Sinomenine, a natural dextrorotatory morphinan analog, is anti-inflammatory and neuroprotective through inhibition of microglial NADPH oxidase

Abstract

Background: The mechanisms involved in the induction and regulation of inflammation resulting in dopaminergic (DA) neurotoxicity in Parkinson's disease (PD) are complex and incompletely understood. Microglia-mediated inflammation has recently been implicated as a critical mechanism responsible for progressive neurodegeneration.

Methods: Mesencephalic neuron-glia cultures and reconstituted cultures were used to investigate the molecular mechanisms of sinomenine (SN)-mediated anti-inflammatory and neuroprotective effects in both the lipopolysaccharide (LPS)- and the 1-methyl-4-phenylpyridinium (MPP+)-mediated models of PD.

Results: SN showed equivalent efficacy in protecting against DA neuron death in rat midbrain neuron-glial cultures at both micro- and sub-picomolar concentrations, but no protection was seen at nanomolar concentrations. The neuroprotective effect of SN was attributed to inhibition of microglial activation, since SN significantly decreased tumor necrosis factor-alpha (TNF-alpha, prostaglandin E2 (PGE2) and reactive oxygen species (ROS) production by microglia. In addition, from the therapeutic point of view, we focused on sub-picomolar concentration of SN for further mechanistic studies. We found that 10(-14) M of SN failed to protect DA neurons against MPP+-induced toxicity in the absence of microglia. More importantly, SN failed to show a protective effect in neuron-glia cultures from mice lacking functional NADPH oxidase (PHOX), a key enzyme for extracellular superoxide production in immune cells. Furthermore, we demonstrated that SN reduced LPS-induced extracellular ROS production through the inhibition of the PHOX cytosolic subunit p47phoxtranslocation to the cell membrane.

Conclusion: Our findings strongly suggest that the protective effects of SN are most likely mediated through the inhibition of microglial PHOX activity. These findings suggest a novel therapy to treat inflammation-mediated neurodegenerative diseases.

Figure 1

Both micromolar and sub-picomolar concentrations of SN are neuroprotective against LPS-induced neurotoxicity. Rat primary mesencephalic neuron-glia cultures seeded in a 24-well culture plate with 5 × 10⁵ rat midbrain cells, then pretreated with SN (10^-17 to 10^-5 M) for 30 min before the addition of 10 ng/ml LPS. Eight days later, the LPS-induced dopaminergic neurotoxicity was quantified by the [³H]-DA uptake assay (A); by immunocytochemical analysis, including TH-IR neuron counts (B); and by representative pictures of immunostained sections (C). Results are expressed as percentage of vehicle-treated control cultures and represent the mean ± SE. for four (A) or three (B, C) independent experiments in triplicate. The mean absolute values of [³H]-DA uptake for vehicle-treated cultures range from 5000 to 7000 cpm. *P < 0.05, **P < 0.01 compared with the LPS-treated cultures.

Figure 2

Effect of SN on LPS-induced production of pro-inflammatory factors and their mRNA expression. Enriched microglia cells were pretreated with vehicle or SN (10^-5, 10^-10, and 10^-14 M) for 30 min before LPS (10 ng/ml) stimulation. Effects of SN are shown on LPS-induced production of superoxide (A, expressed as % of control); and on intracellular ROS (B, expressed as absolute absorbance). Extracellular superoxide was measured as SOD-inhibitable reduction of WST-1, and intracellular ROS was determined by probe DCFH-DA. Supernatant was collected at 24 h for nitrite assay (C), at 3 h for TNF-α assay (D), and at 24 h for PGE₂ assay (E). RNA were extracted at 3 h after LPS stimulation; the effect of SN, at sub-picomolar concentrations, on LPS-induced iNOS, TNF-α, and COX-2 mRNA expression (C-E respectively, open bars) are shown. Results are expressed as mean ± SE for three independent experiments performed in triplicate. *P < 0.05, **P < 0.01 compared with the LPS-treated cultures.

Figure 3

SN protects against MPP⁺-elicited DA neurodegeneration through microglia. SN (10^-14 M) or MPP⁺ (0.2 μM) were added to the following types of cell cultures: (NG): original neuron-glial cultures; (N): neuron-enriched cultures; (N+10%MG): cultures reconstituted by adding 10% of microglia to the neuron-enriched cultures; (N+50%AS): cultures reconstituted by adding 50% of astroglia to the neuron-enriched cultures. Two and 4 days after MPP⁺ treatment, SN (10^-14 M) was added again to the SN-treated cultures. On day 8, the MPP⁺-induced dopaminergic neurotoxicity was quantified by the [³H]-DA uptake assay (A), and on day 4, the release of superoxide was determined as described in Materials and methods section. Results are expressed as percentage of the vehicle-treated control cultures and represent the mean ± SE. for three independent experiments performed in triplicate. *P < 0.05, compared with MPP⁺ treated cultures. # P < 0.05, ## P < 0.01 compared with the vehicle-treated control cultures.

Figure 4

Microglial PHOX is critical for sub-picomolar SN neuroprotection. PHOX^+/+ and PHOX^-/- mouse neuron-glia cultures were pretreated with vehicle or SN (10^-14 M) for 30 min, followed by LPS treatment. Neurotoxicity was assessed by measuring DA uptake (A), TNF-α production (B) and intracelluar ROS (C), respectively. Results are expressed as % of the control culture (A and C) and as pg/ml (B), and represent the mean ± SE for 3 individual experiments performed in triplicate in each experiment. *P < 0.05 compared with LPS culture. # P < 0.05, ## P < 0.01 compared with the vehicle-treated control cultures.

Figure 5

Immunofluorescence and confocal microscopical analysis of p47^phox localization in LPS-stimulated microlgia cells. HAPI cells were treated with LPS for 10 min in the absence or presence of SN pretreatment for 0.5 h. Cells were immunostained with a rabbit polyclonal antibody against p47^phox, then washed and incubated with FITC-conjugated goat anti-rabbit antibody. The signal of p47^phox (FITC-p47^phox; on left) and the merge view of cell morphology and p47^phox (Phase plus FITC-p47^phox, on right) are shown. The inset shown in the right corner of each treatment condition shows the location of FITC-p47^phox in a single, randomly selected cell. Focal planes spaced at 0.4-μm intervals were imaged with a Zeiss 510 laser scanning confocal microscope (63 × PlanApo 1.4 numerical aperture objective) equipped with LSM510 digital imaging software. Three adjacent focal planes were averaged using Metamorph software. The signal of p47^phox (FITC-p47^phox; green) and the merge view of cell morphology and p47^phox (Phase plus FITC-p47^phox) are shown. Scale bar, 50 μm.

Figure 6

Membrane fraction analysis of the effect of SN on cytosolic p47^phoxprotein translocation. HAPI cells were pretreated with vehicle or SN (10^-14 M) for 30 min, followed by LPS treatment for 10 min. Membrane fractions were isolated to perform western blot analysis, using gp91^phox as an internal membrane control. Each experiment was performed three times. *P < 0.05, compared with the LPS-treated cultures.