Immunostimulatory Mechanisms of Water Extracts of Cirsium nipponicum Leaves and Roots: TLR2/4-ROS-p38 and JNK Signaling Pathways Mediating NO Production and iNOS Expression in RAW264.7 Cells

Abstract

Considering the reported immunostimulatory properties of other Cirsium species, it is highly plausible that Cirsium nipponicum exhibits similar immunostimulatory effects. However, definitive scientific evidence supporting the immunostimulatory activity of C. nipponicum remains insufficiently established. This study aimed to elucidate the immunostimulatory activity of C. nipponicum and its underlying mechanisms by investigating the effects of leave extracts (CNL) and root extract (CNR) from C. nipponicum on macrophage activation and the associated signaling pathways. The results demonstrated that CNL and CNR significantly enhanced NO production, iNOS expression, and phagocytic activity in RAW264.7 macrophages. However, the inhibition of TLR2 and TLR4 by C29 and TAK-242 reduced the CNL- and CNR-mediated increases in NO production, iNOS expression, and phagocytic activity. Additionally, CNL and CNR were found to induce the phosphorylation of p38 and JNK, and inhibition of these pathways by SB203580 and SP600125 attenuated the NO production and iNOS expression induced by CNL and CNR. Furthermore, TLR2 and TLR4 inhibition by C29 and TAK-242 suppressed the CNL- and CNR-mediated phosphorylation of p38 and JNK, while ROS inhibition by NAC also reduced both p38 and JNK phosphorylation as well as NO production and iNOS expression. These findings suggest that CNL and CNR activate macrophages through the TLR2/4-ROS-p38 and JNK signaling pathways, thereby regulating NO production and iNOS expressions. This study highlights the potential of CNL and CNR as an immunostimulatory agent and provides a scientific foundation for the development of natural product-based immunostimulatory agents.