According to the methods described in Ref. 13, cells or explants were serum-starved overnight prior to stimulation. Chondrocyte cultures were supplemented with physiological concentrations estrogen (synthetic; 0.1nM; Sigma-Aldrich, shanghai, China), and with or without estrogen receptor inhibitors-ERαinhibitor MPP25 (link) (20uM; Sigma-Aldrich, shanghai, China) and ERβ inhibitor PHTPP26 (link),27 (20uM; Sigma-Aldrich, shanghai, China). Later, recombinant human TGF-β1 (10 ng/mL; Peprotech, shanghai, China) and IL-1β (1 ng/mL; Peprotech, shanghai, China) were added into the culture medium, respectively. Then, E2 (1 nM) was added into the training systems including TGF-β1 or IL-1β. DMSO (Sigma-Aldrich, shanghai, China) was used as vehicle control in the experiments. And chondrocytes were pre-incubated with inhibitors for 0.5 h prior to the addition of E2. estrogen was added 30 min prior to TGF-β1 and IL-1β. Cultures were maintained for 24 h, at which point medium was harvested for ELISA, chondrocytes were harvested for qPCR or WB.