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Resveratrol potently reduces prostaglandin E2 production and free radical formation in lipopolysaccharide-activated primary rat microglia.

Candelario-Jalil E, de Oliveira AC, Gräf S, Bhatia HS, Hüll M, Muñoz E, Fiebich BL - J Neuroinflammation (2007)

Bottom Line: Our results indicate that resveratrol potently reduced LPS-induced PGE2 synthesis and the formation of 8-iso-PGF2 alpha, a measure of free radical production.Interestingly, resveratrol dose-dependently reduced the expression (mRNA and protein) of mPGES-1, which is a key enzyme responsible for the synthesis of PGE2 by activated microglia, whereas resveratrol did not affect the expression of COX-2.These findings suggest that the naturally occurring polyphenol resveratrol is able to reduce microglial activation, an effect that might help to explain its neuroprotective effects in several in vivo models of brain injury.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neurochemistry Research Group, Department of Psychiatry, University of Freiburg Medical School, Hauptstrasse 5, D-79104 Freiburg, Germany. ecandelario-jalil@salud.unm.edu

ABSTRACT

Background: Neuroinflammatory responses are triggered by diverse ethiologies and can provide either beneficial or harmful results. Microglial cells are the major cell type involved in neuroinflammation, releasing several mediators, which contribute to the neuronal demise in several diseases including cerebral ischemia and neurodegenerative disorders. Attenuation of microglial activation has been shown to confer protection against different types of brain injury. Recent evidence suggests that resveratrol has anti-inflammatory and potent antioxidant properties. It has been also shown that resveratrol is a potent inhibitor of cyclooxygenase (COX)-1 activity. Previous findings have demonstrated that this compound is able to reduce neuronal injury in different models, both in vitro and in vivo. The aim of this study was to examine whether resveratrol is able to reduce prostaglandin E2 (PGE2) and 8-iso-prostaglandin F2alpha (8-iso-PGF2 alpha) production by lipopolysaccharide (LPS)-activated primary rat microglia.

Methods: Primary microglial cell cultures were prepared from cerebral cortices of neonatal rats. Microglial cells were stimulated with 10 ng/ml of LPS in the presence or absence of different concentrations of resveratrol (1-50 microM). After 24 h incubation, culture media were collected to measure the production of PGE2 and 8-iso-PGF2 alpha using enzyme immunoassays. Protein levels of COX-1, COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1) were studied by Western blotting after 24 h of incubation with LPS. Expression of mPGES-1 at the mRNA level was investigated using reverse transcription-polymerase chain reaction (RT-PCR) analysis.

Results: Our results indicate that resveratrol potently reduced LPS-induced PGE2 synthesis and the formation of 8-iso-PGF2 alpha, a measure of free radical production. Interestingly, resveratrol dose-dependently reduced the expression (mRNA and protein) of mPGES-1, which is a key enzyme responsible for the synthesis of PGE2 by activated microglia, whereas resveratrol did not affect the expression of COX-2. Resveratrol is therefore the first known inhibitor which specifically prevents mPGES-1 expression without affecting COX-2 levels. Another important observation of the present study is that other COX-1 selective inhibitors (SC-560 and Valeroyl Salicylate) potently reduced PGE2 and 8-iso-PGF2 alpha production by LPS-activated microglia.

Conclusion: These findings suggest that the naturally occurring polyphenol resveratrol is able to reduce microglial activation, an effect that might help to explain its neuroprotective effects in several in vivo models of brain injury.

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Cyclooxygenase enzymatic activity in microglial cells is inhibited by resveratrol treatment. For the COX-1 activity assay (A), cells were left untreated and different concentrations of resveratrol were added for 15 min. After this incubation time, 15 μM of arachidonic acid was added and PGE2 measured after 15 min. For total COX activity assay (COX-1 + COX-2), cells were either left untreated or were stimulated with LPS (10 ng/ml) for 24 h. After removal of medium, cells were treated with different concentrations of resveratrol for 30 min in absence or presence of 15 μM of arachidonic acid. PGE2 in the supernatants was measured by an enzyme immunoassay as described in Materials and Methods. Data are expressed as mean ± S.E.M. Statistical analysis was performed using one-way ANOVA followed by the Student-Newman-Keuls post-hoc test. *p < 0.05 and **p < 0.01 with respect to untreated control (without resveratrol).
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Figure 2: Cyclooxygenase enzymatic activity in microglial cells is inhibited by resveratrol treatment. For the COX-1 activity assay (A), cells were left untreated and different concentrations of resveratrol were added for 15 min. After this incubation time, 15 μM of arachidonic acid was added and PGE2 measured after 15 min. For total COX activity assay (COX-1 + COX-2), cells were either left untreated or were stimulated with LPS (10 ng/ml) for 24 h. After removal of medium, cells were treated with different concentrations of resveratrol for 30 min in absence or presence of 15 μM of arachidonic acid. PGE2 in the supernatants was measured by an enzyme immunoassay as described in Materials and Methods. Data are expressed as mean ± S.E.M. Statistical analysis was performed using one-way ANOVA followed by the Student-Newman-Keuls post-hoc test. *p < 0.05 and **p < 0.01 with respect to untreated control (without resveratrol).

Mentions: These findings prompted us to investigate the ability of resveratrol to reduce COX enzymatic activity in activated microglia. Results from this experiment are shown in Fig. 2. Significant inhibition of COX-1 activity was observed when microglial cells were pre-incubated for 30 min with 25 and 50 μM of resveratrol (Fig. 2A). The effect of resveratrol on total COX activity (COX-1 + COX-2) was also investigated. In this in vitro assay, cells were pre-incubated with LPS for 24 h before resveratrol was added for 30 min, and PGE2 levels were measured in the supernatant. The addition of resveratrol produced a significant inhibition of COX activity starting at 10 μM and showing a 50% inhibition with the highest dose of 50 μM (Fig. 2B).


Resveratrol potently reduces prostaglandin E2 production and free radical formation in lipopolysaccharide-activated primary rat microglia.

Candelario-Jalil E, de Oliveira AC, Gräf S, Bhatia HS, Hüll M, Muñoz E, Fiebich BL - J Neuroinflammation (2007)

Cyclooxygenase enzymatic activity in microglial cells is inhibited by resveratrol treatment. For the COX-1 activity assay (A), cells were left untreated and different concentrations of resveratrol were added for 15 min. After this incubation time, 15 μM of arachidonic acid was added and PGE2 measured after 15 min. For total COX activity assay (COX-1 + COX-2), cells were either left untreated or were stimulated with LPS (10 ng/ml) for 24 h. After removal of medium, cells were treated with different concentrations of resveratrol for 30 min in absence or presence of 15 μM of arachidonic acid. PGE2 in the supernatants was measured by an enzyme immunoassay as described in Materials and Methods. Data are expressed as mean ± S.E.M. Statistical analysis was performed using one-way ANOVA followed by the Student-Newman-Keuls post-hoc test. *p < 0.05 and **p < 0.01 with respect to untreated control (without resveratrol).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2100038&req=5

Figure 2: Cyclooxygenase enzymatic activity in microglial cells is inhibited by resveratrol treatment. For the COX-1 activity assay (A), cells were left untreated and different concentrations of resveratrol were added for 15 min. After this incubation time, 15 μM of arachidonic acid was added and PGE2 measured after 15 min. For total COX activity assay (COX-1 + COX-2), cells were either left untreated or were stimulated with LPS (10 ng/ml) for 24 h. After removal of medium, cells were treated with different concentrations of resveratrol for 30 min in absence or presence of 15 μM of arachidonic acid. PGE2 in the supernatants was measured by an enzyme immunoassay as described in Materials and Methods. Data are expressed as mean ± S.E.M. Statistical analysis was performed using one-way ANOVA followed by the Student-Newman-Keuls post-hoc test. *p < 0.05 and **p < 0.01 with respect to untreated control (without resveratrol).
Mentions: These findings prompted us to investigate the ability of resveratrol to reduce COX enzymatic activity in activated microglia. Results from this experiment are shown in Fig. 2. Significant inhibition of COX-1 activity was observed when microglial cells were pre-incubated for 30 min with 25 and 50 μM of resveratrol (Fig. 2A). The effect of resveratrol on total COX activity (COX-1 + COX-2) was also investigated. In this in vitro assay, cells were pre-incubated with LPS for 24 h before resveratrol was added for 30 min, and PGE2 levels were measured in the supernatant. The addition of resveratrol produced a significant inhibition of COX activity starting at 10 μM and showing a 50% inhibition with the highest dose of 50 μM (Fig. 2B).

Bottom Line: Our results indicate that resveratrol potently reduced LPS-induced PGE2 synthesis and the formation of 8-iso-PGF2 alpha, a measure of free radical production.Interestingly, resveratrol dose-dependently reduced the expression (mRNA and protein) of mPGES-1, which is a key enzyme responsible for the synthesis of PGE2 by activated microglia, whereas resveratrol did not affect the expression of COX-2.These findings suggest that the naturally occurring polyphenol resveratrol is able to reduce microglial activation, an effect that might help to explain its neuroprotective effects in several in vivo models of brain injury.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neurochemistry Research Group, Department of Psychiatry, University of Freiburg Medical School, Hauptstrasse 5, D-79104 Freiburg, Germany. ecandelario-jalil@salud.unm.edu

ABSTRACT

Background: Neuroinflammatory responses are triggered by diverse ethiologies and can provide either beneficial or harmful results. Microglial cells are the major cell type involved in neuroinflammation, releasing several mediators, which contribute to the neuronal demise in several diseases including cerebral ischemia and neurodegenerative disorders. Attenuation of microglial activation has been shown to confer protection against different types of brain injury. Recent evidence suggests that resveratrol has anti-inflammatory and potent antioxidant properties. It has been also shown that resveratrol is a potent inhibitor of cyclooxygenase (COX)-1 activity. Previous findings have demonstrated that this compound is able to reduce neuronal injury in different models, both in vitro and in vivo. The aim of this study was to examine whether resveratrol is able to reduce prostaglandin E2 (PGE2) and 8-iso-prostaglandin F2alpha (8-iso-PGF2 alpha) production by lipopolysaccharide (LPS)-activated primary rat microglia.

Methods: Primary microglial cell cultures were prepared from cerebral cortices of neonatal rats. Microglial cells were stimulated with 10 ng/ml of LPS in the presence or absence of different concentrations of resveratrol (1-50 microM). After 24 h incubation, culture media were collected to measure the production of PGE2 and 8-iso-PGF2 alpha using enzyme immunoassays. Protein levels of COX-1, COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1) were studied by Western blotting after 24 h of incubation with LPS. Expression of mPGES-1 at the mRNA level was investigated using reverse transcription-polymerase chain reaction (RT-PCR) analysis.

Results: Our results indicate that resveratrol potently reduced LPS-induced PGE2 synthesis and the formation of 8-iso-PGF2 alpha, a measure of free radical production. Interestingly, resveratrol dose-dependently reduced the expression (mRNA and protein) of mPGES-1, which is a key enzyme responsible for the synthesis of PGE2 by activated microglia, whereas resveratrol did not affect the expression of COX-2. Resveratrol is therefore the first known inhibitor which specifically prevents mPGES-1 expression without affecting COX-2 levels. Another important observation of the present study is that other COX-1 selective inhibitors (SC-560 and Valeroyl Salicylate) potently reduced PGE2 and 8-iso-PGF2 alpha production by LPS-activated microglia.

Conclusion: These findings suggest that the naturally occurring polyphenol resveratrol is able to reduce microglial activation, an effect that might help to explain its neuroprotective effects in several in vivo models of brain injury.

Show MeSH
Related in: MedlinePlus