Neuroprotective Effects of Sinapine on PC12 Cells Apoptosis Induced by Sodium Dithionite

Chinese Journal of Natural Medicines 2008, 6(3): 205209

Chinese Journal of Natural Medicines

Neuroprotective Effects of Sinapine on PC12 Cells Apoptosis Induced by Sodium Dithionite YANG Chun-Yan, HE Ling* Department of Pharmacology, China Pharmaceutical University, Nanjing 210009, China Available online: 20 May 2008 ABSTRACT€ AIM: To evaluate the effects of sinapine on sodium dithionite (Na2 S 2O 4 ) induced cytotoxicity in differentiated rat pheochromocytoma (PC12) cells. METHODS: PC12 cells hypoxia damage model was established by exposure to Na 2 S 2O 4, and the protective effect of sinapine was assayed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase(LDH), malondialdehyde (MDA) and flow cytometric (FCM) for apoptosis rate and mitochondrial transmembrane potential. RESULTS: Sinapine increased cell viability and showed its significant effect on protecting PC12 cells against Na 2 S 2 O 4 -induced apoptosis and the disruption of mitochondrial transmembrane potential by flow cytometric (FCM) analysis. In addition, sinapine also markedly attenuated the leakage of LDH and the lipid peroxidation products malondialdehyde (MDA) of Na2 S2 O 4 -induced in PC12 cells. CONCLUSION: It suggests that sinapine may be a candidate chemical for the treatment of hypoxia neurodegenerative disease thro ugh anti-oxidation and anti-apoptosis. KEY WORDS€Sinapine; Na2S2O4; PC12 cells; Apoptosis

CLC Number€ 285.5

Document code€ A

Article ID€1672-3651(2008)06-0205-05

Introduction  Hypoxic/ischemic brain injury is a serious cause of death and disability in humans. Oxygen free radicals such as superoxide (O2) and hydroxyl radicals (·OH) as well as non-free radical species such as hydrogen peroxide (H2O2) which can attack nucleic acids proteins and membrane phospholipids are thought to play a crucial role in the pathophysiology of hypoxic ischemic brain injury[1,2]. Hypoxic/ischemic condition induced generation of reactive oxygen species (ROS), loss of mitochondrial transmembrane potential (MMP), release of cytochrome c from the impaired mitochondria to cytosol. The released cytochrome c forms complex with Apaf-1 in the presence of dATP, which recruits and activates caspase-9. Activated caspase-9 leads to the activation of caspase-3, which subsequently contributes to apoptotic cell death [3, 4]. Sinapine (Fig. 1) is a natural small molecular phenolic

ǏReceived onǐ10-Feb-2008 ǏFoundation ItemsǐThis project was supported by the National Natural Science Foundation of China (No.30771690); China Postdoctoral Science Foundation (No. 20070410343); Jiangsu Planned Projects for Postdoctoral Research Funds (No. 0601050B); The Drug Efficacy Study and Evaluation Service Center of Jiangsu Province (BM2005103). Ǐ Corresponding authorǐHE Ling: E-mail: [email protected] Copyright©2008, China Pharmaceutical University. Published by Elsevier B.V.All rights reserved.

compound distributed in Cruciferous Plants. Previous studies have suggested that sinapine or its derivatives has a strong radiation protection effect which can scavenge ·OH and protect DNA against base damage and strand scissions of X-ray protect plants against radiation [5]. It was also reported that sinapine has antioxidative and antiaging effect [6, 7]. In contrast to the antioxidative effects of sinapine demonstrated in non-neuronal aspects, very little research has been done on the neuroprotective properties. Therefore, in the present study, we examined the protective effects of sinapine on Na2S2O4induced cytotoxicity in cultured PC12 cells. It has been reported that Na2S2O4 could cause a chemical hypoxia which can mimic the hypoxic/ischemic condition [8, 9]. PC12 cells, a commonly used cell line that retains the features of dopaminergic neurons and have been widely used for neurobiological and neurochemical studies in vitro model. PC12 cells are O2-sensitive and when subjected to hypoxia, it displays immediate responses such as release of catecholamines, elevation of intracellular calcium and membrane depolarization. In the present study, we used Na2S2O4-treated PC12 cells as a hypoxia neuronal model and firstly investigated the neuroprotective effects of sinapine on Na2S2O4-induced injury in PC12 cells.

1

Materials

Sinapine (purityı98.68%, HPLC) was kindly provided by Prof. LIU Li-Fang (China Pharmaceutical University),

YANG Chun-Yan, et al. /Chinese Journal of Natural Medicines 2008, 6(3): 205209

which was dissolved in PBS as stock solutions and stored at 20 °C before using. Cell culture reagents were obtained from Gibco (BRL, USA). 3-(4, 5-dimethylthio-azol-2-yl) 2, 5-diphenyltetrazolium bromide (MTT), AnnexinV-FITC (AV), propidium iodide (PI), rhodamine 123 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). LDH and MDA diagnostic kit were bought from Jiancheng (Nanjing, China). All other chemicals were analytic grade.

Fig. 1 Structure of sinapine

2 2.1

Methods

Cell culture Rat pheochromocytoma cells (PC12 cells) were obtained from Chinese Academy of Sciences (Shanghai, China), and were cultured in Dulbeccoƍs modified Eagleƍs medium (DMEM), supplemented with 15% fetal bovine serum and 100 units/mL penicillin and 100 units/mL streptomycin in a humidified cell incubator at 37 °C in 5% CO2 atmosphere. In all experiments, cells in logarithmic growth phase were treated with sinapine and Na2S2O4, which were dissolved in sodium phosphate buffer (PBS). 2.2 Assessment of cell viability PC12 cells were seeded in 96-well plates at 5 × 104 cells per well. After overnight incubation, exposure to Na2S2O4 (5 mmol·L1) alone or pretreated with different concentrations of sinapine (0.1, 1, 10 ȝmol·L1) for different time intervals. After treatment, 20 ȝL MTT was added and cells were continuously incubated for a further 4 h. The medium was removed and the cells were lysed in 150 mL DMSO with shaking for 10 min. The absorbance at 570 nm was measured in a microplate reader (Bio-Rad, USA). Cell viability was expressed as the percentage to the untreated control. 2.3 Lactate dehydrogenase (LDH) assay The lactate dehydrogenase activity assay measures the leakage of the soluble cytoplasmic lactate dehydrogenase enzyme into the extra-cellular medium due to cellular lysis. The LDH Assay kit quantitatively measured the activity of LDH, a stable cytosolic enzyme that is released after cell injury. PC12 cells were washed with ice-cold PBS, harvested by centrifugation at 1 000 × r·min1 for 5 min, pooled in 0.5 mL of phosphate buffer (pH 7.4) and homogenized. The homogenate was centrifuged at 3 000 × r·min1 for 20 min at 4 °C, and the supernatant was used for the activity assay according to the manufacturer instructions. Data for LDH activity were normalized to the values measured in control cultures. Absorbance data was collected using a BMG mi-

croplate reader (BMG Labtechnologies, Inc., Durham, NC, USA) at 490 nm. LDH leakage was expressed as the percentage (%) of the maximum LDH release in the cells treated with Na2S2O4 alone (100%). 2.4 Malondialdehyde˄MDA) measurement Malondialdehyde, a terminal product of lipid peroxidation, was measured to estimate the content of lipid peroxidation in PC12 cells. Malondialdehyde concentration in cell homogenates was determined with commercial kits purchased from Jiancheng Bioengineering Institute (Nanjing, China), using the thiobarbituric acid method. The assay was based on the conjugation ability of malondialdehyde with thiobarbituric acid, to form a red product which has maximum absorbance at 532 nm. 2.5 Flow cytometry (FCM) analysis for apoptosis To quantify the apoptotic/necrotic cells, PC12 cells were harvested after treatment. The concentration was adjusted to 1 × 106 cells/mL. After centrifugation and washing with cold PBS, cells were re-suspended in binding buffer, double stained with FITC-conjugated annexin V and PI for 15 min at room temperature in the dark, and analyzed with flow cytometer (Beckman-Coulter, USA). Positioning of quadrants on Annexin V/PI dot, plots was performed and live cells (Annexin V/PI), early/primary apoptotic cells (Annexin V+/PI), late/secondary apoptotic cells (Annexin V+/PI+) and necrotic cells (Annexin V/PI+) were distinguished. Therefore, the total apoptotic proportion included the percentage of cells with fluorescence Annexin V+/PI and Annexin V+/PI+. 2.6 Flow cytometry (FCM) analysis for mitochondrial transmembrane potential Mitochondrial transmembrane potential was measured using the dye rhodamine 123. It has been shown that the uptake of rhodamine 123 into mitochondria is a function of mitochondrial transmembrane potential. PC12 cells (1 × 106 cells/mL) were incubated with 10 ȝmol·L1 rhodamine and analyzed with flow cytometer (Beckman-Coulter, USA). 2.7 Statistical analysis Data are expressed as the x ± s. The significance of inter-group difference was evaluated by one-way analyses of variance (ANOVA: Duncanƍs test for post hoc comparisons). Differences were considered significant at P<0.05.

3

Results

3.1 Effect of sinapine on Na2S2O4-induced cell viability loss in PC12 cells Mitochondrial function-based MTT conversion to formazan crystals is often used as cell viability test in cell culture systems. MTT assay showed a dose and time-dependent decrease in cell viability after PC12 cells were incubated with Na2S2O4 (Fig. 2). A significant inhibition of formazan formation was observed within 2 h after incubation with Na2S2O4 (10 mmol·L1) or with 4 h after treated Na2S2O4 (5 mmol·L1).

YANG Chun-Yan, et al. /Chinese Journal of Natural Medicines 2008, 6(3): 205209

At the lower concentrations, however, there was no signifycant inhibition even after 2 h (Fig. 2). Therefore, it was decided to use a concentration of Na2S2O4 (5 mmol·L1) for the subsequent experiments. PC12 cells were incubated with different concentrations of sinapine (0.1, 1, 10 ȝmol·L1) for 2 h prior to Na2S2O4 exposure. It was observed that pretreatments with sinapine (0.1, 1, 10 ȝmol ·L-1) significantly decreased Na2S2O4-induced cell death. After incubation for up to 6 h, in comparison to cells treated with Na2S2O4 alone (66.7%), cell viability was recovered to 70.5%, 75.6%, 88.9% respectively. Up to 12 h, compared to cells treated with Na2S2O4 alone (51.2%), cell viability was recovered to 73.3%, 77.8%, 79.3% respectively. Up to 24 h, compared to cells treated with Na2S2O4 alone (39.4%), cell viability was recovered to 52.4%, 57%, 61.2% (Fig. 3).

Fig. 2 Na 2S 2O 4 -induced injury in pc 12 cells. These results were expressed as the % of optical density observed in the control (untreated neurons). Data were expressed as the x ± s of three independent experiments. ##

P<0.01, #P<0.05 vs control

Fig. 3 Sinapine inhibited Na2S2O4-induced injury in PC12 cells. Cells were treated with 5 mmol·L1 Na2S2O4 in the presence or absence of sinapine for indicated times. After treatment, cell viability was estimated by MTT method. Results expressed as percentage of control. Data were expressed as the x ± s of three independent experiments. ##

P<0.01, #P < 0.05 vs control; **P<0.01, *P<0.05 vs Na2S2O4

3.2 Effects of sinapine on Na2S2O4-induced LDH leakage and MDA metabolism levels in PC12 cells As shown in Fig. 4, the incubation of PC12 cells with Na2S2O4 (5 mmol·L1) for 4 h could produce an obvious increase in the LDH release. When the cells were pretreated with sinapine (0.1, 1, 10 ȝmol·L1) for 2 h, Na2S2O4-induced cell toxicity was significantly reduced. Malonaldehyde (MDA) is a by-product of a free radical attack on lipids. MDA levels increased significantly after PC12 cells were exposed to Na2S2O4 compared to normal control. Preincubation with sinapine for 2 h could decrease MDA levels significantly.

Fig. 4 Effects of sinapine on Na2S2O4-induced LDH and MDA metabolism in PC12 cells. Cells were pretreated with sinapine(0.1, 1, 10 ȝmol·L1) for 2 h and incubated with 5 mmol·L1 Na2S2O4 for 4 h, then changed for new cultured medium incubated for 24 h. LDH and MDA level were measured as experimental procedures. Data are expressed as percent of values in untreated control cultures, and are x ± s of three independent experiments. *

P<0.05, **P<0.01 vs group treated by Na2S2O4 alone; P<0.01 vs control (untreated group).

##

3.3 Effect of sinapine against Na2S2O4-induced apoptosis in PC12 cells by flow cytometry. It is known that phosphatidylserine (PS) is flipped from the inner to the outer plasma membrane leaflet during the early stages of apoptosis. Annexin V, with a high affinity for PS, can therefore be used as a sensitive marker for earlier apoptosis. On the other hand, propidium iodide (PI) can conjugate to necrotic cells. Double staining of treated PC12 cells with annexinV-FITC and PI was assayed with flow cytometry to further determine the apoptotic rate in each group. It was observed that a typical quadrant analysis of PC12 cells treated with or without sinapine 2 h before Na2S2O4 treatment. The results indicate that, after Na2S2O4 (5 mmol·L1) treatment alone, the percentage of apoptotic cells (33.9%) (Fig. 5b) was increased in comparison with the normal control (6.7%) (Fig. 5a), but was dropped to 13.6% (Fig. 5c), after 10 ȝmol·L1 sinapine pretreatment. 3.4 Effect of sinapine on mitochondrial membrane potential measured by flow cytometry Mitochondrial membrane permeabilization constitutes an

YANG Chun-Yan, et al. /Chinese Journal of Natural Medicines 2008, 6(3): 205209

early event of apoptotic process, which leads to the dissipation of the inner transmembrane potential and release of soluble intermembrane proteins (Zamzami and Kroemer, 2004). In order to characterize the changes in mitochondrial events induced by Na2S2O4 and/or sinapine treatments, we observed the collapse of mitochondrial membrane potential in PC12 cells with the probe rhodamine 123. When incubated PC12 cells with 5 mmol·L1 Na2S2O4 for 4 h caused a significant transmembrane potential loss compared with control (Fig. 5d). Sinapine (10 ȝmol·L1) attenuated the transmembrane potential changes (Fig. 5f) compared with the group treated with Na2S2O4 alone (Fig. 5e).

Fig. 5 Effects of sinapine on Na2S2O4-induced apoptosis in PC12 cells. Cells were pretreated with sinapine (10 ȝmol·L1) for 2 h and incubated with 5 mmol·L1 Na2S2O4 for 4 h then changed for new cultured medium incubated for 24 h. The apoptosis data are expressed as (a, b, c) described. Effects of sinapine on Na2S2O4-induced in PC12 cells collapse of mitochondrial membrane potential by flow cytometric are expressed as (d, e, f) described.

4

Discussion

Our study investigated the neuroprotective effects of sinapine against Na2S2O4-induced toxicity of PC12 cells. The results showed that pretreated with sinapine (0.1, 1, 10 ȝmol·L1) significantly reduced the cell death of PC12 cells induced by Na2S2O4 concentration dependently compared to treatments with Na2S2O4 alone. Flow cytometry results indicated that Na2S2O4 induced cell death via early/later apoptosis in PC12 cells, whereas sinapine reduced the apoptotic rate. In addition, sinapine also reduced LDH release, the lipid peroxidation (the excessive production of MDA) and mitochondrial membrane potential depolarization. These findings suggest that sinapine may exert a protective effect against the neurotoxicity induced by Na2S2O4 in PC12 cells. Previous studies have clearly demonstrated that Sinapine is a potent free radical scavenger and a nature anti-radiation chemical. Numerous studies indicate that antioxidative and free radical scavenger such as Grape seed extracts, carotenoids, epigallocatechin and Ginkgo biloba have neuron pro-

tective effects[10-12]. All of these means sinapine may have a neuroprotective effect potential. Many studyies have been generated to support the premise that hypoxia lead to the formation of reactive oxygen species and mitochondrial dysfunction. The production of reactive oxygen species can lead to cell injury through cell membrane lipid destruction and cleavage of DNA[13], which plays a critical role in apoptosis. Oxidative stress caused by Na2S2O4 at least in part, responsible for the collapse of mitochondrial membrane potential. Antioxidants that modulate reactive oxygen species have been shown to prevent the age-related increase in the protein carbonyl content in mouse synaptic mitochondria[14]. A body of work demonstrates sinapine is a potent antioxidative chemical compound. Our data are further confirmed that sinapine is highly effective in inhibiting the by Na2S2O4. The mitochondria play a key role in the death-signaling pathway[15]. In general, collapse of the mitochondrial membrane potential results in the rapid release of caspase activators such as cytochrome c into the cytoplasm [16] . Cytochrome c activates APAF-1, which oligomerizes to form an apoptosome that in turn recruits and activates caspase-9. The activated caspase-9 cleaves and activates executioner caspases-3. In accordance with this finding, it was found that Na2S2O4 induces the loss of mitochondrial membrane potential. It suggests that signaling through mitochondria plays a key role in Na2S2O4-induced apoptosis. Sinapine inhibited the loss of mitochondrial membrane potential and reduced the apoptosis rate. Therefore, these results support that sinapine may offer the protection against neuronal toxicity by suppressing the effectors involved in mitochondria-mediated apoptosis pathway and mitochondrial membrane potential loss in PC12 cells. Up to now, there was little research concerning the neuroprotective effect of sinapine. This study firstly reported the neuroprotective effects and the related mechanisms of sinapine. In conclusion, sinapine can efficiently protect PC12 cells against Na2S2O4-induced neuronal injury. The protective effects are mediated by inhibiting the leakage of the soluble cytoplasmic lactate dehydrogenase enzyme, reducing the lipid peroxidation, stabilizing mitochondrial membrane potential and reducing cells apoptotic rate. Our work is primary research for the neuroprotective effect of sinapine on Na2S2O4-induced hypoxia cytotoxicity. Additional experimental and clinical studies will be required to assess the potential for clinical application of sinapine or its derivatives.

References [1]

[2]

Wang GX, Li GR, Wang YD, et al. Characterization of neuronal cell death in normal and diabetic rats following experimental focal cerebral ischemia [J]. Life Sci, 2001, 69(23): 2801-2810. Cao YJ, Shibata T, Rainov NG, et al. Hypoxia-inducible transgene expression in differentiated human NT2N neurons-a cell culture model for gene therapy of postischemic neuronal loss[J]. Gene Ther, 1999, 8(17): 1357-1362.

YANG Chun-Yan, et al. /Chinese Journal of Natural Medicines 2008, 6(3): 205209 [3]

Li P, Nijhawan D, Budihardjo I, et al. Cytochrome c and dATP-dependent formation of Aparf-1/caspase-9 complex initiates an apoptotic protease cascade[J]. Cell, 1997, 91(4): 479-489.

cose deprivation in neonatal rats[J]. Chin J Contemp Pediatr, 2007, 9(5): 445-8. [10] Jung JY, Mo HC, Yang KH, et al. Inhibition by epigallocatechin gallate of CoCl2-induced apoptosis in rat PC12

[4]

Araya R, Uehara T, Nomura Y, et al. Hypoxia induces apoptosis in human neuroblastoma SK-N-MC cells by caspase activation accompanying cytochrome c release from mitochondria[J]. FEBS Lett, 1998, 439(1-2): 168-172. LI WY, LI Q, GUO FQ, et al. DNA damage induced by X-irradiation and reactive oxygen species and the protection against it by sinapine[J]. Acta Phytophysiol Sin, 1997, 23(4):

cells[J]. Life Sci, 2007, 80(4): 1355-1363. [11] Kang XG, Chen JZ, Xu Z. Protective effects of Ginkgo

[5]

[6]

[7]

[8]

[9]

319-323. LI Q, GU RQ. The effects of sinapine from crucifers plants on the life-span of drosophila melanogaster[J]. Chin J Appl Environ Biol, 1999, 5(1): 32-35. Matthaus B. Antioxidant activity of extracts obtained from residues of different oilseeds[J]. J Agric Food Chem, 2002, 5(12): 3444-3452. Pan YX, Lin L, Yuan WJ, et al. Preventive effect of endothelin-1 pretreatment on hypoxia-induced injury in cultured neonatal rat cardiomyocytes[J]. Acta Physiol Sin, 2003, 25(2): 171-176. Tang J, Yao YJ, Zhong L, et al. NgR expression in oligodendrocyte precursor cells and its changes after oxygen & glu-

biloba extract on paraquat-inducedapoptosis of PC12 cells[J]. Toxicol In Vitro, 2007, 21(14): 1003-1009. [12] Feng Y, Liu YM, Fratkins JD, et al. Grape seed extract suppresses lipid peroxidation and reduces hypoxic ischemic brain injury in neonatal rats[J]. Brain Res Bull, 2005, 66(2): 120-127. [13] Vincent AM, Maiese K. Directed temporal analysis of apoptosis induction in living adherent neurons[J]. J Histochem Cytochem, 1999, 47(5): 661-672. [14] Renzi MJ, Farrell FX, Bittner A, et al. Erythropoietin induces changes in gene expression PC-12 cells[J]. Brain Res Mol Brain Res, 2002, 104(1): 86-95. [15] Mignotte B, Vayssiere JL. Mitochondria and apoptosis[J]. Eur J Biochem, 1998, 252(1): 1-15. [16] Green D, Kroemer G. The central executioners of apoptosis: caspases or mitochondria[J]. Trends Cell Biol, 1999, 8(7): 267-271.