Psychopharmacology
https://doi.org/10.1007/s00213-022-06194-6

ORIGINAL INVESTIGATION

Duloxetine ameliorates lipopolysaccharide‑induced microglial
activation by suppressing iNOS expression in BV‑2 microglial cells
Yoshihiko Nakatani1· Manami Yaguchi1 · Kazuki Ogino1 · Risako Noguchi1 · Naoki Yamamoto1,2,3 · Taku Amano4

Received: 9 May 2022 / Accepted: 11 July 2022
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022

Abstract
Rationale It is known that both selective serotonin and serotonin noradrenaline reuptake inhibitors (SSRI, SNRI) are firstline drugs for the treatment of major depressive disorder. It has also been considered that both SSRI and SNRI can improve
the symptoms of major depressive disorder by increasing the concentration of monoamine in the synaptic cleft based on themonoamine hypothesis. However, accumulating evidence has indicated that inflammation in the brain may be a key factor
in the pathophysiological mechanisms that underlie the development of major depressive disorder.
Objectives It has been advocated that microglial cells may regulate the inflammatory response under pathological conditions
such as major depressive disorder. In this study, we focused on whether duloxetine can ameliorate the inflammatory response
induced by lipopolysaccharide (LPS) in BV-2 microglial cells.
Results Our results indicated that duloxetine significantly decreased the NO production induced by LPS. The increase in theprotein expression level of iNOS induced by LPS was significantly decreased by treatment with duloxetine. Moreover, theincreases in the protein expression levels of phosphorylated-IκBα, phosphorylated-Akt and Akt induced by LPS were alsosignificantly decreased. Unexpectedly, the protein expression levels of other pro-inflammatory factors such as COX-2 andthe phosphorylation ratios for various molecules including IκBα and Akt were not changed by treatment with duloxetine.
Conclusions These findings suggest that duloxetine may have an anti-inflammatory effect, which could contribute to its
therapeutic effectiveness for major depressive disorder.
Keywords BV-2 · Duloxetine · LPS · Nitric oxide · iNOS
Abbreviations
NO	Nitric oxide
DUX	Duloxetine
LPS	Lipopolysaccharide
CORT	Corticosterone
FXT	Fluoxetine
SET	Sertraline* Yoshihiko Nakatani
ynakatani@iuhw.ac.jp
1

Department of Pharmacotherapeutics, School of Pharmacy,
International University of Health and Welfare,
2600‑1 Kitakanemaru, Ohtawara, Tochigi 324‑8501, Japan

2

Center for Basic Medical Research,
International University of Health and Welfare,
2600‑1 Kitakanemaru, Ohtawara, Tochigi 324‑8501, Japan

3

Laboratory of Neurobiology, Tokyo Metropolitan University,
1‑1 Minami‑Osawa, Hachioji‑shi, Tokyo 192‑0397, Japan

4

Tochigi Prefectural Okamotodai Hospital, 2162
Shimookamotomachi, Utsunomiya, Tochigi 329‑1104, Japan

CIT	Citalopram
ESCIT	Escitalopram
MCP	Milnacipran
VEN	Venlafaxine
ATX	Atomoxetine
BUP	Bupropion
iNOS	Inducible nitric oxide synthase
G6PD	Glucose-6 phosphate dehydrogenase
COX-2	Cyclooxygenase-2
MAPKs	Mitogen-activated protein kinases
NFκB	Nuclear factor-kappa B
IκBα	Nuclear factor of kappa light polypeptide
gene enhancer in B-cells inhibitor alpha
TNF-α	Tumor necrosis factor alphaIL	Interleukin
JNK	C-Jun N-terminal kinase
JAK/STAT​	Janus kinase/signal transduction and activator of transcription

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Psychopharmacology

Introduction
Numerous reports in the literature, including both clinical and pre-clinical studies, have demonstrated that thedevelopment of depression may involve inflammatory
processes (Dantzer et al. 2008; Dowlati et al. 2010; Han
and Ham 2021; Steiner et al. 2011). In particular, it has
been considered that microglial cells, which are brain
macrophages that play a role in immune defense andinflammatory responses in the brain, regulate the brain
microenvironment including neuronal network activation(Hinwood et al. 2012). In addition, it has been suggested
that stress-induced microglial activation may contributeto depression by increasing pro-inflammatory cytokines
or their mRNA expression, such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6 (Yirmiya
et al. 2015). Considering these reports, it is reasonable
that microglial activation followed by irregularity of thebrain microenvironment is related to the onset of major
depression disorder.
In general, microglial cells are in a “resting” state in thehealthy brain. However, once subjected to unusual stimuli,
microglial cells change to classical (M1) or alternative(M2) types (Guo et al. 2022). Whereas M2 microglia haveneuroprotective effects by producing anti-inflammatory
cytokines such as IL4- and IL-13, M1 microglia induce
inflammation and neurotoxicity by secreting several proinflammatory cytokines such as TNF-α, IL-1β, and IL-6
(Colonna and Butovsky 2017). The change in the phenotype of microglial cells from a “resting” state to M1 istypically induced by treatment with interferon-γ (IFN-γ) or
lipopolysaccharide (LPS) (Colonna and Butovsky 2017).
Activated microglial cells are also linked to the expressionof several pro-inflammatory factors including nitric oxide(NO), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and nuclear factor-kappa B (NFκB)(Fu et al. 2017). In addition, several studies have indicated that NFκB, protein kinase B (Akt), and compounds
in the mitogen-activated protein kinase (MAPK) signaling pathway such as p38 and extracellular signal-regulated
kinase (ERK) are involved in the process of inflammationthat accompanies microglial activation (Chu et al. 2021;
Qin et al. 2018; You et al. 2018). Since the neurotoxicity produced by microglial activation may be involved in
the initiation and progression of irregularity of the brain
microenvironment, the inhibition of microglial activation
may be an approach to improve depression.
Selective serotonin and serotonin noradrenaline reuptake inhibitors (SSRI, SNRI) are considered to be firstline drugs for the treatment of major depressive disorder.
Whereas it is indisputable that the therapeutic effectiveness of both SSRI and SNRI for major depression disorder

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is due to the selective inhibition of monoamine reuptake
in the synaptic cleft, it is also possible that an anti-inflammatory effect via the inhibition of microglial activation
produced by both SSRI and SNRI may contribute to theirpathophysiological effects as antidepressants. It has beenreported that fluoxetine and escitalopram, which are typical SSRIs that are used to treat major depression disorder,
inhibited M1 activation and promoted M2 activation of
microglial cells in vitro (Su et al. 2015). A comparison of
the anti-inflammatory effects of SSRI and SNRI in BV-2
microglial cells treated with LPS demonstrated that fluoxetine, sertraline, and paroxetine significantly decreased the
TNF-α secretion and NO production induced by treatment
with LPS (Liu et al. 2011; Lu et al. 2019; Tynan et al.
2012). In addition, fluvoxamine and reboxetine inhibited
INF-γ-induced microglial production of IL-6 and NO(Hashioka et al. 2007). Considering the studies mentioned
above, it is reasonable that the anti-inflammatory effect of
SSRI on microglia may be associated with its function as
an antidepressant. In contrast, several reports have demonstrated that SNRI also has an anti-inflammatory effecton microglial cells. It has been reported that venlafaxine exhibited an anti-inflammatory effect in an astrogliamicroglia co-culture model by reducing IL-6 and IFN-γ
secretion (Vollmar et al. 2008). Moreover, it has been
demonstrated that both venlafaxine and duloxetine could
decrease the serum TNF-α level that has been elevated bya single administration of LPS in mice (Ohgi et al. 2013).
However, it is still unknown whether an SNRI, such as
duloxetine, venlafaxine, or milnacipran, has anti-inflammatory effects on microglial cells activated by treatment
with LPS.
In this study, we examined the effects of duloxetine,
venlafaxine, and milnacipran on NO production inducedby treatment of BV-2 microglial cells with LPS. We also
explored the molecular mechanisms underlying these effects.

Materials and methods
Cell culture
BV-2 microglial cells were cultured as described previously (Nakatani et al. 2012). Briefly, cells were cultured in Dulbecco’s modified Eagle’s medium with highglucose (Wako, Osaka, Japan) supplemented with 10%
Fetal Bovine Serum (Biowest, Nuaillé, France), 100u/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator under a 95%/5% mixture of air and ­C O 2.
Cells were generally given fresh culture media on thesecond day, and then passaged on the third day.

Psychopharmacology

BV-2 microglial cell proliferation was investigated by a
WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt]
assay using a Cell Counting Kit-8 (Dojindo, Kumamoto,Japan). Once cells became confluent, they were plated into
96-well microplates at a density of 2 × ­105/mL (2 × ­104/well)and incubated with various psychotropic drugs, including
SSRI or SNRI, which were first dissolved in dimethyl sulfoxide (DMSO). When cells were treated with these compounds, the final concentration of DMSO in the culture
medium was set at 0.1%. When cells were treated withlipopolysaccharide (LPS, from E. coli O55, Wako), which
was first dissolved in phosphate buffer saline (PBS), the final
concentration of PBS was also set at 0.1%. In general, thisassay was performed after cells were treated with LPS andeach compound simultaneously and then cultured for 24 h.
After culture, the cells were incubated with WST-8 solution at 37 °C for 2 h. The spectrophotometric absorbanceof WST-8-formazan produced by dehydrogenase activity
in living cells at 450 nm was measured using a VersaMax(Molecular Devices, Sunnyvale, CA, USA). Absorbance at
650 nm was also measured as a reference.

ice. After the sample was centrifuged, the supernatant wascollected as a total protein extract. For western blotting, protein extracts were placed on SDS–polyacrylamide gel, transferred to a polyvinylidene difluoride membrane (BioRad,
Hercules, CA, USA), and immunoblotted with anti-iNOS(ABclonal Technology, MA, USA) antibody, anti-COX-2(Cell Signaling Technology, Danvers, MA, USA) antibody,anti-glucose-6-phosphate dehydrogenase (G6PD, Cell Signaling Technology) antibody, anti-phospho Akt (Ser473, Cell
Signaling Technology) antibody, anti-Akt (Cell Signaling
Technology) antibody, anti-phospho NFκB p65 (Ser536,
Cell Signaling Technology) antibody, anti-NFκB p65 (Cell
Signaling Technology) antibody, anti-phospho IκBα (Ser32,
Cell Signaling Technology) antibody, anti- IκBα (Cell Signaling Technology) antibody, anti-phospho-ERK1/2 (Thr202/
Tyr204, Cell Signaling Technology) antibody, anti-ERK1/2(Cell Signaling Technology) antibody, anti-phospho-p38
MAPK (Thr180/Tyr182, Cell Signaling Technology) antibody, anti-p38 MAPK (Cell Signaling Technology) antibody, and anti-β-tubulin (Wako) antibody. The blots weredeveloped with horseradish peroxidase-conjugated goatanti-rabbit or anti-mouse IgG (Jackson ImmunoResearch,West Grove, PA, USA). Western BLoT Quant HRP Substrate(TaKaRa, Ootsu, Japan) was used for detection.

Nitric oxide measurement

Statistical analysis

NO production was determined by measuring the amountof nitrite using a method that included Griess reagent as
described previously with slight modification (Fu et al. 2017;
Ju et al. 2018). Briefly, 100 µL of supernatant from cells cultured for 24 h with LPS and various psychotropic drugs at
the concentration indicated was isolated and then mixed withan equal volume of Griess reagent (1% sulfanilamide and0.1% N-1-naphtylethylendiamine dihydrochloride in 2.5%
phosphoric acid). After incubation for 20 min at room temperature in the dark, the absorbance at 535 nm was measured
using a VersaMax (Molecular Devices). Nitrite concentrations were calculated according to a nitrite standard curve
generated using sodium nitrite solution at 0–100 µM.

The results are presented as the mean ± S.D. One-way analysis of variance was used to detect significant differences
between the control and treatment conditions. A statisticalanalysis was performed using the Dunnett multiple comparisons test. A p value < 0.05 was considered to be statisticallysignificant.

WST‑8 assay

Western blotting
Protein samples were obtained from BV-2 microglial cells
that had been treated with 1 µg/mL LPS, various concentrations of duloxetine or the vehicle used to dissolve LPS or
duloxetine for 30 min or 24 h. For the preparation of protein
extract from BV-2 microglial cells, the cells were washedwith ice-cold homogenizing buffer (pH7.4; 250 mM sucrose,
20 mM Tris–HCl, 10 mM EGTA, and 2 mM EDTA 2Na),
and centrifuged to obtain a cell pellet. The cell pellet was
treated with lysis buffer (homogenizing buffer with 1% Triton X-100 and a protease inhibitor cocktail) and sonicated on

Results
Effect of LPS on NO production and cell viability
in BV‑2 microglial cells
First, we investigated whether LPS affected NO production
by BV-2 microglial cells. As shown in Fig. 1A and 1B, NO
production by BV-2 cells was observed under treatment withLPS at a concentration greater than 1­ 0–1 µg/mL for 24 h andthis production increased with the further addition of LPS
in a concentration-dependent manner. We also investigatedwhether LPS affected the viability of BV-2 microglial cells,
and found that treatment with 10 µg/mL LPS slightly but
significantly decreased cell proliferation (Fig. 1C). According to these results, we decided to use LPS at a concentrationof 1 µg/mL in further experiments.

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Psychopharmacology
Fig. 1  Effects of LPS on NO
production and cell viability in
BV-2 microglia cells. Nitrite
measurement in LPS-stimulated
BV-2 microglial cells. The
supernatants were obtained andthe concentration of nitrite wasmeasured as NO production in
BV-2 microglial cells treatedwith A ­10–2, ­10–1, and 1 µg/mL
LPS and B 1, 3, 5, and 10 µg/
mL LPS for 24 h. The histogram shows the means ± SD
(n = 3, *; p < 0.05 vs. without
LPS treatment). C WST-8 assay
of BV-2 microglial cells treatedwith 1, 3, 5, and 10 µg/mL LPS
for 24 h. The vertical axis shows
the proliferation rate relative
to that in the control without
LPS. The histogram shows themeans ± SD (n = 3, *; p < 0.05
vs. without LPS treatment)

Effects of psychotropic drugs on NO production
and cell viability in LPS‑stimulated BV‑2 microglialcells
To assess the potential anti-inflammatory effects of psychotropic drugs, we examined which psychotropic drugs could
decrease NO production in LPS-stimulated BV-2 microglial
cells. In this experiment, four SSRIs (fluoxetine, sertraline,
citalopram, and escitalopram), three SNRIs (duloxetine,
milnacipran, and venlafaxine), one NRI (atomoxetine), andone NDRI (bupropion) were examined. Since glucocorticoids inhibited NO production induced by the treatment ofprimary rat microglial cells with LPS, CORT was used asa positive control to decrease NO production induced by
the treatment of BV-2 microglial cells with LPS. All compounds were used at a concentration of 10 µM since 10 µM
CORT significantly decreased NO production in LPS-stimulated BV-2 microglial cells (data not shown). As shownin Fig. 2A, treatment with CORT significantly decreased
NO production to 73.8 ± 7.5% compared to that in LPSstimulated BV-2 microglial cells. Among the SSRIs, both
fluoxetine and sertraline also decreased NO production in
LPS-stimulated BV-2 microglial cells to 78.3 ± 1.4 and63.7 ± 3.8%, respectively, and these results corresponded
to previous reports (Tynan et al. 2012). In contrast, duloxetine significantly decreased NO production to 52.8 ± 7.9%

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compared to an LPS-treated control, which is the greatest
decrease among all of the compounds in this study. Of thecompounds tested, only venlafaxine affected cell viability in
LPS-stimulated BV-2 microglial cells (Fig. 2B).

Effects of duloxetine on NO production and cell
viability in LPS‑stimulated BV‑2 microglial cells
Since only duloxetine had an anti-inflammatory effect, which
manifested as an inhibitory effect on NO production, in LPSstimulated BV-2 microglial cells among various psychotropic drugs, except for some compounds that have alreadybeen shown to have anti-inflammatory effects, we focusedon the anti-inflammatory effect of duloxetine in detail. As
shown in Fig. 3A, duloxetine reduced NO production by
LPS-stimulated BV-2 microglial cells in a concentrationdependent manner at concentrations between 10 µM and20 µM, but not at 1 µM. When we also checked whether
the morphological change caused in LPS-stimulated BV-2
restore by 10 µM duloxetine treatment, duloxetine could
not restore (supplementary Figure S1). In contrast, treatment of LPS-stimulated BV-2 microglial cells with 20 µM
duloxetine decreased cell viability slightly but significantly(Fig. 3B). Since the cell viability of BV-2 microglial cells
that were treated with more than 20 µM duloxetine significantly decreased without any effect on NO production (dataPsychopharmacology

Fig. 2  Effects of corticosterone (CORT) and psychotropic drugs on
NO production and cell viability in LPS-stimulated BV-2 microglial
cells. A Nitrite measurement in LPS-stimulated BV-2 microglial cells
treated with CORT and various psychotropic drugs. The supernatants were obtained and the concentration of nitrite was measured as
NO production in LPS-stimulated BV-2 microglial cells treated with
10 µM of CORT and psychotropic drugs for 24 h. The vertical axis
shows nitrite production relative to that in the control treated with
LPS. The histogram shows the means ± SD (n = 3–4, *; p < 0.05 vs.
LPS treatment). B WST-8 assay of LPS-stimulated BV-2 microglial
cells treated with 10 µM of CORT and various psychotropic drugs for
24 h. The vertical axis shows the proliferation rate relative to that in
the control treated with LPS. The histogram shows the means ± SD
(n = 3–4, *; p < 0.05, **; p < 0.01 vs. with LPS treatment). DUX,
duloxetine; FXT, fluoxetine; SET, sertraline; CIT, citalopram; ESCIT,
escitalopram; MCP, milnacipran; VEN, venlafaxine; ATX, atomoxetine; BUP, bupropion

not shown), we decided to examine the effect of duloxetineby using this range of duloxetine concentrations.

Effects of duloxetine on iNOS expressionin LPS‑stimulated BV‑2 microglial cells
To investigate the molecular mechanisms underlying theanti-inflammatory effect, as reflected by the inhibitory
effect on NO production, in LPS-stimulated BV-2 microglial cells, we examined the effect of duloxetine on theprotein expression level of iNOS in LPS-stimulated BV-2
microglial cells. As shown in Fig. 4, increased protein
expression of iNOS induced by LPS treatment was significantly decreased by treatment with more than 10 µM

Fig. 3  Effects of duloxetine on NO production and cell viability in
LPS-stimulated BV-2 microglial cells. A Nitrite measurement in
LPS-stimulated BV-2 microglial cells treated with various concentrations of duloxetine. The supernatants were obtained and the concentration of nitrite was measured as NO production in LPS-stimulated
BV-2 microglial cells treated with duloxetine at concentrations of 1
to 20 µM for 24 h. The vertical axis shows nitrite production relative to that in the control treated with LPS. The histogram shows themeans ± SD (n = 5, *; p < 0.05, **; p < 0.01vs. LPS treatment). B
WST-8 assay of LPS-stimulated BV-2 microglial cells treated with
duloxetine at concentrations of 1 to 20 µM for 24 h. The vertical axis
shows the proliferation rate relative to that in the control treated with
LPS. The histogram shows the means ± SD (n = 5, **; p < 0.01 vs.
with LPS treatment)

duloxetine. Compared to the results with LPS treatment,
10 µM duloxetine decreased the protein expression levelof iNOS to 72.1 ± 16.8% in a concentration-dependent
manner. In addition, the observation that 1 µM duloxetine
did not induce a decrease in the protein expression levelof iNOS in LPS-stimulated BV-2 microglial cells wasconsistent with the results regarding the inhibitory effectof duloxetine on NO production, as mentioned above.

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Psychopharmacology

evaluate the potential of a further anti-inflammatory effectof duloxetine, we examined whether duloxetine affected theprotein expression levels of both COX-2 and G6PD in LPSstimulated BV-2 microglial cells. As shown in Fig. 5A, while
duloxetine did not decrease the protein expression level of
COX-2 compared to treatment with LPS, the COX-2 expression level was increased in LPS-stimulated BV-2 microglial
cells. In contrast, the protein expression level of G6PD did
not change in the presence of LPS and duloxetine (Fig. 5B).

Effects of duloxetine on activated‑NFκB signalingin LPS‑stimulated BV‑2 microglial cells

It is well known that COX-2 is linked to the activation of
microglial cells via the increase in NO production inducedby treatment with LPS (Fu et al. 2017). In addition, it has
been reported that the induction of G6PD by LPS helps
to prevent NO-mediated glutathione depletion in cultured
astrocytes (García-Nogales et al. 1999). Therefore, to

It has been considered that NO production induced by
LPS via an increase in the iNOS protein expression levelis associated with NFκB signaling. Actually, NFκB regulates several genes involved in the inflammatory response
including iNOS and COX-2 (Ju et al. 2018; Kempe et al.
2005). In the inflammatory condition, NFκB is released
from nuclear factor of kappa light polypeptide geneenhancer in B-cells inhibitor alpha (IκBα) via phosphorylation induced by the action of specific kinase proteinssuch as the IkB kinase complex followed by the degradation of IκBα. In addition, it is well known that the PI3K/
Akt signaling pathway regulates NFκB activation as an
upstream molecule in the NFκB pathway (Cianciulli et al.
2016; Wang et al. 2015). To investigate the molecularmechanisms underlying the anti-inflammatory effect of
duloxetine, NFκB signaling and associated molecules
were examined with respect to whether they were affectedby duloxetine. As shown in Fig. 6A, the protein expression levels of phosphorylated-NFκB that were elevatedby treatment with LPS and NFκB did not change in theFig. 5  Protein expressions of COX-2 and G6PD in LPS-stimulated
BV-2 microglial cells treated with duloxetine. Protein expressions of
A COX-2 and B G6PD were detected in LPS-stimulated BV-2 microglial cells treated with duloxetine at concentrations of 1 to 20 µM for

24 h. The relative expressions of COX-2 and G6PD are shown as therelative ratio based on the respective expression in the control treatedwith LPS. The histogram normalized by the expression of β-actin
shows means ± SD (n = 4, **; p < 0.01 vs. with LPS treatment)

Fig. 4  Protein expression of iNOS in LPS-stimulated BV-2 microglial
cells treated with duloxetine at concentrations of 1 to 20 µM for 24 h.
The relative expressions of iNOS are shown as the relative ratio basedon the respective expression in the control treated with LPS. The histogram normalized by the expression of β-actin shows means ± SD
(n = 4, **; p < 0.01 vs. with LPS treatment)

Effects of duloxetine on COX‑2 and G6PD expressionin LPS‑stimulated BV‑2 microglial cells

13Psychopharmacology
Fig. 6  Expressions of phosphorylated and total proteinsof NFκB and IκBα in LPSstimulated BV-2 microglial cells
treated with duloxetine. The
expressions of phosphorylated
and total proteins of A NFκB
and B IκBα in cells treated with
duloxetine at concentrationsof 1 to 20 µM for 30 min. The
relative expressions of phosphorylated and total proteinsof NFκB and IκBα are shownas the relative ratio based on
the respective expression in
the control treated with LPS.
The histogram normalized
by the expression of β-actin
shows means ± SD (n = 5, *;p < 0.05, **; p < 0.01 vs. with
LPS treatment). The phosphorylation ratios of (C) NFκB and(D) IκBα were calculated in
LPS-stimulated BV-2 microglial
cells treated with duloxetine atconcentrations of 1 to 20 µM for
30 min. The relative phosphorylation ratios of NFκB andIκBα are shown as the relative
ratio based on the respective
expression in the control treatedwith LPS. The histogram showsmeans ± SD (n = 5, **; p < 0.01
vs. with LPS treatment)

presence of duloxetine. In Fig. 6C, the phosphorylation
ratio of NFκB increased in LPS-stimulated BV-2 microglial cells compared to the condition without LPS treatment. However, duloxetine did not alter this increased
phosphorylation ratio of NFκB induced by treatment withLPS even though the range of concentration of duloxetinethat showed the decrease of protein expression level of
iNOS significantly. In contrast, the protein expression
levels of phosphorylated-IκBα elevated by treatment with
LPS significantly decreased in the presence of more than
15 µM duloxetine to 77.1 ± 17.0% compared to that under
treatment with LPS (Fig. 6B). However, duloxetine did notaffect the increase in the phosphorylation ratio of IκBαinduced by treatment with LPS (Fig. 6D).

Effects of duloxetine on activated‑Akt signalingin LPS‑stimulated BV‑2 microglial cells
It has been considered that Akt is regulated as a pro-inflammatory factor as an upstream molecule of NFκB signalingor via a mechanistic target of rapamycin (mTOR) pathway.
As shown in Fig. 7A, the protein expression levels of phosphorylated-Akt elevated by treatment with LPS significantly
decreased in the presence of more than 12 µM duloxetine to13

Psychopharmacology
Fig. 7  Expressions of phosphorylated and total proteinsof Akt in LPS-stimulated BV-2
microglial cells treated with
duloxetine. A The expressionsof phosphorylated and total proteins of Akt in cells treated with
duloxetine at concentrations of 1
to 20 µM for 24 h. The relative
expressions of phosphorylated
and total proteins of Akt are
shown as the relative ratio basedon the respective expression in
the control treated with LPS.
The histogram normalized by
the expression of β-actin showsmeans ± SD (n = 4, *; p < 0.05,**; p < 0.01 vs. with LPS treatment). B The phosphorylation
ratios of Akt were calculated in
LPS-stimulated BV-2 microglial
cells treated with duloxetine atconcentrations of 1 to 20 µM for
24 h. The relative phosphorylation ratios of Akt are shown as
the relative ratio based on therespective expression in thecontrol treated with LPS. The
histogram shows means ± SD
(n = 4, *; p < 0.05 vs. with LPS
treatment)

80.1 ± 4.8%. Moreover, the protein expression levels of Akt
also significantly decreased in the presence of more than
15 µM duloxetine to 79.7 ± 5.5% (Fig. 7B). When the phosphorylation ratio of Akt was calculated, the results revealed
that duloxetine did not alter the increase in the Akt phosphorylation ratio in LPS-stimulated BV-2 microglial cells like
NFκB or IκBα (Fig. 7C).

Effects of duloxetine on MAPKs phosphorylationin LPS‑stimulated BV‑2 microglial cells
It has also been considered that MAPKs plays a role inregulating the expression of inflammatory genes. Indeed,some reports have demonstrated that the inhibition of both
p38 MAPK and ERK1/2 activation blocked iNOS expression in LPS-stimulated murine macrophages or microglial
cells (Ajizian et al. 1999; Zhang et al. 2012). Therefore, toinvestigate whether duloxetine decreased the protein expression level of iNOS through MAPK signaling, the effect of
duloxetine on the phosphorylation ratios of p38 MAPK and
ERK1/2 was examined in LPS-stimulated BV-2 microglial
cells. As shown in Fig. 8A and 8B, LPS treatment itself did
not change the phosphorylation ratio of either p38 MAPK
or ERK1/2 in LPS-stimulated BV-2 microglial cells. The

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phosphorylation ratios of both p38 MAPK and ERK1/2 also
did not change in the presence of LPS and duloxetine in
BV-2 microglial cells.

Discussion
In this study, we first demonstrated that duloxetine has ananti-inflammatory effect via NO production in LPS-stimulated BV-2 microglial cells. In addition, since duloxetine
decreased the protein expression level of iNOS in LPS-stimulated BV-2 microglial cells, it is considered that the reduction of iNOS expression induced by duloxetine contributed
to the inhibitory effect of duloxetine on NO production.
To date, several studies have indicated that psychotropic
drugs, such as SSRI or SNRI, may be able to prevent theactivation of inflammatory factors in LPS-stimulated microglial cells (Hashioka et al. 2007; Liu et al. 2011, 2014; Lu
et al. 2019; Mariani et al. 2022; Ohgi et al. 2013; Su et al.
2015; Tynan et al. 2012). Among various psychotropic
drugs, it has been reported that fluoxetine, an SSRI, has
potential to show an anti-inflammatory effect in vivo (Ohgiet al. 2013) and in vitro (Liu et al. 2011). Importantly, it
has been demonstrated that the anti-inflammatory effectPsychopharmacology
Fig. 8  Phosphorylation ratios
of ERK1/2 and p38 MAPK in
LPS-stimulated BV-2 microglial
cells treated with duloxetine.
The phosphorylation ratios of
A ERK1/2 and B p38 MAPK
were detected in LPS-stimulated
BV-2 microglial cells treatedwith duloxetine at concentrations of 1 to 20 µM for 30 min.
The relative phosphorylation
ratios of ERK and p38 MAPK
are shown as the relative ratio
based on the respective expression in the control treated with
LPS. The histogram showsmeans ± SD (n = 5)

produced by fluoxetine in LPS-stimulated microglia cells
is regulated by the inhibition of NFκB, MAPKs, and ERK
signaling (Liu et al. 2011; Yang et al. 2014). Furthermore,
fluoxetine prevented LPS-induced NO production in primary
microglia cells or BV-2 microglial cells by suppressing theprotein expression level of iNOS (Lieb et al. 2003; Liu et al.
2011). Paroxetine and sertraline have also been shown tohave these anti-inflammatory effects (Liu et al. 2014; Lu
et al. 2019; Ohgi et al. 2013). These results suggest that psychotropic drugs, such as antidepressants, particularly SSRI
and SNRI, may have an anti-inflammatory effect by suppressing NO production via a decrease in iNOS expression.
While the anti-inflammatory effects of SSRIs have been well
documented, whether or not SNRIs, such as duloxetine, venlafaxine, and milnacipran, have anti-inflammatory effects
has not yet been adequately investigated. One study indicated that both venlafaxine and duloxetine attenuated LPSinduced increases in TNFα, but increased serum levels of
IL-10, in mice treated with LPS in vivo (Ohgi et al. 2013).
Moreover, it has been reported that venlafaxine could reducethe increase in the production of TNF-α under treatment
with LPS at only certain concentrations but did not affectthe increased NO production induced by LPS treatment in
an in vitro study (Tynan et al. 2012).
Our results demonstrated that duloxetine, but not venlafaxine or milnacipran, decreased NO production in LPSstimulated BV-2 microglial cells. Since the result that venlafaxine did not reduce LPS-induced NO production wasconsistent with the results of a previous study, it is considered that duloxetine has an anti-inflammatory effect, at least
with regard to NO production. Considering that epinephrine
is potently anti-inflammatory including the inhibitory effectof NO production (de Barros et al. 2012; Dello Russo et al.
2004), the difference in the effect of NO production among
three SSRI might be induced by the difference in the potencyof inhibitory effect of norepinephrine reuptake. Duloxetine isused for the treatment of neuropathic pain as an inhibitor of­P2X4 receptor (Yamashita et al. 2016). Since some reports
have indicated that NO release is mediated by ­P2X4 receptor(Smith et al. 2013; Tu et al. 2017), this might explain the difference between duloxetine and other SNRIs with regard totheir inhibitory effect on NO production in LPS-stimulated
BV-2 microglial cells.
In an attempt to clarify the mechanisms underlying thedecrease in LPS-induced NO production, we found that
duloxetine could reduce the increased protein expression
level of iNOS induced by treatment with LPS. Unexpectedly, duloxetine did not decrease the increase in the protein
expression level of COX-2 induced by treatment with LPS
like a SSRI such as fluoxetine. Moreover, duloxetine also
did not affect the phosphorylation ratios of NFκB, IκBα,Akt, p38 MAPK, or ERK1/2, although these molecules havebeen considered to be part of the main signaling pathways tomediate the expression of inflammatory factor such as iNOS,
COX-2, or NO and to be target molecules for expression of
the anti-inflammatory effects of SSRIs such as fluoxetine(Lieb et al. 2003; Liu et al. 2011; Lu et al. 2019; Yang et al.
2014; Zhang et al. 2012).
Nonetheless, it is interesting to note that the protein
expression level of phosphorylated-IκBα was significantly
decreased by treatment with more than 15 µM duloxetine.
In general, phosphorylated-IκBα proteins are subsequently
ubiquitinated and degraded, which results in the translocation of NFκB from the cytoplasm to the nucleus (Viatour
et al. 2005; Zhang et al. 2020). Duloxetine might preventthe increase in the protein expression of iNOS in LPS-stimulated BV-2 microglial cells by reducing IκBα phosphorylation although there was no change in its phosphorylation
ratio. However, if we consider the phosphorylation level of
NFκB under the influence of duloxetine in LPS-stimulated
BV-2 microglial cells, other mechanisms may also involve
to contribute to suppress iNOS expression by duloxetine in13

Psychopharmacology

LPS-stimulated BV-2 microglial cells. In addition, the protein expression levels of both phosphorylated-Akt and Akt
were also significantly decreased by treatment with duloxetine. It has been reported that activation of Akt via phosphorylation activates IκB kinase followed by IκBα phosphorylation, or induces the phosphorylation of mTOR followedby the regulation of transcription factors including thoserelated to iNOS expression (Fang et al. 2017). Although thephosphorylation ratio of Akt in LPS-stimulated BV-2 microglial cells was unaffected by treatment with duloxetine, thedecrease in phosphorylated-Akt and total Akt might reduce
the pro-inflammatory signal itself induced by LPS, followedby the protein expression of iNOS.
As candidate molecules to modify iNOS expression
except for molecules analyzed in present study, it is possible to be involved c-Jun N-terminal kinase (JNK) or
Janus kinase/signal transduction and activator of transcription (JAK/STAT). Some studies have indicated that
inflammatory effects induced by LPS were mediated by
the activation of JNK (Lim et al. 2018; Liu et al. 2014).
In addition, it has been reported that the inhibitor of JAK/STAT pathway prevented both NO production and protein
expression of iNOS in LPS-stimulated BV-2 microglial
cells (Liu et al. 2014). Thus, it is possible that duloxetine
affects both NO production and iNOS expression via JNKor JAK/STAT signaling modulation. Differences in thesignaling molecules affected by each psychotropic drug
may explain the differences between their effects.
In summary, duloxetine significantly decreased theincrease in the NO production induced by LPS in BV-2
microglial cells. In addition, while the protein expression
levels of iNOS, phosphorylated-IκBα, phosphorylated-Akt
and Akt increased by LPS were also significantly decreased,
the phosphorylation ratio was unaffected in all of these molecules. This anti-inflammatory effect due to the inhibition of
NO production by duloxetine may contribute to its antidepressant effect. Further studies will be needed to clarify themechanisms underlying the regulatory effects of duloxetineon NO production induced by LPS.
Supplementary Information The online version contains supplementary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 00213-0​ 22-0​ 6194-6.
Acknowledgements We thank the members of the Department ofPharmacotherapeutics, School of Pharmacy, International University of
Health and Welfare, for their helpful discussions throughout this study.
Funding This work was supported in part by grants from the Japan
Society for the Promotion of Science (Grant-in-Aid for Scientific
Research (C), 21K07557).

Declarations
Conflict of interest The authors declare no competing interests.

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