Chondroprotective effects of a proanthocyanidin rich Amazonian genonutrient reflects direct inhibition of matrix metalloproteinases and upregulation of IGF-1 production by human chondrocytes

Abstract

Background: The Amazonian medicinal plant Sangre de grado (Croton palanostigma) has traditional applications for the treatment of wound healing and inflammation. We sought to characterize two extracts (progrado and zangrado) in terms of safety and oligomeric proanthocyanidin chain length. Additionally progrado was evaluated for antioxidant activity and possible chondroprotective actions.

Methods: Acute oral safety and toxicity was tested in rats according under OECD protocol number 420. The profile of proanthocyanidin oligomers was determined by HPLC and progrado's antioxidant activity quantified by the ORAC, NORAC and HORAC assays. Human cartilage explants, obtained from surgical specimens, were used to assess chondroproteciton with activity related to direct inhibitory effects on human matrix metalloproteinase (MMP, gelatinolytic) activity using synovial fluid and chondrocytes activated with IL-1beta (10 ng/ml). Additionally, progrado (2-10 mug/ml) was tested for its ability to maintain optimal IGF-1 transcription and translation in cartilage explants and cultured chondrocytes.

Results: Both progrado and zangrado at doses up to 2000 mg/kg (po) displayed no evidence of toxicity. Oligomeric proanthocyanidin content was high for both progrado (158 mg/kg) and zangrado (124 mg/kg), with zangrado almost entirely composed of short oligomers (<6 mer), whereas the majority of oligomers in progrado exceeded 10 mers. Progrado was a remarkably potent antioxidant in the standardized tests ORAC, NORAC and HORAC. Progrado was exceptionally effective in reducing both basal and IL-1beta induced glycosaminoglycan release from human cartilage explants at concentrations that also directly blocked the gelatinolytic activity of MMP-2 and MMP-9. Progrado prevented IL-1beta induced suppression of IGF-1 production from human cartilage explants as well as stimulating basal IGF-1 production (P < 0.05). Comparable changes in IGF-1 gene expression were noted in cultured human chondrocytes.

Conclusion: Progrado has a promising safety profile, significant chondroprotective and antioxidant actions, directly inhibits MMP activity and promotes the production of the cartilage repair factor, IGF-1. This suggests that progrado may offer therapeutic benefits in joint health, wound healing and inflammation.

Figure 1

Proanthocyanidin oligomer length for zangrado and progrado. Oligomer length as determined by HPLC was distinctive for the two extracts, zangrado and progrado, of sangre de grado (Croton palanostigma). Short length oligomers were the main source of proanthocyanidins in zangrado, whereas progrado consisted of long chain oligomers. Results reflect mean values for each oligomer length for triplicate analysis.

Figure 2

Weight gain following a single oral dose: Acute oral toxicity. Body weights of rats treated with either progrado (n = 5) or zangrado (n = 5) at 2000 mg/kg administered orally in accordance with OECD test 420. Weight was measured at baseline and day 7 and 14 and depicted as the mean ± sem. The * represents a significant difference from baseline (P < 0.05) and the ** indicates a significance level of P < 0.01.

Figure 3

Cartilage matrix breakdown as measured by the release of glycosaminoglycan. Treatment of human cartilage explants with IL-1β (10 ng/ml) results in the release of glycosaminoglycans (GAG) into the media (* P < 0.01). Co-administration of progrado (2 or 10 μg/ml) blocked the IL-1β induced GAG release and resulted in a significant reduction in media levels of GAG (** P < 0.01) below the IL-1β and untreated control response. Explants treated with progrado alone (10 μg/ml) also produced a dramatic reduction in media GAG levels to that evident with progrado + IL-1β (** P < 0.01). Results are depicted as the mean ± sem.

Figure 4

Cartilage explant production of IGF-1 and the effects of IL-1β and progrado. Control, untreated human cartilage explants release a defined amount of IGF-1 into the bathing media. Cartilage release of IGF-1 was significantly reduced by IL-1β (10 ng/ml, * P < 0.05). Co-treatment with progrado 2 or 10 μg/ml restored and raised IGF-1 production above basal controls (** P < 0.01). This effect was more pronounced with the lower dose of progrado (2 μg/ml) than the higher dose (10 μg/ml). In the absence of IL-1β, progrado (10 μg/ml) reduced IGF-1 production basal values (* P < 0.01). Results are depicted as the mean ± sem.

Figure 5

IGF-1 mRNA levels in cultured human chondrocytes and the effects of IL-1β and progrado. Primary cultures of human chondrocytes taken from surgical explants were examined for IGF-1 gene expression using real time RT-PCR. Basal expression was normalized to the house keeping gene β-actin. Given that lower doses of progrado were more effective in raising IGF-1 production and cultured chondrocytes do not have cartilage matrux as a barrier to agent penetration only the low dose of progrado (2 μg/ml) was evaluated. Treatment with IL-1β resulted in a significant suppression of IGF-1 mRNA levels (* P < 0.001). However, co-treatment with progrado (2 μg/ml) and IL-1β resulted in a significant increase in IGF-1 gene expression above untreated controls and IL-1β treatment (** P < 0.001). Treatment with progrado alone (2 μg/ml) was able to stimulate IGF-1 mRNA levels above all other groups (** P < 0.001). Results expressed as the mean ± SEM.

Figure 6

MMP Activity from synovial fluid of subjects with osteoarthritis and the inhibitory effects of progrado. Synovial fluid from subjects with active osteoarthritis was used as a source of matrix metalloproteinase gelatinolytic activity using gelatin zymography. Recombinant MMP-2 and MMP-9 was used for identification of gelatinolytic activity. Progrado was associated with a dose-dependent inhibition of both MMP-2 and MMP-9 activity. Complete inhibition of MMP-9 was evident at 4 μg/ml, with MMP-2 activity reduced by approximately 90%.

Figure 7

MMP activity from culture media of IL-1β treated human chondrocytes and the inhibitory effects of progrado. Subconfluent human chondrocytes were treated with IL-1β (10 ng/ml) to stimulate the release of matrix metalloproteinases, measured as MMP and MMP-9 using gelatin zymography. Progrado was associated with a dose-dependent inhibition of both MMP-2 and MMP-9 activity. Complete inhibition of MMP-9 was evident at 4 μg/ml, with MMP-2 activity reduced by approximately 90%.