Protective Effect of Oleanolic Acid on IL-1β-Induced Chondrocyte Injury by Regulating Autophagy Based on AKT/mTOR Signaling Pathway
Objective:To explore the protection of oleanolic acid(OLA)on chondrocyte injury induced by IL-1β and its mechanism.Methods:Chondrocytes were cultured and their purity was determined by toluidine blue staining and COL Ⅱ immunofluorescence staining.The qualified cells were randomly divided into normal control group,model control group and OLA 0.125,0.25 and 0.5 μmol/L groups.The normal con-trol group was cultured with conventional medium.The model control group was cultured in the medium containing 10 ng/mL IL-1β for 24 h to establish the injury model.After OLA groups were pre-treated with OLA medium for 2 h,10 ng/mL IL-1β was added for consecutive cul-ture for 24 h.The cell viability was detected by MTT assay.Flow cytometry was used to detect apoptosis and reactive oxygen species(ROS).The protein expressions of MMP-13,COL Ⅱ,LC3 Ⅱ/Ⅰ,P62,p-AKT,AKT,p-mTOR,mTOR and β-actin in each group were determined by Western blot.The expression of LC3 in chondrocytes was determined by immunofluorescence.In addition,autophagy inhibitor 3-methylade-nine(3-MA)was added to explore the role of autophagy.Results:Compared with the conditions of normal control group,the cell viability of the model control group was reduced to 50.95％,and the apoptosis,ROS,MMP-13 and P62 as well as the ratio of p-AKT/AKT and p-mTOR/mTOR were increased(P＜0.01),while the COL Ⅱ expression and the LC3 Ⅱ/Ⅰ ratio were decreased(P＜0.01).Compared with the model control group,the OLA 0.125,0.25 and 0.5 μmol/L groups had increased cell viability by 26.03％,34.70％and 44.37％,respectively,while decreased apoptosis rate by 3.55％,9.94％and 10.99％,respectively.Additionally,the ROS,MMP-13 and P62 as well as the ratio of p-AKT/AKT and p-mTOR/mTOR were lowered,while the COL Ⅱ expression and the LC3 Ⅱ/Ⅰ ratio were elevated(P＜0.01).The fluores-cence intensity of LC3 was gradually enhanced with the increase of OLA concentration.Compared with the OLA groups,the 3-MA+OLA group had decreased cell viability to 47.03％,down-regulated COL Ⅱ expression and LC3 Ⅱ/Ⅰ ratio,while up-regulated MMP-13 and P62(P＜0.01).Conclusion:OLA has protective effect on chondrocyte injury induced by IL-1β,and its mechanism may be related to inhibiting AKT/mTOR signaling pathway and improving autophagy.
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