Celastrol suppresses neovascularization in rat aortic vascular endothelial cells stimulated by inflammatory tenocytes via modulating the NLRP3 pathway

Shoulder rotator cuff injuries are prevalent, causing significant pain and mobility impairment. Current treatments include surgical and non-surgical methods, but surgical repair has a high re-tear rate. Tendon-bone healing is complex, involving stem cells, biological factors, immune-inflammatory responses, and vascular regeneration. Inflammation is strongly linked to tendinopathy, with elevated pro-inflammatory mediators. Uncontrolled angiogenesis exacerbates scar formation and impairs healing. Vascular endothelial growth factor A (VEGFA) is a key regulator of angiogenesis, upregulated by inflammatory mediators like TNF-α and IL-6. Celastrol, from *Tripterygium wilfordii*, has anti-inflammatory and immunosuppressive properties, used in treating various inflammatory conditions. This study aimed to investigate the role of celastrol in regulating angiogenesis in a rotator cuff tear model, using an in vitro model of LPS-induced tenocyte inflammation and its effect on RAOEC angiogenesis, and an in vivo rat rotator cuff tear (RCT) model to assess its impact on tendon-bone healing.

The literature extensively documents the high incidence and significant impact of rotator cuff injuries, highlighting the limitations of current treatment strategies. The role of inflammation in tendinopathy is well-established, with studies demonstrating elevated levels of pro-inflammatory cytokines. The importance of angiogenesis in tendon healing is acknowledged, but the potential negative effects of excessive angiogenesis, leading to scar formation and impaired healing, have also been described. VEGF's role in angiogenesis and its regulation by inflammatory mediators are well-characterized. Celastrol's anti-inflammatory and immunosuppressive effects have been reported in various studies, but its role in tendon healing and angiogenesis required further investigation. This study builds on the existing literature by investigating the interplay between inflammation, angiogenesis, and celastrol's therapeutic effect in a rotator cuff tear model.

This study used both in vitro and in vivo models. In vitro, primary rat tenocytes were isolated and treated with LPS (0.5 or 1 µg/mL) for 6 or 12 h to induce inflammation. The mRNA and protein expression of NLRP3, TNF-α, IL-1β, and VEGFA were assessed using RT-qPCR and Western blotting. VEGFA secretion was measured by ELISA. Conditioned medium from LPS-treated tenocytes was then used to treat RAOECs in a tube formation assay to assess angiogenesis. The effect of celastrol on LPS-induced inflammation and angiogenesis was also evaluated in vitro. In vivo, a rat RCT model was created. Rats were divided into three groups: sham, RCT, and RCT + celastrol (1 mg/kg intra-articularly per week). After 4 and 8 weeks, biomechanical testing (ultimate load and stiffness) was performed on harvested tendons. mRNA expression of VEGFA and NLRP3 was analyzed by RT-qPCR. Immunocytochemistry was used to visualize NLRP3 expression in tenocytes. Statistical analysis was performed using Student's t-test and one-way ANOVA.

In vitro, LPS significantly upregulated the mRNA and protein expression of NLRP3, TNF-α, IL-1β, and VEGFA in tenocytes, leading to increased VEGFA secretion. Conditioned medium from LPS-treated tenocytes promoted angiogenesis in RAOECs. Celastrol significantly suppressed LPS-induced upregulation of NLRP3 and IL-1β, reduced VEGFA secretion, and inhibited angiogenesis. In vivo, celastrol improved biomechanical properties (ultimate load and stiffness) of tendons in RCT rats after 4 and 8 weeks. Celastrol also significantly downregulated the mRNA expression of VEGFA and NLRP3 in RCT rat tendons. These findings consistently demonstrate that celastrol effectively suppresses inflammation and angiogenesis, promoting tendon-bone healing.

This study demonstrates that celastrol effectively targets the NLRP3 pathway to suppress inflammation-induced angiogenesis, promoting tendon-bone healing in a rat RCT model. The in vitro findings show a direct link between LPS-induced tenocyte inflammation and subsequent RAOEC angiogenesis. Celastrol's ability to counteract this process highlights its potential as a therapeutic agent. The in vivo results confirm the beneficial effects of celastrol on tendon biomechanics and gene expression, further supporting its therapeutic role. These findings align with previous research highlighting the detrimental effects of excessive angiogenesis on tendon healing, indicating that controlling angiogenesis is crucial for improved outcomes. Celastrol's action on the NLRP3 pathway provides a mechanistic explanation for its anti-angiogenic and pro-healing effects.

This study demonstrates celastrol's therapeutic potential in treating rotator cuff tears by suppressing inflammation-induced angiogenesis through modulation of the NLRP3 pathway. Both in vitro and in vivo data support this conclusion. Future research could investigate the detailed molecular mechanisms of celastrol's action on the NLRP3/IL-1β signaling pathway and explore strategies to improve celastrol's bioavailability and reduce its potential side effects for improved clinical applications. Further studies could also explore the broader therapeutic potential of celastrol in other tendon injuries.

This study has some limitations. The detailed molecular mechanisms underlying NLRP3/IL-1β signaling beyond the observed effects on collagen production, ECM disorganization, and cytoskeleton dynamics were not fully investigated. The study did not examine all inflammatory response factors or secreted compounds beyond VEGFA. Celastrol's limitations, including poor water solubility and low bioavailability, need to be addressed for clinical translation. The long-term effects of celastrol's immunosuppressive properties also warrant further investigation.