Mechanistic Investigation of a Novel Astragalus Polysaccharide Compound in the Treatment of Iron-Deficiency Anemia Based on Gut Microbiota Modulation

<title>Abstract</title> <p> To develop a novel polysaccharide-ferrous iron supplement with high efficacy and low toxicity, <italic>Astragalus</italic> polysaccharides (APS) and Fe ²⁺ were employed as primary substrates to synthesize an <italic>Astragalus</italic> polysaccharide-iron(II) complex (APS-iron(II)). The contents of neutral sugars, proteins, and Fe ²⁺ were quantitatively determined using the phenol-sulfuric acid method, Coomassie Brilliant Blue assay, and o-phenanthroline method, respectively. An iron-deficiency anemia (IDA) rat model was established to evaluate therapeutic efficacy. Hematological parameters, serum iron metabolism indices, and antioxidant enzyme activities were assessed across multiple groups, including the normal control, model, ferrous succinate (FS), Polysaccharide Iron Complex Capsules (PIC), APS, APS-iron(III), and low-, medium-, and high-dose APS-iron(II) groups. Histopathological alterations in the liver, spleen, and kidney were examined, and 16S rRNA sequencing was conducted to characterize gut microbiota composition, thereby elucidating the anti-anemic effects and underlying mechanisms.The APS-iron(II) complex contains 75.32 ± 4.28% neutral sugars and 0.94 ± 0.74% protein, with an iron content of 7.83 ± 0.64%. Its monosaccharide composition consists of glucose (Glc), galactose (Gal), and arabinose (Ara) in a molar ratio of 85.26:10.69:4.05. APS-iron(II) significantly improved erythrocyte parameters in IDA rats, exhibiting superior iron supplementation and hematopoietic effects compared with the APS-iron(III) group. Notably, the medium- and high-dose APS-iron(II) groups outperformed the positive control treatments. In addition, APS-iron(II) markedly alleviated histopathological damage in hepatic, splenic, and renal tissues and enhanced hepatic iron storage.Gut microbiota analysis revealed that IDA markedly reduced microbial richness and diversity, characterized by a decrease in the relative abundance of Firmicutes and an increase in Bacteroidetes. Treatment with APS-iron(II) restored microbial diversity, increased the abundance of beneficial genera such as <italic>Lactobacillus</italic> and <italic>Bifidobacterium</italic> , and decreased the abundance of potentially pathogenic genera, including <italic>Desulfovibrio</italic> and <italic>Clostridium sensu stricto 1</italic> . Correlation analysis further indicated that <italic>Lactobacillus</italic> and <italic>Bifidobacterium</italic> were positively associated with erythrocyte parameters, whereas genera such as <italic>Romboutsia</italic> exhibited significant negative correlations. Collectively, these findings suggest that APS-iron(II) exerts pronounced anti-iron-deficiency anemia effects by improving iron metabolism, enhancing antioxidant capacity, and modulating the composition and abundance of the gut microbiota. Its efficacy surpasses that of commonly used clinical iron supplements, thereby providing robust experimental evidence and a theoretical foundation for the development of novel polysaccharide-based iron formulations. </p>