Mitigation of carbon tetrachloride-induced hepatic injury by methylene blue, a repurposed drug, is mediated by dual inhibition of GSK3β downstream of PKA.

BACKGROUND AND
PURPOSE: 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. EXPERIMENTAL APPROACH: 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. KEY
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 AND IMPLICATIONS: 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.