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Original Article

Immune Modulation Effects of Curcumin in Pristane‑induced Lupus Mice

Handono Kalim, Kusworini Handono1, Takhta Khalasha2, Mirza Zaka Pratama2,
Tri Wahyudi Iman Dantara2, Ayu Pramitha Wulandari2, Fahimma Albinsaid2, Sofi Nur Fitria2,
Muhammad Vardian Mahardika2
Department of Internal Medicine, Rheumatology and Immunology Division, Faculty of Medicine Brawijaya University, Saiful Anwar General Hospital,
Departments of 1Clinical Pathology, and 2Biomedical Sciences, Faculty of Medicine Brawijaya University, Saiful Anwar General Hospital, Malang,
East Java 65111, Indonesia

Received: December, 2016

Accepted: April, 2017
Published: May, 2017
Address for correspondence:
Prof. Handono Kalim,
Department of Internal Medicine,
Rheumatology and Immunology
Division, Faculty of Medicine
Brawijaya University, Saiful Anwar
General Hospital, Jalan JA Suprapto
No. 2, Malang, East Java 65111,
Indonesia.
E‑mail: hkalim333@gmail.com
Abstract
Background: Curcumin, a polyphenolic compound derived from food spice turmeric has
been widely used in Asian traditional medicine for its medicinal properties as antitumor,
antioxidant, and anti‑inflammatory properties. Meanwhile, intraperitoneal  (i.p.) injection of
the hydrocarbon oil pristane into normal mice leads to a lupus‑like autoimmune syndrome.
We aimed to investigate the effects of curcumin on systemic lupus erythematosus  (SLE)
clinical manifestation, adaptive immune system components, proinflammatory cytokines, and
autoantibody production in pristane‑induced lupus mice.
Methods: Fifty female BALB/c mice, 6–8  weeks old were divided into 2 groups: Forty mice
received a single i.p. injection of 0.5 cc pristane for lupus induction and ten mice as healthy
controls. Starting at 16  weeks after injection, forty pristane‑induced lupus mice were divided
into four groups based on doses of curcumin received intragastrically: 0, 12.5, 50, and 200
mg/kg bw/day daily for 16  weeks. At 32  weeks after injection, all of mice were assessed for
arthritis score, proteinuria level, body weights, adaptive immune system components  (Th1,
Th2, Th17, and Treg percentages) from spleen using flow cytometry; proinflammatory
cytokines and autoantibody production, including interleukin‑6 (IL‑6), interferon‑alpha (IFN‑α),
and antinuclear antibody (ANA) from serum using enzyme‑linked immunosorbent assay.
Results: Arthritis score and proteinuria level were decreased in curcumin‑treated mice.
However, body weights were not significantly different between the groups. The decreased
of Th1, Th2, and Th17 percentages were seen after treatment with 200 mg/kg bw/day of
curcumin  (P  =  0.031, P =  0.017, and P =  0.005, respectively). However, only slight increase
of Treg percentages was seen after curcumin treatment. Treatment with 200 mg/kg bw/day
of curcumin decreased serum IL‑6 and IFN‑α levels  (P  =  0.007 and P =  0.003). Furthermore,
ANA levels were also decreased significantly after treatment with 200 mg/kg bw/day of
curcumin (P = 0.013).
Conclusion: Our findings suggested that curcumin could prove useful as a therapeutic
intervention in SLE.
Key Words: Curcumin, pristane, systemic lupus erythematosus

Introduction Th17 subsets and decreased regulatory of T‑cells  (Treg)

have been reported in many studies to be correlated with
Systemic lupus erythematosus  (SLE) is a systemic activity index, tissue damage, and autoantibodies synthesis
autoimmune disease with complex pathogenesis.[1] In in SLE patients.[2‑4]
previous decades, many studies found that abnormal
function and proportion of CD4+  T‑cells subsets had major There are numerous murine models that have long
roles in the development of SLE. Increased Th1, Th2, and been studied in an effort to understand pathogenesis
and advances in SLE treatment. One SLE animal model
is the pristane‑induced lupus mice model. Pristane is
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DOI:
How to cite this article: Kalim H, Handono K, Khalasha T, Pratama MZ,
Iman Dantara TW, Wulandari AP, et al. Immune modulation effects of
10.4103/injr.injr_95_16
curcumin in pristane-induced lupus mice. Indian J Rheumatol 0;0:0.

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Kalim, et al.: Immunomodulation effects of curcumin
an isoprenoid alkane found at high concentration in
mineral oil and can induce autoantibodies and the clinical
manifestations of SLE in murine models.[5] Studies have
shown that these mice have disparate T‑cell requirements
of two subsets of lupus‑specific autoantibodies as well as
the toll‑like receptor 7‑dependent and FcγR‑independent
production of Type I interferon.[6]
The rapid advance in the treatment and diagnosis of SLE
in recent few years has resulted in greater life expectancy
of SLE patients. Current therapeutics used to treat SLE
including glucocorticoids are directed at suppressing
humoral immunity and the production of autoantibodies
as well as helper T‑cells  (Th) and B‑cells.[7] However, the
use of long‑term glucocorticoids and immunosuppressant
therapies for SLE may result in various side effects.[8]
Immunosuppressant agents are not widely available and
affordable, especially in the developing country.
One of the current developments in autoimmune disease
treatment field in developing country is nutraceutical
product. One example of bioactive compound from
plants that have immunomodulatory properties is
curcumin, a phenolic compound which mainly can be
found in turmeric.[9] It has been shown that curcumin
possesses many biological effects on cells, including
anti‑inflammatory, antioxidant, immunomodulator, and
anticancer.[9‑12] Curcumin is also proved to be able to
modulate cellular response and the growth of various
cell types in the immune system, including T‑cells, B‑cells,
dendritic cells, macrophages, and natural killer cells.[13]
Despite the wide range of curcumin effects on various
biologic processes, the role of curcumin in SLE is rarely
discussed. Therefore, this research aimed to investigate
the role of curcumin on the clinical manifestations and
adaptive immune responses as well as the production of
proinflammatory cytokines in SLE animal model.
Methods
Mice and lupus induction
Totally, fifty female BALB/c mice that were 6–8  weeks
old were obtained from Pusvetma Surabaya, East Java,
Indonesia. All mice were housed in standard cages
(5 mice/cage) in a climate‑controlled environment.
The mice were maintained on 12‑h light/dark cycle
and provided food and water ad libitum. Mice were
habituated to the holding room for a minimum of
1  week before undergoing experimental procedures.
Fifty female BALB/c mice were randomly divided into the
following two groups:  (1) pristane‑induced mice group:
Forty BALB/c mice received a single intraperitoneal
injection of 0.5 ml of pristane  (Sigma‑Aldrich, USA)
for lupus induction and  (2) normal control group: 10
BALB/c mice not injected with pristane and not fed with
curcumin.
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Preparation and administration of curcumin
The curcumin was acquired from Sigma, St Louis, MO.
Curcumin solutions of  (1 or 5 mg/ml in filtered water)
were prepared by dissolving it in hot distilled water (about
90°C); and subsequently heated for 10  min in a boiling
water bath, and filtered using Sigma‑Aldrich sintered glass
funnel number three (pore size 16–40 μm). The precipitate
was washed with additional amounts of hot distilled water
then the solution was cooled. This procedure allowed
the curcumin solubility to increase 12‑fold in water
based.[14] Afterward, the solutions were emulsified with
0.5% carboxymethyl cellulose for intragastric administration
to mice.[15]
Sixteen weeks after pristane injection, pristane‑induced
mice group were randomly divided into the following
four groups:  (1) Curcumin A group: Ten pristane‑induced
mice were given curcumin 12.5 mg/kg bw/day daily for
16  weeks;  (2) Curcumin B group: Ten pristane‑induced
mice were given curcumin 50 mg/kg bw/day daily for
16  weeks;  (3) Curcumin C group: Ten pristane‑induced
mice were given curcumin 200 mg/kg bw/day daily for
16  weeks;  (4) Model control group: Ten pristane‑induced
mice were not given curcumin. Intragastric administration
of curcumin was carried out daily until 32  weeks
postpristane injection. The mice were euthanized at the
end of 32  weeks postpristane injection, then their spleen
and serum had been obtained for further analyses.
Measurement of proteinuria
Beginning at 32  weeks postpristane injection, proteinuria
was measured semi‑quantitatively by impregnating wool
paper test strips with spot urine sample  (URiSCAN,
Yeongdong Pharmaceutical Company). The color
change of the strip infiltrated with the urine sample
was visually judged against a standard strip and scored
from 0 to 5+, according to manufacturer’s instructions.
The urinary protein content was graded according to
the score as follows: 0  ≤100 mg/L, 1+ = 100 mg/L,
2+ = 300 mg/L, 3+ = 1000 mg/L, 4+ = 3000 mg/L, and
5+ = 10,000 mg/L. Significant proteinuria was characterized
by more than positive two score on dipstick score
(equivalent to >300 mg/dl protein in urine).
Scoring of arthritis
At 32  weeks postpristane injection, mice were examined
for arthritis development after curcumin treatment.
Scoring of animals was done blindly using a scoring
system  (arthritis score) based on the number of inflamed
joints in each paw, inflammation being defined by swelling
and redness. In this scoring system, each inflamed toe
or knuckle gave one point, whereas an inflamed wrist
or ankle gave five points, resulting in a score of 0–15
(five toes  +  five knuckles  +  one wrist/ankle) for each
paw and 0–60 points for each mice.[16] The individual
2
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Kalim, et al.: Immunomodulation effects of curcumin

mice arthritis score was obtained by summing the
scores recorded for each limb. Clinical evaluations were
performed by two investigators  (anatomical pathology
specialist) unaware of mice identity, and the mean of both
scores was calculated.
Cell preparation
Preparation from spleen tissue was done using previous
protocol methods. Spleen tissues were harvested and
teased apart into single‑cell suspension by pressing them
with the plunger of a 3 ml syringe. Tissues were collected
in 10 ml of staining buffer and passed cell suspension
through a cell strainer to eliminate clumps and debris,
then collected cell suspension in a conical tube. The cell
suspension was centrifuged for 4–5  min  (300–400 xg) at
4°C and the supernatant was removed. Red blood cell lysis
was performed. Samples were resuspended in 50 ml of
staining buffer and cell count, and viability analyses were
performed using Trypan Blue.[17]
Flow cytometry analysis
Cells that had been taken from spleen were stained
using antibody markers to assess CD4+  T cells subsets
percentages, including Th1, Th2, Th17, and Treg by flow
cytometry. Before Th1, Th2, and Th17 staining, cells were
stimulated with phorbolmyristate acetate  (50 ng/ml;
Sigma, St. Louis, MO, USA) and ionomycin (1 μg/ml; sigma)
in the presence of Brefeldin A  (BD Pharmingen, San
Diego, CA, USA) for at least 4 h. Subsequently, the cells
were labeled with PE antimouse CD4  (Biolegend, USA).
Intracellular staining was performed using FITC antimouse
IFN‑γ  (Biolegend, USA), PerCP antimouse IL‑4  (Biolegend,
USA), and PerCP antimouse IL‑17A to detect Th1, Th2, and
Th17, respectively.
For the detection of Treg, cells were labeled with PE
antimouse CD4  (Biolegend, USA), PerCP antimouse CD25
(Biolegend, USA), and FITC antimouse FoxP3  (Biolegend,
USA). Staining was performed according to Biolegend
manufacture protocols. All cells were analyzed using
Cellquestpro software.
Proinflammatory cytokines and autoantibody
assay
At 32  weeks postpristane injection, whole blood samples
of the mice were collected from their heart following
inhalation of chloroform anesthesia. The blood samples
were allowed to clot by leaving it at room temperature for
15–30  min, and the clot was removed by centrifuging it
at 1000–2000  ×  g for 10  min. Proinflammatory cytokines
levels  (IL‑6 and interferon‑alpha  [IFN‑α]) from serum
were determined using enzyme‑linked immunosorbent
assay  (ELISA) kits  (from Biolegend, USA). Antinuclear
antibody  (ANA) from serum was also determined using
ELISA kits (MyBioSource, San Diego, CA, USA).
Statistical analysis
Comparison of all data between groups was done
by one‑way analysis of variance test followed by
post hoc analysis. Parametric data were described as
mean  ±  standard deviation; the statistical significance of
the various tests was examined by two‑sided hypothesis
testing. P < 0.05 was considered statistically significant.
Statistical analysis was performed using SPSS for Windows
version 16.0 (SPSS, Chicago, IL, USA).
Ethical approval
All of the experiments were undertaken in certified in
vivo laboratories at Pharmacology Laboratory of Brawijaya
University Research Centre, Malang, East Java, Indonesia.
The studies and animal care have been approved by the
Health Research Ethics Committee, Faculty of Medicine,
University of Brawijaya.
Results
Outcome
Initially, ten mice were included in each group;
however, four mice in model control group and one
mice in curcumin A group were dead before reaching
32  weeks after pristane injection. Data from these mice
were excluded from the analysis of treatment efficacy.
Pristane‑induced mice treated with curcumin gained
weight in a manner similar to that of model control and
normal control groups. There was no difference in body
weight between various groups of mice. Mice treated with
curcumin 200 mg/kg bw/day had the highest body weight
[Table 1].
Effect of curcumin on proteinuria
At the end of the study, urinalysis revealed that 2 out
of 6 mice in model control group and 2 out of 9 mice in

Table 1: Clinical Characteristics of Pristane Induced Lupus Mice on Each Group (*P<0.05, **P<0.01, and ***P<0.001
compared to model control group)
Normal Control Model Control Curcumin A Curcumin B Curcumin C
group (n=10) Group (n=6) Group (n=9) Group (n=10) Group (n=10)
Body weight (gr) 35.1±0.7 30.7±4.1 33.3±2.9 33.2±0.9 34.5±3.9
Significant proteinuria >300 mg/dl (n present/n total) 0/10 2/6 2/9 1/10 0/10
Arthritis score 0 50.7±14.5 21.8±11.4*** 19.8±6.4*** 17.2±5.6***
ANA levels (ng/dl) 2.95±1.52 39.6±5.7 27.4±16.7 15.9±14.2* 17.2±9.7*

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Kalim, et al.: Immunomodulation effects of curcumin

curcumin A group had significant proteinuria. One out
of 10 mice in curcumin B group also had a significant
proteinuria. Whereas, there was no mice from curcumin
C group that had proteinuria. Therefore, curcumin
can prevent the development of proteinuria in this
pristane‑induced lupus model.
Effect of curcumin on arthritis and autoantibody
production
The presence of joint swelling and redness on all of the
paws of the mice was assessed using arthritis score.
Curcumin treatment reduced the arthritis score. With a
dose‑dependent manner  [Table  1],  (P  <  0.001, P <  0.001,
and P <  0.001 in groups treated with curcumin 12.5, 50,
and 200 mg/kg bw/day, respectively, compared to model
control group).
Curcumin, especially with administration of
50 mg/kg bw/day and 200 mg/kg bw/day, showed
significantly lower serum ANA level compared to model
control group [Table  1] (P  =  0.008 and P =  0.013,
respectively).
Effects of curcumin treatment on Th1 and Th2
percentages
Th1  (CD4+  IFNγ +) and Th2  (CD4+  IL4+) percentages were
measured from spleen samples using flowcytometry
[Figure  1a and b]. Daily dose of curcumin treatment on
pristane‑induced lupus mice decreased both Th1 and Th2
percentages on dose‑dependent manner. Only in curcumin C
group that was able to decrease Th1 percentages significantly
lower compared to model control group [Figure 2a], (P = 0.031).
However, in curcumin B and C group was able to decreased

d
Figure 1: Representative dot plots which illustrate (a) Th1 expressing CD4+ interferon γ+, (b) Th2 expressing CD4+ interleukin 4+, (c) Th17 expressing
CD4+ interleukin‑17A+, and (d) Treg expressing CD25+ FoxP3+

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Kalim, et al.: Immunomodulation effects of curcumin

Th2 percentages significantly compared to model control group
[Figure 2b], (P = 0.045 and P = 0.017).
Curcumin did not alter the balance of Th1 and Th2
[Figure 2c], (P = 0.898). These results indicated that curcumin
reduced Th1 and Th2 percentages on pristane‑induced lupus
mice without altering Th1/Th2 balance.
Effect of curcumin treatment on Th17 and Treg
percentages
A test had been conducted to prove whether
curcumin could affect Th17 and Treg differentiation on
pristane‑induced lupus mice. Figure  1c showed Th17
expressed CD4+  IL‑17A+, whereas Figure  1d showed Treg
expressed CD4+ CD25+ FoxP3+.
Daily curcumin administration for 16  weeks decreased Th17
(CD4+ IL‑17A+) percentages in the spleen of pristane‑induced
lupus mice in a dose‑dependent manner [Figure  3a].
Administration 50 and 200 mg/kg bw/day of curcumin
decreased Th17 percentages significantly compared to model
control group [Figure 3a], (P = 0.039 and P = 0.005). In contrast,
curcumin tended to increase Treg (CD4+  CD25+  FoxP3+)
percentages in the spleen of pristane‑induced lupus mice in
a dose‑dependent manner [Figure 3b].

b
b

c
Figure 2: Effect curcumin on (a) Th1 percentages, (b) Th2 percentages,
(c) ratio of Th1/Th2 percentages in spleen. The curcumin group
showed a significantly decreased in Th1 and Th2 percentages when
compared with model control group. Values are means, with their
standard errors represented by vertical bars (n = 7–8). Mean values were
significantly different from that of model control group mice: *P < 0.05,
**P < 0.01 (analysis of variance)
c
Figure 3: Effect curcumin on (a) Th17 percentages, (b) Treg percentages,
and (c) ratio of Th17/Treg percentages in spleen. The curcumin group
showed a significantly decreased in Th17 percentages and ratio of
Th17/Treg percentages. However, the increased of Treg percentages were
not significantly different between the groups. Values are means, with their
standard errors represented by vertical bars (n = 7–8). Mean values were
significantly different from that of model control group mice: *P < 0.05,
**P < 0.01 (analysis of variance)

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Kalim, et al.: Immunomodulation effects of curcumin
We also found that curcumin altered Th17/Treg balance by
shifting it toward Treg differentiation in a dose‑dependent
manner [Figure 3c]. Th17/Treg ratios decreased significantly
in curcumin C group compared to model control group
(P = 0.024).
Effects of curcumin treatment on proinflammatory
cytokines
Imbalance of CD4+  T‑cells subsets in SLE might be the
result of an increase in proinflammatory cytokines, such as
IL‑6 and IFN‑α. We assessed serum IL‑6 and IFN‑α levels
of each group to evaluate the role of curcumin on these
cytokines production.
Doses of 12.5, 50, and 200 mg/kg bw/day curcumin
reduced serum IL‑6 levels significantly compared to model
control group in a dose‑dependent manner [Figure  4a]
(P  =  0.013, P =  0.011, and P =  0.007, respectively).
Administration of 200 mg/kg bw/day showed significantly
decreased IFN‑α serum levels compared to model control
group (P = 0.003) [Figure 4b].
Discussion
Curcumin is an active principle component isolated from
the rhizome of Curcuma longa[18] which have been proven
a
b population and Th17/Treg ratios significantly compared
Figure 4: Effect curcumin on (a) interleukin‑6 (b) interferon‑alpha levels
in serum. The curcumin group showed a significantly decreased in
interleukin‑6 and interferon‑alpha levels when compared with the model
control group. Values are means, with their standard errors represented
by vertical bars mean values were significantly different from that of model
control group mice: *P < 0.05 (analysis of variance)
Indian Journal of Rheumatology  ¦  Volume XX  ¦  Issue XX ¦  Month 2017
to be having anti‑inflammatory, antioxidant, and antitumor
activities.[19] Various studies also found that curcumin could
inhibit the complement cascade and alleviate a number of
immune‑mediated diseases.[20‑22] In this study, we explored
the therapeutic effects of curcumin on SLE animal models.
This is the first study that aimed to investigate in vivo
effects of curcumin on pristane‑induced lupus mice.
We found that mice that were treated with curcumin
showed significant decrease of arthritis score and ANA
level compared to model control group. Our study results
showed that there was no significant difference in body
weight between various groups of curcumin‑treated mice,
similar with the finding of another studies.[23,24]
Although SLE pathophysiology is a complex process,
many studies found that CD4+  T‑cells have major roles in
the pathogenesis of SLE.[2‑4] Clinical deteriorations of SLE
have been reported to be correlated with the increase of
Th1, Th2, and Th17 subsets and also the decrease of Treg
populations.[5] This study discovered that administration of
high doses of curcumin  (200 mg/kg bw/day) for 16  weeks
decreased Th1, Th2, and Th17 populations in the spleen of
pristane‑induced lupus mice, but not significantly increased
Treg populations.
Previously, SLE was thought to be a Th2‑driven disease;[25]
however, recent reports showed that besides Th2, Th1 also
has an abnormal response in SLE.[2,26] Interestingly, in this
study, curcumin reduced both Th1 and Th2 populations
simultaneously in the same manner, which was showed
by similar Th1/Th2 ratios to model control group. This
result was somewhat different from the previous studies,
where most of them indicate that curcumin regulates the
shift from Th1 to Th2.[27] However, these previous studies
results were obtained from studies on Th1 dominant
diseases.[28,29] In contrast, in Th2‑driven disease, such as
allergic asthma, curcumin might reduce Th2 polarization.[30]
Added with our study results, it is possible that curcumin
may act differently in Th1/Th2 balance, depending on the
situation, in which component is dominant, Th1 or Th2. In
fact, it confirms that curcumin does not only inhibiting Th1
but also inhibiting Th2. Therefore, in Th1‑  and Th2‑driven
disease such as SLE, curcumin may decrease both Th1 and
Th2 populations simultaneously.
Other subsets of CD4+ T‑cells which have important roles in
the pathogenesis of SLE are Th17 and Treg. Recent reports
suggested that improving Th17/Treg balance might help to
reduce disease activity in SLE patients.[31] From our study,
we found that daily curcumin treatment for 16  weeks,
especially 200 mg/kg bw/day, could decrease Th17
to control. However, there was only slight enhancement
of Treg population that can be seen after curcumin
treatment. Previously, curcumin had been shown to be
able to attenuate airway inflammation on mice models by
increasing Treg and reducing Th17 functions.[32] Another
6
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Kalim, et al.: Immunomodulation effects of curcumin
study also showed that curcumin might reduce the
severity of acute graft‑versus‑host disease by regulating
of Th17 and Treg functions.[33] Another study investigating
the effect of low doses curcumin treatment in human also
revealed similar results.[34] Despite the facts, the roles of
curcumin treatment in mice model of SLE have never been
published.
Imbalance of Th cells and Treg subsets on SLE are
thought to be a complex phenomenon influenced by
abnormal production of proinflammatory cytokines.[35]
Several of the proinflammatory cytokines that mediate
the early and late immune response in SLE are IL‑6 and
Type I interferons, such as IFN‑α.[36] The raise in these
cytokines levels had been reported to correlate with the
increase in autoantibody productions, aggravating clinical
manifestations, and tissue destructions in SLE.[37]
Our present study has proved that curcumin might
also decrease IL‑6 and IFN‑α serum concentration on
pristane‑induced lupus mice model, which was consistent
with the previous studies. Das and Vinayak[38] reported
that curcumin could regulate IL‑6 expression through
a regulation of nuclear factor‑kB transcription factor.
Another research carried out by Sordillo and Helson[39] also
showed that curcumin suppressed cytokine release such
as IL‑6 and IFN‑α in Ebola and other severe viral infections
patients. Curcumin’s ability to modulate various types
of transcription factors is believed to be the molecular
mechanisms of curcumin which may suppress cytokines
production.[38,40]
Despite being an interesting subject to be studied,
this study still had many limitations, such as immune
modulation mechanism of curcumin which still need to be
elucidated. Further research should be aimed particularly
to monitor the effects of curcumin on organs, other
proinflammatory cytokines on serum, and also the risk of
long‑term use of curcumin on the body. Although there
are still many problems that need to be investigated, the
use of curcumin as an immunomodulatory agent is very
interesting to be developed as for the complementary
treatment of SLE.
This report showed that curcumin protected
pristane‑induced lupus mice from arthritis and
overproduction of ANA. Curcumin also modulated the
abnormal immune response in mice model of SLE, shown
by reduction of Th1 and Th2 population on spleen of
pristane‑induced lupus mice without affecting the balance
between Th1 and Th2 subsets. Interestingly, administration
of curcumin on pristane‑induced lupus mice might
also decrease Th17 percentages and Th17/Treg ratio.
However, there was only slight raise of Treg percentages
after curcumin treatment. Proinflammatory cytokines
productions of IL‑6 and IFN‑α were also inhibited by
curcumin. Thus, our observation revealed that curcumin
could prove useful as a therapeutic intervention in SLE.
7 Indian Journal of Rheumatology  ¦  Volume XX  ¦  Issue XX ¦  Month 2017
Financial support and sponsorship
We are grateful to Brawijaya University and Directorate
general of higher education  (DIKTI), for providing the
research funding.
Conflicts of interest
There are no conflicts of interest.
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