Iron deficiency is a global nutritional problem that adversely affects the functional regulation of the immune system. In the process of treatment through iron supplementation, the problem of excessive iron intake often occurs, which increases the level of inflammation in the body. Excessive iron can also lead to an increase in intestinal iron-requiring pathogenic bacteria and an imbalance of intestinal flora. In this study, we aim to explore the effect of Ejiao peptide–iron (EPI) chelates on the intestinal flora and inflammation of ICR mice having iron-deficiency anemia (IDA). The mice were given low, medium, and high doses of EPI and FeSO₄ (1.0, 2.0 and 3.0 mg Fe per kg weight, respectively) daily for 4 weeks by intragastric administration. IDA mice showed increased inflammation levels and decreased sIgA secretion, which were restored after intervention with EPI at different doses. Intestinal mucosal ulcers, inflammatory cell infiltration, and oxidative stress in the colon tissue were reduced, and intestinal permeability was improved. Furthermore, 16S rDNA gene sequencing revealed that EPI increased microbial diversity and richness, changing the community structure, therefore, alleviating microbiota dysbiosis caused by IDA (e.g. the proportion of Firmicutes/Bacteroides). Different from the traditional iron supplement FeSO₄, when the pathogenic bacteria (e.g. Helicobacter and Erysipelatoclostridium) increase and the beneficial bacteria (e.g. Bifidobacterium and Blautia) decrease at high doses, EPI shows higher safety at a high dose, thereby maintaining a healthier intestinal homeostasis.

Immunodeficiency can disrupt normal physiological activity and function. In this study, donkey bone collagen peptide (DP) and its iron chelate (DPI) were evaluated their potential as immunomodulators in cyclophosphamide (Cytoxan^®, CTX)-induced Balb/c mice. The femoral tissue, lymphocytes, and serum from groups of mice were subjected to hematoxylin and eosin (H&E) staining, methylthiazolyldiphenyl-tetrazolium bromide (MTT) cell proliferation assays, and enzyme-linked immunosorbent assay (ELISA), respectively. Furthermore, a non-targeted metabolomics analysis based on UPLC–MS/MS and a reverse transcription polymerase chain reaction (RT-qPCR) technology were used to explore the specific metabolic pathways of DPI regulating immunocompromise. The results showed that CTX was able to significantly reduce the proliferative activity of mouse splenic lymphocytes and led to abnormal cytokine expression. After DP and DPI interventions, bone marrow tissue damage was significantly improved. In particular, DPI showed the ability to regulate the levels of immune factors more effectively than Fe²⁺ and DP. Furthermore, metabolomic analysis in both positive and negative ion modes showed that DPI and DP jointly regulated the levels of 20 plasma differential metabolites, while DPI and Fe²⁺ jointly regulated 14, and all 3 jointly regulated 10. Fe²⁺ and DP regulated energy metabolism and pyrimidine metabolism pathways, respectively. In contrast, DPI mainly modulated the purine salvage pathway and the JAK/STAT signaling pathway, which are the key to immune function. Therefore, DPI shows more effective immune regulation than Fe²⁺ and DP alone, and has good application potential in improving immunosuppression.