Nitric oxide induces apoptosis in human gingival fibroblast through mitochondria-dependent pathway and JNK activation

• Published in: International endodontic journal Vol. 48; no. 3; pp. 287 - 297
• Main Authors: Baek, M.-W., Seong, K.-J., Jeong, Y.-J., Kim, G.-M., Park, H.-J., Kim, S.-H., Chung, H.-J., Kim, W.-J., Jung, J.-Y.
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01-03-2015
• Subjects: Anthracenes - pharmacology; apoptosis; Apoptosis - drug effects; Bcl-2 family; bcl-2-Associated X Protein - metabolism; Blotting, Western; Butadienes - pharmacology; Cell Survival - drug effects; Cells, Cultured; Cytochromes c - metabolism; Dentistry; Enzyme Inhibitors - pharmacology; Fibroblasts - drug effects; Gingiva - cytology; human gingival fibroblast; Humans; Imidazoles - pharmacology; JNK; JNK Mitogen-Activated Protein Kinases - metabolism; Membrane Potentials - drug effects; Mitochondria - metabolism; nitric oxide; Nitric Oxide - pharmacology; Nitriles - pharmacology; Nitroprusside - pharmacology; Pyridines - pharmacology; Signal Transduction - drug effects; apoptosis; nitric oxide; human gingival fibroblast; JNK; Bcl-2 family
• ISSN: 0143-2885; 1365-2591
• DOI: 10.1111/iej.12314

Aim
To investigate the molecular mechanisms of nitric oxide (NO)‐induced cytotoxic effect in human gingival fibroblast (HGF) cells.
Methodology
After sodium nitroprusside (SNP), as NO donor, was treated to HGF, viability was measured by MTT assay and apoptosis was determined by TUNEL and DNA fragmentation assay. Mitochondrial membrane potential was detected using confocal microscopy, and caspase activity assay was measured by spectrophotometer. Mitogen‐activated protein kinases (MAPK) activation, Bax/Bcl‐2 ratio and cytochrome c release were analysed by Western blot analyses. Cells were exposed to MAPK inhibitors (U0126, SB203580 and SP600125) before SNP treatment to investigate the effects of MAPK kinases on the NO‐induced apoptosis in HGF. Statistical analysis was performed using one‐way analysis of variance with the Student–Newman–Keuls post hoc test for multiple group comparison.
Results
Apoptosis was significantly increased (P = 0.011 and 0.0004, respectively) in the presence of SNP (1 and 3 mmol L−1) after 12 h in HGF. However, 1H‐[1,2,4] oxadiatolo [4, 3‐a] cluinoxaline‐1‐one (ODQ), a soluble guanylate cyclase inhibitor, did not block the decrement of cell viability by NO. SNP treatment induced the loss of mitochondrial membrane potential, release of cytochrome c, increased Bax/Bcl‐2 ratio and activation of caspases in HGF. Also, SNP treatment increased phosphorylation of MAPKinases and c‐Jun N‐terminal kinase (JNK) inhibitor (5 and 10 μmol L−1) rescued cell viability decreased by SNP in HGF (P = 0.024 and 0.0149, respectively).
Conclusion
Nitric oxide induced apoptosis in human gingival fibroblast through the mitochondria‐mediated pathway by regulation of Bcl‐2 family and JNK activation.