Lupeol Alleviates Chondrocytes Senescence in Osteoarthritis by Regulating Autophagy via the Sirtuin 3/Mechanistic Target of Rapamycin Kinase Pathway
- VernacularTitle:羽扇豆醇通过SIRT3/mTOR通路调节自噬以改善骨关节炎软骨细胞衰老
- Author:
Yunfeng MA
1
;
Yujing CAO
;
Xiaofei HAN
Author Information
- Keywords: Lupeol; SIRT3/mTOR pathway; Autophagy; Senescence; Chondrocytes
- From: Journal of Sichuan University (Medical Sciences) 2025;56(1):83-93
- CountryChina
- Language:Chinese
- Abstract: Objective To investigate the role of lupeol in mitigating chondrocyte senescence in osteoarthritis(OA)by regulating autophagy through the sirtuin 3(SIRT3)/mechanistic target of rapamycin kinase(mTOR)pathway.Methods Knee articular chondrocytes from primary-generation mice were isolated and divided into different groups,including a control group,a lupeol group(given 2.5,5,10,20,and 40 μmol/L lupeol),a tert-butyl hydrogen peroxide(TBHP)group(receiving 50 μmol/L TBHP),TBHP+lupeol group,TBHP+lupeol+chloroquine(CQ)group(receiving 20 μmol/L CQ,an autophagy inhibitor),TBHP+lupeol+si-NC group,and TBHP+lupeol+si-SIRT3 group.Cell proliferation,reactive oxygen species(ROS)levels,and apoptosis were determined by CCK-8,DCFH-DA probe,and flow cytometry.Cell senescence was evaluated by β-gal staining.Western blot was used to determine the expressions of SIRT3,mTOR,senescence marker proteins(p21 and p16),extracellular matrix(ECM)degradation-related proteins(aggrecan,collagen Ⅱ,ADAMTS5,and MMP13),and autophagy-related proteins(LC3B Ⅰ,LC3BⅡ,and P62).RT-qPCR was used to determine the mRNA levels of senescence-associated secretory phenotypes(SASP),including IL-6,Cxcl10,MCP1,and MMP3.The expression of LC3 was detected by immunofluorescence.Autophagosomes were observed by transmission electron microscopy.A total of 30 male wild-type C57BL/6 mice were divided into different groups(n=10),including a Sham group,an OA group,and an OA+lupeol group receiving 50 mg/(kg·d)lupeol via gastric gavage.Cartilage damage was evaluated by safranin O-fast green staining.Results Based on the results of cell viability assay,20 μmol/L lupeol treatment for 24 h was identified as the optimal intervention concentration and duration.Compared with that in the TBHP group,cell viability was elevated in the TBHP+lupeol group(P＜0.05);ROS production,the proportion of β-gal-positive cells,the protein expression levels of p21 and p16,and the mRNA levels of SASP were decreased(P＜0.05);the protein levels of aggrecan and collagen Ⅱ were elevated and the protein levels of ADAMTS5 and MMP13 were decreased(P＜0.05);apoptosis was reduced(P＜0.05);P62 protein levels were reduced and the LC3B Ⅱ/LC3B Ⅰ ratio,the intensity of LC3B fluorescence spots,and the number of autophagosomes were increased(P＜0.05);the expression level of SIRT3 was elevated and the level of mTOR phosphorylation was reduced(P＜0.05)in the TBHP+Lupeol group.CQ treatment effectively abolished the promotion effects of lupeol on cell viability and autophagy,and the inhibitory effects of lupeol on ROS level,cell senescence,ECM degradation,and apoptosis(P＜0.05).Silencing of SIRT3 reversed the inhibitory effect of lupeol on mTOR phosphorylation level and the promotion effect of lupeol on autophagy(P＜0.05).In the in vivo experiment,compared with the OA group,the OA+lupeol group showed reduced cartilage degeneration and lower scores for the Osteoarthritis Research Society International grading system(P＜0.05).The OA+lupeol group also showed up-regulated SIRT3 expression,reduced mTOR phosphorylation level,increased LC3B Ⅱ/LC3B Ⅰ ratio,reduced MMP13 protein level,and reduced mRNA level of SASP(P＜0.05).Conclusion Lupeol alleviates chondrocyte senescence in osteoarthritis by regulating autophagy through the SIRT3/mTOR pathway.
