Bone marrow mesenchymal stem cell-derived TSG-6 regulates microglial pyroptosis and alleviates bone cancer pain by inhibiting the NLRP3 inflammasome.

Abstract

Background: The central sensitization mechanism of bone cancer pain is related to neuroinflammation, glial cell activation, and an acidic environment. Among them, the NLRP3 inflammasome is involved in the occurrence of bone cancer pain. Studies have shown that an intrathecal injection of bone marrow mesenchymal stem cells (BMSCs) alleviates bone cancer pain by inhibiting microglial cell activation. Upon inflammatory stimulation, BMSCs produce increased levels of Tumor necrosis factor-α-stimulated gene 6 (TSG-6), which regulates intercellular signaling and inflammatory responses; however, whether the secretion of TSG-6 from BMSCs inhibits NLRP3-mediated microglial pyroptosis to alleviate bone cancer pain is unknown. Therefore, this study aimed to evaluate the analgesic effect of TSG-6 released by BMSCs on bone cancer pain (BCP) and explore its potential mechanisms through in vitro and in vivo experiments.

Methods: In vivo, Walker 256 breast cancer cells were injected into the bone marrow cavity of the left tibia of rats to establish a BCP model. On days 7 and 14 after surgery, intrathecal injections of BMSCs or exogenous recombinant TSG-6 were administered, and the analgesic effects were observed at 2, 4, 8, 24, and 48 h after administration. Spinal cord tissues were collected for Western blotting and immunofluorescence staining to assess the activation of the NLRP3 signaling pathway. In vitro, BV2 microglia were cocultured with BMSCs or recombinant TSG-6, and the same detection methods were performed to evaluate changes in the levels of proteins involved in the NLRP3 signaling pathway. BMSCs transfected with TSG-6-targeting shRNA were intrathecally injected in vivo or cocultured with microglia in vitro to investigate whether TSG-6 is involved in the inhibition of microglial pyroptosis and the inflammatory response mediated by BMSCs to alleviate BCP.

Results: BMSC transplantation or treatment with exogenous recombinant TSG-6 significantly improved BCP-related behaviors, inhibited the expression of key proteins in the NLRP3 signaling pathway in spinal dorsal horn microglia in rats, and reduced the release of inflammatory factors. Experiments revealed that coculturing BMSCs with BV2 microglia or treating BV2 cells with exogenous recombinant TSG-6 inhibited the LPS-induced activation of NLRP3 in BV2 cells and regulated microglial pyroptosis. Transfection of BMSCs with TSG-6-targeting shRNA significantly weakened the inhibitory effect on microglial pyroptosis.

Conclusion: In vivo and in vitro experiments revealed that bone marrow mesenchymal stem cells inhibit the NLRP3 signaling pathway to regulate the pyroptosis of microglia through the secretion of TSG-6, thereby alleviating bone cancer pain.