Mailuo Shutong pills alleviate limb swelling in femoral fracture rats by orchestrating mitophagic clearance and ferroptotic suppression.
MLSTP may reduce muscle swelling in femoral fracture rats by enhancing mitophagy and suppressing ferroptosis, but human relevance is unclear.
Where it sits
this study against the rest of the mots-c corpusSummary and findings
The study investigated the effects of Mailuo Shutong Pill (MLSTP) on limb swelling in a rat model of femoral fracture. It focused on the mechanisms of mitophagy and ferroptosis regulation. MLSTP was found to improve muscle pathology and reduce iron deposition in these rats.
Abstract
<h4>Ethnopharmacological relevance</h4>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.<h4>Aim of the study</h4>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.<h4>Materials and methods</h4>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.<h4>Results</h4>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.<h4>Conclusions</h4>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.
Background
The study addresses the pharmacological effects of Mailuo Shutong Pill (MLSTP), a traditional Chinese medicine, on limb swelling due to femoral fractures. Previous research indicated MLSTP's protective effects against such swelling, but the underlying mechanisms remained unclear. Understanding these mechanisms could provide insights into its multi-target actions and potential therapeutic applications.
Methods
The study used a femoral fracture model in male SD rats, dividing them into Sham, Model, MLSTPL, MLSTPH, and Aescuven forte groups. Muscle pathology and iron deposition were assessed using H&E and Prussian blue staining. ELISA kits measured muscle ATP, SOD, MDA, GSH, and CAT levels. Proteomic analysis identified signaling pathways, and mitochondrial morphology was observed via TEM. Immunofluorescence and Western blot analyses assessed proteins related to various pathways, including NRF2/HO-1/NQO1 and AMPK/mTOR.
Results
MLSTP improved muscle pathology and reduced iron deposition in SCFF rats. It increased ATP, SOD, GSH, and CAT levels while decreasing MDA content. Proteomic analysis associated its effects with oxidative phosphorylation. TEM showed reduced mitochondrial damage, and immunofluorescence and Western blot indicated activation of the NRF2/HO-1/NQO1 pathway, enhanced mitophagy via AMPK/mTOR, and suppression of the NLRP3 inflammasome and ferroptosis.
Interpretation
The findings suggest that MLSTP may have a protective role in muscle pathology associated with femoral fractures through mechanisms involving oxidative phosphorylation and mitophagy regulation. While the study provides a theoretical basis for MLSTP's application, the clinical significance remains uncertain due to the lack of human data. The study's reliance on a rat model and absence of detailed quantitative results limit the generalizability of the findings.
Key findings
- MLSTP increased ATP, SOD, GSH, and CAT levels in muscle tissue.
- MLSTP decreased MDA content in muscle tissue.
- Proteomic analysis linked MLSTP's effects to oxidative phosphorylation.
- MLSTP activated the NRF2/HO-1/NQO1 pathway.
- MLSTP enhanced PINK1/Parkin-mediated mitophagy via AMPK/mTOR.
Limitations
- rat model only, no human data
- lack of detailed quantitative results
- no reported statistical significance
- mechanistic focus, unclear clinical relevance