In Vitro Effect of Ferrum phosphoricum D12 on Translation of Inflammation and Iron Metabolism Related Proteins in LPS-stimulated J774A.1 Mouse Macrophages

Background: Ferrum phosphoricum (FP) is a homeopathic remedy used to treat early stages of fever and colds or flu, and iron deficiency anemia. Previously, we analyzed the effects of FP D12 in vitro on cell proliferation and transcription levels of inflammation, oxidative stress, and iron metabolism-related genes in mouse J774A.1 macrophages ± bacterial lipopolysaccharides (LPS, E. coli O26:B6). In this study, we aimed to evaluate the effects of FP D12 on the translation of selected proteins, including NFκB p65, ferritin, CD86, and NADPH oxidase, in the same cell culture model. Methods: Translation levels of selected proteins were analyzed using polyacrylamide gel electrophoresis followed by Western blot analysis. Relative protein levels were quantified densitometrically using ImageJ software, with β-actin serving as an endogenous control. Statistical analyses were conducted using GraphPad Prism V6 software. The effects of FP were compared with untreated control cells and LPS-stimulated cells. Results: FP alone significantly increased protein levels of NFκB p65 (up to 3.2-fold, p < 0.001), CD86 (up to 2.9-fold, p < 0.001), and ferritin (up to 12-fold, p < 0.001) compared with untreated cells. These effects were comparable to, and in some cases exceeded, those of 200 ng/mL LPS treatment. However, FP alone did not affect NADPH oxidase levels. Under LPS stimulation, FP further enhanced the translation of CD86 (up to 2.7-fold, p < 0.001) and ferritin (up to 3.8-fold, p < 0.001) but significantly reduced LPS-induced NFκB p65 and NADPH oxidase levels. Conclusion: Our findings align with previous studies reporting immunostimulatory and iron uptake potential of FP by affecting gene expression in LPS-stimulated macrophages. This study highlights the ability of FP to modulate translation of key proteins involved in the immune response and iron metabolism. The results provide further evidence supporting FP as a potential immunomodulator and therapeutic agent for managing iron deficiency anemia, particularly through its action on beneficial protein synthesis. Acknowledgements: Alphen Pharma AG Ltd., Bulgaria, provided financial support for this study.