The chrondoprotective actions of a natural product are associated with the activation of IGF-1 production by human chondrocytes despite the presence of IL-1beta

Abstract

Background: Cartilage loss is a hallmark of arthritis and follows activation of catabolic processes concomitant with a disruption of anabolic pathways like insulin-like growth factor 1 (IGF-1). We hypothesized that two natural products of South American origin, would limit cartilage degradation by respectively suppressing catabolism and activating local IGF-1 anabolic pathways. One extract, derived from cat's claw (Uncaria guianensis, vincaria), is a well-described inhibitor of NF-kappaB. The other extract, derived from the vegetable Lepidium meyenii (RNI 249), possessed an uncertain mechanism of action but with defined ethnomedical applications for fertility and vitality.

Methods: Human cartilage samples were procured from surgical specimens with consent, and were evaluated either as explants or as primary chondrocytes prepared after enzymatic digestion of cartilage matrix. Assessments included IGF-1 gene expression, IGF-1 production (ELISA), cartilage matrix degradation and nitric oxide (NO) production, under basal conditions and in the presence of IL-1beta.

Results: RNI 249 enhanced basal IGF-1 mRNA levels in human chondrocytes by 2.7 fold, an effect that was further enhanced to 3.8 fold by co-administration with vincaria. Enhanced basal IGF-1 production by RNI 249 alone and together with vincaria, was confirmed in both explants and in primary chondrocytes (P < 0.05). As expected, IL-1beta exposure completely silenced IGF-1 production by chondrocytes. However, in the presence of IL-1beta both RNI 249 and vincaria protected IGF-1 production in an additive manner (P < 0.01) with the combination restoring chondrocyte IGF-1 production to normal levels. Cartilage NO production was dramatically enhanced by IL-1beta. Both vincaria and RNI 249 partially attenuated NO production in an additive manner (p < 0.05). IL-1beta - induced degradation of cartilage matrix was quantified as glycosaminoglycan release. Individually RNI 249 or vincaria, prevented this catabolic action of IL-1beta.

Conclusion: The identification of agents that activate the autocrine production of IGF-1 in cartilage, even in the face of suppressive pro-inflammatory, catabolic cytokines like IL-1beta, represents a novel therapeutic approach to cartilage biology. Chondroprotection associated with prevention of the catabolic events and the potential for sustained anabolic activity with this natural product suggests that it holds significant promise in the treatment of debilitating joint diseases.

Figure 1

Enhanced expression of IGF-1 gene in human chondrocytes. IGF-1 gene expression referenced by the activity of β – actin was examined in human chondrocytes (n = 3 for all groups) under basal (control) conditions and after treatment with RNI 249 50 μg/ml (R50), vincaria 10 μg/ml (V10) and their combination (R50 + V10). The * refers to a significant difference from control levels (P < 0.05), and the ** refers to a significant difference from all other groups (P < 0.001), indicating that the combination of R50 and V10 produced significantly greater increases in IGF-1 expression than either V10 or R50 alone.

Figure 2

Production of IGF-1 from human cartilage explants as measured by media IGF-1 levels. RNI 249 produced a dose-dependent increase in media IGF-1 levels, as determined by ELISA. Only the 50 μg/ml concentration of RNI 249 produced a significant increase over basal, untreated explants (* P < 0.05, n = 6 for all groups).

Figure 3

IGF-1 production from human cartilage explants in response to IL-1β, vincaria and RNI 249 and their combination. Control, untreated cartilage explants (n = 6) release a defined amount of IGF-1 into the bathing media. However, in IL-1β (5 ng/ml) treated explants IGF-1 levels were immeasurable (n = 6). Co-treatment with either vincaria 10 μg/ml (V10, n=3), or RNI 249 50 μg/ml (R50, n = 6), partially restored IGF-1 production. The combination of vincaria and RNI 249 (IL-1 + V + R, n = 3) produced additive effects that resulted in the restoration of IGF-1 levels to control values despite the presence of IL-1β. The * denotes a significant difference from all other groups (P < 0.01).

Figure 4

Normalization of IL-1β enhanced glycosaminoglycan release from human cartilage explants by RNI 249. Treatment of human cartilage explants (n = 6) with IL-1β (5 ng/ml) results in the release of glycosaminoglycans (GAG) into the media. Administration of RNI 249 produced a dose-dependent decrease of IL-1β induced GAG release that was significant at the 10 μg/ml (R10, * P < 0.05) and 50 μg/ml (** P < 0.01) concentrations. The level of GAG release from control, untreated explants was indistinguishable from those explants treated with IL-1β + RNI 249 (50 μg/ml).

Figure 5

Effects of vincaria and RNI 249 on basal and IL-1β stimulated nitrite production in human cartilage explants. Media nitrite levels, a reflection of nitric oxide production, released from human cartilage explants (n = 3) was markedly enhanced by treatment with IL-1β (5 ng/ml, P <0.01). Co-treatment with either vincaria 10 μg/ml (V10) or RNI 249 50 μg/ml (R50) reduced nitrite levels although these changes were not significant. However, the combination of V10 and R50 produced additive effects and a significant reduction in nitrite levels (* P < 0.01 vs. IL-1β). Both V10 and R50 alone, reduced basal nitrite levels when compared to control untreated values but these differences were not significant.