Vitamin B12-Loaded Chitosan Nanoparticles Promote Skeletal Muscle Injury Repair in Aged Rats via Amelioration of Aging-Suppressed Efferocytosis

Abstract

Muscle gradually loses its regenerative capacity with aging. Recent evidence highlights age-related immune dysregulation as a key driver of satellite cell dysfunction and reduced muscle regeneration. Timely elimination of apoptotic cells by phagocytes through efferocytosis is essential for tissue repair. Therefore, exploring age-related alterations in the molecular machinery of efferocytosis and their impact on muscle regeneration is of great relevance. This study examined the efferocytic machinery in the gastrocnemius muscle tissue of young and aged rats after doxorubicin-induced acute myotoxicity and assessed the potential of Vitamin B12-loaded chitosan nanoparticles (B12 CS NPS) to enhance efferocytosis and promote skeletal muscle injury repair in aged rats. Aged rats exhibited impaired efferocytosis with a significant reduction in MerTK, PPARγ, and miR-124 expression, and increased ADAM17 expression. B12 CS NPS administration significantly improved efferocytosis and reduced necrotic tissue areas, accompanied by increased MerTK, PPARγ, and miR-124, and reduced ADAM17 expression. Supplementation with B12 CS NPS significantly enhanced satellite cell proliferation and differentiation, which was indicated by upregulated expression of Pax7, Myog, and MyoD. These findings reveal that age-related alterations in regulatory molecules impair efferocytosis in aged muscle and demonstrate the potential of B12 CS NPs to enhance efferocytosis and improve skeletal muscle repair.

Keywords: aging; doxorubicin; efferocytosis; muscle regeneration; skeletal muscle; vitamin B12.

Figure 1

Schematic representation summarizing the study design. Six groups of rats (n = 7 per group) were used, and gastrocnemius muscles were collected for biochemical, histological, and molecular analyses.

Figure 2

Characterization of B12 CS NPS. (A) Scanning electron microscopy examination showing the spherical nature of the B12-CS-NPS. (B) In vitro release analysis of Vit B12 from chitosan nanoparticles.

Figure 3

(A) BAX and (B) BCL-2 gene expression in the gastrocnemius muscle tissue samples from experimental groups (n = 7/group), as determined by qPCR, normalized for the house-keeping gene B-actin, and expressed relative to controls. Results are expressed as mean ± SD. ****, significant at (p < 0.0001). (C) Representative photomicrographs showing immunohistochemical detection of caspase-3 (counterstained with hematoxylin). (D) Quantitative representation of cleaved caspase-3 area%.

Figure 4

(A) Photoplate (a–l) representing L/S and T/S of skeletal muscles from different study groups stained with (H&E) at magnification ×200. (m–o) Representing L/S sections from Old + Doxo + B12-CS NPS group with higher magnification (×400). (B) Necrotic tissue area, (C) spacing between muscle fibers, and (D) fiber average area in the gastrocnemius muscle tissue samples from experimental groups (n = 7/group). Results are expressed as mean ± SD. ****, significant at (p < 0.0001).

Figure 5

Representativetransmission electron photomicrographs (a–k) of skeletal muscle from the five study groups: Young control (a), old control (b), Old + B12-CS NPS (c), Young + Doxo group (d–f), and Old + Doxo group (g–k).

Figure 6

A photoplate (a–j) representing sections of skeletal muscles from different study groups stained with (toluidine blue) at magnification ×1000. Young control group (a): C/S showing myofibers with peripheral flat nuclei (n) beneath the basal lamina ($), while the satellite cells are located between the fibers outside the basal lamina (▲). The perimysium surrounds the bundle fibers (p). Old control (b) and Old + B12-CS NPS (c) displayed the normal histological features as control group. After induction of doxorubicin toxicity, Young + Doxo group (d) showed large spacing between fibers, these spaces are invaded by inflammatory cells, especially macrophages, which appeared large with irregular nuclei (dotted lines). Some satellite cells were apparent (▲). The Old + Doxo group (e) showed displaced myofibrils (thick ↑). Some fibers appeared as necrotic tissue (#), with apoptotic nuclei (↑). Old + Doxo + B12-CS NPS group (f–j) showed many efferocytic and regenerative features. (f,g) The areas between fibers are occupied by large numbers of satellite cells with euchromatic pale nuclei (▲) invading macrophages displaying irregular outlines (dotted lines). In many sites, the macrophages are located coupled with satellite cells (rectangles). (h) Prominent feature of newly formed myotube (MT) with aligned nuclei (▲) sealed with new basal lamina (curved arrow) was noticed. (i,j) Some active proliferating satellite cells can be noticed with two adjacent nuclei (oval) and some fibers showed centrally located nuclei (yellow ↑).

Figure 7

(A) MerTK, (B) ADAM17, (C) PPARγ, and (D) miR-124 gene expression in the gastrocnemius muscle tissue samples from experimental groups (n = 7/group), as determined by qPCR, normalized for the house-keeping gene B-actin, and expressed relative to controls. Results are expressed as mean ± SD. *, significant at (p < 0.05); **, significant at (p < 0.01), ***, significant at (p < 0.001) and ****, significant at (p < 0.0001).

Figure 8

(A) BAX and (B) BCL-2 gene expression in the gastrocnemius muscle tissue samples from experimental groups (n = 7/group), as determined by qPCR, normalized for the house-keeping gene B-actin, and expressed relative to controls. (C) Quantitative representation of cleaved caspase-3 area%. (D) Necrotic tissue area, (E) spacing between muscle fibers, and (F) fiber average area in the gastrocnemius muscle tissue samples from experimental groups (n = 7/group). Results are expressed as mean ± SD. ****, significant at (p < 0.0001).

Figure 9

A photoplate (a–h) representing electron micrographs of skeletal muscles from Old + Doxo + B12-CS NPS group. When comparing Old + Doxo + B12-CS NPS group (a–h) to the previously described Old + Doxo group (Figure 5g–k), we found less inflammatory features accompanied by efferocytic and regenerative changes and (a) distorted and widely spaced myofibrils with vacoulated mitochondria (circles), and less-aligned z-lines (z). (b) Large apoptotic bodies are detached from the surrounding necrotic muscle tissue (red ↑). Many features of phagocytic activity indicate efferocytosis: (c) large macrophage with irregular nucleus (Q) appear to be invading the necrotic area. (d) Another large irregular macrophage displaying irregular nucleus (Q) at the site of inflammation near the detached necrotic debris (red ↑), showing multivesicular bodies as remnants of phagocytic activity (✸). The adjacent sarcoplasm showing shrunken nucleus (N) and large vacuole (v). (e) Large macrophages (Q) displayed well developed lysosomal apparatus (y) and phagosomes (ph) representing engulfed apoptotic bodies. Additionally, some features of regeneration are obvious. (f) Large satellite cell (S) in active stage of dividing nucleus into two small nuclei (n1 and n2). (g) Two adjacent satellite nuclei (S) are forming myotube (blue ↑) beneath the basal lamina representing proliferative activity. (h) Large macrophage (Q) with irregular nucleus in close contact with adjacent satellite cells (S).

Figure 10

(A) MerTK, (B) ADAM17, (C) PPARγ, and (D) miR-124 gene expression in the gastrocnemius muscle tissue samples from experimental groups (n = 7/group), as determined by qPCR, normalized for the house-keeping gene B-actin, and expressed relative to controls. Results are expressed as mean ± SD. *, significant at (p < 0.05); **, significant at (p < 0.01), and ****, significant at (p < 0.0001).

Figure 11

Immunohistochemical (IHC) detection of M1 and M2 polarization in skeletal muscles from different experimental groups, counterstained with hematoxylin. (A) Photoplate representing IHC detection of M1 macrophages based on anti-iNOS immune expression in all samples. (B) Quantitative representation of iNOS area%. (C) Photoplate representing IHC detection of M1 macrophages based on anti-CD86 immune expression in all samples. (D) Quantitative representation of CD86+ cells%. Little iNOS+ expression and few CD86+ M1 cells (arrowheads) are seen in Old + Doxo + B12-CS NPS group, indicating cessation of inflammation. ((A): scale bar = 100 μm, Magnification = ×200), ((C): scale bar = 50 μm, Magnification = ×400). (E) Detection of M2 macrophages based on anti-CD163 immune expression. The highest number of CD163⁺ M2 cells (arrowheads) are seen in Old + Doxo + B12 CS NPS group. Large number of large CD163⁺ M2 cells were distributed mainly in the endomysium and perimysium of muscle fibers and around the blood vessels. Note the large foamy appearance of M2 cells (small inset) representing phagocytic activity ((E): scale bar = 50 μm, Magnification = ×400). (F) Quantitative representation of CD163⁺ cells%. *, significant at (p < 0.05) and ****, significant at (p < 0.0001).

Figure 12

Effect of Vit B12 on oxidative stress, inflammatory markers and extracellular matrix deposition: (A) MDA, (B) TAC, (C) TNF-α, (D) IL-1β, (E) IL-6, (F) IL-10, (G) A photoplate representing collagen deposition in skeletal muscles. Detection of extracellular matrix collagen fibers stained with Masson’s trichrome stain. Green–blue color indicates positive results. (Scale bar = 100 μm, Magnification = ×200). (H) Analysis of positive Masson’s trichrome area%. *, significant at (p < 0.05); **, significant at (p < 0.01), ***, significant at (p < 0.001) and ****, significant at (p < 0.0001).

Figure 13

(A) Pax7, (B) MyoD, and (C) Myog gene expression in the gastrocnemius muscle tissue samples from experimental groups (n = 7/group), as determined by qPCR, normalized for the house-keeping gene B-actin, and expressed relative to controls. Results are expressed s at (p < 0.05); **, significant at (p < 0.001) and ****, significant at (p < 0.0001).