Mailuo Shutong pills alleviate limb swelling in femoral fracture rats by orchestrating mitophagic clearance and ferroptotic suppression.

Mailuo Shutong Pill (MLSTP) is a well-established formula developed by National Grand Master of Traditional Chinese Medicine Tang Zuxuan for treating thrombotic superficial phlebitis of the lower limbs. Clinically, it is commonly used for conditions caused by damp-heat and blood stasis obstructing the meridians, including thrombotic superficial phlebitis, subacute deep vein thrombosis, varicose veins, diabetic foot, as well as limb swelling and pain. Previous studies have demonstrated that MLSTP protected against limb swelling caused by femoral fracture (SCFF). This study further investigated the regulatory mechanisms of MLSTP on mitophagy and ferroptosis in the SCFF model, aiming to elucidate its multi-target pharmacological actions. Male SD rats were subjected to a femoral fracture procedure to establish the SCFF model. Rats were randomly divided into Sham, Model, MLSTPL, MLSTPH, and Aescuven forte groups. Muscle pathology and iron deposition were assessed by hematoxylin-eosin (H&E) and Prussian blue staining. Muscle ATP, SOD, MDA, GSH, and CAT were assayed using Enzyme-linked immunosorbent assay (ELISA) kits. Proteomic analysis of muscle tissues was performed to identify differentially regulated signaling pathways. Mitochondrial morphology was observed by transmission electron microscopy (TEM). Mitophagy and ferroptosis-related proteins were assessed by immunofluorescence. Western blot analysis was performed to detect the expression of proteins related to the NRF2/HO-1/NQO1 pathway, NLRP3 inflammasome signaling, mitochondrial respiratory chain complexes, AMPK/mTOR axis, mitophagy, and ferroptosis. MLSTP ameliorated muscle pathology and reduced iron deposition in SCFF rats, while increasing ATP, SOD, GSH, and CAT levels and decreasing MDA content. Proteomic analysis associated its protective effect with oxidative phosphorylation. TEM revealed attenuated mitochondrial damage. Immunofluorescence and Western blot suggested that MLSTP activated NRF2/HO-1/NQO1, regulated respiratory chain components including NDUFB8, SDHB, UQCRC2, MTCO1, ATP5A, enhanced PINK1/Parkin-mediated mitophagy via AMPK/mTOR, and suppressed NLRP3 inflammasome and ferroptosis. This study demonstrates that MLSTP alleviates muscle swelling in SCFF rats by regulating the mitochondrial respiratory chain, ameliorating disordered mitophagy, and inhibiting ferroptosis. The present investigation provides a theoretical framework supporting the application of MLSTP for managing fracture-induced muscle edema.