Mitohormesis and Regeneration: Natural Compounds Chlorogenic Acid (CGA) and Isovanillic Acid 3-O-sulfate (IVAS) Boost Muscle Cell Recovery in the Equine Athlete Model.
Abstract: Skeletal muscle satellite cells ( SCs), essential for muscle regeneration, are a valuable model for studying exercise-induced stress relevant to human athletes. This study examined the effects of two natural compounds-chlorogenic acid (CGA) and isovanillic acid 3-O-sulfate (IVAS)-increasingly recognized as components of modern, nature-based recovery strategies. Their combination (Hybrid) was also tested on equine model of skeletal muscle satellite cells (ESCs) exposed to heat shock (40 °C, 1 h), mimicking exercise stress. Cells were treated with CGA (0.005%), IVAS (0.0005%), or both for 24 h post-stress. Cell viability (MTS), mitochondrial membrane potential, apoptosis (Annexin V/7-AAD), nitric oxide (NO) production, and gene expression (RT-qPCR) were assessed. CGA significantly improved viability under both normothermia and heat stress (216-227%, p < 0.05), while IVAS was effective only without stress. Only the Hybrid group maintained elongated morphology post-heat shock. CGA increased NO levels (p < 0.05), with no effect from IVAS or Hybrid. Antioxidant gene expression remained unchanged, but proinflammatory cytokines IL-6 and IL-1β were upregulated in the Hybrid group (2.74- and 5.64-fold, p < 0.01), suggesting a controlled, adaptive immune response. Early apoptosis rose in CGA and Hybrid groups (~ 34%, p < 0.05), but total cell death was lowest in the Hybrid group (6.26%). BCL2 was downregulated (p < 0.05), while BAX increased only in the Hybrid group (8.14-fold, p < 0.01). Mitochondrial genes MFN2, TFAM, and PUSL1 were significantly upregulated in the Hybrid group; MIRO1 expression increased in all treated groups. CGA and IVAS synergistically promote mitochondrial stability and ESC survival via mitochondrial activation and inflammation regulation-supporting the growing trend of using natural compounds in muscle recovery strategies.
© 2025. The Author(s).
Publication Date: 2025-09-03 PubMed ID: 40900286PubMed Central: PMC12504121DOI: 10.1007/s12015-025-10959-9Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
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Overview
- This study investigates how two natural compounds, chlorogenic acid (CGA) and isovanillic acid 3-O-sulfate (IVAS), individually and combined, affect muscle satellite cells from horses under heat stress, simulating exercise-induced damage.
- The research focuses on cell recovery, mitochondrial function, apoptosis, and inflammatory responses to understand their potential for enhancing muscle regeneration and recovery in athletes.
Background and Purpose
- Skeletal muscle satellite cells (SCs) are crucial for muscle repair and regeneration, making them an important model for exercise-related stress studies.
- Exercise induces cellular stress, including heat shock, which challenges muscle cell viability and function.
- Natural compounds such as CGA and IVAS have gained attention for their possible roles in natural recovery and regeneration strategies.
- The purpose was to test these compounds’ effects on equine skeletal muscle satellite cells (ESCs), a relevant model for human muscle recovery due to physiological similarities.
- The study specifically explored how CGA, IVAS, and their combination (Hybrid) influence cell viability, mitochondrial activity, apoptosis, nitric oxide production, and gene expression after heat-induced stress.
Methods
- ESCs were exposed to heat shock at 40 °C for 1 hour to simulate exercise stress conditions.
- Post-heat shock, cells were treated with CGA (0.005%), IVAS (0.0005%), or their combination for 24 hours.
- Various assays and measurements were conducted:
- Cell viability via MTS assay
- Mitochondrial membrane potential assessment
- Apoptosis detection through Annexin V/7-AAD staining
- Nitric oxide (NO) production measurement
- Gene expression profiling using RT-qPCR for antioxidant, inflammatory, apoptotic, and mitochondrial genes
Key Findings
- Cell Viability:
- CGA significantly enhanced cell viability under both normal and heat stress conditions (increase of ~216-227%).
- IVAS improved viability only in cells not exposed to heat stress.
- The combination (Hybrid) maintained cell viability beyond individual treatments.
- Cell Morphology:
- Only the Hybrid treatment preserved elongated morphology of ESCs after heat shock, indicating better structural preservation.
- Nitric Oxide Production:
- CGA increased NO levels, which are involved in cellular signaling and muscle repair.
- No significant NO change was seen with IVAS or Hybrid treatments.
- Gene Expression:
- Antioxidant gene expression did not significantly change, suggesting these compounds do not primarily act through antioxidant pathways.
- Proinflammatory cytokines IL-6 and IL-1β were significantly upregulated in the Hybrid group (2.74- and 5.64-fold increases), suggesting an adaptive immune response rather than harmful inflammation.
- Apoptotic genes showed differential regulation:
- BCL2 (anti-apoptotic) was downregulated.
- BAX (pro-apoptotic) was strongly upregulated only in the Hybrid group (8.14-fold).
- Mitochondrial-related genes MFN2, TFAM, and PUSL1 were significantly increased in the Hybrid group, indicating enhanced mitochondrial function and biogenesis.
- MIRO1, involved in mitochondrial transport, was upregulated in all treated groups.
- Apoptosis and Cell Death:
- Early apoptosis increased in CGA and Hybrid groups (~34%), implying activation of controlled cell turnover processes.
- The Hybrid group showed the lowest total cell death (6.26%), reflecting a protective effect possibly through mitochondrial and inflammatory regulation.
Interpretation and Implications
- The study suggests that CGA and IVAS, especially when combined, synergistically enhance the recovery and survival of muscle satellite cells after stress.
- The combination supports mitochondrial stability and function, crucial for energy production and cell survival during recovery.
- The controlled upregulation of proinflammatory cytokines may represent a beneficial, adaptive immune response that facilitates regeneration rather than damaging inflammation.
- Activation of apoptotic pathways alongside increased viability points to a fine-tuned balance between removing damaged cells and promoting muscle repair.
- These findings support the use of natural bioactive compounds in muscle recovery therapies and interventions, potentially applicable to human athletes based on the equine model.
Conclusion
- Natural compounds CGA and IVAS promote muscle cell recovery after exercise-like stress by enhancing mitochondrial function and regulating inflammatory and apoptotic responses.
- Their combined use (Hybrid) provides superior benefits, maintaining cell viability and morphology, and activating adaptive mechanisms involved in muscle regeneration.
- This research underlines the potential of nature-based compounds as effective elements in muscle recovery strategies for athletes.
Cite This Article
APA
Witkowska-Piłaszewicz O, Nowicka-Kazmierczak M, Pietrzak P, Marycz K.
(2025).
Mitohormesis and Regeneration: Natural Compounds Chlorogenic Acid (CGA) and Isovanillic Acid 3-O-sulfate (IVAS) Boost Muscle Cell Recovery in the Equine Athlete Model.
Stem Cell Rev Rep, 21(8), 2654-2666.
https://doi.org/10.1007/s12015-025-10959-9 Publication
Researcher Affiliations
- Department of Large Animals Diseases and Clinic, Institute of Veterinary Medicine, Warsaw University of Life Sciences, Nowoursynowska 166, Warsaw, 02- 787, Poland. olga_witkowska_pilaszewicz@sggw.edu.pl.
- International Institute of Translational Medicine (MIMT), ul. Jesionowa 11, Malin Wisznia Mała, 55-114, Poland.
- International Institute of Translational Medicine (MIMT), ul. Jesionowa 11, Malin Wisznia Mała, 55-114, Poland.
- International Institute of Translational Medicine (MIMT), ul. Jesionowa 11, Malin Wisznia Mała, 55-114, Poland. krzysztofmarycz@gmail.com.
- Veterinary Institute for Regenerative Cures, School of Veterinary Medicine, University of California-Davis, Davis, CA, USA. krzysztofmarycz@gmail.com.
MeSH Terms
- Animals
- Horses
- Chlorogenic Acid / pharmacology
- Apoptosis / drug effects
- Membrane Potential, Mitochondrial / drug effects
- Regeneration / drug effects
- Cell Survival / drug effects
- Mitochondria / drug effects
- Mitochondria / metabolism
- Muscle, Skeletal / drug effects
- Nitric Oxide / metabolism
Grant Funding
- INNOGLOBO/2/myo(miR)egen/10/2022 / Narodowe Centrum Badań i Rozwoju
Conflict of Interest Statement
Declarations. Ethics Approval: Muscles sampling procedures were conducted post mortem. Following the European Directive 2010/63/EU and Polish regulations on animal experimentation, ethical approval was not required and are therefore exempt from the directive. Consent to Participate: n/a. Consent for Publication: n/a. Competing Interests: The authors declare no competing interests. Conflict of interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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