Sinomenine, a natural dextrorotatory morphinan analog, is anti-inflammatory and neuroprotective through inhibition of microglial NADPH oxidase

Abstract

Background: The mechanisms involved in the induction and regulation of inflammation resulting in dopaminergic (DA) neurotoxicity in Parkinson's disease (PD) are complex and incompletely understood. Microglia-mediated inflammation has recently been implicated as a critical mechanism responsible for progressive neurodegeneration.Methods: Mesencephalic neuron-glia cultures and reconstituted cultures were used to investigate the molecular mechanisms of sinomenine (SN)-mediated anti-inflammatory and neuroprotective effects in both the lipopolysaccharide (LPS)- and the 1-methyl-4-phenylpyridinium (MPP⁺)-mediated models of PD.Results: SN showed equivalent efficacy in protecting against DA neuron death in rat midbrain neuron-glial cultures at both micro- and sub-picomolar concentrations, but no protection was seen at nanomolar concentrations. The neuroprotective effect of SN was attributed to inhibition of microglial activation, since SN significantly decreased tumor necrosis factor-α (TNF-α, prostaglandin E₂(PGE₂) and reactive oxygen species (ROS) production by microglia. In addition, from the therapeutic point of view, we focused on sub-picomolar concentration of SN for further mechanistic studies. We found that 10^-14M of SN failed to protect DA neurons against MPP⁺-induced toxicity in the absence of microglia. More importantly, SN failed to show a protective effect in neuron-glia cultures from mice lacking functional NADPH oxidase (PHOX), a key enzyme for extracellular superoxide production in immune cells. Furthermore, we demonstrated that SN reduced LPS-induced extracellular ROS production through the inhibition of the PHOX cytosolic subunit p47^phoxtranslocation to the cell membrane.Conclusion: Our findings strongly suggest that the protective effects of SN are most likely mediated through the inhibition of microglial PHOX activity. These findings suggest a novel therapy to treat inflammation-mediated neurodegenerative diseases.