RESEARCH ARTICLE

Effect of diclofenac and antidepressants on the inflammatoryresponse in astrocyte cell culture

Md. Mamun Al-Amin • Mir Muhammad Nasir Uddin •

Md. Mahbubur Rahman • Hasan Mahmud Reza •

Md. Sohel Rana

Received: 30 May 2013 / Accepted: 17 July 2013

� Springer Basel 2013

Abstract Central nervous system (CNS) has a com-

pletely separate immune system that communicates with

the neurons by small molecules called cytokines. Cytokines

are involved in many crucial processes in neuron including

cell metabolism and neurotransmitter synthesis. It has been

reported that cytokine imbalance is involved in the pro-

gression of many CNS diseases such as neuropsychiatric

disorders (depression, schizophrenia, autism, and bipolar

disorder) and neurodegenerative disorders (Parkinson’s and

Alzheimer’s disease). Here, the effects of diclofenac, dif-

ferent antidepressants (sertraline, venlafaxine, and

fluvoxamine), and vitamin B6 (pyridoxine) on IL-10 and

tumor necrosis factor-a (TNF-a) change with and without

immune challenges with lipopolysaccharide (LPS) were

investigated in in vitro culture of astrocytes from 2-day-old

Swiss-Albino mice. Diclofenac and Sertraline significantly

(p \ 0.05) improves anti-inflammatory cytokine (IL-10)

while suppress (p \ 0.05) LPS-induced elevated level of

pro-inflammatory mediators (TNF-a) in astrocyte culture.

Pyridoxine was not able to reduce (p [ 0.05) TNF-a in the

astrocyte culture. Antidepressant (sertraline) showed posi-

tive effects (increased IL-10 and reduced TNF-a level)

possibly through the suppression of Th1 lymphocytes and

monocytes and stimulation of Th2 lymphocytes and

monocytes/macrophages. NSAID (diclofenac) showed

positive immune regulation effect possibly through the

inhibition of cyclo-oxygenase enzyme. Based on these

findings, it may conclude that, diclofenac and antidepres-

sants (sertraline) may positively contribute in the cytokine

production in astrocyte cell culture.

Keywords Astrocyte � Serotonin � Tryptophan �Lipopolysaccharide

Introduction

Central nervous system (CNS) is an immunologically

privileged site of the body. Glial cells such as astrocyte

regulate inflammatory response in CNS disorders. Astro-

cyte cells have the ability to produce pattern recognition

receptors (PRR). PRR recruitment in the astrocyte cell

causes the production of many cytokines that promote the

blood–brain barrier (BBB) leakage. Leakage BBB enhan-

ces the engagement of immune cells from the blood

circulation into the CNS. Therefore, overall innate and

adaptive immune system enhances. In results, infectious

agent removed from the injured sites and inflamed or

injured site are repaired. Adaptive immune system exerts

immune function via B and T cells. Th subtype of T cells is

categorized into two types (Th1 and Th2). Th1 produces

Type-1 cytokines such as tumor necrosis factor-a (TNF-a),

Interferon-c (IFN-c), etc., while Th2 cell produces Inter-

leukin-10 (IL-10), Interleukin-4 (IL-4), etc. TNF-a and

Md. M. Al-Amin (&) � H. M. Reza

Department of Pharmacy, North South University, Plot-15,

Block-B, Bashundhara, Dhaka 1229, Bangladesh

e-mail: alamin@northsouth.edu; bd_pharmacy@yahoo.com

M. M. N. Uddin

Department of Pharmacy, Chittagong University, Chittagong,

Bangladesh

Md. M. Rahman

Institute of Pharmacology, University of Lubeck, House 50,

Ratzeburger Allee 160, 23538 Lubeck, Germany

Md. S. Rana

Department of Pharmacy, Jahangirnagar University, Savar,

Dhaka 1342, Bangladesh

Inflammopharmacol

DOI 10.1007/s10787-013-0181-9 Inflammopharmacology

123

IL-10 are the two important cytokines that possesses totally

opposite roles in immune system. TNF-a is a pro-inflam-

matory compound while IL-10 is an anti-inflammatory

compound. Astrocyte cells are able to express TNF-a (Li

et al. 2012). In many cases such as injured brain, infections,

stroke, and cerebral inflammatory and neurodegenerative

diseases, the concentration of TNF-a can be increased in

the brain. Astrocyte cells have also the ability to produce

anti-inflammatory cytokine IL-10. Thus, astrocyte cell

plays an active role in inflammatory disease progression in

the CNS by promoting inflammatory response and many

other ways such as neurodegeneration and providing neu-

roprotective action.

Neuroinflammation and associated disorders

Inflammation in the CNS is developed due to the acti-

vation of astrocyte and microglial cells that produce

major inflammatory mediators (cytokines and chemo-

kines) as well as neurotoxic-free radicals. Cytokines are

involved in many crucial processes in neuron including

cell metabolism and neurotransmitter synthesis. Neuro-

degenerative disorder such as stroke, multiple sclerosis

(MS), Alzheimer’s disease (AD), and neuropsychiatric

disorders such as depression, schizophrenia, autism, nar-

colepsy, anorexia nervosa (AN), obsessive compulsive

disorder (OCD), and bipolar disorder (BD) are often

associated with the modulation of cytokine system in

different brain regions. Several important things that

are affected by the immune system in the CNS are:

(a) indoleamine-2,3 dioxygenase (IDO enzyme) in the

kynurenine pathway, (b) neurotransmitter synthesis (for

instance; dopamine and serotonin), (c) various neuronal

networks (e.g., ACC, basal ganglia), (d) cytokine signals

(such as, NFkB).

Rationale of selecting TNF-a and IL-10

We were especially interested in studying TNF-a and

IL-10 level in astrocyte cells in animal model. There are

many inflammatory diseases in the CNS where these

cytokines are involved. TNF-a was chosen due to its

involvement as a trait marker in neuropsychiatric disorders

such as major depression, bipolar disorders, schizophrenia,

and OCD (Najjar et al. 2013). Moreover, IL-10 was

selected because of its active role in modulating the syn-

thesis of pro-inflammatory cytokines in the brain. For

example, animal study reported that IL-10 imbalance

(especially low level) causes depressive-like behavior

(Mesquita et al. 2008). Many chemical pathways and

immune regulation might be affected by this cytokine.

TNF-a particularly interrupts intracerebral immune

response demyelination associated with inflammation.

Rationale of using diclofenac, antidepressants, and

pyridoxine

A large number of studies have been reported that IDO

enzyme in the kynurenine pathway is linked with many

inflammatory CNS disorders. We thought that the aforesaid

drugs might affect IDO enzyme by influencing the pro-

duction of cytokines in the astrocyte cells. In this study,

several antidepressants, pyridoxine (vitamin B6), and an

anti-inflammatory drug (diclofenac) were selected to

investigate their influence on LPS-induced IL-10 and TNF-

a level in astrocyte culture. It is known that TNF-a induces

IDO enzyme activation in the tryptophan metabolism into

kynurenine pathway (Connor et al. 2008; Li et al. 2011).

Previous study showed that cyclo-oxygenase inhibitor

might have the ability to inhibit IDO enzyme activation

(Lee et al. 2009). Therefore, we may expect that cyclo-

oxygenase inhibitor such as diclofenac might have an

active role to downregulate IDO enzyme activation through

the reduction of cytokine production. In result, sufficient

level of tryptophan would be available for serotonin pro-

duction. Beside diclofenac, antidepressants may also

reduce pro-inflammatory cytokine and enhance anti-

inflammatory cytokines. We thought that astrocyte cell

culture would provide us similar result. Moreover, pyri-

doxine is a vitamin, which has long been claimed to have

antidepressant action. It may inhibit cytokine production.

Thus, pyridoxine may stop the activation of IDO enzyme

by pro-inflammatory cytokines. We wanted to see the

effect of pyridoxine on inflammatory response in LPS-

stimulated astrocyte culture.

Methods

Sample collection

Two-day-old Swiss-Albino mice (total 20) were killed by

decapitation. The brain was dissected carefully using an

applicator and the scissors. Afterward it was removed

immediately (\60 s) from the skull and placed in a 60 mm

Petri dish and immersed in medium. Each pup’s brain was

separately placed in the Petri dish. The brain tissue was

washed and homogenized gently and seeded into 75 cm2

culture flasks in RPMI with supplements. Ethical Com-

mittee of Department of Pharmacy, North South University

approved the animal study.

Cell culture

After 10–12 days incubation at 37 �C with change of

medium at day 3, 5, and 8, the cell culture was shaked for

6 h to remove the microglial cells. The remaining

Md. M. Al-Amin et al.

123

astrocytes were subcultivated by trypsinization to passage

1 and incubated at 37 �C for 3–4 days. Following tryp-

sinization to passage 2 and seeded 100,000 cells/well in a

24-well plate (1 ml RPMI culture medium/well). After

24 h incubation, stimulation started with LPS (5 lg/ml

final) and the treated with diclofenac (100 ng/ml), three

antidepressants; fluvoxamine (300 ng/ml), sertraline

(200 ng/ml), venlafaxine (250 ng/ml), and pyridoxine

(300 ng/ml). Pure LPS medium was used as a control

condition. Incubation was done at 37 �C for 72 h. Then

supernatant and the cell layer (in Trypsin) were collected

and stored in a freezer at a temperature of -80 �C until

analysis.

We used ELISA method to determine the IL-10 and

TNF-a content. ELISA is a well-established method to

measure neuroinflammatory response in astrocyte culture

(Qin and Benveniste 2012). The concentrations of IL-10

and TNF-a in the astrocyte culture media were determined

using commercially available ELISA kits (R&D Systems,

Minneapolis, MO, USA) according to the manufacturer’s

instructions. In our study, intra-assay CV values were

\8 % and the limits of detection (LOD) were: IL-10:

10 pg/ml; TNF-a: 5 pg/ml.

Statistical analysis

The results of statistical analysis for animal experiment

were expressed as mean ± SEM and were evaluated by

ANOVA followed by Dunnet’s multiple comparisons. The

p \ 0.05 were considered to be statistically significant. All

the statistical tests were carried out using SPSS (version

16.0).

Results and discussion

Effect of medications on IL-10 levels

Initially negligible amount of IL-10 was found while LPS

stimulation significantly (p \ 0.05) increases IL-10 level in

astrocyte culture. Diclofenac and sertraline both signifi-

cantly (p \ 0.05) stimulates IL-10 level in astrocyte culture

(Fig. 1). Other two antidepressants and pyridoxine did not

induce significant (p [ 0.05) change in IL-10 level.

Effect of medications on TNF-a level

In the beginning, insignificant amount of TNF-a was found

while LPS stimulation significantly (p \ 0.05) increases

TNF-a level in astrocyte culture. TNF-a level were

significantly (p \ 0.05) decreased by diclofenac- and

sertraline-treated astrocyte culture (Fig. 2). Other two

antidepressants and pyridoxine did not significantly

(p [ 0.05) stimulate TNF-a level.

Several conclusions can be drawn from the present

study. Diclofenac and sertraline improves anti-inflamma-

tory cytokine (IL-10) in one hand and reduces pro-

inflammatory cytokine (TNF-a) on the other hand in the

LPS-stimulated astrocyte culture. Pyridoxine was not able

to reduce pro-inflammatory cytokine (TNF-a) in the

astrocyte culture.

In astrocyte, Th1 lymphocytes and M1 phenotype

monocyte secretes TNF-a level while Th2 lymphocyte and

IL-10

0

50

100

150

200

250

No_S S_L L_D L_F L_V L_S L_P

Co

nce

ntr

atio

n (

pg

/ml)

Fig. 1 Effect of different medications on IL-10 level in astrocyte cell

culture. Culture conditions were No_S, no stimulation only Medium;

S_L, stimulation with LPS; L_D, stimulation with LPS and treated

with diclofenac; L_F, stimulation with LPS and treated with

fluvoxamine; L_P, stimulation with LPS and treated with pyridoxine;

L_S, stimulation with LPS and treated with sertraline; L_V,

stimulation with LPS and treated with venlafaxine

TNF-

0

20

40

60

80

No_S S_L L_D L_F L_V L_S L_P

Co

nce

ntr

atio

n (

pg

/ml)

Fig. 2 Effect of different medications on TNF-a level in astrocyte

cell culture. Culture conditions were No_S, no stimulation only

medium; S_L, stimulation with LPS; L_D, stimulation with LPS and

treated with diclofenac; L_F, stimulation with LPS and treated with

fluvoxamine; L_P, stimulation with LPS and treated with pyridoxine;

L_S, stimulation with LPS and treated with sertraline; L_V,

stimulation with LPS and treated with venlafaxine

Effect of diclofenac and antidepressants

123

M2 phenotype monocyte secrets IL-10. Both types of

monocyte phenotype may interact with the tryptophan

metabolism by inducing TDO, IDO enzymes in many CNS

disorders including major depressive disorders, schizo-

phrenia. Anti-inflammatory cytokines those are secreted by

astrocytes, Th2 lymphocytes, T regs, and M2 phenotype

monocytes/macrophages can control neuroinflammation by

converting M1 phenotype into beneficial anti-inflammatory

M2 phenotype. This conversion process is supported by

animal study where it has been reported that IL-10 inacti-

vation enhanced LPS-induced TNF-a release. Mechanism

of IL-10 action also can be explained as IL-10 is probably

interfere pro-inflammatory cytokine production by dimin-

ishing the expression of pro-inflammatory cytokine

receptor (Sawada et al. 1999).

In our study, reduction of pro-inflammatory compound

with the treatment of diclofenac might be achieved by

blocking cyclo-oxygenase (COX) enzyme in the astrocyte

culture. Reduction of pro-inflammatory cytokine could ulti-

mately stop the induction of IDO enzyme and hence

tryptophan metabolism into the kynurenine pathway.

Therefore, sufficient level of tryptophan will be available for

serotonin production and neurotoxic metabolites synthesis

might be reduced. Here, our findings are consistent with the

previous findings. Akhondzadeh et al. (2009) showed that

COX inhibitors have significant role in the treatment of

depressive symptoms by reducing the production of pro-

inflammatory cytokines. Guo et al. (2009) found that the

chronic treatment with COX inhibitor reverses chronic

unpredictable stress-induced depressive-like behavior via

reducing cyclo-oxygenase-2 expression in rat brain.

This study demonstrated that sertraline reduces LPS-

induced elevated TNF-a level while stimulates IL-10 level.

Sertraline could diminish the release of pro-inflammatory

cytokine from M1 phenotype monocytes and macrophages

while boost up the release of anti-inflammatory cytokines.

In vitro study supports our present findings where sertraline

causes increased level of IL-10 production while inhibiting

pro-inflammatory cytokines (Maes et al. 1999). Patient

with MDD was reported to have higher TNF-a level. This

increased TNF-a level was decreased following the treat-

ment with Sertraline (Sutcigil et al. 2007). The stimulation

of IL-10 level by sertraline can be mediated by non-specific

mechanisms through inhibitory effects on lymphocyte

blastogenesis, such as DNA synthesis in lymphocytes or

alterations in second messenger cyclic AMP (Xia et al.

1996).

We have not found immunomodulatory effect of pyri-

doxine in the LPS challenged astrocyte culture. This study

confirms the previous report that pyridoxine does not

improve inflammatory compounds (Chiang et al. 2005).

In the present study, a question may arise in ones mind

that concentration of diclofenac and sertraline used in this

study might be higher than normal to get this effect. But,

we treated astrocyte cultures with diclofenac and antide-

pressants at a concentration that are clinically relevant with

regard to the therapeutic plasma level. We used LPS for the

astrocyte culture stimulation. LPS is a bacterial toxin,

which can bind with the TLR-4 (Toll-like receptor) in the

cell membrane. TLR-4 binding with the LPS causes the

activation of second messenger system and finally inflam-

matory compounds are produced from the cell. Another

important fact is the relevancy of in vitro findings with

in vivo data. Responses of the immune inflammatory sys-

tem to antidepressants have been clearly interpreted in this

paper.

In the present study, TNF-a was selected because it

might contribute to the pathogenesis in many CNS disor-

ders. TNF-a might influence the progression of

pathogenesis by the activation of hypothalamo-pituitary-

adrenocortical (HPA) axis, neuronal serotonin transporters,

and IDO enzymes. Activation of IDO enzyme leads to the

tryptophan deficiency (Wichers and Maes 2002) and the

production of neurotoxic metabolites such as 3-hydroxy-

kynurenine (3-HK) and quinolinic acid (QUIN) that causes

damage to the neurons. The diminish production of TNF-aby diclofenac and sertraline might stop the progression of

neuroinflammation, which can give an idea of developing

inflammatory drug target in CNS disorders. In addition, IL-

10 is able to diminish the effects of pro-inflammatory

cytokine (such as TNF-a). Our results may explain in a

way that, diclofenac and antidepressants (specifically ser-

traline) might decrease TNF-a level possibly by increasing

IL-10 level.

Based on the previous and our present findings, it may

conclude that diclofenac and antidepressants both could

have beneficial activity in improving inflammatory cyto-

kine levels in the astrocyte cell. However, further in vivo

study needed to conduct to confirm our present findings.

Conclusion

In our present study, diclofenac, antidepressants, and pyr-

idoxine were used to investigate their effect on TNF-a and

IL-10 level in the LPS-stimulated astrocyte culture. Dic-

lofenac and Sertraline might be a promising option for

controlling cytokine production in the astrocyte cells. Due

to the financial limitation and limited facilities, we were

not able to investigate few more cytokine. However, in

future study, we will recruit more ELISA Kit to analyze

other important cytokines such as IL-4, IL-6, IFN-c, etc.

Moreover, the study of pro- and anti-inflammatory cyto-

kine balance, their ratio present in the CNS could give us a

better idea to develop strategy in designing treatment

strategies.

Md. M. Al-Amin et al.

123

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