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Protective effect of trihexyphenidyl on hydrogen peroxide-induced oxidative damage in PC12 cells
Neuroscience Letters 437 (2008) 50–54
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Protective effect of trihexyphenidyl on hydrogen peroxide-induced oxidative damage in PC12 cells Bian-Sheng Ji a,∗ , Yuan Gao b a b
Institute of Pharmacy, Henan University, Kaifeng 475001, China Department of Pharmacology, Zhengzhou University, Zhengzhou 450052, China
a r t i c l e
i n f o
Article history: Received 15 January 2008 Received in revised form 27 March 2008 Accepted 28 March 2008 Keywords: Trihexyphenidyl Antioxidant PC12 cells
a b s t r a c t The protective effect of trihexyphenidyl (THY) on hydrogen peroxide-induced oxidative damage was investigated in the rat pheochromocytoma line PC12 cells. Following the exposure of PC12 cells to H2 O2 , there was a reduction in cell survival, activities of superoxide dismutase (SOD) and mitochondria membrane potential (MMP), in contrast, the increased levels in Lactate dehydrogenase (LDH) release, malondialdehyde (MDA) production and intracellular reactive oxygen species (ROS), as well as intracellular [Ca2+ ]i level were observed. However, preincubation of cells with THY prior to H2 O2 exposure attenuated all the changes mentioned above, THY exhibited protective effect against H2 O2 -induced toxicity in PC12 cells, indicating that the compound may be a potential therapeutic agent for the diseases influenced by oxidative damage. © 2008 Elsevier Ireland Ltd. All rights reserved.
When neural cells are under oxidative stress, excessive reactive oxygen species (ROS) are produced that induce neuronal death. It has been well demonstrated that oxidative stress was associated with both physiological process of aging and pathological progression in the central nervous system (CNS) leading usually to some neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases [3,10], thus, removal of excess ROS or suppression of their generation by antioxidants may be effective in preventing oxidative cell death. As the major component of ROS, H2 O2 has been extensively used as an inducer of oxidative stress in vitro models [19]. The delineation of biochemical pathways involved in neuronal cell death due to H2 O2 may aid in the development of drugs for treatment of various neurodegenerative diseases. Previous works have showed that trihexyphenidyl (THY), a agent for the treatment of Parkinson’s disease, exerted markedly protective effect on acute forebrain ischemia reperfusion injury in rats [14,13], the purpose of the present study is to determine the antioxidant activity of THY and its mechanism of action in PC12 cells. PC12 cells (purchased from Shanghai Institutes for Biological Science, Chinese Academy of Sciences) were grown in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO Life Technologies) supplemented with 15% (v/v) heat-inactivated fetal calf serum at 37 ◦ C in a humidified atmosphere of 5% CO2 . THY was prepared as stock solution in dimethylsulfoxide (DMSO) and diluted with phosphate-
∗ Tel.: +86 378 3880680. E-mail address: [email protected] (B.-S. Ji). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.03.089
buffered saline (PBS) before the experiment, 0.1% (v/v) DMSO had no protective or toxic effect by itself. Control cultures were performed in the presence of DMSO under the same culture conditions. Experiments were carried out 72 h after cells were seeded. The medium was changed every other day and cells were plated at an appropriate density according to each experimental scale. The PC12 cells were preincubated with indicated concentrations of THY (Sigma, USA) and Vitamine E for 60 min before H2 O2 was added to the medium. Assays for cell viability, LDH, ROS, Ca2+ , lipid peroxide, MMP and apoptosis were performed at different times after H2 O2 was added. PC12 cells were plated at a density of 1 × 104 cells/well in 96-well plates, and the cell viability was determined by the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. After exposure to 200 M H2 O2 for 6 h, the cells were treated with the MTT (Sigma) solution (final concentration, 0.5 mg/ml) for 4-h. The medium was removed and 50 l of DMSO was added to each well. The formazan dye crystals were solubilized for 15 min, and absorbance at 570 nm was measured with an ELISA plate reader (Hua Dong Electronic Co., Nanjing, China). Results were expressed as the percentage of MTT reduction, assuming that the absorbance of control cells was 100%. After cells were exposed to 200 M H2 O2 in the presence of THY or Vitamine E for 6 h, the medium was collected, and the amount of LDH released by cells was determined using an assay kit (Nanjing Jiancheng Co., China) according to the manufacturer’s instruction. The absorbance of samples was read at 440 nm. LDH activity in the cells was determined after incubation in a hypotonic solution
B.-S. Ji, Y. Gao / Neuroscience Letters 437 (2008) 50–54
containing 15 mmol/l Tris, at pH 7.4. LDH leakage was expressed as the percentage (%) of the total LDH activity (LDH in the medium +LDH in the cell), according to the equation %LDH released = (LDH activity in the medium/total LDH activity) × 100. After treated with H2 O2 for 6 h, the cells were rinsed with PBS for two times, every well was added 1% Triton-X 100 50 l. The protein was precipitate by adding 25% H3 PO4 100 l. The homogenate was centrifugated at 10,000 × g at 4 ◦ C for 1 h. Then, SOD activities and MDA contents were determined according to the direction of the assay kit (Nanjing Jiancheng Co.). Intracellular ROS was estimated by using a fluorescent probe, 2 ,7 -dichlorofluorescein diacetate (DCFH-DA). DCFH-DA readily diffuses through the cell membrane and is enzymatically hydrolyzed by intracellular esterases to form non-fluorescent 2 ,7 dichlorofluorescein (DCFH2 ), which is then rapidly oxidized to form highly fluorescent 2 ,7 -dichlorofluorescin (DCF) in the presence of ROS. The DCF fluorescence intensity is believed to be parallel to the amount of ROS formed intracellularly. Here, after the exposure to 200 M H2 O2 for 60 min, cells (105 cells/ml) were collected and resuspensed with PBS. 195 l of the suspension was loaded into a 96-well plate and then 5 l DCFH-DA (Sigma) was added (final concentration 10 M). The fluorescence intensity (relative fluorescence units) was determined with 1420 Victor2 V (PerkinElmer Life Science, USA). The percentage increase in fluorescence per well was calculated by the formula [(Ft60 − Ft0 )/Ft0 × 100], where Ft0 is the fluorescence at time 0 min and Ft60 is the fluorescence at time 60 min in the presence of H2 O2 . MMP was monitored using a flow cytometer (Becton Dickinson), and the fluorescent dye Rh123 (Sigma), a cell permeable cationic dye, which preferentially enters into mitochondria based on the highly negative MMP. Depolarization of MMP results in the loss of Rh123 from the mitochondria and a decrease in intracellular fluorescence. After the exposure to 200 M H2 O2 for 60 min, PC12 cells were collected, centrifuged at 1200 × g for 5 min, and resuspended in DMEM. Cells were counted and 1 × 106 cells were used for flowcytometry analysis. Briefly, Rh123 was added to the cell samples at the final concentration of 1 mM. After 1 h of incubation at 37.8 ◦ C, the intracellular mean fluorescence intensity (MFI) associated with Rh123 was determined by flow cytometry (Becton Dickinson) with excitation at 480 and 530 nm long-pass filter. All data acquisition and analysis was done using Cell Quest software. [Ca2+ ]i was monitored using the fluorescent Ca2+ -sensitive dye, Fura 2-acetoxymethy ester (Fura 2-AM) [16]. The confluent monolayer of PC12 cells was preloaded with 5 M Fura 2-AM (Sigma) for 60 min at 37 ◦ C in a humidified incubator. Afterwards, cells were then rinsed three times with phosphate-buffered saline (pH 7.4) and incubated in the presence of 200 M H2 O2 for 60 min, then, cells were digested with 0.125% trypsin (w/v) and 0.04% edetic acid (w/v) in phosphate-buffered saline (pH 7.4), suspended in 1 ml
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Fig. 1. Effect of THY on intracellular ROS in H2 O2 -induced PC12 cells. Intracellular ROS were determined based on the peroxide-sensitive DCF. Each value represents the mean ± S.D. from four experiments and was # p < 0.05 vs. control, * p < 0.05 vs. H2 O2 group.
Fig. 2. Effect of THY on the loss of MMP in H2 O2 -induced PC12 cells. MMP were determined based on the Rh123-associated MFI. Each value represents the mean ± S.D. from four experiments and was measured as described in paragraph 2. # p < 0.05 vs. control, * p < 0.05 vs. H2 O2 group.
Fig. 3. Effect of THY on intracellular [Ca2+ ]i in H2 O2 -induced PC12 cells. Intracellular [Ca2+ ]i were measured by Fura-2/AM 340/380 nm fluorescence ratio (F340/F380). Each value represents the mean ± S.D. from four experiments and was measured as described in paragraph 2. # p < 0.05 vs. control, * p < 0.05 vs. H2 O2 group.
Table 1 Protective effect of THY on H2 O2 -induced PC12 cells damage Group
A570
Reduction of MTT (%)
LDH released (% of total)
MDA (mol g−1 pro)
SOD (kU g−1 pro)
Control THY 10 M alone H2 O2
0.878 ± 0.06 0.876 ± 0.05 0.336 ± 0.03 #
0.2 61.7
6.42 ± 2.12 6.87 ± 1.56 85.62 ± 7.63#
3.3 ± 0.2 3.2 ± 0.4 23.4 ± 4.1#
8.9 ± 0.5 9.0 ± 0.8 0.63 ± 0.14#
THY 0.1 M 1 M 10 M
0.438 ± 0.05 * 0.577 ± 0.03 * 0.757 ± 0.07 *
50.1* 34.2* 13.8*
61.21 ± 5.48 * 42.36 ± 6.17* 33.60 ± 5.14 *
15.6 ± 2.1* 8.1 ± 1.5* 5.0 ± 0.6*
1.9 ± 0.22* 4.2 ± 0.62* 5.77 ± 0.6*
Vitamine E 10 M
0.771 ± 0.09 *
12.1*
27.54 ± 4.11 *
4.2 ± 0.4*
7.12 ± 1.8*
The cells were exposed to 200 M H2 O2 for 6 h, for MTT, LDH, SOD as well as MDA assay in the presence of Vitamine E and different levels of THY. Each value represents the mean ± S.D. from four experiments and was measured as described in paragraph 2. # p < 0.05 vs. control. * p < 0.05 vs. H2 O2 group.
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B.-S. Ji, Y. Gao / Neuroscience Letters 437 (2008) 50–54
Kreb’s solution containing calcium or calcium-free Kreb’s solution with 1% BSA, respectively. The cells suspension was transferred to 24-well plates. THY and Vitamine E were added into the medium 60 min prior to H2 O2 addition. The fluorescence intensity was measured at emission wavelength of 510 nm and excitation wavelengths of 340 and 380 nm on a 1420 Victor2 V, respectively. The ratio of fluorescence intensity (F340/F380) was calculated as the contents of [Ca2+ ]i, the ratio of fluorescence intensity (F340/F380) in control was taken as 100%, the ratio of fluorescence intensity (F340/F380) in other groups was compared with the ratio in control. The cells were collected by centrifugation (350 × g) 12 h after H2 O2 was added and washed with PBS. the cells were collected, fixed with 70% ethanol (−20 ◦ C) and stored at −20 ◦ C for 24 h, The internuleosomally fragmented DNA were removed from apoptotic cells by incubation in 0.2 M citrate-phosphate buffer (pH 7.8) containing 0.2 mg/ml RNase for 30 min, and then 100 g/ml propidium iodide (Sigma) was added. The remaining DNA content was mea-
sured using FACScan flow cytometry (Becton Dickinson) [11]. Data acquisition and analysis were controlled by Cell Quest software. All data were presented as mean ± S.D. and analyzed using analysis of variance (ANOVA) followed by q test. As shown in Table 1, after the PC12 cells were exposed with 200 M H2 O2 for 6 h, cell viability as MTT reduction was decreased, and a significant increase in LDH release reflecting cytotoxicity was also observed,. THY attenuated significantly all the changes mentioned above. As shown in Tab 1, after PC12 cells were exposed to H2 O2 , the elevated contents of MDA and decreased activities of SOD in PC12 cells were observed, preincubation with THY at different concentrations and Vitamine E significantly attenuated lipid peroxidation in a concentration-dependent manner. As shown in Fig. 1, cultures exposed to H2 O2 for 60 min displayed increased intensity of DCF-labeled cells when compared to untreated control cultures. Cultures pretreated with 0.1, 1.0,
Fig. 4. Effect of THY on H2 O2 -induced apoptosis. The H2 O2 -induced apoptosis in PC12 cells was measured by determining the remaining DNA content using FACScan flow cytometry. Data acquisition and analysis were controlled by Cell Quest software (mean ± S.D., n = 6).
B.-S. Ji, Y. Gao / Neuroscience Letters 437 (2008) 50–54
and 10 M THY or 10 M Vitamine E showed reduced intensity of DCF-labeled cells when compared to H2 O2 -treated cultures. As shown in Fig. 2, after incubation of PC12 cells with 200 M H2 O2 for 60 min, the MMP significantly decreased. Pretreatment with different concentrations of THY protected cells against the H2 O2 -induced lowering of MMP. In Krebs’ solution containing calcium, 200 M H2 O2 exposure induced an increase in the fluorescence ratio (F340/F380). When the cells were pretreated with 0.1, 1, and 10 M THY, F340/F380 was significantly reduced. In calcium-free Krebs’ solution, 200 M H2 O2 did not induce the increase of F340/F380, and THY at 0.1, 1, 10 M did not further decrease F340/F380 (Fig. 3). The H2 O2 -induced increase in intracellular Ca2+ was dependent on extracellular Ca2+ , since removal of extracellular Ca2+ prevented the H2 O2 -induced increase in intracellular Ca2+ . The apoptosis rate of the cells treated with H2 O2 was about 44% ± 7%, after preconditioned with 0.1, 1 and 10 M THY for 60 min as above described, the numbers of apoptosis cells were reduced to 22% ± 4% (p < 0.05 vs. H2 O2 group), 12% ± 5% (p < 0.05 vs. H2 O2 group) and 5% ± 2% (p < 0.05 vs. H2 O2 group), respectively, indicating that THY preconditioning can obviously protect against PC12 cells apoptosis induced by H2 O2 (shown in Fig. 4). Oxidative stress has been emphasized as a common mechanism implicated in several neurodegenerative diseases [6,9,12]. Administration of antioxidant has been shown to protect neuronal cells from oxidative stress in several in vitro models of neurodegenerative diseases [2,4,5]. Recent studies indicated that THY, a agent used for the treatment of Parkinson’s disease via blocking cholinoceptor in locus niger and corpora striatum, attenuated the brain edema formation and inhibited the decrease of monoamines contents of brain tissues in cerebral ischemia reperfusion rats [14,13], however, the exact mechanism of those actions was not well elucidated, thus, based on the various in vitro experiments, we observed the effect of THY on hydrogen peroxide-induced oxidative damage in PC12 cells. In the present report, exposure of PC12 cells to H2 O2 followed a decreased cell viability and a increased LDH leakage, the phenomenon of which was attenuated by THY in a concentrationdependent manner (shown in Table 1). The results also showed that treatment with THY significantly reduced the characteristic of apoptotic cells, suggesting that THY protected PC12 cells against H2 O2 -induced apoptosis (Fig. 4). Although the mechanisms of how ROS worsen neural functions are complex and are not well understood, cellular Ca2+ mishandling seems to be a common endpoint. The interactions between Ca2+ and oxidative stress are complex since free radicals may impair the neuronal ionic homeostasis, actually, many Ca2+ regulatory proteins can be influenced by ROS [21]. Conversely, Ca2+ may enhance the production of free radicals such as nitric oxide. The lowering of Ca2+ diminishes the free radical concentrations and enhances the survival of cultured neurons exposed to glutamate [8,20]. Sustained elevated Ca2+ levels in cells may impair mitchondrial function and activate phospholipase, protease, and endonucleases leading to irreversible membrane, organelles, and chromatin damage and eventually to cell death [17,22]. Recent reports have demonstrated that ROS can also affect mitochondrial function through the mitochondrial ATP-sensitive potassium (mito KATP ) channels and the mitochondrial permeability transition pore (mPTP) [7,23]. The irreversible opening of mPTP has been recognized as an early event in lethal cell insult (e.g., apoptosis) [7]. Previous study also reported that oxidative stress by H2 O2 exposure led to a loss of MMP [15]. A significant decrease in MMP induced may release an apoptosisinducing factor which activates caspase protease(s), causes nuclear condensation, cytoplasmic fragmentation, and secondary generation of ROS [24,25,18]. On the other hand, maintenance of MMP is
53
necessary for production of energy (ATP) and preservation of cellular homeostasis. Akao et al. showed that maintenance of MMP is a critical primary determinant of myocyte survival [1]. In the present report, after H2 O2 exposure, there was a marked increase of intracellular ROS formation accompanying loss of MMP and elevation of intracellular free Ca2+ level that was considered as the result of membrane depolarization leading to the opening of ion channels and increased Ca2+ influx through Ca2+ channels, pretreatment with THY significantly reduced the intracellular ROS formation following a increased SOD activities and a decreased MDA contents, blocked H2 O2 -induced Ca2+ influx, as well as diminished the loss of MMP, indicating that THY can attenuate ROS-triggered loss of MMP and disturbance of intracellular Ca2+ . THY may have prevented the signaling mechanism between high ROS levels and the opening of Ca2+ channels, which remains to be investigated. In conclusion, THY, a known agent for the treatment of Parkinson’s disease, exhibited potent protective effect in vitro on oxidative damage, it may be a potential therapeutic agent for diseases influenced by oxidative damage. Acknowledgement This project was supported by Nature Science Foundation of Education Office of Henan Province, China (No. 2007310001). References ´ Mechanistically [1] M. Akao, B. O’Rourke, Y. Teshima, J. Seharaseyon, E. Marban, distinct steps in the mitochondrial death pathway triggered by oxidative stress in cardiac myocytes, Circ. Res. 92 (2003) 186–194. [2] L.R. Barlow-Walden, R.J. Reiter, M. Abe, M. Pablos, A. Menendez-Pelaez, L.D. Chen, B. Poeggeler, Melatonin stimulates brain glutathione peroxidase activity, Neurochem. Int. 26 (1995) 497–502. [3] G. Benzi, A. Moretti, Age and peroxidative stress-related modifications of the cerebral enzymatic activities linked to mitochondria and glutathione system, Free Radic. Biol. Med. 19 (1995) 77–101. [4] K.C. Chan, C.C. Hsu, M.C. Yin, Protective effect of three diallyl sulphides against glucose-induced erythrocyte and platelet oxidation, and ADP-induced platelet aggregation, Thromb. Res. 108 (2002) 317–322. [5] Y.J. Chyan, B. Poeggeler, R.A. Omar, D.G. Chain, B. Frangione, J. Ghiso, Potent neuroprotective properties against the Alzheimer beta-amyloid by an endogenous melatonin-related indolestructure, indole-3-propionic acid, J. Biol. Chem. 274 (1999) 21937–21942. [6] J.T. Coyle, P. Puttfarcken, Oxidative stress, glutamate and neurodegenerative disorders, Science 262 (1993) 689–695. [7] M. Crompton, The mitochondrial permeability transition pore and its role in cell death, Biochem. J. 341 (1999) 233–249. [8] N. Dutrait, M. Culcasi, C. Cazevieille, S. Pietri, P. Tordo, C. Bonne, A. Muller, Calcium-dependent free radical generation in cultured retinal neurons injured by kainate, Neurosci. Lett. 198 (1995) 13–16. [9] S. Fahn, G. Cohen, The oxidant stress hypothesis in Parkinson’s disease: evidence supporting it, Ann. Neurol. 32 (1992) 804–812. [10] T. Finkel, N.J. Holbrook, Oxidants, oxidative stress and the biology of aging, Nature 408 (2000) 239–247. [11] J. Gong, F. Traganos, Z. Darzynkiewicz, A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry, Anal. Biochem. 218 (1994) 314–319. [12] B. Halliwell, Reactive oxygen species and the central nervous system, J. Neurochem. 59 (1992) 1609–1623. [13] M.X. He, Neuroprotective Effects of Trihexyphenidyl on acute cerebral ischemia and reperfusion injury of rats, Chin. J. Neuroimmunol. Neurol. 5 (1998) 102–105. [14] X.J. Hu, Protection of trihexpyphenidyl against acute forebrain ischemia reperfusion injury in rats, Chin. Pharmacol. Bull. 13 (August) (1997) 364–366. [15] S.P.Y. Jones, Akao M. Teshima, E. Marba’n, Simvastatin attenuates oxidantinduced mitochondrial dysfunction in cardiac myocytes, Circ. Res. 93 (2003) 669–697. [16] W.B. Liu, Y.P. Wang, Magnesium lithospermate B inhibits hypoxia-induced calcium influx and nitric oxide release in endothelial cell, Acta Pharmacol. Sin. 22 (2001) 1135–1142. [17] S. Orrenius, M.J. Burkitt, G.E. Kass, J.M. Dypbukt, P. Nicotera, Calcium ions and oxidative cell injury, Ann. Neurol. 32 (1992) S33–S42. [18] P.X. Petit, S.A. Susin, N. Zamzami, B. Mignote, G. Kroemer, Mitochondria and programmed cell death: back to the future, FEBS Lett. 396 (1996) 3–7. [19] I. Satoh, N. Sakai, Y. Enokido, Y. Uchiyama, H. Hatanaka, Free radical independent protection by nerve growth factor and Bcl-2 of PC12 cells from hydrogen peroxide triggered-apoptosis, J. Biochem. 120 (1996) 540–546.
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[20] S. Tan, Y. Sagara, Y. Liu, P. Maher, D. Schubert, The regulation of reactive oxygen species production during programmed cell death, J. Cell. Biol. 141 (1998) 1423–1432. [21] K. Tasaki, C.A. Ruezler, T. Ohstuki, Lipopolysaccharide pre-treatment induces resistance against subsequent focal cerebral ischemic damage in spontaneously hypertensive rats, Brain Res. 748 (1997) 267–270. [22] B.F. Trump, I.K. Berezesky, Calcium-mediated cell injury and cell death, FASEB J. 9 (1995) 219–228. [23] T.L. Vanden Hoek, L.B.Z. Shao, C. Li, P.T. Schumacker, Reactive oxygen species released from mitochondria during brief hypoxia induce preconditioning in cardiomyocytes, J. Biol. Chem. 273 (1998) 18092–18098.
[24] N. Zamzami, P. Marchetti, M. Castedo, D. Decaudin, A. Macho, T. Hirsch, S.A. Susin, C. Zanin, P.X. Petit, B. Mignotte, G. Kroemer, Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death, Exp. Med. 182 (1995) 367– 377. [25] N. Zamzami, P. Marchetti, M. Castedo, C. Zanin, L. Vayssiere, P.X. Petit, G. Kroemer, Reduction in mitochondria potential constitute an early step of programmed lymphocyte death in vitro, Exp. Med. 181 (1995) 1661– 1672.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Protective effect of trihexyphenidyl on hydrogen peroxide-induced oxidative damage in PC12 cells Bian-Sheng Ji a,∗ , Yuan Gao b a b
Institute of Pharmacy, Henan University, Kaifeng 475001, China Department of Pharmacology, Zhengzhou University, Zhengzhou 450052, China
a r t i c l e
i n f o
Article history: Received 15 January 2008 Received in revised form 27 March 2008 Accepted 28 March 2008 Keywords: Trihexyphenidyl Antioxidant PC12 cells
a b s t r a c t The protective effect of trihexyphenidyl (THY) on hydrogen peroxide-induced oxidative damage was investigated in the rat pheochromocytoma line PC12 cells. Following the exposure of PC12 cells to H2 O2 , there was a reduction in cell survival, activities of superoxide dismutase (SOD) and mitochondria membrane potential (MMP), in contrast, the increased levels in Lactate dehydrogenase (LDH) release, malondialdehyde (MDA) production and intracellular reactive oxygen species (ROS), as well as intracellular [Ca2+ ]i level were observed. However, preincubation of cells with THY prior to H2 O2 exposure attenuated all the changes mentioned above, THY exhibited protective effect against H2 O2 -induced toxicity in PC12 cells, indicating that the compound may be a potential therapeutic agent for the diseases influenced by oxidative damage. © 2008 Elsevier Ireland Ltd. All rights reserved.
When neural cells are under oxidative stress, excessive reactive oxygen species (ROS) are produced that induce neuronal death. It has been well demonstrated that oxidative stress was associated with both physiological process of aging and pathological progression in the central nervous system (CNS) leading usually to some neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases [3,10], thus, removal of excess ROS or suppression of their generation by antioxidants may be effective in preventing oxidative cell death. As the major component of ROS, H2 O2 has been extensively used as an inducer of oxidative stress in vitro models [19]. The delineation of biochemical pathways involved in neuronal cell death due to H2 O2 may aid in the development of drugs for treatment of various neurodegenerative diseases. Previous works have showed that trihexyphenidyl (THY), a agent for the treatment of Parkinson’s disease, exerted markedly protective effect on acute forebrain ischemia reperfusion injury in rats [14,13], the purpose of the present study is to determine the antioxidant activity of THY and its mechanism of action in PC12 cells. PC12 cells (purchased from Shanghai Institutes for Biological Science, Chinese Academy of Sciences) were grown in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO Life Technologies) supplemented with 15% (v/v) heat-inactivated fetal calf serum at 37 ◦ C in a humidified atmosphere of 5% CO2 . THY was prepared as stock solution in dimethylsulfoxide (DMSO) and diluted with phosphate-
∗ Tel.: +86 378 3880680. E-mail address: [email protected] (B.-S. Ji). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.03.089
buffered saline (PBS) before the experiment, 0.1% (v/v) DMSO had no protective or toxic effect by itself. Control cultures were performed in the presence of DMSO under the same culture conditions. Experiments were carried out 72 h after cells were seeded. The medium was changed every other day and cells were plated at an appropriate density according to each experimental scale. The PC12 cells were preincubated with indicated concentrations of THY (Sigma, USA) and Vitamine E for 60 min before H2 O2 was added to the medium. Assays for cell viability, LDH, ROS, Ca2+ , lipid peroxide, MMP and apoptosis were performed at different times after H2 O2 was added. PC12 cells were plated at a density of 1 × 104 cells/well in 96-well plates, and the cell viability was determined by the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. After exposure to 200 M H2 O2 for 6 h, the cells were treated with the MTT (Sigma) solution (final concentration, 0.5 mg/ml) for 4-h. The medium was removed and 50 l of DMSO was added to each well. The formazan dye crystals were solubilized for 15 min, and absorbance at 570 nm was measured with an ELISA plate reader (Hua Dong Electronic Co., Nanjing, China). Results were expressed as the percentage of MTT reduction, assuming that the absorbance of control cells was 100%. After cells were exposed to 200 M H2 O2 in the presence of THY or Vitamine E for 6 h, the medium was collected, and the amount of LDH released by cells was determined using an assay kit (Nanjing Jiancheng Co., China) according to the manufacturer’s instruction. The absorbance of samples was read at 440 nm. LDH activity in the cells was determined after incubation in a hypotonic solution
B.-S. Ji, Y. Gao / Neuroscience Letters 437 (2008) 50–54
containing 15 mmol/l Tris, at pH 7.4. LDH leakage was expressed as the percentage (%) of the total LDH activity (LDH in the medium +LDH in the cell), according to the equation %LDH released = (LDH activity in the medium/total LDH activity) × 100. After treated with H2 O2 for 6 h, the cells were rinsed with PBS for two times, every well was added 1% Triton-X 100 50 l. The protein was precipitate by adding 25% H3 PO4 100 l. The homogenate was centrifugated at 10,000 × g at 4 ◦ C for 1 h. Then, SOD activities and MDA contents were determined according to the direction of the assay kit (Nanjing Jiancheng Co.). Intracellular ROS was estimated by using a fluorescent probe, 2 ,7 -dichlorofluorescein diacetate (DCFH-DA). DCFH-DA readily diffuses through the cell membrane and is enzymatically hydrolyzed by intracellular esterases to form non-fluorescent 2 ,7 dichlorofluorescein (DCFH2 ), which is then rapidly oxidized to form highly fluorescent 2 ,7 -dichlorofluorescin (DCF) in the presence of ROS. The DCF fluorescence intensity is believed to be parallel to the amount of ROS formed intracellularly. Here, after the exposure to 200 M H2 O2 for 60 min, cells (105 cells/ml) were collected and resuspensed with PBS. 195 l of the suspension was loaded into a 96-well plate and then 5 l DCFH-DA (Sigma) was added (final concentration 10 M). The fluorescence intensity (relative fluorescence units) was determined with 1420 Victor2 V (PerkinElmer Life Science, USA). The percentage increase in fluorescence per well was calculated by the formula [(Ft60 − Ft0 )/Ft0 × 100], where Ft0 is the fluorescence at time 0 min and Ft60 is the fluorescence at time 60 min in the presence of H2 O2 . MMP was monitored using a flow cytometer (Becton Dickinson), and the fluorescent dye Rh123 (Sigma), a cell permeable cationic dye, which preferentially enters into mitochondria based on the highly negative MMP. Depolarization of MMP results in the loss of Rh123 from the mitochondria and a decrease in intracellular fluorescence. After the exposure to 200 M H2 O2 for 60 min, PC12 cells were collected, centrifuged at 1200 × g for 5 min, and resuspended in DMEM. Cells were counted and 1 × 106 cells were used for flowcytometry analysis. Briefly, Rh123 was added to the cell samples at the final concentration of 1 mM. After 1 h of incubation at 37.8 ◦ C, the intracellular mean fluorescence intensity (MFI) associated with Rh123 was determined by flow cytometry (Becton Dickinson) with excitation at 480 and 530 nm long-pass filter. All data acquisition and analysis was done using Cell Quest software. [Ca2+ ]i was monitored using the fluorescent Ca2+ -sensitive dye, Fura 2-acetoxymethy ester (Fura 2-AM) [16]. The confluent monolayer of PC12 cells was preloaded with 5 M Fura 2-AM (Sigma) for 60 min at 37 ◦ C in a humidified incubator. Afterwards, cells were then rinsed three times with phosphate-buffered saline (pH 7.4) and incubated in the presence of 200 M H2 O2 for 60 min, then, cells were digested with 0.125% trypsin (w/v) and 0.04% edetic acid (w/v) in phosphate-buffered saline (pH 7.4), suspended in 1 ml
51
Fig. 1. Effect of THY on intracellular ROS in H2 O2 -induced PC12 cells. Intracellular ROS were determined based on the peroxide-sensitive DCF. Each value represents the mean ± S.D. from four experiments and was # p < 0.05 vs. control, * p < 0.05 vs. H2 O2 group.
Fig. 2. Effect of THY on the loss of MMP in H2 O2 -induced PC12 cells. MMP were determined based on the Rh123-associated MFI. Each value represents the mean ± S.D. from four experiments and was measured as described in paragraph 2. # p < 0.05 vs. control, * p < 0.05 vs. H2 O2 group.
Fig. 3. Effect of THY on intracellular [Ca2+ ]i in H2 O2 -induced PC12 cells. Intracellular [Ca2+ ]i were measured by Fura-2/AM 340/380 nm fluorescence ratio (F340/F380). Each value represents the mean ± S.D. from four experiments and was measured as described in paragraph 2. # p < 0.05 vs. control, * p < 0.05 vs. H2 O2 group.
Table 1 Protective effect of THY on H2 O2 -induced PC12 cells damage Group
A570
Reduction of MTT (%)
LDH released (% of total)
MDA (mol g−1 pro)
SOD (kU g−1 pro)
Control THY 10 M alone H2 O2
0.878 ± 0.06 0.876 ± 0.05 0.336 ± 0.03 #
0.2 61.7
6.42 ± 2.12 6.87 ± 1.56 85.62 ± 7.63#
3.3 ± 0.2 3.2 ± 0.4 23.4 ± 4.1#
8.9 ± 0.5 9.0 ± 0.8 0.63 ± 0.14#
THY 0.1 M 1 M 10 M
0.438 ± 0.05 * 0.577 ± 0.03 * 0.757 ± 0.07 *
50.1* 34.2* 13.8*
61.21 ± 5.48 * 42.36 ± 6.17* 33.60 ± 5.14 *
15.6 ± 2.1* 8.1 ± 1.5* 5.0 ± 0.6*
1.9 ± 0.22* 4.2 ± 0.62* 5.77 ± 0.6*
Vitamine E 10 M
0.771 ± 0.09 *
12.1*
27.54 ± 4.11 *
4.2 ± 0.4*
7.12 ± 1.8*
The cells were exposed to 200 M H2 O2 for 6 h, for MTT, LDH, SOD as well as MDA assay in the presence of Vitamine E and different levels of THY. Each value represents the mean ± S.D. from four experiments and was measured as described in paragraph 2. # p < 0.05 vs. control. * p < 0.05 vs. H2 O2 group.
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Kreb’s solution containing calcium or calcium-free Kreb’s solution with 1% BSA, respectively. The cells suspension was transferred to 24-well plates. THY and Vitamine E were added into the medium 60 min prior to H2 O2 addition. The fluorescence intensity was measured at emission wavelength of 510 nm and excitation wavelengths of 340 and 380 nm on a 1420 Victor2 V, respectively. The ratio of fluorescence intensity (F340/F380) was calculated as the contents of [Ca2+ ]i, the ratio of fluorescence intensity (F340/F380) in control was taken as 100%, the ratio of fluorescence intensity (F340/F380) in other groups was compared with the ratio in control. The cells were collected by centrifugation (350 × g) 12 h after H2 O2 was added and washed with PBS. the cells were collected, fixed with 70% ethanol (−20 ◦ C) and stored at −20 ◦ C for 24 h, The internuleosomally fragmented DNA were removed from apoptotic cells by incubation in 0.2 M citrate-phosphate buffer (pH 7.8) containing 0.2 mg/ml RNase for 30 min, and then 100 g/ml propidium iodide (Sigma) was added. The remaining DNA content was mea-
sured using FACScan flow cytometry (Becton Dickinson) [11]. Data acquisition and analysis were controlled by Cell Quest software. All data were presented as mean ± S.D. and analyzed using analysis of variance (ANOVA) followed by q test. As shown in Table 1, after the PC12 cells were exposed with 200 M H2 O2 for 6 h, cell viability as MTT reduction was decreased, and a significant increase in LDH release reflecting cytotoxicity was also observed,. THY attenuated significantly all the changes mentioned above. As shown in Tab 1, after PC12 cells were exposed to H2 O2 , the elevated contents of MDA and decreased activities of SOD in PC12 cells were observed, preincubation with THY at different concentrations and Vitamine E significantly attenuated lipid peroxidation in a concentration-dependent manner. As shown in Fig. 1, cultures exposed to H2 O2 for 60 min displayed increased intensity of DCF-labeled cells when compared to untreated control cultures. Cultures pretreated with 0.1, 1.0,
Fig. 4. Effect of THY on H2 O2 -induced apoptosis. The H2 O2 -induced apoptosis in PC12 cells was measured by determining the remaining DNA content using FACScan flow cytometry. Data acquisition and analysis were controlled by Cell Quest software (mean ± S.D., n = 6).
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and 10 M THY or 10 M Vitamine E showed reduced intensity of DCF-labeled cells when compared to H2 O2 -treated cultures. As shown in Fig. 2, after incubation of PC12 cells with 200 M H2 O2 for 60 min, the MMP significantly decreased. Pretreatment with different concentrations of THY protected cells against the H2 O2 -induced lowering of MMP. In Krebs’ solution containing calcium, 200 M H2 O2 exposure induced an increase in the fluorescence ratio (F340/F380). When the cells were pretreated with 0.1, 1, and 10 M THY, F340/F380 was significantly reduced. In calcium-free Krebs’ solution, 200 M H2 O2 did not induce the increase of F340/F380, and THY at 0.1, 1, 10 M did not further decrease F340/F380 (Fig. 3). The H2 O2 -induced increase in intracellular Ca2+ was dependent on extracellular Ca2+ , since removal of extracellular Ca2+ prevented the H2 O2 -induced increase in intracellular Ca2+ . The apoptosis rate of the cells treated with H2 O2 was about 44% ± 7%, after preconditioned with 0.1, 1 and 10 M THY for 60 min as above described, the numbers of apoptosis cells were reduced to 22% ± 4% (p < 0.05 vs. H2 O2 group), 12% ± 5% (p < 0.05 vs. H2 O2 group) and 5% ± 2% (p < 0.05 vs. H2 O2 group), respectively, indicating that THY preconditioning can obviously protect against PC12 cells apoptosis induced by H2 O2 (shown in Fig. 4). Oxidative stress has been emphasized as a common mechanism implicated in several neurodegenerative diseases [6,9,12]. Administration of antioxidant has been shown to protect neuronal cells from oxidative stress in several in vitro models of neurodegenerative diseases [2,4,5]. Recent studies indicated that THY, a agent used for the treatment of Parkinson’s disease via blocking cholinoceptor in locus niger and corpora striatum, attenuated the brain edema formation and inhibited the decrease of monoamines contents of brain tissues in cerebral ischemia reperfusion rats [14,13], however, the exact mechanism of those actions was not well elucidated, thus, based on the various in vitro experiments, we observed the effect of THY on hydrogen peroxide-induced oxidative damage in PC12 cells. In the present report, exposure of PC12 cells to H2 O2 followed a decreased cell viability and a increased LDH leakage, the phenomenon of which was attenuated by THY in a concentrationdependent manner (shown in Table 1). The results also showed that treatment with THY significantly reduced the characteristic of apoptotic cells, suggesting that THY protected PC12 cells against H2 O2 -induced apoptosis (Fig. 4). Although the mechanisms of how ROS worsen neural functions are complex and are not well understood, cellular Ca2+ mishandling seems to be a common endpoint. The interactions between Ca2+ and oxidative stress are complex since free radicals may impair the neuronal ionic homeostasis, actually, many Ca2+ regulatory proteins can be influenced by ROS [21]. Conversely, Ca2+ may enhance the production of free radicals such as nitric oxide. The lowering of Ca2+ diminishes the free radical concentrations and enhances the survival of cultured neurons exposed to glutamate [8,20]. Sustained elevated Ca2+ levels in cells may impair mitchondrial function and activate phospholipase, protease, and endonucleases leading to irreversible membrane, organelles, and chromatin damage and eventually to cell death [17,22]. Recent reports have demonstrated that ROS can also affect mitochondrial function through the mitochondrial ATP-sensitive potassium (mito KATP ) channels and the mitochondrial permeability transition pore (mPTP) [7,23]. The irreversible opening of mPTP has been recognized as an early event in lethal cell insult (e.g., apoptosis) [7]. Previous study also reported that oxidative stress by H2 O2 exposure led to a loss of MMP [15]. A significant decrease in MMP induced may release an apoptosisinducing factor which activates caspase protease(s), causes nuclear condensation, cytoplasmic fragmentation, and secondary generation of ROS [24,25,18]. On the other hand, maintenance of MMP is
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necessary for production of energy (ATP) and preservation of cellular homeostasis. Akao et al. showed that maintenance of MMP is a critical primary determinant of myocyte survival [1]. In the present report, after H2 O2 exposure, there was a marked increase of intracellular ROS formation accompanying loss of MMP and elevation of intracellular free Ca2+ level that was considered as the result of membrane depolarization leading to the opening of ion channels and increased Ca2+ influx through Ca2+ channels, pretreatment with THY significantly reduced the intracellular ROS formation following a increased SOD activities and a decreased MDA contents, blocked H2 O2 -induced Ca2+ influx, as well as diminished the loss of MMP, indicating that THY can attenuate ROS-triggered loss of MMP and disturbance of intracellular Ca2+ . THY may have prevented the signaling mechanism between high ROS levels and the opening of Ca2+ channels, which remains to be investigated. In conclusion, THY, a known agent for the treatment of Parkinson’s disease, exhibited potent protective effect in vitro on oxidative damage, it may be a potential therapeutic agent for diseases influenced by oxidative damage. Acknowledgement This project was supported by Nature Science Foundation of Education Office of Henan Province, China (No. 2007310001). References ´ Mechanistically [1] M. Akao, B. O’Rourke, Y. Teshima, J. Seharaseyon, E. Marban, distinct steps in the mitochondrial death pathway triggered by oxidative stress in cardiac myocytes, Circ. Res. 92 (2003) 186–194. [2] L.R. Barlow-Walden, R.J. Reiter, M. Abe, M. Pablos, A. Menendez-Pelaez, L.D. Chen, B. Poeggeler, Melatonin stimulates brain glutathione peroxidase activity, Neurochem. Int. 26 (1995) 497–502. [3] G. Benzi, A. Moretti, Age and peroxidative stress-related modifications of the cerebral enzymatic activities linked to mitochondria and glutathione system, Free Radic. Biol. Med. 19 (1995) 77–101. [4] K.C. Chan, C.C. Hsu, M.C. Yin, Protective effect of three diallyl sulphides against glucose-induced erythrocyte and platelet oxidation, and ADP-induced platelet aggregation, Thromb. Res. 108 (2002) 317–322. [5] Y.J. Chyan, B. Poeggeler, R.A. Omar, D.G. Chain, B. Frangione, J. Ghiso, Potent neuroprotective properties against the Alzheimer beta-amyloid by an endogenous melatonin-related indolestructure, indole-3-propionic acid, J. Biol. Chem. 274 (1999) 21937–21942. [6] J.T. Coyle, P. Puttfarcken, Oxidative stress, glutamate and neurodegenerative disorders, Science 262 (1993) 689–695. [7] M. Crompton, The mitochondrial permeability transition pore and its role in cell death, Biochem. J. 341 (1999) 233–249. [8] N. Dutrait, M. Culcasi, C. Cazevieille, S. Pietri, P. Tordo, C. Bonne, A. Muller, Calcium-dependent free radical generation in cultured retinal neurons injured by kainate, Neurosci. Lett. 198 (1995) 13–16. [9] S. Fahn, G. Cohen, The oxidant stress hypothesis in Parkinson’s disease: evidence supporting it, Ann. Neurol. 32 (1992) 804–812. [10] T. Finkel, N.J. Holbrook, Oxidants, oxidative stress and the biology of aging, Nature 408 (2000) 239–247. [11] J. Gong, F. Traganos, Z. Darzynkiewicz, A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry, Anal. Biochem. 218 (1994) 314–319. [12] B. Halliwell, Reactive oxygen species and the central nervous system, J. Neurochem. 59 (1992) 1609–1623. [13] M.X. He, Neuroprotective Effects of Trihexyphenidyl on acute cerebral ischemia and reperfusion injury of rats, Chin. J. Neuroimmunol. Neurol. 5 (1998) 102–105. [14] X.J. Hu, Protection of trihexpyphenidyl against acute forebrain ischemia reperfusion injury in rats, Chin. Pharmacol. Bull. 13 (August) (1997) 364–366. [15] S.P.Y. Jones, Akao M. Teshima, E. Marba’n, Simvastatin attenuates oxidantinduced mitochondrial dysfunction in cardiac myocytes, Circ. Res. 93 (2003) 669–697. [16] W.B. Liu, Y.P. Wang, Magnesium lithospermate B inhibits hypoxia-induced calcium influx and nitric oxide release in endothelial cell, Acta Pharmacol. Sin. 22 (2001) 1135–1142. [17] S. Orrenius, M.J. Burkitt, G.E. Kass, J.M. Dypbukt, P. Nicotera, Calcium ions and oxidative cell injury, Ann. Neurol. 32 (1992) S33–S42. [18] P.X. Petit, S.A. Susin, N. Zamzami, B. Mignote, G. Kroemer, Mitochondria and programmed cell death: back to the future, FEBS Lett. 396 (1996) 3–7. [19] I. Satoh, N. Sakai, Y. Enokido, Y. Uchiyama, H. Hatanaka, Free radical independent protection by nerve growth factor and Bcl-2 of PC12 cells from hydrogen peroxide triggered-apoptosis, J. Biochem. 120 (1996) 540–546.
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