Inflammation and oxidative stress play important roles in a number of acute neurodegenerative conditions such as stroke and traumatic injury, as well as in chronic neurodegenerative conditions such as Parkinson disease and Alzheimer disease. Microglia are the resident macrophages in the brain and spinal cord that mediate innate defense system mechanisms in the central nervous system. Under pathological conditions, microglia are activated and switch to promote inflammatory responses, which result in production of inflammatory factors and oxidative stress including reactive oxygen species (ROS) (1), tumor necrosis factor α (2), and nitric oxide (NO) (3). Although an exaggerated response of microglia occurs, neurons can be indirectly damaged by cytokines or inflammatory and oxidative stress factors released from activated microglia. Therefore, studies involving microglia-associated inflammation and neuronal damage have thus been central to many neurodegenerative diseases studies.
Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram-negative bacteria. Microglia respond to LPS-mediated activation of several pathways and transcription factors; therefore, LPS is commonly used as a microglial activator to stimulate the production of neurotoxic factors and leads to neuronal damage. On the other hand, 6-hydroxydopamine (6-OHDA) is a neurotoxin commonly used in experimental models to induce oxidative stress both in vitro and in vivo. As an ROS-producing agent, 6-OHDA has also been shown to reduce glutathione and superoxide dismutase (SOD) enzyme activity.
Paeonol is a main component isolated from Moutan cortex, which has been used as a traditional herbal medicine for thousands of years. Also, many pharmacological properties of paeonol have been reported, including antiproliferative (4), anti-inflammatory (5, 6), antioxidative (7), antiallergic (8), anxiolytic-like (9), vasodilation (10), and osteoclastogenesis effects (11). Besides, it has been proved that paeonol quickly passed through blood-brain barrier by analyzing paeonol in different rat tissues including brain after oral administration (12). Studies also suggest that paeonol protects cultured rat hippocampal neurons against oxygen-glucose deprivation–induced injury (13) and attenuates neurotoxicity and ameliorates cognitive impairment induced by D-galactose in ICR mice (14). However, studies about the effects of paeonol on microglia-mediated inflammation and direct oxidative damage on neurons are not well documented.
Strategies to inhibit inflammation and oxidative stress may reduce production of neurotoxic factors and be valuable to control neurodegeneration (15, 16). Paeonol has been shown to enter into the brain tissue across the blood-brain barrier. The aim of this study was to characterize the neuroprotective mechanisms of paeonol against LPS-activated microglia-induced inflammation and 6-OHDA–induced oxidative stress in cortical neurons.
MATERIALS AND METHODS
Paeonol; bovine serum albumin (BSA); arabinoside; dimethyl sulfoxide; LPS (L8274) from Escherichia coli (O26:B6); 2′,7′-dichloro-dihydrofluorescein diacetate (H2DCF-DA); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT); poly-L-lysine; Triton X-100; mouse antibody against inducible NO synthase (iNOS); and β-actin were obtained from Sigma-Aldrich (St Louis, Mo). Dulbecco modified Eagle medium, Ham F-12 medium, minimum essential medium (MEM), fetal bovine serum (FBS), horse serum, glutamine, B27, nonessential amino acids, sodium pyruvate, penicillin, amphotericin B, streptomycin, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), and Alexa Fluor 488 goat anti–rabbit immunoglobulin G (H+L) were obtained from Invitrogen (Carlsbad, Calif). Anti-Iba1 antibody was from Wako Pure Chemical (Osaka, Japan). All materials for sodium dodecyl sulfate–polyacrylamide gel electrophoresis were obtained from Bio-Rad (Hercules, Calif). Mouse antibody against B-cell lymphoma 2 (Bcl-2) and phosphorylated extracellular signal–regulated kinase (ERK); goat antibody against cyclooxygenase 2 (COX-2) and heme oxygenase 1 (HO-1); rabbit antibody against p47phox, p67phox, ERK, Jun N-terminal kinase (JNK), phosphorylated JNK, MAP-2; and all horseradish peroxidase–conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif). Plasma membrane protein extraction kit was from BioVision Foundation (Mountain View, Calif). Enzyme-immunoassay kit was from Cayman Chemical (Ann Arbor, Mich). Enhanced chemiluminescence reagent and polyvinylidene difluoride membranes were purchased from PerkinElmer Life and Analytical Sciences (Boston, Mass). Superoxide dismutase activity kit was purchased from Assay Designs (Ann Arbor, Mich). All other chemicals were purchased from Sigma Chemical Co.
Pregnant Sprague-Dawley (SD) rats were purchased from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan) and then fed in the Animal Center of Kaohsiung Medical University under constant temperature and 12-h light-dark cycle. Animal handling was conducted in accordance with the National Institutes of Health guidelines (17). The Animal Care and Use Committee at the Kaohsiung Medical University approved the animal use and methods of anesthesia/sacrifice (approval ID: 98096).
Primary microglia cultures
Primary microglia cultures were prepared from cortices dissected from postnatal day 1 SD rats. The cortices were dissociated and triturated. Cells were then plated onto 20 μg/mL poly-L-lysine–coated tissue culture flasks and cultured in Dulbecco modified Eagle medium and Ham F12 mixed medium 1:1, which contained 10% FBS, 4 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B at 37°C in a humidified atmosphere containing 5% CO2. Culture medium was changed weekly. After 14 days, microglia were removed by 3-h shaking at 200 revolutions per minute on a rotary shaker. Isolated microglia were plated onto precoated 6- or 96-well plates in culture medium as previous described. Cells were treated with paeonol (0.75, 1, and 1.5 μM) or vehicle (0.1% ethanol) 1 h before or after addition of LPS (100 ng/mL) for 24 h.
Primary cortical neuron cultures
Primary cortical neuron cultures were prepared from cortices dissected from 15-day-old embryos of SD rats. The cortices were dissociated and triturated. Cells were plated onto 6-, 12-, or 96-well plates previously coated with 30 μg/mL poly-L-lysine and cultured in MEM medium containing 10% FBS, 10% horse serum, 1 g/L glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 μM nonessential amino acids, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B at 37°C in a humidified atmosphere containing 5% CO2. After 24-h incubation, culture medium was replaced by MEM supplemented with 2% B27 and 10 μM cytosine arabinoside and then replaced with MEM containing 2% B27 after 42 h. After 6 days in vitro, cultured cells were treated with paeonol (0.75, 1, and 1.5 μM) or vehicle (0.1% ethanol) 1 h before addition of 6-OHDA (40 μM) for 24 h.
Microglia were stained with an antibody against Iba1 (a marker for microglia), and neurons were stained with an antibody against MAP-2 (a marker for both the cell body and neurites). Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min then washed with PBS incubated in 0.2% Triton X-100 for 10 min. After two washes, cells were blocked by PBS incubated with 2% BSA for 1 h. The cells were then incubated overnight at 4°C with the anti-Iba1 or anti–MAP-2 antibody (1:1,000). Alexa Fluor 488 goat anti–rabbit immunoglobulin G (H+L) was used as the secondary antibody. Cell nuclei were then counterstained with DAPI. Images were collected under a fluorescence microscope to ensure the purity of primary microglia (Fig. 1A) and cortical neurons (Fig. 1B).
Preparation of LPS-treated condition medium
To investigate the protective effect of paeonol on microglia-mediated neurotoxicity, LPS-treated condition medium (LCM) was used. Primary microglia were treated with paeonol (0.75, 1, and 1.5 μM) or vehicle (0.1% ethanol) for 1 h before or after addition of LPS (100 ng/mL). After 24 h, medium was collected as LCM and then added to the cortical neurons cultures for 24 h.
Cell viability assay
Cell viability was determined by the quantitative colorimetric MTT assay. Cells were plated in 96-well culture plates. After treatment with various concentrations of paeonol ranging from 0.75 to 1.5 μM or vehicle with or without LPS (100 ng/mL) or 6-OHDA (40 μM) for 24 h, the medium was replaced with MTT at a final concentration of 0.5 mg/mL for 3 h at 37°C and 5% CO2 incubation. The formazan crystals in the cells were solubilized with 100 μL dimethyl sulfoxide. Absorbance was read at 560 nm on a microplate reader.
Western blotting analysis
After 24-h treatment, cells were collected and lysed to determine the expression of iNOS, COX-2, pERK1/2, ERK1/2, pJNK, JNK, HO-1, and Bcl-2. The lysates were centrifuged at 15,000g for 30 min at 4°C, and the supernatant was collected. Cell membranes were obtained by plasma membrane protein extraction kit (BioVision) according to the manufacturer’s instructions to determine the expression of NOX2, p47phox, and p67phox. Collected protein was used for sodium dodecyl sulfate–polyacrylamide gel electrophoresis. A Bio-Rad protein assay kit was used to determine protein concentration. Equal amounts ofprotein (20 μg per lane) were separated on a 10% polyacrylamide gel and transferred to polyvinylidene difluoride membranes (PerkinElmer Life and Analytical Sciences). Nonspecific binding was blocked with tris-buffered saline and tween 20 (50 mM Tris-HCl, pH 7.6; 150 mM NaCl; 0.1% Tween 20) containing 5% nonfat milk for 1hat room temperature. The membranes were then each incubated with mouse anti-iNOS (1:500), mouse anti–Bcl-2 (1:1,000), mouse anti-pERK (1:1,000), mouse anti–β-actin (1:10 000), mouse anti-NOX2 (1:1,000), rabbit anti-p47phox (1:1,000), rabbit anti-p67phox (1:1,000), rabbit anti-ERK (1:1,000), rabbit anti-JNK (1:1,000), rabbit anti-pJNK (1:1,000), goat anti–COX-2 (1:1,000), and goat anti–HO-1 (1:1,000) primary antibodies overnight at 4°C. Membranes were washed six times for 5 min with TBST. The appropriate dilutions of secondary antibodies (1:1,000) were incubated for 1 h. After six washes with TBST, the protein bands were detected with the enhanced chemiluminescence reagent (PerkinElmer Life and Analytical Sciences).
Nitrite generation by primary microglia was an indicator of NO release and measured by the Griess reagent (1% sulfanilamide and 0.1% N-1-naphthylethylenediamide in 5% phosphoric acid). Briefly, the medium from treated cells was collected. Fifty microliters of this medium was incubated for 10 min at room temperature in the dark with 50 μL of Griess reagent. The absorbance was measured at 540 nm (OD540), and the concentration was calculated from a sodium nitrite standard reference curve.
Measurement of prostaglandin E2 production
Prostaglandin E2 (PGE2) production in supernatant was detected with an enzyme-immunoassay kit from Cayman Chemical, following the manufacturer’s instructions. Supernatant was collected after 24 h of LPS treatment, and the absorbance was measured at 405 nm (OD405).
Measurement of intracellular ROS
The level of intracellular ROS was detected by fluorescence with H2DCF-DA staining. After 24 h of drug treatments, microglia or neurons were stained with 10 μM H2DCF-DA at 37°C. After 30-min incubation, cells were detached and washed with PBS. One hundred thousand cells were analyzed by a Coulter CyFlow Cytometer (Partec, Münster, Germany). Dichlorofluorescein fluorescence was determined by an excitation of 495 nm and emission of 520 nm.
Superoxide dismutase activity assay
Superoxide dismutase activity was measured by an SOD activity kit (Assay Designs). The principle of this kit is based on xanthine oxidase and the conversion of WST-1 to WST-1 formazan. Neurons were grown on six-well plates and treated for 24 h. Then, cells were detached, and cytosolic protein was extracted. Superoxide dismutase activity was determined by adding the master mix and xanthine solution according to the manufacturer’s instructions of this kit. And the absorbance was measured by enzyme-linked immunosorbent assay reader at 450 nm for 10 min at 1-min intervals right after addition of xanthine solution. Protein concentration was quantified by Bio-Rad protein assay kit. Superoxide dismutase activity was calculated versus an SOD standard curve and normalized to the protein concentration.
All data were expressed as means ± SEM. Statistical significance was analyzed using one-way analysis of variance followed by Dunnett test for all pair comparisons. P < 0.05 or less was considered statistically significant. Statistical analysis was performed using InStat version 3.0 (GraphPad Software, San Diego, Calif).
Paeonol downregulates LPS-induced inflammatory iNOS/NO and COX-2/PGE2 pathways in primary microglia cultures
Because iNOS/NO and COX-2/PGE2 are important markers for inflammation resulting to neurodegeneration, the anti-inflammatory effects of paeonol were investigated in LPS-activated microglia. Microglia were pretreated with paeonol (0.75, 1, 1.5 μM) for 1 h and further treated with LPS (100 ng/mL) for an additional 24 h. As shown in Figure 2, LPS induced iNOS overexpression and markedly increased NO production in primary microglia. Pretreatment of paeonol significantly decreased LPS-induced iNOS overexpression (Fig. 2A) and NO production (Fig. 2B). Similarly, paeonol also attenuated LPS-induced COX-2 overexpression (Fig. 2C) and PGE2 (Fig. 2D) production in primary microglia.
Paeonol suppresses LPS-induced phosphorylation of ERK and JNK in primary microglia
Mitogen-activated protein kinase (MAPK) pathway is an important signaling related to LPS-induced inflammation. To further evaluate the anti-inflammatory effects of paeonol on LPS-activated microglia, we further examined the effects of paeonol on MAPK molecules ERK and JNK. As shown in Figure 3A and B, paeonol significantly inhibited LPS-induced phosphorylation of ERK and JNK.
Paeonol increases antioxidative HO-1 expression
Heme oxygenase 1 is a cytoprotective protein that protects against oxidative stress and inflammation. In the present study, paeonol significantly increased HO-1 expression in LPS-treated microglia (Fig. 3C). This result suggests that HO-1 upregulation may also be involved in the protective effects of paeonol.
Paeonol attenuates LPS-induced NADPH oxidase activation and ROS production in primary microglia
Microglia-induced NADPH oxidase (NOX) activation, especially phagocyte NADPH oxidase (NOX2), is a major mechanism to cause oxidative stress– and inflammation-related neurodegeneration. Therefore, we examined the effects of paeonol on the activation of NOX2 and its two cytosolic subunits, p47phox and p67phox, on the cell membrane of microglia. Results indicated that LPS upregulated NOX2 expression on the cell membrane of microglia, and paeonol treatment could attenuate LPS-induced NOX2 activation (Fig. 4A). Moreover, paeonol inhibited LPS-induced translocation of p47phox and p67phox from the cytosol to the cell membrane in microglia (Fig. 4, B and C). Furthermore, NOX2 activation–induced ROS overproduction has been shown to be an important cause of oxidative stress–induced neuronal damage. To investigate the antioxidative properties of paeonol, effects of paeonol on LPS-induced ROS production in primary microglia were evaluated. As shown in Figure 4D, LPS significantly increased ROS production, which was attenuated by paeonol treatment.
Paeonol decreases ROS production, increases SOD activity, and enhances Bcl-2 expression in 6-OHDA–treated cortical neurons
We further examined the effects of paeonol on 6-OHDA–induced oxidative damage by measurement of ROS production and SOD activity in cortical neurons. As shown in Figure 5A, 6-OHDA significantly increased ROS production, which was inhibited by paeonol treatment. Moreover, enzymatic antioxidant SOD is an important component of the cellular defense against oxidative stress, and paeonol reversed 6-OHDA–induced attenuation of SOD activity in neuron cultures (Fig. 5B). To further ascertain the cytoprotective effects of paeonol, we examined the effects of paeonol on antiapoptotic protein Bcl-2 expression in 6-OHDA–treated cortical neurons. Results indicated that paeonol could enhance Bcl-2 expression in 6-OHDA–treated primary cortical neurons (Fig. 5C).
Paeonol protects against LPS-activated microglia and 6-OHDA–induced neuronal cells death
We examined the cytoprotective effects of paeonol on microglia-mediated neurotoxicity and 6-OHDA–induced neuronal death. First we examined the direct effects of paeonol (0.75, 1, and 1.5 μM) and vehicle (0.1% ethanol) on cell viability of primary microglia and cortical neurons by using an MTT test. Results indicated both vehicle and paeonol (0.75–1.5 μM) did not affect cell viability of primary microglia (Fig. 6A) and cortical neurons (Fig. 6B). Then, we examined the effects of paeonol on LCM-induced neuronal death, a model used for LPS-activated microglia-mediated neurotoxicity. Results indicated that paeonol attenuated LCM-induced neuron cells death (Fig. 6C). Furthermore, we examined the effects of paeonol on 6-OHDA–induced cells death. Results indicated that paeonol attenuated 6-OHDA–induced neuron cells death at the concentration of 1 and 1.5 μM (Fig. 6D).
Posttreatment of Paeonol attenuates LPS-activated microglia-induced inflammation and neuronal cells death
Finally, we examined the anti-inflammatory and neuroprotective effects of paeonol after the treatment of LPS. Microglia cultures were treated with paeonol (0.75–1.5 μM) for 1 h after the treatment of LPS (100 ng/mL) for 24 h. Results indicated paeonol (1 and 1.5 μM) attenuated LPS-induced increase in iNOS/NO and COX-2/PGE2 (Fig. 7). To further ensure the neuroprotective effect of paeonol, cortical neurons were treated with paeonol (0.75–1.5 μM) for 1 h after the treatment of LPS-treated condition medium (LCM) for 24 h. Results indicated that, at higher concentration (1.5 μM), paeonol protected against LCM-induced neuronal cell death (Fig. 8).
Activated microglia-mediated inflammation and oxidative stress are known to be involved in the progression of acute and chronic neurodegenerative disorders (18–20). Lipopolysaccharide-induced iNOS and COX-2 overexpression results in the production of NO and prostaglandins, which contribute to damage of cells (21–22). Evidence suggests that iNOS overexpression and NO overproduction by activated microglia are involved in neurodegeneration, whereas inhibition of them contributes to neuroprotection (23). In addition, COX-2 and PGE2 play an important role in initiation and progression of many neurodegenerative diseases such as Alzheimer disease (24) and Parkinson disease (25). In this study, paeonol significantly downregulated iNOS/COX-2 expression and NO/PGE2 production induced by LPS in primary microglia. Furthermore, LPS can activate MAPK pathways leading to the inflammatory response. Mitogen-activated protein kinases, including ERK and JNK, are a group of molecules that play key roles in inflammatory responses (26). The present study indicates that paeonol can suppress LPS-induced ERK and JNK activation in microglia. This finding is similar to earlier report that paeonol can inhibit LPS-induced MAPK activation in murine macrophage-like RAW 264.7 cells (6). Taken together, the present results suggest that paeonol exerts protective effects against LPS-activated microglia-induced inflammation.
It has been reported that activated microglia-induced oxidative stress through NOX2 activation leads to death of neighboring neurons (27) and contributes to dopaminergic neurodegeneration (28). Decrease in LPS-induced neuronal loss can be observed in NOX2-deficient mice (29). In the present study, paeonol decreased LPS-induced NOX2 activation and ROS overproduction in microglia. Moreover, the results also indicated that paeonol inhibited translocation of p47phox and p67phox from cytosol to the membrane in activated microglia. p47phox and p67phox are cytosolic subunits of NOX2, which can regulate NOX2 activation (30). Furthermore, paeonol increased expression of HO-1, which plays an important role in antioxidant defense. Therefore, our results suggested that paeonol decreases microglia-mediated oxidative stress by attenuation of NOX2 activation and enhancement of HO-1 expression.
Oxidative stress–induced cytotoxicity can be attenuated by the regulation of antioxidant enzyme activity and ROS production. 6-Hydroxydopamine is known as a neurotoxin, and ROS plays a critical role in 6-OHDA–induced neuronal death (31). Superoxide dismutases, including copper-zinc SOD, manganese SOD, and extracellular SOD, are important antioxidant enzymes catalyzing O2− dismutase to H2O2, implicated in several neurodegenerative diseases (32–33). According to our results, paeonol not only decreased ROS production and neuronal death, but also increased SOD activity in 6-OHDA–treated cortical neurons. Furthermore, paeonol upregulated Bcl-2 expression, which is known as a cytoprotective protein that can stabilize mitochondrial function (34–35) and contribute to cell survival. Therefore, our results suggested that paeonol protects neurons from oxidative stress–induced cytotoxicity via decreasing ROS production, increasing SOD activity, and upregulating Bcl-2 expression.
In conclusion, the beneficial protective effects and mechanisms of paeonol against microglia-mediated inflammation and oxidative stress–induced neurotoxicity suggest that paeonol might be a potential agent used for neuroprotection (Fig. 9).
The authors thank Belinda Wilson for her editorial assistance.
1. Tanaka M, Sotomatsu A, Yoshida T, Hirai S, Nishida A: Detection of superoxide production by activated microglia
using a sensitive and specific chemiluminescence assay and microglia
-mediated PC12h cell death. J Neurochem 63 (1): 266–270, 1994.
2. Sawada M, Kondo N, Suzumura A, Marunouchi T: Production of tumor necrosis factor-alpha by microglia
and astrocytes in culture. Brain Res 491 (2): 394–397, 1989.
3. Moss DW, Bates TE: Activation of murine microglial cell lines by lipopolysaccharide and interferon-gamma causes NO-mediated decreases in mitochondrial and cellular function. Eur J Neurosci 13 (3): 529–538, 2001.
4. Sun GP, Wang H, Xu SP, Shen YX, Wu Q, Chen ZD, Wei W: Anti-tumor effects of paeonol
in a HepA-hepatoma bearing mouse model via induction of tumor cell apoptosis and stimulation of IL-2 and TNF-alpha production. Eur J Pharmacol 584 (2–3): 246–252, 2008.
5. Chou TC: Anti-inflammatory and analgesic effects of paeonol
in carrageenan-evoked thermal hyperalgesia. Br J Pharmacol 139 (6): 1146–1152, 2003.
6. Chae HS, Kang OH, Lee YS, Choi JG, Oh YC, Jang HJ, Kim MS, Kim JH, Jeong SI, Kwon DY: Inhibition of LPS-induced iNOS, COX-2 and inflammatory mediator expression by paeonol
through the MAPKs inactivation in RAW 264.7 cells. Am J Chin Med 37 (1): 181–194, 2009.
7. Hsieh CL, Cheng CY, Tsai TH, Lin IH, Liu CH, Chiang SY, Lin JG, Lao CJ, Tang NY: Paeonol
reduced cerebral infarction involving the superoxide anion and microglia
activation in ischemia-reperfusion injured rats. J Ethnopharmacol 106 (2): 208–215, 2006.
8. Lee B, Shin YW, Bae EA, Han SJ, Kim JS, Kang SS, Kim DH: Antiallergic effect of the root of Paeonia lactiflora
and its constituents paeoniflorin and paeonol
. Arch Pharm Res 31 (4): 445–450, 2008.
9. Mi XJ, Chen SW, Wang WJ, Wang R, Zhang YJ, Li WJ, Li YL: Anxiolytic-like effect of paeonol
in mice. Pharmacol Biochem Behav 81 (3): 683–687, 2005.
10. Li YJ, Bao JX, Xu JW, Murad F, Bian K: Vascular dilation by paeonol
—a mechanism study. Vascul Pharmacol 53 (3–4): 169–176, 2010.
11. Tsai HY, Lin HY, Fong YC, Wu JB, Chen YF, Tsuzuki M, Tang CH: Paeonol
inhibits RANKL-induced osteoclastogenesis by inhibiting ERK, p38 and NF-kappaB pathway. Eur J Pharmacol 588 (1): 124–133, 2008.
12. Li H WS, Yang Q, Xin Y, Cao W, Zang B, Wang JB, Wang TP, Wang M: LC tissue distribution study of paeonol
in rats after oral administration. Chromatographia 73 (5–6): 495–500, 2011.
13. Wu JB, Song NN, Wei XB, Guan HS, Zhang XM: Protective effects of paeonol
on cultured rat hippocampal neurons against oxygen-glucose deprivation-induced injury. J Neurol Sci 264 (1–2): 50–55, 2008.
14. Zhong SZ, Ge QH, Qu R, Li Q, Ma SP: Paeonol
attenuates neurotoxicity and ameliorates cognitive impairment induced by d-galactose in ICR mice. J Neurol Sci 277 (1–2): 58–64, 2009.
15. Qian L, Flood PM, Hong JS: Neuroinflammation is a key player in Parkinson’s disease and a prime target for therapy. J Neural Transm 117 (8): 971–979, 2010.
16. Agostinho P, Cunha RA, Oliveira C: Neuroinflammation, oxidative stress and the pathogenesis of Alzheimer’s disease. Curr Pharm Des 16 (25): 2766–2778, 2010.
17. Grossblatt N. Guide for the Care and Use of Laboratory Animals. Washington, DC: Institute of Laboratory Animal Research, Commission on Life Sciences, National Research Council, 1996.
18. Sun AY, Chen YM: Oxidative stress and neurodegenerative disorders. J Biomed Sci 5 (6): 401–414, 1998.
19. Wyss-Coray T, Mucke L: Inflammation in neurodegenerative disease—a double-edged sword. Neuron 35 (3): 419–432, 2002.
20. Block ML, Hong JS: Microglia
and inflammation-mediated neurodegeneration: multiple triggers with a common mechanism. Prog Neurobiol 76 (2): 77–98, 2005.
21. Laubach VE, Shesely EG, Smithies O, Sherman PA: Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death. Proc Natl Acad Sci U S A 92 (23): 10688–10692, 1995.
22. Liu HE, Chang AS, Teng CM, Chen CC, Tsai AC, Yang CR: Potent anti-inflammatory effects of denbinobin mediated by dual inhibition of expression of inducible no synthase and cyclooxygenase 2. Shock 35 (2): 191–197, 2011.
23. Brown GC: Mechanisms of inflammatory neurodegeneration: iNOS and NADPH oxidase. Biochem Soc Trans 35 (Pt 5): 1119–1121, 2007.
24. Hoozemans JJ, Rozemuller JM, van Haastert ES, Veerhuis R, Eikelenboom P: Cyclooxygenase-1 and -2 in the different stages of Alzheimer’s disease pathology. Curr Pharm Des 14 (14): 1419–1427, 2008.
25. Teismann P, Tieu K, Choi DK, Wu DC, Naini A, Hunot S, Vila M, Jackson-Lewis V, Przedborski S: Cyclooxygenase-2 is instrumental in Parkinson’s disease neurodegeneration. Proc Natl Acad Sci U S A 100 (9): 5473–5478, 2003.
26. Shi Y, Tu Z, Tang D, Zhang H, Liu M, Wang K, Calderwood SK, Xiao X: The inhibition of LPS-induced production of inflammatory cytokines by HSP70 involves inactivation of the NF-kappaB pathway but not the MAPK pathways. Shock 26 (3): 277–284, 2006.
27. Qin B, Cartier L, Dubois-Dauphin M, Li B, Serrander L, Krause KH: A key role for the microglial NADPH oxidase in APP-dependent killing of neurons. Neurobiol Aging 27 (11): 1577–1587, 2006.
28. Block ML, Li G, Qin L, Wu X, Pei Z, Wang T, Wilson B, Yang J, Hong JS: Potent regulation of microglia
-derived oxidative stress and dopaminergic neuron survival: substance P vs. dynorphin. FASEB J 20 (2): 251–258, 2006.
29. 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 279 (2): 1415–1421, 2004.
30. Bedard K, Krause KH: The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev 87 (1): 245–313, 2007.
31. Kulich SM, Chu CT: Role of reactive oxygen species in extracellular signal–regulated protein kinase phosphorylation and 6-hydroxydopamine cytotoxicity. J Biosci 28 (1): 83–89, 2003.
32. Reiter RJ: Oxidative processes and antioxidative defense mechanisms in the aging brain. FASEB J 9 (7): 526–533, 1995.
33. Choi J, Rees HD, Weintraub ST, Levey AI, Chin LS, Li L: Oxidative modifications and aggregation of Cu,Zn–superoxide dismutase associated with Alzheimer and Parkinson diseases. J Biol Chem 280 (12): 11648–11655, 2005.
34. Mattson MP, Culmsee C, Yu ZF: Apoptotic and antiapoptotic mechanisms in stroke. Cell Tissue Res 301 (1): 173–187, 2000.
35. Niizuma K, Endo H, Chan PH: Oxidative stress and mitochondrial dysfunction as determinants of ischemic neuronal death and survival. J Neurochem 109 (Suppl 1): 133–138, 2009.