LIPUS combined with mild hyperthermia promotes rotator cuff healing via TRPV1/Ca2+/CaMKII-driven BACH2 nuclear translocation and inhibition of M1 polarization

Background Rotator cuff injuries often lead to impaired shoulder function, and tendon–bone healing remains challenging due to the complex structure of the enthesis and persistent remodeling-associated pro-inflammatory signaling. Low-intensity pulsed ultrasound (LIPUS) and mild hyperthermia (MH) have shown potential in tissue repair, but the molecular mechanisms underlying their combined effects are unclear. Objective This study aimed to investigate whether LIPUS and MH synergistically promote tendon–bone healing by regulating macrophage polarization through the TRPV1/Ca 2+ /CaMKII/BACH2 signaling pathway. Methods A rabbit supraspinatus tendon–bone injury model and in vitro macrophage polarization assays were employed. Histology, immunohistochemistry, MRI, and biomechanical testing were performed to assess repair outcomes. RNA-seq with qPCR and Western blot validation was used to identify key signaling pathways. Functional assays, including Ca 2+ flux detection, pharmacological inhibition, and siRNA knockdown, were conducted to examine the role of TRPV1, CaMKII, and BACH2. Results LIPUS and MH significantly suppressed M1 polarization (CD86, iNOS) and enhanced M2 markers (CD163, Arg1), reducing ROS levels and inflammatory cytokine expression. Combined intervention yielded the most pronounced effects, showing improved fibrocartilage formation, collagen alignment, and bone remodeling, with superior biomechanical strength approaching native tissue. Transcriptomic and protein analyses revealed increased TRPV1 activation, enhanced Ca 2+ influx, CaMKII phosphorylation, and promoted BACH2 nuclear translocation. Mechanistically, TRPV1 activation promoted Ca 2+ entry, CaMKII activation, and BACH2 nuclear translocation, thereby inhibiting M1 polarization. Inhibition of TRPV1, Ca 2+ influx, or CaMKII significantly reduced BACH2 nuclear translocation and diminished the anti-inflammatory effects. Conclusion Combined LIPUS and MH synergistically enhance tendon--bone healing, potentially through modulating the TRPV1/Ca 2+ /CaMKII/BACH2 pathway, which appears to modulate macrophage polarization during the later stages of tendon–bone remodeling. The translational potential of this article This study identifies the TRPV1/Ca 2+ /CaMKII/BACH2 signaling axis as a novel mechanistic target for non-invasive physical therapy in tendon-bone healing. Our findings provide a strong preclinical rationale for combining LIPUS and mild hyperthermia as a promising, non-pharmacological strategy to improve immune microenvironment and outcomes after rotator cuff repair. This paves the way for future clinical trials to translate this combinatorial biophysical therapy into adjuvant treatment protocols.