608248

research-article2015
IJI0010.1177/0394632015608248International Journal of Immunopathology and PharmacologySannegowda et al.

Original article

International Journal of

Tinospora cordifolia inhibits autoimmune Immunopathology and Pharmacology

2015, Vol. 28(4) 521­–531
© The Author(s) 2015
arthritis by regulating key immune Reprints and permissions:
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mediators of inflammation and DOI: 10.1177/0394632015608248

iji.sagepub.com

bone damage

KM Sannegowda,1,2 SH Venkatesha1 and KD Moudgil1,3

Abstract
Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joints leading to tissue
damage. Despite the availability of potent drugs including the biologics, many patients fail to respond to them, whereas
others suffer adverse effects following long-term use of these drugs. Accordingly, the use of natural herbal products by
RA patients has been increasing over the years. However, limited information about the mechanism of action of these
natural products is a major shortcoming that prevents the widespread acceptance of herbal therapy by professionals
and patients alike. In this study, we demonstrated the anti-arthritic activity of Tinospora cordifolia extract (TCE) using
the rat adjuvant-induced arthritis model of human RA and elaborated the immune mechanisms underlying this effect.
TCE treatment suppressed arthritic inflammation and bone and cartilage damage. The anti-inflammatory effect of TCE
was mediated via reduction of the pro-inflammatory cytokines such as: IL-1β, TNF-α, IL-6, and IL-17; the frequency of
IL-17-producing T cells; and the production of chemokines such as RANTES. Furthermore, TCE treatment limited bone
damage by shifting the balance of mediators of bone remodeling (e.g., receptor activator of nuclear factor-kB ligand
[RANKL] and MMP-9) in favor of anti-osteoclastic activity. Our results suggest that TCE and its bioactive components
should be evaluated for their utility as therapeutic adjuncts to conventional drugs against RA.

Keywords
adjuvant arthritis, autoimmunity, bone remodeling, chemokines, cytokines, natural products, rheumatoid arthritis,
Tinospora cordifolia

Date received: 4 June 2015; accepted: 2 September 2015

Introduction
Rheumatoid arthritis (RA) is a chronic inflamma- damage, or infections.6–9 Accordingly, increasing
tory disease that affects about 1% of the population numbers of RA patients are resorting to the use of
and its prevalence is higher in women than in
men.1,2 The major pharmacological agents currently
being used for the treatment of RA are the non- 1Department of Microbiology and Immunology, University of Maryland
School of Medicine, Baltimore, MD, USA
steroidal anti-inflammatory drugs (NSAIDs) such 2Department of Biochemistry, Government College for Women,
as aspirin; the disease-modifying anti-rheumatic Mandya, Karnataka, India
drugs (DMARDs) like methotrexate; the immuno- 3Division of Rheumatology, Department of Medicine, University of

suppressive agents such as prednisone; and the anti- Maryland School of Medicine, Baltimore, MD, USA

cytokine agents like anti-TNFα receptor antibody.3–6 Corresponding author:

These drugs are quite effective, but their prolonged KD Moudgil, Department of Microbiology and Immunology, University
of Maryland School of Medicine, 685 W. Baltimore Street, Baltimore,
use may be associated with significant adverse MD 21201, USA.
effects such as gastrointestinal toxicity, kidney Email: [email protected]
522 International Journal of Immunopathology and Pharmacology 28(4)

natural herbal products.10 Such products have been the commercial source was 10%, and well-defined
used in the Indian traditional system of medicine standardization procedures were performed for
(Ayurveda) and traditional Chinese medicine purity and identity of the extract. The level of
(TCM) for a long period of time and generally dis- heavy metals such as lead, arsenic, cadmium, and
play minimal side effects. However, there is limited mercury was not more than 3 ppm, 1 ppm, 1 ppm,
information about the mechanisms of action of and 0.1 ppm, respectively, and the level of total
many of these natural products. heavy metals was not more than 20 ppm. Chemical
Tinospora cordifolia (TC) is a climbing shrub standardization, nature of bioactive components,
that belongs to the family Menispermaceae. It is and toxicity of TCE are described previously by
native to India, but is also found in neighboring other investigators.14–17 Several bioactive phyto-
countries.11,12 The Tinospora cordifolia extract chemical constituents belonging to the sub-catego-
(TCE) has been used in many herbal formulations ries of alkaloids (e.g., berberine, tinosporin),
to treat various inflammatory conditions.13,14 TCE glycosides (e.g., cordiofolioside A, palmatosides
and the compounds isolated from T. cordifolia have C), sesquiterpenoids (e.g., tinocordifolin), diterpe-
been shown to possess immunomodulatory, anti- noid lactones (e.g., tinosporon, columbin), aliphatic
proliferative, and anti-angiogenic effects in various compounds (e.g., octacosanol), and steroids (e.g.,
in vitro models.15 The anti-bacterial activity of T. makisterone A, giloinsterone) have been identified
cordifolia has also been reported.16 It has previ- in T. cordifolia.14–17
ously been shown in the rat collagen-induced
arthritis (CIA) model that T. cordifolia reduced the Induction and evaluation of adjuvant
severity of arthritis and protected against joint arthritis (AA)
destruction.17 In this study, we validated the arthri-
tis-suppressive effect of TCE using the rat adju- Lewis rats were immunized subcutaneously at the
vant-induced arthritis (AA) model of RA and base of the tail with 1.5 mg of grinded heat-killed
elaborated the immunological and biochemical M. tuberculosis H37Ra (Mtb) (Difco, Detroit, MI,
mechanisms involved therein. Our results show USA) suspended in 200 μL of mineral oil for each
that TCE inhibits key pro-inflammatory cytokines rat. The rats were observed regularly for erythema,
and chemokines along with mediators of bone swelling, and induration in each paw after Mtb
remodeling and matrix degradation. injection and the severity of arthritis in each paw
was graded on a scale of 0 to 4.18,19 A typical dis-
ease course consisted of incubation, onset, peak,
Materials and methods
and recovery phase of AA.
Animals
Male Lewis rats (LEW/Hsd, RT.1l), aged 5–6 weeks, Treatment of arthritic Lewis rats with
were purchased from Harlan Sprague-Dawley T. cordifolia extract (TCE)
(Indianapolis, IN, USA) and housed in the vivarium TCE was suspended in water and fed to Lewis rats
of University of Maryland School of Medicine, (1 g/kg in a 2 mL volume) daily using a gavage
Baltimore, MD (UMB). UMB Institutional Animal needle (FNC-16-3, Kent Scientific Corp.,
Care and Use Committee (IACUC) specifically Torrington, CT, USA). TCE was fed from the onset
approved this study via IACUC protocol #0514012. of AA (day 9) and then continued uninterrupted for
All experimental procedures were conducted in the entire duration of the observation period. The
accordance with IACUC guidelines. control rats received vehicle (water). All rats were
graded regularly for the severity of arthritis in this
Tinospora cordifolia extract: Source period.
and characteristics
Histological examination of hind paws
The methanol extract of aerial part of T. cordifolia
extract was a kind gift from Sami Labs Limited,
of TCE-/vehicle-treated rats
Bangalore, India/ Sabinsa Corporation, NJ, USA The hind paws were harvested from the Lewis rats
(Product code: 2020, Batch No. C111811). The treated with vehicle/TCE on day 19 and immersed
percentage of yield of the extract as specified by in Cal-Ex decalcifying solution CS510-1D (Fisher)
Sannegowda et al. 523

for 9 days. Thereafter, the paws were immersed in Preparation and restimulation of
70% ethanol for 5 days followed by embedding in synovial-infiltrating cells (SIC)
paraffin as described elsewhere.20 The embedded
Hind paws of the TCE-fed and water-fed rats
tissue was then sectioned serially using the
were collected on day 19 after Mtb immunization
microtome and mounted on microscope slides. The
and SIC were harvested by opening the ankle
microtome sections were then stained with hema-
joint using a sterile blade as described else-
toxylin and eosin (H&E) (Histology Core,
where.19 These SIC were then washed three times
University of Maryland School of Medicine,
with HBSS and cultured (5 × 105 cells/well) in a
Baltimore, MD, USA)21 or safranin-O22 and exam-
flat-bottomed 96-well plate in HL-1 serum-free
ined under a microscope for arthritis-related
medium (Ventrex Laboratories, Portland, ME,
changes such as synovial hyperplasia, pannus for-
USA) supplemented with 2 mM L-glutamine, 100
mation, and cartilage and bone damage, and digital
units/mL penicillin G sodium, and 100 μg/mL
images were obtained.21
streptomycin sulfate in an atmosphere of 95% air
and 5% CO2. The cells were re-stimulated with
Preparation and restimulation of Mtb sonicate for 24–48 h and their RNA or cul-
lymph node cells (LNC) ture supernate was used for cytokine/chemokine
Arthritic Lewis rats treated with TCE/vehicle testing.
were euthanized on day 19 after disease induc-
tion and their draining lymph nodes (para-aortic, Methods of testing of cytokines,
inguinal, and popliteal) were harvested. A single chemokines, and mediators of bone
cell suspension of LNC was prepared. These remodeling
LNC were washed three times with Hanks’ bal-
anced salt solution (Invitrogen) and cultured (5 × Measurements of cytokines / chemokines / media-
105 cells/well) in a flat-bottomed 96-well plate in tors of bone remodeling were performed using
HL-1 serum-free medium (Ventrex Laboratories, LNC, SAC, SIC, and sera of TCE- and vehicle-
Portland, ME, USA) supplemented with 2 mM treated arthritic Lewis rats. Specific mediators
L-glutamine, 100 units/mL penicillin G sodium, of bone remodeling tested included receptor
and 100 μg/mL streptomycin sulfate in an activator of nuclear factor-kB (RANK), receptor
atmosphere of 95% air and 5% CO2.19 The cells activator of NF-kB ligand (RANKL), osteoprote-
were re-stimulated with antigen (Mtb sonicate, gerin (OPG), osteopontin (OPN), osteocalcin
10 ug/mL) for 24–48 h and then their RNA or (OSC, also known as OCN), and macrophage
culture supernate was used for cytokine/ colony-stimulating factor (M-CSF). The testing
chemokine testing.19,23 was done employing mRNA expression by quanti-
tative real-time PCR (qRT-PCR) and/or protein
levels by Multiplex assay (Cytokine Core facility,
Preparation and restimulation of
UMB). The preparation and restimulation of vari-
spleen adherent cells (SAC)
ous cell types are described above. These cells
Spleens were harvested from rats treated as were used for RNA isolation. RNA was isolated
described above under LNC and a single cell sus- by Trizol reagent (Invitrogen) and cDNA prepared
pension was prepared.24 The spleen cells (2.0 × 106 from it using an iScript cDNA synthesis kit (Bio-
cells/well) were placed in a six-well plate (Corning, Rad).19 The cDNA was amplified by quantitative
Corning, NY, USA) and incubated at 37°C for 90 RT-PCR using SYBR Green PCR Master Mix
min in RPMI 1640 medium supplemented with 5% (Applied Biosystems) and appropriate primers for
FBS, 2 mM L-glutamine, 100 units/mL penicillin different cytokines in an ABI PRISM 7900HT
G sodium, and 100 μg/mL streptomycin sulfate. cycler (Applied Biosystems, Foster City, CA,
The non-adherent cells were removed. The remain- USA). The expression of hypoxanthine-guanine
ing cells consisted of SAC. These cells were res- phosphoribosyltransferase (HPRT) gene served as
timulated with Mtb sonicate (10 μg/mL) for 6–24 h a reference for normalization of the test values.19
and then their RNA or culture supernate was used The relative gene expression levels were expressed
for cytokine/chemokine testing. as “fold change”.
524 International Journal of Immunopathology and Pharmacology 28(4)

Flow cytometric analysis Results

Spleen and hind paws were harvested from water- TCE reduces the severity of AA in
fed arthritic control rats and TCE-fed rats at peak Lewis rats
phase of adjuvant arthritis. Single-cell suspensions
from the spleens and synovial-infiltrating cells To assess the efficacy of TCE in reducing the
(SIC) from the joints were prepared and stimulated severity of arthritis, we tested this natural product
under in vitro conditions for 6 h with 5 μg/mL extract in the rat AA model. Arthritic Lewis rats
phorbol 12-myristate 13-acetate (PMA) (Promega) were fed TCE (test group) or water (vehicle) (con-
and 100 μM ionomycin (Sigma-Aldrich) in the trol group) orally by gavage after the onset of
presence of 100 μg/mL Brefeldin A (Life disease (beginning on day 9 and then continuing
Technologies) for the final 4 h of culture.25 Cells throughout the course of AA), and thereafter these
were surface-stained with anti-CD3 APC and anti- rats were observed daily for the signs of disease.
CD4 FITC (all from eBioscience). Thereafter, cells The TCE-fed group consistently had lower arthritic
were fixed and permeabilized using a BD Fixation/ scores when compared with the water-fed group
Permeabilization Kit (BD Bioscience) and stained (Figure 1a). For example, at the peak phase of dis-
for intracellular cytokines using anti-IL-17A ease, day 18 post-Mtb immunization, the swelling
eFluor 450 (eBioscience) and anti-IFN-γ PE and redness of the hind paws were markedly
(Biolegend), and then analyzed on an LSRII flow reduced in the TCE-treated rats compared to con-
cytometer (BD Biosciences). The data were ana- trol rats (Figure 1b). To further validate the effec-
lyzed with FlowJo Software (TreeStar). tiveness of TCE in suppressing arthritis severity,
we examined the hind paws of the two groups of
rats for histological changes in the joints using
Matrix metalloproteinase (MMP) H&E and safranin-O staining. The sections from
activity TCE-treated rat had decreased pannus formation,
The SIC were collected from vehicle-/TCE-treated reduced cellular infiltrates, and reduced bone and
rats, washed, and cultured in a 6-well plate follow- cartilage damage compared with that of the control
ing the method described elsewhere.19 After 24 h, rat (Figure 1c, d). Taken together, these results
the supernates were collected and their MMP activ- show that TCE is effective in reducing both clinical
ity was analyzed by a zymogram assay.26,27 Briefly, and histological features of arthritis severity.
the supernatant (10 uL) was separated on a gelatin-
coated, precast SDS-polyacrylamide gel under TCE treatment inhibits key
non-reducing conditions at constant voltage. The pro-inflammatory cytokines and
SDS from the gel was removed by incubating it chemokines involved in arthritis
with Triton X-100 (2.5%) at room temperature for pathogenesis
1 h. After thorough washing, the gel was incubated
overnight at 37°C in a developing buffer (Tris- The RNA prepared from SAC and LNC of the
HCl, pH 7.4) containing 5 mM CaCl2, 0.2 M NaCl, TCE- and water-fed rats was tested by qRT-PCR
and 0.02% Brij 35 followed by staining with for various cytokines and chemokines. In the case
Coomassie Brilliant Blue R-250. The MMP activ- of SAC (Figure 2a), there was a marked decrease
ity was evident in the form of translucent bands (P <0.05) in the expression of key pro-inflamma-
after de-staining. The gel was then scanned and the tory cytokines interleukin-1β (IL-1β), IL-6, IL-23,
intensity of bands was quantitated by densitometry and tumor necrosis factor-α (TNF-α) as well as the
using Image J software. chemokine “regulated on activation, normal T cell
expressed and secreted” (RANTES) in the TCE-
treated rats when compared with the control rats.
Statistical analysis In the case of LNC, the cytokine IL-17 was reduced
The data were expressed as mean ± SEM. Student’s significantly (P <0.05) without much change in
t test was used to assess the significance of the dif- IFN-γ in the TCE-treated rats compared to the con-
ference between vehicle and TCE group. Two-way trol rats (Figure 2b). In addition, IL-23 and TNF-α
analysis of variance with the Bonferroni post-test showed no significant change, while RANTES was
was carried out for in vivo clinical experiments. decreased (data not shown).
Sannegowda et al. 525

IL-10 was not altered following TCE treatment.

Furthermore, the chemokines RANTES and
MCP-1 showed a reduction (P <0.05) in TCE-
treated rats.

TCE treatment reduces the frequency

of the IL-17/IFN-γ double-producing
cells in the spleen
To gain more insight into the modulation of the
pro-inflammatory cytokines by TCE, we deter-
mined the frequency of CD3+ and CD4+ T cells
producing IL-17 and/or IFN-γ in splenic cells of
TCE-fed arthritic rats compared with water-fed
control rats using flow cytometry (Figure 4).
IL-17+IFN-γ+ CD4+ T cells (double producers)
were found to be significantly reduced from 2.2%
in water-fed control rats to 0.6% in TCE-fed rats.
Although, there was a slight decrease in the
percentages of IL-17-producing only and IFN-γ-
producing only (single producers) cells, the differ-
ence was not statistically significant (Figure 4a).
The testing of SIC revealed a similar trend of
reduced double producers, but the difference
was not statistically significant (Figure 4b).
Figure 1.  Suppression of AA in Lewis rats by TCE treatment.
Groups (n = 4 per group) of Mtb-immunized Lewis rats
Nevertheless, as both IL-17 and IFN-γ have pro-
were fed with either TCE or vehicle (water) beginning at the inflammatory properties, the reduction of double
onset of the disease. The arthritic scores of rats (a), hind paw producers in the spleen along with the results
photographs (b), H&E stained sections (c), and Safranin-O- of cytokine testing (Figures 2 and 3) reveal one
Stained sections (d) of hind paws of the two groups of rats on of the mechanisms by which TCE suppresses
day 19 after Mtb injection are shown. The histology sections
show the joint space (JS), the pannus (P) containing the arthritis.
mononuclear cell infiltrate, bone (Bo), and cartilage (Cart)
(c) and the extent of cartilage damage (shown by arrow)
(d) (*, P <0.05, statistically significant difference between
TCE treatment alters the balance
TCE-treated and control groups from day 17 to day 29). of immune mediators of bone
The results are representative of two independent remodeling
experiments. In (c) and (d), each section is at 10X.
To examine the mechanism underlying TCE-
induced protection against bone damage in rats
In another set of experiments, we tested various with AA, we determined the effect of TCE on the
cytokines and chemokines in the culture supernates immune mediators of bone remodeling such as
of SIC and in sera of TCE-treated and control rats. RANK, RANKL, OPG, OPN, OSC (also known
In the case of SIC (Figure 3a), the levels of the as OCN), and M-CSF in TCE-fed and control
cytokines IL-1β, IL-17, IL-6, and TNF-α were rats. The expression levels of these mediators
decreased significantly (P <0.05) in TCE-fed rats were measured in both SAC and SIC by qRT-
compared to control rats. The chemokines mono- PCR. SAC (Figure 5a) and SIC (Figure 5b)
cyte chemoattractant protein-1 (MCP-1) and mac- displayed a similar profile: OPN, M-CSF, and
rophage inflammatory protein-1α (MIP-1α) were RANKL were decreased, whereas OSC and
also decreased, but the difference was statistically RANK were increased in TCE-fed rats compared
significant for MCP-1 but not MIP-1α (Figure 3a). with control rats, and these alterations were
In the case of serum (Figure 3b), IL-1β and IL-17 statistically significant. Although OPG was
were decreased significantly (P <0.05), whereas not altered much, the RANKL/OPG ratio was
526 International Journal of Immunopathology and Pharmacology 28(4)

Figure 2.  The influence of TCE treatment on cytokine and chemokine expression in SAC and LNC. Spleen adherent cells (SAC)
(a) and lymph node cells (LNC) (b) were harvested from arthritic Lewis rats fed either with TCE or with water, on day 19 after
Mtb challenge. These cells were restimulated for 6 and 24 h, respectively with Mtb sonicate (10 ug/mL) (n = 3 each). The cytokine/
chemokine mRNA expression in these cells was quantified by qRT-PCR and the results were expressed as “fold over medium” after
normalization to HPRT (*, P <0.05, when comparing TCE and control samples).

Figure 3.  Alteration of cytokines and chemokines in SIC and sera following TCE treatment. Synovial-infiltrating cells (SIC) (a) and
sera (b) were collected from arthritic rats fed either with TCE or with water (n = 3/each group), on day 19 after Mtb injection.
SIC were restimulated for 24 h with Mtb sonicate (10 ug/mL) and culture supernates were collected. The levels of cytokines (top
panels and TNF-α in lower panel) and chemokines (lower panel, except TNF-α) in the culture supernates of SIC and sera were
measured by using a Multiplex assay. The results are expressed as pg/mL (*, P <0.05, when comparing TCE and control samples).

decreased significantly in both SAC and SIC in OSC, a marker of osteoblastic activity, the
in TCE group. This altered ratio favors anti- reduced RANKL/OPG ratio offers insight into
osteoclastic activity. Combined with an increase the bone damage-protective effect of TCE.
Sannegowda et al. 527

synovial tissue.1,2 Pro-inflammatory cytokines

such as TNF-α, IL-17, IL-6, and IL-1β; chemokines
such as RANTES and MCP-1; as well as biochem-
ical mediators such as RANKL and MMPs initiate
and propagate joint inflammation and tissue dam-
age.28–30 Among the pro-inflammatory cytokines,
IL-17 has been shown to play a vital role in arthri-
tis pathogenesis.31,32 This cytokine is produced by
Th17 cells and it can facilitate the production of
other pro-inflammatory cytokines such as TNF-α,
IL-1β, and IL-6.
Our results showed that the pro-inflammatory
cytokines were significantly reduced in SAC (e.g.,
IL-1β, IL-6, IL-23, and TNFα) and draining LNC
(e.g., IL-17) of arthritic rats treated with TCE.
These results were reinforced by those showing
Figure 4.  Treatment of arthritic rats with TCE reduces the that the pro-inflammatory cytokines were also
frequency of IL-17 and IFN-γ double-producing cells. Spleen reduced in SIC culture supernatants (e.g., IL-1β,
cells (a) and synovial-infiltrating cells (SIC) (b) were prepared IL-17, IL-6, and TNFα) and sera (e.g., IL-1β and
from TCE-fed and control arthritic rats (n = 3 each), on day IL-17) of TCE-treated rats. However, there was not
19 after Mtb immunization. These cells were then stimulated
with PMA and ionomycin for 6 h, followed by staining for much change in the levels of anti-inflammatory
CD3, CD4, IL-17, and IFN-γ. The cells were analyzed by flow IL-10 (Figure 3b) and other cytokines (data not
cytometry for IL-17- and/or IFN-γ-expressing CD4+ T cells shown). Thus, TCE altered the balance of pro-
(*, P <0.05, when comparing TCE and control samples). versus anti-inflammatory cytokines primarily by
downregulating the pro-inflammatory cytokines,
TCE suppresses matrix particularly IL-17 and IL-1β.
metalloproteinase-9 (MMP-9) activity Activated synovial fibroblasts and monocytes/
We also evaluated the effect of TCE on the MMPs, macrophages in the inflamed arthritic joint are the
which mediate damage to the cartilage and bone in source of chemokines, such as RANTES, MCP-1,
arthritic joints, in culture supernate of SIC obtained and MIP-1α.35,36 Our study revealed TCE-induced
from the joints of TCE-treated and control rats. reduction in RANTES in SAC, the draining LNC,
Treatment with TCE reduced MMP-9 activity, but and serum, as well as decrease in MCP-1 in both
without any effect on MMP-2 activity (Figure 6). serum and SIC. These results suggest that TCE-
induced suppression of arthritis might be mediated
in part by altering the chemotactic cellular migra-
Discussion
tion of immune cells into the joints.
We describe in this study that T. cordifolia extract Th17 cells produce IL-17, which plays a critical
(TCE) can effectively inhibit the severity of auto- role in arthritis pathogenesis. IL-17 upregulates
immune arthritis in the rat AA model. We also other pro-inflammatory cytokines, chemokines,
unraveled the immunological and molecular mech- and additional mediators of bone damage. IL-17
anisms underlying the anti-inflammatory and anti- facilitates neutrophil influx into the inflamed syno-
bone resorptive effects of TCE. It has previously vial joints and increases synoviocyte survival,
been reported that TCE can inhibit arthritis in the angiogenesis, osteoclast differentiation, and
CIA model17 and modulate the mediators of immune matrix-degrading enzymes.32,33,37 Other investiga-
response in other diseases.14,15 However, there is tors have reported that Th17 cells were signifi-
limited information on the arthritis-related immune cantly increased at all stages of the disease in RA
mechanisms influenced by TCE. The results of our patients.38,39 Similarly, increased frequency of
study discussed below have filled this gap. Th17 in mice/rats with arthritis has been reported
RA is a chronic inflammatory disease leading to by others and us.40,41 Studies based on the testing
joint destruction mediated by the migration of of natural products in animal models of RA have
CD4+ T cells and macrophages infiltrating the revealed that Th17 frequency was reduced in
528 International Journal of Immunopathology and Pharmacology 28(4)

Figure 5.  TCE alters the levels of mediators of bone remodeling. Spleen adherent cells (SAC) (a) and synovial-infiltrating cells (SIC)
(b) were harvested from arthritic Lewis rats fed either with TCE or with water, on day 19 after Mtb challenge. The SIC and SAC
were then restimulated for 24 h with Mtb sonicate (10 ug/mL) (n = 3 each). The mRNA expression of the bone remodeling-related
mediators in these cells was quantified by qRT-PCR and the results were expressed as “fold over medium” after normalization to
HPRT (*, P <0.05, when comparing TCE and control samples).

animals treated with defined natural products. For Nevertheless, the reduced frequency of IL-17+
example, the treatment of arthritic animals by IFN-γ+ (double producer) cells correlates with
grape seed proanthocyanidin extract or pristimerin reduced production of IL-17 as measured by qPCR
reduces the Th17 frequency in splenocytes and SIC and Multiplex assay.
of mice/rats with CIA and AA, respectively.25,40 In We described above the anti-inflammatory
our present study based on TCE, a statistically sig- activity of TCE as evident from attenuation of the
nificant decrease in IL-17-producing T cells was severity of clinical arthritis. We further investigated
observed, but that difference was primarily in whether TCE also limits bone damage in arthritic
IL-17+IFN-γ+ (double producer) cells of spleen rats, and also examined the mechanisms involved.
but not in IL-17+ (single producer) cells in the Bone homeostasis is maintained by a balance
periphery (spleen) or the target organ (the joints). between bone resorption and bone formation. The
Sannegowda et al. 529

Among the MMPs, MMP-9 plays an important

role in cartilage and bone destruction in arthritic
joints.44 TCE-treated rats showed marked reduc-
tion in MMP-9 activity, but without much effect on
MMP-2 activity. TCE might inhibit MMP-9 either
by a direct effect on the enzyme or by an indirect
effect via inhibiting the key inducers of MMP-9. In
regard to the latter, TCE reduced three of the posi-
tive inducers of MMP-9, namely RANKL, IL-6,
and IL-17. Similar to TCE, other herbal products
have previously been shown to protect against
osteoclastogenesis and bone loss. For example,
Celastrus extract and its bioactive component
Celastrol have been shown to protect against bone
damage in AA by inhibiting pro-inflammatory
cytokines, RANKL production, and MMP-9 activ-
Figure 6.  TCE reduces the level of MMP-9. Synovial- ity, but increasing RANKL/OPG ratio.20 Similarly,
infiltrating cells (SIC) from the joints of TCE-fed and water-fed green tea protects against inflammation-induced
rats were collected on day 19 after Mtb injection. The SIC bone loss in rats by reducing TNF-α production as
were then restimulated for 24 h in vitro with or without Mtb
well as inflammation.45
sonicate (10 ug/mL) and the activity of MMP-9 and MMP-2
was measured in the culture supernatant by using a gelatin In summary, the TCE treatment of arthritic rats
zymogram assay. A representative pattern of MMP activity is inhibited two inter-related features of arthritis,
shown. The graph above the gel shows the normalized units namely inflammation and bone damage compared
obtained by densitometric analysis. with the control rats. These effects were the inte-
grated outcomes of TCE-induced changes in
RANKL/RANK/OPG system regulates bone defined cytokines, chemokines, and mediators of
resorption by osteoclasts.30 Osteoclastogenesis and bone remodeling, which play critical roles in arthri-
bone resorption can be inhibited by decreased pro- tis pathogenesis. On the basis of our results, we
duction of RANKL and consequently decreased suggest that TCE should be further examined as a
activation of RANK. The reduction in RANKL potential anti-arthritic therapy in conjunction with
may or may not be accompanied by an increase in conventional medications in patients with RA.
OPG, resulting in a decrease in RANKL/OPG
ratio.20,30 In our study, we observed that TCE- Acknowledgements
treated rats showed reduction in the expression of The authors thank Steven Dudics, Bodhraj Acharya,
RANKL as well as the RANKL/OPG ratio in both Chithrashree Chamegowda, and Rajesh Rajaiah for dis-
SAC and SIC compared with control rats. Thus, cussions and/or help in experimental work. The authors
TCE restored the RANKL/OPG balance in AA. In also thank Dr. SN Vogel for providing the real-time PCR
addition, TCE treatment resulted in a marked facility, Lisa Hester for Multiplex assay, and Sabinsa
reduction in the osteoclastic mediators, namely Corporation, New Jersey for the kind gift of Tinospora
M-CSF and OPN, coupled with an increase in cordifolia extract.
OSC, which is required for osteoblastic activity.
Taken together, TCE shifted the balance of Declaration of conflicting interests
mediators of bone remodeling in favor of anti- The author(s) declared no potential conflicts of interest
osteoclastic activity. A previous report by other with respect to the research, authorship, and/or publication
investigators has shown the anti-osteoporotic of this article.
effects of TCE in an animal model of osteoporosis.42
Furthermore, a recent study has shown that TCE Funding
promotes the proliferation of osteoblast cells and This work was supported by grants from the Department
mineralization of bone-like matrix on human of Biotechnology, Government of India, New Delhi, India
osteoblast-like cells MG-63 and rat primary cul- (to KMS) and the National Institutes of Health, Bethesda,
ture of osteoblasts.43 MD (to KDM).
530 International Journal of Immunopathology and Pharmacology 28(4)

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