Background/Purpose: There is growing evidence that shows the involvement of IL-6 in cartilage degradation during OA. Significant correlation between IL-6 levels in serum as well as synovial fluid and OA severity has been reported. IL-6 stimulates the expression of MMP-13 and inhibits the expression of type II collagen. Harpagide is a low molecular weight compound isolated from the secondary roots of Harpagophytum procumbens (Hp). In the present study we used an in vitromodel of inflammation in OA to investigated the therapeutic potential of Harpagide in OA.

Methods: Primary human chondrocytes were isolated from the non-affected cartilage obtained from OA patients who underwent total knee arthroplasty. Human OA chondrocytes were cultured and pre-treated with Harpagide (500 µM) and then cultured with and without IL-1β (5 ng/ml). Chondrocyte viability was assayed using Trypan blue exclusion assay. Secreted levels of IL-6 in the culture supernatants were quantified by ELISA. Total protein levels and phosphorylation levels of CEBPβ and AP-1 in human OA chondrocytes were measured by Western blot analysis using specific antibodies. Nuclear extracts were prepared and used to study the effect on the nuclear localization and activation of NF-κB, CEBPβ and AP-1 in OA chondrocytes by Western blotting and DNA binding activity. IL-6 mRNA levels were quantified using the TaqMan assays. Data were derived using Origin 6.1 software and P<0.05 was considered significant.

Results: Harpagide had no effect on OA chondrocyte viability in vitro.Treatment of primary human OA chondrocytes with IL-1β markedly stimulated the mRNA expression of IL-6 and protein secretion in the culture supernatants which was inhibited significantly (p<0.05) by pre-treatment with Harpagide. Harpagide did not inhibit the IL-1b-induced degradation of IκB and the activation of NF-κB but suppressed the IL-1β-triggered nuclear localization and activation of CEBPβ and AP-1 in human OA chondrocytes. There was a significant decrease in IL-1β-induced phosphorylation of CEBPβ, c-Fos and ATF-2 by Harpagide which also inhibited the IL-1β-induced enhancement in cytoplasmic levels of total c-FOS protein in OA chondrocytes. While there was a significant decrease in the nuclear levels of CEBPβ and c-Fos, there was no effect of pre-treatment of the cells with Harpagide on nuclear levels of NF-kB-p-65. Activation of p38 MAPK, which phosphorylates CEBPβ, ATF-2 and c-Fos resulting in their activation was significantly inhibited by Harpagide in human OA chondrocytes. Two small molecule inhibitors of p38MAPK (SB203580 and SB202190) also significantly inhibited the IL-1b-induced activation of p38-MAPK and the expression and secretion of IL-6 in human OA chondrocytes.

Conclusion: Taken together, the data presented here suggests that Harpagide may have a significant chondroprotective and OA suppressive effect by inhibiting the expression and production of IL-6. Importantly, we also identify a novel mechanism of IL-6 suppression which bypasses the activation of NF-kB.These data provide strong evidence with mechanistic details in support of the possible use of Harpagide as a therapeutic choice to prevent/retard the progression of OA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/harpagide-a-low-molecular-weight-natural-product-suppresses-il-1a-induced-il-6-expression-by-blocking-the-activation-of-p38-mapk-and-transcription-factors-cebpa-and-ap-1-in-primary-human-osteoarthri/