Fig. 1Ghrelin inhibits p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) activation in BV-2 microglia after lipopolysaccharide (LPS) stimulation. BV-2 cells were seeded in 6-well plates (5×105 cells/well) and treated with ghrelin (1, 10, 100, and 1,000 nM) for 30 minutes prior to LPS (100 ng/mL) treatment. (A) Western blots of phosphor-p38MAPK (p-p38MAPK), p-JNK, and p-c-Jun at 30 minutes after LPS treatment. (B-D) Quantitative analysis of Western blots shows that ghrelin significantly inhibited the level of p-p38MAPK, p-JNK, and p-c-Jun as compared with LPS-treated cells. Values are expressed as mean±SD of three separate experiments. aP<0.05 vs. control; bP<0.05 vs. LPS-treated control; cP<0.01 vs. LPS-treated control.
Fig. 2Ghrelin inhibits pro-nerve growth factor (proNGF) and reactive oxygen species (ROS) production in BV-2 microglia after lipopolysaccharide (LPS) stimulation. BV-2 cells were seeded in 6-well or 24-well plates and treated with ghrelin (1, 10, 100, and 1,000 nM) 30 minutes before LPS (100 ng/mL) treatment. (A) Western blot of proNGF at 4 hours after LPS treatment. (B) Quantitative analysis of Western blots shows that ghrelin treatment significantly inhibited the expression of proNGF in a dose dependent manner. (C) Dichlorodihydrofluorescein (DCF) fluorescence in BV-2 at 12 hours was increased by LPS and decreased by ghrelin (1,000 nM). Scale bar, 10 µm. (D) Quantitative analysis of DCF fluorescence shows that ghrelin significantly decreased ROS production when compared to the LPS-treated control (CTR). Values are expressed as mean±SD of three separate experiments. aP<0.05 vs. control; bP<0.05 vs. LPS-treated control; cP<0.01 vs. LPS-treated control.
Fig. 3Ghrelin inhibits cell death of oligodendrocytes cocultured with BV-2 cells activated by lipopolysaccharide (LPS). For oligodendrocyte/microglia cocultures, BV-2 cells were grown on porous upper inserts of transwell chambers in 24-well plates. After treatment with LPS (100 ng/mL) for 30 minutes, the inserts containing BV-2 were placed above mature oligodendrocyte culture in 24-well plates, allowing diffusion of soluble molecules. Treatment with ghrelin occured 30 minutes before LPS treatment. (A) Cell viability measured by MTT reduction assay at 48 hours. (B) Quantitative analysis of transferase-mediated deoxyuridine triphosphate-biotin nick end labeling: terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling/myelin basic protein (TUNEL)/MBP-positive oligodendrocytes at 48 hours. Values are expressed as mean±SD of three separate experiments. OL, oligodendrocytes. aP<0.05 vs. control; bP<0.05 vs. LPS-treated control.