Ribes fasciculatum Leaf Water Extract Enhances Macrophage Immune Responses via TLR4-Mediated JNK and NF-κB Activation in RAW264.7 Cells

Abstract

This study investigated the immunostimulatory activity and underlying mechanism of Ribes fasciculatum leaf water extract in RAW264.7 macrophages. Among extracts prepared using water or ethanol-containing solvents, only the water extract induced NO production. Further evaluation of extraction temperature and time identified the extract prepared at 20°C for 24 h (RFL-DW20) as the most active sample under the tested conditions, with NO-inducing activity increasing after 12 h of extraction and reaching a plateau by 24 h. RFL-DW20 increased NO and PGE2 production, upregulated iNOS and COX-2 expression, and enhanced IL-6 and TNF-α production without cytotoxicity. RFL-DW20 also increased IL-1β mRNA expression but did not significantly alter extracellular IL-1β levels. RFL-DW20 also increased neutral red uptake, indicating enhanced macrophage functional activity. The immunostimulatory effects of RFL-DW20 were strongly inhibited by TAK-242, SP600125, and BAY 11-7082, suggesting the involvement of TLR4, JNK, and NF-κB signaling. RFL-DW20 activated MAPK and NF-κB signaling, including rapid IκBα phosphorylation/degradation and p65 phosphorylation, while pharmacological inhibition identified JNK and NF-κB as major functional mediators of the NO response. TAK-242 markedly suppressed RFL-DW20-induced JNK and p65 phosphorylation. Ethanol precipitation further enriched its NO-inducing activity. These findings suggest that RFL-DW20 activates macrophages through the TLR4/JNK/NF-κB signaling axis and may serve as a natural immune-stimulating material.