Abstract: Objective: This study investigated the regulatory effects and underlying mechanisms of a preparation comprising Astragali Radix, Moslae Herba, and Saccharomyces cerevisiae fermentation broth (AMSFP) on immune function and intestinal homeostasis in rats exposed to a composite stress environment simulating space microgravity and potential microbial risks, with a view to evaluating its application potential for long-duration spaceflight mission. Methods: A rat model of gut disorders was established under simulated microgravity conditions via tail suspension combined with oral gavage with Escherichia coli (E. coli). During the 4-week intervention period, rats received continuous administration of AMSFP at low, medium, or high doses. Immune function was assessed by measuring footpad swelling, 50% hemolytic complement (HC50) values, complete blood counts, and thymus and spleen indices. Hematoxylin and eosin (HE) staining was employed to observe histopathological changes in the spleen and jejunum, and 16S rRNA gene sequencing was performed to analyze variations in the gut microbiota composition. Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to quantify fecal short-chain fatty acids (SCFAs) levels. To evaluate the role of AMSFP in regulating the gut microbiota and repairing intestinal damage, enzyme-linked immunosorbent assay (ELISA) was applied to determine the serum and jejunal inflammatory cytokine levels and the expression of jejunal zonula occludens-1 (ZO-1), Toll-like receptor 4 (TLR4) and reticuloendotheliosis viral oncogene homolog A (RelA). The chemical components in AMSFP were identified by using UPLC-quadrupole time of flight/mass spectrometry (UPLC-Q-TOF MS). Results: Compared with the model group, the medium and high doses of AMSFP significantly improved the general conditions and body mass of rats, increased the thymus and spleen indices, enhanced the delayed-type hypersensitivity responses (P < 0.05), and elevated the serum HC50 value (P < 0.05). Histopathological examination revealed that AMSFP alleviated splenic white pulp atrophy and jejunal mucosal damage. Gut microbiota analysis showed that AMSFP intervention upregulated the relative abundance of Romboutsia and induced an increase in the levels of SCFAs such as acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid. Additionally, AMSFP significantly attenuated proinflammatory cytokine levels in both serum and jejunal tissue, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interferon-γ (IFN-γ), interleukin-1β (IL-1β), and transforming growth factor-β1 (TGF-β1). High-dose AMSFP significantly upregulated the expression of ZO-1 (P < 0.000 1) and downregulated the expression of RelA and TLR4 (P < 0.01, P < 0.001). A total of 25 chemical components were identified from AMSFP, including amino acids, isoflavones and triterpenoid saponins. Conclusion: AMSFP demonstrates efficacy in improving survival status and optimizing gut microbiota composition in rats exposed to simulated space composite stress environments through a multi-component and multi-target mechanism. By promoting the production of SCFAs, it alleviates intestinal inflammation and repairs the intestinal mucosal barrier through inhibition of the TLR4/RelA signaling pathway, while exerting synergistic immunomodulatory effects. Mechanistically, AMSFP reduces inflammatory responses by lowering the serum levels of pro-inflammatory cytokines TNF-α, IL-6, and IFN-γ; it also exerts intestinal protective effects by increasing SCFAs levels, inhibiting the expression of TLR4 and RelA proteins, promoting ZO-1 protein expression, and alleviating intestinal mucosal damage, thereby achieving dual regulation of immune function and intestinal homeostasis.

Key words: simulated microgravity; fermentation broth-based preparation; immune regulation; intestinal tight junction proteins; Toll-like receptor 4/nuclear transcription factor-B signaling pathway