Impact of melatonin on inflammatory cytokine profiles in 24-hour cultured equine uterine explants’.

Overview

• This study investigated whether melatonin modulates inflammatory cytokine gene expression in cultured equine uterine tissue over a 24-hour period.
• The researchers found that melatonin did not significantly affect cytokine expression, suggesting limited anti-inflammatory activity under the tested conditions.

Introduction and Purpose

• Melatonin is known to influence inflammatory processes in several body tissues.
• Its potential role in regulating inflammation within the mare’s uterus (endometrium) was previously unclear.
• The main goal was to determine how melatonin affects gene expression of key cytokines involved in inflammation: IL-1β, IL-6, IL-8, IL-10, and TNF-α in equine uterine explants.
• The cytokines chosen represent both pro-inflammatory (e.g., IL-1β, IL-6, IL-8, TNF-α) and anti-inflammatory (IL-10) mediators.

Methods

• The research was conducted in two parts:
• Part 1: Tested whether melatonin at different concentrations (0, 0.5, 1, 2, 4, 8 mM) was toxic to endometrial cells, ensuring cell viability for part 2 experiments.
• Part 2: Evaluated inflammatory cytokine gene expression in uterine explants cultured for 6, 12, or 24 hours.
• Fifty-four uterine explants were cultured using William’s medium under controlled oxygen (5% O₂) and carbon dioxide (5% CO₂) conditions.
• Treatments included supplementation with melatonin at 0.5 mM or 1.0 mM, or a no melatonin control, each with or without sperm to induce inflammation:
• MEL0.5- and MEL1.0-: melatonin treated without sperm.
• MEL0.5+ and MEL1.0+: melatonin treated with sperm-induced inflammation.
• MEL0- and MEL0+: no melatonin control, with or without sperm respectively.
• The study monitored expression of inflammatory cytokines at three time points: 6, 12, and 24 hours of culture.

Results

• No significant differences (P > 0.05) were found in relative gene expression of cytokines IL-6, IL-8, IL-10, and TNF-α across treatments or time points.
• IL-1β showed a significant difference only at 24 hours, where the no-treatment, no sperm control (MEL0-) had higher expression compared to 0.5 mM melatonin treated explants both with (MEL0.5+) and without sperm (MEL0.5-).
• This isolated change suggests melatonin at 0.5 mM might slightly reduce IL-1β expression after 24 hours, but the effect was not robust or consistent across other cytokines or conditions.
• Overall, melatonin supplementation did not demonstrate a clear anti-inflammatory effect on cytokine gene expression in the uterine explants exposed to sperm-induced inflammation within the 24-hour culture period.

Conclusions and Future Directions

• The study concludes that melatonin did not significantly modulate inflammatory cytokine expression in equine uterine explants under the experimental conditions used.
• Limitations may include:
• Short culture duration (24 hours may not be sufficient to observe melatonin’s effects).
• The inflammatory stimulus used (sperm) might not be ideal for eliciting cytokine responses sensitive to melatonin modulation.
• Recommendations for future research include:
• Exploring longer-term cultures to assess potential delayed effects of melatonin on uterine inflammation.
• Using alternative inflammatory challenges such as lipopolysaccharides (LPS), which are known to strongly induce immune responses.
• Further characterizing melatonin’s immunomodulatory role specifically in the mare’s endometrium to possibly identify therapeutic applications.