Sinomenine, a natural dextrorotatory morphinan analog, is anti-inflammatory and neuroprotective through inhibition of microglial NADPH oxidase
© Qian et al; licensee BioMed Central Ltd. 2007
Received: 16 May 2007
Accepted: 19 September 2007
Published: 19 September 2007
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.
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.
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-α (TNF-α, 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 p47 phox translocation to the cell membrane.
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.
Increasing evidence has shown that the production and accumulation of pro-inflammatory and cytotoxic factors by over-activated microglia are closely associated with the pathogenesis of several neurological disorders, such as Parkinson's disease (PD), Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, and the AIDS dementia complex [1–4]. Microglia are the main immune cells in the brain, which provides the CNS immune protection against infection under normal physiologic conditions . However, in pathological conditions microglia may easily become over-activated and produce large amounts of pro-inflammatory factors, such as cytokines, prostaglandins, and reactive oxygen species (ROS), which result in neuronal death [6–8]. Microglia are found in great abundance in the midbrain region that encompasses the substantia nigra . Thus, it has been suggested that activation of nigral microglia and subsequent release of these pro-inflammatory neurotoxic factors is a critical component of the degenerative process of dopaminergic (DA) neurons in PD. Unfortunately, therapies using specific inhibitors to prevent the production of individual pro-inflammatory and neurotoxic factors have not been particularly successful, suggesting that the mechanism of inflammation-mediated damage to DA neurons has yet to be elucidated. In addition, current DA replacement therapy for PD is only able to treat the symptoms, and fails to slow down the progression of the disease. Therefore, there is an urgent need to develop drugs that have wide spectrum anti-inflammatory effects and which are able to slow down or curtail the progression of the degenerative process.
The alkaloid sinomenine (SN) is a pure compound extracted from the Chinese medicinal plant, sinomenium acutum, which has been utilized to treat inflammatory diseases for many centuries [10, 11]. Clinical trials have demonstrated that purified SN has significant therapeutic efficacy for patients who suffer from rheumatoid arthritis [10, 12]. Previous pharmacological studies have demonstrated that the pharmacological profile of SN includes immunosuppression , arthritis amelioration , anti-inflammation  and protection against hepatitis induced by lipopolysaccharide (LPS) . In addition, in studies using intramuscular injection and multiple dosing, a combination of SN and cyclosporin A showed immunomodulatory effects in a cardiac transplant model . Up to now, little is known about the molecular mechanism by which SN exhibits immunomodulatory effects.
Based on its molecular structure, SN belongs to the family of morphinans. Morphinans are a series of compounds structurally similar to morphine, but lacking the E ring as well as the 6-OH and the 7, 8-double bond. Our previous studies have clearly shown that several morphinan compounds, including naloxone  and dextromethorphan , are neuroprotective anti-inflammatory agents. Given the reported anti-inflammatory properties of SN, the record of its effective use in clinical therapy, its natural abundance, and its relative low cost in purification and preparation, we theorized that this compound might be a better alternative than synthetic morphinan compounds in inhibiting inflammatory destruction of DA neurons. Moreover, the finding that it is a naturally occurring dextrorotatory morphinan isomer indicates that it would have minimal interaction with opiate receptors, and therefore prove to be a much safer alternative than some other morphinan compounds when used chronically to treat PD. In this study, we report that SN shows a significant neuroprotective effects against both LPS- and MPP+-induced DA neurotoxicity, and that this protection is mediated through microglia. The finding that SN is acting to inhibit NADPH oxidase (PHOX) activity, which then results in the inhibition of a wide array of pro-inflammatory mediators produced by activated microglia, suggests that SN may be a potential novel and safe therapeutic agent for the treatment of inflammatory-mediated neurodegenerative diseases.
PHOX-deficient (gp91phox-/-) and wild-type C57BL/6J (gp91phox+/+) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Breeding of the mice was performed to achieve timed pregnancy with the accuracy of ± 0.5 days. Timed-pregnant Fisher F344 rats were obtained from Charles River Laboratories (Raleigh, NC). Housing and breeding of the animals were performed in strict accordance with the National Institutes of Health guidelines.
SN was obtained from Sigma-Aldrich (St. Louis, MO). LPS (strain O111:B4) was purchased from Calbiochem (San Diego, CA). Cell culture reagents were obtained from Invitrogen (Carlsbad, CA). [3H]-DA (30 Ci/mmol) was obtained from Perkin-Elmer Life Sciences (Boston, MA), and the polyclonal anti-tyrosine hydroxylase antibody was a generous gift from Dr. John Reinhard (GlaxoSmithKline, Research Triangle Park, NC). The Vectastain ABC kit and biotinylated secondary antibodies were purchased from Vector Laboratories (Burlingame, CA). The fluorescence probe Dichlorodihydro-fluorescein Diacetate (DCFH-DA) was obtained from Calbiochem (La Jolla, CA).
Microglial cell line
The rat microglia HAPI cell line was a generous gift from Dr. James R. Connor . Briefly, cells were maintained at 37°C in DMEM supplemented with 10% FBS, 50 U/ml penicillin, and 50 μg/ml streptomycin in a humidified incubator with 5% CO2 and 95% air.
Primary mesencephalic neuron-glia culture
Neuron-glia cultures were prepared from the ventral mesencephalic tissues of embryonic day 14–15 rats or day 13–14 mice, as described previously . Briefly, dissociated cells were seeded at 1 × 105/well and 5 × 105/well to poly-D-lysine-coated 96-well and 24-well plates respectively. Cells were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air, in MEM containing 10% fetal bovine serum, 10% horse serum, 1 g/L glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 μM nonessential amino acids, 50 U/ml penicillin, and 50 μg/ml streptomycin. Seven-day-old cultures were used for treatment. At the time of treatment, immunocytochemical analysis indicated that the rat neuron-glia cultures were made up of 11% microglia, 48% astrocytes, 41% neurons, and 1% tyrosine hydroxylase immunoreacitve (TH-IR) neurons. The composition of the neuron-glia cultures of PHOX-deficient mice was very similar to that of the wild-type mice consisting of 12% microglia, 48% astrocytes, 40% neurons, and 1% TH-IR neurons.
Primary mesencephalic neuron-enriched and microglia-enriched Cultures
Midbrain neuron-enriched cultures were established as described previously . Briefly, 24 h after seeding the cells, cytosine β-D-arabinocide was added to a final concentration of 5 μM to suppress glial proliferation. Three days later, cultures were changed back to maintenance medium and were used for treatment 7 days after initial seeding. Rat microglia-enriched cultures, with a purity of > 98%, were prepared from whole brains of 1-day-old Fischer 344 rat pups as described previously .
[3H]-DA uptake assay
[3H]-DA uptake assays were performed as described . Briefly, cells were incubated for 20 min at 37°C with 1 μM [3H]-DA in Krebs-Ringer buffer (16 mM sodium phosphate, 119 mM NaCl, 4.7 mM KCl, 1.8 mM CaCl2, 1.2 mM MgSO4, 1.3 mM EDTA, and 5.6 mM glucose; pH 7.4). After washing three times with ice-cold Krebs-Ringer buffer, the cells were collected in 1 N NaOH. Radioactivity was determined by liquid scintillation counting. Nonspecific DA uptake observed in the presence of mazindol (10 μM) was subtracted.
DA neurons were recognized with the anti-TH antibody as described previously . Briefly, formaldehyde (3.7%)-fixed cultures were treated with 1% hydrogen peroxide (10 min) followed by sequential incubation with blocking solution for 30 min, primary antibody overnight at 4°C, biotinylated secondary antibody for 2 h, and ABC reagents for 40 min. Color was developed with 3,3'-diaminobenzidine. For morphological analysis, the images were recorded with an inverted microscope (Nikon, Tokyo, Japan) connected to a charge-coupled device camera (DAGE-MTI, Michigan City, IN) operated with the MetaMorph software (Universal Imaging Corporation, Downingtown, PA). For visual counting of TH-IR neurons, nine representative areas per well of the 24-well plate were counted under the microscope at 100 × magnification by three individuals. The average of these scores was reported.
Nitrite and tumor necrosis factor (TNF-α assays)
The production of NO was determined by measuring the accumulated levels of nitrite in the supernatant with Griess reagent and the release of TNF-α was measured with a rat TNF-α enzyme-linked immunosorbent assay kit from R and D System (Minneapolis, MN, USA), as described .
Prostaglandin E2 (PGE2) production
PGE2 in supernatant was measured with a PGE2 EIA kit from Cayman (Ann Arbor, MI, USA) according to the manufacturer's instructions.
The production of superoxide was determined by measuring the superoxide dismutase (SOD)-inhibitable reduction of the tetrazolium salt WST-1 [24, 25]. Microglia-enriched cultures in 96-well culture plates were washed twice with HBSS without phenol red. Cultures were then incubated at 37°C for 30 min with vehicle control (water) or SN in HBSS (50 μl/well). Then, 50 μl of HBSS with and without SOD (50 U/ml, final concentration) was added to each well along with 50 μl of WST-1 (1 mM) in HBSS, and 50 μl of vehicle or LPS (10 ng/ml). To measure superoxide production induced by 0.2 uM MPP+, seven days-old mesencephalic neuron-glia cultures grown in 96-well plates were treated with SN in the presence or absence of MPP+, or vehicle in 150 μl of phenol red-free treatment medium. Four days after treatment, 50 μl of HBSS with and without SOD (50 U/ml, final concentration) was added to each well along with 50 μl of WST-1 (1 mM) in HBSS. Fifteen minutes later, absorbance at 450 nm was read with a SpectraMax Plus microplate spectrophotometer (Molecular Devices Corp, Sunnyvale, CA). The difference in absorbance observed in the presence or absence of SOD was considered to be the amount of superoxide produced, and results were expressed as percentage of vehicle-treated control cultures.
Assay of intracellular reactive oxygen species
Intracellular oxidative stress was measured by DCFH Oxidation. DCFH-DA enters cells passively and is deacetylated by esterase to nonfluorescent DCFH. DCFH reacts with ROS to form dichlorodifluorescein, the fluorescent product. DCFH-DA was dissolved in methanol at 10 mM and was diluted 500-fold in HBSS to give DCFH-DA at 20 μM. The cells were exposed to DCFH-DA for 1 h and then treated with HBSS containing the corresponding concentrations of LPS for 2 h. The fluorescence was read immediately at wavelengths of 485 nm for excitation and 530 nm for emission using a SpectraMax Gemini XS fluorescence microplate reader (Molecular Devices). The value subtracted by control group was viewed as the increase of intracellular ROS.
Real time RT-PCR analysis
Real time quantitative PCR was performed as described previously , The relative differences between control and treatment groups were calculated and expressed as relative increases setting control as 100%. The sequence of the oligonucleotide primers from rats were: TNF-α: 5'-TCGTAGCAAACCACCAAGCA-3' and 5'-CCCTTGAAGAGAACCTGGGAGTA-3'; Inducible nitric oxide synthase (iNOS): 5'-ACATCAGGTCGGCCATCACT-3', and 5'-CGTACCGGATGAGCTGTGAATT-3'; Cyclo-oxygenases-2 (COX-2): 5'-CCAGCAG GCTCATACTGATAGGA-3' and 5'-GCAGGTCTGGGTCGAACTTG-3'; GAPDH: 5'-CCTGGAGAAACCTGCCAAGTAT-3' and 5'-AG CCCAGGATGCCCTTTAGT-3'.
Confocal analysis was performed as described previously . HAPI cells seeded in dish at 5 × 104 cells/well were treated with LPS for 10 min in the absence or presence of SN (10-14 M) pretreatment for 30 min. Cells were fixed with 3.7% paraformaldehyde in PBS for 10 min. After a washing with PBS, cells were incubated with rabbit polyclonal antibody against p47 phox . Cells were then washed and incubated with FITC-conjugated goat anti-rabbit antibody. 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. 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.
Membrane fractionation and western blot analysis
Membrane fractionation was performed as described (34). HAPI cells were lysed in hypotonic lysis buffer (1 mM EGTA, 1 mM EDTA, 10 mM β-lycerophosphate, 10 mM NaF, 1 mM sodium orthovanadate, 2 mM MgCl2, 10 mM DTT, 1 mM PMSF, and 10 ug/ml each leupeptin, aprotinin, and pepstatin A), incubated on ice for 30 min, and then subjected to Dounce homogenization (20~25 stokes, tight pestle A). The lysates were loaded onto sucrose in lysis buffer (final 0.5 M) and centrifuged at 1600 × g for 15 min, The supernatant above the sucrose gradient was centrifuged at 150,000 × g for 30 min. The resulting pellets were solubilized in 1% NP-40 hypotonic lysis buffer and used as membranous fraction. Equal amounts of protein (20 μg per lane) were separated by 4~12% Bis-Tris Nu-PAGE gel and transferred to polyvinylidene difluoride membranes (Novex, San Diego, CA). Membranes were blocked with 5% nonfat milk and incubated with rabbit anti-p47 phox antibody (1: 2000 dilution) or mouse anti-gp91phox (1:1000 dilution) for 1 h at 25°C. Horseradish peroxidase-linked anti-rabbit or mouse IgG (1:3000 dilution) for 1 h at 25°C, ECL+Plus reagents (Amersham Biosciences Inc., Piscataway, NJ) was used as a detection system.
The data are presented as mean ± SE. For multiple comparisons of groups, two-way ANOVA was used. Statistical significance of differences between groups was assessed using paired Student's t test, followed by Bonferroni correction using the JMP program (SAS Institute, Cary, NC, USA). A value of P < 0.05 was considered statistically significant.
Effect of SN on LPS-induced DA neurotoxicity
SN pretreatment suppresses LPS-induced pro-inflammatory factors production and gene expression
SN also suppresses MPP+-induced neurotoxicity by inhibition of microglia activation
We then sought to determine the cellular target of SN-mediated neuroprotection. Results showed that SN had no effect on the toxic effects of MPP+ in neuron-enriched cultures (Fig. 3A – N), demonstrating that SN does not function by directly protecting neurons from MPP+-mediated toxicity. We then investigated if glial cells were the target for the neuroprotective activity of SN by reconstituting neuron-enriched cultures with either 10% purified microglia (Fig. 3A – N + 10% MG) or 50% astroglia (Fig. 3A – N+50% AS). The percentage of glial cells chosen for reconstitution was based on the fact that our standard mesencephalic neuron-glia cultures contain ~10% microglia and ~50% astrocytes in addition to DA and other neurons. We found that while SN is not able to protect DA neurons in neuron-astroglia reconstituted cultures, SN was able to reduce neurotoxicity in neuron-microglia reconstituted cultures to a level similar to that seen in the original neuron-glia cultures. This result indicates that there is an additional, indirect microglia-mediated neurotoxicity by MPP+ through reactive microgliosis, a process initiated when the death or damage of neurons triggers further activation of microglia .
Previous work from our laboratory and others have indicated that oxidative stress plays a very important role in the progressive neurodegeneration, and MPTP/MPP+-induced reactive microgliosis has been clearly linked with microglial activation and closely associated with increased production of oxygen free radicals [27, 29]. Therefore, we sought to determine whether SN is able to reduce MPP+-induced ROS production. Rat mesencephalic neuron-glia cultures were pretreated with SN (10-14 M) before the addition of MPP+. Release of superoxide from activated microglia was then determined on day 4 after treatment. As shown in Fig. 3B, treatment of cultures with SN significantly inhibited MPP+-induced superoxide production. Interestingly, we found that while significant superoxide production was detected following MPP+-induced reactive microgliosis, the production of other inflammatory mediators, including TNF-α and nitrite, were not detectable at any time during the 7-day MPP+ treatment in neuron-glial cultures (data not shown). Taken together, these results demonstrated that SN can inhibit ROS production induced either directly by LPS stimulation or indirectly by MPP+-mediated reactive microgliosis, and lend further evidence to the idea that superoxide production, rather than TNF-α or nitrite production, is the mechanism of neurotoxicity in reactive microgliosis.
PHOX is essential for SN mediated neuroprotection
The main findings in this study are the elucidation of anti-inflammatory and neuroprotective effects of SN at both the cellular and molecular level. Using both LPS and MPP+-mediated PD models, we are the first to demonstrate that SN could be effective in diminishing inflammation-induced neurodegeneration at both micro- and sub-picomolar concentrations in primary midbrain neuron-glia cultures. Mechanistic studies revealed that inhibition of microglial PHOX activity is the target for SN-mediated neuroprotection in both LPS- and MPP+-induced DA neurotoxicity. The mechanism underlying SN-mediated inhibition of PHOX activity occurs at least in part through the inhibition of the translocation of PHOX cytosolic component p47 phox to the plasma membrane, a key event required for extracellular ROS production . We are currently investigating the effects of SN on the activity of the other subunits of the PHOX enzyme, including p67 phox , p40 phox , gp91 phox , p22 phox and Rac2. The inhibition of PHOX leads to a subsequent reduction in the production of other pro-inflammatory mediators, such as intracellular ROS, TNF-α, NO and PGE2. Our studies suggest that reduction in PHOX activity by SN at the site of inflammation diminishes host tissue damage, thereby underlying the neuroprotective effect of SN for both LPS- and MPP+-induced DA neurotoxicity.
Although SN has been used clinically as an anti-inflammatory agent in several inflammation-related diseases [10, 11], so far only a few studies have addressed the immunomodulatory mechanism of this herbal medicine. For example, it has been reported that SN inhibits the production of pro-inflammatory mediators, such as TNF-α, IL-1, PGE2, leukotriene C4 and NO from macrophages [15, 32, 33]. Our observations have not only extended the above findings to microglia, but more importantly, our studies indicate that the reduction in the release of these pro-inflammatory factors by SN could be due to the inhibition of superoxide production through the inhibition of microglial PHOX activity. Our earlier studies established that inhibition of microglial production of superoxide was most effective in protecting neurons, indicating that superoxide was a dominant degenerative factor for the DA neurons in the culture . Our evidence that SN significantly inhibits the production of superoxide induced by either LPS or MPP+, as well as the observation that ROS production is the only inflammatory mediator that can be detected after exposure of neuron-glial cultures to MPP+, led us to examine the role of PHOX in neurotoxicity in greater detail by using PHOX-deficient mutant mice. Our findings that sub-picomolar SN could significantly lessen the LPS-induced DA uptake reduction in wild-type mice, has no significant protective effect in PHOX-/- mice (Fig. 4A) strongly support the conclusion that PHOX activity is critical to SN-mediated DA neuroprotection. Moreover, the production of intracellular ROS and release of TNF-α are both reduced in PHOX-/- mice, suggesting PHOX can indirectly regulate intracellular ROS concentration and ultimately the production of pro-inflammatory mediators. We can not rule out the possibility that cells other than microglia are producing superoxide, and that PHOX is not the only enzyme which may play a role in ROS production. However, only microglia express LPS receptors and are activated by LPS, and therefore no other cell is likely to produce ROS upon stimulation with LPS. In addition, no extracellular superoxide production has been detected in cultures from PHOX-/- mice (Qin et al., 2004), suggesting that PHOX is the only enzyme involved in superoxide production in these cultures. It has been found that an increase of intracellular ROS can intensify the activation of NF-kB, which leads to higher TNF-α and PGE2 production [2, 28]. In addition, it has been reported that PHOX inhibitors prevent LPS/IFNγ-induced degradation of IkB and, thus, inhibit the activation of NF-kB . These data are consistent with the notion that PHOX plays a central role in both inflammation-induced neurotoxicity, as well as in the regulation of the inflammatory response by microglial cells, and that SN can inhibit these inflammatory responses by inhibiting PHOX activity.
The bimodal dose response of SN in the inhibition of LPS-induced superoxide production and neuroprotection adds SN to the list of compounds which show the same pattern of responsiveness, including dynorphins , enkephalins , endorphin , pituitary adenylate cyclase-activating polypeptide (4–6) . Currently it is still not clear how both micro- and sub-picomolar concentrations of SN and the other compounds inhibit PHOX activation. To further understand how SN inhibits PHOX activity, we performed the translocation experiment using confocal microscopy. We found that the translocation of the p47 phox subunit from the cytosol to the plasma membrane following LPS stimulation was inhibited by SN. As the activation of a functional PHOX enzyme requires the phorsphorylation of p47 phox followed by the translocation of the p47 phox , p67 phox , and p40 phox complex to the plasma membrane, our results suggest that SN works on one of the earliest stages of PHOX activation to inhibit the assembly of the PHOX enzyme complex. Presently the molecular mechanisms by which SN leads to the inhibition of p47 phox translocation are under investigation.
It is interesting to note that SN has protective effects in both the LPS and the MPP+ model of PD, although the mode of action for these two agents to produce neurotoxicity is different. In the LPS model, it is clear that direct activation of microglia by LPS leads to the activation of PHOX, which results in the production of superoxide that mediates the neurotoxicity, and that direct inhibition of PHOX by SN in microglia can prevent this destruction. However, even though MPP+ directly targets DA neurons to produce toxicity, SN still shows a significant protective effect in these cultures as well. This is most likely due to the fact that a significant portion of the MPP+-mediated neurotoxicity requires the presence of microglial cells, suggesting that MPP+ works both directly to kill a subset of DA neurons, but also indirectly to activate microglia through reactive microgliosis. Recent evidence indicates that neuronal death or damage triggers activation of microglia through either release of soluble factors or loss of cell-cell contact inhibition between neurons and microglia [27, 39]. This activation of microglia by dying neurons then continuously damages the remaining DA neurons, and creates a self-propelling inflammatory cycle, which may underlie the progressive nature of neurodegenerative diseases such as PD. Reports from our laboratory and others have indicated that MPP+ can cause reactive microgliosis, and that oxidative stress is involved in MPTP/MPP+ -induced neurotoxicity [27, 40]. Our observation that MPP+-mediated neurotoxicity is significantly less in PHOX-/- animals , and that SN does not protect DA neurons from MPP+-mediated toxicity in neuron-enriched or neuron-astroglial cultures (Fig. 3), supports the notion that SN protects MPP+-induced neurotoxicity mainly through the inhibition of ROS production, which in turn slows down the self-propelling cycle and prevents further neuronal death. In addition, although LPS and MPP+ work in different ways to produce neurotoxicity, the death of neurons induced by both LPS and MPP+ will further activate microglia through the process of reactive microgliosis, in which superoxide plays a critical role.
In summary, this study is the first report indicating that SN is a potent anti-inflammatory and neuroprotective agent that acts through inhibition of microglial PHOX-generated superoxide by inhibiting the translocation of p47 phox to the plasma membrane. We have previously reported that several morphinan compounds, such as naloxone, exert their neuroprotective effects independent of the conventional opioid receptors [18, 22], because the dextrorotatory form of this compound, which binds poorly to the opioid receptors, is equipotent with its levorotatory isomer. SN, being a natural dextrorotatory form of morphinan that can be easily extracted and purified from its herb plant, is an ideal alternative for other synthesized dextrorotatory form morphinan compounds since the dextrorotatory isomers are extremely expensive to fabricate. In view of its potent anti-inflammatory and neuroprotective effects, its safe record of clinical usage to treat rheumatoid arthritis, and its relatively low cost, SN is an ideal candidate for animal and clinical trial studies to evaluate its therapeutic potential as a neuroprotective agent for PD and other inflammation-related neurodegenerative diseases.
We thank Dr. Jie Liu at NIEHS for his help in performing the real time PCR studies, and thank Dr. Brian Mill at NIEHS for his help in editing this paper. This work was supported by NIH grant DE-13079 from the National Institute for Dental and Craniofacial Research, and was also supported in part by the Intramural Research Program of the NIH/NIEHS.
- McGeer PL SI, Boyes BE, McGeer EG: Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains. Neurology. 1988, 38: 1285-1291.View ArticlePubMedGoogle Scholar
- Liu B, Hong JS: Role of microglia in inflammation-mediated neurodegenerative diseases: mechanisms and strategies for therapeutic intervention. J Pharmacol Exp Ther. 2003, 304 (1): 1-7. 10.1124/jpet.102.035048.View ArticlePubMedGoogle Scholar
- Rosi S, Ramirez-Amaya V, Vazdarjanova A, Worley PF, Barnes CA, Wenk GL: Neuroinflammation alters the hippocampal pattern of behaviorally induced Arc expression. J Neurosci. 2005, 25 (3): 723-731. 10.1523/JNEUROSCI.4469-04.2005.View ArticlePubMedGoogle Scholar
- Chen LC, Smith A, Ben Y, Zukic B, Ignacio S, Moore D, Lee N: Temporal gene expression patterns in G93A/SOD1 mouse. Amyotroph Lateral Scler Other Motor Neuron Disord. 2004, 5 (3): 164-171. 10.1080/14660820410017091.View ArticlePubMedGoogle Scholar
- Kreutzberg GW: Microglia: a sensor for pathological events in the CNS. Trends Neurosci. 1996, 19 (8): 312-318. 10.1016/0166-2236(96)10049-7.View ArticlePubMedGoogle Scholar
- McGuire SO, Ling ZD, Lipton JW, Sortwell CE, Collier TJ, Carvey PM: Tumor necrosis factor alpha is toxic to embryonic mesencephalic dopamine neurons. Exp Neurol. 2001, 169 (2): 219-230. 10.1006/exnr.2001.7688.View ArticlePubMedGoogle Scholar
- Sriram K, Matheson JM, Benkovic SA, Miller DB, Luster MI, O'Callaghan JP: Mice deficient in TNF receptors are protected against dopaminergic neurotoxicity: implications for Parkinson's disease. Faseb J. 2002, 16 (11): 1474-1476.PubMedGoogle Scholar
- Chao CC, Hu S, Molitor TW, Shaskan EG, Peterson PK: Activated microglia mediate neuronal cell injury via a nitric oxide mechanism. J Immunol. 1992, 149 (8): 2736-2741.PubMedGoogle Scholar
- Kim WG, Mohney RP, Wilson B, Jeohn GH, Liu B, Hong JS: Regional difference in susceptibility to lipopolysaccharide-induced neurotoxicity in the rat brain: role of microglia. J Neurosci. 2000, 20 (16): 6309-6316.PubMedGoogle Scholar
- Yamasaki H: Pharmacology of sinomenine, an anti-rheumatic alkaloid from Sinomenium acutum. Acta Med Okayama. 1976, 30 (1): 1-20.PubMedGoogle Scholar
- Feng CI, Chin Y, Wang NC, Chang SS: [The pharmacology of sinomenine. VII. Effect of sinomenine on the gastro-intestinal movement and its mechanism]. Yao Xue Xue Bao. 1965, 12 (8): 492-495.PubMedGoogle Scholar
- Wang Y, Zhou L, Li R: [Study progress in Sinomenium acutum (Thunb.) Rehd. et Wils.]. Zhong Yao Cai. 2002, 25 (3): 209-211.PubMedGoogle Scholar
- Vieregge B, Resch K, Kaever V: Synergistic effects of the alkaloid sinomenine in combination with the immunosuppressive drugs tacrolimus and mycophenolic acid. Planta Med. 1999, 65 (1): 80-82. 10.1055/s-2006-960446.View ArticlePubMedGoogle Scholar
- Liu L, Buchner E, Beitze D, Schmidt-Weber CB, Kaever V, Emmrich F, Kinne RW: Amelioration of rat experimental arthritides by treatment with the alkaloid sinomenine. Int J Immunopharmacol. 1996, 18 (10): 529-543. 10.1016/S0192-0561(96)00025-2.View ArticlePubMedGoogle Scholar
- Liu L, Riese J, Resch K, Kaever V: Impairment of macrophage eicosanoid and nitric oxide production by an alkaloid from Sinomenium acutum. Arzneimittelforschung. 1994, 44 (11): 1223-1226.PubMedGoogle Scholar
- Kondo Y, Takano F, Yoshida K, Hojo H: Protection by sinomenine against endotoxin-induced fulminant hepatitis in galactosamine-sensitized mice. Biochem Pharmacol. 1994, 48 (5): 1050-1052. 10.1016/0006-2952(94)90378-6.View ArticlePubMedGoogle Scholar
- Candinas D, Mark W, Kaever V, Miyatake T, Koyamada N, Hechenleitner P, Hancock WW: Immunomodulatory effects of the alkaloid sinomenine in the high responder ACI-to-Lewis cardiac allograft model. Transplantation. 1996, 62 (12): 1855-1860. 10.1097/00007890-199612270-00030.View ArticlePubMedGoogle Scholar
- Liu B, Du L, Hong JS: Naloxone protects rat dopaminergic neurons against inflammatory damage through inhibition of microglia activation and superoxide generation. J Pharmacol Exp Ther. 2000, 293 (2): 607-617.PubMedGoogle Scholar
- Liu Y, Qin L, Li G, Zhang W, An L, Liu B, Hong JS: Dextromethorphan protects dopaminergic neurons against inflammation-mediated degeneration through inhibition of microglial activation. J Pharmacol Exp Ther. 2003, 305 (1): 212-218. 10.1124/jpet.102.043166.View ArticlePubMedGoogle Scholar
- Cheepsunthorn P, Radov L, Menzies S, Reid J, Connor JR: Characterization of a novel brain-derived microglial cell line isolated from neonatal rat brain. Glia. 2001, 35 (1): 53-62. 10.1002/glia.1070.View ArticlePubMedGoogle Scholar
- Qian L, Block ML, Wei SJ, Lin CF, Reece J, Pang H, Wilson B, Hong JS, Flood PM: Interleukin-10 protects lipopolysaccharide-induced neurotoxicity in primary midbrain cultures by inhibiting the function of NADPH oxidase. J Pharmacol Exp Ther. 2006, 319 (1): 44-52. 10.1124/jpet.106.106351.View ArticlePubMedGoogle Scholar
- Liu B, Du L, Kong LY, Hudson PM, Wilson BC, Chang RC, Abel HH, Hong JS: Reduction by naloxone of lipopolysaccharide-induced neurotoxicity in mouse cortical neuron-glia co-cultures. Neuroscience. 2000, 97 (4): 749-756. 10.1016/S0306-4522(00)00057-9.View ArticlePubMedGoogle Scholar
- Liu Y, Qin L, Wilson BC, An L, Hong JS, Liu B: Inhibition by naloxone stereoisomers of beta-amyloid peptide (1-42)-induced superoxide production in microglia and degeneration of cortical and mesencephalic neurons. J Pharmacol Exp Ther. 2002, 302 (3): 1212-1219. 10.1124/jpet.102.035956.View ArticlePubMedGoogle Scholar
- Peskin AV, Winterbourn CC: A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1). Clin Chim Acta. 2000, 293 (1–2): 157-166. 10.1016/S0009-8981(99)00246-6.View ArticlePubMedGoogle Scholar
- Tan AS, Berridge MV: Superoxide produced by activated neutrophils efficiently reduces the tetrazolium salt, WST-1 to produce a soluble formazan: a simple colorimetric assay for measuring respiratory burst activation and for screening anti-inflammatory agents. J Immunol Methods. 2000, 238 (1–2): 59-68. 10.1016/S0022-1759(00)00156-3.View ArticlePubMedGoogle Scholar
- Liu J, Shen HM, Ong CN: Role of intracellular thiol depletion, mitochondrial dysfunction and reactive oxygen species in Salvia miltiorrhiza-induced apoptosis in human hepatoma HepG2 cells. Life Sci. 2001, 69 (16): 1833-1850. 10.1016/S0024-3205(01)01267-X.View ArticlePubMedGoogle Scholar
- Block ML, Hong JS: Microglia and inflammation-mediated neurodegeneration: Multiple triggers with a common mechanism. Prog Neurobiol. 2005, 76 (2): 77-98. 10.1016/j.pneurobio.2005.06.004.View ArticlePubMedGoogle Scholar
- Qin L, Liu Y, Wang T, Wei SJ, Block ML, Wilson B, Liu B, Hong JS: NADPH oxidase mediates lipopolysaccharide-induced neurotoxicity and proinflammatory gene expression in activated microglia. J Biol Chem. 2004, 279 (2): 1415-1421. 10.1074/jbc.M307657200.View ArticlePubMedGoogle Scholar
- Pazdernik TL, Emerson MR, Cross R, Nelson SR, Samson FE: Soman-induced seizures: limbic activity, oxidative stress and neuroprotective proteins. J Appl Toxicol. 2001, 21 (Suppl 1): S87-94. 10.1002/jat.818.View ArticlePubMedGoogle Scholar
- Groemping Y, Rittinger K: Activation and assembly of the NADPH oxidase: a structural perspective. Biochem J. 2005, 386 (Pt 3): 401-416.PubMed CentralView ArticlePubMedGoogle Scholar
- Johnson JL, Park JW, Benna JE, Faust LP, Inanami O, Babior BM: Activation of p47(PHOX), a cytosolic subunit of the leukocyte NADPH oxidase. Phosphorylation of ser-359 or ser-370 precedes phosphorylation at other sites and is required for activity. J Biol Chem. 1998, 273 (52): 35147-35152. 10.1074/jbc.273.52.35147.View ArticlePubMedGoogle Scholar
- Wang WJ, Wang PX, Li XJ: [The effect of sinomenine on cyclooxygenase activity and the expression of COX-1 and COX-2 mRNA in human peripheral monocytes]. Zhongguo Zhong Yao Za Zhi. 2003, 28 (4): 352-355.PubMedGoogle Scholar
- Wang Y, Fang Y, Huang W, Zhou X, Wang M, Zhong B, Peng D: Effect of sinomenine on cytokine expression of macrophages and synoviocytes in adjuvant arthritis rats. J Ethnopharmacol. 2005, 98 (1–2): 37-43. 10.1016/j.jep.2004.12.022.View ArticlePubMedGoogle Scholar
- Pawate S, Shen Q, Fan F, Bhat NR: Redox regulation of glial inflammatory response to lipopolysaccharide and interferongamma. J Neurosci Res. 2004, 77 (4): 540-551. 10.1002/jnr.20180.View ArticlePubMedGoogle Scholar
- Qin L, Block ML, Liu Y, Bienstock RJ, Pei Z, Zhang W, Wu X, Wilson B, Burka T, Hong JS: Microglial NADPH oxidase is a novel target for femtomolar neuroprotection against oxidative stress. Faseb J. 2005, 19 (6): 550-557. 10.1096/fj.04-2857com.View ArticlePubMedGoogle Scholar
- Zaitsev SV, Sazanov LA, Koshkin AA, Sud'ina GF, Varfolomeev SD: Respiratory burst inhibition in human neutrophils by ultra-low doses of [D-Ala2]methionine enkephalinamide. FEBS Lett. 1991, 291 (1): 84-86. 10.1016/0014-5793(91)81109-L.View ArticlePubMedGoogle Scholar
- Williamson SA, Knight RA, Lightman SL, Hobbs JR: Effects of beta endorphin on specific immune responses in man. Immunology. 1988, 65 (1): 47-51.PubMed CentralPubMedGoogle Scholar
- Yang S, Yang J, Yang Z, Chen P, Fraser A, Zhang W, Pang H, Gao X, Wilson B, Hong JS, et al: Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) 38 and PACAP4–6 Are Neuroprotective through Inhibition of NADPH Oxidase: Potent Regulators of Microglia-Mediated Oxidative Stress. J Pharmacol Exp Ther. 2006, 319 (2): 595-603. 10.1124/jpet.106.102236.View ArticlePubMedGoogle Scholar
- Wu DC, Teismann P, Tieu K, Vila M, Jackson-Lewis V, Ischiropoulos H, Przedborski S: NADPH oxidase mediates oxidative stress in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease. Proc Natl Acad Sci USA. 2003, 100 (10): 6145-6150. 10.1073/pnas.0937239100.PubMed CentralView ArticlePubMedGoogle Scholar
- Jackson-Lewis V, Smeyne RJ: MPTP and SNpc DA neuronal vulnerability: role of dopamine, superoxide and nitric oxide in neurotoxicity. Minireview. Neurotox Res. 2005, 7 (3): 193-202.View ArticlePubMedGoogle Scholar
- Gao HM, Liu B, Zhang W, Hong JS: Critical role of microglial NADPH oxidase-derived free radicals in the in vitro MPTP model of Parkinson's disease. Faseb J. 2003, 17 (13): 1954-1956.PubMedGoogle Scholar
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