Sinomenine inhibits microglial activation by Aβ and confers neuroprotection
© Shukla and Sharma; licensee BioMed Central Ltd. 2011
Received: 7 April 2011
Accepted: 14 September 2011
Published: 14 September 2011
Neuroinflammation is an important contributor to the development of neurodegenerative diseases, including Alzheimer's disease. Thus, there is a keen interest in identifying compounds, especially from herbal sources, that can inhibit neuroinflammation. Amyloid-β (Aβ) is a major component of the amyloid plaques present in the brains of Alzheimer's disease patients. Here, we examined whether sinomenine, present in a Chinese medicinal plant, prevents oligomeric Aβ-induced microglial activation and confers protection against neurotoxicity.
Oligomeric amyloid-β was prepared from Aβ(1-42). Intracellular reactive oxygen species production was determined using the dye 2',7'-dichlorodihydrofluorescin diacetate. Nitric oxide level was assessed using the Griess reagent. Flow cytometry was used to examine the levels of inflammatory molecules. BV2-conditioned medium was used to treat hippocampal cell line (HT22) and primary hippocampal cells in indirect toxicity experiments. Toxicity was assessed using MTT reduction and TUNEL assays.
We found that sinomenine prevents the oligomeric Aβ-induced increase in levels of reactive oxygen species and nitric oxide in BV2 microglial cells. In addition, sinomenine reduces levels of Aβ-induced inflammatory molecules. Furthermore, sinomenine protects hippocampal HT22 cells as well as primary hippocampal cells from indirect toxicity mediated by Aβ-treated microglial cells, but has no effect on Aβ-induced direct toxicity to HT22 cells. Finally, we found that conditioned medium from Aβ-treated BV2 cells contains increased levels of nitric oxide and inflammatory molecules, but the levels of these molecules are reduced by sinomenine.
Sinomenine prevents oligomeric Aβ-induced microglial activation, and confers protection against indirect neurotoxicity to hippocampal cells. These results raise the possibility that sinomenine may have therapeutic potential for the treatment of Alzheimer's diseases as well as other diseases that involve neuroinflammation.
Alzheimer's disease (AD) is a devastating neurodegenerative disorder that eventually leads to severe cognitive impairment. Although AD is typically a late onset disease, in a small number of familial cases it occurs early in life. Extracellular amyloid plaques and intracellular neurofibrillary tangles are the pathological hallmarks of AD. Amyloid-β (Aβ) is a major component of the plaques. Aβ is produced by processing of amyloid precursor protein, and plays important roles in the pathogenesis of AD. Aβ exists in several forms, including oligomeric forms. Oligomeric Aβ is thought to play an important role in the development of the disease [1, 2]. Several studies have shown that oligomeric Aβ causes neuronal cell death, impairment in synaptic plasticity and memory deficits [e.g. [3–6]].
The available evidence suggests that neuroinflammation contributes to the development of neurodegenerative diseases, including AD [7, 8]. Microglia are the resident immune cells in the brain. They are normally in a resting state, but they become activated in response to pathogens, toxins or cellular damage. Microglia are found in close association with the neuritic plaques in AD brain , and Aβ-induced inflammatory responses mediated by microglia are thought to contribute to neuronal toxicity . Treatment of microglia with Aβ leads to release of inflammatory and toxic factors including reactive oxygen species (ROS) and nitric oxide (NO) [11, 12], which may lead to neuronal cell damage and eventual death. Aβ inhibits long-term potentiation (LTP), which is considered a promising cellular mechanism for memory formation. Importantly, inhibition of LTP by Aβ also involves microglia . Thus, in addition to direct neuronal cell death, Aβ causes indirect neuronal cell death due to neuroinflammation, and inhibits synaptic plasticity.
Considering the available supporting literature regarding the role of microglial activation in neurodegenerative disorders, there is keen interest in identifying compounds from natural sources that can reduce or prevent neuroinflammation, and which thus could be beneficial in neurodegenerative diseases, including AD. Sinomenine is an alkaloid isolated from Sinomenium acutum, a Chinese medicinal plant. It is a dextrorotatory morphinan analog which shares structural similarity with morphine, and weakly binds to the opioid μ-receptor . Qian and colleagues  have shown that sinomenine protects dopaminergic neurons against lipopolysaccharide (LPS)-induced cell death in neuron-glia cultures. NADPH oxidase (PHOX) activity is involved in the protective effects of sinomenine. In addition, this compound confers protection against 1-methyl-4-phenylpyridinium (MPP+)-induced cell death. Wang and colleagues  found that sinomenine reduces advanced glycation end products-induced increases in the levels of cytokines in retinal microglial cells. Furthermore, this compound shows beneficial effects in rheumatoid arthritis and mesangial proliferative nephritis , inhibits morphine withdrawal symptoms , and shows protective effects against cold ischemia/reperfusion injury . In this study, we have examined the effects of sinomenine on oligomeric Aβ-induced microglial activation. In addition, we have investigated the protective effects of this compound on neuronal toxicity caused by Aβ.
Preparation of oligomeric amyloid beta
Oligomeric amyloid-β (Aβ-derived diffusible ligands, ADDL) was prepared using amyloid-β 1-42 peptide (American Peptide) as described previously  with minor modifications. The peptide was dissolved in 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP, Fluka), aliquoted, dried in fume hood and stored at -80°C. The peptide film was dissolved in DMSO to 5 mM concentration and further diluted in phosphate-buffered saline (PBS) to make a 100 μM solution. This preparation was incubated at 4°C for 24 h. To remove insoluble material, the preparation was centrifuged at 14,000 g for 10 min at 4°C. The soluble fraction (ADDL) was stored at -80°C until use. Protein concentration was determined using BCA reagent with bovine serum albumin as standard. ADDL was used at a 2 μM final concentration .
Cell culture and treatments
Sinomenine (Sigma-Aldrich) was dissolved in DMSO and diluted to different concentrations in DMEM such that the final DMSO concentration was 0.1%. BV2 microglial cells were obtained from Dr. A. Basu of our Centre and cultured in DMEM with 10% fetal bovine serum (FBS). The cells were serum starved for 4-8 h before treatment. The control cultures received vehicle for ADDL. For the analysis of reactive oxygen species and nitric oxide, and for assaying the levels of inflammatory molecules described under "sinomenine reduces A-beta-induced increases in inflammatory molecules", cells were cultured in 24-well plates (3.5 × 104cells per well). After serum starvation, BV2 cells were treated with sinomenine for 1.5 h, then with ADDL for 12 h, co-incident with ADDL treatment of a sister culture. For "pre-treatment condition" ADDL and sinomenine treatment was done as described above, whereas for "simultaneous addition", ADDL and sinomenine were added to the culture at the same time. After treatment, samples were used for different assays. For the indirect toxicity experiments and for the analysis of NO and inflammatory molecules in the BV2 conditioned media, BV2 cells grown in 24-well plates (3 × 104 cells per well) were treated with sinomenine for 1.5 h, then with ADDL for 6 h, co-incident with ADDL treatment of a sister culture. After treatment, the cells were washed and fresh medium was added without ADDL or sinomenine. The conditioned medium was collected after a 12 h period and then centrifuged to obtain cell-free supernatant. In all cases sinomenine was present throughout ADDL treatment. Where sinomenine alone was used, the cultures were treated with sinomenine (without ADDL) similar to the sinomenine + ADDL condition.
Hippocampal HT22 cells were a kind gift from Dr. D. Schubert, The Salk Institute, La Jolla, California. The cells were cultured in DMEM with 10% FBS [in a 96-well plate (5 × 103 cells per well) for MTT assay or in poly-D-lysine-coated 4-well chamber slide (1 × 104 cells per well) for TUNEL assay]. For the indirect toxicity experiments, HT22 cells were serum-starved for 4 h and then treated with a mixture of 50% BV2-conditioned medium and 50% fresh DMEM. For MTT assay, cells were treated for 44 h (before addition of MTT), and for the TUNEL assay, cells were treated for 48 h. For direct toxicity experiments HT22 cells were serum-starved for 2.5 h, treated with sinomenine for 1.5 h, then treated with ADDL, co-incident with ADDL treatment of a sister culture. Sinomenine was present throughout the ADDL treatment. ADDL treatment was for 20 h (before addition of MTT) for MTT assay and 24 h for TUNEL assay.
For primary hippocampal cultures, Sprague Dawley pregnant female rats were sacrificed according to a protocol approved by the Institutional Animal Ethics Committee and hippocampal cultures were prepared from E18-E20 embryos as described previously  with minor modifications. Briefly, hippocampi were isolated and triturated to obtain dissociated cells which were then seeded in 90 mm dishes in DME\F12 medium with 10% FBS. After 16-20 h, the medium was replaced with Neurobasal medium containing B27 supplement, glutamax (all from Invitrogen) and glutamic acid (Sigma-Aldrich). After 4 days in vitro (DIV), cells were detached from the plates and seeded in 8-well chamber slides (6 × 104 cells per well) in 50% fresh Neurobasal maintenance medium (Neurobasal medium containing B27 supplement and glutamax) mixed with 50% neuronal conditioned medium. Ara C (5 μM; Sigma-Aldrich) was added to reduce glial cell proliferation. Fifty percent of the medium was replaced every 2 days with Neurobasal maintenance medium, and cultures were used for treatment on DIV 8-9. For indirect toxicity experiments, cells were treated for 24 h with 50% BV2-conditioned medium that was mixed with 50% fresh Neurobasal maintenance medium.
Reactive oxygen species assay
BV2 cells were treated under different conditions, and the level of intracellular ROS was measured fluorimetrically using the dye 2',7'-dichlorodihydrofluorescin diacetate (DCFDA; Sigma-Aldrich) as described previously [21, 22]. The cells were incubated with DMEM containing 5 μM DCFDA for 1 h at 37°C, washed with PBS and lysed in lysis buffer (10 mM Tris pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.2 mM EGTA, 0.2 mM NaVO3, 0.5% NP-40 and 1% Triton X-100). The lysate was centrifuged at 10,000 g for 15 min. A 10-μl aliquot of supernatant was mixed with 90 μl of PBS in a 96-well black plate and fluorescence was measured using a Varioskan Flash multimode Reader (Thermo Electron Corporation, Finland) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The readings obtained were normalized with the amount of protein in each sample. Data are expressed as a percentage of control cultures.
Nitric oxide assay
After different treatments of BV2 cells, released nitric oxide was measured in the culture medium using Griess reagent (Sigma-Aldrich). A 100-μl aliquot of cell-free culture medium was incubated with 100 μl of Griess reagent in the dark at room temperature for 15 min. The intensity of color developed was measured at 540 nm using a Benchmark Plus 96-well ELISA plate Reader (BioRad). Data are expressed as a percentage of control samples.
Cytokine bead array assay
BV2 cells were treated under different conditions, and the levels of inflammatory molecules were measured in cell free culture medium using a Mouse Inflammation cytokine bead array kit (Becton Dickinson) as described previously  with minor modifications. Briefly, a 30-μl bead mix was incubated with an aliquot of cell-free culture medium and 30 μl of phycoerythrin detection reagent for 2 h at room temperature in the dark. The beads were then washed with wash buffer (provided with the kit), re-suspended in 300 μl of the wash buffer and analyzed in FACS Calibur using Cell Quest Pro Software and BD CBA software (Becton Dickinson, San Diego, CA). The standard curve was prepared according to the kit's manual. Data are expressed as fold relative to control.
Cell viability of HT22 cells was assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma-Aldrich) assay. After treatment, MTT reagent was added to the wells, incubated for 4 h, and the samples were processed for MTT assay as described previously . The absorbance was measured at 570 nm. The mean of readings of triplicate wells was taken as one value. The OD value for the control cultures was considered as 100% viability and viability in other samples is expressed as a percentage of viability in the control cultures.
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay
After treatments, cells were fixed and processed for TUNEL assay as described previously . The total number of DAPI (4'6 diamidino-2-phenylindole)-stained or TUNEL-positive cells, in 5 different frames, were counted. The average number of cells (DAPI-stained) per frame in experiments ranged from 162-213.6 (control), 170.8-215.6 (ADDL) and 175.2-217 (ADDL + sinomenine) in HT22 indirect toxicity experiments, 123.4-130.2 (control), 124-135.2 (ADDL) and 125.8-134.4 (ADDL + sinomenine) in primary hippocampal cell indirect toxicity experiments, and 87.4-121 (control), 81-105.6 (ADDL) and 82.2-101.8 (ADDL + sinomenine) in HT22 direct toxicity experiments. Data are expressed as percent TUNEL-positive cells.
Data were analyzed using a paired Student's t-test. Differences were considered significant when the p value was less than 0.05. Data are expressed as mean ± SEM.
Sinomenine inhibits amyloid-β-induced increase in level of reactive oxygen species in microglial cells
In these experiments, BV2 cells were treated with sinomenine before addition of ADDL (Pre-treatment condition). Thus, we next asked whether simultaneous treatment of sinomenine and ADDL has any effect on ADDL-induced ROS generation. We found that whereas pre-treatment with sinomenine inhibited ADDL-induced ROS generation, simultaneous treatment with sinomenine did not reduce ADDL-induced ROS level (Figure 1B). Thus, pretreatment with sinomenine is required for its effect on ADDL-induced ROS level. In subsequent experiments, pretreatment with sinomenine was used to examine its effects in different assays.
Sinomenine inhibits amyloid-β-induced increase in level of nitric oxide in BV2 cells
Sinomenine reduces A-beta-induced increases in inflammatory molecules
Sinomenine confers protection to hippocampal HT22 cells against indirect toxicity
We used another measure, TUNEL assay, to examine the protective effect of sinomenine against indirect neurotoxicity. This assay is based on labeling of fragmented DNA during cell death. In this assay also, we found that treatment of HT22 cells with conditioned medium from ADDL-treated BV2 cells led to significant toxicity as evident by increased number of cells that were positive for TUNEL staining. In contrast, conditioned medium from ADDL plus sinomenine-treated BV2 cells did not increase the number of TUNEL-positive cells (Figure 5B). These results suggest that the neuronal toxicity was mediated by factors released from the microglial cells after treatment with ADDL, and that sinomenine confers protection to HT22 cells against indirect toxicity by oligomeric Aβ.
Sinomenine confers protection to primary hippocampal cells against indirect toxicity
Sinomenine does not confer protection to hippocampal cells against direct toxicity
In this study, we show that sinomenine, an alkaloid from a Chinese medicinal plant, inhibits oligomeric Aβ-induced increases in levels of ROS, NO and inflammatory molecules. In addition, sinomenine confers protection to hippocampal cells (HT22) against indirect toxicity. Furthermore, sinomenine also protects primary hippocampal cells from indirect toxicity.
Considerable evidence points to an important role for Aβ in the pathogenesis of AD. With regards to neurotoxicity, Aβ can directly cause neuronal cell death (direct toxicity) or Aβ can affect microglial cells to produce inflammatory and toxic factors that then affect the viability of neurons. The second mode of neuronal toxicity is referred to as indirect toxicity. Both kinds of toxicity mechanisms have been described in the literature. Microglia are the brain's resident immune cells that offer defense against pathogens. These cells are associated with the amyloid plaques in the brains of both human AD patients and AD transgenic animals . Microglia-mediated inflammation has been implicated in the pathogenesis of neurodegenerative disorders including AD [11, 35]. Thus, while microglial function is important for normal functioning of the brain, over-activation of microglia could have deleterious effects [10, 12].
Aβ induces ROS generation in microglial cells . In addition, Aβ induces the production of NO in these cells [26–28]. Reactive oxygen species and nitric oxide have been implicated in the pathogenesis of AD. The analysis of AD brain samples reveals evidence of ROS and NO production . In addition, Aβ activates microglia leading to the release of inflammatory molecules . An enhancement in cytokine levels is observed in AD transgenic animals [37, 38]. Collectively, several studies suggest that Aβ activates microglia which may contribute to AD pathology by promoting inflammation and neuronal toxicity. We found that sinomenine inhibits ADDL-induced production of ROS, NO and inflammatory molecules. Importantly, we also showed that sinomenine confers protection against indirect toxicity to hippocampal cells. Our results are consistent with the study of Wang et al , who showed that sinomenine inhibits advanced glycation end products-induced release of cytokines, and enhancement of ROS production, in retinal microglial cells. In addition, Qian and colleagues showed that sinomenine inhibits LPS-induced NO and ROS production . These authors showed also that sinomenine confers protection to dopaminergic neurons against LPS- and MPP+-induced toxicity in neuron-glia cultures.
Although sinomenine was effective in reducing indirect toxicity, it did not confer protection to hippocampal HT22 cells in direct toxicity experiments. This finding is consistent with that of Qian et al  who showed that although sinomenine protects dopaminergic neurons against MPP+-induced toxicity in neuron-glia cultures, it has no effects in neuron-enriched cultures.
Since inhibition of microglia-mediated damage could be helpful in at least delaying the progression of AD, anti-inflammatory therapy is considered a promising strategy in this disease. It has been shown that intraperitoneally injected or orally administered sinomenine can reach the brain [39, 40] suggesting that it can cross blood-brain barrier. In addition, intraperitoneally injected sinomenine confers protection in ischemic brain injury . It would be interesting to examine whether sinomenine reduces inflammation and confers neuroprotection in vivo in a model of Alzheimer's disease. Since sinomenine does not confer protection against Aβ-induced direct neuronal toxicity, the protection observed would likely be due to its effects on microglia. Our results, along with those of other studies, suggest that sinomenine may have therapeutic potential in neurodegenerative diseases that involve neuroinflammation.
Our results show that sinomenine inhibits oligomeric amyloid-β-induced increases in levels of ROS, NO and inflammatory molecules in BV2 microglial cells. Moreover, this compound protects immortalized as well as primary hippocampal cells from indirect toxicity mediated by amyloid-β-treated BV2 cells. Thus, sinomenine may have therapeutic value in neurodegenerative diseases, including Alzheimer's disease.
We thank Dr. D Schubert of Salk Institute for providing us the hippocampal HT22 cell line. We thank Jyoti Chhibber and Jeet Bahadur Singh for their help in the experiments, and Justin Shobe, Mike Sutton and Shara Stough for their thoughts on an earlier version of the manuscript. This work was supported by core grant to National Brain Research Centre from Department of Biotechnology, India. Shilpa Mishra Shukla was supported by a Senior Research Fellowship from Council of Scientific and Industrial Research, India.
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