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Soybean-derived Bowman-Birk inhibitor inhibits neurotoxicity of LPS-activated macrophages.

Li J, Ye L, Cook DR, Wang X, Liu J, Kolson DL, Persidsky Y, Ho WZ - J Neuroinflammation (2011)

Bottom Line: In contrast, BBI pretreatment (1-100 μg/ml) of macrophages significantly inhibited LPS-mediated induction of these cytokines and ROS.Further, supernatant from BBI-pretreated and LPS-activated macrophage cultures was found to be less cytotoxic to neurons than that from non-BBI-pretreated and LPS-activated macrophage cultures.BBI, when directly added to the neuronal cultures (1-100 μg/ml), had no protective effect on neurons with or without LPS-activated macrophage supernatant treatment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology & Laboratory Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania, USA.

ABSTRACT

Background: Lipopolysaccharide (LPS), the major component of the outer membrane of gram-negative bacteria, can activate immune cells including macrophages. Activation of macrophages in the central nervous system (CNS) contributes to neuronal injury. Bowman-Birk inhibitor (BBI), a soybean-derived protease inhibitor, has anti-inflammatory properties. In this study, we examined whether BBI has the ability to inhibit LPS-mediated macrophage activation, reducing the release of pro-inflammatory cytokines and subsequent neurotoxicity in primary cortical neural cultures.

Methods: Mixed cortical neural cultures from rat were used as target cells for testing neurotoxicity induced by LPS-treated macrophage supernatant. Neuronal survival was measured using a cell-based ELISA method for expression of the neuronal marker MAP-2. Intracellular reactive oxygen species (ROS) production in macrophages was measured via 2', 7'-dichlorofluorescin diacetate (DCFH2DA) oxidation. Cytokine expression was determined by quantitative real-time PCR.

Results: LPS treatment of macrophages induced expression of proinflammatory cytokines (IL-1β, IL-6 and TNF-α) and of ROS. In contrast, BBI pretreatment (1-100 μg/ml) of macrophages significantly inhibited LPS-mediated induction of these cytokines and ROS. Further, supernatant from BBI-pretreated and LPS-activated macrophage cultures was found to be less cytotoxic to neurons than that from non-BBI-pretreated and LPS-activated macrophage cultures. BBI, when directly added to the neuronal cultures (1-100 μg/ml), had no protective effect on neurons with or without LPS-activated macrophage supernatant treatment. In addition, BBI (100 μg/ml) had no effect on N-methyl-D-aspartic acid (NMDA)-mediated neurotoxicity.

Conclusions: These findings demonstrate that BBI, through its anti-inflammatory properties, protects neurons from neurotoxicity mediated by activated macrophages.

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Related in: MedlinePlus

Effect of BBI on LPS-induced cell aggregation and production of reactive oxygen species (ROS). Macrophages were preincubated with or without BBI (100 μg/mL) for 24 h and then treated with LPS (100 ng/mL) for an additional 24 h. Cells were washed with serum-free medium and DCFH2DA was then added to the cultures, which were further incubated at 37°C for 30 min. ROS production in macrophages was examined using a fluorescence microscope (magnification: х100). Macrophage aggregation was assessed using a phase contrast microscope. Images presented are representative of three independent experiments. Values on the images indicate fluorescence intensity, as quantified by Image J 1.43.
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Figure 5: Effect of BBI on LPS-induced cell aggregation and production of reactive oxygen species (ROS). Macrophages were preincubated with or without BBI (100 μg/mL) for 24 h and then treated with LPS (100 ng/mL) for an additional 24 h. Cells were washed with serum-free medium and DCFH2DA was then added to the cultures, which were further incubated at 37°C for 30 min. ROS production in macrophages was examined using a fluorescence microscope (magnification: х100). Macrophage aggregation was assessed using a phase contrast microscope. Images presented are representative of three independent experiments. Values on the images indicate fluorescence intensity, as quantified by Image J 1.43.

Mentions: Activated macrophages/microglia can produce ROS that cause neurotoxicity [3]. Thus, we examined whether BBI treatment of macrophages could reduce ROS production in LPS-activated macrophages. As shown in Figure 5, BBI pretreatment of macrophages significantly attenuated ROS production in LPS-activated macrophages. Morphologically, LPS, when added to macrophage cultures, induced cell aggregation (Figure 5B). However, BBI pretreatment of macrophages suppressed LPS-induced cell aggregation (Figure 5C). BBI treatment alone had no effect on macrophage aggregation (Figure 5D). The aggregated macrophages were highly positive for DCFH2DA (indicative of ROS production) (Figure 5B and 5F). BBI treatment significantly reduced LPS-induced ROS production of macrophages as indicated by the fluorescence intensity quantitated by Image J (Figure 5E-H).


Soybean-derived Bowman-Birk inhibitor inhibits neurotoxicity of LPS-activated macrophages.

Li J, Ye L, Cook DR, Wang X, Liu J, Kolson DL, Persidsky Y, Ho WZ - J Neuroinflammation (2011)

Effect of BBI on LPS-induced cell aggregation and production of reactive oxygen species (ROS). Macrophages were preincubated with or without BBI (100 μg/mL) for 24 h and then treated with LPS (100 ng/mL) for an additional 24 h. Cells were washed with serum-free medium and DCFH2DA was then added to the cultures, which were further incubated at 37°C for 30 min. ROS production in macrophages was examined using a fluorescence microscope (magnification: х100). Macrophage aggregation was assessed using a phase contrast microscope. Images presented are representative of three independent experiments. Values on the images indicate fluorescence intensity, as quantified by Image J 1.43.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3046894&req=5

Figure 5: Effect of BBI on LPS-induced cell aggregation and production of reactive oxygen species (ROS). Macrophages were preincubated with or without BBI (100 μg/mL) for 24 h and then treated with LPS (100 ng/mL) for an additional 24 h. Cells were washed with serum-free medium and DCFH2DA was then added to the cultures, which were further incubated at 37°C for 30 min. ROS production in macrophages was examined using a fluorescence microscope (magnification: х100). Macrophage aggregation was assessed using a phase contrast microscope. Images presented are representative of three independent experiments. Values on the images indicate fluorescence intensity, as quantified by Image J 1.43.
Mentions: Activated macrophages/microglia can produce ROS that cause neurotoxicity [3]. Thus, we examined whether BBI treatment of macrophages could reduce ROS production in LPS-activated macrophages. As shown in Figure 5, BBI pretreatment of macrophages significantly attenuated ROS production in LPS-activated macrophages. Morphologically, LPS, when added to macrophage cultures, induced cell aggregation (Figure 5B). However, BBI pretreatment of macrophages suppressed LPS-induced cell aggregation (Figure 5C). BBI treatment alone had no effect on macrophage aggregation (Figure 5D). The aggregated macrophages were highly positive for DCFH2DA (indicative of ROS production) (Figure 5B and 5F). BBI treatment significantly reduced LPS-induced ROS production of macrophages as indicated by the fluorescence intensity quantitated by Image J (Figure 5E-H).

Bottom Line: In contrast, BBI pretreatment (1-100 μg/ml) of macrophages significantly inhibited LPS-mediated induction of these cytokines and ROS.Further, supernatant from BBI-pretreated and LPS-activated macrophage cultures was found to be less cytotoxic to neurons than that from non-BBI-pretreated and LPS-activated macrophage cultures.BBI, when directly added to the neuronal cultures (1-100 μg/ml), had no protective effect on neurons with or without LPS-activated macrophage supernatant treatment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology & Laboratory Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania, USA.

ABSTRACT

Background: Lipopolysaccharide (LPS), the major component of the outer membrane of gram-negative bacteria, can activate immune cells including macrophages. Activation of macrophages in the central nervous system (CNS) contributes to neuronal injury. Bowman-Birk inhibitor (BBI), a soybean-derived protease inhibitor, has anti-inflammatory properties. In this study, we examined whether BBI has the ability to inhibit LPS-mediated macrophage activation, reducing the release of pro-inflammatory cytokines and subsequent neurotoxicity in primary cortical neural cultures.

Methods: Mixed cortical neural cultures from rat were used as target cells for testing neurotoxicity induced by LPS-treated macrophage supernatant. Neuronal survival was measured using a cell-based ELISA method for expression of the neuronal marker MAP-2. Intracellular reactive oxygen species (ROS) production in macrophages was measured via 2', 7'-dichlorofluorescin diacetate (DCFH2DA) oxidation. Cytokine expression was determined by quantitative real-time PCR.

Results: LPS treatment of macrophages induced expression of proinflammatory cytokines (IL-1β, IL-6 and TNF-α) and of ROS. In contrast, BBI pretreatment (1-100 μg/ml) of macrophages significantly inhibited LPS-mediated induction of these cytokines and ROS. Further, supernatant from BBI-pretreated and LPS-activated macrophage cultures was found to be less cytotoxic to neurons than that from non-BBI-pretreated and LPS-activated macrophage cultures. BBI, when directly added to the neuronal cultures (1-100 μg/ml), had no protective effect on neurons with or without LPS-activated macrophage supernatant treatment. In addition, BBI (100 μg/ml) had no effect on N-methyl-D-aspartic acid (NMDA)-mediated neurotoxicity.

Conclusions: These findings demonstrate that BBI, through its anti-inflammatory properties, protects neurons from neurotoxicity mediated by activated macrophages.

Show MeSH
Related in: MedlinePlus