基于AKT/mTOR通路探讨齐墩果酸调控自噬对IL-1β诱导软骨细胞损伤的保护作用

Protective Effect of Oleanolic Acid on IL-1β-Induced Chondrocyte Injury by Regulating Autophagy Based on AKT/mTOR Signaling Pathway

目的:探讨齐墩果酸对白细胞介素-1β(IL-1β)诱导软骨细胞损伤的保护作用及机制。方法:培养软骨细胞,利用甲苯胺蓝染色法及COLⅡ免疫荧光染色对软骨细胞进行纯度鉴定。将合格细胞随机分为空白对照组、模型对照组及齐墩果酸0.125、0.25、0.5μmol/L组。空白对照组用常规培养液培养;造模组用含10 ng/mL IL-1β常规培养液培养24 h建立损伤模型;齐墩果酸各浓度组以常规培养液预处理2 h后,再用IL-1β继续培养24 h。采用MTT法检测细胞存活率;流式细胞术检测细胞凋亡及活性氧(ROS)含量;Western blot法检测细胞中MMP-13、COLⅡ、LC3Ⅱ/Ⅰ、P62、p-AKT、AKT、p-mTOR、mTOR和β-actin的蛋白表达;免疫荧光法检测LC3在软骨细胞中的表达;加入自噬抑制剂3-甲基腺嘌呤(3-Methyladenine,3-MA)探究自噬在其中作用。结果:与空白对照组相比,模型对照组细胞存活率降低至50.95%,凋亡率、ROS水平、MMP-13、P62的表达及p-AKT/AKT和p-mTOR/mTOR的比值均显著增加(P<0.01),COLⅡ的表达及LC3 II/I的比值均显著降低(P<0.01);与模型对照组相比,齐墩果酸0.125、0.25、0.5μmol/L组存活率显著升高,凋亡率显著降低,ROS水平、MMP-13、P62的表达及p-AKT/AKT和p-mTOR/mTOR的比值均显著降低,COLⅡ表达及LC3 II/I的比值均显著升高(P<0.01),免疫荧光可见LC3荧光强度随着齐墩果酸浓度增加逐渐增强;与齐墩果酸组相比,3-MA+齐墩果酸组细胞存活率降低至47.03%,COL II表达与LC3Ⅱ/Ⅰ的比值均降低,MMP-13、P62的表达均上调(P<0.01)。结论:齐墩果酸对IL-1β引起的软骨细胞损伤具有保护作用,其作用机制与抑制AKT/mTOR信号通路及改善自噬有关。

Objective:To explore the protection of oleanolic acid(OLA)on chondrocyte injury induced by IL-1βand its mechanism.Methods:Chondrocytes were cultured and their purity was determined by toluidine blue staining and COLⅡimmunofluorescence staining.The qualified cells were randomly divided into normal control group,model control group and OLA 0.125,0.25 and 0.5μmol/L groups.The normal control group was cultured with conventional medium.The model control group was cultured in the medium containing 10 ng/mL IL-1βfor 24 h to establish the injury model.After OLA groups were pre-treated with OLA medium for 2 h,10 ng/mL IL-1βwas added for consecutive culture 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,COLⅡ,LC3Ⅱ/Ⅰ,P62,p-AKT,AKT,p-mTOR,mTOR andβ-actin in each group were determined by Western blot.The expression of LC3 in chondrocytes was determined by immunofluorescence.In addition,autophagy inhibitor 3-methyladenine(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 COLⅡexpression and the LC3Ⅱ/I ratio were decreased(P<0.01).Compared with the model control group,the OLA 0.125,0.25 and 0.5μmol/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 COLⅡexpression and the LC3Ⅱ/I ratio were elevated(P<0.01).The fluorescence 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 COLⅡexpression and LC3Ⅱ/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.