Low-level Nd:YAG laser inhibiting inflammation and oxidative stress in human gingival fibroblasts via AMPK/SIRT3 axis

Abstract

ObjectivePhotobiomodulation is extensively employed in the management of chronic inflammatory diseases such as periodontitis because of its anti-inflammatory and antioxidant effects. This study used low-level Nd:YAG laser to investigate the mechanism of photobiomodulation as well as the role of adenosine monophosphate-activated protein kinase (AMPK) and Sirtuins (SIRT) 3 in it, providing new clues for the treatment of periodontitis. MethodsHuman gingival fibroblasts (HGFs) were extracted from gingiva and stimulated with LPS. The suitable parameters of Nd:YAG laser were chosen for subsequent experiments by detecting cell viability. We assessed the level of inflammation and oxidative stress as well as AMPK and SIRT3. The mechanism for AMPK targeting SIRT3 modulating the anti-inflammatory and antioxidant effects of photobiomodulation was explored by the AMPK inhibitor (Compound C) test, cell transfection, western blot, and immunofluorescence. ResultsHGFs were isolated and identified, followed by the identification of optimal Nd:YAG laser parameters (60 mJ, 15 Hz, 10s) for subsequent experimentation. With this laser, inflammatory factors (IL-6, TNF-α, COX2, and iNOS) decreased as well as the phosphorylation and nuclear translocation of NFκB-P65. SOD2 was up-regulated but reactive oxygen species (ROS) was down-regulated. The laser treatment exhibited enhancements in AMPK phosphorylation and SIRT3 expression. The above effects could all be reversed by Compound C. Silencing AMPK or SIRT3 by siRNA, the down-regulation of COX2, iNOS, and ROS by laser was inhibited. SIRT3 was down-regulated when the AMPK was silenced. ConclusionLow-level Nd:YAG laser activated AMPK-SIRT3 signaling pathway, facilitating the anti-inflammatory and antioxidative activity.