Targeting TGF ‐β to Attenuate Ischemia–Reperfusion Associated Muscle Fibrosis and Enhance Regeneration: A Preclinical Study

Ischemia-reperfusion (IR) injury induces a pro-inflammatory cascade that disrupts inflammation resolution and exacerbates skeletal muscle fibrosis, leading to impaired regeneration through fibrosis and compromised myofiber regeneration. This study aimed to: (1) Demonstrate IR-induced dysregulation of muscle regeneration following acute injury; (2) Elucidating how TGF-β1 signaling within the FAP-associated inflammatory niche mediates fibrosis and impairs myofiber repair; and (3) Evaluating anti-TGF-β neutralization as a mechanism-based strategy to preserve regenerative capacity. Male C57BL6 mice (8 weeks) received CTX injections into the tibialis anterior (TA) to induce skeletal muscle injury. Transient clamping of femoral artery and vein was induced at 3 days post-injury to induce IR. Mice were stratified into CTX group and CTX-IR group depending on the induction of IR injury. TGF-β neutralizing antibody (TGF-β NAb) was administered in vivo to evaluate therapeutic potential. HE and Sirius red staining was used to assess cross-sectional area (CSA) of myofibers and percentage of fibrotic tissue. Western blotting was used to assess the expression of Collagen I, Collagen III and TGF-β. Flow cytometry was used to assess the number of fibro-adipogenic progenitors (FAPs). Collagen I and CD90 were assessed using immunostaining of TA cryosections and FAPs isolated from muscle tissues. The result shows that CTX-IR mice showed smaller myofiber cross-sectional area and more fibrotic tissue than CTX mice. Western blots revealed higher levels of Collagen I, Collagen III and TGF-β1 in CTX-IR samples. TGF-β1 stimulated the expression of collagen I in FAPs. TGF-β NAb application ameliorated the fibrosis of skeletal muscle and improved myofiber regeneration after IR. In conclusion, IR impairs muscle regeneration through TGF-β-associated FAP activation and collagen deposition. Inhibition of TGF-β attenuates fibrosis and increases the CSA of myofibers, demonstrating it as a potential target for promoting structural recovery and regenerative architecture in co-existing muscle injuries during orthopedic trauma.