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 PurposeMethylene 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 ApproachBlood biochemistry and histopathology were assessed in mice injected with CCl4 (0.5mLkg(-1)) following MB administration (3mgkg(-1)day(-1), 3 days). Immunoblottings were performed to measure protein levels. Cell survival, H2O2, and mitochondrial superoxide and membrane permeability transition were determined in HepG2 cells. Key ResultsMB protected cells from oxidative stress induced by arachidonic acid plus iron; it restored GSH content and decreased the production of H2O2. It consistently attenuated mitochondria dysfunction, as indicated by inhibition of superoxide production and mitochondrial permeability transition. MB inhibited glycogen synthase kinase-3 (GSK3) and protected the liver against CCl4. Using siRNA, the inhibition of GSK3 was shown to depend on AMPK. MB increased the activation of AMPK in vitro (3-24h) 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 (1h) facilitated PKA-mediated GSK3 serine phosphorylation independently of AMPK. Conclusions and ImplicationsMB has antioxidant and mitochondria-protecting effects and protects the liver from toxicants, which results from the dual inhibition of GSK3 by AMPK downstream of PKA-activated LKB1, and PKA itself. Our findings reveal a novel pharmacological effect of MB and its molecular basis.