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

Lee EJ, Ko HM, Jeong YH, Park EM, Kim HS - J Neuroinflammation (2015)

Bottom Line: The effects of β-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.Thus, β-LAP inhibited microglial activation and the expressions of iNOS, proinflammatory cytokines, and MMPs in the LPS-injected mouse brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Tissue Injury Defense Research Center, School of Medicine, Ewha Womans University, Mok-6-dong 911-1, Yangchun-Ku, Seoul, 158-710, South Korea. 1sagain@ewhain.net.

ABSTRACT

Background: β-Lapachone (β-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 β-LAP has anti-inflammatory effects under in vitro and in vivo neuroinflammatory conditions.

Methods: The effects of β-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 β-LAP was analyzed by electrophoretic mobility shift assay, reporter gene assay, Western blot, and RT-PCR analysis.

Results: β-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, β-LAP upregulated the expressions of anti-inflammatory molecules such as IL-10, heme oxygenase-1 (HO-1), and the tissue inhibitor of metalloproteinase-2 (TIMP-2). The anti-inflammatory effect of β-LAP was confirmed in an LPS-induced systemic inflammation mouse model. Thus, β-LAP inhibited microglial activation and the expressions of iNOS, proinflammatory cytokines, and MMPs in the LPS-injected mouse brain. Further mechanistic studies revealed that β-LAP exerts anti-inflammatory effects by inhibiting MAPKs, PI3K/AKT, and NF-κB/AP-1 signaling pathways in LPS-stimulated microglia. β-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 β-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 β-LAP may provide preventive therapeutic potential for various neuroinflammatory disorders.

No MeSH data available.


Related in: MedlinePlus

Effects of β-LAP on iNOS and pro-/anti-inflammatory cytokines in LPS-stimulated BV2 cells and primary cultured microglia. BV2 cells (a) or primary cultured microglia (b) were pretreated with β-LAP (0.5, 1, and 2 μM) for 1 h and incubated with LPS (100 ng/ml for BV2, 10 ng/ml for primary microglia). After incubation for 16 h, the conditioned media were collected, and the amounts of NO, TNF-α, IL-1β, IL-6, and IL-10 were measured using Griess reagent or ELISA. The data are the mean ± SEM of three independent experiments. *P < 0.05, significantly different from LPS-treated samples. BV2 cells (c) or primary microglia (d) were pretreated with β-LAP (0.5, 1, and 2 μM) for 1 h, followed by LPS (100 ng/ml) for 6 h, and total RNA was isolated. The mRNA expressions of iNOS and cytokines were analyzed by RT-PCR. Representative gels are shown on the left panel, and quantifications of three independent experiments are shown in the right panel. Values correspond to the mean ± SEM of three independent experiments. *P < 0.05, significantly different from LPS-treated samples
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Fig1: Effects of β-LAP on iNOS and pro-/anti-inflammatory cytokines in LPS-stimulated BV2 cells and primary cultured microglia. BV2 cells (a) or primary cultured microglia (b) were pretreated with β-LAP (0.5, 1, and 2 μM) for 1 h and incubated with LPS (100 ng/ml for BV2, 10 ng/ml for primary microglia). After incubation for 16 h, the conditioned media were collected, and the amounts of NO, TNF-α, IL-1β, IL-6, and IL-10 were measured using Griess reagent or ELISA. The data are the mean ± SEM of three independent experiments. *P < 0.05, significantly different from LPS-treated samples. BV2 cells (c) or primary microglia (d) were pretreated with β-LAP (0.5, 1, and 2 μM) for 1 h, followed by LPS (100 ng/ml) for 6 h, and total RNA was isolated. The mRNA expressions of iNOS and cytokines were analyzed by RT-PCR. Representative gels are shown on the left panel, and quantifications of three independent experiments are shown in the right panel. Values correspond to the mean ± SEM of three independent experiments. *P < 0.05, significantly different from LPS-treated samples

Mentions: To investigate the anti-inflammatory effect of β-LAP, BV2 or primary microglial cells were treated with LPS in the presence or absence of β-LAP for 16 h. Consecutively, NO and cytokine levels in CM were measured. We observed that β-LAP significantly inhibited the LPS-induced production of NO and proinflammatory cytokines such as TNF-α, IL-1β, and IL-6, whereas it raised anti-inflammatory IL-10 in BV2 cells and primary microglia (Fig. 1a, b). β-LAP did not have any cytotoxicity in the concentrations used in this study in both the BV2 and primary microglial cells, at least for 48 h (MTT assay, data not shown). Subsequent RT-PCR analysis showed that β-LAP reduced the mRNA expression of iNOS, TNF-α, IL-1β, and IL-6, and increased IL-10 (Fig. 1c, d). The data suggest that β-LAP modulates the expressions of iNOS and cytokines at the transcriptional level.Fig. 1


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

Lee EJ, Ko HM, Jeong YH, Park EM, Kim HS - J Neuroinflammation (2015)

Effects of β-LAP on iNOS and pro-/anti-inflammatory cytokines in LPS-stimulated BV2 cells and primary cultured microglia. BV2 cells (a) or primary cultured microglia (b) were pretreated with β-LAP (0.5, 1, and 2 μM) for 1 h and incubated with LPS (100 ng/ml for BV2, 10 ng/ml for primary microglia). After incubation for 16 h, the conditioned media were collected, and the amounts of NO, TNF-α, IL-1β, IL-6, and IL-10 were measured using Griess reagent or ELISA. The data are the mean ± SEM of three independent experiments. *P < 0.05, significantly different from LPS-treated samples. BV2 cells (c) or primary microglia (d) were pretreated with β-LAP (0.5, 1, and 2 μM) for 1 h, followed by LPS (100 ng/ml) for 6 h, and total RNA was isolated. The mRNA expressions of iNOS and cytokines were analyzed by RT-PCR. Representative gels are shown on the left panel, and quantifications of three independent experiments are shown in the right panel. Values correspond to the mean ± SEM of three independent experiments. *P < 0.05, significantly different from LPS-treated samples
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig1: Effects of β-LAP on iNOS and pro-/anti-inflammatory cytokines in LPS-stimulated BV2 cells and primary cultured microglia. BV2 cells (a) or primary cultured microglia (b) were pretreated with β-LAP (0.5, 1, and 2 μM) for 1 h and incubated with LPS (100 ng/ml for BV2, 10 ng/ml for primary microglia). After incubation for 16 h, the conditioned media were collected, and the amounts of NO, TNF-α, IL-1β, IL-6, and IL-10 were measured using Griess reagent or ELISA. The data are the mean ± SEM of three independent experiments. *P < 0.05, significantly different from LPS-treated samples. BV2 cells (c) or primary microglia (d) were pretreated with β-LAP (0.5, 1, and 2 μM) for 1 h, followed by LPS (100 ng/ml) for 6 h, and total RNA was isolated. The mRNA expressions of iNOS and cytokines were analyzed by RT-PCR. Representative gels are shown on the left panel, and quantifications of three independent experiments are shown in the right panel. Values correspond to the mean ± SEM of three independent experiments. *P < 0.05, significantly different from LPS-treated samples
Mentions: To investigate the anti-inflammatory effect of β-LAP, BV2 or primary microglial cells were treated with LPS in the presence or absence of β-LAP for 16 h. Consecutively, NO and cytokine levels in CM were measured. We observed that β-LAP significantly inhibited the LPS-induced production of NO and proinflammatory cytokines such as TNF-α, IL-1β, and IL-6, whereas it raised anti-inflammatory IL-10 in BV2 cells and primary microglia (Fig. 1a, b). β-LAP did not have any cytotoxicity in the concentrations used in this study in both the BV2 and primary microglial cells, at least for 48 h (MTT assay, data not shown). Subsequent RT-PCR analysis showed that β-LAP reduced the mRNA expression of iNOS, TNF-α, IL-1β, and IL-6, and increased IL-10 (Fig. 1c, d). The data suggest that β-LAP modulates the expressions of iNOS and cytokines at the transcriptional level.Fig. 1

Bottom Line: The effects of β-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.Thus, β-LAP inhibited microglial activation and the expressions of iNOS, proinflammatory cytokines, and MMPs in the LPS-injected mouse brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Tissue Injury Defense Research Center, School of Medicine, Ewha Womans University, Mok-6-dong 911-1, Yangchun-Ku, Seoul, 158-710, South Korea. 1sagain@ewhain.net.

ABSTRACT

Background: β-Lapachone (β-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 β-LAP has anti-inflammatory effects under in vitro and in vivo neuroinflammatory conditions.

Methods: The effects of β-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 β-LAP was analyzed by electrophoretic mobility shift assay, reporter gene assay, Western blot, and RT-PCR analysis.

Results: β-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, β-LAP upregulated the expressions of anti-inflammatory molecules such as IL-10, heme oxygenase-1 (HO-1), and the tissue inhibitor of metalloproteinase-2 (TIMP-2). The anti-inflammatory effect of β-LAP was confirmed in an LPS-induced systemic inflammation mouse model. Thus, β-LAP inhibited microglial activation and the expressions of iNOS, proinflammatory cytokines, and MMPs in the LPS-injected mouse brain. Further mechanistic studies revealed that β-LAP exerts anti-inflammatory effects by inhibiting MAPKs, PI3K/AKT, and NF-κB/AP-1 signaling pathways in LPS-stimulated microglia. β-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 β-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 β-LAP may provide preventive therapeutic potential for various neuroinflammatory disorders.

No MeSH data available.


Related in: MedlinePlus