Ipriflavone Prevents The Degeneration Of Cultured Human Osteoarthritis Chondrocytes And Cartilage Explants Through Blocking Indian Hedgehog Pathway

Objective:(1). To reseach whether indian hedgehog pathway inhibitor-ipriflavone(IP)can attenuate human osteoarthritis(OA) chondrocytes hypertrophy and cartilage explants matrix degeneration in vitro;(2). To reseach the influence of IP on the proliferation and apoptosis of human OA chondrocytes;(3). To reseach the effects of IP on the biomechanical properties of human OA chondrocytes in vitro;Methods: The human OA chondrocytes and cartilage explants, obtained from subjects with OA undergoing total knee arthroplasty, were cultured in the presence of DMSO, IP 5μM(50ug/ml IP for OA cartilage explants),IP 10μM(100ug/ml IP for OA cartilage explants). SMO, Gli-1, Gli-2, Gli-3, Runx-2, MMP-13, COL II, Aggrecan m RNA were determined by real-time q PCR after 48 h of incubation. Protein expression of COL II, MMP-13, COL X were analyzed by Western blotting. CCK-8, Ed U staining, FCM(flow cytometry) were used to check the influence of IP on the proliferation and apoptosis of the chondrocytes. Micropipette aspiration combined with a standard linear viscoelastic solid model was used to quantify changes in the viscoelastic properties of OA chondrocytes after 48 h cultured in the presence of DMSO, IP 5μM,IP 10μM. The proteoglycan and COL II, Runx-2, COL X changes of IP on the OA cartilage explants were measured by Safranin-O/Fast Green staining and immunofluorescence histochemistry, respectively.Results: Treatment of OA chondrocytes with IP(both 5μM and 10μM) blocked the indian hedgehog pathway, with less SMO, Gli-1, Gli-2, Gli-3(P<0.05) m RNA levels. It also decresed the expression of Runx-2, MMP-13 m RNA(P<0.01), and increased COL II, Aggrecan m RNA(P<0.05) in the chondrocytes. The protein expression of COL II production was increased 100%, and MMP-13, COL X were decreased 75% and 87% respectively in IP 5μM-treated chondrocytes, compared to the DMSO treatment. IP also increased the proliferation of OA chondrocytes, decresed the apoptosis after 3 days cultured in the presence of IP 5μM or IP 10μM. In response to an applied constant 0.7-0.8 k Pa of negative pressure, all chondrocytes exhibited standard linear viscoelastic solid properties. The viscoelastic properties-the average equilibrium modulus(E∞), instantaneous modulus(E0), and the apparent viscosity(μ) of OA chondrocytes after 48 h of cultured in the presence of IP 5μM, IP 10μM were significantly lower than that observed in DMSO treatment chondrocytes respectively(P<0.001). Safranin-O/Fast Green staining indicated that more PG staining and less cartilage damage was found in the OA cartilage explants treated with IP compared with DMSO group. Mankin scores of histology quantity changes from DMSO, 50ug/ml IP and 100ug/ml IP were 8.34 ± 0.58,4.67 ± 0.57(P<0.01), 2.34 ± 0.28(P<0.001) respectively. Immunofluorescence histochemistry indicated more staining of COL II, less production of Runx-2 and COL X in IP-treated OA cartilage explants.Conclusion: Our data show that ipriflavone, by blocking indian hedgehog pathway, decreaed gene and protien expression consistent with chondrocyte hypertrophy and cartilage matrix degeneration seen in OA chondrocytes and cartilage explants in vitro. Ipriflavone is also able to incresed the proliferation of cultured human OA chondrocytes, decreased chondrocytes apoptosis, and improved the viscoelastic properties of human OA chondrocytes. All data suggested that ipriflavone may be a possible way to delay or reduce the severity of OA development.