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1East-West Bone & Joint Research Institute, East-West Neo Medical Center, Kyung Hee University, 149 Sangil-dong, Gangdong-gu, Seoul, Republic
of Korea
2Acupuncture and Meridian Science Research Center, Kyung Hee University, Hoeggidong, Dongdaemoon-gu, Seoul, Republic of Korea
3Department of Pathology, East-West Neo Meidcal Center, Kyung Hee University, 149 Sangil-dong, Gangdong-gu, Seoul, Republic of Korea
4Department of Pathology, Inje University Sanggye Paik Hospital, Sanggye 7 dong 761-7, Nowon-gu, Seoul, Republic of Korea
5Department of Internal Medicine, East-West Neo Medical Center, Kyung Hee University, 149 Sangil-dong, Gangdong-gu, Seoul, Republic of Korea
6Department of Orthopedic Surgery, East-West Neo Medical Center, Kyung Hee University, 149 Sangil-dong, Gangdong-gu, Seoul, Republic of
Korea
* Contributed equally
Received: 30 Dec 2008 Revisions requested: 9 Feb 2009 Revisions received: 4 Mar 2009 Accepted: 30 Mar 2009 Published: 30 Mar 2009
Abstract
Introduction The objective of this study was to determine the Results Piperine inhibited the expression of IL6 and MMP13
anti-inflammatory, nociceptive, and antiarthritic effects of and reduced the production of PGE2 in a dose dependant
piperine, the active phenolic component in black pepper extract. manner at concentrations of 10 to 100 μg/ml. In particular, the
production of PGE2 was significantly inhibited even at 10 μg/ml
Methods The in vitro anti-inflammatory activity of piperine was of piperine. Piperine inhibited the migration of activator protein
tested on interleukin 1β (IL1β)-stimulated fibroblast-like 1 (AP-1), but not nuclear factor (NF)κB, into the nucleus in IL1β-
synoviocytes derived form patients with rheumatoid arthritis. The treated synoviocytes. In rats, piperine significantly reduced
levels of IL6, matrix metalloproteinase (MMPs), cyclo-oxygenase nociceptive and arthritic symptoms at days 8 and 4,
2 (COX-2), and prostaglandin E2 (PGE2) were investigated by respectively. Histological staining showed that piperine
ELISA and RT-PCR analysis. The analgesic and antiarthritic significantly reduced the inflammatory area in the ankle joints.
activities of piperine were investigated on rat models of
carrageenan-induced acute paw pain and arthritis. The former Conclusions These results suggest that piperine has anti-
were evaluated with a paw pressure test, and the latter by inflammatory, antinociceptive, and antiarthritic effects in an
measuring the squeaking score, paw volume, and weight arthritis animal model. Thus, piperine should be further studied
distribution ratio. Piperine was administrated orally to rats at 20 with regard to use either as a pharmaceutical or as a dietary
and 100 mg/kg/day for 8 days. supplement for the treatment of arthritis.
ELISA: enzyme-linked immunosorbent assay; FLS: fibroblast-like synoviocytes; H&E: hematoxylin and eosin; IL: interleukin; MMP: matrix metallopro-
teinase; OA: osteoarthritis; RA: rheumatoid arthritis; WDR: weight distribution ratio.
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Arthritis Research & Therapy Vol 11 No 2 Bang et al.
anti-inflammatory agents carry the risk of gastrointestinal toxic- (DMEM, low glucose) (Gibco-BRL, Grand Island, NY, USA)
ity; thus, their use is limited. In an attempt to avoid adverse gas- supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco-
trointestinal effects, a new generation of non-steroidal anti- BRL) and 1 × antibiotic-antimycotic (Gibco-BRL). After the
inflammatory drugs (NSAIDs) was developed that selectively cells had grown to confluence, they were split at a 1:4 ratio.
inhibited cyclo-oxygenase (COX)-2 selective inhibitors (for FLS passages 3 to 6 from three patients were used for all
example, celecoxib, rofecoxib, and valdecoxib) [3]. Celecoxib experiments. Piperine, prednisolone, corn oil and carrageenan
and valdecoxib appear to show satisfactory cardiovascular were obtained from Sigma-Aldrich Korea (Young-In, Korea).
safety, however, rofecoxib was withdrawn from the market due Celecoxib was purchased in the form of the commercial drug,
to cardiovascular toxicity. However, side effects remain one of Celebrex (capsules; Pfizer Korea, Seoul, Korea).
the problems for long-term use; thus, there is a need for anti-
inflammatory drugs with less severe side effects. In addition, Semiquantitative RT-PCR
recent interest in alternative treatments for arthritis [4,5] has FLSs (2.5 × 105 cells) were cultured overnight in 60 mm
promoted their use in the US, but scientific evidence of antiar- dishes containing 2 ml of media. Cells were incubated with
thritic efficacy is lacking. serum-free media for 2 h and new serum-free media was
replaced just prior to the addition of piperine and cultured for
Black pepper (Piper nigrum) is commonly used as a spice in 24 h. Supernatants were collected for ELISA and the cells
human diets, but it is also used as a medicine, a preservative, were used for semiquantitative RT-PCR. Trizol was used to
and a perfume in many Asian countries. An extract of the active extract total RNA from the cells. Complementary DNA was
phenolic component, piperine, is well known to provide bene- synthesized from 1 μg of total RNA in a 20 μl reverse transcrip-
ficial physiological effects [6]. It stimulates the digestive tion reaction mixture. For semiquantitative PCR, aliquots of
enzymes of pancreas, protects against oxidative damage, low- cDNA were amplified in a 25 μl PCR mixture according to the
ers lipid peroxidation, and enhances the bioavailability of a protocol provided by the manufacturer (TaKaRa Bio, Kyoto,
number of therapeutic drugs. In addition, its anti-inflammatory Japan), as described previously [13]. The PCR conditions for
activities have been demonstrated in rat models of carra- the MMPs, IL6, and COX-2 were as follows: 30 to 33 cycles
geenan-induced rat paw edema, cotton pellet-induced granu- of 95°C for 45 s, 55 to 60°C for 45 s, and 72°C for 45 s. PCR
loma, and a croton oil-induced granuloma pouch [7]. products were subjected to electrophoresis on 1.5% agarose
Constituents of the piper species have shown in vitro inhibi- gels containing ethidium bromide, and the bands were visual-
tory activity against the enzymes responsible for leukotriene ized under ultraviolet (UV) light.
and prostaglandin biosynthesis, 5-lipoxygenase and COX-1,
respectively [8]. These effects of piperine seem to be benefi- ELISA
cial for inflammatory diseases that are accompanied by severe Synovial cells (2.5 × 105 cells/60 mm dish/2 ml serum-free
pain; for example, rheumatoid arthritis. media) were treated with various concentrations of piperine
30 minutes prior to IL1β stimulation. Conditioned media was
The excellent therapeutic properties of piperine have been collected 24 h later. Briefly, FLS cultures were centrifuged and
demonstrated in various cell types [9-12]. Nevertheless, little the supernatants were collected and analyzed for IL6, PGE2,
is known about the effect of piperine on the production of MMP1, and MMP13 with an ELISA kit (R&D Systems, Minne-
proinflammatory mediators in FLSs. Furthermore, to our knowl- apolis, MN, USA). For mRNA analysis, the cells were lysed and
edge, its antiarthritic efficacy has never been evaluated. In this total RNA was extracted. The mRNA levels of IL6 and COX-2
study, the anti-inflammatory effects of piperine were tested in were measured by semiquantitative RT-PCR analysis. The
IL1β-stimulated rheumatoid arthritis fibroblast-like synovio- COX-2 protein expression was measured by western blot.
cytes derived from patients with rheumatoid arthritis. Its antiar- Three independent experiments were performed in duplicate.
thritic efficacy was evaluated in animal models of experimental Each experiment was performed using synovial cells from dif-
arthritis. ferent patients. The collected supernatants were analyzed for
IL6, PGE2, MMP1 and MMP13 using commercial kits (ELISA;
Materials and methods R&D Systems). For the measurement of transcription factors,
Cell culture and reagents nuclear factor (NF)κB and activator protein 1 (AP-1), in the
All in vitro experiments were carried out with fibroblast-like nucleus, FLSs were seeded (5 × 106 cells) into 100 mm
synoviocytes derived from patients with rheumatoid arthritis dishes and grown to 80% confluence. The cells were serum-
(RA). After obtaining informed consent, synovial tissues were starved overnight and stimulated by IL1β (10 ng/ml) for 90
collected from RA patients. They met the 1987 American Col- minutes in the presence or absence of piperine. Subsequently,
lege of Rheumatology (ACR) criteria for the diagnosis of RA the cells were washed twice in phosphate-buffered saline
and had been treated with non-biological disease-modifying (PBS) and treated with lysis buffer and the extraction of tran-
antirheumatic drugs (DMARDs) and were underwent thera- scription factors from the nucleus was performed according to
peutic joint surgery. FLSs were isolated as described previ- the manufacturer's protocol (Active Motif, Seoul, Korea).
ously [13] and grown in Dulbecco's Modified Eagle Medium
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Western blot analysis Care and Use of Laboratory Animals, published by the Korean
FLSs cultured (2.5 × 105 cells) in 60 mm dishes were serum- National Institute of Health.
starved overnight and stimulated by IL1β (10 ng/ml) for 10 or
30 minutes in the presence or absence of piperine. The cells To induce paw hyperalgesia, rats were given an intraplantar
were subsequently washed twice in PBS and treated with 50 injection of 1% carrageenan (0.1 ml) in the posterior right paw
μl of lysis buffer (20 mM Tris-Cl pH 8.0, 150 mM NaCl, 1 mM as described previously [14]. After 3 hr of the injection, the
ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 20 pain threshold was measured using a paw pressure analgesia
μg/ml chymostatin, 2 mM phenylmethylsulfonyl fluoride instrument (UGO-BASIL Biological Research Apparatus,
(PMSF), 10 μM leupeptin, and 1 mM 4-(2-aminoethyl)benze- Comerio-Varese, Italy) for the Randall-Selitto test paw. A total
nesulfonyl fluoride (AEBSF)). As described previously [13], of 10 rats were studied per group and the test was performed
the samples were separated using 12% SDS-PAGE, and blind. Rats were starved overnight and piperine was evaluated
were then transferred to Hybond-ECL membranes (Amer- at doses of 20 and 100 mg/kg. Piperine dissolved in corn oil
sham, Arlington Heights, IL, USA). The membranes were first was fed orally 1 h before carrageenan injection. To evaluate
blocked with 6% non-fat milk dissolved in Tris-buffered saline/ paw hyperalgesia, we measured the tolerance to increasing
Tween (TBST) buffer (10 mM Tris-Cl pH 8.0, 150 mM NaCl, mild pressure on the affected paw between a flat surface and
0.05% Tween 20). The blots were then probed with various a blunt pointer of the instrument, as manufacturer's protocols.
rabbit polyclonal antibodies for inhibitor of κB (IκB)α, p-ERK1/ The effects of piperine were compared to the effects of Cele-
2, p-P38, p-Jun N-terminal kinase (JNK) and β-actin (Cell Sig- brex (Pfizer), a selective COX-2 inhibitor (100 mg/kg).
naling Technology, Beverly, MA, USA) diluted 1:1,000 in TBS
at 4°C for overnight, and incubated with 1:1,000 dilutions of The carrageenan-induced arthritic rat model was prepared as
goat anti-rabbit IgG secondary antibody coupled with horse- described previously [15]. Animals were briefly anesthetized
radish peroxidase. The blots were developed using the ECL with 3% isoflurane in a mixed N2O/O2 gas. Arthritic inflamma-
method (Amersham). For re-probing, the blots were incubated tion was induced by a single injection of 3% carrageenan sus-
in the stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, pended in 100 μl of pyrogen-free sterile saline, into the left
62.5 mM Tris-HCl pH 6.7) at 50°C for 30 minutes with occa- tibiotarsal ankle joint. The effects of piperine were compared
sional agitation. to the effects of prednisolone (10 mg/kg), a corticosteroid.
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et al. [15]. To evaluate arthritic pain, the rat was placed in the Results
test box of an incapacitance meter in which a slanted plank is Effect of piperine on FLS production of inflammatory
located. The bearing force of each hind limb was quantified by mediators
two mechanotransducers, separately placed below the two To test the anti-inflammatory efficacy of piperine, FLSs were
hind limbs: one is normal and the other is the arthritic limb. The stimulated with IL1β at 10 ng/ml in the presence or absence
bearing force of each hind limb was estimated as a 5-s aver- of piperine. The addition of IL1β significantly increased the
age, and the mean bearing force was calculated from four sep- production of IL6 and PGE2 compared to that of controls (no
arate estimations. The WDR percentage was calculated as IL1β). The addition of piperine greatly inhibited the IL6 and
percentage WDR = 100 × (weight borne by ipsilateral limb/ PGE2 response to IL1β in dose-dependent manner (Figure 1).
total weight borne by both limbs). The WDR of the hind paws Piperine also inhibited both the protein and mRNA expression
in the normal group was 50:50 (data not shown), indicating levels of IL6 and COX-2. In particular, piperine inhibited the
that 50% of the weight was carried in each hind paw. As the production of PGE2 more potently than the production of IL6.
pain and swelling of the ankle progressed due to induction of Interestingly, piperine inhibited the expression of the COX-2
arthritis, the balance of weight was disrupted, resulting in a protein more significantly than the COX-2 mRNA.
reduction of the WDR in the arthritic leg. All behavioral tests
were performed blinded. Next, we tested whether piperine inhibited the expression of
the extracellular matrix degradation enzymes (MMPs). MMP1
Statistical analysis and MMP13 play an important role in degrading cartilage in
The in vitro experimental data are expressed as the mean ± IL1β-stimulated FLSs. We found that piperine inhibited
standard error of the mean (SEM) of three independent exper- MMP13 expression at both the protein and mRNA levels, but
iments. The in vivo experimental data are presented as the not MMP1 (Figure 2). To understand the molecular mecha-
mean ± SEM. The differences between groups were assessed nisms underlying piperine inhibition of IL6, COX-2 and MMP
by repeated analysis of variance (ANOVA), followed by the expression, we investigated the MAP kinase and IκB kinase
Tukey "honestly significantly different" (HSD) post hoc analy- signaling pathways by western blot (Figure 3a). Interestingly,
sis. The degree of inflammation observed in H&E stained sec- piperine did not significantly affect the IκB kinase signaling
tions was compared between groups with the Mann-Whitney pathway or the MAP kinase mediated phosphorylation of JNK
test. Differences were considered significant at P < 0.05. and P38; however, piperine slightly inhibited the MAP kinase
mediated phosphorylation of ERK1/2. In addition, piperine
reduced the level of AP-1 that migrated to the nucleus (in
response to IL1β) in a dose dependent manner, but did affect
Figure 1
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Figure 2
the levels of NFκB in nucleus (Figure 3b). This suggested that Analgesic effect of piperine in carrageenan-induced paw
piperine inhibition of the ERK1/2 signaling pathway blocked hyperalgesia
the migration of AP-1 into the nucleus. Because piperine significantly inhibited the production of
PGE2 and the protein levels of COX-2, we tested whether pip-
Figure 3
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erine had antinociceptive effects in a rat model of carra- The vocalizations caused by flexion or extension of the
geenan-induced paw hyperalgesia. We found in paw pressure inflamed ankle reached a maximum point on day 1 after the
tests that rats treated with piperine could tolerate higher pres- carrageenan injection and was sustained at a maximum level in
sures on the affected paw (Figure 4a). The efficacy at 100 mg/ untreated rats through the end of the experiment (Figure 4c).
kg was better than that of celecoxib, and at a dose of 20 mg/ In the100 mg/kg piperine treated group, the number of vocal-
kg, piperine showed a mild analgesic effect. izations started to decrease at 5 days post-carrageenan injec-
tion. At 20 mg/kg, piperine exhibited little analgesic effect.
Antiarthritic effect of piperine on the carrageenan- Next, we measured the analgesic effect of piperine (20 and
induced arthritis rat model 100 mg/kg) on the weight distributed on the hind paws
To demonstrate the in vivo antiarthritic effect of piperine, the (WDR) of rats with carrageenan-induced arthritis in one paw
efficacy of piperine was tested in a rat model of carrageenan- (Figure 4d). Before the carrageenan injection (day 0), the
induced arthritis. The piperine (100 mg/kg) group showed a mean WDR did not differ significantly among the experimental
significant reduction in paw volume compared to the vehicle- groups (the WDR was 50:50, thus controls carried 50% of the
treated arthritic group (Figure 4). At this dose, piperine weight on each hind paw). However, significant changes in the
showed almost the same efficacy as prednisolone (10 mg/kg), ratio were observed on day 1 after the carrageenan injection,
which was used as a positive control. Piperine also provided a and the weight carried by the affected leg in the vehicle-
mild antiedema effect at 20 mg/kg, although it was not statis- treated arthritic group (CON) reached 20% at day 9. Distinct
tically significant. recovery of WDR was observed in groups that received 20
and 100 mg/kg piperine on days 8 and 9, despite the statisti-
cally insignificant analgesic effect of 20 mg/kg piperine.
Figure 4
Analgesic and antiarthritic effects of piperine in rat models of paw edema and arthritic ankle
ankle. (a) Piperine showed analgesic effects in carrageenan-
induced paw edema. The y axis indicated the pressure (g) that was tolerated before the rat exhibited signs of pain. Arthritic symptoms were meas-
ured by (b) relative paw volume, expressed as a function of the unaffected paw (100%). (c) The ankle flexion pain score (a value of 0 represents no
indication of pain); and (d) the weight distribution ratio (a value of 50% indicated that weight was equally distributed between the two hind paws).
The results indicated that piperine had antiarthritic effects. Con = control mice, Cele = celecoxib (100 mg/kg), PI-20/PI-100 = pierine at 20/100
mg/kg, Pre = prednisolne. Values are expressed ± standard error of the mean (SEM). *P < 0.05, **P < 0.01, ***P < 0.001 vs control group.
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Anti-inflammatory effect of piperine by histological meric. Recently, curcumin has been shown to possess diverse
evaluation pharmacological properties, including anti-inflammation, anti-
To evaluate the anti-inflammatory effects of piperine, samples proliferation, and antiangiogenesis. Currently, curcumin is in
of the ankle joints from each experimental group were exam- phase I of clinical trials [18].
ined by H&E staining. We found that the group that received
piperine (100 mg/kg) had significantly smaller areas of lym- Piperine is also a promising natural source with potential for
phocyte infiltration into the joints compared to the corn oil clinical use. Piper longum Linn. has been used in Asia as a nat-
treated group (Figure 5). The degree of inflammation in five ural treatment for poor peripheral blood circulation [19]. Piper
specimens was evaluated by three different pathologists. The longum Linn. and Piper nigrum Linn. are conventionally used
scores indicated that piperine significantly reduced the inflam- as immune enhancers in Indian traditional medicine [20].
mation induced by carrageenan (Figure 5b). Therefore, piperine has been proven effective indirectly, but its
mechanism of action remains unknown. In the present study,
Discussion we evaluated the anti-inflammatory and antiarthritic effects of
Anti-inflammatory drugs used for treating chronic inflammatory piperine to determine whether it had therapeutic potential for
diseases such as rheumatoid arthritis are typically prescribed the treatment of arthritis.
long term to properly control the disordered immune system.
Thus, there is a strong need to develop safe and effective We found that piperine significantly inhibited the production of
drugs for the long-term use. Many groups have studied non- two important proinflammatory mediators, IL6 and PGE2, in
steroidal anti-inflammatory small molecules that were derived IL1β-stimulated human FLS. This result was consistent with
from natural sources with the aim of developing new treat- other studies that showed potent anti-inflammatory effects in
ments for clinical use [17]. For example, curcumin is a other systems. The inhibition of PGE2 production is important
polyphenolic compound derived from the dietary spice, tur- due to its central role in triggering pain. In addition, MMP1 and
Figure 5
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MMP13 collagenases play dominant roles in RA and osteoar- well tolerated, with few or no clinical adverse effects, and the
thritis because they are the rate-limiting components of the mean maximum plasma concentration of nevirapine was
collagen degradation process. The significant inhibition of increased when combined with piperine. In another clinical
MMP13 expression is particularly important because it study, piperine was shown to increase the plasma levels of
degrades a wide range of collagenous and non-collagenous coenzyme Q10 [28]. Therefore, piperine may improve the ther-
extracellular matrix macromolecules and is remarkably active apeutic effect or lower the dose requirements of other drugs
against collagen type II, the predominant collagen in cartilage. when administrated with DMARDs as a therapeutic drug or
To our knowledge, this is the first report to show that piperine dietary supplement. In addition, combinations of DMARDs
inhibited the expression of MMP13 in IL1β-stimulated FLSs. with piperine may reduce the side effects of DMARDs.
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