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

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: Inflammation; Low-intensity pulsed ultrasound; Macrophage polarization; Muscle injury; WNT signaling.

Fig 1

Experimental design for both the in vitro. and in vivo. studies. (A) Experimental design for LPS-induced RAW264.7. (B) Both the normal contusion injury model and macrophages depletion contusion were presented. Molecular Analysis included Antibody Array analysis, Western Blot, ELISA, qPCR, and flow cytometry. The histological evaluation included Laser speckle contrast analysis, HE staining, Masson staining, and immunofluorescence. Muscle function tests included the Water Maze test, Rotarod test, Treadmill test, CatWalk test, and Tetanus strength test.

Fig 2

LIPUS promoted muscle functional recovery. (A-C) gastrocnemius muscles were directly observed on Day 3 after dissecting. The ratio of bilateral gastrocnemius muscles weight at different time points. (n=4) Red*P<0.05(Contusion+Ultrasound^L group compared with Control group). Bule**P<0.01(Contusion +Ultrasound^H group compared with Control group). Statistics of passive strength of isolated gastrocnemius muscles. (n=4) *P<0.05. (D-F) A photo was recorded during the Water Maze test and a representation of the movement trajectories of mice over 1 minute in different four groups. The swimming speed of mice in 1 minute was compared. (n=4) *P<0.05. (G-H) Photos were taken during the Rotarod test. The duration time of mice on the accelerated rod was recorded and compared among different groups. (n=4) *P<0.05. (I-J) Photos were taken during the Treadmill test. From left to right, there are two mice from the Control group, two mice from the Contusion group, two mice in the Contusion +Ultrasound^L group, and two mice from the Contusion +Ultrasound^H group. (n=4) *P<0.05.**P<0.01. The duration time till fatigue of mice on the acceleration band was recorded and compared among different groups (n=4) *P<0.05. (K-L) A photograph of the apparatus used to conduct the gait experiment and representative images of three-dimensional paw pressure distribution of mice in different groups. Quantitative analysis of four gait parameters of hind limbs, including the ratio of bilateral swing time, bilateral standing time, bilateral paw pressure mean intensity, and bilateral paw print area. (n=4) *P<0.05.**P<0.01.

Fig 3

LIPUS relieved inflammation, enhanced angiogenesis, inhibited fibrosis, and improved myogenesis in vivo. (A-B) Representative blood perfusion images of bilateral gastrocnemius muscle. Circle marked the aimed injured area. The ratio of blood perfusion (injured/uninjured) of lower limbs in different groups was calculated and compared vertically and horizontally. (n=4) Bule**P<0.01(Contusion +ultrasound^H group compared with Control group). (C-D) Representative HE staining images of injured muscle among different groups on Day 3. The scale bar from top to bottom is 200μm. Cellularity percentage on Day 3 was calculated in different groups. (n=4) *P<0.05. The scale bar from top to bottom is 250μm, 100μm, and 50μm. (E-J) Representative Masson staining images of injured muscle among different groups on Day 7 and Day14. The scale bar from top to bottom is 250μm, 100μm, and 50μm. Fibrosis percentage on Day 7 and Day 14 among different groups was compared. (n=4) *P<0.05 **P<0.01 ***P<0.001. CSA on Day 7 and Day 14 among different groups was compared. (n=4) *P<0.05 **P<0.01.

Fig 4

LIPUS regulated macrophage polarization and inhibited the inflammatory microenvironment in vivo. (A-B) A diagram to show the location of all the cytokines on the array. The original images showed significantly decreased antibodies which were also highlighted with a red box. Relative protein expression of significantly changed inflammatory cytokines after LIUPS treatment. (n=4) *P<0.05 **P<0.01 ***P<0.001 ****P<0.0001. (C-D) The expression level of CD206, Arg 1, iNOS, and CD86 in injured gastrocnemius muscles from different groups on Day 3 and Day7 were determined by Western Blot. (n=4) *P<0.05 **P<0.01. (E) The gene expression levels of CD86, iNOS, ARG1, and CD206 in injured gastrocnemius muscles from different groups on Day 3 were detected by qPCR. (n=3) *P<0.05 **P<0.01. (F-H) Immunofluorescence was utilized to measure the relative expression level and distribution of CD68(green) and iNOS(red) among the three groups on Day 3, including the Control group, the Contusion group, and the Contusion +Ultrasound^H group. The nuclei were dyed with Dapi (blue). Arrows indicate iNOS positive and CD68 positive cells. The percentage of CD68 (macrophage marker) positive cells were counted. The percentage of both CD68 and iNOS positive cells was counted. (n=4) *P<0.05. (I-J) Immunofluorescence was used to detect the relative expression and distribution of CD206(green) among the three groups on Day 3, including the Control group, the Contusion group, and the Contusion +Ultrasound^H group. The nuclei were dyed with Dapi (blue). Arrows indicate CD206 positive cells. The percentage of CD206 positive cells was counted. (n=4) *P<0.05.

Fig 5

The therapeutic effect of LIPUS was suppressed in macrophage-depleted mice. (A) Representative flow cytometry plots showing the percentages of F4/80 and CD11b phenotype in macrophage-depleted mice on Day 1, Day 3, Day 7, and normal mice. The proportion of macrophages in spleen after macrophage depletion was compared with normal mice. (n=4) ****P < 0.0001. (B-C) The 3D footprint intensities images of the uninjured left hind foot and injured right hind foot in macrophage-depleted mice on Day 14. (n=3) No statistically significant improvement was found between the Contusion +Ultrasound^H group and the Contusion group. (n=4) ns P>0.05. (D-E) Representative blood perfusion images of the bilateral gastrocnemius muscle in macrophage-depleted mice on Day 14. Circle marked the aimed injured area. The ratio of blood perfusion (injured/uninjured) of lower limbs in different groups was calculated and compared. No statistically significant improvement was found between the Contusion +Ultrasound^H group and the Contusion group. (n=4) ns P>0.05. (F-J) Representative HE staining images and Masson staining of injured muscle with macrophages depletion among different groups on Day 14. The scale bar from top to bottom is 250μm, 100μm, and 50μm. Muscle fibers on Day 14 were calculated in different groups. (n=4) *P<0.05. Cross-sectional area and fibrosis percentage on Day 14 among different groups were compared. (n=4) ns P>0.05.

Fig 6

LIPUS promoted M2 macrophage polarization in vitro. (A) Representative flow cytometry plots showing the percentages of M1 (CD86 + CD4/80+) and M2 (CD163 + CD4/80+) phenotype in macrophages after LIPUS treatment. Quantification of flow cytometry data after two ultrasound stimulation sessions with LPS-induced RAW 264.7 cells. (n=4) *P < 0.05. (C) The gene expression level of CD86, iNOS, ARG1, and CD206 in LPS-induced RAW 264.7 cells were detected by qPCR. (n=3) *P<0.05 **P<0.01. (D) The concentration of cytokine IL-6, IL-1α, TNF-α, and IL-10 in supernatants of LPS-stimulated RAW 264.7 cells after treated were measured by Elisa assay (n=4). *P < 0.05 **P<0.01 ****P < 0.0001. (E-F) The protein expression of CD206, Arg 1, iNOS, and CD86 in LPS-stimulated RAW 264.7 was determined by Western Blot. (n=4) *P<0.05 **P<0.01 ***P<0.001. (G-H) Immunofluorescence intensity changes of iNOS (green), a pro-inflammatory cytokine after treatment with different intensity of ultrasound. The nuclei were dyed with Dapi (blue). Scale=25μm Relative fluorescence intensity (normalized to Dapi) of iNOS was determined. (n=4). *P < 0.05. ***P < 0.001.

Fig 7

LIPUS regulated macrophage polarization through the WNT pathway in LPS-induced M1 macrophages. (A-B) The changes in protein expression of FZD5 and β-catenin in LPS-stimulated RAW 264.7 cells were exposed and recorded over two times of LIPUS treatment. Relative protein expression of β-catenin was calculated after normalized with Lamin B1. (n=4). *P < 0.05. Relative protein expression of FZD5 was calculated after normalized with GAPDH. (n=4). **P < 0.01. (C) Immunofluorescence staining of FZD5(green) expressed on the cell membrane and Phalloidin (red) of macrophage in different groups after treatment with LIPUS. The nuclei were dyed with Dapi (blue). Scale bar=25μm. The relative fluorescence intensity of FZD5 was compared among different groups. (n=4). *P < 0.05. ***P < 0.001. (D)Images showed Immunofluorescence staining of β-catenin(green) expressed in the nucleus, and Phalloidin (red) of macrophages in different groups after treatment with LIPUS. The nuclei were dyed with Dapi (blue). Scale bar=25μm. The nuclear translocation percentage for β-catenin was compared among different groups. (n=4) *P < 0.05 ***P < 0.001 ****P<0.0001. (E-F) Immunofluorescence staining of β-catenin(green) expressed in the nucleus, and Phalloidin (red) of macrophage in different groups after treating different combinations of UltrasoundH, XAV-939, and QS11. The nuclei were dyed with Dapi (blue). Scale bar=25μm. The nuclear translocation percentage for β-catenin was compared among different groups. (n=4) *P < 0.05 **P < 0.01 ***P<0.001. No significant changes were found when LIPUS and XAV-939 were combined. (G) The gene expression level of CD86, iNOS, ARG1, and CD206 in different groups after treating different combinations of LIPUS, ultrasoundH, XAV-939, and QS11 were detected by qPCR. (n=3)

Fig 8

LIPUS promoted M2 macrophage polarization through the Wnt pathway in vivo. (A-B) The protein expressions of FZD5 and β-catenin after being treated with LIPUS in contusion-injured mice were determined by Western Blot. (n=4) *P<0.05 **P<0.01 ***P<0.001. (C-D) Immunofluorescence was used to detect the relative expression and distribution of FZD5/β-catenin on Day 3 after acute injury with or without LIPUS treatment. Scale bar=200, 50, or 25μm.

Fig 9

Schematic depiction of this work. The pathophysiological mechanism of LIPUS treatment of muscle injury mainly includes relieving inflammation, enhancing angiogenesis, inhibiting fibrosis, improving myogenesis, and promoting functional recovery. At the level of molecular mechanism, LIPUS regulates macrophage polarization by activating the WNT pathway and finally reversing the hyperinflammatory microenvironment.