Influence of Cannabinoid on Interleukin-1β Treated Cartilage: An In Vitro Study

Abstract

Category:Basic Sciences/BiologicsIntroduction/Purpose:Cannabinoids have been reported to possess the analgesic, immunomodulatory and anti-inflammatory properties. Recent studied further shown that cannabinoids attenuated joint damage in animal models of arthritis. However, the underlying mechanism has been completely understood. Interleukin-1β (IL-1β), a proinflammatory cytokine that can result in the degradation of cartilage, is associated with the pathogenesis of osteoarthritis. In this study, we hypothesize that cannabinoid can mitigate the detrimental effect of IL-1β on cartilage, thus reduce the progression of osteoarthritis. To test the hypothesis, we insulted human chondrocyte-derived cartilage with IL-1 β for 48 hours and then applied a synthetic cannabinoid agonist, Win- 55,212-2(Win-55), into the culture. The tissue phenotypes were assessed by real time polymerase chain reaction (PCR), histology and immunostaining.Methods:With the approval from CORID, human chondrocytes were isolated from healthy articular cartilage. P2 cells were used. MTS assay were employed to test the half-maximal (50%) inhibitory concentration (IC50). To generate cartilage in vitro, chondrocytes were pelleted and subjected to 14 days chondrogenic culture. The engineered cartilages were stimulated with 10 ng/ml IL-1β for 48 hours and then treated with different concentration of Win-55 (0.01, 0.1, or 1 µM) for another 48 hours. The tissue phenotype was assessed by glycosaminoglycan (GAG) assay, real-time PCR and histology.Results:We tested 10 doses, from 0.001µM up to 10 µM, and determined that the IC 50 of Win-55 on human chondrocytes for 2 days was ˜ 2 µM. Interestingly, this dose is significantly lower than the doses reported in similar studies. As shown in Figure 1, treatment with 2µM Win-55 causes the complete loss of GAG from engineer cartilage. In a relatively safe dose (<=1 µM), we did not observe obvious changes in all tested genes after the treatments of Win-55 (Figure 2).Conclusion:High dose of Win-55 may directly cause the degeneration of cartilage, while low dose of Win-55 doesn’t show beneficial influence on the phenotype of IL1-β-insulted cartilage. The reported anti-inflammatory effect of Win 55 on chondrocytes may due to the cytotoxicity or global inhibition of high dose Win 55 on cell activities. Therefore, if cannabinoid can be used to treat OA requires further investigation.