Osteopontin May Improve Postinjury Muscle Repair Via Matrix Metalloproteinases And tgf-β Activation in Regular Exercise

Abstract

Skeletal muscle injuries are commonly observed during sports and trauma. Regular exercise promotes muscle repair; however, the underlying mechanisms require further investigation. In addition to exercise, osteopontin (OPN) contributes to skeletal muscle regeneration and fibrosis following injury. However, whether and how OPN affects matrix proteins to promote post-injury muscle repair remains uncertain. We recruited regular exercise (RE) and sedentary control (SC) groups to determine plasma OPN levels. Additionally, we developed a murine model of muscle contusion injury and compared the extent of damage, inflammatory state, and regeneration-related proteins in OPN knockout (OPN KO) and wild-type (WT) mice. Our results show that regular exercise induced the increase of OPN, matrix metalloproteinases (MMPs), and transforming growth factor-β (TGF-β) expression in plasma. Injured muscle fibers were repaired more slowly in OPN-KO mice than in WT mice. The expression levels of genes and proteins related to muscle regeneration were lower in OPN-KO mice after injury. OPN also promotes fibroblast proliferation, differentiation, and migration. Additionally, OPN upregulates MMP expression by activating TGF-β, which promotes muscle repair. OPN can improve post-injury muscle repair by activating MMPs and TGF-β pathways. It is upregulated by regular exercise. Our study provides a potential target for the treatment of muscle injuries and explains why regular physical exercise is beneficial for muscle repair.

Keywords: Matrix metalloproteinases; Muscle repair; Osteopontin; Regular exercise; TGF-β.

Figure 1

Regular Exercise Induces the Increase of OPN and MMPs Expression in Plasma. (A) People in the regular exercise (RE) group showed increased OPN expression in plasma than sedentary control (SC). (B-F) MMP2, MMP3, MMP7, MMP12, and MMP13 levels in plasma were upregulated in the RE group. The results are expressed as means ± range. *p < 0.05, determined by two-tailed Student's t-test.

Figure 2

OPN Promoted Muscle Repair after Contusion Injury. (A)H&E staining of the gastrocnemius muscle of the OPN-KO and WT mice before and 4, 48, and 72 h after contusion-induced muscle injury. Arrows indicate regenerating muscle fibers (centrally placed nuclei). Scale bar = 100 µm. (B) Quantification of regenerating muscle fibers at 72 hours post-injury. (C) Gastrocnemius muscle of the OPN-KO and WT mice in the figure before or 48h after muscle injury (MI). The mice were randomly assigned to four groups (6-10 mice/group). Data were analyzed using a two-tailed Student's t-test. Values are presented as mean ± SD, *p < 0.05.

Figure 3

OPN Improved Muscle Fiber Regeneration. (A) IHC staining of desmin protein in muscle tissue before and after injury. Arrows indicate desmin-positive fibers. Scale bar = 100 µm. (B) Quantification of desmin-positive fibers at 48 hours post-injury. (C-D) qPCR analysis of fibronectin and tenascin-C (TN-C) in OPN-KO and WT mice before and 48h after muscle injury respectively. Data were analyzed using a two-tailed Student's t-test or one-way ANOVA. Values are mean ± SD, *p < 0.05.

Figure 4

Myofibroblasts Got Activated after OPN Cultivation and TGF-β Expression Was Enhanced. (A-B) Myofibroblasts were cultured in vitro and stained 72 h after OPN cultivation (DAPI=blue, tubulin, or vimentin=green). Fluorescence micrographs were pictured under different multiples. (C) The proliferation of myofibroblasts cultured with OPN for 48h, and 72h with a concentration of 100ng/ml was promoted. Data were analyzed with one-way ANOVA. (D) OPN promoted the secretion of TGF-β1 in myofibroblasts, and this effect was blocked by TGF-β inhibitors. This was determined by a two-tailed Student's t-test. (E) qPCR analysis of TGF-β1 in OPN-KO and WT mice before and 48h after muscle injury. Data were analyzed with one-way ANOVA. Values are mean ± SD, *p < 0.05. (F-H) Plasma levels of the TGF-β family were elevated in the RE group compared to the SC group. The results are expressed as means ± range. *p < 0.05, determined by two-tailed Student's t-test.

Figure 5

MMPs Were Upregulated by OPN through TGF-β Signaling. (A-F) The levels of MMP2, MMP3, MMP7, MMP9, MMP12, and MMP13 were increased after OPN cultivation, which was blocked by TGF-β inhibitors. (G) Serum levels of OPN in RE and SC group of mice. (H-K) Serum levels of TGF-β1, MMP3, MMP7, and MMP12 in SC, RE, and OPNKO-RE group of mice. 6-10 mice/group. Results are expressed as means ± SD. *p < 0.05, determined by two-tailed Student's t-test.