Capsaicin Modulates Proliferation, Migration, and Activation of Hepatic Stellate Cells

ORIGINAL PAPER

Capsaicin Modulates Proliferation, Migration,and Activation of Hepatic Stellate Cells

Shanna Bitencourt • Fernanda Mesquita • Bruno Basso • Julia Schmid •

Gabriela Ferreira • Lucas Rizzo • Moises Bauer • Ramon Bartrons •

Francesc Ventura • Jose Luis Rosa • Inge Mannaerts •

Leo Adrianus van Grunsven • Jarbas Oliveira

� Springer Science+Business Media New York 2013

Abstract Capsaicin, the active component of chili pep-

per, has been reported to have antiproliferative and anti-

inflammatory effects on a variety of cell lines. In the cur-

rent study, we aimed to investigate the effects of capsaicin

during HSC activation and maintenance. Activated and

freshly isolated HSCs were treated with capsaicin. Prolif-

eration was measured by incorporation of EdU. Cell cycle

arrest and apoptosis were investigated using flow cytome-

try. The migratory response to chemotactic stimuli was

evaluated by a modified Boyden chamber assay. Activation

markers and inflammatory cytokines were determined by

qPCR, immunocytochemistry, and flow cytometry. Our

results show that capsaicin reduces HSC proliferation,

migration, and expression of profibrogenic markers of

activated and primary mouse HSCs. In conclusion, the

present study shows that capsaicin modulates proliferation,

migration, and activation of HSC in vitro.

Keywords Hepatic stellate cell � Capsaicin �Proliferation � Migration � Activation

Introduction

The recruitment of HSCs to sites of injury is thought to be

an early step in their function in repair and matrix

remodeling during liver fibrosis. These cells undergo a

process of activation headed for a phenotype characterized

by increased proliferation, motility, contractility, and syn-

thesis of ECM components. Stimulation of HSCs is regu-

lated by several soluble factors, including cytokines,

chemokines, and growth factors [1]. HSC activation is the

major contributor for the development of hepatic fibrosis.

Based on this knowledge, antifibrotic therapies for the liver

could be based on the inhibition or reversion of HSC

activation [2, 3]. Triggering the activity of interstitial col-

lagenases, such as matrix metalloproteinases (MMPs),

could be also an attractive strategy to deactivate HSC from

the view point that the imbalance between MMPs and

tissue inhibitors of metalloproteinases (TIMPs) is the main

obstacle to the reversion of activated HSC [4].

Previously published work demonstrates the efficacy of

natural products for the treatment of hepatic fibrosis. The

use of nutraceuticals that can inhibit the proliferation of

activated HSCs has a great potential in reversing fibrosis

[5]. Capsaicin, a naturally occurring phytochemical, is the

major ingredient of hot peppers and it is well known to have

anti-inflammatory and antiproliferative properties [6–8].

Leo Adrianus van Grunsven and Jarbas Oliveira have contributed

equally to this study.

S. Bitencourt � F. Mesquita � B. Basso � J. Schmid �G. Ferreira � J. Oliveira (&)

Laboratorio de Pesquisa em Biofısica Celular e Inflamacao,

Pontifıcia Universidade Catolica do Rio Grande do Sul

(PUCRS), Avenida Ipiranga 6681, predio 12, bloco C, sala 221,

Porto Alegre, RS CEP 90619-900, Brazil

e-mail: [email protected]

S. Bitencourt � I. Mannaerts � L. A. van Grunsven

Liver Cell Biology Lab, Department of Cell Biology,

Vrije Universiteit Brussel, Brussels, Belgium

L. Rizzo � M. Bauer

Laboratorio de Imunologia do Envelhecimento, Instituto de

Pesquisas Biomedicas, Pontifıcia Universidade Catolica do Rio

Grande do Sul, Porto Alegre, RS, Brazil

R. Bartrons � F. Ventura � J. L. Rosa

Departament de Ciencies Fisiologiques II, Campus de Bellvitge,

Universitat de Barcelona, L’Hospitalet de Llobregat, Barcelona,

Spain

123

Cell Biochem Biophys

DOI 10.1007/s12013-013-9719-0

In order to clarify the mechanism underlying the anti-

proliferative effects of capsaicin, we investigated whether

capsaicin alters cell cycle or induces programmed cell

death and examined its effect on cell migration and acti-

vation of HSCs, which play key roles in liver fibrogenesis.

Here, we show that capsaicin reduces cell proliferation

through cell cycle arrest, regulates profibrogenic/anti-

fibrogenic molecules balance, and inhibits activation and

cell migration in mouse HSCs.

Methods

Cell Culture and Treatment

The murine HSC line GRX was obtained from the Rio de

Janeiro Cell Bank (HUCFF, UFRJ, Rio de Janeiro, Brazil).

Cells were cultured in Dulbecco’s Modified Eagle’s Med-

ium supplemented with 5 % fetal bovine serum (Invitrogen,

Carlsbad, CA), 2 g/L HEPES buffer, 3.7 g/L NaHCO3, and

1 % penicillin and streptomycin (Invitrogen) in a humidi-

fied atmosphere of 5 % CO2 at 37 �C. Mouse HSCs were

isolated from normal livers of male Balb/c mice (aged

20–27 weeks) by the pronase-collagenase method followed

by a Nycodenz gradient as described elsewhere [9, 10].

After isolation, cells were plated on plastic for several days.

Animals were used in accordance with institutional ethical

guidelines. Cells were incubated with 100 lM capsaicin

(M2028, Sigma-Aldrich, St. Louis, MO) for different time

points as indicated in the figure legends.

Viable Cell Counting

GRX cells were seeded into 24-well plates at a density of

5 9 105 cells/well and treated with capsaicin for 24 h. For

determination of cell number, cells were counted using a

hemocytometer. Trypan blue analyses were performed to

determine cellular viability.

Proliferation Assay

Cell proliferation was measured as active DNA synthesis

with the Click-iT EdU Cell Proliferation Assay Kit

(Invitrogen). GRX and freshly isolated mouse HSCs were

plated in the presence or absence of capsaicin and/or the

mitogen PDGF-bb (20 ng/mL; R&D Systems, Minneapo-

lis, MN). After 48 h, EdU labeling was initiated. After 24

or 48 h, for GRX and primary mouse HSC, respectively,

cells were formalin-fixed. Visualization of the EdU incor-

poration was obtained according to the manufacturer’s

instructions. The ratio of total cells and EdU incorporated

cells was calculated.

Cell Cycle Analysis

To determine the effect of capsaicin on the cell cycle,

defined as G0/G1, S, and G2/M phase, GRX cells were first

synchronized by 24 h serum starvation and then exposed to

capsaicin for 24 h. Cell cycle phase analysis was per-

formed using the FITC BrdU Flow Kit (BD Biosciences,

San Jose, CA). In brief, after treatment and BrdU labeling,

cells were harvested by trypsinization and adjusted to

1 9 106 cells/mL. Samples were fixed with BD Cytofix/

Cytoperm Buffer (BD Biosciences). Following fixation,

cells were treated with DNase for 20 min to expose BrdU

epitopes. Later on, DNA was stained for cell cycle analysis

using 7-AAD dye. The DNA profiles were determined by

FACSCanto II flow cytometer (BD Biosciences) and ana-

lyzed using the FlowJo 7.2.5 software (Tree Star Inc.).

Quantification of Apoptosis

Apoptosis was assessed using the FITC Annexin V

Apoptosis Detection Kit I (BD Bioscience). In brief, after

treating with capsaicin for 24 h, GRX cells were washed

twice with PBS and resuspended in binding buffer before

addition of annexin V-FITC and propidium iodide (PI).

Cells were vortexed and incubated for 15 min in the dark at

room temperature. A total of 10,000 events were acquired

for each assayed sample. All data were acquired with a

FACSCanto II flow cytometer (BD Biosciences). Data

were analyzed using the FlowJo 7.2.5 software (Tree Star

Inc., Ashland, OR). Results are displayed as scatter dots

allowing discrimination among viable cells, apoptotic cells

with an intact membrane, and cells undergoing secondary

necrosis.

Analysis of Mitochondrial Membrane Potential (DWm)

Breakdown of DWm was determined by FACS analysis

using the MitoScreen Kit (BD Biosciences). JC-1 (5,5,6,

6-tetra-chloro-1,1,3,3-tetraethylbenzimidazol-carbocyanine

iodide) dye, which is selectively incorporated into mito-

chondria, is a sensitive and reliable method to detect

changes of the mitochondrial membrane potential (DWm)

[11, 12]. After incubation for 24 h with capsaicin, cells

were stained with 0.5 ml JC-1 solution for 15 min at

37 �C. Stained GRX were washed twice in JC-1 Mito-

Screen wash buffer. A total of 10,000 events were acquired

for each assayed sample. All data were obtained immedi-

ately after staining on a FACSCanto II flow cytometer with

CellQuest PRO v4.0.2 software (BD Biosciences). Results

are displayed as scatter dots allowing discrimination

between polarized and depolarized cells.


123

Cell Migration

Cell motility was determined with a modified Boyden

chamber assay. In brief, cells were suspended in a medium

supplemented with 2.5 % serum (GRX, 2 9 105/well) or

serum-free medium (mouse HSCs, 5 9 104/well) in the

presence or absence of capsaicin. The cells were then

seeded into the upper chambers of transwell inserts

equipped with 8-lM pore polyethylene terephthalate filters

(Millipore, Billerica, MA) previously coated with type I

collagen (4 mg/mL; BD Biosciences). The lower chambers

were filled with medium supplemented with 2.5 % serum.

For the chemotaxis experiment, the lower chambers were

filled with serum-free medium supplemented with PDGF-

BB (20 ng/mL). After 24 h, non-migrated cells on the

upper side of the membrane were rubbed off with a cotton

swab. Migrated cells on the underside of the membrane

were fixed in 100 % methanol, stained with DAPI, and

counted in a fluorescent microscope.

Real Time Quantitative PCR (qPCR)

Total RNA of GRX and primary mouse HSC was extracted

using Reliaprep RNA Cell Miniprep System purification kit

(Promega, Madison, WI). RNA was reverse-transcribed

using RevertAidTM Premium Reverse Transcriptase kit

(Fermentas, St. Leon-Rot, Germany). For real-time PCR,

GoTaq qPCR Master Mix with BRYTE green was used

(Promega), subjected to qPCR in an ABI 7500 Real Time

PCR System and analyzed using System SDS software

(Applied Biosystems, Foster City, CA, USA). The gene-

specific primers used are listed in Table 1. GAPDH was

used as reference gene.

Quantification of Cytokines

To determine cytokine production, GRX cells were cul-

tured for 24 h and 10 days. The supernatants were col-

lected and stored at -20 �C for later analysis. Multiple

soluble cytokines (IL-12 (p70), IFN-c, IL-10 and MCP-1)

were simultaneously measured by flow cytometry using the

cytometric bead array (CBA) Mouse Inflammation Kit (BD

Biosciences). Acquisition was performed with a FACSC-

anto II flow cytometer (BD Biosciences). Quantitative

results were generated using FCAP Array v1.0.1 software

(Soft Flow Inc., Pecs, Hungary). The detection limit was

20–5,000 pg/mL.

Immunocytochemistry

Freshly isolated mouse HSCs were cultivated on glass

coverslips and formalin-fixed after 10 days of culture in the

presence or absence of capsaicin followed by overnight

incubation with primary antibody against a-smooth muscle

actin (a-SMA, 1/1000; Sigma). Antibody binding was

visualized using Alexa 488-labeled antibody (1/250;

Molecular Probes, Eugene, OR). Following mounting with

ProLong� Gold antifade reagent with DAPI (Invitrogen),

cells were analyzed by fluorescent microscope (Carl Zeiss,

Zaventem, Belgium).

Table 1 List of qPCR primersGene Genebank accession number Primers sequence (forward/reverse)

GAPDH NM_008084.2 50-cctgcttcaccaccttcttg-30/

50-tgtccgtcgtggatctgac-30

a-SMA NM_007392 50-ccagcaccatgaagatcaag-30/

50-tggaaggtagacagcgaagc-30

Lox NM_010728 50-tcactgcgctcgttctgat-30/

50-cgatcgaaagtatgagggatg-30

Col1a1 NM_007742 50-cctaagggtaccgctgga-30/

50-tccagcttctccatctttgc-30

Col3a1 NM_009930.1 50-tggtcctgctggaaaggat-30/

50-caggcagtccacgctctc-30

MMP-13 NM_008607.2 50-tgtttgcagagcactacttgaa-30/

50-cagtcacctctaagccaaagaaa-30

MMP-2 NM_008610.2 50-aactttgagaaggatggcaagt-30/

50-tgccacccatggtaaacaa-30

MMP-9 NM_013599.2 50-ctggacagccagacactaaag-30/

50-ctcgcggcaagtcttcagag-30

TIMP-1 NM_001044384.1 50-gcaaagagctttctcaaagacc-30/

50-aagggatagataaacagggaaaca-30


123

Statistical Analysis

Statistical analyses were performed using GraphPad Prism

5.0 (GraphPad Software, San Diego, CA). All experiments

were repeated at least three times and data expressed as

mean ± SEM. Comparisons were performed according to

Student’s t test or one-way ANOVA followed by Bonfer-

roni’s post-test wherever appropriate. A P value\0.05 was

considered statistically significant.

Results

To further characterize the antiproliferative action of cap-

saicin, we investigated the effect of this molecule on cell

cycle and programmed cell death of activated HSCs. GRX,

a murine cell line that presents a myofibroblast transitional

phenotype, was cultured with capsaicin for a 10-day per-

iod. The effect on cell migration and the expression and

release of pro- and anti-fibrogenic markers were also

assessed. To analyze the effects of capsaicin during HSC

activation, freshly isolated mouse HSCs were also cultured

with capsaicin. The dose and period of incubation of cap-

saicin were based on our previous work [8].

Capsaicin Inhibits Activated HSC Proliferation

and Migration

As shown in Fig. 1a, a 24 h capsaicin (100 lM) exposure

significantly reduced the number of viable GRX cells by

approximately 32 % compared with untreated control. In

order to analyze whether the above effect of capsaicin on

cell viability was accompanied by decreased entry into S

phase and DNA synthesis, incorporation of EdU, a thy-

midine analog, was assessed in serum-starved GRX cells.

EdU incorporation was measured after a 24 h treatment

with PDGF-BB in the presence or absence of capsaicin. As

shown in Fig. 1b, capsaicin led to a significant decrease in

the proportion of EdU? cells when compared with non-

treated cells. The effects of capsaicin on PDGF-BB-

induced mitogenesis were also evaluated. PDGF-induced

HSC proliferation was drastically reduced after capsaicin

administration. While PDGF-BB-treated cells showed

about 25 % DNA incorporation of EdU, the co-treatment

of PDGF-BB and capsaicin showed only 2 %, indicating

less proliferation (Fig. 1b). We also analyzed the cell cycle

by flow cytometry. The analysis showed that the antipro-

liferative effect of capsaicin was associated with a G0/G1

cell cycle arrest (Fig. 1c). The percentage of cells in phase

G0/G1 increased from 31.2 % in control cells to 44.7 % in

cells treated with capsaicin.

The above-mentioned findings raised the question

whether apoptosis was changed in capsaicin-treated cells.

The potential effect of capsaicin on apoptosis was analyzed

by two different methods, one detecting apoptotic cells by

measuring the translocation of phosphatidylserine to the

outer cell membrane surface and a second measuring the

impact on mitochondrial transmembrane potential (DWm).

Flow cytometric analysis of the Annexin V labeling assay

Fig. 1 Effects of capsaicin on activated HSC proliferation. a GRX

cells were treated with capsaicin for 24 h and cell viability assessed

by direct cell counting. Results are expressed as cell number. b GRX

cells were cultured for 24 h in the presence or absence of CPS and/or

PDGF-BB. Proliferation rate was assessed by EdU incorporation

assays. Results are expressed as percentage of EdU? cells. c Effect of

24-h capsaicin treatment on the GRX cell cycle measured by flow

cytometry. Results are expressed as percentage of cell number. All

data represent the mean ± SEM (n = 3). *P \ 0.05, **P \ 0.01,

***P \ 0.001. CPS capsaicin


123

did not detect apoptosis in the capsaicin-treated cells. As

shown in Fig. 2a, the number of apoptotic cells did not

increase compared to control experiments. To assess the

effects of capsaicin on mitochondrial injury, we analyzed

the DWm of treated cells. Changes of DWm were deter-

mined by JC-1 staining of GRX cells treated with capsaicin

for 24 h. Capsaicin did not provoke significant loss of

DWm (Fig. 2b).

To examine the effects of capsaicin on the migration of

activated HSCs, a modified Boyden chamber migration

assay was performed. PDGF-BB was used as chemoat-

tractant. As shown in Fig. 3, GRX cells treated with cap-

saicin showed a reduced cell motility even in the presence

of PDGF-BB.

MMPs and Inflammatory Cytokines are Modulated

by Capsaicin

We had previously proposed that capsaicin might de-acti-

vate HSCs via decreasing profibrotic and proinflammatory

mediators. HSCs dedifferentiate when exposed to capsaicin

for 10 days [8]. To complement our studies, we investi-

gated the effect of capsaicin on MMP gene expression. As

shown in Fig. 4, capsaicin upregulates the expression of

MMP-13 after 10 days of capsaicin exposure, while the

expression of MMP-2 and MMP-9 is downregulated.

TIMP-1 was not affected by capsaicin.

We additionally investigated whether capsaicin could

also modulate soluble agents involved in HSC activation.

Multiple inflammatory cytokines (IL-12 (p70), IFN-c, IL-

10, and MCP-1) were assessed in GRX culture supernatants

by CBA. All cytokines were analyzed after 24 h and

10 days of 100 lM capsaicin exposure (Fig. 5). IL-12,

known to be the trigger of other cytokines (Kong et al.

[27]), had its bioactive form (p70) secretion increased only

after 10-day exposure to capsaicin. Consequently, the

release of IFN-c increased two times. In addition, anti-

inflammatory cytokine IL-10 levels were substantially

unaffected in the first 24 h. However, IL-10 release had a

4.3-fold increase after 10-day treatment with capsaicin.

Fig. 2 Effects of capsaicin on

programmed cell death. a Flow

cytometric scatter plot of FITC-

annexin V/PI stained GRX

control and capsaicin-treated

cells for 24 h. The lower left

quadrant shows the viable cells,

which are negative for annexin

V and PI. The lower right

quadrant represents the

apoptotic cells, annexin V

positive, and PI negative. The

upper right quadrant contains

the late apoptotic or dead cells

that are positive for annexin V

and PI. Representative

experiment out of three. b Flow

cytometric scatter plot of the

capsaicin impact on DWm. GRX

cells non-treated or treated with

capsaicin for 24 h. The upper

quadrant represents the

polarized cells and the lower

quadrant the depolarized cells.

Representative experiment out

of three. CPS capsaicin


123

MCP-1 levels were increased in the first 24 h and then,

drastically reduced up to ninefold in day 10.

Effects of Capsaicin During Activation of HSCs

Our results suggest that capsaicin is capable of reducing

proliferation, migration, and expression and release of

profibrogenic markers in activated HSCs. This prompted us

to investigate whether this molecule is able to impair the

activation of HSCs. To this end, we incubated freshly

isolated mouse HSCs with capsaicin. In vitro activation of

HSC is characterized by a phenotypic transdifferentiation

from a small star-shaped cell filled with lipid droplets at

day 1 toward a large myofibroblast-like cell depleted of

Fig. 3 Migratory response of GRX cells cultured for 24 h in the

presence or absence of CPS and/or PDGF-BB. a Representative

images out of three experiments showing migrated cells stained with

DAPI. b Direct cell counting of migrated cells in control conditions or

in the presence of PDGF-BB with or without CPS. Results are

presented as cell number. Data are expressed as mean ± S.E.M.

(n = 3). *P \ 0.05, **P \ 0.01, ***P \ 0.001. CPS capsaicin

Fig. 4 Timecourse of MMP

gene expression after capsaicin

exposure. mRNA levels of

MMP-13, TIMP-1, MMP-2, and

MMP-9 in capsaicin-treated

GRX cells were determined by

RT-qPCR. Results are presented

as relative expression of mRNA

levels at day 1. Data are

expressed as mean ± S.E.M.

(n = 3). *P \ 0.05,

**P \ 0.01, ***P \ 0.001.

CPS capsaicin


123

retinoid-containing lipid droplets at day 10 (Fig. 6a).

Therefore, we examined the short (24 h) and long term

(10 days) effects of capsaicin in primary mouse HSCs.

qPCR analysis shows the expression of a-SMA, Col1a1,

Col3a1 and TIMP-1 was downregulated in 24 h and

10 days (Fig. 6b). MMP-13 showed an upregulation in

both timepoints evaluated. MMP-2 was significantly

downregulated at day 1, while MMP-9 mRNA levels were

only decreased at day 10. Next, we evaluated by immu-

nocytochemistry whether a-SMA protein expression was

also influenced by capsaicin in primary cultures. As seen in

Fig. 6c, a-SMA protein expression is clearly downregu-

lated by capsaicin.

Proliferation and chemotaxis are also key features of

HSC activation. Hence, to complement our findings, we

investigated whether capsaicin could reduce the prolifera-

tion and migration of primary mouse HSCs. Figure 7a, b

shows that cells treated with capsaicin for 48 h have a

drastically decreased DNA incorporation of EdU. The

migratory capacity of primary HSCs was inhibited by

capsaicin, even in condition where the chemotactic stim-

ulation was enhanced with PDGF-BB (Fig. 7c).

Discussion

The murine GRX cell line presents several characteristics

of activated HSCs. They are in a transitional state between

quiescent lipocyte and fully activated myofibroblast [13]. It

has been previously demonstrated that GRX cells can be

induced to express the lipocyte phenotype with an overall

increase of lipid storage by a variety of phytochemicals

[8, 14–16]. Capsaicin, a naturally occurring alkaloid from

capsicum fruits, is known to have antiproliferative and anti-

inflammatory properties [17]. Several studies support that

capsaicin can inhibit proliferation by inducing apoptosis or

halting cell cycle in a variety of cell lines, especially tumor

cells [7, 18–20].

Our previous work showed that capsaicin deactivates

HSCs and leads to a more quiescent cell morphology by

suppressing fibrotic mediators probably via PPARc path-

way [8]. In this current study, we showed that capsaicin

treatment inhibited cell proliferation of activated HSCs.

Since PPARc is implicated in growth arrest and apoptosis

[21], we thought that capsaicin, as a PPARc-ligand, may

act via the apoptosis pathway. In the early stages of

apoptosis changes occur at the cell surface, one of these

alterations is the externalization of phosphatidylserine [22].

Furthermore, mitochondrial dysfunction has been shown to

participate and perhaps be central to the apoptotic pathway.

Mitochondrial disintegration not only leads to a depolar-

ization of the transmembrane potential (DWm) but also

causes the release of proapoptotic factors. In some apop-

totic systems, loss of DWm may be an earlier event in the

apoptotic process [23]. According to our findings, capsaicin

does not induce change in plasma membrane structure or

disruption of mitochondrial membrane potential.

In established fibrosis, HSCs are not dispersed within

the parenchyma, but rather are present in very character-

istic areas of fibrosis. For this to take place, a mechanism of

Fig. 5 Capsaicin modulates the

release of inflammatory

cytokines. Flow cytometric

analyses of IL-12 (p70), IFN-c,

IL-10, and MCP-1 in GRX cells

supernatant of 24-h and 10-day

treatments. Cytokines levels are

expressed as picograms per

1 9 105 cells. Data represent

the mean ± S.E.M. (n = 3).

*P \ 0.05, ***P \ 0.001. CPS

capsaicin


123

localization such as the ability to undergo chemotaxis must

be present [24]. We demonstrated that capsaicin can reduce

the migration of HSCs. Since cell migration usually begins

in response to extracellular stimuli [25], we decided to

check the effects exerted by PDGF on HSCs and the ability

of capsaicin to impair it. Our findings confirmed that HSCs

increase their proliferation and migration when exposed to

PDGF, while capsaicin is able to negatively modulate both

effects of PDGF.

HSCs are believed to be the major ECM-producing cells

in the liver [26]. In rodents, the main enzyme in the deg-

radation of interstitial collagen is MMP-13 [4]. The

decreased activity of MMPs in activated HSCs is mainly

due to an increased expression of their inhibitors, the

TIMPs [4]. In this study, we observed a significant increase

in gene expression of MMP-13 in the GRX cells treated

with capsaicin. On the other hand, MMP-2 and MMP-9 had

their mRNA levels downregulated, while gene levels of

TIMP-1 did not show a difference between groups.

HSCs are an important source of cytokines in the liver

resulting in autocrine and paracrine signaling [26].

Increased production and activity of cytokines are essential

for the maintenance of HSC activation and liver fibrogen-

esis [27]. IL-12 is a cytokine that coordinates innate and

adaptive response and its major action is the induction of

other cytokines, especially IFN-c [28]. IFN-c is a proin-

flammatory cytokine that is capable to reduce HSC acti-

vation, cell growth and differentiation. IFN-c antifibrotic

effects in the liver are via impairment of TGF-b signaling,

including the blockage of proliferation and induction of

apoptosis [27, 29]. We demonstrate that capsaicin increases

production of IL-12 and IFN-c. The anti-inflammatory


HSC activation. Mouse HSCs

were isolated from healthy mice

and exposed to capsaicin for the

indicated time. a Bright field

images of mouse HSCs cultured

for 10 days. b Capsaicin-treated

primary HSCs were harvested at

the indicated time points and

mRNA levels of a-SMA,

Col1a1, Col3a1, MMP-13,

TIMP-1, MMP-2, and MMP-9

were determined by RT-qPCR.

Results are presented as fold

change to untreated control at

day 1 or day 10 (dotted line).

Data are expressed as

mean ± S.E.M. (n = 3).

c a-SMA protein expression in

cells treated with capsaicin for

10 days. Nuclei were visualized

with DAPI. Scale bar 200 lm.

*P \ 0.05, **P \ 0.01,

***P \ 0.001. CPS capsaicin


123

IL-10 that downregulates the immune response is also

synthesized by activated HSCs. As a key factor for reducing

perpetuation of fibrogenesis, it suppresses inflammation

through several mechanisms, including the reduction of

proinflammatory cytokines [4]. In this study, we showed

that IL-10 levels increased upon capsaicin treatment, sup-

porting the inhibitory effect of IL-10 on fibrogenesis. MCP-

1 is another chemoattractant implicated in direct migration

of activated HSCs to the site of injury. However, quiescent

cells do not express MCP-1, and responsiveness to it is an

indicator of at least a minimum level of HSC activation

[30]. Recent data indicate that MCP-1 expression can be

downregulated by agonists of PPARc [31]. We demon-

strated that capsaicin-treated HSCs had their secretion of

MCP-1 drastically decreased (*90 %).

To confirm that capsaicin could be a candidate to pre-

serve the quiescent HSC phenotype in vitro, we also tested

its effects on freshly isolated mouse HSCs. As expected,

we observed similar trends as in activated HSCs; capsaicin

inhibited the culture-induced activation of primary mouse

HSCs, prevents the upregulation of several activation

markers such as a-SMA, Col1a1, Col3a1, MMP-2, MMP-

9, and TIMP-1, and capsaicin inhibited the PDGF-induced

chemotaxis and proliferation of HSCs.

In conclusion, the present study indicates that capsaicin is

capable of modulating HSC activation. In addition to its capa-

city to induce a decrease in activation features in already

activated HSCs, capsaicin is able to inhibit the first steps of

activation. Further studies shall focus on examining the an-

tifibrotic effects of capsaicin using in vivo models.

Acknowledgments This work was supported by CAPES/DGU

(BEX 4426/10-0) Grant of the Brazilian Ministry of Science and

Technology and Secretaria de Estado de Universidades (PHB2008-

0080-PC) Grant of the Spanish Ministry of Science and Innovation.

SB is receipt of a fellowship from CAPES. LAvG is supported by the

Vrije Universiteit Brussels.

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HSC activation and migration.

a Representative image of the

EdU-staining of freshly isolated

mouse HSCs that were cultured

for 48 h in the presence or

absence of CPS. b Proliferation

rate assessed by EdU

incorporation assay. Results are

expressed as percentage of

EdU? cells. c Direct cell

counting of migrated cells in the

presence of PDGF-BB with or

without CPS. Results are

presented as cell number. Data

are expressed as

mean ± S.E.M. (n = 3).

*P \ 0.05, **P \ 0.01. CPS

capsaicin


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Capsaicin Modulates Proliferation, Migration, and Activation of Hepatic Stellate Cells