Introduction: Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system characterized by inflammation, demyelination, and neuronal dysfunction, mainly affecting young women. Experimental autoimmune encephalomyelitis (EAE) serves as a standard animal model for studying MS pathophysiology and immune responses. Sex hormones like estrogen and vitamin D are implicated in modulating immunity and reducing disease severity. This study evaluated the therapeutic effects of combined vitamin D3 and 17-beta estradiol treatment on clinical symptoms, immune responses, and gene expression in ovariectomized mice with EAE.
Methods: Thirty-two ovariectomized female C57BL/6 mice were randomly assigned to four groups: combination therapy, vitamin D3, estrogen, and control. EAE was induced by subcutaneous injection of 250 µg MOG35-55 peptide. The combination group received daily intraperitoneal vitamin D3 (100 ng/kg) and a 17-beta estradiol pellet (15 mg/21 days). Clinical scores were recorded daily up to day 21. Brain and spinal cord histology, BrdU proliferation assays, cytokine levels by ELISA, and gene expression by real-time PCR were analyzed.
Results: Combination therapy significantly reduced EAE severity at peak disease (mean score 1.1 ± 0.1 vs. 4.5 ± 0.15, p < 0.001), decreased CNS inflammatory infiltration and immune cell proliferation (p < 0.001). Pro-inflammatory cytokines (IL-6, IL-17, TNF-α, IFN-γ) decreased, while anti-inflammatory cytokines (IL-4, IL-10, TGF-β) increased (all p < 0.001). Expression of T-bet and RORγt decreased, whereas Foxp3 and GATA3 increased, favoring anti-inflammatory immune responses.
Conclusion: Combined vitamin D3 and estrogen therapy exerts synergistic immunomodulatory effects in EAE and may offer a promising approach for MS treatment.