Tetrahydropalmatine alleviates cancer induced bone pain by inhibiting TRPV1-SP-mediated macrophage recruitment and promoting M2 polarization.

BACKGROUND: Cancer-induced bone pain (CIBP) remains a debilitating clinical challenge due to its complex pathogenesis and limited therapeutic options. Tetrahydropalmatine (THP), an active alkaloid from Corydalis tuber, has shown analgesic potential, but its specific mechanisms in mitigating CIBP- especially its interactions with neural factors and immune cells-remain incompletely understood. This study aimed to investigate the efficacy of THP in alleviating CIBP and clarify its underlying mechanisms, with a focus on the roles of transient receptor potential vanilloid 1 (TRPV1), substance P (SP), and macrophage dynamics in a mouse model of CIBP. METHODS: Using a multidisciplinary approach, we established a CIBP model in male C57BL/6 mice (and TRPV1-knockout mice) via intramedullary injection of lung cancer cells. Behavioral assessments were performed to evaluate mechanical, thermal, and cold allodynia, as well as spontaneous pain, following daily oral administration of THP (80 mg/kg) from day 7 post-modeling. Molecular and cellular analyses included immunofluorescence staining, real-time PCR, ELISA (to quantify SP, cytokines, and Tac1 expression), calcium imaging (to measure TRPV1-mediated calcium influx in DRG neurons), scratch assays (to assess macrophage migration), and flow cytometry (to analyze macrophage polarization in RAW 264.7 cells). RESULTS: THP significantly alleviated CIBP-related allodynia and spontaneous pain in mice. Mechanistically, THP directly inhibited TRPV1 function in the primary phase (≤ 14 days post-modeling) (with an IC(50) of 77.9 µM), reducing SP release from DRG neurons and suppressing macrophage recruitment to DRG and sciatic nerve via the TRPV1-SP pathway. Importantly, THP directly promoted the polarization of recruited macrophages toward the anti-inflammatory M2 phenotype, as evidenced by downregulated iNOS, TNF-α, IL-1β and upregulated CD206, IL-4, IL-10 in vitro (RAW264.7 cells) and in vivo. These effects were abrogated in TRPV1-knockout mice, confirming TRPV1 as a critical mediator in the primary phase. CONCLUSION: In conclusion, THP mitigates CIBP through a dual mechanism: regulating macrophage recruitment via the TRPV1-SP pathway in the early stage to inhibit CIBP initiation and directly modulating the migrated macrophage polarization. This study provides novel insights into THP's analgesic mechanisms and supports its potential as a preclinical candidate for CIBP treatment.