小胶质细胞培养

l ce , L y, D sed ne 3 t in + + disorder characterized by the loss of dopaminergic neurons in dropyridine (MPTP), an inducer of Parkinsonism in humans, monkeys and mice (Hallman et al., 1984; Langston et al., 1984). complex I of the mitochondrial electron-transport chain (Nick- The involvement of mitochondrial dysfunction in Parkin- son's disease is based on the finding that the activity of complex I of the electron-transport chain is significantly decreased in observed in the substantia nigra of postmortem brain (Schapira gy MPTP is a neurotoxin that selectively injures the nigrostriatal system. In the brain, MPTP is metabolized by monoamine the nigrostriatal pathway. Although the mechanism still remains to be elucidated, the combination of mitochondrial dysfunction and increased oxidative stress is hypothesized to contribute to the selective degeneration of nigrostriatal dopaminergic neurons (Jenner and Olanow, 1996; Brown and Yamamoto, 2003; Orth and Schapira, 2002). The neurotoxin 1-methyl-4-phenylpyridi- nium (MPP+) is selectively toxic to nigrostriatal dopaminergic neurons and is widely used in the testing of many antiparkinso- nian agents. MPP+ is a metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahy- las et al., 1985). Several in vivo and in vitro studies have found that MPP+ exerts oxidative stress on cells. MPTP treatment produces an increase in brain hydroxyl radicals in mice (Cassa- rino et al., 1997), and high concentrations of MPP+ have been shown to increase reactive oxygen species in neuroblastoma cells (Cassarino et al., 1997). In animals, overexpression of antioxidant enzymes protects against MPTP toxicity (Przed- borski et al., 1992), and antioxidant molecules protect against MPP+ toxicity in neuronal cell lines and dopaminergic neurons in primary culture (Akaneya et al., 1995). activities of endogenous antioxidants and the lipid peroxide content were measured. The results indicated that catalpol prevented the MPP - induced inhibition of complex I activity and the loss of mitochondrial membrane potential. In addition, catalpol reduced the content of lipid peroxide and increased the activity of glutathione peroxidase and superoxide dismutase. Taken together, the above results suggest that catalpol may be a candidate drug for the treatment of oxidative stress-induced neurodegenerative disease. © 2007 Elsevier B.V. All rights reserved. Keywords: Neuroprotective; Catalpol; Complex I; Oxidative stress; Mitochondria 1. Introduction Parkinson's disease is an aged-related neurodegenerative oxidase B (MAO-B) to form the active neurotoxic metabolite MPP+, which is then taken up into dopaminergic neurons via the dopaminergic transporter, and inhibits the multienzyme dopaminergic neuron death in a dose-dependent manner. In order to clar (MPP )-induced oxidative stress in cultured mesencephalic neurons, especially dopaminergic neurons, were investigated. Exposure of mesencephalic neurons to 10μM MPP+ induced a leakage of lactate dehydrogenase (LDH) and decreased cell viability, measured with the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Catalpol increased neuron viability and markedly attenuated MPP+-induced ify the neuroprotective mechanism of catalpol, mitochondrial function, the Neuroprotective effect of catalpo stress in mesen Yuan-Yuan Tian, Bo Jiang ⁎ Department of environmental and biological science and technolog Received 29 September 2006; received in revi Available onli Abstract The neuroprotective effects of catalpol, an iridoid glycoside presen European Journal of Pharmacolo ⁎ Corresponding author. Tel.: +86 411 84706355; fax: +86 411 84706365. E-mail address: bojiang0411@yahoo.com.cn (B. Jiang). 0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.04.039 against MPP+-induced oxidative phalic neurons i-Jia An, Yong-Ming Bao alian University of Technology, Dalian, Liaoning, 116024, China form 11 April 2007; accepted 19 April 2007 0 April 2007 the roots of Rehmannia glutinosa, on 1-methyl-4-phenylpyridinium 568 (2007) 142–148 www.elsevier.com/locate/ejphar et al., 1990). The impairment of mitochondrial activity contri- butes to both reactive oxygen species generation and nigral cell loss. Excessive production of reactive oxygen species, such as superoxide anion, hydroxyl radical and hydrogen peroxide, may either directly damage the cellular macromolecule to cause cell necrosis or indirectly affect normal cellular signaling pathways and gene regulation to induce apoptosis (Facchinetti et al., 1998; Sugawara and Chan, 2003). Malondialdehyde, a stable metabolite of the free radical-mediated lipid peroxidation cascade, is widely used as a marker of oxidative stress. Studies Y.-Y. Tian et al. / European Journal of P have shown that biological systems have evolved with endo- genous defense mechanisms to help protect against reactive oxygen species-induced cell damage. Superoxide dismutase (SOD) and glutathione peroxidase are endogenous antioxidant enzymes which play pivotal roles in preventing cellular damage caused by reactive oxygen species (Barlow et al., 2005; Husain et al., 2005). Catalpol, an iridiod glucoside (Fig. 1) isolated from the roots of Rehmannia glutinosa, has been reported to induce neuronal differentiation in PC12 cells through activation of the in- tracellular signal transduction pathway (Yamazaki et al., 1996) and to attenuate apoptosis induced by H2O2 in PC12 cells in vitro (Jiang et al., 2004). The antioxidant property of catalpol is also well documented (Li et al., 2004). In addition, mitochon- dria are involved as producers of reactive oxygen species. Oxidative stress at the level of the mitochondria will result in the failure of enzymatic, transport and receptor systems (Ozawa, 1995). Moreover, mitochondrial dysfunction and oxidative stress are thought to play a role in the etiology of Parkinson's disease. Owing to the lack of evidence explaining the effects of catalpol on mitochondrial activity and oxidative stress, this study sought to determine whether catalpol could protect dopa- minergic neurons from toxicity induced in MPP+. Furthermore, the effects of catalpol on complex activity and antioxidative enzymes in MPP+-treated mesencephalic neuron-enriched cul- tures were investigated in order to elucidate the neuroprotective mechanism. 2. Materials and methods 2.1. Materials Catalpol was of analytical grade (purityN98%) and was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) and dissolved in physiological saline. MPP+, antimycin A, coen- zyme Q1, and NADH were purchased from Sigma. The mono- clonal anti-tyrosine hydroxylase (TH) antibody was purchased from Chemicon. SABC compound kits were from Sino-Ameri- can Biotechnology Company. 2′, 7′-Dichlorofluorescein diace- Fig. 1. The chemical structure of catalpol. tate was obtained from Beyotime. Tissue culture media and fetal bovine serum were obtained from Gibco. 2.2. Primary mesencephalic neuron-enriched cultures and treatment Mesencephalic neuron-enriched cultures were prepared from the ventral mesencephalic tissues of embryonic day 13/14 mice as described previously (Gao et al., 2003; Qin et al., 2004). Briefly, dissociated cells were seeded at 5 × 105/well and 6 × 106 onto poly-D-lysine-coated 24-well plates and 75-cm2 T-flask. Cells were maintained at 37°C in a humidified atmosphere of 5 % CO2 and 95 % air in Dulbecco's modified Eagle's medium/ nutrient F12 (DMEM/F12) containing 10% fetal bovine serum (FBS), 50 U/ml penicillin, 1.2 g/l sodium bicarbonate and 2 mM L-glutamine. Glial proliferation was suppressed by the inclusion of cytosine β-D-arabinofuranoside (Ara-C, 10 μM) at 48 h. Two days later, the β-D-arabinofuranoside-containing medium was replaced with fresh complete medium. Seven-day-old cultures were used. Immunocytochemical analysis indicated that the purity of neurons was ≥95% The cells were cultured in the presence or absence of 10μMMPP+ for 48 h.When the effects of catalpol on cells were studied, the various concentrations of catalpol were added for 30 min prior MPP+ treatment. Thirty minutes later, MPP+ and different concentrations of catalpol were added and incubated for 48 h in growth media. 2.3. Analysis of cell viability After the above cell treatment protocol, the level of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasolium bromide (MTT) was quantified as previously described (Mosmann, 1983; Vian et al., 1995). Briefly, cells in 96-well plates were rinsed with phosphate-buffered saline, and MTT (0.5 mg/ml) was added to each well. The microplate was incubated at 37 °C for 3 h. At the end of the incubation period, the medium with MTTwas removed and 200 μl dimethyl sulfoxide (DMSO) was added to each well. The plate was shaken on a microplate shaker to dissolve the blue MTT-formazan. Absorbance was read at 570 nm on a microplate reader. Cell viability is expressed as a percentage of that of the control culture. 2.4. Immunocytochemistry Immunostaining was performed as previously described (Zhou et al., 2005). Dopaminergic (DA) neurons were recog- nized with a rat monoclonal anti-TH antibody (1:300, Chemi- con). Briefly, formaldehyde-fixed cultures were treated with 1% hydrogen peroxide followed by sequential incubation with blocking solution for 30 min. Cells were incubated overnight at 4 °C or 37 °C for 2 h with primary anti-TH diluted in antibody diluent. The bound primary anti-TH antibody was visualized after incubation with biotinylated secondary antibody, followed by the ABC reagents and color was developed with 3, 3′- 143harmacology 568 (2007) 142–148 diaminobenzidine. For morphological analysis, the images were recorded with an inverted microscope (OLYMPUS CK40) con- nected to a camera. leakage was calculated as the percentage of LDH in the medium versus total LDH activity in the cells. The assay of SOD activity was based on its ability to inhibit the oxidation of oxymine by O2 − produced from the xanthine–xanthinoxidase system. One unit of SOD activity was defined as the amount that reduced the absorbance at 550nm by 50%. Glutathione peroxidase activity was assayed by quantifying the rate of oxidation of the reduced glutathione to the oxidized glutathione by H2O2 catalyzed by glutathione peroxidase. One unit of glutathione peroxidase was defined as the amount that reduced the level of GSH by 1 μmol L−1. Lipid peroxidation was assessed by measuring the con- centration of malondialdehyde, which can be measured at a wavelength of 532nm, formed by reaction with thiobarbituric acid. 2.9. Statistical analysis Data are expressed as the means±S.E.M. Statistical evalua- tion of the data was performed by ANOVA. All estimates were conducted in triplicate. A value of p less than 0.05 was consi- dered statistically significant. of Pharmacology 568 (2007) 142–148 2.5. Isolation of mitochondria The mitochondrial fraction was prepared as previously de- scribed (Menzies et al., 2002). Cells were washed in phosphate- buffered saline at the end of the treatment period, homogenized on ice in 10 volumes of 250 mM sucrose with 0.1 mM EGTA and 2 mM HEPES, pH 7.4, and the homogenates were cen- trifuged at 500 ×g for 5 min at 4 °C. The mitochondrial pellet and cytosolic fraction were obtained by centrifugation of the supernatant at 12,000 ×g for 10 min. The mitochondrial pellet was resuspended in sucrose medium containing 130 mM suc- rose, 50 mM KCl, 5 mM MgCl2, 5 mM KH2PO4, and 5 mM HEPES, pH 7.4, at a concentration of 2 μg protein/μl, and used for the measurement of complex I activity. 2.6. Measurement of complex I activity Complex I activity was determined by monitoring the de- crease in absorbance at 340 nm due to the oxidation of NADH (Helmerhorst et al., 2002; Schapira et al., 1990). The reaction mixture contained 250 mM sucrose, 1 mM EDTA, 50 mM Tris– HCl, pH 7.4, 2 μg/ml antimycin A, 2 mM KCN, 0.15 mM coenzyme Q1, and 20–40 μg mitochondrial homogenate. The total assay volume was 1 ml and the reagents were pre-warmed for 2 min at 30 °C. The reaction was initiated by addition of 0.1 mM NADH and the rate of decrease in absorbance was monitored spectrophotometrically at 340 nm for 3 min. Rote- none (10 μg/ml) was used to inhibit complex I activity. Absor- bance was monitored for the indicated time period before and after addition of rotenone, using a microplate spectro-photo- meter (JASCO, V-560). 2.7. Measurement of mitochondrial membrane potential Mitochondrial membrane potential was monitored using the fluorescent dye Rhodamine 123 (Rh 123), a cell-permeable cationic dye, which preferentially partitions into mitochondria because of the highly negative mitochondrial membrane poten- tial. Depolarization of the mitochondrial membrane potential results in the loss of Rh 123 from the mitochondria and a decrease in intracellular fluorescence (Satoh et al., 1997). Rh 123 was added to cultures to attain a final concentration of 10 μM for 30 min at 37 °C after cells were treated and washed with phosphate-buffered saline. The cells were collected and washed twice with phosphate-buffered saline. Fluorescence was read at 480 nm for excitation and 530 nm for emission with a fluorescence plate reader. (Genios, TECAN). 2.8. Biochemical assays The activities of lactate dehydrogenase (LDH), SOD, gluta- thione peroxidase as well as the concentration of the malon- dialdehyde in the supernatant were all determined by using commercially available kits (Jiancheng Bioengineering), ac- 144 Y.-Y. Tian et al. / European Journal cording to the manufacturer's instructions. After treatment with catalpol (0.05–0.5 mM) and MPP+ (10 μM), the culture super- natants were collected for measuring enzyme activity. LDH Fig. 2. Effects of catalpol on MPP+-induced cell damage. Mesencephalic neurons were treated with 10 μM MPP+ in the absence or presence of catalpol. LDH leakage (A) and viability of the cells (B) were determined after 48 h. Data are expressed as percent of values in untreated control cultures and are means±S.E.M. of three experiments. #Pb0.05 in comparison with control, ⁎Pb0.05 and ⁎⁎Pb0.01 in comparison with cells exposed to MPP+ alone. 3. Results 3.1. Effects of catalpol on MPP+-induced neuron damage The effect of catalpol on cell viability was evaluated by MTT assay. The maximum concentrations of catalpol that did not affect cell viability was determined (0.5mM) before examining protective effect on neurons treated with MPP+. The results in Fig. 2A showed that pretreatment of mesencephalic neurons with catalpol dose-dependently increased cell viability, and these findings were further verified by LDH assay (Fig. 2B). Thus, catalpol is effective in protecting mesencephalic neurons. 3.2. Catalpol protects dopaminergic neurons against MPP+- induced neurotoxicity inmesencephalic neuron-enriched cultures Mesencephalic neuron-enriched cultureswere used to evaluated the effect of catalpol on MPP+-induced dopaminergic neurode- generation. Neuron-enriched cultures were pretreated with the desired concentrations of catalpol for 30min, thenwere treatedwith 10μMMPP+ for an additional 48h. The extent of the degeneration of dopaminergic neurons was assessed by counting the number of TH-positive neurons and by morphological inspection. Immuno- cytochemical analysis of TH-positive neurons demonstrated that MPP+ induced a significant decrease in the number of dopami- nergic neurons. Pretreatment with catalpol (0.05–0.5mM) effec- tively reduced the degeneration of TH-positive cell bodies. Morphological inspection revealed that MPP+ treatment not only decreased the number of TH-positive neurons, but also had an apparent inhibitory effect on the outgrowth of neurites. These Fig. 4. Effects of catalpol on mitochondrial complex I activity and the loss of mitochondrial membrane potential in MPP+-treated mesencephalic neuron- enriched cultures. Neuron-enriched cultures were pretreated with different con- centrations of catalpol for 30 min prior to exposure to MPP+. A. The activity of complex I. B. Mitochondrial membrane potential was measured with Rh 123 as described in Materials and methods. Results are expressed as mean±S.E.M of three experiments performed in triplicate. #Pb0.05 in comparison with control, ⁎Pb0.05 and ⁎⁎Pb0.01 in comparison with cells exposed to MPP+ alone. Y.-Y. Tian et al. / European Journal of P Fig. 3. Neuroprotective effects of catalpol against MPP+-induced neurotoxicity. Mesencephalic neuron-enriched cultures were pretreated for 30 min with vehicle or the indicated concentrations of catalpol prior to treatment for 48 h with MPP+(10 μM). After immunostaining, the number of TH-positive neurons and the average length of TH-positive neurites were quantified as described in the Materials and methods (A). Results are means±S.E.M from three independent experiments. #Pb0.05 in comparison with control, ⁎Pb0.05 and ⁎⁎Pb0.01 in comparison with cells exposed to MPP+ alone. After immunostaining, the images were recorded with an inverted microscope connected to a camera (B). Healthy TH-positive neurons in the control cultures had extensive neurites and the MPP+-induced loss of cell bodies and neuronal processes was reversed by catalpol pretreatment. Scale bar, 25 μm. 145harmacology 568 (2007) 142–148 characteristics were reversed by catalpol in a dose-dependent manner. Treatment with catalpol (0.5mM) alone had no effect on the morphology or number of TH-positive neurons (Fig. 3). 3.3. Effects of catalpol on mitochondrial function After incubation of cells with 10 μM MPP+ for 48 h, the mitochondrial membrane potential decreased to 61.1±3.97% of control. Pretreatmentwith low concentrations of catalpol failed to significantly (Pb0.05) affect the membrane potential (Fig. 4B). However, a higher concentration of catalpol (0.5 mM) protected cells against the MPP+-induced lowering of mitochondrial membrane potential (82.3±7.45 %). The activity of complex I in the mitochondrial fraction was measured spectrophotometri- cally as described in Materials and methods. As shown in Fig. 4A, 10 μMMPP+ caused a significant decrease in complex I activity and pretreatment with catalpol blocked the effect of MPP+. These characteristics were reversed by catalpol in a dose- dependent manner. 3.4. Effects of catalpol on the content of lipid peroxide in MPP+-treated mesencephalic neurons and glutathione peroxidase. Pretreatment with catalpol (0.05– 0.5 mM) significantly and dose-dependently increased the acti- vities of SOD and glutathione peroxidase. 4. Discussion The generation of free radicals is considered to be a major factor in the pathogenesis and progression of Parkinson's dis- ease (Chiueh et al., 1994). The use of antioxidants has been reported to protect dopaminergic neurons againstMPP+-induced neurodegeneration (Le, 1994). Oxidative stress is a harmful condition that occurs when there is an excess of reactive oxygen species and/or a decrease in antioxidant levels. Therefore, re- moval of excess reactive oxygen species or suppression of their generation by antioxidants may be effective in preventing oxi- dative cell death. Recently, researchers have made considerable effort to search for natural substances with neuroprotective po- tential, and attention has been focused on a wide array of Fig. 6. Effects of catalpol on activities of antioxidant enzymes. Neuron-enriched cultures were pretreated with indicated concentrations of catalpol for 30 min prior to treatment with MPP+. After 48 h, the effect of catalpol on activities of antioxidant enzymes was determined by detecting SOD (A) and glutathione peroxidase (B) released into supernatant. Results are means±S.E.M at least three independent experiments. #Pb0.05 in comparison with control, ⁎Pb0.05 in comparison with cells exposed to MPP+ alone. 146 Y.-Y. Tian et al. / European Journal of P T