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

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.

Figure 1

A: Resveratrol dose-dependently inhibits LPS-induced PGE₂ production in primary rat microglial cells. B: Effects of Trolox C and α-tocopherol on PGE₂ production by microglia challenged with LPS. The amounts of PGE₂ in the culture medium were determined using an enzyme immunoassay after 24 h incubation with LPS. Substances were added 30 min before stimulating the cells with LPS. Each column and error bar represents the mean ± S.E.M. of 4 independent experiments. Asterisks indicate significant difference between the treatments. *p < 0.05, **p < 0.01 and ***p < 0.001 with respect to LPS control (One-way ANOVA followed by the Student-Newman-Keuls post-hoc test).

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).

Figure 3

A: Representative photomicrographs showing RT-PCR products of COX-1, COX-2, mPGES-1, and β-actin mRNAs. Microglial cells were treated with LPS (10 ng/ml) in the absence or presence of different concentrations of resveratrol. The mRNA expression levels were tested for each condition. There is a constitutive COX-1 mRNA expression that is observed under all conditions. However, COX-2 and mPGES-1 are undetectable in untreated microglial cells, while their expression is dramatically increased in the presence of LPS. Resveratrol treatment significantly reduced mPGES-1, but not COX-2 expression. RT-PCR analysis was performed after 4 h of incubation with LPS. Resveratrol was added to the cultures 30 min before LPS. B: Semi-quantitative analysis of the effect of resveratrol on mPGES-1 expression. This analysis was done with results from 4 different RT-PCR experiments performed independently. Relative expression of rat β-actin was used for normalization. The amplicon size of the PCR product appears below the name of each transcript. *p < 0.05 and **p < 0.01 with respect to untreated LPS control.

Figure 4

A: Immunoblot analysis of protein levels of COX-1, COX-2, mPGES-1, and actin in LPS-activated microglia treated with different concentrations of resveratrol (1–50 μM). Microglial protein extracts were prepared after 24 h of incubation with LPS, and subjected to SDS-PAGE followed by immunoblot analysis using specific antibodies for each protein. B: Quantitative densitometric analysis of mPGES-1 and COX-2 protein expression normalized to actin loading control. Resveratrol potently reduced mPGES-1 protein expression induced by LPS. *p < 0.05 and **p < 0.01 with respect to untreated LPS control. Bars represent mean ± S.E.M. of 4 independent experiments.

Figure 5

A: Effect of resveratrol on 8-iso-prostaglandin F_2α (8-iso-PGF_2α) production in response to 10 ng/ml LPS. 8-iso-PGF_2α was determined in the culture medium 24 h following stimulation of microglial cells with LPS alone, or in combination with resveratrol at the given concentrations. B: Moderate reduction in LPS-mediated 8-iso-PGF_2α formation by the antioxidants Trolox C and α-tocopherol. *p < 0.05, **p < 0.01 and ***p < 0.001 with respect to LPS alone. For both panels, histograms represent mean ± S.E.M. of 4 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Student-Newman-Keuls post-hoc test.

Figure 6

Potent inhibition of LPS-induced PGE₂ (A and C) and 8-iso-PGF_2α formation (B and D) by two highly selective COX-1 inhibitors (SC-560 and VAS). After 30 min pre-stimulation with the inhibitors, LPS (10 ng/mL) was added to the culture medium, and the amounts of PGE₂ and 8-iso-PGF_2α were determined using specific enzyme immunoassays after 24 h incubation with LPS. For all panels, histograms represent mean ± S.E.M. of 4 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Student-Newman-Keuls post-hoc test. *p < 0.05, **p < 0.01 and ***p < 0.001 with respect to LPS control.

Figure 7

A: Effects of the antioxidant Trolox C and the COX-1 inhibitors (SC-560 and VAS) on the expression of COX-1, COX-2, and mPGES-1 mRNAs in rat microglial cells stimulated with LPS. RT-PCR analysis was performed after 4 h of incubation with LPS. There are no significant effects of the COX-1 inhibitors on mPGES-1 expression as demonstrated by the semi-quantitative analysis shown in Panel B. Trolox C produced a moderate reduction in mPGES-1 expression that did not attain statistical significance. Semi-quantitative analysis was done with results from 3 different RT-PCR experiments performed independently. Relative expression of rat S12 was used for normalization.