Celastrol Promotes Chondrocyte Autophagy by Regulating mTOR Expression

Celastrol Promotes Chondrocyte Autophagy by Regulating mTOR Expression

Osteoarthritis (OA) is a debilitating disease characterized by the progressive deterioration of cartilage, leading to pain, stiffness, and reduced mobility. Despite its prevalence, treatment options for OA remain limited, and there is a pressing need for novel therapeutic strategies. Autophagy, a cellular process that degrades and recycles damaged organelles and proteins, has emerged as a critical protective mechanism against cartilage degradation in OA. The mammalian target of rapamycin (mTOR) is a key regulator of autophagy, and its inhibition has been shown to promote autophagy and protect chondrocytes, the cells responsible for maintaining cartilage.

Celastrol, a triterpenoid quinine methide isolated from the root of Tripterygium wilfordii Hook F, has demonstrated promising therapeutic potential in various forms of arthritis, including OA. Previous studies have suggested that celastrol can promote autophagy in different cell types, but the specific mechanisms underlying its effects on OA chondrocytes remain unclear. This study aims to elucidate the role of celastrol in promoting autophagy in primary human OA chondrocytes and to explore its potential therapeutic effects.

The study began by determining the optimal dose of celastrol for chondrocyte cultures. Using the Cell Counting Kit-8 (CCK-8) assay, researchers identified 200 nmol/L as the optimal concentration of celastrol for a 24-hour treatment period. This dose was selected for subsequent experiments to ensure maximal efficacy while minimizing potential cytotoxicity.

Based on preliminary transcriptome sequencing and network pharmacology analysis, mTOR was identified as a potential direct target of celastrol in OA chondrocytes. To investigate this hypothesis, the researchers treated primary OA chondrocytes with celastrol and measured the protein levels of mTOR, P62, and LC3 using Western blotting. P62 is a marker of autophagy turnover, with its expression inversely related to autophagic activity. LC3, on the other hand, indicates autophagosome formation, and its conversion from LC3-I to LC3-II is a widely accepted marker of autophagy. The results showed that celastrol treatment significantly reduced mTOR levels, decreased P62 expression, and increased LC3-II levels, indicating that celastrol promotes autophagy in OA chondrocytes.

To further validate the role of mTOR in celastrol-induced autophagy, the researchers compared the effects of celastrol with rapamycin, a well-known mTOR inhibitor. Both celastrol and rapamycin treatment increased the levels of phosphorylated pancreatic ER kinase (PERK), a marker of endoplasmic reticulum (ER) stress activation, and LC3-II while decreasing P62 levels. Additionally, both treatments reduced the expression of caspase 12 (CASP12) and DNA damage-inducible transcript 3 (DDIT3), markers of proapoptotic effects. These findings suggest that celastrol, like rapamycin, promotes autophagy and inhibits chondrocyte apoptosis, potentially offering therapeutic benefits for OA.

Interestingly, the study also explored the synergistic effects of combining celastrol and rapamycin. The combined treatment resulted in a stronger suppression of mTOR and enhanced autophagy compared to either treatment alone. This suggests that celastrol and rapamycin may act through complementary mechanisms to promote autophagy in OA chondrocytes.

To further investigate the role of mTOR in celastrol-induced autophagy, the researchers used MHY1485, an mTOR agonist, in combination with celastrol. The results showed that MHY1485 reduced the inhibitory effect of celastrol on mTOR signaling and blocked the activation of PERK. Moreover, the combination of celastrol and MHY1485 increased the expression of CASP12 and DDIT3, indicating a proapoptotic effect, and increased P62 levels while decreasing LC3-II, suggesting reduced autophagy. These findings confirm that mTOR is a key intracellular target of celastrol and that the pharmacological effects of celastrol on autophagy are dependent on mTOR inhibition.

To further confirm the role of mTOR in celastrol-induced autophagy, the researchers used small interfering RNA (siRNA) to silence the mTOR gene in OA chondrocytes. Chondrocytes treated with celastrol after mTOR silencing showed significantly increased autophagy, as indicated by reduced P62 and increased LC3-II levels. However, there was no significant difference in the outcomes between celastrol treatment and mTOR silencing alone, indicating that mTOR is the primary target of celastrol in promoting autophagy in OA chondrocytes.

In conclusion, this study demonstrates that celastrol promotes autophagy in primary human OA chondrocytes by inhibiting mTOR. The reduction in mTOR levels leads to increased autophagic activity, which protects chondrocytes from apoptosis and may offer therapeutic benefits for OA. The study also highlights the potential synergistic effects of combining celastrol with rapamycin, suggesting that this combination could be a promising therapeutic strategy for OA. Conversely, the use of an mTOR agonist, such as MHY1485, can block the effects of celastrol on autophagy, further confirming the central role of mTOR in celastrol’s mechanism of action.

Future studies using an OA mouse model will be necessary to further validate the therapeutic potential of celastrol and to explore its underlying mechanisms in vivo. The findings of this study provide valuable insights into the role of autophagy in OA and suggest that targeting mTOR with celastrol or similar compounds could be a promising approach for the treatment of this debilitating disease.

doi.org/10.1097/CM9.0000000000001552

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