[Indirubin relieves inflammatory injury of chondrocytes in a mouse model of osteoarthritis].

Objective: To investigate the effect of indirubin for relieving joint inflammation and injury in a rat model of osteoarthritis.
Methods: Articular cartilage chondrocytes were isolated from adult rat knee joint and cultured in the presence of interleukin-1β (IL-1β) and 0.1, 0.5, 1.0, or 2.0 μmol/L indirubin. The cells were transfected with NPAS2 siRNA or a non-specific siRNA, and the cell proliferation and apoptosis were evaluated using tetramethylthiazole blue staining and flow cytometry. The protein expression levels of Bax, Bcl-2, ACAN, COL2A1, MMP-13 and NPAS2 were detected with Western blotting, and the levels of NO, PGE2 and TNF-α in the culture supernatant were determined with ELISA. The mRNA expression levels of NPAS2, ACAN, COL2A1 and MMP-13 were detected using fluorescence quantitative PCR. In a C57BL/6 mouse model of osteoarthritis, the effect of indirubin on BAX, Bcl-2, ACAN and MMP-13 protein expressions in the bone and joint tissues were evaluated with Western blotting.
Results: Treatment with 0.1 μmol/L indirubin produced no significant changes in chondrocyte proliferation, apoptosis, caspase-3 activity, or BAX and Bcl-2 protein expressions. At higher doses (0.5, 1.0 and 2.0 μmol/L), indirubin significantly promoted cell proliferation, increased Bcl-2 protein expression, and lowered cell apoptosis rate, caspase-3 activity and Bax protein expression ( P < 0.05). Indirubin treatment at 0.5 μmol/L up-regulated the protein and mRNA expressions of NPAS2, ACAN and COL2A1, and down-regulated the expressions of MMP-13, NO, PGE2 and TNF-α ( P < 0.05). Interference of NPAS2 expression significantly attenuated the protective effect of 0.5 μmol/L indirubin against IL-1β-induced chondrocyte injury. The mouse model of osteoarthritis showed obviously increased protein levels of BAX and MMP-13 ( P < 0.01) and decreased levels of Bcl-2 ( P < 0.05) and ACAN ( P < 0.01) in the knee joint, and indirubin treatment of the mouse models significantly inhibited the increase of BAX and MMP-13 protein expressions ( P < 0.01) and up-regulated the protein expressions of Bcl-2 and ACAN ( P < 0.05).
Conclusion: Indirubin has a protective effect on osteoarthritis tissue and alleviates inflammation and damage of osteoarthritis chondrocytes possibly through NPAS2.