Studies On The Mechanisms Of Oridonin-enhanced Phagocytosis Of Apoptotic Cells By U937Cell

Macrophages rapidly engulf and remove apoptotic cells to limit the release of noxious cellular contents and to restrict autoimmune diseases or inflammation, which is important for organism’s homeostasis. This dissertation reports the mechanisms of oridonin-induced phagocytosis of UV-irradiated apoptotic U937cells (human histocytic lymphoma) by differentiated U937macrophage-like cells.The results showed that pre-incubation of U937cell-derived macrophages with low doses of oridonin (0.6-2.5μM) significantly augmented phagocytosis of UV-irradiated (52.1J/m2) U937cells undergoing apoptosis. This effect of oridonin may contribute to its antitumor activities. Further, oridonin-induced phagocytic stimulation was significantly suppressed by inhibitors, for a phosphoinositide3kinases (PI3K)(wortmannin), a phospholipase C (PLC)(U73122), Ras or Raf, extracellular signal-related kinase (ERK)(PD98059), nuclear factor (NF-кB)(PDTC) and COX-2(NS398). Exposure of U937cells to oridonin caused an increase in expression of PI3K-Akt and PLC y. Oridonin also activated Ras-Raf-ERK and NF-кB/COX-2pathways, mediating the release of IL-1β. These results demonstrated that PI3K-Akt, Ras/Raf/ERK, and NF-кB/COX-2/IL-1β partially contributed to phagocytic activity of oridonin.Meanwhile,2.5μM oridonin induced autophagy in U937macrophage-like cells through activating ERK-NF-кB pathway. Autophagic inhibitor3-methyladenine (3MA) and Beclin-1(known as autophagic regulator) siRNA decreased phagocytosis. Autophagy enhanced oridonin-induced phagocytosis through feedback regulation of ERK, NF-кB-and caspase-1-mediated IL-1β release.In addition, oridonin induced the generation of reactive oxygen species (ROS) in U937cells in a time dependent manner, and hydrogen peroxide (H2O2) and hydroxyl free radical (OH) were the major players. Phagocytosis and autophagy were suppressed by ROS scavengers of N-acetyl-L-cysteine (NAC), catalase or Glutathione (GSH). H2O2and OH were positive regulators involving in oridonin-enhanced engulfment of apoptotic cells through down-regulating mitochondrial membrane potential (MMP) and inducing autophagy. ROS mediated phagocytosis was independent on cellular adenosine triphosphate (ATP) levels. H2O2and OH generation also activated PI3K-Akt and PLC y-Ras-Raf-ERK signaling pathways, which were essential for oridonin-induced engulfment of apoptotic cells. Moreover,2.5μM oridonin caused up-regulation of inducible nitric oxide synthase (iNOS) expression and continuous endogenous generation of nitric oxide (NO), which was reversed by pre-treatment with the inhibitors of nitric oxide synthase1400W or L-NAME.1400W or L-NAME impaired oridonin-induced stimulation of efferocytosis and autophagy. NO donor sodium nitroprusside (SNP) and efferocytosis irritant lipopolysaccharide (LPS) could also exert NO generation, iNOS expression and phagocytosis as well as autophagy, similar to the effect of oridonin. NO augmented the oridonin-induced efferocytosis of apoptotic cells by mediating autophagy and activating the NF-KB-COX-2-IL-1β pathway. There existed a positive feedback loop between NO generation and NF-кB-COX-2-IL-1β pathway. However, autophagy functions as regulators to clear the overmuch inflammation and maintains inter environment homeostasis.