beta-Lapachone suppresses neuroinflammation by modulating the expression of cytokines and matrix metalloproteinases in activated microglia

Abstract

Background: beta-Lapachone (beta-LAP) is a natural naphthoquinone compound isolated from the lapacho tree (Tabebuia sp.), and it has been used for treatment of rheumatoid arthritis, infection, and cancer. In the present study, we investigated whether beta-LAP has anti-inflammatory effects under in vitro and in vivo neuroinflammatory conditions. Methods: The effects of beta-LAP on the expression of inducible nitric oxide synthase (iNOS), cytokines, and matrix metalloproteinases (MMPs) were examined in lipopolysaccharide (LPS)-stimulated BV2 microglial cells and rat primary microglia by ELISA, reverse transcription polymerase chain reaction (RT-PCR), and Western blot analysis. Microglial activation and the expression levels of proinflammatory molecules were measured in the LPS-injected mouse brain by immunohistochemistry and RT-PCR analysis. The detailed molecular mechanism underlying the anti-inflammatory effects of beta-LAP was analyzed by electrophoretic mobility shift assay, reporter gene assay, Western blot, and RT-PCR analysis. Results: beta-LAP inhibited the expression of iNOS, proinflammatory cytokines, and MMPs (MMP-3, MMP-8, MMP-9) at mRNA and protein levels in LPS-stimulated microglia. On the other hand, beta-LAP upregulated the expressions of antiinflammatory molecules such as IL-10, heme oxygenase-1 (HO-1), and the tissue inhibitor of metalloproteinase-2 (TIMP-2). The anti-inflammatory effect of beta-LAP was confirmed in an LPS-induced systemic inflammation mouse model. Thus, beta-LAP inhibited microglial activation and the expressions of iNOS, proinflammatory cytokines, and MMPs in the LPS-injected mouse brain. Further mechanistic studies revealed that beta-LAP exerts anti-inflammatory effects by inhibiting MAPKs, PI3K/AKT, and NF-kappa B/AP-1 signaling pathways in LPS-stimulated microglia. beta-LAP also inhibited reactive oxygen species (ROS) production by suppressing the expression and/or phosphorylation of NADPH oxidase subunit proteins, such as p47(phox) and gp91(phox). The anti-oxidant effects of beta-LAP appeared to be related with the increase of HO-1 and NQO1 via the Nrf2/anti-oxidant response element (ARE) pathway and/or the PKA pathway. Conclusions: The strong anti-inflammatory/anti-oxidant effects of beta-LAP may provide preventive therapeutic potential for various neuroinflammatory disorders.