Neurol Sci (2014) 35:875–881 DOI 10.1007/s10072-013-1618-z
ORIGINAL ARTICLE
Cinepazide maleate protects PC12 cells against oxygen–glucose deprivation-induced injury Jun Zhao • Ya Bai • Chen Zhang • Xiao Zhang Yun-Xia Zhang • Jing Chen • Lize Xiong • Ming Shi • Gang Zhao
•
Received: 19 November 2013 / Accepted: 18 December 2013 / Published online: 29 December 2013 Ó Springer-Verlag Italia 2013
Abstract Our previous study showed that cinepazide maleate (CM) was as effective and safe as mildronate in the treatment of acute ischemic stroke in a randomized, double-blind, active-controlled phase II multicenter trial, but underlying mechanism(s) is not well understood. As an extending study, here we demonstrated that CM could protect neuronal cells by affecting mitochondrial functions. PC12 cells were exposed to 2.5 h oxygen–glucose deprivation (OGD) followed by a 24 h reoxygenation, and then treated with different concentrations (1, 10, 100 lM) of CM. Among various concentrations, 10 lM CM exhibited most significant protection on PC12 cells against OGD injury. CM was found to suppress OGD-induced oxidative stress, as supported by its capability of reducing intracellular reactive oxygen species and malondialdehyde production and enhancing superoxide dismutase activity.
J. Zhao and Y. Bai contributed equally to this work. J. Zhao Y. Bai C. Zhang X. Zhang Y.-X. Zhang J. Chen M. Shi (&) G. Zhao (&) Department of Neurology, Xijing Hospital, Fourth Military Medical University, No. 15 Changle-Xi Road, Xi’an 710032, China e-mail: [email protected]; [email protected] G. Zhao e-mail: [email protected] J. Zhao e-mail: [email protected] Y. Bai e-mail: [email protected]
Importantly, our results showed that CM could preserve mitochondrial functions, as revealed by its capability of stabilizing mitochondrial membrane potential, improving OGD-induced suppression of mitochondrial respiratory complex activities and enhancing ATP production. In summary, our present study provides the first evidence that CM can protect neuronal cells against OGD injury by preserving mitochondrial functions. Keywords Cinepazide maleate Neuroprotection Oxidative stress Mitochondria PC12 cells
Introduction A previous study showed that cinepazide could increase blood flow in the myocardium, muscle, and especially in the cerebral cortex. Cinepazide also produced a mild and sustained fall in blood pressure [1]. There is evidence Y.-X. Zhang e-mail: [email protected] J. Chen e-mail: [email protected] C. Zhang Department of Neurology, Second Artillery General Hospital of PLA, Beijing 100088, China L. Xiong Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China e-mail: [email protected]
C. Zhang e-mail: [email protected] X. Zhang e-mail: [email protected]
123
876
indicating that cinepazide potentiated the effects of adenosine by preventing both its degradation by deaminase and its accumulation by atrial tissue [2]. In addition, cinepazide was found to augment the artery relaxing responses to ATP, adenosine and cAMP [3]. In our latest randomized, double-blind, active-controlled phase II multicenter trial in China, we showed that cinepazide maleate (CM) injection was as effective and safe as mildronate injection in the treatment of acute cerebral ischemic stroke [4]. However, the mechanism(s) underlying the effects of cinepazide on neuroprotection is still largely unknown. Ischemic stroke, a leading cause for human disability, causes neuronal cell death by activating a cascade of events, such as excessive release of excitatory amino acid, intracellular calcium accumulation, oxidative stress, mitochondrial dysfunction and inflammation [5–7]. Because of its high intrinsic metabolic activity, brain is susceptible to these events, especially oxidative stress after ischemic insult. Reactive oxygen species, which are mainly generated from abnormal mitochondria, disrupt antioxidant defense, impair mitochondrial homeostasis and energy production, and mediate mitochondrial-dependent apoptotic pathways, consequently leading to neuronal cell death [8]. In the present study, we investigated the effects of CM on PC12 cells, a model of neuron-like cells, subjected to oxygen–glucose deprivation (OGD) injury. Our results revealed that CM could protect PC12 cells against OGD injury by suppressing oxidative stress and preserving mitochondrial functions.
Neurol Sci (2014) 35:875–881
maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal bovine serum in a humidified atmosphere of 5 % CO2 at 37 °C. Experiments were carried out 72 h after the cells were seeded onto plates or dishes at an appropriate density according to each experimental protocol. OGD injury model on PC12 cells was performed according to previous studies [9, 10]. In brief, the culture medium was replaced with pre-warmed Hepes buffer (10 mM Hepes, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2) without glucose, and then the cells were transferred into an anaerobic chamber with 95 % N2 and 5 % CO2 at 37 °C. After 2.5 h, PC12 cells were transferred to the normal incubator and refueled with normal culture medium to allow reoxygenation for 24 h. Different concentrations (1, 10, 100 lM) of CM were added to the medium from OGD onset. For the control, the same volume of saline was added to the culture medium. MTT and LDH assays MTT assay was used to quantitatively assess cell survival as described before with microplate reader at 490 nm absorbance [11]. The cell viabilities were represented as the percentages relative to the control. LDH assay was performed according to the manufacturer’s instructions. Ratios of LDH leakage were represented as the folds of control. At least three to five independent experiments were performed for each assay. Hoechst 33342/PI double staining
Materials and methods Materials Cinepazide maleate was supplied by Beijing Sihuan Pharmaceutical Co., Ltd and the stock solution was prepared in saline. Hoechst33342, rhodamine123 (Rh123), propidium iodide (PI), and 2,7-dichlorofluorescein diacetate (DCFHDA) were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Lipid peroxidation MDA assay kit, ATP Bioluminescence assay kit, and Mitochondria/ cytosol fractionation kit were purchased from Beyotime Institute of Biotechnology. Superoxide dismutase (SOD) detection kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Mitochondrial respiration complex activity detection kits were purchased from Genmed Scientifics Corp. (Shanghai, China). Cell culture and OGD injury Rat PC12 pheochromocytoma cells (Shanghai Institute of Biochemistry and Cell Biology, Shanghai, China) were
123
To determine the portion of dead cells, PC12 cells were analyzed with PI and Hoechst 33342 double staining. As a nucleic acid staining, Hoechst 33342 was utilized to stain nuclei of normal and apoptotic cells while PI was used to visualize the necrotic cells. At 24 h post-OGD, PI (1 mM) and Hoechst 33342 (5 mM) were added to the culture medium and incubated in the dark at 37 °C for 30 min. For quantitation of dead cells, the numbers of PI? cells plus Hoechst 33342? cells with condensed nuclei were counted in a visual field (about 0.3 mm2) at 2009 and at least five different visual fields were included in each group. The percentages of surviving cells (excluding apoptotic and necrotic cells) to total cells were calculated. ROS detection Intracellular reactive oxygen species (ROS) production was determined using a fluorescent probe DCFH-DA, which can cross cell membranes and be hydrolyzed by intracellular esterase to non-fluorescent DCFH. In the presence of ROS, DCFH was oxidized to highly fluorescent
Neurol Sci (2014) 35:875–881
dichlorofluorescein (DCF) [12]. At 24 h post-OGD, the culture medium was changed to flesh DMEM containing 10 lM DCFH-DA for 30 min at 37 °C in the dark. After washing twice with DMEM without FBS, the cells were observed under a DMR fluorescence microscope (Leica, Germany). For quantitation of ROS levels, the intensities of fluorescence signals were measured using the NIH image-J software and normalized to that of the controls. Measurement of MDA level, SOD activity Lipid peroxidation MDA assay kit and SOD detection kit were used to examine intracellular MDA levels and SOD activities, respectively, according to the manufacturer’s instructions. Determination of mitochondrial membrane potential The mitochondrial membrane potential (MMP) was measured by fluorescent dye Rh123 [13], which binds specifically to mitochondria due to its highly negative MMP. The reduction of MMP causes the release of Rh123 and its fluorescence intensity will be decreased. At 24 h postOGD, PC12 cells were incubated with Rh123 (5 mM) in D-Hanks for 30 min at 37 °C in the dark. 105 cells were examined and their fluorescence intensity was detected under a flow cytometer. The excitation wavelength was 488 nm and the emission wavelength was 525 nm.
877
Results CM prevents against OGD-induced cell death First, we examined whether CM had a toxic effect on PC12 cells. Different concentrations (0.1, 1, 10, 100, and 1000 lM) of CM were added. MTT results showed that up to 100 lM, viabilities of PC12 cells were not affected whereas 1000 lM caused an obvious decrease in cell survival (data not shown). In addition, we found that the cell viabilities of PC12 cells treated with 0.1–100 lM CM were comparable to that of normally cultured cells, indicating that CM itself did not affect PC12 cell proliferation (data not shown). Then, we tested whether CM could protect PC12 cells against OGD injury. After 2.5 h of OGD and 24 h reoxygenation, cell viabilities of PC12 cells indicated by MTT assay were reduced by [40 %, when compared with the control (Fig. 1a), and LDH leakage was about 2.1-fold of control (Fig. 1b). CM with 10 lM but not 1 and 100 lM significantly attenuated OGD-induced cell death from 40 % to 18 % and LDH leakage from 2.1- to 1.5-fold of control (Fig. 1a, b). Hoechst 33342 and PI double staining showed that after 24 h post-OGD, about 50 % PC12 cells were dead while 10 lM CM could decrease OGD-induced cell death to 26 % (Fig. 1c, d). Considering a significant neuroprotective effect of CM at 10 lM, we chose this concentration to complete the following studies. CM reduces OGD-induced oxidative stress
Measurement of mitochondrial respiration complex activities and ATP levels A commercial mitochondria/cytosol fractionation kit was used to isolate the mitochondria. Before measurement, mitochondrial samples were exposed to three freeze–thaw cycles to disrupt membranes. The enzymatic activities were examined by the mitochondrial respiration complex activity kit according to the manufacturer’s instructions. For measurement of intracellular ATP levels, an ATP Bioluminescence assay kit was used according to the manufacturer’s instructions. The data were normalized to that of the controls. Statistical analysis Statistical analysis was performed using the GraphPad Prism software. Difference comparisons were analyzed with one-way ANOVA followed by Newman–Keuls’ multiple comparison test. All data were expressed as mean ± SD. Values were considered to be significant when P \ 0.05.
OGD-induced oxidative stress is one of important causes for neuronal cell death. We tested whether CM could suppress oxidative stress to exert its protective effects. As expected, at 24 h post-OGD, ROS and MDA levels were significantly increased in PC12 cells, which can be, however, noticeably reversed by CM treatment (Fig. 2a–c). By contrast, OGD-induced decreased activity of intracellular total SOD could be ameliorated by CM treatment (Fig. 2d). In addition, CM did not affect either ROS and MDA levels or SOD activities of normal cultured PC12 cells (data not shown). CM preserves mitochondrial function Mitochondrial dysfunction is often responsible for the generation of ROS. Then, we investigated whether CM exerted its effects through affecting mitochondrial function by measuring MMP and mitochondrial respiratory complex activities. At 24 h post-OGD, the intensity of Rh123, a fluorescent dye for detection of MMP, lowered to 45 % of the control. However, CM treatment significantly alleviated OGD-induced decreased MMP from 45 % to 76 %
123
878
Neurol Sci (2014) 35:875–881
Fig. 1 The effects of CM on PC12 cells injured by OGD insult. At 24 h post-OGD, cell viability and cell death were assessed by MTT assay (a) and LDH leakage assay (b), respectively. c Representative photomicrographs showing Hoechst 33342/PI double stained PC12
cells with or without 10 lM CM treatment. Scale bar 50 lm. d The percentages of surviving cells in different groups. **P \ 0.01; ***P \ 0.001 vs. OGD group
(Fig. 3a). For the activities of mitochondrial respiratory complexes, we showed that OGD insult significantly decreased the activities of complexes I, II, III and IV by 45 %, 65 %, 51 % and 61 %, respectively, as compared to the control. After treatment with CM, however, the activities of complexes I, III and IV were recovered to 63 %, 68 %, and 78 %, respectively. It is noted that CM did not affect OGD-induced decreased activity of complex II (Fig. 3b–e).
treatment made a marked recovery in ATP levels after OGD (Fig. 4a, b).
CM inhibits OGD-induced ATP depletion Finally, we showed that immediately and at 4 h post-OGD, ATP levels were decreased by 50 % and 43 %, respectively, compared with the control group. However, CM
123
Discussion In the present study, we investigated the effects of CM on OGD-injured PC12 cells. Our data showed that CM protected PC12 cells against OGD insult, and this effect may be due to its capability of suppressing oxidative stress and preserving mitochondrial function. Available evidence suggests that oxidative stress plays a crucial role in ischemia/reperfusion process [14]. ROS causes intracellular lipid peroxidation and oxidative DNA,
Neurol Sci (2014) 35:875–881
879
Fig. 2 The effects of CM on OGD-induced oxidative stress. a Representative photomicrographs showing intracellular ROS accumulation revealed by DCFH-DA probe in the control, OGD and CM treatment group. Scale bar 50 lm. b Relative intensity of DCF
fluorescence was calculated using Image-J software. c, d Relative MDA levels (d) and relative SOD activities (d) in PC12 cells with or without treatment of CM after OGD. *P \ 0.05; ***P \ 0.001 vs. OGD group
Fig. 3 The effects of CM on mitochondrial function after OGD. a The changes of MMP in PC12 cells with or without treatment of CM after OGD. b–e The changes of activities of mitochondrial
respiratory chain complexes I (b), II (c), III (d), and IV (e). *P \ 0.05; **P \ 0.01; ***P \ 0.001 vs. OGD group
further resulting in the loss of cell membrane integrity, mitochondrial dysfunction and apoptosis [8]. In addition, oxidants also activate redox-sensitive signal transduction pathways, causing post-ischemia inflammatory injury [15]. Therefore, free radical-scavenging drugs and the existence of SOD, an essential enzyme eliminating superoxide radical, can reduce oxidative stress [16]. At present, we observed that CM not only reduced ROS accumulation and
lipid peroxidation production, but also ameliorated the activity of SOD after OGD injury. Thus, we proposed that this anti-oxidative effect of CM may be partly related to its capability of maintaining the activities of endogenous antioxidant enzyme. Furthermore, the protective effect of CM was also evidenced by the preserving of mitochondrial function. Ischemia limits the supplying of glucose and oxygen,
123
880
Neurol Sci (2014) 35:875–881
Fig. 4 The effects of CM on intracellular ATP levels. a Relative ATP levels in PC12 cells after 2.5 h OGD. b Relative ATP levels in PC12 cells after 2.5 h OGD followed by 4 h reoxygenation. ***P \ 0.001 vs. OGD group
causes excessive ROS production and strongly affects production of high energy phosphate, which is likely to be important in maintaining MMP [17]. The decrease in MMP can generate secondary ROS and trigger cytochrome c releasing from the inner membrane space to the cytoplasm, which mediated the apoptosis pathway [18]. Therefore, MMP is a sensitive indicator reflecting the mitochondrial function. In addition, the activities of mitochondrial respiratory chains, which are both source and target of reactive oxygen in cerebral ischemia [19, 20], can also reflect the changes of mitochondrial function. In the present study, we showed that CM could ameliorate MMP and the activities of mitochondrial respiratory chain complexes I, III, and IV, suggesting that CM can preserve mitochondrial function after OGD. Interestingly, we noted that the decrease of complex II activity seemed not to be affected by CM treatment. We still lacked knowledge about how to address this issue. However, given that complexes I and III are major sources of mitochondria-derived ROS generation in cytosol while Complex II mainly serves as a modulator in this process [21], we proposed that CM may mainly influence on ROS-produced complexes. Finally, ATP production is another index for mitochondrial function, and our present results showed that CM could maintain the ATP levels after OGD.
Conclusion In summary, our present results provided the first evidence that CM could protect neuronal cells against OGD injury. This protective effect may be due to its ability to inhibit oxidative stress and ameliorate mitochondrial function. Admittedly, the present study still unanswered how CM acted on oxidative stress and mitochondria, and thus, further studies should be done to clarify underlying protective mechanisms of CM. Acknowledgments We thank Ms. Dongyun Feng and Ms. Rui Wu for the technique support. This work was supported by the National Natural Science Foundation of China (Nos. 31170801 and 81371365) and by Program for Changjiang Scholars and Innovative Research Team in University (No. IRT1053).
123
References 1. Hirohashi M, Hagihara Y (1979) Effect of 1-[(1-pyrrolidynylcarbonyl) methyl]-4-(3, 4, 5-trimethoxycinnamoyl)piperazine maleate (cinepazide) on cerebral and peripheral circulation in cats (author’s transl). Nihon Yakurigaku Zasshi 75(5):495–506 2. Moritoki H, Takei M, Fujita S, Ishida Y, Akashi A (1980) Interaction of cinepazide with adenosine on guinea-pig atria. Arch Int Pharmacodyn Ther 248(2):212–224 3. Muramatsu I, Sakakibara Y, Hong SC, Fujiwara M (1984) Effects of cinepazide on the purinergic responses in the dog cerebral artery. Pharmacology 28(1):27–33 4. Zhu Y, Zhang G, Zhao J, Li D, Yan X, Liu J, Liu X, Zhao H, Xia J, Zhang X, Li Z, Zhang B, Guo Z, Feng L, Zhang Z, Qu F, Zhao G (2013) Efficacy and safety of mildronate for acute ischemic stroke: a randomized, double-blind, active-controlled phase II multicenter trial. Clin Drug Investig 33(10):755–760 5. Moskowitz MA, Lo EH, Iadecola C (2010) The science of stroke: mechanisms in search of treatments. Neuron 67(2):181–198 6. Serteser M, Ozben T, Gumuslu S, Balkan S, Balkan E (2002) The effects of NMDA receptor antagonist MK-801 on lipid peroxidation during focal cerebral ischemia in rats. Prog Neuropsychopharmacol Biol Psychiatry 26(5):871–877 7. Xu L, Sun J, Lu R, Ji Q, Xu JG (2005) Effect of glutamate on inflammatory responses of intestine and brain after focal cerebral ischemia. World J Gastroenterol 11(5):733–736 8. Chen H, Yoshioka H, Kim GS, Jung JE, Okami N, Sakata H, Maier CM, Narasimhan P, Goeders CE, Chan PH (2011) Oxidative stress in ischemic brain damage: mechanisms of cell death and potential molecular targets for neuroprotection. Antioxid Redox Signal 14(8):1505–1517 9. Gundimeda U, McNeill TH, Elhiani AA, Schiffman JE, Hinton DR, Gopalakrishna R (2012) Green tea polyphenols precondition against cell death induced by oxygen–glucose deprivation via stimulation of laminin receptor, generation of reactive oxygen species, and activation of protein kinase Cepsilon. J Biol Chem 287(41):34694–34708 10. Larsen EC, Hatcher JF, Adibhatla RM (2007) Effect of tricyclodecan-9-yl potassium xanthate (D609) on phospholipid metabolism and cell death during oxygen–glucose deprivation in PC12 cells. Neuroscience 146(3):946–961 11. Schabitz WR, Kollmar R, Schwaninger M, Juettler E, Bardutzky J, Scholzke MN, Sommer C, Schwab S (2003) Neuroprotective effect of granulocyte colony-stimulating factor after focal cerebral ischemia. Stroke 34(3):745–751 12. Cen J, Liu L, He L, Liu M, Wang CJ, Ji BS (2012) N(1)-(quinolin-2-ylmethyl)butane-1,4-diamine, a polyamine analogue, attenuated injury in in vitro and in vivo models of cerebral ischemia. Int J Dev Neurosci 30(7):584–595 13. Zhao Y, Wang B, Gao Y, Xiao Z, Zhao W, Chen B, Wang X, Dai J (2007) Olfactory ensheathing cell apoptosis induced by hypoxia and serum deprivation. Neurosci Lett 421(3):197–202
Neurol Sci (2014) 35:875–881 14. Rodrigo R, Fernandez-Gajardo R, Gutierrez R, Matamala JM, Carrasco R, Miranda-Merchak A, Feuerhake W (2013) Oxidative stress and pathophysiology of ischemic stroke: novel therapeutic opportunities. CNS Neurol Disord Drug Targets 12(5):698–714 15. Zhang N, Komine-Kobayashi M, Tanaka R, Liu M, Mizuno Y, Urabe T (2005) Edaravone reduces early accumulation of oxidative products and sequential inflammatory responses after transient focal ischemia in mice brain. Stroke 36(10):2220–2225 16. Warner DS, Sheng H, Batinic-Haberle I (2004) Oxidants, antioxidants and the ischemic brain. J Exp Biol 207(Pt 18):3221–3231 17. Iijima T (2006) Mitochondrial membrane potential and ischemic neuronal death. Neurosci Res 55(3):234–243
881 18. Ly JD, Grubb DR, Lawen A (2003) The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update. Apoptosis 8(2):115–128 19. Turrens JF (2003) Mitochondrial formation of reactive oxygen species. J Physiol 552(Pt 2):335–344 20. Moro MA, Almeida A, Bolanos JP, Lizasoain I (2005) Mitochondrial respiratory chain and free radical generation in stroke. Free Radic Biol Med 39(10):1291–1304 21. Drose S, Brandt U (2012) Molecular mechanisms of superoxide production by the mitochondrial respiratory chain. Adv Exp Med Biol 748:145–169
123
ORIGINAL ARTICLE
Cinepazide maleate protects PC12 cells against oxygen–glucose deprivation-induced injury Jun Zhao • Ya Bai • Chen Zhang • Xiao Zhang Yun-Xia Zhang • Jing Chen • Lize Xiong • Ming Shi • Gang Zhao
•
Received: 19 November 2013 / Accepted: 18 December 2013 / Published online: 29 December 2013 Ó Springer-Verlag Italia 2013
Abstract Our previous study showed that cinepazide maleate (CM) was as effective and safe as mildronate in the treatment of acute ischemic stroke in a randomized, double-blind, active-controlled phase II multicenter trial, but underlying mechanism(s) is not well understood. As an extending study, here we demonstrated that CM could protect neuronal cells by affecting mitochondrial functions. PC12 cells were exposed to 2.5 h oxygen–glucose deprivation (OGD) followed by a 24 h reoxygenation, and then treated with different concentrations (1, 10, 100 lM) of CM. Among various concentrations, 10 lM CM exhibited most significant protection on PC12 cells against OGD injury. CM was found to suppress OGD-induced oxidative stress, as supported by its capability of reducing intracellular reactive oxygen species and malondialdehyde production and enhancing superoxide dismutase activity.
J. Zhao and Y. Bai contributed equally to this work. J. Zhao Y. Bai C. Zhang X. Zhang Y.-X. Zhang J. Chen M. Shi (&) G. Zhao (&) Department of Neurology, Xijing Hospital, Fourth Military Medical University, No. 15 Changle-Xi Road, Xi’an 710032, China e-mail: [email protected]; [email protected] G. Zhao e-mail: [email protected] J. Zhao e-mail: [email protected] Y. Bai e-mail: [email protected]
Importantly, our results showed that CM could preserve mitochondrial functions, as revealed by its capability of stabilizing mitochondrial membrane potential, improving OGD-induced suppression of mitochondrial respiratory complex activities and enhancing ATP production. In summary, our present study provides the first evidence that CM can protect neuronal cells against OGD injury by preserving mitochondrial functions. Keywords Cinepazide maleate Neuroprotection Oxidative stress Mitochondria PC12 cells
Introduction A previous study showed that cinepazide could increase blood flow in the myocardium, muscle, and especially in the cerebral cortex. Cinepazide also produced a mild and sustained fall in blood pressure [1]. There is evidence Y.-X. Zhang e-mail: [email protected] J. Chen e-mail: [email protected] C. Zhang Department of Neurology, Second Artillery General Hospital of PLA, Beijing 100088, China L. Xiong Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China e-mail: [email protected]
C. Zhang e-mail: [email protected] X. Zhang e-mail: [email protected]
123
876
indicating that cinepazide potentiated the effects of adenosine by preventing both its degradation by deaminase and its accumulation by atrial tissue [2]. In addition, cinepazide was found to augment the artery relaxing responses to ATP, adenosine and cAMP [3]. In our latest randomized, double-blind, active-controlled phase II multicenter trial in China, we showed that cinepazide maleate (CM) injection was as effective and safe as mildronate injection in the treatment of acute cerebral ischemic stroke [4]. However, the mechanism(s) underlying the effects of cinepazide on neuroprotection is still largely unknown. Ischemic stroke, a leading cause for human disability, causes neuronal cell death by activating a cascade of events, such as excessive release of excitatory amino acid, intracellular calcium accumulation, oxidative stress, mitochondrial dysfunction and inflammation [5–7]. Because of its high intrinsic metabolic activity, brain is susceptible to these events, especially oxidative stress after ischemic insult. Reactive oxygen species, which are mainly generated from abnormal mitochondria, disrupt antioxidant defense, impair mitochondrial homeostasis and energy production, and mediate mitochondrial-dependent apoptotic pathways, consequently leading to neuronal cell death [8]. In the present study, we investigated the effects of CM on PC12 cells, a model of neuron-like cells, subjected to oxygen–glucose deprivation (OGD) injury. Our results revealed that CM could protect PC12 cells against OGD injury by suppressing oxidative stress and preserving mitochondrial functions.
Neurol Sci (2014) 35:875–881
maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal bovine serum in a humidified atmosphere of 5 % CO2 at 37 °C. Experiments were carried out 72 h after the cells were seeded onto plates or dishes at an appropriate density according to each experimental protocol. OGD injury model on PC12 cells was performed according to previous studies [9, 10]. In brief, the culture medium was replaced with pre-warmed Hepes buffer (10 mM Hepes, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2) without glucose, and then the cells were transferred into an anaerobic chamber with 95 % N2 and 5 % CO2 at 37 °C. After 2.5 h, PC12 cells were transferred to the normal incubator and refueled with normal culture medium to allow reoxygenation for 24 h. Different concentrations (1, 10, 100 lM) of CM were added to the medium from OGD onset. For the control, the same volume of saline was added to the culture medium. MTT and LDH assays MTT assay was used to quantitatively assess cell survival as described before with microplate reader at 490 nm absorbance [11]. The cell viabilities were represented as the percentages relative to the control. LDH assay was performed according to the manufacturer’s instructions. Ratios of LDH leakage were represented as the folds of control. At least three to five independent experiments were performed for each assay. Hoechst 33342/PI double staining
Materials and methods Materials Cinepazide maleate was supplied by Beijing Sihuan Pharmaceutical Co., Ltd and the stock solution was prepared in saline. Hoechst33342, rhodamine123 (Rh123), propidium iodide (PI), and 2,7-dichlorofluorescein diacetate (DCFHDA) were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Lipid peroxidation MDA assay kit, ATP Bioluminescence assay kit, and Mitochondria/ cytosol fractionation kit were purchased from Beyotime Institute of Biotechnology. Superoxide dismutase (SOD) detection kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Mitochondrial respiration complex activity detection kits were purchased from Genmed Scientifics Corp. (Shanghai, China). Cell culture and OGD injury Rat PC12 pheochromocytoma cells (Shanghai Institute of Biochemistry and Cell Biology, Shanghai, China) were
123
To determine the portion of dead cells, PC12 cells were analyzed with PI and Hoechst 33342 double staining. As a nucleic acid staining, Hoechst 33342 was utilized to stain nuclei of normal and apoptotic cells while PI was used to visualize the necrotic cells. At 24 h post-OGD, PI (1 mM) and Hoechst 33342 (5 mM) were added to the culture medium and incubated in the dark at 37 °C for 30 min. For quantitation of dead cells, the numbers of PI? cells plus Hoechst 33342? cells with condensed nuclei were counted in a visual field (about 0.3 mm2) at 2009 and at least five different visual fields were included in each group. The percentages of surviving cells (excluding apoptotic and necrotic cells) to total cells were calculated. ROS detection Intracellular reactive oxygen species (ROS) production was determined using a fluorescent probe DCFH-DA, which can cross cell membranes and be hydrolyzed by intracellular esterase to non-fluorescent DCFH. In the presence of ROS, DCFH was oxidized to highly fluorescent
Neurol Sci (2014) 35:875–881
dichlorofluorescein (DCF) [12]. At 24 h post-OGD, the culture medium was changed to flesh DMEM containing 10 lM DCFH-DA for 30 min at 37 °C in the dark. After washing twice with DMEM without FBS, the cells were observed under a DMR fluorescence microscope (Leica, Germany). For quantitation of ROS levels, the intensities of fluorescence signals were measured using the NIH image-J software and normalized to that of the controls. Measurement of MDA level, SOD activity Lipid peroxidation MDA assay kit and SOD detection kit were used to examine intracellular MDA levels and SOD activities, respectively, according to the manufacturer’s instructions. Determination of mitochondrial membrane potential The mitochondrial membrane potential (MMP) was measured by fluorescent dye Rh123 [13], which binds specifically to mitochondria due to its highly negative MMP. The reduction of MMP causes the release of Rh123 and its fluorescence intensity will be decreased. At 24 h postOGD, PC12 cells were incubated with Rh123 (5 mM) in D-Hanks for 30 min at 37 °C in the dark. 105 cells were examined and their fluorescence intensity was detected under a flow cytometer. The excitation wavelength was 488 nm and the emission wavelength was 525 nm.
877
Results CM prevents against OGD-induced cell death First, we examined whether CM had a toxic effect on PC12 cells. Different concentrations (0.1, 1, 10, 100, and 1000 lM) of CM were added. MTT results showed that up to 100 lM, viabilities of PC12 cells were not affected whereas 1000 lM caused an obvious decrease in cell survival (data not shown). In addition, we found that the cell viabilities of PC12 cells treated with 0.1–100 lM CM were comparable to that of normally cultured cells, indicating that CM itself did not affect PC12 cell proliferation (data not shown). Then, we tested whether CM could protect PC12 cells against OGD injury. After 2.5 h of OGD and 24 h reoxygenation, cell viabilities of PC12 cells indicated by MTT assay were reduced by [40 %, when compared with the control (Fig. 1a), and LDH leakage was about 2.1-fold of control (Fig. 1b). CM with 10 lM but not 1 and 100 lM significantly attenuated OGD-induced cell death from 40 % to 18 % and LDH leakage from 2.1- to 1.5-fold of control (Fig. 1a, b). Hoechst 33342 and PI double staining showed that after 24 h post-OGD, about 50 % PC12 cells were dead while 10 lM CM could decrease OGD-induced cell death to 26 % (Fig. 1c, d). Considering a significant neuroprotective effect of CM at 10 lM, we chose this concentration to complete the following studies. CM reduces OGD-induced oxidative stress
Measurement of mitochondrial respiration complex activities and ATP levels A commercial mitochondria/cytosol fractionation kit was used to isolate the mitochondria. Before measurement, mitochondrial samples were exposed to three freeze–thaw cycles to disrupt membranes. The enzymatic activities were examined by the mitochondrial respiration complex activity kit according to the manufacturer’s instructions. For measurement of intracellular ATP levels, an ATP Bioluminescence assay kit was used according to the manufacturer’s instructions. The data were normalized to that of the controls. Statistical analysis Statistical analysis was performed using the GraphPad Prism software. Difference comparisons were analyzed with one-way ANOVA followed by Newman–Keuls’ multiple comparison test. All data were expressed as mean ± SD. Values were considered to be significant when P \ 0.05.
OGD-induced oxidative stress is one of important causes for neuronal cell death. We tested whether CM could suppress oxidative stress to exert its protective effects. As expected, at 24 h post-OGD, ROS and MDA levels were significantly increased in PC12 cells, which can be, however, noticeably reversed by CM treatment (Fig. 2a–c). By contrast, OGD-induced decreased activity of intracellular total SOD could be ameliorated by CM treatment (Fig. 2d). In addition, CM did not affect either ROS and MDA levels or SOD activities of normal cultured PC12 cells (data not shown). CM preserves mitochondrial function Mitochondrial dysfunction is often responsible for the generation of ROS. Then, we investigated whether CM exerted its effects through affecting mitochondrial function by measuring MMP and mitochondrial respiratory complex activities. At 24 h post-OGD, the intensity of Rh123, a fluorescent dye for detection of MMP, lowered to 45 % of the control. However, CM treatment significantly alleviated OGD-induced decreased MMP from 45 % to 76 %
123
878
Neurol Sci (2014) 35:875–881
Fig. 1 The effects of CM on PC12 cells injured by OGD insult. At 24 h post-OGD, cell viability and cell death were assessed by MTT assay (a) and LDH leakage assay (b), respectively. c Representative photomicrographs showing Hoechst 33342/PI double stained PC12
cells with or without 10 lM CM treatment. Scale bar 50 lm. d The percentages of surviving cells in different groups. **P \ 0.01; ***P \ 0.001 vs. OGD group
(Fig. 3a). For the activities of mitochondrial respiratory complexes, we showed that OGD insult significantly decreased the activities of complexes I, II, III and IV by 45 %, 65 %, 51 % and 61 %, respectively, as compared to the control. After treatment with CM, however, the activities of complexes I, III and IV were recovered to 63 %, 68 %, and 78 %, respectively. It is noted that CM did not affect OGD-induced decreased activity of complex II (Fig. 3b–e).
treatment made a marked recovery in ATP levels after OGD (Fig. 4a, b).
CM inhibits OGD-induced ATP depletion Finally, we showed that immediately and at 4 h post-OGD, ATP levels were decreased by 50 % and 43 %, respectively, compared with the control group. However, CM
123
Discussion In the present study, we investigated the effects of CM on OGD-injured PC12 cells. Our data showed that CM protected PC12 cells against OGD insult, and this effect may be due to its capability of suppressing oxidative stress and preserving mitochondrial function. Available evidence suggests that oxidative stress plays a crucial role in ischemia/reperfusion process [14]. ROS causes intracellular lipid peroxidation and oxidative DNA,
Neurol Sci (2014) 35:875–881
879
Fig. 2 The effects of CM on OGD-induced oxidative stress. a Representative photomicrographs showing intracellular ROS accumulation revealed by DCFH-DA probe in the control, OGD and CM treatment group. Scale bar 50 lm. b Relative intensity of DCF
fluorescence was calculated using Image-J software. c, d Relative MDA levels (d) and relative SOD activities (d) in PC12 cells with or without treatment of CM after OGD. *P \ 0.05; ***P \ 0.001 vs. OGD group
Fig. 3 The effects of CM on mitochondrial function after OGD. a The changes of MMP in PC12 cells with or without treatment of CM after OGD. b–e The changes of activities of mitochondrial
respiratory chain complexes I (b), II (c), III (d), and IV (e). *P \ 0.05; **P \ 0.01; ***P \ 0.001 vs. OGD group
further resulting in the loss of cell membrane integrity, mitochondrial dysfunction and apoptosis [8]. In addition, oxidants also activate redox-sensitive signal transduction pathways, causing post-ischemia inflammatory injury [15]. Therefore, free radical-scavenging drugs and the existence of SOD, an essential enzyme eliminating superoxide radical, can reduce oxidative stress [16]. At present, we observed that CM not only reduced ROS accumulation and
lipid peroxidation production, but also ameliorated the activity of SOD after OGD injury. Thus, we proposed that this anti-oxidative effect of CM may be partly related to its capability of maintaining the activities of endogenous antioxidant enzyme. Furthermore, the protective effect of CM was also evidenced by the preserving of mitochondrial function. Ischemia limits the supplying of glucose and oxygen,
123
880
Neurol Sci (2014) 35:875–881
Fig. 4 The effects of CM on intracellular ATP levels. a Relative ATP levels in PC12 cells after 2.5 h OGD. b Relative ATP levels in PC12 cells after 2.5 h OGD followed by 4 h reoxygenation. ***P \ 0.001 vs. OGD group
causes excessive ROS production and strongly affects production of high energy phosphate, which is likely to be important in maintaining MMP [17]. The decrease in MMP can generate secondary ROS and trigger cytochrome c releasing from the inner membrane space to the cytoplasm, which mediated the apoptosis pathway [18]. Therefore, MMP is a sensitive indicator reflecting the mitochondrial function. In addition, the activities of mitochondrial respiratory chains, which are both source and target of reactive oxygen in cerebral ischemia [19, 20], can also reflect the changes of mitochondrial function. In the present study, we showed that CM could ameliorate MMP and the activities of mitochondrial respiratory chain complexes I, III, and IV, suggesting that CM can preserve mitochondrial function after OGD. Interestingly, we noted that the decrease of complex II activity seemed not to be affected by CM treatment. We still lacked knowledge about how to address this issue. However, given that complexes I and III are major sources of mitochondria-derived ROS generation in cytosol while Complex II mainly serves as a modulator in this process [21], we proposed that CM may mainly influence on ROS-produced complexes. Finally, ATP production is another index for mitochondrial function, and our present results showed that CM could maintain the ATP levels after OGD.
Conclusion In summary, our present results provided the first evidence that CM could protect neuronal cells against OGD injury. This protective effect may be due to its ability to inhibit oxidative stress and ameliorate mitochondrial function. Admittedly, the present study still unanswered how CM acted on oxidative stress and mitochondria, and thus, further studies should be done to clarify underlying protective mechanisms of CM. Acknowledgments We thank Ms. Dongyun Feng and Ms. Rui Wu for the technique support. This work was supported by the National Natural Science Foundation of China (Nos. 31170801 and 81371365) and by Program for Changjiang Scholars and Innovative Research Team in University (No. IRT1053).
123
References 1. Hirohashi M, Hagihara Y (1979) Effect of 1-[(1-pyrrolidynylcarbonyl) methyl]-4-(3, 4, 5-trimethoxycinnamoyl)piperazine maleate (cinepazide) on cerebral and peripheral circulation in cats (author’s transl). Nihon Yakurigaku Zasshi 75(5):495–506 2. Moritoki H, Takei M, Fujita S, Ishida Y, Akashi A (1980) Interaction of cinepazide with adenosine on guinea-pig atria. Arch Int Pharmacodyn Ther 248(2):212–224 3. Muramatsu I, Sakakibara Y, Hong SC, Fujiwara M (1984) Effects of cinepazide on the purinergic responses in the dog cerebral artery. Pharmacology 28(1):27–33 4. Zhu Y, Zhang G, Zhao J, Li D, Yan X, Liu J, Liu X, Zhao H, Xia J, Zhang X, Li Z, Zhang B, Guo Z, Feng L, Zhang Z, Qu F, Zhao G (2013) Efficacy and safety of mildronate for acute ischemic stroke: a randomized, double-blind, active-controlled phase II multicenter trial. Clin Drug Investig 33(10):755–760 5. Moskowitz MA, Lo EH, Iadecola C (2010) The science of stroke: mechanisms in search of treatments. Neuron 67(2):181–198 6. Serteser M, Ozben T, Gumuslu S, Balkan S, Balkan E (2002) The effects of NMDA receptor antagonist MK-801 on lipid peroxidation during focal cerebral ischemia in rats. Prog Neuropsychopharmacol Biol Psychiatry 26(5):871–877 7. Xu L, Sun J, Lu R, Ji Q, Xu JG (2005) Effect of glutamate on inflammatory responses of intestine and brain after focal cerebral ischemia. World J Gastroenterol 11(5):733–736 8. Chen H, Yoshioka H, Kim GS, Jung JE, Okami N, Sakata H, Maier CM, Narasimhan P, Goeders CE, Chan PH (2011) Oxidative stress in ischemic brain damage: mechanisms of cell death and potential molecular targets for neuroprotection. Antioxid Redox Signal 14(8):1505–1517 9. Gundimeda U, McNeill TH, Elhiani AA, Schiffman JE, Hinton DR, Gopalakrishna R (2012) Green tea polyphenols precondition against cell death induced by oxygen–glucose deprivation via stimulation of laminin receptor, generation of reactive oxygen species, and activation of protein kinase Cepsilon. J Biol Chem 287(41):34694–34708 10. Larsen EC, Hatcher JF, Adibhatla RM (2007) Effect of tricyclodecan-9-yl potassium xanthate (D609) on phospholipid metabolism and cell death during oxygen–glucose deprivation in PC12 cells. Neuroscience 146(3):946–961 11. Schabitz WR, Kollmar R, Schwaninger M, Juettler E, Bardutzky J, Scholzke MN, Sommer C, Schwab S (2003) Neuroprotective effect of granulocyte colony-stimulating factor after focal cerebral ischemia. Stroke 34(3):745–751 12. Cen J, Liu L, He L, Liu M, Wang CJ, Ji BS (2012) N(1)-(quinolin-2-ylmethyl)butane-1,4-diamine, a polyamine analogue, attenuated injury in in vitro and in vivo models of cerebral ischemia. Int J Dev Neurosci 30(7):584–595 13. Zhao Y, Wang B, Gao Y, Xiao Z, Zhao W, Chen B, Wang X, Dai J (2007) Olfactory ensheathing cell apoptosis induced by hypoxia and serum deprivation. Neurosci Lett 421(3):197–202
Neurol Sci (2014) 35:875–881 14. Rodrigo R, Fernandez-Gajardo R, Gutierrez R, Matamala JM, Carrasco R, Miranda-Merchak A, Feuerhake W (2013) Oxidative stress and pathophysiology of ischemic stroke: novel therapeutic opportunities. CNS Neurol Disord Drug Targets 12(5):698–714 15. Zhang N, Komine-Kobayashi M, Tanaka R, Liu M, Mizuno Y, Urabe T (2005) Edaravone reduces early accumulation of oxidative products and sequential inflammatory responses after transient focal ischemia in mice brain. Stroke 36(10):2220–2225 16. Warner DS, Sheng H, Batinic-Haberle I (2004) Oxidants, antioxidants and the ischemic brain. J Exp Biol 207(Pt 18):3221–3231 17. Iijima T (2006) Mitochondrial membrane potential and ischemic neuronal death. Neurosci Res 55(3):234–243
881 18. Ly JD, Grubb DR, Lawen A (2003) The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update. Apoptosis 8(2):115–128 19. Turrens JF (2003) Mitochondrial formation of reactive oxygen species. J Physiol 552(Pt 2):335–344 20. Moro MA, Almeida A, Bolanos JP, Lizasoain I (2005) Mitochondrial respiratory chain and free radical generation in stroke. Free Radic Biol Med 39(10):1291–1304 21. Drose S, Brandt U (2012) Molecular mechanisms of superoxide production by the mitochondrial respiratory chain. Adv Exp Med Biol 748:145–169
123
