Effect and mechanism of novel mTOR inhibitor cedrelone for osteoarthritis treatment

Abstract:

Objective：To investigate the effect and mechanism of cedrelone for osteoarthritis treatment. Methods：C28/I2 cells were used to establish the cell model. MTT was used to detect the toxicity of cedrelone. Western blot and immunofluorescence were used to detect the effect of cedrelone on mTOR inhibition and autophagy activation. The right knees of C57BL/6J mice（n=40） were used to establish the osteoarthritis model，and the left knees of the same mice were used as the control；the mice were given cedrelone（n=20） and normal saline（n=20） treatment through intraperitoneal injection. The effects of cedrelone on mTOR inhibition and synovial in－flammation，activation of autophagy and protection of cartilage were detected through Western blot，safranin O stain，immunohisto－chemistry，immunofluorescence，semi-quantitative scoring sys－tem and inflammation grading system. Results：①For the C28/I2 cell，cedrelone could inhibit the mTOR phosphorylation in the site of Ser-2448，and suppress the phosphorylation of ribo－somal protein S6（rpS6） in the site of Thr-389 and Ser-371. The optimum dose was 40 μmol/L（Ser-2448：t=13.42，P=0.000；Thr-389：t=19.06，P=0.000；Ser-371：t=9.52，P=0.000），and the best time was 24 h（Ser-2448：t=34.82，P=0.000；Thr-389：t=3.14，P=0.007；Ser-371：t=19.32，P=0.000）. ②The value of LC3II/LC3I（t=8.15，P=0.002） and the number of autophagosome positive cells（t=9.71，P=0.000） were significantly increased by cedrelone treatment in C28/I2 cells. ③In the osteoarthritis model，cedrelone could inhibit the phosphorylation level of mTOR（t=19.03，P=0.005） and the number of rpS6 positive cells（t=27.97，P=0.000），as well as enhance the level of LC3II/LC3I（t=24.65，P=0.000） and the number of LC3 positive cells（t=33.89，P=0.000）. ④Cedrelone also could reduce the degradation of extracellular matrix through ADAMTS-5 in－hibition（F=1.82，P=0.002），and then reduce the severity of osteoarthritis（t=8.32，P=0.009），increase the number of chondrocytes（F=3.59，P=0.013）. ⑤In addition，in the synovial tissue，synovial inflammation was suppressed（F=5.44，P=0.029） by cedrelone through IL-1β inhibition（F=3.20，P=0.000）. Conclusion：Cedrelone acts as a potential mTOR inhibitor，not only reducing the severity of osteoarthritis and protection chondrocytes through autophagy activation，but also inhibiting the degradation of extracellular matrix and synovial inflammation by ADAMTS-5 and IL-1β inhibition in mice，however，the protective effect and mechanism of cedrelone for cartilage injury in osteoarthritis need to be further studied.