title: Mitigation of carbon tetrachloride-induced hepatic injury by methylene blue, a repurposed drug, is mediated by dual inhibition of GSK3β downstream of PKA.
abstract: OBJECTIVE
  Methylene blue (MB) has recently been considered for new therapeutic applications. In this study, we investigated whether MB has antioxidant and mitochondria-protecting effects and can prevent the development of toxicant-induced hepatitis. In addition, we explored the underlying basis of its effects.
  
  
  METHODS
  Blood biochemistry and histopathology were assessed in mice injected with CCl4 (0.5 mL·kg(-1)) following MB administration (3 mg·kg(-1) ·day(-1), 3 days). Immunoblottings were performed to measure protein levels. Cell survival, H2 O2 , and mitochondrial superoxide and membrane permeability transition were determined in HepG2 cells.
  
  
  RESULTS
  MB protected cells from oxidative stress induced by arachidonic acid plus iron; it restored GSH content and decreased the production of H2 O2 . It consistently attenuated mitochondria dysfunction, as indicated by inhibition of superoxide production and mitochondrial permeability transition. MB inhibited glycogen synthase kinase-3β (GSK3β) and protected the liver against CCl4. Using siRNA, the inhibition of GSK3β was shown to depend on AMPK. MB increased the activation of AMPK in vitro (3-24 h) and in vivo. MB also increased the phosphorylation of liver kinase B1 (LKB1) via cAMP-dependent PKA. SiRNA knockdown of LKB1 eliminated phosphorylation of AMPK and inhibited MB activation of AMPK. In addition, MB treatment (≤1 h) facilitated PKA-mediated GSK3β serine phosphorylation independently of AMPK.
  
  
  CONCLUSIONS
  MB has antioxidant and mitochondria-protecting effects and protects the liver from toxicants, which results from the dual inhibition of GSK3β by AMPK downstream of PKA-activated LKB1, and PKA itself. Our findings reveal a novel pharmacological effect of MB and its molecular basis.
authors.affiliation: College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Korea.
authors.forename: Hong Min
authors.initials: HM
authors.lastname: Wu
authors.forename: Chan Gyu
authors.initials: CG
authors.lastname: Lee
authors.forename: Se Jin
authors.initials: SJ
authors.lastname: Hwang
authors.forename: Sang Geon
authors.initials: SG
authors.lastname: Kim
mesh_terms: D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases / Q000378:metabolism; D000818:Animals; D002251:Carbon Tetrachloride; D002470:Cell Survival / Q000187:drug effects; D056486:Chemical and Drug Induced Liver Injury / Q000188:drug therapy / Q000209:etiology / Q000378:metabolism* / Q000473:pathology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D058492:Drug Repositioning; D005978:Glutathione / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008297:Male; D008751:Methylene Blue / Q000494:pharmacology* / Q000627:therapeutic use; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D020011:Protective Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D020011:Protective Agents; D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide; D002251:Carbon Tetrachloride; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D017346:Protein Serine-Threonine Kinases; C109790:STK11 protein, human; D017868:Cyclic AMP-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases; D005978:Glutathione; D008751:Methylene Blue
keywords: AMPK; GSK3β; LKB1; PKA; methylene blue