Abstract
Osteoarthritis (OA) is a chronic joint disease characterized by cholesterol accumulation in chondrocytes, cartilage degeneration, as well as extracellular matrix (ECM) destruction, and joint dysfunction. Curcumin, a chemical that can reduce cholesterol levels in OA patients, also can inhibit the progression of OA. However, a high concentration of curcumin may also trigger apoptosis in normal chondrocytes. Besides curcumin, probucol that is found can also effectively decrease the cholesterol level in OA patients. Considering that high cholesterol is a risk factor of OA, it is speculated that the combination treatment of curcumin and probucol may be effective in the prevention of OA. To investigate the possible effects of such two chemicals on OA pathophysiology, chondrocyte apoptosis and autophagy behavior under inflammatory cytokine stress were studied, and specifically, the PI3K-Akt-mTOR signaling pathway was studied. Methods. Cell proliferation, colony formation, and EdU assay were performed to identify the cytotoxicity of curcumin and probucol on chondrocytes. Transwell assay was conducted to evaluate chondrocyte migration under TNF-α inflammation stress. Immunofluorescence, JC-1, flow cytometry, RT-PCR, and western blot were used to investigate the signal variations related to autophagy and apoptosis in chondrocytes and cartilage. A histological study was carried out on OA cartilage. Glycosaminoglycan (GAG) release was determined to evaluate the ECM degradation under stress. Results. Compared with a single intervention with curcumin or probucol, a combined treatment of these two chemicals is more effective in terms of protecting chondrocytes from stress injury induced by inflammatory cytokines. The promoted protection may be attributed to the inhibition of apoptosis and the blockage of the autophagy-related PI3K/Akt/mTOR pathway. Such results were also verified in vitro by immunofluorescence staining of OA chondrocytes and in vivo by immunohistochemistry staining of cartilage. Besides, in vivo studies also showed that when applied in combination, curcumin and probucol could block the PI3K-AKT-mTOR signaling pathway; promote COL-II expression; suppress P62, MMP-3, and MMP-13 expression; and inhibit TNF-α-stimulated cartilage degradation. Moreover, the combined medication could help reduce the release of ECM GAGs in OA cartilage and alleviate the severity of OA. Conclusion. A combined treatment of curcumin and probucol could be used to protect chondrocytes from inflammatory cytokine stress via inhibition of the autophagy-related PI3K/Akt/mTOR pathway both in vitro and in vivo, which might be of potential pharmaceutical value for OA prevention and therapy.
Highlights
OA is a chronic inflammatory disease closely related to cartilage degeneration
Considering the possible reported side effects of such substances [19], a combination of curcumin 25 μM + probucol 50 μM was used in this study, and the results suggest that such a combination can promote chondrocyte proliferation (Figures 1(c))
Colony formation assays further confirmed that they play a promotive role in chondrocyte proliferation (Figures 1(d) and 1(e)), and such effect is in a synergistic way by the two chemicals
Summary
OA is a chronic inflammatory disease closely related to cartilage degeneration. Researchers have found that a high level of total cholesterol is related to the OA process. In a prospective cohort study, total cholesterol and triglycerides are verified to be associated with new bone marrow lesion formation in asymptomatic middle-aged women [1] and result in cartilage defect and OA eventually Another possible explanation could be lipid embolism caused by serum cholesterol, which may cause osteonecrosis leading to OA. The interrelationship between high cholesterol levels and increased risk of OA has been studied extensively in recent years [3, 4], and previous reports have shown that inhibition of de novo cholesterol synthesis may provide better OA remiment outcome [5, 6] In this context, OA should be considered as a syndrome rather than merely a joint disease
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