Cell culture and reagents

All in vitro experiments were carried out with fibroblast-like synoviocytes derived from patients with rheumatoid arthritis (RA). After obtaining informed consent, synovial tissues were collected from RA patients. They met the 1987 American College of Rheumatology (ACR) criteria for the diagnosis of RA and had been treated with non-biological disease-modifying antirheumatic drugs (DMARDs) and were underwent therapeutic joint surgery. FLSs were isolated as described previously [13] and grown in Dulbecco's Modified Eagle Medium (DMEM, low glucose) (Gibco-BRL, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco-BRL) and 1 × antibiotic-antimycotic (Gibco-BRL). After the cells had grown to confluence, they were split at a 1:4 ratio. FLS passages 3 to 6 from three patients were used for all experiments. Piperine, prednisolone, corn oil and carrageenan were obtained from Sigma-Aldrich Korea (Young-In, Korea). Celecoxib was purchased in the form of the commercial drug, Celebrex (capsules; Pfizer Korea, Seoul, Korea).

Semiquantitative RT-PCR

FLSs (2.5 × 10⁵ cells) were cultured overnight in 60 mm dishes containing 2 ml of media. Cells were incubated with serum-free media for 2 h and new serum-free media was replaced just prior to the addition of piperine and cultured for 24 h. Supernatants were collected for ELISA and the cells were used for semiquantitative RT-PCR. Trizol was used to extract total RNA from the cells. Complementary DNA was synthesized from 1 μg of total RNA in a 20 μl reverse transcription reaction mixture. For semiquantitative PCR, aliquots of cDNA were amplified in a 25 μl PCR mixture according to the protocol provided by the manufacturer (TaKaRa Bio, Kyoto, Japan), as described previously [13]. The PCR conditions for the MMPs, IL6, and COX-2 were as follows: 30 to 33 cycles of 95°C for 45 s, 55 to 60°C for 45 s, and 72°C for 45 s. PCR products were subjected to electrophoresis on 1.5% agarose gels containing ethidium bromide, and the bands were visualized under ultraviolet (UV) light.

ELISA

Synovial cells (2.5 × 10⁵ cells/60 mm dish/2 ml serum-free media) were treated with various concentrations of piperine 30 minutes prior to IL1β stimulation. Conditioned media was collected 24 h later. Briefly, FLS cultures were centrifuged and the supernatants were collected and analyzed for IL6, PGE₂, MMP1, and MMP13 with an ELISA kit (R&D Systems, Minneapolis, MN, USA). For mRNA analysis, the cells were lysed and total RNA was extracted. The mRNA levels of IL6 and COX-2 were measured by semiquantitative RT-PCR analysis. The COX-2 protein expression was measured by western blot. Three independent experiments were performed in duplicate. Each experiment was performed using synovial cells from different patients. The collected supernatants were analyzed for IL6, PGE₂, MMP1 and MMP13 using commercial kits (ELISA; R&D Systems). For the measurement of transcription factors, nuclear factor (NF)κB and activator protein 1 (AP-1), in the nucleus, FLSs were seeded (5 × 10⁶ cells) into 100 mm dishes and grown to 80% confluence. The cells were serum-starved overnight and stimulated by IL1β (10 ng/ml) for 90 minutes in the presence or absence of piperine. Subsequently, the cells were washed twice in phosphate-buffered saline (PBS) and treated with lysis buffer and the extraction of transcription factors from the nucleus was performed according to the manufacturer's protocol (Active Motif, Seoul, Korea).

Western blot analysis

FLSs cultured (2.5 × 10⁵ cells) in 60 mm dishes were serum-starved overnight and stimulated by IL1β (10 ng/ml) for 10 or 30 minutes in the presence or absence of piperine. The cells were subsequently washed twice in PBS and treated with 50 μl of lysis buffer (20 mM Tris-Cl pH 8.0, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 20 μg/ml chymostatin, 2 mM phenylmethylsulfonyl fluoride (PMSF), 10 μM leupeptin, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF)). As described previously [13], the samples were separated using 12% SDS-PAGE, and were then transferred to Hybond-ECL membranes (Amersham, Arlington Heights, IL, USA). The membranes were first blocked with 6% non-fat milk dissolved in Tris-buffered saline/Tween (TBST) buffer (10 mM Tris-Cl pH 8.0, 150 mM NaCl, 0.05% Tween 20). The blots were then probed with various rabbit polyclonal antibodies for inhibitor of κB (IκB)α, p-ERK1/2, p-P38, p-Jun N-terminal kinase (JNK) and β-actin (Cell Signaling Technology, Beverly, MA, USA) diluted 1:1,000 in TBS at 4°C for overnight, and incubated with 1:1,000 dilutions of goat anti-rabbit IgG secondary antibody coupled with horseradish peroxidase. The blots were developed using the ECL method (Amersham). For re-probing, the blots were incubated in the stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.7) at 50°C for 30 minutes with occasional agitation.

Histological assessment of inflammation

The rats were killed after 9 days of carrageenan and control treatments. Immunohistochemical staining was performed to determine the degree of immune cells infiltration into the joints. Rat ankles were dissected, fixed in 10% formalin, dehydrated through a graded ethanol series, cleared in xylene, and processed for embedding in paraffin wax with routine protocols. A microtome was used to cut 4 μm-thick sections that were subsequently stained with hematoxylin and eosin (H&E) stain. The degree of inflammation was evaluated on a scale from 0 to 5 by three different pathologists that had been blinded to the treatments. The scale was defined as follows: 0 = no inflammation, 1 = mild inflammation, 2 = mild/moderate inflammation, 3 = moderate inflammation, 4 = moderate/severe inflammation, and 5 = severe inflammation.

Rat models of paw hyperalgesia and arthritis

Sprague-Dawley 5-week-old to 6-week-old male rats, purchased from SLC (Shizuoka, Japan), were used in this study. All animals were maintained in plastic cages at 21 to 24°C under a 12 h light/dark cycle and were given free access to pellet food and water. They were adapted for at least 1 week prior to the start of the experiment. All subjects were habituated to the behavioral test chambers and handled with special care to minimize stress. All methods were approved by the Animal Care and Use Committee of Kyung Hee University. All procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, published by the Korean National Institute of Health.

To induce paw hyperalgesia, rats were given an intraplantar injection of 1% carrageenan (0.1 ml) in the posterior right paw as described previously [14]. After 3 hr of the injection, the pain threshold was measured using a paw pressure analgesia instrument (UGO-BASIL Biological Research Apparatus, Comerio-Varese, Italy) for the Randall-Selitto test paw. A total of 10 rats were studied per group and the test was performed blind. Rats were starved overnight and piperine was evaluated at doses of 20 and 100 mg/kg. Piperine dissolved in corn oil was fed orally 1 h before carrageenan injection. To evaluate paw hyperalgesia, we measured the tolerance to increasing mild pressure on the affected paw between a flat surface and a blunt pointer of the instrument, as manufacturer's protocols. The effects of piperine were compared to the effects of Celebrex (Pfizer), a selective COX-2 inhibitor (100 mg/kg).

The carrageenan-induced arthritic rat model was prepared as described previously [15]. Animals were briefly anesthetized with 3% isoflurane in a mixed N₂O/O₂ gas. Arthritic inflammation was induced by a single injection of 3% carrageenan suspended in 100 μl of pyrogen-free sterile saline, into the left tibiotarsal ankle joint. The effects of piperine were compared to the effects of prednisolone (10 mg/kg), a corticosteroid.

To evaluate the arthritic progression of carrageenan-induced arthritis in the rat, three different parameters were measured: paw volume, squeaking score in the ankle flexion test, and weight distribution ratio (WDR). These were considered behavioral indicators of carrageenan-induced arthritis and checked daily for 9 days. With progression of arthritis, redness and swelling of the ankle joints and arthritic pain started to appear and reached a maximum on day 1 after the carrageenan injection. At that time, piperine and prednisolone dissolved in corn oil was administrated orally once a day for 8 days.

The paw volumes were measured using a digital plethysmometer (UGO-BASIL Biological Research Apparatus), as described by Kwon et al. [16]. Paw volumes were expressed as relative values to that of day 0 when carrageenen was injected. The ankle flexion test involved gentle flexion and extension of the carrageenan-injected ipsilateral hind limb, as described by Kwon et al. [16]. This elicited vocalizations (squeaking) that were scored on a scale (squeaking score) as a measure of hyperalgesia. The procedure of flexion and extension were repeated 10 times in every 5 s and the rating of 0 (null) or 1 (vocalization) was given to each hind limb. This test was performed only once a day in each animal. The WDR is a ratio of the percentage of weight carried on each hind leg in which the weight-bearing forces of both hind limbs were measured with an incapacitance meter (UGO-BASIL Biological Research Apparatus), as previously described by Hwang et al. [15]. To evaluate arthritic pain, the rat was placed in the test box of an incapacitance meter in which a slanted plank is located. The bearing force of each hind limb was quantified by two mechanotransducers, separately placed below the two hind limbs: one is normal and the other is the arthritic limb. The bearing force of each hind limb was estimated as a 5-s average, and the mean bearing force was calculated from four separate estimations. The WDR percentage was calculated as percentage WDR = 100 × (weight borne by ipsilateral limb/total weight borne by both limbs). The WDR of the hind paws in the normal group was 50:50 (data not shown), indicating that 50% of the weight was carried in each hind paw. As the pain and swelling of the ankle progressed due to induction of arthritis, the balance of weight was disrupted, resulting in a reduction of the WDR in the arthritic leg. All behavioral tests were performed blinded.

Statistical analysis

The in vitro experimental data are expressed as the mean ± standard error of the mean (SEM) of three independent experiments. The in vivo experimental data are presented as the mean ± SEM. The differences between groups were assessed by repeated analysis of variance (ANOVA), followed by the Tukey "honestly significantly different" (HSD) post hoc analysis. The degree of inflammation observed in H&E stained sections was compared between groups with the Mann-Whitney test. Differences were considered significant at P < 0.05.

Effect of piperine on FLS production of inflammatory mediators

To test the anti-inflammatory efficacy of piperine, FLSs were stimulated with IL1β at 10 ng/ml in the presence or absence of piperine. The addition of IL1β significantly increased the production of IL6 and PGE₂ compared to that of controls (no IL1β). The addition of piperine greatly inhibited the IL6 and PGE₂ response to IL1β in dose-dependent manner (Figure 1). Piperine also inhibited both the protein and mRNA expression levels of IL6 and COX-2. In particular, piperine inhibited the production of PGE₂ more potently than the production of IL6. Interestingly, piperine inhibited the expression of the COX-2 protein more significantly than the COX-2 mRNA.

Next, we tested whether piperine inhibited the expression of the extracellular matrix degradation enzymes (MMPs). MMP1 and MMP13 play an important role in degrading cartilage in IL1β-stimulated FLSs. We found that piperine inhibited MMP13 expression at both the protein and mRNA levels, but not MMP1 (Figure 2). To understand the molecular mechanisms underlying piperine inhibition of IL6, COX-2 and MMP expression, we investigated the MAP kinase and IκB kinase signaling pathways by western blot (Figure 3a). Interestingly, piperine did not significantly affect the IκB kinase signaling pathway or the MAP kinase mediated phosphorylation of JNK 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 the levels of NFκB in nucleus (Figure 3b). This suggested that piperine inhibition of the ERK1/2 signaling pathway blocked the migration of AP-1 into the nucleus.

Analgesic effect of piperine in carrageenan-induced paw hyperalgesia

Because piperine significantly inhibited the production of PGE₂ and the protein levels of COX-2, we tested whether piperine had antinociceptive effects in a rat model of carrageenan-induced paw hyperalgesia. We found in paw pressure tests that rats treated with piperine could tolerate higher pressures on the affected paw (Figure 4a). The efficacy at 100 mg/kg was better than that of celecoxib, and at a dose of 20 mg/kg, piperine showed a mild analgesic effect.

Antiarthritic effect of piperine on the carrageenan-induced arthritis rat model

To demonstrate the in vivo antiarthritic effect of piperine, the efficacy of piperine was tested in a rat model of carrageenan-induced arthritis. The piperine (100 mg/kg) group showed a significant reduction in paw volume compared to the vehicle-treated arthritic group (Figure 4). At this dose, piperine showed almost the same efficacy as prednisolone (10 mg/kg), which was used as a positive control. Piperine also provided a mild antiedema effect at 20 mg/kg, although it was not statistically significant.

The vocalizations caused by flexion or extension of the inflamed ankle reached a maximum point on day 1 after the carrageenan injection and was sustained at a maximum level in untreated rats through the end of the experiment (Figure 4c). In the100 mg/kg piperine treated group, the number of vocalizations started to decrease at 5 days post-carrageenan injection. At 20 mg/kg, piperine exhibited little analgesic effect. Next, we measured the analgesic effect of piperine (20 and 100 mg/kg) on the weight distributed on the hind paws (WDR) of rats with carrageenan-induced arthritis in one paw (Figure 4d). Before the carrageenan injection (day 0), the mean WDR did not differ significantly among the experimental groups (the WDR was 50:50, thus controls carried 50% of the weight on each hind paw). However, significant changes in the ratio were observed on day 1 after the carrageenan injection, and the weight carried by the affected leg in the vehicle-treated arthritic group (CON) reached 20% at day 9. Distinct recovery of WDR was observed in groups that received 20 and 100 mg/kg piperine on days 8 and 9, despite the statistically insignificant analgesic effect of 20 mg/kg piperine.

Anti-inflammatory effect of piperine by histological evaluation

To evaluate the anti-inflammatory effects of piperine, samples of the ankle joints from each experimental group were examined by H&E staining. We found that the group that received piperine (100 mg/kg) had significantly smaller areas of lymphocyte infiltration into the joints compared to the corn oil treated group (Figure 5). The degree of inflammation in five specimens was evaluated by three different pathologists. The scores indicated that piperine significantly reduced the inflammation induced by carrageenan (Figure 5b).