Ribes fasciculatum Leaf Water Extract Enhances Macrophage Immune Responses via TLR4-Mediated JNK and NF-κB Activation in RAW264.7 Cells.
Park So Jung SJ, Choi Jeong Won JW, Choi Hyeok Jin HJ, Im Gyeong Eun GE et al.
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.