Study on the structure-activity relationship, action pathway and site of lipopolysaccharide protecting macrophage...

Chemotherapy remains a cornerstone of cancer treatment, but its toxicity toward immune cells can compromise host defense. In this study, we investigated the structural features and cellular processes associated with the effects of low-dose lipopolysaccharide (LPS) on chemotherapy-injured macrophages. Treatment with 100 ng/mL LPS markedly restored the chemotherapy-reduced normalized CCK-8 metabolic signal in Adriamycin (ADR)- and vincristine (VCR)-treated RAW264.7 macrophages, and this response was reproduced in NR8383 rat alveolar macrophages and primary mouse peritoneal macrophages. In an indirect RAW264.7-4T1 co-culture system, LPS did not measurably alter the ADR-associated CCK-8 response or cell-cycle distribution of 4T1 tumor cells. LPS preparations from different bacterial sources produced comparable responses, while Kdo₂-lipid A partially reproduced the LPS-associated effect, supporting a contribution of lipid A-associated bioactivity. FITC-LPS imaging and flow cytometry demonstrated cellular association and an intracellular fluorescence component, and pharmacological inhibition implicated actin-dependent and other endocytosis-related processes in LPS handling by macrophages. Colchicine sensitivity further implicated microtubule-related processes, while chloroquine attenuated the LPS-associated response and qRT-PCR and western blotting revealed increased expression of multiple lysosome-related genes and proteins. Together, these findings support a model in which lipid A-associated bioactivity, endocytosis-related cellular processing, microtubule-related trafficking, and lysosome-related processes contribute to the macrophage response to low-dose LPS during chemotherapeutic injury. This study provides a theoretical basis for further exploration of the underlying molecular mechanisms of LPS and supports its potential application in immune-cell preservation during chemotherapy.