Immunomodulatory activity of a water-soluble polysaccharide extracted from mussel on cyclophosphamide-induced immunosuppressive mice models

Cyclophosphamide (Cy), a potent anticancer drug, causes gastrointestinal mucosal damage and intestinal microbiota dysbiosis, leading to immunosuppression. The gut microbiota is crucial for intestinal health, protecting against pathogens and producing short-chain fatty acids (SCFAs) that nourish intestinal cells and regulate the immune response. Polysaccharides, possessing diverse biological activities including immunomodulatory effects, are gaining attention as potential prebiotics. Mussels, a rich source of bioactive polysaccharides (MP), have shown immune-regulatory and antioxidant properties. This study hypothesized that MP could alleviate Cy-induced immunosuppression and improve intestinal health by modulating the gut microbiota.

Existing literature demonstrates that high doses of Cy damage the gastrointestinal mucosa and alter the composition of the intestinal microbiota, leading to immunosuppression. Studies have shown the immunomodulatory effects of various polysaccharides, including their ability to regulate intestinal mucosal immunity and act as prebiotics. Previous research highlights the immune-regulating, antioxidant, and cholesterol-lowering properties of mussel polysaccharides in mice. This study builds upon these findings by investigating the specific immunomodulatory effects of MP on Cy-induced immunosuppression and dysbiosis.

Forty male BALB/c mice were divided into four groups: control, Cy-treated, Cy-treated + low-dose MP (300 mg/kg), and Cy-treated + high-dose MP (600 mg/kg). Cy was administered intraperitoneally for four days to induce immunosuppression. Body weight, immune organ indices (liver, thymus, spleen), and intestinal barrier integrity were assessed. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to evaluate ileum morphology. ELISA was employed to measure SIgA, DAO, LPS, IL-2, IFN-γ, IL-4, and IL-10 levels. RT-PCR analyzed mRNA expression of cytokines and tight junction proteins. Western blotting assessed NF-κB pathway protein expression. 16S rRNA sequencing analyzed gut microbiota composition and diversity. SCFAs were quantified using gas chromatography-mass spectrometry (GC-MS). Data were analyzed using one-way ANOVA.

MP treatment significantly mitigated Cy-induced body weight loss and prevented the reduction in immune organ indices. SEM and TEM revealed that MP partially restored the damaged ileum microvilli. MP significantly reduced LPS and DAO levels. MP significantly increased the secretion and mRNA expression of IL-2, IFN-γ, IL-4, and IL-10. MP upregulated the mRNA expression of occludin, claudin-1, ZO-1, and mucin-2. Western blotting showed that MP reversed Cy-induced inhibition of the NF-κB pathway. 16S rRNA sequencing demonstrated that MP increased gut microbiota diversity, increased the abundance of *Lactobacillus*, and decreased the abundance of *Desulfovibrio*. MP increased the levels of various SCFAs. Correlation analysis revealed a positive correlation between beneficial bacteria (*Lactobacillus*, *Rikenella*) and improved immune parameters, and a negative correlation between *Desulfovibrio* and immune parameters.

The findings indicate that MP protects against Cy-induced immunosuppression by multiple mechanisms. Restoration of the intestinal barrier integrity, as evidenced by improved tight junction protein expression and reduced intestinal permeability markers, is a key aspect. The immunomodulatory effects of MP, including the increased production of Th1 and Th2 cytokines and the activation of the NF-κB pathway, contribute to enhanced immune function. Furthermore, MP's ability to modulate the gut microbiota towards a more beneficial composition, increasing beneficial bacteria and decreasing harmful ones, further supports the observed improvements in immune function. The increase in SCFAs further contributes to improved intestinal health.

This study demonstrates that MP possesses significant immunomodulatory activity, protecting against Cy-induced immunosuppression in mice. MP achieves this by restoring intestinal barrier function, modulating cytokine production, regulating the NF-κB pathway, and shaping gut microbiota composition towards a healthier state. Future research could investigate the specific mechanisms underlying MP's immunomodulatory effects and explore its potential as a therapeutic agent for treating immunosuppression.

The study was conducted in a mouse model, and the findings may not directly translate to human populations. Further studies are needed to confirm the efficacy and safety of MP in humans. The sample size (n=8 per group) could be considered relatively small, though this is a common sample size in this kind of study. Further investigations are needed to elucidate the detailed mechanisms and optimal dosage of MP for different applications.