Endocrinol Metab.  2014 Sep;29(3):371-378. 10.3803/EnM.2014.29.3.371.

Ghrelin Inhibits Oligodendrocyte Cell Death by Attenuating Microglial Activation

• Lee JY
• Yune TY

• KMID: 2169475
• DOI: http://doi.org/10.3803/EnM.2014.29.3.371

Abstract

BACKGROUND
Recently, we reported the antiapoptotic effect of ghrelin in spinal cord injury-induced apoptotic cell death of oligodendrocytes. However, how ghrelin inhibits oligodendrocytes apoptosis, is still unknown. Therefore, in the present study, we examined whether ghrelin inhibits microglia activation and thereby inhibits oligodendrocyte apoptosis.
METHODS
Using total cell extracts prepared from BV-2 cells activated by lipopolysaccharide (LPS) with or without ghrelin, the levels of p-p38 phosphor-p38 mitogen-activated protein kinase (p-p38MAPK), phospho-c-Jun N-terminal kinase (pJNK), p-c-Jun, and pro-nerve growth factor (proNGF) were examined by Western blot analysis. Reactive oxygen species (ROS) production was investigated by using dichlorodihydrofluorescein diacetate. To examine the effect of ghrelin on oligodendrocyte cell death, oligodendrocytes were cocultured in transwell chambers of 24-well plates with LPS-stimulated BV-2 cells. After 48 hours incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate nick end labeling staining were assessed.
RESULTS
Ghrelin treatment significantly decreased levels of p-p38MAPK, p-JNK, p-c-Jun, and proNGF in LPS-stimulated BV-2 cells. ROS production increased in LPS-stimulated BV-2 cells was also significantly inhibited by ghrelin treatment. In addition, ghrelin significantly inhibited oligodendrocyte cell death when cocultured with LPS-stimulated BV-2 cells.
CONCLUSION
Ghrelin inhibits oligodendrocyte cell death by decreasing proNGF and ROS production as well as p38MAPK and JNK activation in activated microglia as an anti-inflammatory hormone.

Keyword

Ghrelin; p38MAPK; c-Jun N-terminal kinase; Pro-nerve growth factor; Reactive oxygen species; Oligodendroglia; BV-2 microglia cell

MeSH Terms

Apoptosis
Blotting, Western
Cell Death*
Cell Extracts
DNA Nucleotidylexotransferase
Ghrelin*
JNK Mitogen-Activated Protein Kinases
Microglia
Oligodendroglia*
Phosphotransferases
Protein Kinases
Reactive Oxygen Species
Spinal Cord
Cell Extracts
DNA Nucleotidylexotransferase
Ghrelin
JNK Mitogen-Activated Protein Kinases
Phosphotransferases
Protein Kinases
Reactive Oxygen Species

Figure

• 

  Fig. 1 Ghrelin 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. 2 Ghrelin 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. 3 Ghrelin 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.

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