Resveratrol protects dopamine neurons against lipopolysaccharide-induced neurotoxicity through its anti-inflammatory actions

Abstract

Parkinson's disease (PD) is the second most common neurodegenerative disease characterized by a progressive loss of dopamine (DA) neurons in the substantia nigra. Accumulating evidence indicates that inhibition of microglia-mediated neuroinflammation may become a reliable protective strategy for PD. Resveratrol, a nonflavonoid polyphenol naturally found in red wine and grapes, has been known to possess antioxidant, anticancer, and anti-inflammatory properties. Although recent studies have shown that resveratrol provided neuroprotective effects against ischemia, seizure, and neurodegenerative disorders, the mechanisms underlying its beneficial effects on dopaminergic neurodegeneration are poorly defined. In this study, rat primary midbrain neuron-glia cultures were used to elucidate the molecular mechanisms underlying resveratrol-mediated neuroprotection. The results clearly demonstrated that resveratrol protected DA neurons against lipopolysaccharide (LPS)-induced neurotoxicity in concentration- and time-dependent manners through the inhibition of microglial activation and the subsequent reduction of proinflammatory factor release. Mechanistically, resveratrol-mediated neuroprotection was attributed to the inhibition of NADPH oxidase. This conclusion is supported by the following observations. First, resveratrol reduced NADPH oxidase-mediated generation of reactive oxygen species. Second, LPS-induced translocation of NADPH oxidase cytosolic subunit p47 to the cell membrane was significantly attenuated by resveratrol. Third and most importantly, resveratrol failed to exhibit neuroprotection in cultures from NADPH oxidase-deficient mice. Furthermore, this neuroprotection was also related to an attenuation of the activation of mitogen-activated protein kinases and nuclear factor-kappaB signaling pathways in microglia. These findings suggest that resveratrol exerts neuroprotection against LPS-induced dopaminergic neurodegeneration, and NADPH oxidase may be a major player in resveratrol-mediated neuroprotection.

Fig. 1.

Resveratrol protected DA neurons against LPS-induced neurotoxicity. Rat primary mesencephalic neuron-glia cultures were seeded in 24-well culture plates at 5 × 10⁵/well and then pretreated with various concentrations of resveratrol (15–60 μM) for 30 min before the addition of 10 ng/ml LPS. Seven days later, the LPS-induced dopaminergic neurotoxicity was determined by [³H]DA uptake assay (A) and the quantification of TH-positive neurons after immunostaining of DA neurons with an anti-TH antibody (B). Representative images of immunostaining from three independent experiments are shown (C). Scale bar, 100 μm. Results are the mean ± S.E.M. from three independent experiments performed in triplicate, and data from [³H]DA uptake assay are expressed as a percentage of the control cultures. #, p < 0.05 compared with control cultures; *, p < 0.05 compared with LPS-treated cultures.

Fig. 2.

Resveratrol afforded neuroprotection against LPS-induced dopaminergic neurotoxicity in a time-dependent manner. Primary neuron-glia cultures were pretreated with resveratrol (60 μM) and then stimulated with LPS (10 ng/ml). The neurotoxicity was quantified by [³H]DA uptake assay 1, 3, 5, and 7 days after LPS treatment. Results are expressed as a percentage of the control cultures and are the mean ± S.E.M. from three independent experiments performed in triplicate. #, p < 0.05 compared with control cultures; *, p < 0.05 compared with LPS-treated cultures.

Fig. 3.

Microglia are essential for resveratrol-mediated neuroprotection. Four types of cultures including neuron-glia, neuron-enriched, neuron-astrocyte, and neuron-microglia reconstituted cultures were pretreated with resveratrol (60 μM) followed by the treatment of 0.5 μM MPP⁺. The [³H]DA uptake analysis was performed 7 days after MPP⁺ addition. Results are expressed as a percentage of the control cultures and are the mean ± S.E.M. from three independent experiments performed in triplicate. #, p < 0.05 compared with control cultures; *, p < 0.05 compared with MPP⁺-treated cultures.

Fig. 4.

Resveratrol attenuated LPS-induced microglia activation. Rat primary mesencephalic neuron-glia cultures were pretreated with resveratrol (60 μM) for 30 min before LPS (10 ng/ml) stimulation. After 1, 4, and 7 days of LPS treatment, the total cell protein was harvested, respectively, and the level of Iba-1 protein was determined by Western blotting analysis (A). The ratio of densitometry values of Iba-1 and β-actin was accessed and normalized to each respective control group (B). Results were the mean ± S.E.M. from three independent experiments performed in triplicate. #, p < 0.05 compared with control cultures; *, p < 0.05 compared with LPS-treated cultures. Seven days after LPS treatment, the cultures were immunostained with anti-Iba-1 antibody (C). Activated microglia exhibited an enlarged cell body and irregular shapes, a shift from resting round and small cells to the highly activated amoeboid status. Images presented were representative of three independent experiments. Scale bar, 200 μm.

Fig. 5.

Resveratrol inhibited the levels of proinflammatory factors produced by LPS-activated microglia. Primary neuron-glia cultures were pretreated with resveratrol for 30 min before 10 ng/ml LPS stimulation. The supernatant was collected in the following time point after LPS treatment: 3 h for TNFα assay; and 24 h for nitrite (an indicator of NO production) and IL-1β assays. The release of TNFα and IL-1β was detected by enzyme-linked immunosorbent assay and the production of NO was accessed by Griess reagent (A). The mRNA expression of proinflammatory factors was measured by real-time RT-PCR at different time after LPS treatment: 1 h for TNFα and 3 h for IL-1β and iNOS (B). Twelve and 24 h after LPS treatment, the total protein was harvested, respectively, to detect the levels of iNOS using Western blotting assay (C). Results were the mean ± S.E.M. from three independent experiments performed in triplicate. #, p < 0.05 compared with control cultures; *, p < 0.05 compared with LPS-treated cultures.

Fig. 6.

NADPH oxidase participated in resveratrol-mediated neuroprotection against LPS-induced neurotoxicity. Primary microglia-enriched cultures were pretreated with resveratrol for 30 min before LPS treatment. The production of extracellular superoxide was detected by SOD-inhibitable reduction of WST-1 (A), and the levels of intracellular ROS were measured with DCFH-DA (B). After LPS treatment for 15 min, subcellular fractions were isolated to perform Western blot analysis for p47^PHOX levels in membrane and cytosolic fractions of microglia. β-Actin and gp91^PHOX were used as internal cytosolic and membrane controls, respectively (C). Primary neuron-glia cultures from PHOX^+/+ and PHOX^−/− mice were pretreated with resveratrol (60 μM) for 30 min and then stimulated with 10 ng/ml LPS. Seven days later, the neurotoxicity was accessed by [³H]DA uptake assay (D). Results are expressed as a percentage of the control cultures and are the mean ± S.E.M. from three independent experiments performed in triplicate. #, p < 0.05 compared with control cultures; *, p < 0.05 compared with LPS-treated cultures.

Fig. 7.

Resveratrol inhibited LPS-induced MAPKs activation in microglia. Primary microglia-enriched cultures were pretreated with resveratrol (60 μM) for 30 min followed by LPS (10 ng/ml) treatment for 15 min. The whole-cell protein was harvested to perform Western blotting assay (A). The ratio of densitometry values of phosphorylated MAPKs compared with total MAPKs was analyzed and normalized to each respective control group (B). Results are the mean ± S.E.M. from three independent experiments. #, p < 0.05 compared with control cultures; *, p < 0.05 compared with LPS-treated cultures.

Fig. 8.

Resveratrol prevented LPS-induced activation of microglia NF-κB signaling pathway. Primary microglia-enriched cultures were pretreated with resveratrol (60 μM) for 30 min and then incubated with LPS (10 ng/ml) for 15 min. The whole-cell lysates were analyzed by Western blotting assay (A). The ratio of densitometry values of phosphorylated p65 and IKKβ compared with total p65 and IKKβ and phosphorylated IκBα and total IκBα relative to β-actin was analyzed and normalized to each respective control group (B). Results are the mean ± S.E.M. from three independent experiments. #, p < 0.05 compared with control cultures; *, p < 0.05 compared with LPS-treated cultures.