Exosomal miR-24-3p mediates myoblast-macrophage crosstalk to promote abdominal muscle repair

Abstract

Objective: The objective of this study was to explore the role of exosomal miR-24-3p in facilitating communication between myoblasts and macrophages, and to assess its potential in promoting abdominal muscle repair.

Methods: We utilized C2C12 myoblasts and RAW 264.7 macrophages, inducing the latter into an M2 phenotype. miR-24-3p levels were manipulated via transfection, and exosomes were isolated from M2 macrophages using ultracentrifugation. Exosome characterization was performed using TEM and Western blot. In vitro assays evaluated C2C12 cell proliferation, migration, and differentiation. In vivo, a cardiotoxin-induced mouse model of muscle injury was used to assess the effects of exosomal miR-24-3p on muscle repair, including histological assessment and analysis of cytokine and metabolic markers.

Results: Our results demonstrated that exosomal miR-24-3p, when isolated from M2 macrophages, was effectively internalized by C2C12 cells and significantly enhanced their metabolic activity, proliferation, and migratory capabilities. Moreover, it induced cellular differentiation, as observed under microscopic examination. In the abdominal muscle injury model, the administration of exosomal miR-24-3p led to a reduction in muscle fiber damage, fibrosis, and inflammation. It also promoted the restoration of glucose and lipid metabolism, which is critical for the energy demands of regenerating muscle. Furthermore, exosomal miR-24-3p upregulated the expression of genes associated with muscle cell proliferation and differentiation, suggesting its potential role in muscle repair.

Conclusion: In conclusion, exosomal miR-24-3p plays a significant role in facilitating abdominal muscle repair by mediating the interaction between myoblasts and macrophages.

Keywords: abdominal muscle repair; exosomal miR-24-3p; macrophage; muscle regeneration; myoblast.

Schematic illustration of exosomal miR-24-3p-mediated crosstalk between M2 macrophages and C2C12 myoblasts, promoting abdominal muscle repair via enhanced proliferation, differentiation, and metabolic regulation.

FIGURE 1

Preparation and characterization of M2 macrophage-derived exosomes (M2-exos). (A) Flow cytometry analysis of RAW 264.7 macrophages induced with IL-4/IL-10 (10 ng/mL, 7 days) for M2 polarization, gated on CD11b⁺ CD68⁺ CD11c⁻/CD206⁺ populations. (B) Validation of miR-24-3p inhibitor transfection efficiency in M2 macrophages via qRT-PCR. (C) Transmission electron microscopy (TEM) imaging of isolated exosomes (scale bar: 100 nm). (D) Western blot analysis of exosomal markers (ALIX, CD63, TSG101) in M2-exos. (E) mRNA levels of miR-24-3p were analyzed using quantitative RT-PCR. (F) Confocal microscopy imaging of PKH26-labeled M2-exos (red) internalized by DAPI-stained C2C12 myoblasts (blue; scale bar: 20 μm). n = 3, ***p < 0.001.

FIGURE 2

Effects of M2-exosomal miR-24-3p on myoblast proliferation and migration. (A) CCK-8 assay to assess metabolic activity of C2C12 cells treated with PBS, exo-NC (control exosomes), or Exo-inhibitor (miR-24-3p-suppressed exosomes). (B) EdU staining (red) for quantification of proliferating C2C12 cells (DAPI counterstain, blue; scale bar: 50 μm). (C) qRT-PCR analysis of proliferation-related genes (PCNA, Cyclin D1, CDK2) in C2C12 cells. (D) Wound healing assay to evaluate C2C12 cell migration post-treatment (images captured at 0 h and 24 h; scale bar: 200 μm). (E) Luciferase reporter assays using Notch wild-type (Notch-WT) and mutant (Notch-MUT) reporter vectors co-transfected with miR-24-3p mimic or miR-NC mimic. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3

Exosomal miR-24-3p induces myoblast differentiation. (A) Microscopic evaluation of C2C12 myoblast differentiation under indicated experimental conditions (Control, DM, exo-inhibitors-DM, exo-NC) (scale bar: 200 μm). (B) Western blot analysis of myogenic markers MYOD, MYOG, and MYH6 in C2C12 cells following experimental treatments. Loading control: GAPDH. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4

Exosomal miR-24-3p ameliorates abdominal muscle injury in C57BL/6J male mice. (A) Hematoxylin and eosin (HE) staining was performed on paraffin-embedded sections of injured abdominal muscle tissues to evaluate histological architecture and inflammatory cell infiltration (scale bar: 50 μm). (B) Evans Blue dye uptake assay was conducted to assess muscle fiber integrity and regeneration status at the injury site (scale bar: 100 μm). (C) Masson’s trichrome staining was employed to quantify collagen deposition and fibrosis in regenerating abdominal muscle tissues (scale bar: 100 μm). (D) Oil Red O staining of cryosectioned muscle tissues was used to visualize lipid accumulation and metabolic alterations in the injured region (scale bar: 100 μm). (E) Enzyme-linked immunosorbent assay (ELISA) measured serum levels of pro-inflammatory cytokines (TNF-α, IL-6) in experimental and control groups. n = 3, **p < 0.01, ***p < 0.001.

FIGURE 5

Restoration of glucose and lipid metabolism in muscle-injured mice by exosomal miR-24-3p. (A,B) Western blot analysis of glucose metabolism markers (GLUT4, AKT2, G6PC, GYS1) and lipid metabolism-related proteins (PLIN4, FABP4, FASN) in muscle tissues across experimental groups (AM, AM-Exo-miR-NC, AM-Exo-miR-inhibitor). GAPDH was used as a loading control. n = 3, **p < 0.01, ***p < 0.001.

FIGURE 6

Metabolic-related experiments in muscle-injured mice. (A) qRT-PCR was performed to detect the mRNA expression of genes involved in fatty acid oxidation (ACOX1, CPT1, FATP) and glucose uptake (GLUT4, HK), as well as PFK and PGC-1α. (B) Western blot was used to analyze the protein expression of ATP5A and COX IV. (C) ELISA was conducted to measure ATP concentration and CPT activity. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7

Exosomal miR-24-3p promotes muscle repair by enhancing myoblast proliferation and differentiation. (A) mRNA levels of MYH6, MYOD, CyclinD1, MYPT1, and VEGFR-1 were analyzed using quantitative RT-PCR. (B) mRNA levels of MYOD, MYOG, and MYH6 were analyzed using quantitative RT-PCR. n = 3, **p < 0.01, ***p < 0.001.