Methylene blue targets PHD3 expression in murine microglia to mitigate lipopolysaccharide-induced neuroinflammation and neurocognitive impairments

Methylene blue (MB) has anti-inflammatory properties, however, its underlying molecular mechanism remains elusive. This study aimed to investigate whether and how MB could attenuate lipopolysaccharide (LPS)-induced microglial activation, neuroinflammation, and neurobehavioral deficits. We measured the expression of pro-inflammatory factors and performed three neurobehavioral tests to assess the effect of MB on neuroinflammation and neurocognitive dysfunction in LPS-treated adult C57BL/6N male mice or LPS-stimulated microglia cells. In vitro and in vivo experiments were further performed to investigate the molecular mechanism underlying MB inhibition of neuroinflammation using various experimental methods, including western blot, RT-qPCR, immunofluorescence, seahorse measurement, positron emission tomography (PET) scan, and flow cytometry analyses. Our results demonstrated that microglial activation and M1 polarization were induced by LPS exposure, resulting in an inflammatory response and neuronal apoptosis. Furthermore, LPS induced metabolic reprogramming in microglial cells. However, MB treatment substantially inhibited LPS-induced elevated levels of pro-inflammatory factors and reversed metabolic activation in vivo, which eventually led to the resolution of neuroinflammation and neurobehavioral improvement. Mechanistically, MB specifically inhibited the LPS-induced overexpression of PHD3 in vitro and in vivo. The pharmacological and genetic manipulations unveiled that the Siah2/Morg1/PHD3 signaling pathway may mediate MB protection against LPS-induced neuroinflammation and neurotoxicity. Therefore MB inhibited PHD3-dependent neuroinflammation may via Siah2/Morg1/PHD3 pathway, and that PHD3 expressed in microglia may be a drug target for the treatment of neuroinflammation-related brain disorders.