Transcriptomic and Proteomic Analysis of the Skeletal Muscle Revealed the Effects and Mechanism of Mulberry Leaf Flavonoids on Alleviating Exercise-Induced Muscle Damage in Mongolian Horses.

Overview

• This study investigates how mulberry leaf flavonoids (MLFs), natural antioxidant compounds, help protect Mongolian horses from muscle damage caused by intense exercise.
• The research combines transcriptomic and proteomic analyses to understand the biological mechanisms behind MLFs’ protective effects on skeletal muscle during and after exercise.

Background and Purpose

• Exercise-induced muscle damage (EIMD) is a common problem in horses subjected to high-intensity exercise, leading to inflammation, oxidative stress, and muscle structure damage.
• Plant-derived extracts like MLFs are known for their antioxidant properties but scientific evidence on their effectiveness in horses is limited.
• The goal of this study was to determine if MLFs could alleviate muscle damage in Mongolian horses and elucidate the underlying molecular mechanisms.

Study Design and Methodology

• Twelve Mongolian horses participated in the study using a 3 × 3 Latin square design, allowing each horse to receive different dosages or placebo in a structured sequence.
• Various biomarkers associated with immune response, metabolism, muscle damage, and antioxidant capacity were measured in blood samples taken at different exercise distances and recovery periods.
• Transcriptomic analysis was performed to identify changes in gene expression related to muscle structure, antioxidant defense, and muscle cell proliferation.
• Quantitative proteomics was used to analyze protein expression and signaling pathways involved in muscle repair and metabolism.

Key Findings

• High-intensity exercise caused negative effects including:
  • Elevated white blood cells (WBC) and monocytes (MON), indicating inflammation.
  • Increased enzymes like AST and CK, markers of muscle damage.
  • Higher levels of malondialdehyde (MDA), a marker of oxidative stress.
  • Disruption of myofibrillar (muscle fiber) structure and reduced antioxidant capacity.
• MLF supplementation resulted in:
  • Significant reduction of WBC, MON, AST, CK, and MDA levels across exercise times and recovery, indicating reduced inflammation, muscle damage, and oxidative stress.
  • Improvement in antioxidant defense, shown by increased serum glutathione peroxidase (GPx), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC).

Mechanistic Insights from Transcriptomics and Proteomics

• Transcriptomics revealed that MLFs upregulated genes involved in:
  • Myofibrillar structural proteins, which are essential for muscle fiber integrity and repair.
  • Antioxidant defense systems, enhancing the muscle’s ability to neutralize oxidative damage.
  • Skeletal muscle satellite cells’ proliferation and differentiation, key processes in muscle regeneration.
• Proteomic analysis highlighted the involvement of specific signaling pathways:
  • PI3K-Akt pathway – linked to protein synthesis, cell survival, and muscle growth.
  • TGF-β signaling pathway – important for regulating inflammation and tissue remodeling.
  • Extracellular matrix (ECM)-receptor interactions – facilitating muscle structural support and repair.

Conclusions and Practical Implications

• MLFs effectively reduce inflammation, oxidative stress, and muscle damage caused by high-intensity exercise in Mongolian horses.
• The protective effects are mediated by enhancing antioxidant defenses, supporting muscle fiber structure, and promoting muscle cell regeneration through specific molecular pathways.
• A daily dose of 10 g of MLFs showed the most significant protective benefits under the experimental conditions.
• These findings support the use of mulberry leaf flavonoids as a natural therapeutic agent for managing exercise-induced muscle damage in athletic horses, potentially improving their performance and recovery.