Resveratrol confers protection against rotenone-induced neurotoxicity by modulating myeloperoxidase levels in glial cells

Abstract

Myeloperoxidase (MPO) functions as a key molecular component of the host defense system against diverse pathogens. We have previously reported that increased MPO levels and activity is a distinguishing feature of rotenone-exposed glial cells, and that either overactivation or deficiency of MPO leads to pathological conditions in the brain. Here, we provide that modulation of MPO levels in glia by resveratrol confers protective effects on rotenone-induced neurotoxicity. We show that resveratrol significantly reduced MPO levels but did not trigger abnormal nitric oxide (NO) production in microglia and astrocytes. Resveratrol-induced down-regulation of MPO, in the absence of an associated overproduction of NO, markedly attenuated rotenone-triggered inflammatory responses including phagocytic activity and reactive oxygen species production in primary microglia and astrocytes. In addition, impaired responses of primary mixed glia from Mpo (-/-) mice to rotenone were relieved by treatment with resveratrol. We further show that rotenone-induced neuronal injury, particularly dopaminergic cell death, was attenuated by resveratrol in neuron-glia co-cultures, but not in neurons cultured alone. Similar regulatory effects of resveratrol on MPO levels were observed in microglia treated with MPP(+), another Parkinson's disease-linked neurotoxin, supporting the beneficial effects of resveratrol on the brain. Collectively, our findings provide that resveratrol influences glial responses to rotenone by regulating both MPO and NO, and thus protects against rotenone-induced neuronal injury.

Figure 1. Resveratrol suppresses both MPO and NO production in rotenone-exposed microglia.

A–B. Mouse BV2 microglial cells (A) and rat primary microglia (B) were pre-treated with or without the indicated concentrations of resveratrol (RESV) for 1 h, and then incubated with 30 nM rotenone (Rot) for 24 h. The levels of MPO were analyzed by FACS using an anti-MPO antibody. The graph represents the fold changes in MFI (mean fluorescence intensity) ± SD from more than three independent experiments. The graph represents the fold changes in Mean ± SD of three independent experiments. *P<0.05, **P<0.01 compared with rotenone-treated cells. C. Rat primary microglia were mock-treated or treated with 30 nM rotenone in the presence or absence of the indicated concentrations of resveratrol for 24 h, and supernatants were assayed for nitrate concentration. *P = 0.01; **P = 0.001; ***P = 0.001.

Figure 2. Resveratrol reduces MPO levels and its activity in MPO-treated rat primary microglia and astrocytes.

A. Rat primary microglia were pre-treated with or without 10 µM resveratrol for 1 h, and then treated with 100 ng/ml MPO for 24 h, after which MPO levels were determined by FACS analyses. The graph represents the fold changes in MFI ± SD from three independent experiments. *P = 0.027; **P = 0.049. B. Rat primary microglia (PM, upper) and astrocytes (PA, lower) were pretreated with 10 µM (PM) or 20 µM (PA) of resveratrol for 1 h. The cells were incubated with 100 ng/ml MPO for 24 h and MPO levels were observed by confocal microscopy using an anti-MPO antibody. MPO, green; DAPI, blue; scale bar = 20 µm. The data shown are representative of at least three independent experiments. C–D. Primary microglia were mock-treated or treated with 100 ng/ml MPO in the presence of the indicated concentrations of resveratrol, 4-aminobenzoylhydrazide (ABAH), or salichylhydroxamic acid (SHA). Total cell lysates (C) and media (D) were collected, and then MPO activity was measured fluorometrically (SpectraMax Gemini EM spectrofluorometer, Molecular Devices) using an EnzChek® Myeloperoxidase activity assay kit. The results are the fold changes in Mean SD of three experiments performed in triplicate. *P<0.05, **P<0.01, ***P<0.001 compared with MPO-treated cells.

Figure 3. MPO-dependent increase of MPO levels are attenuated by resveratrol in rat primary microglia and astrocytes.

A. Rat primary microglia (PM, upper) and astrocytes (PA, lower) were pretreated with resveratrol (RESV), ethyl pyruvate (EtPy), or 15d-PGJ2 for 1 h, followed by incubation with 100 ng/ml MPO for 18 h. MPO levels were determined by Western blot analyses. B. Concentration-dependent effects of resveratrol were observed in MPO-treated primary microglia. C. Rat primary microglia were treated with 20 µM resveratrol and/or 100 ng/ml MPO at various time points. The cells were further incubated for 18 h and then MPO levels were examined by western blot analyses. The data shown are representative of at least three independent experiments.

Figure 4. Either rotenone- or MPO-stimulated increase of phagocytic activity of microglia was markedly reduced by resveratrol.

A. BV2 microglia were treated with 30 nM rotenone (Rot, a) or 100 ng/ml MPO (b) in the presence of 5 µM resveratrol (RESV) for 24 h, and the cellular uptake of FITC-conjugated fluorescent beads was determined by flow cytometric analyses. The graph represents the fold changes in MFI ± SD from three independent experiments. *P<0.05, **P<0.01, ***P<0.001 when compared with rotenone-or MPO-treated cells. B. BV2 microglia were pretreated with 5 µM resveratrol for 1 h, and followed by exposure to 30 nM rotenone (left) or 100 ng/ml MPO (right) for 24 h. The expression of Fcγ receptors was determined by FACS analyses using an anti-CD16/CD32 antibody directed against FcγIII/II receptors. The graph represents the fold changes in MFI ± SD from three independent experiments. *P<0.05, **P<0.01, ***P<0.001 when compared with rotenone-or MPO-treated cells.

Figure 5. Resveratrol attenuates the expression of inflammation-associated genes and ROS under rotenone- or MPO-exposed microglia.

A. Primary microglia were stimulated with rotenone (upper) or MPO (lower) in the presence or absence of resveratrol, after which the mRNA levels of iNOS, COX-2, and TNF-α were determined by RT-PCR (left) and quantitative real-time PCR analyses (right). The data are representative of three independent experiments with similar results. The graph represents the fold changes in mean ± SD of more than three independent experiments performed in triplicate. *P<0.05, **P<0.01 when compared with rotenone-or MPO-treated cells. B. Primary microglia were pretreated with or without 10 µM resveratrol for 1 h before exposure to 30 nM rotenone for 3 h, and then incubated with 5 µM DCF for 30 min at 37°C. DCF fluorescence was measured by FACS analyses. The graph represents the fold changes in MFI ± SD from four independent experiments. *P<0.05, **P<0.01 when compared with rotenone-treated cells. C. Primary microglia were pretreated with 10 µM resveratrol for 1 h, and then treated with 30 nM rotenone for 24 h. The intracellular levels of gp91 phox were determined by Western blot analyses. The bar graph represents quantitative analysis of protein band intensity from three independent experiments using ImageJ. **P<0.01, ***P<0.001 when compared with rotenone-treated cells.

Figure 6. Resveratrol relieves the impaired inflammatory responses of MPO-deficient primary mixed glia to rotenone exposure.

A. Primary mixed glial cells from Mpo^−/− mice were mock-treated or treated with the indicated concentrations of resveratrol (RESV) for 1 h before exposure to 30 nM rotenone (Rot) for 24 h, and the supernatants were assayed for nitrate concentration. The results are the fold changes in mean ± SD of three experiments performed in triplicate.*P<0.05, **P<0.01 when compared with rotenone-treated cells. B. Primary mixed glial cells from Mpo^−/− mice were mock-treated or treated with 20 µM resveratrol prior exposure to 30 nM rotenone. The levels of IL-1β, COX-2 and TNF-α mRNA in primary mixed glia from Mpo^−/− mice were determined by RT-PCR-based analyses. The data are representative of more than three independent experiments. C–D. Primary mixed glial cells from Mpo^−/− mice were incubated with the indicated concentrations of resveratrol and/or rotenone for 3 days. Cell viability was determined using the LDH assay (C) and the CCK-8 assay (D). Data were expressed as the percentage of cell death or cell viability to mock-treated cells from more than three independent experiments (C and D, respectively). *P<0.05, **P<0.01 when compared with rotenone-treated cells.

Figure 7. Rotenone-triggered neuronal injury was attenuated by resveratrol in neuron-microglia co-cultures.

A. Rat primary mesencephalic neurons were incubated with or without rat primary microglia (PM) using transwell chambers, and the cells were treated with 10 µM resveratrol and/or 30 nM rotenone for 3 days. Cell viability was analyzed using the LDH assay. Data were expressed as the percentage of cell death relative to maximum LDH control. The results are the mean ± SD of three experiments performed in triplicate. ***P<0.001 when compared with rotenone-treated cell. B. TH-positive dopaminergic neurons were determined in rotenone- and/or resveratrol-treated rat primary mesencephalic cultures with or without primary microglia. The results for TH positive cells are expressed as a percentage of the mock-treated control cultures. *P = 0.002 when compared with rotenone-treated cell; N.S. no significant difference. C-E. Primary neuron-microglia co-cultures were treated with 30 nM rotenone and/or 10 µM resveratrol for 1 day. The morphological changes of dopaminergic neuronal cells were evaluated immunocytochemically using antibodies specific for TH (green) (C). Scale bar = 20 µm. The primary dendrite number (D) and maximal dendrite length (E) of TH-positive neurons were measured using TH immunocytochemistry and AxioVision software. The results expressed as a mean percent change ± SD of the mock-treated control values from three independent experiments. *P = 0.005, **P = 0.031, ***P = 0.002; N.S., no significant difference.

Figure 8. MPP⁺-induced MPO levels and ROS were reduced by resveratrol in microglia.

A. BV2 microglia were treated with 0.1 µM 1-methyl-4-ph9enylpyridinium (MPP⁺) and/or 5 µM resveratrol (RESV) for 1 day, after which the intracellular levels of MPO were analyzed by FACS using an anti-MPO antibody. *P = 0.002, **P = 0.014 when compared with MPP⁺-treated cell. B. BV2 microglia were treated with 01 µM MPP⁺ and/or 5 µM resveratrol for 3 h, and then incubated with 5 µM DCF for 30 min at 37°C. DCF fluorescence was measured by FACS analyses. The data are representative of three independent experiments with similar results. The graph represents the fold changes in MFI (mean fluorescence intensity) ± SD from three independent experiments in triplicate. *P = 0.006, **P = 0.018 when compared with MPP⁺-treated cell.