secretion (Fig. 2f, g,
respectively). Although 5 and 10 µM of compound 3 did
not signifcantly inhibit IL-6, there was noticeable activ-
ity occurred at 20 µM. Similarly, signifcant reduction of
TNF-α secretion was observed at 10 and 20 µM in LPS-
activated BV-2 cells. Te Inter assay CV for all the elisa was
confrmed less than 10% while the intra assay CV was less
than 15% as shown in Additional fle 2: Fig. S2.

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Fig. 3 Efect of compound 3 on MAPK pathway in LPS-induced BV-2 cells. BV-2 cells were treated with LPS and with or without 5–20 µM com-
pound 3 for 30 min. Cell lysates were prepared to evaluate the protein levels of MAPKs. The phosphorylation of p38, JNK, and ERK was measured by
western blotting (a–c). Similarly, the cells were activated with LPS for 24 h as chronic activation and the expression pf MAPK was observed (d–f). The
densitometric analysis of bands was performed as described in the methods section. **P < 0.01, ***P < 0.001 indicates signifcant diferences com-
pared with treatment with LPS alone; and ###P < 0.001 indicates signifcant diferences compared with untreated control group. Here Ctl indicates
untreated control and LPS indicates lipopolysaccharide
Efect of conditioned media (CM) treated with compound 3
Efect of compound 3 on cell viability of BV2 and N2a cells
on N2a cells
against LPS and TNF‑α induced direct toxicity
Here, we investigated whether the CM of compound 3 Here, we have investigated whether the compound 3
had any protective efects on neurons against activated has any direct protective efects on microglia and neu-
microglia-induced MAPK signaling, NF-κB-mediated rons against LPS and TNF-α induced direct toxicity. As
transcription, pro-infammatory cytokines, and infam- the aim of this study is to check the efectiveness of com-
matory mediators. N2a cell viability decreased when pound 3 against neurodegenerative conditions, here we
treated with CM containing only LPS (Fig. 4a). Te CM checked the efect of this compound to protect microglial
of LPS induced signifcant cytotoxicity via the induc- cells against β-amyloid induced cytotoxicity as shown
tion of apoptosis (Fig. 4b–h). Te MTT assay showed in Fig. 5a. Compound-3 signifcantly prevented micro-
that the CM of compound 3 increased cell viability to glial cell death against β-amyloid providing its cytopro-
199.1 and 397.7% of the only LPS treated control value tective efects. Tis result was further supported by the
at concentrations of 10 µM and 20 µM, respectively. Te prevention of N2a cell survival against TNF-α induced
CM of LPS induced apoptosis and necrosis in N2a cells, N2a cytotoxicity (Fig. 5c). LPS treatment was unable to
as determined by FACS analysis. As shown in Fig. 4g–h, induce cellular toxicity to N2a cells (Fig. 5b). Tis results
treatment with LPS signifcantly increased the concentra- suggests that N2a cell death is not because of the LPS
tion of apoptotic and necrotic cells compared with BV-2 but because of the pro-infammatory cytokines pro-
cells in normal conditions. However, the decrease in con- duced by the activated microglia. Compound 3 mediated
centration of apoptotic and necrotic cells was mediated neuroprotection in N2a cells is due to the inhibition of
by the CM of compound 3 at each concentration tested proinfammatory cytokines produced by LPS activated
in N2a cells.
microglia.

et al. BMC Neurosci (2017) 18:82
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Fig. 4 Neuroprotection of compound 3 in LPS-activated microglia in N2a cells. BV-2 microglial cells were pretreated with 5, 10, and 20 µM com-
pound 3 for 30 min and stimulated with 100 ng/mL of LPS for 24 h. After 24 h of LPS treatment, the conditioned media (CM) were collected and
transferred to N2a cells. After 24 h, MTT assay was done in the same CM treated N2a cells (a) and FACS analysis (b–h). An annexin V/PI apoptosis kit
was used to quantify the percentage of cells undergoing apoptosis and necrosis (x-axis, annexin V; y-axis, PI) by using FACS analysis. CM-LPS treat-
ment alone induced apoptosis and necrosis in N2a cells (g, h). All data are presented as the mean standard error of the mean of three independ-
ent experiments. *P < 0.05, ***P < 0.001; and ###P < 0.001 indicates signifcant diferences compared with the CM-LPS group. Here Ctl indicates
untreated control and LPS indicates lipopolysaccharide
Fig. 5 Cytoprotection of compound 3 in β-amyloid induced BV2 cell and LPS and TNF-α induced N2a cell toxicity

et al. BMC Neurosci (2017) 18:82
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neuroinfammatory conditions induced by LPS activated
BV2 microglia.
Discussion
Te chronic activation of microglia is usually closely
associated with neurodegenerative diseases. Te neuro-
infammatory response caused by the activation of micro-
glia may induce neuronal cell death through its harmful
pro-infammatory cytokines. Activated microglia can
induce production and secretion of many harmful pro-
infammatory mediators such as nitric oxide (NO), PGE2,
TNF-α, and IL-6 [32]. Terefore, fnding a potential can-
didate that can protect neurons against chronic micro-
glia-mediated neurotoxicity is crucial for designing new
therapeutic strategies for neurodegenerative disorders.
According to previous reports, compounds that can
activate autophagy could attenuate the level of iNOS,
IL-6 and cell death in LPS activated microglial cells [33].
Azine compounds are known to regulate autophagy [34].
Lack of strong report on the neuroprotective efects of
azine derivatives in microglia-mediated neurotoxicity
inspired us to perform this experiment. We were to iden-
tify the most active azine derivative with anti-infamma-
tory activity, and to explore its acting mechanisms.
NO is an important infammatory mediator that is
afected by the activation of microglia [11]. Activated
microglia can produce and secrete cytotoxic factors such
as NO. NO is an important biomarker of neuroinfamma-
tion. Because of this reason, we performed nitrite assay
for their anti-neuroinfammatory activity of azine and its
derivatives. Most azine derivatives (except compound
4) inhibited the production of LPS-induced pro-infam-
matory mediators such as NO, and demonstrated no
cytotoxicity as shown in Table 1. However, compounds
7 and 8 exhibited high toxicity in BV-2 cells. Among the
derivatives tested, the compound 3, 4,4′-(1E,1′E,3E,3′E)-
NO and PGE2 production are catalyzed by iNOS and
COX-2, respectively, which are the key proteins in the
regulation of infammatory responses in the brain [15,
36]. As shown in Fig. 2, LPS-induced iNOS and COX-2
protein expressions were signifcantly inhibited by com-
pound 3. Te reduced expression of iNOS by compound
3 might be caused by NO decrease in the same treat-
ment group. Additionally, LPS-activated COX-2 expres-
sion was also lowered by compound 3, followed by the
reduced production of PGE2 against LPS activated BV-2
cells.
Te overproduction of pro-infammatory cytokines has
a harmful efect on microglia and neurons [37, 38]. Tey
are believed to be a key player for the neurodegeneration
as well as neuronal cell death. We tested compound 3 for
inhibition of TNF-α and IL-6 secretion in LPS-activated
BV-2 cells. As shown in Fig. 2, treatment with compound
3 suppressed the infammatory response by decreasing
the secretion of TNF-α and IL-6 in LPS-induced BV-2
cells. Te inactivation of microglial cells by compound 3
may be regulated by infammatory cytokines such as IL-6
and TNF-α.
As LPS is a TLR4 agonist, once it binds to its receptor.
And, it triggers the efector signaling pathways through
the enhanced phosphorylation of MAPK proteins and
followed by NF-κB mediated transcription [29]. With
this notion, we hypothesized that the observed decrease
in pro-infammatory mediators were regulated by the
MAPK pathway. To confrm this, we measured the
expression of MAPKs by western blotting. compound 3
downregulated the LPS-induced phosphorylation of p38
as well as JNK in a dose-dependent manner, but did not
alter the expression of pERK. Tis mechanism of action
suggests that compound 3 non-specifcally downregu-
lated p-p38 and pJNK in LPS activated microglia. Studies
suggested that the stress or endotoxin induced phospho-
rylation of p38 and JNK are major cause for the induction
of apoptosis followed by the increased TNF-α secretion
through NF-κB mediated transcription [27, 39]. And here
we fnd that compound 3 inhibited the phosphorylation
of p38 as well as JNK together with signifcant reduction
of TNF-α. Tis result suggests that compound 3 might
give protection against neuronal cell death induced by
activated microglia. Furthermore, inhibitory efect of
compound 3 on the p-p38 and pJNK was further con-
frmed in 24 h chronic LPS activation. In conclusion,
the results of this study clearly provides evidences that
compound 3 has a potent anti-neuroinfammatory efect
in LPS-activated microglia. Tis efect was achieved
through the decreased expression of pro-infammatory
3,3′-(hydrazine-1,2-diylidene)
bis(prop-1-ene-1-yl-3-
ylidene) bis(2-methoxyphenol), strongly inhibited NO
production without cytotoxicity. Te inhibition was even
greater than that of commercially available well-known
iNOS inhibitor L-NG-monomethylarginine (L-NMMA)
[35]. From this data we hypothesized that, the methoxy-
phenol substituent present in compound 3 made it the
most potential candidate then that of other azine deriva-
tives. It is believed that, a phenolic hydroxyl group has an
important pharmachore potency. Since the azine deriva-
tive 3 substituted with a phenolic hydroxyl group showed
the best activity, it is thought that the phenolic hydroxyl
group is an important pharmacologically active group,
which is consistent with the result that it exhibits various
physiological activities. Terefore, our data may suggest
that the presence of methoxyphenol and the substitution
pattern within hydrazine derivatives infuences the anti-
neuroinfammatory activity. Tese results indicate that
azine and its derivatives have the potential to alter the

et al. BMC Neurosci (2017) 18:82
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Page 10 of 12
mediators via the suppression of the MAPK signaling
pathways.
Chronic activation of microglia can produce several
harmful pro-infammatory factors through MAPK sign-
aling and NF-κB mediated transcription. As a result,
it may induce neuronal cell death through apoptosis
[40]. As compound 3 inhibited MAPK signaling as well
as proinfammatory cytokines, we hypothesized that it
might prevent the neuronal apoptosis induced by LPS-
activated microglia. Terefore, to prove this hypothesis,
we investigated whether compound 3 regulated the
microglia-mediated neuronal cell toxicity. Te condi-
tioned medium (CM) of compound 3 restored micro-
glia-mediated neuronal cell toxicity to normal levels in
N2a cells (Fig. 4), and no adverse efects were observed
in either N2a or BV-2 cells. In addition, after exposure to
compound 3, levels of apoptotic and necrotic cells were
returned to normal, which was attributed to the down-
regulation of pro-infammatory factors by the CM of
compound 3 in N2a cells. As is confrmed in our experi-
ment, B-amyloid can induce microglia activation as well
as microglial death. Compound 3 protected against
B-amyloid-induced BV2 cells toxicity (Fig. 5). For fur-
ther clarifcation of mode of action, TLR4 expression [27]
was measured to answer whether the toxicity was due
to LPS itself or pro-infammatory cytokines. To answer
this question, we treated N2a cells with LPS and TNF-α
directly. We found that direct LPS treatment does not
induced N2a cell death where it was signifcantly induced
with TNF-α treatment (Fig. 5b, c). In support of earlier
results, compound 3 increased cell viability of N2a cells
against TNF-α mediated toxicity. As a result, we can con-
clude that TNF-α produced by LPS activated microglia is
responsible for N2a cell death. Inhibition of TNF-α pro-
duction by compound 3 is one of the major action mech-
anisms giving protection of neurons. Namely, compound
3 protected against activated microglia induced N2a
cell toxicity. Tis result suggests that compound 3 may
protect neuronal cell damage via inhibition of the pro-
infammatory cytokine released from activated micro-
glia through inhibition of p38 and JNK phosphorylation.
Further experiments are required to study the potential
efect of compound 3 on toxin-mediated neuronal cell
damage specially in vivo experiments.
Fig. 6 Summary of the efect of compound 3 on neuroinfammation
and neurodegeneration inhibition
as TNF-α, IL-6, NO, and PGE2) by the regulation of
iNOS and COX-2 expression in acute as well as chronic
LPS-stimulated BV-2 cells. In addition, compound 3
protected N2a cells against microglia-mediated neu-
rotoxicity. Our fndings validated the efectiveness and
safety (low toxicity) of compound 3 as a therapeutic neu-
roprotective agent which can attenuate chronically acti-
vated microglial infammation. Further in vivo studies are
needed to confrm its efcacy and safety.
Additional fles
Additional fle 1: Fig. S1. Diferentiation of N2a cells after serum
starvation. N2a cells were treated in serum free condition, that let cells
for proper diferentiation and with the treatment of LPS, degeneration of
neurite outgrowth can be seen.
Additional fle 2: Fig. S2. Inter and intra assay CV for all the Elisa per-
formed in this experiment.
Conclusions
As shown in the summary Fig. 6, we have demon-
strated for the frst time, the key role of the highly con-
jugated azine derivative compound 3, in the regulation
of activated microglia for the protection of neuronal
cells. Compound 3 modulated over activated micro-
glia by downregulating the MAPK pathway (specially
p-p38 and pJNK) and pro-infammatory factors (such
Abbreviations
CNS: central nervous system; AD: Alzheimer’s disease; PD: Parkinson’s disease;
LPS: lipopolysaccharide; IFN-γ: interferon-γ; NO: nitric oxide; PGE2: prostaglan-
din E2; IL-6: interleukin-6; IL-1β: interleukin-1β; TNF-α: tumor necrosis factor-α;
iNOS: inducible nitric oxide synthase; COX-2: cyclooxygenase 2; ROS: reactive

et al. BMC Neurosci (2017) 18:82
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Page 11 of 12
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progression of neurodegenerative disorders. Med Hypotheses.
2009;73(6):1031–4.
Authors’ contributions
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SYK and HK designed and conceptualized the study in the presence of our
funding source. LS and KOW performed the experiment. LS, CP and SYK
wrote the manuscript. GL, KL and HK synthesized the compound. GL and KL
reviewed the manuscript and suggest some important corrections in the
manuscript. SYK and HK analyzed the data and reviewed the manuscript. All
authors read and approved the fnal manuscript.
Author details
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1 College of Pharmacy, Gachon University, #191, Hambakmoero, Yeonsu-gu,
Incheon 21936, Republic of Korea. 2 Graduate School of East-West Medical
Science, Kyung Hee University, Global Campus, #1732 Deogyeong-daero,
Giheung-gu, Yongin, Gyeonggi-do 446-701, Republic of Korea. 3 Department
of Applied Chemistry and Institute of Natural Sciences, Kyung Hee University,
Global Campus, #1732 Deogyeong-daero, Giheung-gu, Yongin, Gyenggi-do
446-701, Republic of Korea.
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Competing interests
The authors declare that they have no competing interests.
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Availability of data and materials
The dataset during and/or analyzed during the current study are available
from the corresponding author on reasonable request.
Consent for publication
Not applicable.
18. Son D, Lee P, Lee J, Kim H, Kim SY. Neuroprotective efect of wogonin in
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Funding
This research was supported by a grant of High Value-added Food Technol-
ogy Development Program 114006-4, Ministry of Agriculture, Food and Rural
Afairs and Brain Korea 21 Plus. Additionally, it was partly supported by the
GRRC program of Gyeonggi province [GRRC-kyunghee2017(B01)]. Fund-
ing source helped to design the experiments and provide all the required
reagents for the completion of this experiment. They bear all the cost for this
experiment and also for the publication of this manuscript.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional afliations.
Received: 19 May 2017 Accepted: 13 December 2017
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with bis (trifuoroacetoxy) iodobenzene. Tetrahedron letters.
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