Immunostimulatory activity of the aqueous extract from the leaves of Sambucus racemosa subsp. pendula through TLR4-dependent JNK activation in RAW264.7 cells

Sambucus racemosa subsp. pendula (SRP) is an endemic plant of Korea, exclusively found on Ulleungdo Island. SRP is widely used as both a traditional medicine and food source. However, there is a lack of research on the pharmacological activities of SRP. Therefore, the present study aimed to explore the potential use of SRP leaves (SRPL) as a natural immunostimulant by analyzing its macrophage activation properties and the underlying mechanisms of action. Among the various extraction conditions, SRPL (AE20-SRPL) extracted with 100% distilled water at 20 & ring;C induced the highest nitric oxide (NO) production in RAW264.7 cells. Thus, the further studies were performed using AE20-SRPL. AE20-SRPL increased the production of immunostimulatory factors such as NO, prostaglandin E2, inducible nitric oxide synthase, cyclooxygenase-2, IL-1 beta and TNF-alpha and phagocytosis in a dose-dependent manner in RAW264.7 cells without exhibiting cytotoxicity. Among Toll-like receptor (TLR)2 and TLR4, inhibition of TLR4 significantly reduced AE20-SRPL-mediated increases in the production of immunostimulatory factors and phagocytosis in RAW264.7 cells. Furthermore, in RAW264.7 cells, inhibition of JNK, one of the components of MAPK signaling along with ERK1/2 and p38, attenuated the AE20-SRPL-mediated increases in the production of immunostimulatory factors and phagocytosis. Additionally, AE20-SRPL induced the phosphorylation of JNK and inhibition of TLR4 reduced AE20-SRPL-mediated JNK phosphorylation. These results suggested that AE20-SRPL may enhance the production of immunostimulatory factors and phagocytosis through TLR4-dependent activation of JNK in macrophages. Although the present study is limited to in vitro research using a cell model, AE20-SRPL demonstrated potential as a natural material capable of inducing macrophage activation for immune enhancement.