Anti-inflammatory and antiarthritic effects of piperine in human interleukin 1beta-stimulated fibroblast-like synoviocytes and in rat arthritis models

Abstract

Introduction: The objective of this study was to determine the anti-inflammatory, nociceptive, and antiarthritic effects of piperine, the active phenolic component in black pepper extract.

Methods: The in vitro anti-inflammatory activity of piperine was tested on interleukin 1beta (IL1beta)-stimulated fibroblast-like synoviocytes derived form patients with rheumatoid arthritis. The levels of IL6, matrix metalloproteinase (MMPs), cyclo-oxygenase 2 (COX-2), and prostaglandin E2 (PGE2) were investigated by ELISA and RT-PCR analysis. The analgesic and antiarthritic activities of piperine were investigated on rat models of carrageenan-induced acute paw pain and arthritis. The former were evaluated with a paw pressure test, and the latter by measuring the squeaking score, paw volume, and weight distribution ratio. Piperine was administrated orally to rats at 20 and 100 mg/kg/day for 8 days.

Results: Piperine inhibited the expression of IL6 and MMP13 and reduced the production of PGE2 in a dose dependant manner at concentrations of 10 to 100 microg/ml. In particular, the production of PGE2 was significantly inhibited even at 10 microg/ml of piperine. Piperine inhibited the migration of activator protein 1 (AP-1), but not nuclear factor (NF)kappaB, into the nucleus in IL1beta-treated synoviocytes. In rats, piperine significantly reduced nociceptive and arthritic symptoms at days 8 and 4, respectively. Histological staining showed that piperine significantly reduced the inflammatory area in the ankle joints.

Conclusions: These results suggest that piperine has anti-inflammatory, antinociceptive, and antiarthritic effects in an arthritis animal model. Thus, piperine should be further studied with regard to use either as a pharmaceutical or as a dietary supplement for the treatment of arthritis.

Figure 1

Effect of piperine on the production of proinflammatory mediators. (a) ELISA results show that piperine inhibited the production of interleukin (IL)6 and prostaglandin E₂ (PGE₂) in IL1β-stimulated fibroblast-like synoviocytes (FLSs) in a dose-dependent manner. (b) Piperine effects on IL6 and cyclo-oxygenase (COX)-2 mRNA expression measured by semiquantitative RT-PCR. (c) Piperine effects on COX-2 protein expression measured by western blot. Experiments were performed with synovial cells derived from patients with rheumatoid arthritis. Values are expressed ± standard error of the mea (SEM). ***P < 0.001 vs IL1β treated group without piperine.

Figure 2

Effect of piperine on the production of extracelluar matrix degradation enzymes (matrix metalloproteinases (MMPs)). (a) ELISA results show that piperine inhibited the production of MMP13, but not MMP1 proteins, in interleukin (IL)1β-stimulated fibroblast-like synoviocytes (FLSs) in a dose dependent manner. (b) mRNA levels were measured by semiquantitative RT-PCR. Experiments were performed with synovial cells derived from patients with rheumatoid arthritis. Values are expressed ± standard error of the mean (SEM). ***P < 0.001 vs IL1β treated group without piperine.

Figure 3

Effects of piperine on signaling pathways and transnuclear migration. (a) Interleukin (IL)1β-stimulated fibroblast-like synoviocytes (FLSs) treated with piperine were analyzed by western blot. Piperine treatment did not inhibit the degradation of inhibitor of κB (IκB)α, but slightly inhibited the phosphorylation of extracellular-regulated kinase (ERK)1/2 in the MAP kinase signaling pathways was slightly inhibited in the presence of piperine. (b) The nuclear levels of nuclear factor (NF)κB and activator protein 1 (AP-1) were measured by ELISA detecting p65 and c-FOS from nuclear extracts, respectively, on an ELISA. Piperine inhibited the level of AP-1 in the nucleus, but not NFκB levels. Values are expressed ± standard error of the mean (SEM). ***P < 0.001 vs IL1β treated group without piperine or piperine alone.

Figure 4

Analgesic and antiarthritic effects of piperine in rat models of paw edema and arthritic 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 measured 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.

Figure 5

Histological evaluation of the anti-inflammatory effects of piperine. Paraffin sections of rat ankles were stained with hematoxylin and eosin (H&E). (a) Histopathological analysis showed that piperine (100 mg/kg) significantly inhibited ankle inflammation. Each photo is representative of five specimens for each group (original magnification × 100). The insets are enlargements of the regions outlined in black, and show the infiltrates at a magnification of × 200. (b) The degree of inflammation was evaluated on a scale from 0 to 5 by three pathologists that were blinded to the treatments. Values are expressed ± standard error of the mean (SEM). *P < 0.05 vs corn oil treated group.