Low-intensity pulsed ultrasound promotes skeletal muscle regeneration via modulating the inflammatory immune microenvironment

Abstract

<b>Background:</b> Low-intensity pulsed ultrasound (LIPUS, a form of mechanical stimulation) can promote skeletal muscle functional repair, but a lack of mechanistic understanding of its relationship and tissue regeneration limits progress in this field. We investigated the hypothesis that specific energy levels of LIPUS mediates skeletal muscle regeneration by modulating the inflammatory microenvironment. <b>Methods:</b> To address these gaps, LIPUS irritation was applied in <i>vivo</i> for 5 min at two different intensities (30mW/cm² and 60mW/cm²) in next 7 consecutive days, and the treatment begun at 24h after air drop-induced contusion injury. In vitro experiments, LIPUS irritation was applied at three different intensities (30mW/cm², 45mW/cm², and 60mW/cm²) for 2 times 24h after introduction of LPS in RAW264.7. Then, we comprehensively assessed the functional and histological parameters of skeletal muscle injury in mice and the phenotype shifting in macrophages through molecular biological methods and immunofluorescence analysis both <i>in vivo</i> and <i>in vitro</i>. <b>Results:</b> We reported that LIPUS therapy at intensity of 60mW/cm² exhibited the most significant differences in functional recovery of contusion-injured muscle in mice. The comprehensive functional tests and histological analysis <i>in vivo</i> indirectly and directly proved the effectiveness of LIPUS for muscle recovery. Through biological methods and immunofluorescence analysis both <i>in vivo</i> and <i>in vitro</i>, we found that this improvement was attributable in part to the clearance of M1 macrophages populations and the increase in M2 subtypes with the change of macrophage-mediated factors. Depletion of macrophages <i>in vivo</i> eliminated the therapeutic effects of LIPUS, indicating that improvement in muscle function was the result of M2-shifted macrophage polarization. Moreover, the M2-inducing effects of LIPUS were proved partially through the WNT pathway by upregulating FZD5 expression and enhancing β-catenin nuclear translocation in macrophages both <i>in vitro</i> and <i>in vivo</i>. The inhibition and augment of WNT pathway <i>in vitro</i> further verified our results. <b>Conclusion:</b> LIPUS at intensity of 60mW/cm² could significantly promoted skeletal muscle regeneration through shifting macrophage phenotype from M1 to M2. The ability of LIPUS to direct macrophage polarization may be a beneficial target in the clinical treatment of many injuries and inflammatory diseases.