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 â ¡, ADAMTS5, and MMP13), and autophagy-related proteins (LC3Bâ , LC3Bâ ¡, 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 â ¡ 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 LC3Bâ ¡/LC3Bâ 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 LC3Bâ ¡/LC3Bâ 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.