p38 MAPK-associated autophagic response restrains TLR4/MyD88 signaling in RT-treated macrophages.

Ricin toxin (RT) is a highly potent plant-derived toxin that causes severe cellular injury and inflammatory responses, yet effective therapeutic interventions remain limited. Autophagy is a conserved stress-response pathway that maintains intracellular homeostasis and modulates innate immune signaling. However, whether autophagy contributes to macrophage adaptation during RT intoxication and how this process is regulated remain incompletely understood. In this study, we investigated RT-induced autophagic responses and their relationship with TLR4/MyD88 signaling in RAW264.7 macrophages using transmission electron microscopy, immunofluorescence staining, tandem mRFP-eGFP-LC3 reporter analysis, Western blotting, and Atg5 knockdown/overexpression approaches. RT treatment induced autophagic vacuole formation, increased LC3 puncta, and elevated the expression of LC3-II, Atg5, Beclin-1, and p62, with LC3 puncta peaking at 2 h after stimulation. Tandem LC3 reporter analysis further supported activation of autophagic flux during the early response to RT. Pharmacological inhibition of autophagy with 3-methyladenine reduced LC3-II accumulation and aggravated RT-associated loss of cell viability, suggesting a cytoprotective role of autophagy. Mechanistically, RT increased p38 MAPK phosphorylation, and inhibition of p38 MAPK activity with SB203580 attenuated RT-induced LC3 puncta formation, whereas JNK inhibition showed no obvious effect under the same conditions. Furthermore, Atg5 knockdown enhanced RT-induced TLR4 and MyD88 protein expression and IL-1β secretion, whereas Atg5 overexpression suppressed these responses; TRIF expression was not markedly altered. These findings suggest that RT triggers an early p38 MAPK-associated autophagic response in macrophages and that Atg5-dependent autophagy restrains TLR4/MyD88-associated innate immune signaling. This study provides mechanistic insight into the adaptive cellular response to RT exposure and identifies autophagy as a potential modulatory pathway in RT-induced macrophage injury.