Betulinic acid protects against cerebral ischemia–reperfusion injury in mice by reducing oxidative and nitrosative stress

Nitric Oxide 24 (2011) 132–138

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Betulinic acid protects against cerebral ischemia–reperfusion injury in mice by reducing oxidative and nitrosative stress Qing Lu a,c, Ning Xia a, Hui Xu d, Lianjun Guo c, Philip Wenzel b, Andreas Daiber b, Thomas Münzel b, Ulrich Förstermann a, Huige Li a,⇑ a

Department of Pharmacology, University Medical Center, Johannes Gutenberg University, Mainz, Germany Department of Medicine II, University Medical Center, Johannes Gutenberg University, Mainz, Germany Department of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China d Department of Anesthesiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China b c

a r t i c l e

i n f o

Article history: Received 22 February 2010 Revised 24 November 2010 Available online 1 February 2011 Keywords: Betulinic acid Stroke Nitric oxide synthase NADPH oxidase Oxidative stress Nitrosative stress

a b s t r a c t Increased production of reactive oxygen and nitrogen species following cerebral ischemia–reperfusion is a major cause for neuronal injury. In hypercholesterolemic apolipoprotein E knockout (ApoE-KO) mice, 2 h of middle cerebral artery (MCA) occlusion followed by 22 h of reperfusion led to an enhanced expression of NADPH oxidase subunits (NOX2, NOX4 and p22phox) and isoforms of nitric oxide synthase (neuronal nNOS and inducible iNOS) in the ischemic hemisphere compared with the non-ischemic contralateral hemisphere. This was associated with elevated levels of 3-nitrotyrosine, an indicator of peroxynitrite-mediated oxidative protein modification. Pre-treatment with betulinic acid (50 mg/kg/day for 7 days via gavage) prior MCA occlusion prevented the ischemia reperfusion-induced upregulation of NOX2, nNOS and iNOS. In parallel, betulinic acid reduced the levels of 3-nitrotyrosine. In addition, treatment with betulinic acid enhanced the expression of endothelial eNOS in the non-ischemic hemispheres. Finally, betulinic acid reduced infarct volume and ameliorated the neurological deficit in this mouse stroke model. In conclusion, betulinic acid protects against cerebral ischemia–reperfusion injury in mice. This is likely to result from a reduction of oxidative stress (by downregulation of NOX2) and nitrosative stress (by reduction of nNOS and iNOS), and an enhancement of blood flow (by upregulation of eNOS). Ó 2011 Elsevier Inc. All rights reserved.

Introduction Stroke is a leading cause of death and long-term disability in industrialized countries. Excessive production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are implicated in the neuronal damage of stroke [1,2]. Principal sources of ROS in the brain include the mitochondrial respiratory chain, xanthine oxidase and cyclooxygenase [1,2]. Recent studies demonstrate that also NADPH oxidases are important ROS producers. NADPH oxidase is a multicomponent enzyme complex that consists of the membrane-bound cytochrome b558 (gp91phox and p22phox) and cytoplasmic proteins (p40phox, p47phox and p67phox) that translocate, along with the small G-protein Rac1, to the membrane during cellular stimulation to produce superoxide [3]. NADPH oxidase expression and activity are elevated in penumbral arteries [4,5] and in neuronal tissues [6] following ischemic ⇑ Corresponding author. Address: Department of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany. Fax: +49 6131 39 36611. E-mail address: [email protected] (H. Li). 1089-8603/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2011.01.007

stroke. Hypoglycemic neuronal death is triggered by glucose reperfusion and activation of neuronal NADPH oxidase [7]. In animal models of ischemia–reperfusion, the NADPH oxidase inhibitor apocynin attenuates cerebral infarction [8], suggesting a pivotal role for NADPH oxidase in pathogenesis of ischemia–reperfusion injury in the brain. Several homologues of gp91phox (now termed NOX2) have been cloned. These include NOX1, NOX3, NOX4 and NOX5, with NOX3 being mainly expressed in inner ear and NOX5 only found in human tissues but not in rodents [3]. NOX1, NOX2 and NOX4 are all expressed in intracranial arteries (even at much higher levels than in systemic arteries) [9] and in neuronal tissues [10]. The infarct volume induced by transient cerebral ischemia is significantly reduced in mice lacking NOX2 [11,12], whereas increased NOX2 expression in diabetic rats was associated with an aggravated ischemic brain injury [13]. Atorvastatin protects against cerebral infarction via inhibition of NOX2-derived superoxide in ischemic stroke [5]. Nitric oxide (NO) can exert both protective and deleterious effects depending on factors such as the NO synthase (NOS) isoform and the cell type by which NO is produced or the temporal stage after the onset of the ischemic brain injury. Whereas NO release

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from eNOS is protective by promoting vasodilation, NO produced by overactivation of neuronal nNOS and or by inducible iNOS contribute to brain damage [14]. Therefore, compounds that modulate the expression of NADPH oxidases and NOS isoforms are of therapeutic interest. We hypothesized that betulinic acid could be a compound with such potential. Betulinic acid is a pentacyclic triterpenoid widespread in fruit peel, leaves and stem bark. The compound is mainly known for its anti-tumor and anti-inflammatory activities [15]. Recently, we have found that betulinic acid reduces the expression of NADPH oxidases and enhances the expression of eNOS in cultured human endothelial cells [16]. Betulinic acid is also known to be an inhibitor of NF-kB [17,18], which is involved in the expressional regulation of nNOS [19,20] and iNOS [21]. Therefore, we assumed that betulinic acid might also modulate the expression of nNOS and iNOS. The present study was conducted to test the protective potential of betulinic acid in a mouse model of stroke. Materials and methods Animals and compounds Male atherosclerosis-prone apolipoprotein E knockout (ApoEKO) mice (16 weeks of age) on the genetic background of C57BL/ 6J strain were used. The mice were housed in a room where lighting was controlled (12 h on, 12 h off) and room temperature was kept at 25 °C. The animals were kept in filter top cages and handled under a laminar flow. Regular microbiological monitoring of specified pathogens was performed. They were given a standard diet and water ad libitum. Before subjected to ischemia, mice were treated with betulinic acid (Sigma–Aldrich, Deisenhofen, Germany; dissolved in dimethyl sulfoxide, DMSO, at a concentration of 10 mg/ml) via gavage at a dose of 50 mg/kg/day for 7 days. The control group received DMSO. All animal experiments were performed in accordance with the German animal protection law and the guidelines for the use of experimental animals as given by the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Induction of focal cerebral ischemia and reperfusion Transient focal ischemia was produced by intraluminal middle cerebral artery (MCA) occlusion with a nylon filament, as previously described [22,23]. Mice were anesthetized with a mixture of ketamine and xylazine (90/25 mg/kg) administered intraperitoneally [24]. After midline neck incision, the left common carotid artery, external carotid artery and internal carotid artery were carefully separated. The proximal left common carotid artery and the external carotid artery were ligated. A 6-0 nylon monofilament (Ethicon) with a heat-blunted tip was introduced through a small arteriotomy of the common carotid artery into the distal internal carotid artery and was advanced 8–9 mm distal to the origin of the MCA until the MCA was occluded. To verify adequate occlusion, a neurological deficit score was assessed (see below) [25]. The suture was withdrawn from the carotid artery under anesthesia 2 h after insertion to enable reperfusion. Then, the wound was closed. Mice were maintained in an air-conditioned room at 25 °C during the reperfusion period of 22 h. Evaluation of neurological deficit score Neurological deficits of the mice that had undergone stroke surgery were measured on a scale of 0–4 [25]. After 2 h occlusion and 22 h reperfusion, the animals were scored for neurological damage as follows: 0 = normal spontaneous movement; 1 = failure to ex-

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tend forelimb; 2 = circling to affected side; 3 = partial paralysis on affected side; 4 = no spontaneous motor activity. Determination of infarct size After 22 h reperfusion, mice were killed with an overdose of pentobarbital. The brains were immediately removed and frozen at 20 °C for 20 min and sectioned into six coronal slices. The brain slices were incubated in 2% 2,3,5-triphenyltetrazolium chloride monohydrate (TTC) at 37 °C for 15 min, followed by 4% paraformaldehyde overnight. The brain slices were photographed and the area of ischemic damage was measured by an imaging analysis system (NIH Image). The total infarct volume was calculated by integration of the infarct areas in sequential 1-mm-thick brain sections [23,26,27]. Real-time RT-PCR for mRNA expression Total RNA was isolated from mouse brain using E.Z.N.A. total RNA kit (Omega Bio-tek, Norcross, GA, USA). Total RNA (2 lg) was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Forster City, CA, USA) according to the manufacturer’s instructions. Quantitative realtime PCR amplification was performed in an iCyclerTM iQ System (Bio-Rad Laboratories, Munich, Germany) using the ABsolute™ QPCR SYBR Green Fluorescein Mix kit (Thermo Fischer Scientific, Surry, UK). The comparative threshold cycle (Ct) method was used for relative mRNA quantitation [28]. Gene expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA, and the amount of target gene mRNA expression in each sample was expressed relative to that of control. Sequences of used forward and reverse primers: 50 -GGA GGA ATT AGG CAA AAT GGA TT-30 and 50 -GCT GCA TGA CCA GCA ATG TT-30 for NOX1; 50 -CCA ACT GGG ATA ACG AGT TCA-30 and 50 -GAG AGT TTC AGC CAA GGC TTC-30 for NOX2; 50 -TGT AAC AGA GGG AAA ACA GTT GGA30 and 50 -GTT CCG GTT ACT CAA ACT ATG AAG AGT-30 for NOX4; 50 -GGA GCG ATG TGG ACA GAA GTA-30 and 50 -GCA CCG ACA ACA GGA AGT G-30 for p22phox; 50 -CCT TCC GCT ACC AGC CAG A-30 and 50 -CAG AGA TCT TCA CTG CAT TGG CTA-30 for eNOS; 50 -TCC ACC TGC CTC GAA ACC-30 and 50 -TTG TCG CTG TTG CCA AAA AC30 for nNOS; 50 -ACA ACG TGA AGA AAA CCC CTT GTG-30 and 50 ACA GTT CCG AGC GTC AAA GAC C-30 for iNOS; 50 -CTC AAC TAC ATG GTC TAC ATG TTC CA-30 and 50 -CCA TTC TCG GCC TTG ACT GT-30 for GAPDH. Western blot analysis for eNOS, nNOS and 3-nitrotyrosine Western blot analysis was performed as described previously [29,30]. Briefly, total protein of the brain homogenate was isolated and 25 lg of protein samples was separated by SDS–PAGE, and then transferred to the nitrocellulose membrane. The membrane was blocked with 5% powdered milk in TBS (10 mM Tris HCl, pH 7.4, 150 mM NaCl) with 0.1% Tween 20 for 1 h at room temperature. Membranes were then incubated with the first antibodies (rabbit polyclonal antibody against 3-nitrotyrosine, 1:2000, Upstate/Millipore; mouse monoclonal antibody against eNOS, 1:2000, BD Bioscience; rabbit polyclonal antibody against nNOS, 1:2000, Enzo Life Sciences; mouse monoclonal antibody against b-tubulin, 1:20000, Enzo Life Sciences) overnight at 4 °C. Blots were washed three times in TBS/Tween 20 (0.1%) and then incubated with a horseradish peroxidase-conjugated secondary antibody in 5% powdered milk and 0.1% Tween 20 in TBS for 1 h at room temperature. After washing, immunocomplexes were developed using an enhanced horseradish peroxidase/luminol chemiluminescence reagent (PerkinElmer Life Sciences, Boston, MA, USA) according to the manufacturer’s instructions. Quantitative analysis

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was performed after scanning of the X-ray films with the ImageJ software (NIH Image, USA) by normalizing to the b-tubulin control. Statistics Statistical differences between mean values were determined by analysis of variance (ANOVA) followed by Fisher’s protected least-significant-difference test for comparison of different means. Results Effects of betulinic acid on mRNA expression of NADPH oxidases No significant difference in NOX1 mRNA expression was found between the ischemic ipsilateral hemisphere and the non-ischemic contralateral hemisphere in vehicle-treated ApoE-KO mice subjected to 2 h MCA occlusion and 22 h reperfusion. Betulinic acid treatment had no effect on NOX1 mRNA expression, neither in contralateral nor in ipsilateral hemisphere (Fig. 1A). Compared to nonischemic contralateral hemisphere, NOX2 mRNA expression was markedly enhanced in the ischemic hemisphere of ApoE-KO mice subjected to 2 h MCA occlusion and 22 h reperfusion (Fig. 1B). Treatment with betulinic acid (50 mg/kg/day for 7 days) significantly reduced the ischemia reperfusion-induced NOX2 upregulation in the ischemic ipsilateral hemisphere (Fig. 1B). Ischemia– reperfusion also led to an upregulation of NOX4 and p22phox (Fig. 1C and D). Betulinic acid had no significant effect on NOX4 or p22phox expression (Fig. 1C and D).

Effects of betulinic acid on the expression of NOS isoforms ApoE-KO mice subjected to 2 h MCA occlusion and 22 h reperfusion showed a significant upregulation of nNOS (Fig. 2A) and a marked induction of iNOS (Fig. 2B) in the ischemic ipsilateral hemisphere in comparison to the non-ischemic contralateral hemisphere. Treatment with betulinic acid (50 mg/kg/day for 7 days) completely prevented the ischemia reperfusion-induced nNOS upregulation and significantly reduced iNOS induction (Fig. 2A and B). Treatment with betulinic acid (50 mg/kg/day for 7 days) led to an upregulation of eNOS mRNA expression, both in the ischemic ipsilateral hemisphere as well as in the non-ischemic contralateral hemisphere (Fig. 2C). nNOS protein expression was upregulated in the ischemic ipsilateral hemisphere compared to the non-ischemic contralateral hemisphere, an effect that was blocked by betulinic acid (Fig. 2D). Betulinic acid treatment significantly increased the expression of eNOS protein in the non-ischemic contralateral hemisphere (Fig. 2E). Effects of betulinic acid on 3-nitrotyrosine levels Two hours MCA occlusion and 22 h reperfusion of ApoE-KO mice led to a significant increase in 3-nitrotyrosine levels in the ischemic ipsilateral hemisphere compared to the non-ischemic contralateral hemisphere (Fig. 3A). This increase was significantly reduced by pre-treatment with betulinic acid (50 mg/kg/day for 7 days). Effects of betulinic acid on infarct size and neurologic deficit

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Reproducible brain infarcts were observed after 2 h MCA occlusion and 22 h reperfusion in vehicle-treated ApoE-KO mice (Fig. 4A). The infarct area was smaller in betulinic acid-treated mice than in vehicle-treated mice (Fig. 4A and B). Total infarction volume was also significantly reduced by betulinic acid (Fig. 4C). Moreover, neurologic deficit scored after 2 h MCA occlusion and 22 h reperfusion was decreased in betulinic acid-treated animals (Fig. 5).

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Fig. 1. Effects of betulinic acid on mRNA expression of NADPH oxidases. ApoE-KO mice were treated with 50 mg/kg betulinic acid (BA) or dimethyl sulfoxide (DMSO, as control) for 7 days and then subjected to 2 h MCA occlusion and 22 h reperfusion. mRNA expression in the brain was analyzed with quantitative real-time RT-PCR. Columns represent mean ± SEM, n = 9–12. ⁄P < 0.05, ⁄⁄P < 0.01.

We used ApoE-KO mice in the present study because these animals are hypercholesterolemic and may resemble to some extent the pathology of high risk patients for stroke. ApoE-KO mice show oxidative stress and endothelial dysfunction in extracranial vessels as well as in cerebral arterioles [31]. These mice have increased susceptibility to cerebral ischemia and suffer larger infarcts and severer hemiparesis than wild type mice [32,33]. Stroke is the third most common cause of death in industrialized countries after coronary artery disease and cancer. To date, there are no efficient curative treatments, except the thrombolytic recombinant tissue plasminogen activator (rtTPA). To prevent extensive damage after stroke, it is most important to restore the blood supply in the brain as quickly as possible. However, reperfusion (either spontaneously or as a result of rtTPA therapy) induces a massive increase in ROS production, and consequently, causes further damage and neuronal death. Therefore, identifying the sources of these ROS and strategies to reduce the ROS production are of great therapeutic interest [2]. It is now known that NADPH oxidases are a major source of ROS in cerebral ischemia–reperfusion injury [10,12,34]. Therefore, inhibition of the expression and/or activity of NADPH oxidase may become a novel strategy to treat stroke. Unfortunately, there are few compounds with such properties available. The classic NADPH oxidase inhibitors, diphenyleneiodonium and apocynin, are not spe-

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Fig. 2. Effects of betulinic acid on the expression of nitric oxide synthases (NOS). ApoE-KO mice were treated with 50 mg/kg betulinic acid (BA) or dimethyl sulfoxide (DMSO, as control) for 7 days and then subjected to 2 h MCA occlusion and 22 h reperfusion. mRNA expression in the brain was analyzed with quantitative real-time RT-PCR (A–C). Western blot analysis was performed using a polyclonal anti-nNOS antibody and a monoclonal anti-eNOS antibody, respectively. b-tubulin is shown as internal control (D and E). Columns represent mean ± SEM, n = 9–12. ⁄P < 0.05, ⁄⁄P < 0.01.

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Fig. 3. Effects of betulinic acid on peroxynitrite-mediated protein nitration. ApoEKO mice were treated with 50 mg/kg betulinic acid (BA) or dimethyl sulfoxide (DMSO, as control) for 7 days and then subjected to 2 h MCA occlusion and 22 h reperfusion. 3-nitrotyrosine levels in the brain were analyzed by Western blotting. b-tubulin is shown as internal control. Panel A shows representative blots. Panel B illustrates results of densitometric analyses. Columns represent mean ± SEM, n = 9– 12. ⁄P < 0.05.

cific and cannot be used as drugs [35]. We have recently identified two plant-derived pentacyclic triterpenes, ursolic acid and betulinic acid, which reduce vascular NADPH oxidase expression in cultured endothelial cells [16,36]. In the present study, we demonstrate that betulinic acid also suppresses NADPH oxidase induction in response to brain ischemia–reperfusion in vivo. In the brain of ApoE-KO mice, all three NOX isoforms (NOX1, NOX2 and NOX4) and their partner protein, p22phox, were found (Fig. 1). Focal ischemia–reperfusion induced by 2 h MCA occlusion and 22 h reperfusion led to an upregulation of NOX2, NOX4 and p22phox, but not of NOX1. Especially for NOX2, a 4-fold induction was observed (Fig. 1B), indicating the importance of this NOX isoform in brain ischemia–reperfusion injury. Indeed, infarct volume induced by transient cerebral ischemia was reduced in mice lacking NOX2 [11] and NOX2 plays a central role in blood–brain barrier damage in experimental stroke [12]. Importantly, treatment with betulinic acid significantly reduced the upregulation of NOX2 in response to ischemia–reperfusion (Fig. 1B). NOX1 does not appear to contribute to stroke size, and it may limit cortical infarct development following cerebral ischemia [37]. Ischemia induced by MCA occlusion results in a dramatic increase in cortical NOX4 expression [38]. A recent study demonstrated that mice deficient in NOX4 were largely protected from oxidative stress, blood–brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia [39]. These results indicate that NOX4 may represent a major source of oxidative stress and novel class of drug target for stroke therapy.

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Brain slice number Fig. 4. Effects of betulinic acid on infarct size. ApoE-KO mice were treated with 50 mg/kg betulinic acid (BA) or dimethyl sulfoxide (DMSO, as control) for 7 days and then subjected to 2 h MCA occlusion and 22 h reperfusion. Panel A shows representative images of coronal brain sections stained with TTC. The infarct area and calculated infarct volume are illustrated in panels B and C, respectively. Columns represent mean ± SEM, n = 9–12. ⁄P < 0.05, compared with DMSO.

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Fig. 5. Effects of betulinic acid on neurological deficit. ApoE-KO mice were treated with 50 mg/kg betulinic acid (BA) or dimethyl sulfoxide (DMSO, as control) for 7 days and then subjected to 2 h MCA occlusion and 22 h reperfusion. Neurological deficits are scored as described in Materials and methods section. Columns represent mean ± SEM, n = 9–12. ⁄P < 0.05, compared with DMSO.

It is now well established that different NOS isoforms play different roles in cerebral ischemia–reperfusion injury [14]. Mice lacking

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nNOS are more resistant to ischemic brain damage [40]. iNOS null mice show smaller infarcts and better neurological outcome than wild-type littermates in stroke models [41]. eNOS-KO mice, however, are more vulnerable to ischemia damage, an effect associated with a reduction in the cerebral blood flow in the ischemic penumbra [42]. Thus, eNOS-derived NO is beneficial for promoting collateral circulation and microvascular flow, whereas NO produced by nNOS and iNOS is detrimental in the ischemic brain [14]. Recent evidence indicates that most of the cytotoxicity attributed to NO is rather due to peroxynitrite, produced from the reaction between NO and superoxide anion. Peroxynitrite interacts with lipids, DNA and proteins, and commits cells to necrosis or apoptosis. Peroxynitrite generation represents a crucial pathogenic mechanism in conditions such as ischemia–reperfusion injury, myocardial infarction, chronic heart failure, diabetes, circulatory shock, chronic inflammatory diseases, cancer and neurodegenerative disorders [43,44]. Peroxynitrite-derived radicals cause direct nitration of tyrosine residues. These modifications often result in the alteration of protein function or structure and, usually, inhibition of enzyme function. Proteins containing nitrotyrosine residues have been detected in diverse pathologies associated with enhanced oxidative stress [45]. Therefore, 3-nitrotyrosine (proteins containing tyrosine residues modified in position 3 of the phenyl ring by a nitro group) can be used as an indicator of peroxynitrite formation and peroxynitrite-mediated oxidative stress [45]. In the present study, we provide evidence that nNOS and iNOS were upregulated in the ischemic hemisphere compared to the non-ischemic contralateral hemisphere (Fig. 2), which is consistent with previous studies [46,47]. Treatment with betulinic acid markedly blunted the ischemia reperfusion-induced upregulation of nNOS and iNOS (Fig. 2). The cerebral content of 3-nitrotyrosine/peroxynitrite was increased in response to ischemia–reperfusion. Betulinic acid prevented this increase (Fig. 3). This may result from the inhibitory effects of the compound on the expression of nNOS, iNOS and NOX2. The protective effects of betulinic acid on ischemia–reperfusion injury (Figs. 4 and 5) can therefore, at least in part, be attributed to the inhibition of peroxynitrite formation. Another part of the stroke protection by betulinic acid is likely to result from eNOS upregulation (Fig. 2). The current study has some limitations. (i) Our study is performed in ApoE-KO mice. Whether the observed protective effects of betulinic acid also apply to wild type mice or other mouse strains is yet unknown. (ii) Betulinic acid was administrated in the current study before the ischemia event as a pre-treatment. This indicates the potential of the compound in stroke prevention for high risk patients. However, it is unclear whether betulinic acid is also effective when used as a post-treatment after stroke. This needs to be investigated in further studies. (iii) We have observed reduced infarct size and improved neurologic deficits in betulinic acid-treated animals 22 h after reperfusion. The long-term therapeutic benefit of the compound is still unknown. In conclusion, betulinic acid unifies multiple protective properties in one single compound. Moreover, betulinic acid is a safe substance and is non-toxic even at doses up to 500 mg/kg body weight in mice [17]. By upregulating eNOS and downregulating nNOS, iNOS, and NOX2, betulinic acid (and its derivatives) may have therapeutic potential for diseases related to oxidative stress and nitrosative stress, including stroke. Acknowledgments Qing Lu was a member of the Neuroscience Graduate School at the Johannes Gutenberg University of Mainz supported by DFG (Deutsche Forschungsgemeinschaft) Research Training Group

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(GRK 1044). This work was also supported by grant LI-1042/1-1 from the DFG, Bonn, Germany, and by grants from the National Natural Sciences Foundation of China (No. 30772559 to Lianjun Guo and No. 30800445 to Hui Xu). The authors thank Gisela Reifenberg for excellent technical assistance. References [1] D.S. Warner, H. Sheng, I. Batinic-Haberle, Oxidants, antioxidants and the ischemic brain, J. Exp. Biol. 207 (2004) 3221–3231. [2] I. Margaill, M. Plotkine, D. Lerouet, Antioxidant strategies in the treatment of stroke, Free Radic. Biol. Med. 39 (2005) 429–443. [3] K. Bedard, K.H. Krause, The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology, Physiol. Rev. 87 (2007) 245–313. [4] A.A. Miller, G.J. Dusting, C.L. Roulston, C.G. Sobey, NADPH-oxidase activity is elevated in penumbral and non-ischemic cerebral arteries following stroke, Brain Res. 1111 (2006) 111–116. [5] H. Hong, J.S. Zeng, D.L. Kreulen, D.I. Kaufman, A.F. 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