Resveratrol inhibits inflammatory responses via the mammalian target of rapamycin signaling pathway in cultured LPS-stimulated microglial cells

Abstract

Background: Resveratrol have been known to possess many pharmacological properties including antioxidant, cardioprotective and anticancer effects. Although current studies indicate that resveratrol produces neuroprotection against neurological disorders, the precise mechanisms for its beneficial effects are still not fully understood. We investigate the effect of anti-inflammatory and mechamisms of resveratrol by using lipopolysaccharide (LPS)-stimulated murine microglial BV-2 cells.

Methodology/principal findings: BV-2 cells were treated with resveratrol (25, 50, and 100 µM) and/or LPS (1 µg/ml). Nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by Griess reagent and ELISA. The mRNA and protein levels of proinflammatory proteins and cytokines were analysed by RT-PCR and double immunofluorescence labeling, respectively. Phosphorylation levels of PTEN (phosphatase and tensin homolog deleted on chromosome 10), Akt, mammalian target of rapamycin (mTOR), mitogen-activated protein kinases (MAPKs) cascades, inhibitor κB-α (IκB-α) and cyclic AMP-responsive element-binding protein (CREB) were measured by western blot. Resveratrol significantly attenuated the LPS-induced expression of NO, PGE2, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and nuclear factor-κB (NF-κB) in BV-2 cells. Resveratrol increased PTEN, Akt and mTOR phosphorylation in a dose-dependent manner or a time-dependent manner. Rapamycin (10 nM), a specific mTOR inhibitor, blocked the effects of resveratrol on LPS-induced microglial activation. In addition, mTOR inhibition partially abolished the inhibitory effect of resveratrol on the phosphorylation of IκB-α, CREB, extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal protein kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK).

Conclusion and implications: This study indicates that resveratrol inhibited LPS-induced proinflammatory enzymes and proinflammatory cytokines via down-regulation phosphorylation of NF-κB, CREB and MAPKs family in a mTOR-dependent manner. These findings reveal, in part, the molecular basis underlying the anti-inflammatory properties of resveratrol.

Figure 1. Chemical structure of resveratrol.

Figure 2. Effects of resveratrol on the cell viability of BV-2 microglial cells.

BV-2 cells were treated with 25, 50, and 100 µM of resveratrol without LPS treatments or with 1 µg/ml LPS or with 10 nM rapamycin treatments for 18 h. BV-2 cells viabilities were measured and expressed as mean ± SEM for three independent experiments.

Figure 3. Resveratrol activates the Akt/mTOR signaling pathway in BV-2 microglial cells.

Panel A shows that BV-2 cells were treated with resveratrol (50 µM) for the indicated times, Panel B shows that BV-2 cells were pre-treated with resveratrol (50 µM) for 1 h, then exposed to LPS (1 µg/ml) for the indicated times and Panels C and D shows that BV-2 cells were pre-treated with resveratrol (25, 50, and 100 µM) for 1 h, then exposed to LPS (1 µg/ml) for 40 min. Various treated BV-2 cell lysates (50 µg protein) were prepared and subjected to Western blot analysis by using antibodies specific for PTEN and phospho-PTEN, Akt and phospho-Akt, mTOR and phospho-mTOR as described in the methods. The relative protein levels were quantified by scanning densitometry and normalized to total Akt. The values shown are mean ± SEM of data from three independent experiments. ^#Significant compared with control alone, p<0.05. *Significant compared with LPS alone, p<0.05. ^▵Significant compared with resveratrol+LPS, p<0.05.

Figure 4. mTOR is required for resveratrol-inhibited expression of NO (A) and PGE2 (B) induced by LPS in BV-2 cells.

Approximately 1×10⁶ cells/ml were seeded in six-well plates and incubated until 80% confluency. Cells were pre-treated with resveratrol (25, 50, and 100 µM) for 1 h in the absence or presence of rapamycin (10 nM), then exposed to LPS (1 µg/ml) for 8 h. The values shown are mean ± SEM of data from three independent experiments. ^#Significant compared with control alone, p<0.05. ^*Significant compared with LPS alone, p<0.05. ^▵Significant compared with resveratrol+LPS, p<0.05.

Figure 5. mTOR is required for resveratrol-inhibited expression of iNOS protein and mRNA induced by LPS in BV-2 cells.

Panel A shows the immunofluorenscence images for protein expression of iNOS and Panel B shows the corresponding mRNA data. The relative mRNA level was quantified by scanning densitometry and normalized to β-actin mRNA. Note the up-regulated protein and mRNA expression of iNOS by LPS is suppressed by different concentrations of resveratrol; however, in cells pretreated with mTOR inhibitor rapamycin, the suppressive effect of resveratrol is abrogated. The values shown are mean ± SEM of data from three independent experiments. ^#Significant compared with control alone, p<0.05. ^*Significant compared with LPS alone, p<0.05. ^▵Significant compared with resveratrol+LPS, p<0.05.

Figure 6. mTOR is required for resveratrol-inhibited expression of COX-2 protein and mRNA induced by LPS in BV-2 cells.

Panel A shows the immunofluorenscence images for protein expression of COX-2 and Panel B shows the corresponding mRNA data. The relative mRNA level was quantified by scanning densitometry and normalized to β-actin mRNA. Note the up-regulated protein and mRNA expression of COX-2 by LPS is suppressed by different concentrations of resveratrol; however, in cells pretreated with mTOR inhibitor rapamycin, the suppressive effect of resveratrol is abrogated. The values shown are mean ± SEM of data from three independent experiments. ^#Significant compared with control alone, p<0.05. ^*Significant compared with LPS alone, p<0.05. ^▵Significant compared with resveratrol+LPS, p<0.05.

Figure 7. mTOR is involved in resveratrol-attenuated the production of the proinflammatory cytokine TNF-α at the transcriptional and translational levels in BV-2 cells.

Panel A shows the immunofluorenscence images for protein expression of TNF-α and Panel B shows the corresponding mRNA data. The relative mRNA level was quantified by scanning densitometry and normalized to β-actin mRNA. Note the up-regulated protein and mRNA expression of TNF-α by LPS is suppressed by different concentrations of resveratrol; however, in cells pretreated with mTOR inhibitor rapamycin, the suppressive effect of resveratrol is abrogated. The values shown are mean ± SEM of data from three independent experiments. ^#Significant compared with control alone, p<0.05. ^*Significant compared with LPS alone, p<0.05. ^▵Significant compared with resveratrol+LPS, p<0.05.

Figure 8. mTOR is involved in resveratrol-attenuated the production of the proinflammatory cytokine IL-1β at the transcriptional and translational levels in BV-2 cells.

Panel A shows the immunofluorenscence images for protein expression of IL-1β and Panel B shows the corresponding mRNA data. The relative mRNA level was quantified by scanning densitometry and normalized to β-actin mRNA. Note the up-regulated protein and mRNA expression of IL-1β by LPS is suppressed by different concentrations of resveratrol; however, in cells pretreated with mTOR inhibitor rapamycin, the suppressive effect of resveratrol is abrogated. The values shown are mean ± SEM of data from three independent experiments. ^#Significant compared with control alone, p<0.05. ^*Significant compared with LPS alone, p<0.05. ^▵Significant compared with resveratrol+LPS, p<0.05.

Figure 9. mTOR is required for resveratrol-inhibited expression of NF-κB/RelA protein and mRNA induced by LPS in BV-2 cells.

Panel A shows the immunofluorenscence images for protein expression of NF-κB/RelA and Panel B shows the corresponding mRNA data. The relative mRNA level was quantified by scanning densitometry and normalized to β-actin mRNA. Note the up-regulated protein and mRNA expression of NF-κB/RelA by LPS is suppressed by different concentrations of resveratrol; however, in cells pretreated with mTOR inhibitor rapamycin, the suppressive effect of resveratrol is abrogated. The values shown are mean ± SEM of data from three independent experiments. ^#Significant compared with control alone, p<0.05. ^*Significant compared with LPS alone, p<0.05. ^▵Significant compared with resveratrol+LPS, p<0.05.

Figure 10. Inhibition of phosphorylation of IκB-α, CREB, and MAPKs signaling by resveratrol is mTOR-dependent during BV-2 cells activation by LPS.

Approximately 1×10⁶ cells/ml were seeded in six-well plates and incubated until 80% confluency. Cells were pre-treated with resveratrol (25, 50, and 100 µM) for 1 h in the absence or presence of rapamycin (10 nM), then exposed to LPS (1 µg/ml) for 30 min. Cell lysates (50 µg protein) were prepared and subjected to Western blot analysis by using antibodies specific for phosphorylated forms of IκB-α, CREB, ERK1/2, JNK and p38 MAPK (shown as phospho-IκB-α, etc.) as described in the methods. Equivalent loading of cell lysates was determined by reprobing the blots with anti-β-actin, total ERK1/2, JNK or p38 MAPK antibodies. The relative protein levels were quantified by scanning densitometry and normalized to β-actin, total ERK1/2, JNK or p38 MAPK. The values shown are mean ± SEM of data from three independent experiments. ^#Significant compared with control alone, p<0.05. ^*Significant compared with LPS alone, p<0.05. ^▵Significant compared with resveratrol+LPS, p<0.05.