Rotator cuff injuries (RCIs) are prevalent shoulder injuries causing significant pain and mobility impairment, impacting millions annually and leading to substantial economic burdens. Current treatments, including surgical and non-surgical approaches, have limitations, such as high re-tear rates after surgery. Tendon-bone healing is complex, involving stem cells, biological factors, immune-inflammatory responses, and vascular regeneration. Inflammation plays a significant role in tendinopathy, with increased levels of pro-inflammatory mediators. Angiogenesis, while essential for healing, can be detrimental if uncontrolled, exacerbating scar formation and delaying healing. Vascular endothelial growth factor A (VEGFA) is a key regulator of angiogenesis, and inflammatory mediators such as TNF-α and IL-6 can upregulate VEGFA. Celastrol, from *Tripterygium wilfordii*, possesses anti-inflammatory and immunosuppressive properties, showing promise in treating various inflammatory conditions. This study aimed to investigate the role of celastrol in regulating inflammation-induced angiogenesis during RCI using in vitro and in vivo models.

The literature extensively documents the relationship between inflammation and tendinopathy. Studies have shown elevated levels of pro-inflammatory mediators like PGE2, IL-1, and IL-6 in injured tendons compared to healthy tissues. The activation of signaling pathways such as IFN, NF-κB, and STAT-6 is observed in early stages of tendon injury, while M2 microglia and STAT-6 pathway activation is seen in later stages. Inflammation significantly influences angiogenesis, and uncontrolled angiogenesis is linked to various diseases. VEGFA is a crucial proangiogenic factor regulated by inflammatory mediators, highlighting the link between inflammation and angiogenesis in tendon healing. Celastrol's anti-inflammatory effects have been demonstrated across various diseases, including rheumatoid arthritis, systemic lupus erythematosus, and certain cancers. However, its role in regulating RCI remains less explored.

This study used Sprague-Dawley rats. Primary rat tenocytes were isolated and cultured, treated with lipopolysaccharide (LPS) to induce inflammation. Conditioned medium from these LPS-treated tenocytes was then applied to rat aortic vascular endothelial cells (RAOECs). mRNA and protein levels of NLRP3, TNFα, IL-1β, and VEGFA were assessed using RT-qPCR and Western blotting. VEGFA secretion was measured by ELISA. A tube formation assay evaluated angiogenesis in RAOECs. Celastrol was administered to LPS-treated tenocytes to assess its effects on inflammation and angiogenesis. A rotator cuff tear (RCT) rat model was created, and rats were injected intra-articularly with celastrol. Biomechanical testing (ultimate load to failure and stiffness) was performed at 4 and 8 weeks post-RCT. Tendon samples were harvested for RT-qPCR analysis of VEGFA and NLRP3. Immunocytochemistry was used to visualize NLRP3 expression in tenocytes. Statistical analysis used Student's t-test and one-way ANOVA. All animal procedures adhered to ethical guidelines.

LPS treatment significantly upregulated the mRNA and protein levels of NLRP3, TNFα, IL-1β, and VEGFA in tenocytes in a time- and dose-dependent manner, with increased VEGFA secretion. Conditioned medium from LPS-treated tenocytes significantly induced angiogenesis in RAOECs. Celastrol significantly suppressed LPS-induced upregulation of NLRP3 and IL-1β mRNA and protein levels in tenocytes and reduced VEGFA secretion. Treatment with celastrol significantly inhibited RAOEC angiogenesis stimulated by conditioned medium from LPS-treated tenocytes. In the RCT rat model, celastrol administration significantly improved the ultimate load to failure and stiffness of the repaired tendons at both 4 and 8 weeks post-injury. Celastrol also significantly suppressed the upregulation of VEGFA and NLRP3 mRNA levels in RCT rat tendons.

This study demonstrates that inflammation in tenocytes, induced by LPS, leads to increased VEGFA production and subsequent angiogenesis in RAOECs. Celastrol effectively counteracts this process by suppressing the NLRP3 inflammatory pathway, thereby reducing VEGFA levels and angiogenesis. The in vivo results further support these findings, showing that celastrol promotes tendon-bone healing and improves tendon biomechanical properties in an RCT model by modulating VEGFA and NLRP3 expression. These results align with previous studies highlighting the importance of controlling angiogenesis in tendon healing. While promoting angiogenesis is beneficial in the early stages, excessive angiogenesis can hinder long-term healing outcomes.

This study establishes celastrol's role in suppressing inflammation-induced angiogenesis in RCI through modulation of the NLRP3 pathway, both in vitro and in vivo. Celastrol's ability to improve tendon healing and functional recovery in an RCT rat model highlights its potential therapeutic application for RCIs. Future studies should explore the detailed molecular mechanisms of the NLRP3/IL-1β signaling pathways involved, investigate a wider range of inflammatory factors, and address celastrol's limitations regarding bioavailability and potential side effects to optimize its clinical use.

This study has several limitations. First, the detailed molecular mechanisms underlying the NLRP3/IL-1β signaling pathways remain to be fully elucidated. Further investigation is needed to identify other signaling pathways involved. Second, the study did not analyze all inflammatory factors (pro- and anti-inflammatory cytokines) and secreted compounds other than VEGFA. Third, Celastrol's poor water solubility, low bioavailability, and narrow therapeutic index pose challenges to clinical translation. Further optimization strategies are required to mitigate side effects.