Activated Toll-like Receptor 4 is Involved in Oridonin-induced Phagocytosis Via Promotion of Migration and Autophagy-lysosome Pathway in RAW264.7 Macrophages

In our previous study, we demonstrated that oridonin enhances phagocytosis of apoptotic bodies by macrophage-like cells by inducing autophagy. However, the direct sensor of autophagy and the key event controlling phagocytosis remains unknown. Herein, we showed that Toll-like receptor 4 (TLR4), known to mediate immune responses, was activated by oridonin. Activated TLR4 contributes to phagocytosis of apoptotic cells by RAW264.7 macrophages. Indeed, inhibition or small interfering RNA (siRNA) silencing of TLR4 significantly attenuated oridonin-induced phagocytosis. Inhibition of TLR4 also decreased the level of autophagy and its associated proteins, Beclin-1 and light chain 3 (LC3), suggesting that activated TLR4 is involved in activation of autophagy. LPS-induced activation of TLR4 promoted phagocytosis and autophagy progression. Activation of TLR4 accompany increase in activities of lysosome acid phosphatase and cathepsin B as well as in up-regulation of lysosomal-associated membrane protein (LAMP 1 and 2) levels. Furthermore, TLR4 in association with translocation to cytoplasm leads to macrophage motility or migration through increased plasticity of skeleton and/or membrane structure. These results suggest that oridonin-induced phagocytosis of apoptotic bodies by macrophages is TLR4 signal pathway-mediated, via activation of the autophagy-lysosome pathway as well as increase of cell migration.