Radix pseudostellariae Saponins Promote Immunocyte Migration and Chemotaxis via the CCL5/CCR4 Signaling Axis

Radix pseudostellariae saponins (RPS) enhance immune responses in animals; however, the regulatory mechanisms of these effects remain unclear. This study observed that 14 days post-intranasal immunization with RPS and a Mycoplasma gallisepticum-attenuated vaccine (MGAV), MGAV-specific antibody titers were significantly increased in the blood, and chemokine (C-C motif) ligand 5 (CCL5) messenger RNA expression was significantly increased in the trachea and blood of chickens. Transcriptomic analysis demonstrated that RPS treatment significantly upregulated specific Kyoto Encyclopedia of Genes and Genomes pathways, notably the cytokine–cytokine receptor interaction pathway, which is linked to immune cell migration and involves chemokine receptor chemokine (C-C motif) receptor 4 (CCR4). This finding was corroborated at the protein level by immunohistochemical evidence showing increased CCL5 expression in tracheal tissue. In vitro studies showed that RPS enhanced the phagocytic capacity of RAW264.7 macrophages against ovalbumin, with immunofluorescence revealing time-dependent and dose-dependent CCL5 in these cells. Transwell and scratch-healing assays confirmed that RPS promoted this migration of both RAW264.7 cells and CCR4-positive lymphocytes. Collectively, the findings revealed that RPS modulated the activation, chemotaxis, and migration of macrophages and lymphocytes and is associated with the promotion of the CCL5/CCR4 signaling axis, providing novel evidence for the immune-enhancing effects of RPS by enhancing immunogenicity.