Influence of Medical Marijuana on Interleukin-1Β Treated Cartilage: An in Vitro Study

Category: Basic Sciences/Biologics Introduction/Purpose: Medical marijuana is reported to have potent analgesic, immunomodulatory and anti-inflammatory properties. Some recent studies have shown that cannabinoids may attenuate joint damage in animal models of arthritis. However, the underlying mechanism has not been completely elucidated. Interleukin-1β (IL-1β), a proinflammatory cytokine that can result in the degradation of cartilage, is known to be associated with the pathogenesis of osteoarthritis. We hypothesize that cannabinoids can mitigate the detrimental effect of IL-1β on cartilage, thus slowing the progression of osteoarthritis. We exposed human chondrocyte-derived engineered cartilage pellet cultures to IL-1 β for 48 hours or not and then treated the samples with a synthetic cannabinoid agonist, Win-55,212-2(Win). The tissue phenotypes were thereafter assessed by real time polymerase chain reaction (RT-PCR), histology and western blotting. Methods: Medical marijuana is reported to have potent analgesic, immunomodulatory and anti-inflammatory properties. Some recent studies have shown that cannabinoids may attenuate joint damage in animal models of arthritis. However, the underlying mechanism has not been completely elucidated. Interleukin-1β (IL-1β), a proinflammatory cytokine that can result in the degradation of cartilage, is known to be associated with the pathogenesis of osteoarthritis. We hypothesize that cannabinoids can mitigate the detrimental effect of IL-1β on cartilage, thus slowing the progression of osteoarthritis. We exposed human chondrocyte-derived engineered cartilage pellet cultures to IL-1 β for 48 hours or not and then treated the samples with a synthetic cannabinoid agonist, Win-55,212-2(Win). The tissue phenotypes were thereafter assessed by real time polymerase chain reaction (RT-PCR), histology and western blotting. Results: RT-PCR revealed persistent increase in the expression of inflammatory mediators IL-1beta, TNF-alpha and COL2 in IL1beta+ cultures over IL1beta- controls at all levels of WIN exposure. In contrast there was clear decrease in IL-6 expression at high WIN doses coincident with a downward trend the other mediators. WIN was unable to rescue IL-1beta-induced reductions in COL2 and ACN, while inducing ALP, a marker of osteogenesis and cartilage hypertrophy. Safranin O/Fast Green histology revealed progressive loss in GAG deposition with increasing doses of WIN (Figure 2). Western blots confirmed reductions in COL2 and COMP protein, persistent COX2 production, and increasing translation of the osteogenic transcription factor RUNX2 and p-SMAD1/5/8 (Figure 3). Conclusion: At doses below 1µM, Win does not impact IL-1beta-induced chondrocyte inflammation. while inhibiting chondrogenesis and stimulating osteogenesis through RUNX2 as compared to IL-1β controls. In proper context, BMP2 stimulates osteogenesis through SMAD1/5/8 and the induction of RUNX2. We did observe an apparent anti-inflammatory role of WIN at 1µM, but this dose did not rescue a loss in cartilage pellet GAG loss. Our data suggests that cannabinoids are anti-inflammatory, but are unable to rescue cartilage degeneration by IL-1β and promote an osteogenic phenotype. However, the data are not conclusive with regarding the beneficial or detrimental to arthritic bones and cartilage