S Y Park1,2,3, M Y Park1,2,3, H G Park1,2,3, K J Lee4, M S Kook1,3,5, W J Kim1,2,3, J Y Jung2,3. 1. Dental Science Research Institute and Medical Research Center for Biomineralization Disorders, Chonnam National University, Gwangju, Korea. 2. Department of Oral Physiology, Chonnam National University, Gwangju, Korea. 3. School of Dentistry, Chonnam National University, Gwangju, Korea. 4. Department of Convergence Medicine, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea. 5. Department of Oral and Maxillofacial Surgery, Chonnam National University, Gwangju, Korea.
Abstract
AIM: To investigate the role of nitric oxide (NO)-induced autophagy in human dental pulp cells (HDPCs) and the involvement of AMP-activated protein kinase (AMPK) pathway. METHODOLOGY: The MTT assay was used to determine the cytotoxic effect of the NO donor sodium nitroprusside (SNP) in HDPCs. Apoptosis was detected by means of the terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay, and apoptosis- or autophagy-related signal molecules were observed by Western blot analysis. Acidic autophagolysosomal vacuoles were stained with acridine orange to detect autophagy in the presence of 3-methyladenine (3MA) used to inhibit autophagy. To explore the mechanism underlying autophagy and its protective role against apoptosis, compound C, the chemical AMPK inhibitor, was used. Statistical analysis was performed using Student's t-test or analysis of variance (anova) followed by the Student-Newman-Keuls test (P < 0.05). RESULTS: SNP decreased viability of the HDPCs in a dose- and time-dependent manner. Exposing the HDPCs to SNP increased the levels of p62 and LC3-II, the typical markers of autophagy, and increased the number of acidic autophagolysosomal vacuoles, indicating the appearance of autophagy as detected by acridine orange staining (P < 0.05). Pre-treatment with 3MA decreased cell viability but increased cleaved poly(ADP-ribose) polymerase (PARP) and caspase-3, apoptosis indicators, in the SNP-treated HDPCs (P < 0.05). SNP activated AMPK/ULK signalling, whilst the inhibition of AMPK by compound C enhanced apoptotic cell death induced by SNP in the HDPCs (P < 0.05). CONCLUSION: NO induced autophagy with AMPK activation, which plays a role in the survival of HDPCs against NO-induced apoptosis.
AIM: To investigate the role of nitric oxide (NO)-induced autophagy in human dental pulp cells (HDPCs) and the involvement of AMP-activated protein kinase (AMPK) pathway. METHODOLOGY: The MTT assay was used to determine the cytotoxic effect of the NO donorsodium nitroprusside (SNP) in HDPCs. Apoptosis was detected by means of the terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay, and apoptosis- or autophagy-related signal molecules were observed by Western blot analysis. Acidic autophagolysosomal vacuoles were stained with acridine orange to detect autophagy in the presence of 3-methyladenine (3MA) used to inhibit autophagy. To explore the mechanism underlying autophagy and its protective role against apoptosis, compound C, the chemical AMPK inhibitor, was used. Statistical analysis was performed using Student's t-test or analysis of variance (anova) followed by the Student-Newman-Keuls test (P < 0.05). RESULTS: SNP decreased viability of the HDPCs in a dose- and time-dependent manner. Exposing the HDPCs to SNP increased the levels of p62 and LC3-II, the typical markers of autophagy, and increased the number of acidic autophagolysosomal vacuoles, indicating the appearance of autophagy as detected by acridine orange staining (P < 0.05). Pre-treatment with 3MA decreased cell viability but increased cleaved poly(ADP-ribose) polymerase (PARP) and caspase-3, apoptosis indicators, in the SNP-treated HDPCs (P < 0.05). SNP activated AMPK/ULK signalling, whilst the inhibition of AMPK by compound C enhanced apoptotic cell death induced by SNP in the HDPCs (P < 0.05). CONCLUSION: NO induced autophagy with AMPK activation, which plays a role in the survival of HDPCs against NO-induced apoptosis.