Leukotriene B₄(LTB4) is lipid mediator derived from membrane phospholipids during the process of inflammation, having many roles(ie; inducer of chemotaxis, the production of nitric oxide, transepithelial migration of neutrophil). The major activities of LTB4 include the recruitment and activation of leukocytes, suggesting that it may involve the process for transendothelial migration of nuclear cells in bone marrow environment. Reactive Oxygen Species (ROS) have a cell signaling roles that are involved in signal transduction cascades of numerous growth factor-, cytokine-, and hormone-mediated pathways, and regulate many biological systems. In this present study, we focused on the role of LTB4 and ROS on transmigration of bone marrow nuclear cells across endothelial or stromal cell monolayer.

MS-5, murine stromal cell line cells, or bEnd.3, murine microvascular cell line cells, were grown to confluence on microporous transwell membrane. Murine marrow cells were placed on top of the prepared transwell membrane. The transwells were then seated in wells containing media and LTB4 with or without pretreatment of N-acetylcysteine(NAC), an oxygen free radical scavenger, or diphenylene iodonium(DPI), an inhibitor of NADPH oxidase-like flavoproteins. Cells that migrated through the stromal or endothelial layer into the wells were assayed for transendothelial migration.

The numbers of migrated bone marrow nuclear cells through the bEnd.3 were increased by treatment of LTB4(control, 12.5±0.2%; 50nM, 22.7±0.9%; 100nM, 44.3±1.4%; 200 nM, 36.3±0.9%; p<0.05). The numbers of migrated bone marrow nuclear cells through the MS-5 were also increased by treatment of LTB4(control, 11.0±0.9%; 50nM, 25.7±0.9%; 100nM, 35.8±1.8%; 200nM, 32.1±0.9%; p<0.05). However, increasing effect of LTB4 to the transmigration of bone marrow nuclear cells through the MS-5 or bEnd.3 were inhibited by pretreatment of NAC or DPI.

Through our data, it is suggested that LTB4 could induce the transmigration of bone marrow nuclear cells and ROS might be involved on the transendothelial migration of bone marrow nuclear cells by LTB4. It would be very interesting to test the effects of LTB4 and ROS on stem cell mobilization and homing in the future.

Reactive oxygen species (ROS) such as hydrogen peroxide, superoxide anion and hydroky radicals are produced in various physiologic and pathologic conditions and involved in many cellular processes as proliferation, differentiation and apoptosis. Studies investigating the role of ROS in various cellular behaviors especially in proliferation and apoptosis have been widely conducted in many cell types but the role of ROS in nontransformed human hepatocyte differentiation has not been investigated yet. thus we were going to elucidate the roleof ROS on human hepatocyte differentiaiton using sodium butyrate (SB) induced hepatocyte differentiation model of our own establishment.

Intracellular ROS and apoptotic cell death were monitored by flowcytometry using peroxide sensitive probe (Dicholorofluorscein diacetate) and Annexin V/Propidium iodide, respectively. Urea nitrogen in culture media was measured by colorimetric methods. Ornithine transcarbamylase(OTC) and albumin trasncription was evaluated by RT-PCR.

Intracellular ROS production was increased by SB. SB induced urea production was significantly decreased with antioxidant treatment (p<0.05) and SB induced OTC and albumin transcription were also attenuated with antioxidant treatment. SB induced increase in apoptosiswas significantly inhibited by antioxidant treatment (p<0.05).

ROS produced during the process of sodium butyrate induced human hepatocyte differentiation auguments hepatoctye differentiation and apoptosis.