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Int J Biol Sci 2023; 19(4):1123-1145. doi:10.7150/ijbs.79685 This issue Cite

Research Paper

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

Haocheng Qin^1,#, Zhiwen Luo^2,#, Yaying Sun^2,#, Zhong He^1,#, Beijie Qi⁵, Yisheng Chen², Junlong Wang⁶, Ce Li¹, Weiwei Lin^4,✉, Zhihua Han^3,✉, Yulian Zhu^1,✉

1. Department of Rehabilitation Medicine, Huashan Hospital, Fudan University, Shanghai, China.
2. Department of Sports Medicine, Huashan Hospital, Fudan University, Shanghai, China.
3. Department of Orthopedics and Traumatology, Shanghai General Hospital Shanghai Jiaotong University, Shanghai, China.
4. Department of Neurosurgery, Second Affiliated Hospital of Zhejiang University School of Medicine, Zhejiang University, Zhejiang, China.
5. Department of Orthopedics, Shanghai Pudong Hospital, Fudan University, Shanghai, China.
6. Department of Radiology, Huashan Hospital, Fudan University, Shanghai, China.
# These authors make equal contributions to this work.

✉ Corresponding author: Yu-Lian Zhu, MD, Ph.D., Doctor, Professor, Department of Rehabilitation Medicine, Huashan Hospital, Fudan University, No. 12 Wulumuqi road, Jing'an District, Shanghai 200040, China. Email: zyljullycom. Chairman of Shanghai Physical Therapy Association, Vice-chairman of the Chinese Physical Therapy Association. Zhihua Han, MD, Ph.D., Doctor, Trauma Center, Department of Orthopedics and Traumatology, Shanghai General Hospital Shanghai Jiaotong University. Email: hzhdoccom. Weiwei Lin, MD, Ph.D., Doctor, Department of Neurosurgery, Second Affiliated Hospital of Zhejiang University School of Medicine, Zhejiang University, 88 Jiefang Road, Hangzhou, 310009 Zhejiang, China. Email: lindwedu.cn More

Abstract

Background: 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.

Methods: To address these gaps, LIPUS irritation was applied in vivo 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 in vivo and in vitro.

Results: 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 in vivo indirectly and directly proved the effectiveness of LIPUS for muscle recovery. Through biological methods and immunofluorescence analysis both in vivo and in vitro, 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 in vivo 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 in vitro and in vivo. The inhibition and augment of WNT pathway in vitro further verified our results.

Conclusion: 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.

Keywords: Low-intensity pulsed ultrasound, Macrophage polarization, Inflammation, Muscle injury, WNT signaling

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