Oleanolic Acid Plays A Protective Role In IL-1β-induced Chondrocyte Injury By Regulating AKT/mTOR Pathway To Activate Autophagy

ObjectiveTo explore the protection of oleanolic acid(OLA)on the injury of cho ndrocyte induced by IL-1β and its mechanism.MethodsCondylar cartilage of temporomandibular joint(TMJ)was collected from SPF male SD rats(200±10g)and digested by type IV collagenase to isolate primary chondrocytes.The purity of the primary chondrocyte cells was identified by toluidine blue staining and type II collagen immunofluorescence staining.The eligible cells were randomly divided into control group,model group and another three groups that treated by low,medium and high dose of OLA.The control group was cultured in DMEM-F12 complete medium for 24 hours;model group was induced by DMEM-F12 complete medium with 10 ng/ml IL-1β for 24 h.OLA low,medium and high dose groups were pretreated with medium containing 0.13,0.25 and 0.50 μM of OLA respectively for 2 hrs,and then cultured with IL-1β at a final concentration of 10 ng/ml for 24 h.Cell viability was measured by MTT assay.Cell apoptosis and reactive oxygen species(ROS)were detected by flow cytometry.Western Blot was used to detect the contents of MMP-13,COLII,p-AKT,AKT,p-mTOR,mTOR,LC3II/I and P62 in cells of all these groups.The expression of LC3 B in chondrocytes was detected by immunofluorescence assay.In addition,the role of autophagy was explored by adding an autophagy inhibitor,3-methyladenine(3-MA).ResultsThe purity of cultured chondrocytes was more than 98%,which met the criteria of the experiment.Compared with the control group,the survival rate of chondrocytes induced by 10 ng/ml IL-1β for 24 h was significantly decreased(P<0.01),and the expression of MMP-13,P62 and the ratio of p-AKT/AKT and p-mTOR/mTOR were significantly increased(P<0.01).The expression of COLII and the ratio of LC3II/I were significantly decreased(P<0.01).Compared with the model group,the survival rate of chondrocytes that treated by different dose of OLA was significantly increased(P<0.01),while the apoptosis rate and the level of ROS in all these OLA treated cells were significantly decreased(P<0.01).The expression of MMP-13,P62 and the ratio of p-AKT/AKT and p-mTOR/mTOR in OLA treated cells were significantly decreased(P<0.01),and the ratio of COLII and LC3II/I were significantly increased(P<0.01).Immunofluorescence showed that the fluorescence intensity of LC3 B gradually increased with the increase of OLA concentration.Compared with the OLA group,the 3-MA+OLA group had lower cell survival rate(P<0.01),higher expression of MMP-13 and P62(P<0.01),and lower expression of COLII and LC3 II/I(P<0.01).ConclusionsThe results showed that IL-1β can inhibit the autophagy-induced apoptosis in chondrocyte through stimulating ROS production and activating AKT/mTOR signaling pathway.OLA can activate autophagy by inhibiting the generation of ROS and the activating of AKT/mTOR signaling pathway.