Activation of phosphoinositide 3-kinase, protein kinase C, and extracellular signal-regulated kinase is required for oridonin-enhanced phagocytosis of apoptotic bodies in human macrophage-like U937 cells

Our previous study showed that oridonin isolated from Rabdosia rubescens enhanced phagocytosis of apoptotic cells by macrophage-like U937 cells through tumor necrosis factor (TNF) alpha and interleukin (IL)-1 beta release. In this study, we further investigated signaling events involved in oridonin-augmented phagocytosis. Phagocytic stimulation was significantly suppressed by inhibitors, including a phosphoinositide 3-kinases (PI3K) inhibitor (wortmannin), a protein kinase C (PKC) inhibitor (stauroporine), and a phospholipase C (PLC) inhibitor (U73122). Exposure of U937 cells to oridonin caused an increase in PKC activity time-dependently, which was prevented by pretreatment with inhibitors of PI3K and PLC. Simultaneously, the activation of protein kinase B (PKB/Akt) and the increased expression of PLC gamma 2 were also blocked by wortmannin. In addition, an extracellular signal-regulated kinase (ERK) MAPK inhibitor, PD98059, suppressed oridonin-augmented phagocytosis, whereas the p38 MAPK inhibitor (SB203580) and c-Jun N-terminal kinase (JNK) MAPK inhibitor (SP98059) had no inhibitory effect. Furthermore, pretreatment of U937 cells with anti-TNF alpha and anti-IL-1 beta antibodies blocked oridonin-induced phagocytic stimulation as well as phosphorylation of EPK, but did not block the activation of PKC, indicating that these signaling events are triggered by oridonin, whereas secreted TNF alpha or IL-1 beta only activate the ERK-dependent pathway. Taken together, oridonin is suggested to enhance phagocytosis of apoptotic bodies by activating PI3K, PKC, and EPK-dependent pathways.