# iTRAQ-Based Proteomics Reveals the Potential Mechanisms Underlying Diet Supplementation with Stevia Isochlorogenic Acid That Alleviates Immunosuppression in Cyclophosphamide-Treated Broilers

## TL;DR

This study shows that stevia isochlorogenic acid can boost immunity in chickens treated with antibiotics, offering a natural alternative to reduce antibiotic use in farming.

## Contribution

The study identifies specific immune pathways and molecular targets through which SICA alleviates immunosuppression in broilers.

## Key findings

- SICA increased immune organ indices and immune molecule levels in broilers.
- Proteomics revealed SICA activates immune pathways like IgA production and cell adhesion.
- Molecular docking showed strong binding affinity between SICA and p67phox.

## Abstract

Overuse of antibiotics in poultry farming can weaken broiler immune systems, increasing disease susceptibility and threatening food safety. Safe natural alternatives are therefore essential for supporting immune function. In this study, we investigated the immunomodulatory effects of stevia isochlorogenic acid (SICA) in an immunosuppressed broiler model. The results showed that SICA repaired damaged immune organs and increased the levels of key immune molecules in the blood, which are critical for effective disease resistance. Our analyses further indicated that SICA may exert its effects by activating the body’s innate defense pathways, enhancing immune cell communication, and improving the capacity to eliminate harmful pathogens. In summary, SICA effectively restored immunity in broilers through multiple coordinated mechanisms. These findings suggest that SICA could serve as a valuable natural feed additive to help farmers raise healthier broilers and reduce antibiotic dependence.

The extensive use of antibiotics in intensive farming weakens immunity and threatens food safety. Stevia isochlorogenic acid (SICA), a kind of dicaffeoylquinic acid derived from stevia residue, exhibits strong antioxidant activity. This study evaluated the ability of SICA to improve immune function in an immunosuppressed broiler model. SICA significantly increased the spleen, thymus, and bursa of Fabricius indices (p < 0.05), alleviated spleen damage, and elevated serum interleukin-2 (IL-2), IL-4, interferon-γ, IL-1β, tumor necrosis factor-α, immunoglobulins (IgA, IgM, IgG), and complement components C3 and C4 (p < 0.05). Isobaric tags for relative and absolute quantification-based proteomics indicated that SICA enhanced splenic immune function by activating cell adhesion molecules, phagosomes, and the intestinal immune network for IgA production pathways. Quantitative PCR analysis showed upregulation of mRNA and protein levels of B-cell receptor, major histocompatibility complex class II, protein tyrosine phosphatase receptor type C, and neutrophil cytosolic factor 2 (p67phox) and downregulation of C-C motif chemokine receptor 9. Molecular docking demonstrated the strongest binding affinity between SICA and p67phox. Overall, SICA effectively alleviated immunosuppression in broiler chickens and represents a promising natural alternative to antibiotic feed additives.