Effect of a Herbal-Leucine mix on the IL-1β-induced cartilage degradation and inflammatory gene expression in human chondrocytes

Abstract

Background: Conventional treatments for the articular diseases are often effective for symptom relief, but can also cause significant side effects and do not slow the progression of the disease. Several natural substances have been shown to be effective at relieving the symptoms of osteoarthritis (OA), and preliminary evidence suggests that some of these compounds may exert a favorable influence on the course of the disease. The objective of this study was to investigate the anti-inflammatory/chondroprotective potential of a Herbal and amino acid mixture containing extract of the Uncaria tomentosa, Boswellia spp., Lepidium meyenii and L-Leucine on the IL-1β-induced production of nitric oxide (NO), glycosaminoglycan (GAG), matrix metalloproteinases (MMPs), aggrecan (ACAN) and type II collagen (COL2A1) in human OA chondrocytes and OA cartilage explants.

Methods: Primary OA chondrocytes or OA cartilage explants were pretreated with Herbal-Leucine mixture (HLM, 1-10 μg/ml) and then stimulated with IL-1β (5 ng/ml). Effect of HLM on IL-1β-induced gene expression of iNOS, MMP-9, MMP-13, ACAN and COL2A1 was verified by real time-PCR. Estimation of NO and GAG release in culture supernatant was done using commercially available kits.

Results: HLM tested in these in vitro studies was found to be an effective anti-inflammatory agent, as evidenced by strong inhibition of iNOS, MMP-9 and MMP-13 expression and NO production in IL-1β-stimulated OA chondrocytes (p < 0.05). Supporting these gene expression results, IL-1β-induced cartilage matrix breakdown, as evidenced by GAG release from cartilage explants, was also significantly blocked (p < 0.05). Moreover, in the presence of herbal-Leucine mixture (HLM) up-regulation of ACAN and COL2A1 expression in IL-1β-stimulated OA chondrocytes was also noted (p < 0.05). The inhibitory effects of HLM were mediated by inhibiting the activation of nuclear factor (NF)-kB in human OA chondrocytes in presence of IL-1β.

Conclusion: Our data suggests that HLM could be chondroprotective and anti-inflammatory agent in arthritis, switching chondrocyte gene expression from catabolic direction towards anabolic and regenerative, and consequently this approach may be potentially useful as a new adjunct therapeutic/preventive agent for OA or injury recovery.

Figure 1

Chondrocyte viability after treatment with HLM (Uncaria tomentosa., Boswellia spp., Lepidium meyenii and L-Leucine). Human OA chondrocytes were incubated with 1 and 5 μg/ml of HLM for 24 h and viability was determined using release of lactate dehydrogenase (LDH) in culture supernatant. Values given are mean ± SD of 4 experiments.

Figure 2

Effect of HLM (Uncaria tomentosa., Boswellia spp., Lepidium meyenii and L-Leucine) on the IL-1β-induced gene expression of inducible nitric oxide synthase (iNOS) (A) and on the production of nitric oxide (NO) (B) in human OA chondrocytes. Chondrocytes were pretreated for 2 h with HLM (1-5 μg/ml) and then stimulated or not stimulated with IL-1β (5 ng/ml) for 24 h. Relative gene expression of iNOS was determined by Real-time PCR using GAPDH as endogenous control and compared with un-stimulated control. Corresponding culture supernatants were analyzed for total NO levels. Values represent Mean ± SE of three different experiments run in duplicate. * p < 0.05.

Figure 3

Effect of HLM (Uncaria tomentosa., Boswellia spp., Lepidium meyenii and L-Leucine) on the IL-1β-induced expression of MMP-9 and MMP-13 in IL-1β-stimulated OA chondrocytes. Chondrocytes were pretreated for 2 h with HLM (1-10 μg/ml) and then stimulated or not stimulated with IL-1β (5 ng/ml) for 6 h for MMP-9 mRNA (A) and 24 h for MMP-13 mRNA(B). mRNA expression was analyzed by Real time-PCR. Relative gene expression was normalized to GAPDH and compared with un-stimulated control. Levels of MMP-9 (C) and MMP-13 (D) in culture supernatants were quantified by sandwich ELISA at 24 h. Value represents Mean ± SD of three different experiments runs in duplicate. * p < 0.05.

Figure 4

Effect of HLM (Uncaria tomentosa., Boswellia spp., Lepidium meyenii and L-Leucine) on the IL-1β-induced gene expression of ACAN and COL2A1 in IL-1β-stimulated OA chondrocytes. Chondrocytes were pretreated for 2 h with HLM (1 and 5 μg/ml) and then stimulated or not stimulated with IL-1β (5 ng/ml) for 24 h, expression of ACAN (A) and COL2A1 (B) was analyzed by Real time-PCR. Relative gene expression was normalized to GAPDH and compared with un-stimulated control. Value represents Mean ± SD of three different experiments run in duplicate. * p < 0.05.

Figure 5

Cartilage matrix status. HLM inhibited the IL-1β-induced release of GAG from human cartilage explants in vitro. Cartilage pieces (26.7 mg ± 1.3) were incubated with medium alone or medium containing either IL-1β alone or in combination with HLM (1 and 5 μg/ml) for 7 days. Total GAG release from cartilage explants in culture supernatants was quantified by using Proteoglycan detection kit and values are derived from standard curve. Value represents Mean ± SD of three different experiments repeated in duplicate. * p < 0.05.

Figure 6

Effect of HLM (Uncaria tomentosa., Boswellia spp., Lepidium meyenii and L-Leucine) on the IL-1β-induced activation of NF-kB in IL-1β-stimulated OA chondrocytes. Chondrocytes were transfected with NF-kB luciferase plasmid and the NF-kB dependent transcriptional activity was determined by luciferase assay using commercially available kit (Promega). Value represents Mean ± SD of three different experiments repeated in duplicate. * p < 0.05.