Nitric oxide‐induced autophagy and the activation of activated protein kinase pathway protect against apoptosis in human dental pulp cells

Aim To investigate the role of nitric oxide ( NO )‐induced autophagy in human dental pulp cells ( HDPC s) 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 HDPC s. 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 (3 MA ) 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 HDPC s in a dose‐ and time‐dependent manner. Exposing the HDPC s to SNP increased the levels of p62 and LC 3‐ 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 3 MA decreased cell viability but increased cleaved poly( ADP ‐ribose) polymerase ( PARP ) and caspase‐3, apoptosis indicators, in the SNP ‐treated HDPC s ( 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 HDPC s ( P  <   0.05). Conclusion NO induced autophagy with AMPK activation, which plays a role in the survival of HDPC s against NO ‐induced apoptosis.