Neurochemical Research

https://doi.org/10.1007/s11064-021-03369-z

ORIGINAL PAPER

Neuroprotective Effects of Sodium Butyrate through Suppressing

Neuroinflammation and Modulating Antioxidant Enzymes
Al Borhan Bayazid1 · Young Ah Jang2 · Yu Mi Kim3 · Jae Gon Kim4 · Beong Ou Lim1

Received: 19 March 2021 / Revised: 25 May 2021 / Accepted: 31 May 2021

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021

Abstract
The discovery of effective therapeutic agents against neurodegenerative diseases (NDDs) remains challenging. Neurotoxicity,
inflammations, and oxidative stress are associating factors of NDDs. Sodium butyrate (NaB) is a short-chain fatty acid found
in diet and produced in the gut that reportedly protects cancer, inflammation, obesity and so on. Previously, SH-SY5Y cells
were studied as in vitro models of cerebral diseases. We have investigated the neuroprotective effects of NaB in SH-SY5Y
cells stimulated with TNF-α. The expression of inflammatory mediators, including iNOS, COX-2, and mitogen-activated
protein kinases (MAPK) and the apoptotic regulators, including P-53, Bcl-2 associated X (BAX) Protein, and caspase-3 were
analyzed by western blot analysis. The anti-apoptotic gene Bcl-2 and the pro-apoptotic gene BAX translocation were also
investigated. Our results showed that NaB attenuated cell death and inhibited the NO production and decreased the expres-
sion of iNOS and COX-2 in TNF-α-stimulated SH-SY5Y cells. NaB notably ameliorated apoptotic regulatory proteins p-53,
Caspase-3 and caspase-1 level, and reversed phosphorylation of extracellular signal-regulated kinases and p-38 proteins.
NaB ameliorated Glucocorticoid receptor and NLRP3 inflammasome expressions. NaB also suppressed the BAX nuclear
translocation and modulated Nrf-2, HO-1 and MnSOD expression in neuroblastoma cells. In addition, NaB substantially
reversed the reactive oxygen species in ­H2O2 induced SH-SY5Y cells. Altogether, our results suggest that sodium butyrate
has potential therapeutic effects against NDDs.

Keywords Sodium butyrate · Neurodegenerative diseases · SH-SY5Y cells · NLRP3 · Glucocorticoid receptor ·
Nutraceuticals

Introduction
Neuronal cell death leads to neurodegenerative diseases
(NDDs) due to cytotoxic events like apoptosis, cell-death
related pathways, neuroinflammation, synaptic toxicity and
so on [1]. NDDs are one of the leading causes of death all
over the world and the etiology of those diseases is little
known [2]. Reportedly, the inflammatory cascades in the
* Beong Ou Lim
beongou@kku.ac.kr
1
Department of Integrated Biosciences, Graduate School
of Konkuk University, Chungju 27478, Korea
2
Convergence Research Center for Smart Healthcare, R&DB
Foundation of Kyungsung University, Busan, Korea
3
Bio-Nano Technology Co, Daegu, Korea
4
BK21 FOUR, GLOCAL Education Program
for Nutraceutical and Biopharmaceutical Research, Konkuk
University, Chungju 27478, Korea
nervous system are one of the primary factors that lead to the
progression of NDDs, including Alzheimer’s disease (AD),
Parkinson’s disease (PD), and Huntington’s disease (HD)
[2, 3]. Inflammation is the direct result of the overactiva-
tion of immune responses against pathogens, antigens, and
cellular debris [4], where tumor necrosis factor-alpha (TNF-
α) plays a vital role in inducing inflammatory responses.
TNF-α activates cellular signaling by TNF-receptor in the
cell membrane, which further stimulates inflammation and
caused apoptosis [5]. Furthermore, TNF-α is a pleiotropic
cytokine that may lead to cell death. Therefore, protection
against TNF-α-stimulated neuronal cell death and neuro-
inflammation is considered an important characteristic of
therapeutics against NDDs.
Apoptosis in neuronal cells plays a vital role in NDDs
progression. P53-BAX and caspase-3 are the pivotal pro-
apoptotic regulators that play significant roles in neu-
ronal cell death. mostly triggered by cellular oxidative
stress[6–8]. P-53 is a pivotal transcriptional factor that

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causes apoptosis through caspase cascades and plays a cru-
cial role in increasing reactive oxygen species (ROS) lead-
ing to oxidative stress-mediated cell death, while down-
regulating cellular antioxidant enzymes [9, 10]. Therefore,
inhibiting P-53 is an essential step in the neuroprotective
process [11]. According to a previous study, TNF-α eleva-
tion in serum and cerebral spinal fluid (CSF) causes NDDs
[12]. SH-SY5Y cells are widely used to study the neuro-
protective activity, which obtained homogeneity, express
several markers of mature neurons, and widely studied to
investigate neuroprotective effects [8]. TNF-α mediated
SH-SY5Y cell death is an indication of the progression of
NDDs [12]. P-53 transcriptional factor in neuroblastoma
cells trigger cell cycle arrest and apoptosis by upregu-
lating pro-apoptotic gene BAX, which indicates neuronal
cell loss. Oxidative stress-inducing P-53 can also trigger
BAX and caspase cascades [13]. The protective effects in
SH-SY5Y against TNF-α would be a significant indicator
of neuroprotective activities that may reduce the NDDs
progression. The nuclear factor erythroid 2-related fac-
tor 2(Nrf-2), Heme Oxygenase-1(HO-1) and manganese
superoxide dismutase (MnSOD) are crucial cellular anti-
oxidant enzymes that maintain homeostasis of oxidative
stress by reducing ROS and RNS. In addition, oxida-
tive stress can trigger neuroinflammation and apoptosis.
Although nitric oxide (NO) produced by NOS plays impor-
tant roles in promoting vasodilation of cerebral blood ves-
sels and relaxation, overproduction of NO is regarded as
an indication of inflammation. Abnormal NO production
induces the release of inflammatory mediators, such as
iNOS and COX-2, which contributes to the pathogenesis
of NDDs [2]. Mitogen-activated protein kinases (MAPKs)
signaling pathways are one of the crucial mediators that
cause both apoptosis and inflammation. MAPKs trigger
the activation of NF-κB pathways, which later induces the
production of several pro-inflammatory cytokines [14, 15]
including TNF-α. Our study aimed to investigate the pro-
tective activity of NaB against TNF-α-mediated neuronal
cell death with underlying molecular mechanisms.
NaB is a short-chain fatty acid (SCFA) with a molecular
weight of 110.088gm, which shows therapeutic activities
against cancer, aging, cerebral diseases, NDDs [16–19].
NaB is known for its ability to bypass the blood–brain bar-
rier. NaB-producing bacteria are found in the gut [18] that
modulates Nrf-2 antioxidant cellular signaling pathways
including SOD and attenuated AD in obese mice. Moreo-
ver, NaB showed neuroprotective effects against brain
injury and neuronal cell death [20]. However, no reports
regarding the effects of NaB against neuroinflammation has
been found. Therefore, the current study was undertaken
to investigate the neuroprotective effects of NaB and their
underlying molecular mechanisms in TNF-α-induced SH-
SY5Y cells.
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Neurochemical Research
Materials and Methods
Chemicals and Reagents
S o d i u m b u t y ra t e ( Na B ) ch e m i c a l fo r m u l a
­CH3CH2CH2COONa (PubChem ID: 24858121, Molecu-
lar weight: 110.09 g) was purchased from Sigma Aldrich.
Sodium nitrite and MTT(3-(4,5-dimethylthiazol-2-yl)-2,5
diphenyltetrazolium bromide), DMSO (dimethyl sulfox-
ide) were purchased from Sigma Aldrich. DMEM (Dul-
becco’s Modified Eagle’s Medium), DPBS, Penicillin
Streptomycin (P.S), Trypsin EDTA were purchased from
WELGENE Inc. (Seoul, Korea). Western blot primary and
secondary antibodies were collected from Cell Signaling
Technology or Abcam. All the other chemicals and rea-
gents were analytical grade and used without any further
purification.
Cell Culture
Human neuroblastoma SH-SY5Y cells were obtained from
the Korean cell line bank. SH-SY5Y cells were maintained
in DMEM supplemented with 10% FBS and 1% P.S. Cells
were cultured in an incubator at 37 °C with 5% ­CO2 and 95%
humidified condition. Cells were maintained by subculture
and used when confluency reached 80–85%. Cells passage
number 20–30 were used for the experiments.
Cell Viability
The cell viability of NaB was conducted by MTT assay as
previously mentioned [14, 21] with slight modifications.
Briefly, the SH-SY5Y cells were seeded as 5 × ­104 cells per
well in a 96-well plate and after 24 h of incubation, the cells
were treated with various concentrations of NaB with or
without TNF-α (10 ng/mL) for another 24 h. Then, the MTT
was added as 0.5 mg/mL (final concentration) to each well
and kept at 37 °C for 2 h, where live cells form formazan
blue. Afterward, the DMSO was added and the absorbance
was taken at 570 nm in a 96-well plate reader (Molecular
Devices, California, United States). The cell viability was
expressed as 100% of control, where cells were treated with
only Media.
ROS Assay
The reactive oxygen species (ROS) assay of NaB in SH-
SY5Y was performed using the DCF-DA method against
­H 2O 2 stimulation. After 24 h of incubation, cells were
seeded in a black 96-well plate and treated with 01, 0.25,
0.5, and 1 mM NaB. After removing the media on the
Neurochemical Research
following day, 10 μM DCF-DA was added for 30 min and
600 μM of ­H2O2 was added into each well except the con-
trol group for 1 h. Then the fluorescence was measured at
the excitation of 485 nm and the emission of 530.
NO Assay
The nitric oxide (NO) assay of NaB in TNF-α-induced
SH-SY5Y cells was conducted with Griess reagent [15].
An equal number of cells were seeded as 5 × ­104 cells each
well in a 96-well plate. The following day, the cells were
treated with NaB and TNF-α for another 24 h. Then, 80 μL
of supernatant from each group was collected to be mixed
with an equal amount of Griess reagent, and then absorb-
ance was taken at 540 nm. NO production was calculated
from the standard curve of ­NaNO2 by the regression equa-
tion and expressed as μM.
Western Blot Analysis
SH-SY5Y cells were seeded as 1 × ­1 0 6 cells/well in a
6-well plate for western blot analysis [22–24]. The cells
were treated with TNF-α (10 ng/mL) with or without
NaB (0.25, 0.5 and 1 mM) for 24 h and 1 h to investigate
MAPK(P-38) and ERK phosphorylation. Then, the cells
were washed twice by ice-cold PBS and the whole protein
was collected with PRO-PREP (iNtRON Biotechnology))
lysis buffer. The protein concentrations were determined
with Bio-Rad protein assay dye. 30 μg of protein from
each group was subjected to 10% SDS-PAGE gel and
separated by electrophoresis. Then, the proteins were
transferred into a PVDF membrane. After blocking the
membrane with 5%BSA in TBS-T at room temperature
for 1 h, the membranes were cut according to the kDa of
target antibodies. Afterward, the membranes were incu-
bated with corresponding antibodies overnight at 4 °C.
Next, the membranes were incubated with secondary
antibody (anti-rabbit) at room temperature for 1 h after
being washed with TBS-T two times. Finally, the images
of the protein bands were captured using the ECL kit by
ChemiDoc XRS+(Bio-Rad), and the band intensity was
analyzed with ImageJ (NIH).
Table 1  Sequences of primers Name Forward
used for PCR in this study
iNOS 5′-GGT​GTT​GAA​GGC​GTA​GCT​GA-3′
COX-2 5′-ATG​CTC​CTG​CTT​GAG​TAT​GT-3′
MnSOD 5′-TTA​ACG​CGC​AGA​TCA​TGC​A-3′
GPx1 5′-TAT​CGA​GAA​TGT​GGC​GTC​CC-3′
GAPDH 5′-GAG​TCA​ACG​GAT​TTG​GTC​GT-3′
Confocal Microscopy
The SH-SY5Y cells were seeded as 5 × ­104 cells per well in a
6well-plate with coverslips for Confocal Microscopy analysis
as previously reported [14] with some changes. After 24 h of
seeding, the cells were treated with TNF-α (10 ng/mL) with or
without 1 mM NaB, while the control group was treated with
only Media for another 24 h. Afterward, cells were fixed with
4% paraformaldehyde for 15 min, and the cell permeabilization
was done by adding PBS containing 0.3% Triton X-100(Sigma
Aldrich) for 15 min. After blocking with 3% BSA in PBS-T,
the cells were incubated with primary antibody Bcl-2(1:100)
and BAX (1:50) at 4 °C overnight. Then, cells were washed
with PBS-T after removing primary antibodies. Next, the cells
were incubated with secondary antibody (1:1000, Alexa Fluor
568 goat anti-rabbit IgG) for an hour at room temperature in
a dark place. The cells were stained with Hoechst 33342 dye
1 μ/mL (Thermo Fisher Scientific) and the image acquisition
was performed with a confocal microscope.
Extraction of RNA and Reverse Transcriptase (RT)
Polymerase Chain Reaction(PCR)
The RNA was extracted using TRIzol (Invitrogen Life Tech-
nologies, Carlsbad, CA, USA) reagent from the treated cells as
per manufacturer instructions. The extracted RNA was quanti-
fied by nanodrop and an equal amount of RNA was transcribed
by superscript II kit(Invitrogen, San Diego, CA, USA) as previ-
ously mentioned [14, 25]. PCR amplifications were conducted
using emerald Amp 2 × PCR Master Mix (Takara) according
to the manufacturer’s instructions. Then, the mRNA was sub-
jected to 1.5% agarose gel electrophoresis and stained with
EtBr. The mRNA band was detected in ChemiDoc XRS+(Bio-
Rad). The primer sequences have been described in Table 1.
Statistical Analysis
The histograms were presented as mean ± SD for all experi-
mental data. Microsoft Excel 2016 edition and GraphPad
Prism were used to perform data analysis. One-way analy-
sis of variance (ANOVA) as Tukey’s multiple comparison
test in GraphPad Prism, where p-Value less than 0.05 was
expressed as statistically significant. All experimental data
were conducted at least three times.
Reverse
5′-ATC​ATG​GAC​CAC​CAC​ACA​GC-3′
5′-CAC​TAC​ATC​CTG​ACC​CAC​TT-3’
5′-CCT​CGG​TGA​CGT​TCA​GAT​TGT-3′
5′-TCT​TGG​CGT​TCT​CCT​GAT​GC-3′
5′-TGG​GAT​TTC​CAT​TGA​TGA​CA-3′
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Results
Effects of Sodium Butyrate on Cell Viability
and NO Production in TNF‑α Stimulated SH‑SY5Y
Cells
The MTT assay of sodium butyrate (NaB) was performed
to find out the non-toxic dose or safe concentration of NaB
for further assessment. We found no toxic effect of NaB
up to 5 mM in human neuroblastoma as shown in Fig. 1a.
Fig. 1  a The effect of cell viability sodium butyrate (NaB) in the
human neuroblastoma (SH-SY6Y Cells), b The effect NaB on cell
viability in TNF-a induced SH-SY5Y cells and c NO production
against TNF-a induced SH-SY5Y cells. Histograms were presented
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Neurochemical Research
Furthermore, NaB ameliorated TNF-α induced cell death
in Fig. 1b. Cell viability decreased by about 88% of control
when cells were treated with TNF-α alone. NaB restored
cell viability of about 93% at 0.5 mM and of about 100%
at 5 mM. While many studies reported TNF-α may trigger
inflammatory cell death, which leads to several neurode-
generative diseases (NDDs), such as Parkinson’s disease,
Alzheimer’s disease, Huntington disease [26, 27]. Nitric
oxide (NO) production was markedly increased by TNF-α
induction of about 20 μM, while NaB decreased the NO
as Mean ± SD, where n = 3. The p value is determined by ANOVA
with Tukey’s multiple test comparison and p value less than 0.05
was expressed as statistically significant. *p < 0.05, **p < 0.001 and
***p < 0.0001 compared to the only TNF-a-treated group
Neurochemical Research
production notably from the concentration of 0.1 mM. The
decrease of NO by NaB treatment was dose-dependent as
shown in Fig. 1c. Reportedly, NO trigger neuroinflamma-
tion which leads to NDDs, necrosis, and apoptosis [25, 28].
Effects of NaB on Apoptotic Protein Expressions
in TNF‑α Stimulated SH‑SY5Y Cells
P-53, Caspase-3, and BAX (Bcl-2-associated X protein) are
potent apoptotic markers, while increased P-53 is a response
to the genotoxic and pivotal cause of the neuronal loss that
leads to NDDs [4, 29]. Wherein P-53 upregulates pro-apop-
totic BAX protein and down-regulated anti-apoptotic Bcl-2
protein [30]. TNF-α stimulation in SH-SY5Y cells dramati-
cally increased P-53 protein expression from 0.5 to 1.5 fold
and BAX protein from 0.5 to 1.7 fold. NaB reversed P-53
protein significantly from the concentration of 0.25 mM in
a dose-dependent manner. BAX/Bcl-2 ratio was notably
attenuated from 0.25 mM NaB. Moreover, cleaved caspase-3
was blocked by NaB in a dose-dependent manner in TNF-α-
induced SH-SY5Y cells (Fig. 2a).
Effects of NaB on Intercellular Localization of Bcl‑2
and Bax in TNF‑α Stimulated SH‑SY5Y Cells
The human neuroblastoma SH-SY5Y cells were stained
with anti-Bcl-2 and BAX antibodies and observed in a con-
focal microscope. The anti-apoptotic regulator, Bcl-2 was
reduced by TNF-α, while TNF-α increased proapoptotic
regulator BAX in Dapi staining as shown in Fig. 2b. Bcl-2
was remarkably up-regulated when NaB was added to the
TNF-α induced SH-SY5Y cells. On the other hand, NaB
attenuated BAX expression in TNF-α induced SH-SY5Y
cells. The merged image in Fig. 2b shows the relative expres-
sion of Bcl-2 and BAX.
Effects of NaB on Antioxidant Mechanisms in TNF‑α
Stimulated SH‑SY5Y Cells
The nuclear factor erythroid 2-related factor 2(Nrf-2) and
Heme Oxygenase-1(HO-1) are potent cellular antioxidant
mechanisms that maintain homeostasis against excessive ROS
and RNS generation due to metabolism, respiration, and other
physiological processes. NaB modulated TNF-α-influenced
Nrf-2 and HO-1 protein expression as depicted in Fig. 3a.
NaB also modulated the mRNA expression of the antioxi-
dant enzymes, including manganese superoxide dismutase
(MnSOD) and Glutathione peroxidase 1(GPx1) as illustrated
shown in Fig. 3b. Moreover, NaB significantly reduced ROS
production in a dose-dependent manner (Fig. 3c).
Effects of NaB on iNOS and COX‑2 Expressions
in TNF‑α Stimulated SH‑SY5Y Cells
The changes in iNOS and COX-2 expression in TNF-α
stimulated SH-SY5Y cells by NaB treatment were evaluated
through western blotting. TNF-α is a potent cell-mediated
(upon inflammatory response) inflammatory stimuli, which
markedly increased NOS and COX-2 pro-inflammatory
markers in this study. NaB treatment reduced both iNOS
and COX-2 protein expressions in a concentration-dependent
manner as shown in Fig. 4. The mRNA expression of iNOS
and COX-2 by NaB in the TNF-α stimulated SH-SY5y cell
has been added as a supplementary figure.
Effects of NaB on MAPK Activation in TNF‑α
Stimulated SH‑SY5Y Cells
TNF-α is one of the abundantly studied pro-inflamma-
tory cytokines in the nervous system that targets the pro-
inflammatory mediator mitogen-activated protein kinases
(MAPKs) family [31] through TNF receptor. Activation
of extracellular signal-regulated kinase (ERK) and P-38 by
phosphorylation are vital indicators of inflammation. TNF-α
stimulated SH-SY5Y cells had increased P-38 and ERK
phosphorylation whereas NaB dose-dependently decreased
P-38 and ERK phosphorylation (Fig. 5). Noticeably, ERK
activation was significantly suppressed by NaB at 0.25 mM
concentration.
Effects of NaB on Glucocorticoid Receptor
and NLRP3 Inflammasome in TNF‑α Stimulated
SH‑SY5Y Cells
Glucocorticoids receptor (GR) activated as pivotal cell
anti-inflammatory and survival factor which response
against inflammation. GR phosphorylation occurs upon
inflammatory responses, which release glucocorticoids
to restore the inflammatory condition upon inflammation
stimulation. GR phosphorylation rapidly increased when
TNF-α stimulated to inhibiting the cellular inflammations
and NaB altered the GR pathway and restored inflam-
mation. GR pathway and NLRP-3 inflammasome both
respond in inflammation, wherein both target HSP90 [32,
33] and GR showed anti-inflammatory effect by inhibiting
HSP90 protein and NLRP3 modulates HSP90 protein and
leads to inflammation. NaB reduced NLRP3 significantly
in TNF-α stimulated SH-SY5Y cells shown in Fig. 5.
NLRP-3 also triggers apoptotic caspase-1 which leads to
cell death and NaB attenuated remarkably cleaved cas-
pase-1 expression in TNF-α stimulated SH-SY5Y cells.
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Fig. 2  a The effect of NaB on apoptotic protein expressions Bax/
Bcl-2, P-53 and cleaved caspase-3 against TNF-a induced SH-SY5Y
cells. Histograms were presented as Mean ± SD, where n = 3. The p
value is determined by ANOVA with Tukey’s multiple comparison
test and p value less than 0.05 was expressed as statistically signifi-
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Neurochemical Research
cant. *p < 0.05, **p < 0.001 and ***p < 0.0001 compared to the only
TNF-a-treated group. b The effect of NaB on anti-apoptotic Bcl-2 and
proapoptotic BAX expression in Hoechst 33342 staining in TNF-a
stimulated SH-SY5Y cells
Neurochemical Research

Fig. 3  Effect of AG on a Nrf-2, HO-1 protein expression, b MnSOD, the p value is determined by ANOVA with Tukey’s multiple com-
GPx1 mRNA expressions in TNF-a-induced SH-SY5Y cells. c parison test and p value less than 0.05 was expressed as statistically
Effect of AG on ROS generation in H ­ 2O2 stimulated SH-SY5Y significant.*p < 0.05, **p < 0.001 and ***p < 0.0001 compared to the
cells. Histograms were presented as Mean ± SD, where n = 3.and only TNF-a-treated group

Discussions was tremendously studied for its several bio-functional

activities, such as anti-cancer, anti-inflammatory, anti-
In this study, we demonstrated the neuroprotective effects viral and so on [19, 34]. NaB is present in human diet and
of NaB in TNF-α-induced SH-SY5Y cells and showed the produced in the gut, which is absorbed in cells and influ-
underlying mechanisms with molecular experiments. NaB ences cellular signal transductions [18]. The attribution of

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Fig. 4  Effect of AG on TNF-a-induced iNOS and COX-2 expressions
in SH-SY5Y cells. Histograms were presented as Mean ± SD, where
n = 3. and the p value is determined by ANOVA with Tukey’s multi-
NaB in neuroprotective effects by down-regulated apop-
totic proteins and inflammatory cytokines and mediators
could be significant for the use of NaB as a therapeutic
agent against NDDs. We have designed our study in human
neuroblastoma SH-SY5Y cells, which obtained homogene-
ity, express several markers of mature neurons, and widely
studied to investigate neuroprotective effects [8, 35, 36].
Apoptosis plays a crucial role in the progression of
NDDs by neuronal cell loss [35]. We evaluated the protec-
tive actions of NaB against TNF-α induced neuronal cell
death using SH-SY5Y cells that represent many features of
NDDs. TNF-α is a product of cellular response to inflam-
mation against damage, presence of pathogens, debris, etc.
TNF-α attacks cells when inflammation occurs and contrib-
utes to neuronal cell loss that leads to NDDs like Parkin-
son’s disease (PD) [4, 37]. NaB remarkably ameliorated cell
death and significantly reduced NO productions in TNF-α
induced SH-SY5Y cells. To further confirm these results, we
evaluated the expression of potent apoptotic proteins, such
as P-53, BAX and cleaved Caspase-3, where NaB mark-
edly restored anti-apoptotic regulator Bcl-2 against TNF-α
stimulation. Furthermore, NaB modulated BAX localization
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ple comparison test and p-Value less than 0.05 was expressed as sta-
tistically significant. *p < 0.05, **p < 0.001 and ***p < 0.0001 com-
pared to the only TNF-a-treated group
in TNF-α induced SH-SY5Y cells, which was a remarkable
indication of NaB’s effects against neurotoxicity. Excessive
oxidative stress triggers apoptotic pathways and Nrf-2/HO-1
is a potent regulator to maintain homeostasis of oxidative
stress which may be disrupted by TNF-α. NaB modulated
Nrf-2 and HO-1 in TNF-α stimulated SH-SY5Y cells. Also,
NaB modulated antioxidant enzymes and inhibited ROS
generation significantly.
We evaluated the effects of NaB against inflammatory
markers, such as iNOS and COX-2 in TNF-α induced SH-
SY5Y cells. NaB significantly reversed the iNOS and COX-2
expression indicating its anti-inflammatory effects in neu-
ronal cells. When inflammation occurs, TNF-α is released
and induces cellular events that lead to neurodegenerative
diseases. P38-mitogen-activated protein kinase (MAPK)
cascades signaling are widely reported in previous studies
for its crucial role in NDDs [34, 36]. ERK and P-38 are
activated by phosphorylation and target apoptotic regulators
and inflammatory mediators. In this study, NaB significantly
reduced ERK phosphorylation and suppressed pP-38. On the
other hand, MAPK activation produces pro-inflammatory
cytokines, such as interleukins (ILs), interferons (IFNs)
Neurochemical Research

Fig. 5  The effect of NaB on the activation of glucocorticoid receptor, and the p value is determined by ANOVA with Tukey’s multiple
NLRP3 inflammasome and apoptotic caspase1 inhibition and inflam- comparison test and p value less than 0.05 was expressed as statisti-
matory mediator MAPK activations and against TNF-a induced SH- cally significant.*p < 0.05, **p < 0.001 and ***p < 0.0001 compared
SY5Y cells. Histograms were presented as Mean ± SD, where n = 3 to the only TNF-a-treated group

13

TNF-α and so on. The suppression of MAPK cascade sign-
aling by NaB in our study was a potential indicator of the
neuroprotective effects. NaB reduced NLRP3 significantly
and NLRP3 is a pivotal factor of cell death by triggering
caspase cascades and NaB also altered the GR pathway by
attenuating GR phosphorylation. NLRP3 and GR both tar-
get HSP90 [32, 33] wherein GR shower anti-inflammatory
effect by inhibiting HSP90 protein and NLRP3 is modulated
by HSP90 protein and leads to inflammation. TNF-α is a
pro-inflammatory cytokine that has cytotoxicity through
inflammatory pathways. Secretion of TNF-α is a part of the
inflammatory response, which causes inflammation through
TNF receptor (TNFR) in other membranes of cells. In this
study, NaB showed remarkable neuroprotective activity in
SH-SY5Y cells by preventing TNF-α-induced cytotoxicity
and inflammatory responses indicating its therapeutic poten-
tial against NDDs.
Conclusions
NaB ameliorated TNF-α induced cell death and apoptotic
responses through modulating P-53, Bax, and caspase-3
signaling. NaB also enhanced the anti-apoptotic gene Bcl-2
and antioxidant enzymes and suppressed pro-inflammatory
cytokines and MAPK cascade signaling, NLRP3 activation.
These results corroborated previous studies on NaB’s activi-
ties against neuropsychiatric disorders, cerebral ischemia
and others. Our findings suggest that NaB could be a poten-
tial therapeutic agent against NDDs for which further com-
prehensive studies are required.
Supplementary Information The online version contains supplemen-
tary material available at https://​doi.​org/​10.​1007/​s11064-​021-​03369-z.
Acknowledgements This research was financially supported by the
Ministry of Trade, Industry, and Energy (MOTIE), Korea, under the
“Regional Specialized Industry Development Program” supervised by
the Korea Institute for Advancement of Technology (KIAT).
Authors Contributions ABB designed the study, carried out the experi-
ments, and wrote the manuscript. ABB, YAJ, YMK and JGK analyzed
the data and reviewed the manuscript. BOL reviewed and supervised
the study.
Data Availability Data available within the article or its supplementary
materials.
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