Salvianolic acid A inhibits calpain activation and eNOS uncoupling during focal cerebral ischemia in mice

Phytomedicine 25 (2017) 8–14

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Salvianolic acid A inhibits calpain activation and eNOS uncoupling during focal cerebral ischemia in mice Qaisar Mahmood a,1, Guang-Fa Wang b,1, Gang Wu a, Huan Wang a, Chang-Xin Zhou a, Hong-Yu Yang c, Zhi-Rong Liu d, Feng Han a,∗, Kui Zhao b,∗ a

College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China Department of PET/CT Center, The First Affiliated Hospital, School of Medicine, Zhejiang University Zhejiang 310003, China Department of Pharmacy, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, China d Department of Neurology, Second Affiliated Hospital of Zhejiang University, School of Medicine, Hangzhou, Zhejiang 310009, China b c

a r t i c l e

i n f o

Article history: Received 8 June 2016 Revised 21 October 2016 Accepted 11 December 2016

Keywords: Salvianolic acid A Stroke Oxidative stress Neurovascular protection eNOS uncoupling

a b s t r a c t Background: Salvianolic acid A (SAA) is obtained from Chinese herb Salviae Miltiorrhizae Bunge (Labiatae), has been reported to have the protective effects against cardiovascular and neurovascular diseases. Hypothesis: The aim of present study was to investigate the relationship between the effectiveness of SAA against neurovascular injury and its effects on calpain activation and endothelial nitric oxide synthase (eNOS) uncoupling. Study design: SAA or vehicle was given to C57BL/6 male mice for seven days before the occlusion of middle cerebral artery (MCAO) for 60 min. Methods: High-resolution positron emission tomography scanner (micro-PET) was used for small animal imaging to examine glucose metabolism. Rota-rod time and neurological deficit scores were calculated after 24 h of reperfusion. The volume of infarction was determined by Nissl-staining. The calpain proteolytic activity and eNOS uncoupling were determined by western blot analysis. Results: SAA administration increased glucose metabolism and ameliorated neuronal damage after brain ischemia, paralleled with decreased neurological deficit and volume of infarction. In addition, SAA pretreatment inhibited eNOS uncoupling and calpain proteolytic activity. Furthermore, SAA inhibited peroxynitrite (ONOO− ) generation and upregulates AKT, FKHR and ERK phosphorylation. Conclusion: These findings strongly suggest that SAA elicits a neurovascular protective role through the inhibition of eNOS uncoupling and ONOO− formation. Moreover, SAA attenuates spectrin and calcineurin breakdown and therefore protects the brain against ischemic/reperfusion injury. © 2016 Elsevier GmbH. All rights reserved.

Introduction

Abbreviations: BH4, tetrahydrobiopterin; Ca2+ , calcium; CaM, calmodulin; CaN, calcineurin; CCA, common carotid artery; CT, computed tomography; ECA, external carotid artery; eNOS, endothelial nitric oxide synthase; 18F-FDG, [18F]-fluoro2-deoxy-d-glucose; ICA, internal carotid artery; MCI, Mitsubishi Chemical Industry; NF-kB, nuclear factor kappa B; NADPH, nicotinamide adenine dinucleotide phosphate; NIH, National Institutes of Health; NMDARs, N-methyl-d-aspartate receptors; NO, nitric oxide; O2 ·- , superoxide; ONOO- , peroxynitrite; PKB, protein kinase B; PET, positron emission tomography; RNS, reactive nitrogen species; ROS, reactive oxygen species; rtTPA, recombinant tissue plasminogen activator; SAA, Salvianolic acid A; SDS, sodium dodecyl sulfate; SUV, standardized uptake value; tMCAO, transit middle cerebral artery occlusion. ∗ Corresponding author. Present address: Institute of Pharmacology and Toxicology, Zhejiang University, 866 Yu hang-Tang Road, Hangzhou, 310058, China. E-mail addresses: [email protected] (F. Han), [email protected] (K. Zhao). 1 Both authors contributed equally to this work. http://dx.doi.org/10.1016/j.phymed.2016.12.004 0944-7113/© 2016 Elsevier GmbH. All rights reserved.

Stroke is a devastating disease, a massive socio-economic burden in all societies. Multiple deleterious pathological events during stroke caused death and disability (Lopez et al., 2006). Ischemic stroke subjected to deprivation of glucose and oxygen, reduction in blood flow linked to excitotoxicity, energy loss, and ionic imbalances, rapid apoptosis to endothelial cells (Alfieri et al., 2013; Manevich et al., 2001). The cerebral ischemia caused an excessive influx of calcium (Ca2+ ) by increased glutamate release and activation of N-methyl-d-aspartate receptors (NMDARs) (Du et al., 2010). Increased neuronal-free Ca2+ by activation of NMDA receptors and other Ca2+ channels participate in signal transduction pathways leading to cell survival or cell death (Tauskela and Morley, 2004). The Ca2+ induces the binding of calmodulin (CaM) to the enzyme nitric oxide synthases (NOS) and activated CaM is important for the regulation of endothelial nitric oxide synthase (eNOS) (Förstermann and Sessa, 2012). The cerebral ischemia caused loss

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of tetrahydrobiopterin (BH4) and altered eNOS function, with increased superoxide (O2 ·− ) production due to activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase induced reactive oxygen species (ROS) (Kukaya et al., 2003). The nitric oxide (NO) imbalance and to react with O2 ·− generates peroxynitrite (ONOO− ) which plays a key role in ischemic injury and triggers numerous pathological events via induction of nitrosative damage to lipids, DNA, and proteins (Han et al., 2011). The multiple pathophysiological cascades activated as a result of ischemia-like excitotoxicity, nitrosative stress, inflammation, and blood-brain barrier (BBB) leakage (Mitsios et al., 2006). The calpain belongs to proteases that play an important role in ischemic cell death. The overload of Ca2+ disrupt the membrane integrity following activation of Ca2+ -dependent calpains, in turn, triggers apoptosis via activation of caspases (Bano and Nicotera, 2007). Salvianolic acid A (SAA) is a caffeic acid derivative, obtained from Chinese herb Salviae miltiorrhizae Bunge (Labiatae), also known as Chinese sage, or Danshen in Chinese literature (Xu, 1990). Traditional Chinese medicine has been used for the treatment of a wide range of vascular diseases. SAA is one of the major active components of Danshen with strong antioxidant properties, a multi-target agent, used for the treatment of cardiovascular and cerebrovascular diseases (Du and Zhang, 1997; Fan et al., 2015; Ji et al., 20 0 0; Wang et al., 2012; Wang et al., 2009). SAA inhibits inflammatory/oxidative stress-mediated neuronal and vascular damage by impairing nuclear factor kappa B (NF-kB) signaling during ischemic brain injury (Chien et al., 2016). The distinct ischemic factors trigger multiple intracellular signaling that converges into several pathways (Chien et al., 2016). Based on the above description and multi-target agent, we investigated for the first time the effects of SAA on inhibition of calpain activity and eNOS uncoupling in mice subjected to transit middle cerebral artery occlusion (tMCAO). Materials and methods Experimental animals Male C57BL/6 mice, weighing 20–28 g, were obtained from the Zhejiang Medical Animal Centre (Hangzhou, China). Mice were housed under climate-controlled conditions with a 12 h light/dark cycle and provided with standard food and water. Animals were acclimated before initiating the experimental procedure. All experimental protocols and animal handling procedures were performed in accordance with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals and were approved by the Committee for Animal Experiments at the Zhejiang University in China. Extraction and isolation of SAA Slices of roots and rhizomes of Salvia miltiorrhiza (10 kg) was purchased from East China Pharmaceutical Group Co. Ltd. (Hangzhou, China). For chromatographic analysis column was prepared by d-101 macroporous resin (Chemical Plant of Nankai University, Tianjin), MCI gel (CHP20P, 75–150 μm, Mitsubishi Chemical Industries Ltd.), and C18 reversed-phase silica gel (20–45 μm, Fuji Silysia Chemical LTD). The material was extracted with water under reflux three times each for at least 2 h For chromatographic analysis the extracted solution was then subjected to macroporous resin d-101 column (ϕ 10 cm × 50 cm), eluted with MeOH/H2 O (10%, 30%, 50%, 70%, 95%) to afford five fractions (Fr-A∼Fr-E). Fr-C was found to have a high SAA content (Monitored by TLC), which was subsequently subjected to MCI gel column (ϕ 5 cm × 40 cm) eluted with MeOH/H2 O (15%, 50%, 75%) to obtain the crude product.

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Drug administration and transit middle cerebral artery occlusion mice model Animals were divided into four groups sham, vehicle and treatment groups. SAA (1 mg and 5 mg per kg body weight) or vehicle (saline) was given intragastric by i.p. to mice for seven days before the MCAO for 60 min. The tMCAO model which has resemblances to stroke in humans was used for the study (Braeuninger and Kleinschnitz, 2009; Carmichael, 2005) and the surgery was performed as previously described by our lab (Tao et al., 2014). Animal procedures were approved by the Committee on Animal Experiments at the Zhejiang University. Mice were weighed prior to surgery. Anesthesia was induced with 3% trichloroacetaldehyde hydrate. Rectal temperature was monitored throughout the surgery, and the body temperature was maintained at 37 °C ± 0.5 °C with a heating pad. Mice were subjected to tMCAO with the use of an operating microscope; the left common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) were surgically exposed. A 6–0 silicon-coated nylon surgical suture (Ethicon (XZW8305.032), Lot no. HJB388, Johnson and Johnson Intl., Livingston, Scotland) was inserted into the ICA through the CCA and gently advanced to the orifice of MCA. After 1 h of MCA occlusion, the suture was removed to restore blood flow, the neck incision was closed, and the mice were allowed to recover. Neurologic evaluation Neurological deficits, including neurological scores and rotarod test, were examined at 24 h after tMCAO. Neurological scores were determined using a previously described scoring system (Shioda et al., 2007), with slight modifications: 0 = Normal motor function, 1 = failure to extend left forepaw fully, 2 = Circling to the contralateral side but normal posture at rest, 3 = Leaning to the contralateral side at rest, 4 = No spontaneous motor activity. Rotarod test was performed to examine the motor coordination, started 3 days before the surgery five times every day (Tao et al., 2014). The persisted time (s) on the rotarod after ischemia was recorded; the data were expressed as the mean duration of five trials. Nissl staining to assess infarct volume The brain infarct was measured from the vehicle and SAAtreated groups. Twenty-four hours after reperfusion, mice were anesthetized and transcardially perfused with phosphate-buffered saline (PBS) followed by 10% paraformaldehyde as described previously (Tao et al., 2014). Sections of the brains 45 μm thick were cut with the aid of a cryostat (Leica VT10 0 0S). For Nissl-staining, slices were hydrated in 0.1% cresyl violet for 3–5 min. Then they were dehydrated in ethanol and cleaned with xylene. The slides were next examined via light microscopy; pictures were taken with a digital camera (Leica MZ95 and Leica application suite). The brain infarct area was evaluated from digital images of Nissl-stained brain sections using Image J software (NIH). Immunoblotting analysis Immunoblotting was carried out using penumbra brain region of mice after determination of protein concentrations by the Brad-Ford’s solution. The brain lysates containing equivalent amounts of protein were applied to 10%–12.5% acrylamide denaturing gels (SDS-polyacrylamide gel electrophoresis) (Wang et al., 2015a). Proteins were then transferred to an immobilon polyvinylidene difluoride membrane for 1 h at 50 V. Membranes were blocked in 20 mM Tris hCl (pH 7.4), 150 mM NaCl, and 0.1% Tween 20 (TBS-T) containing 5% fat-free milk powder for 1 h and immunodetected with antibodies to spectrin (1:30 0 0, monoclonal

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antibody; Millipore), calcineurin (1:30 0 0) (Fukunaga et al., 1986), anti-eNOS (1;30 0 0, rabbit polyclonal antibody Sigma, St Louis, MO, USA), Nitrotyrosine (1:10 0 0, monoclonal antibody; Millipore), AKT (1:10 0 0, polyclonal antibody; Cell Signaling Technology), PhosphoAKT (1:10 0 0, polyclonal antibody; Cell Signaling Technology), FKHR (1:20 0 0, polyclonal antibody; Cell Signaling Technology), Phospho-FKHR (1:30 0 0, polyclonal antibody; Cell Signaling Technology), ERK (1:30 0 0, polyclonal antibody; Santa Cruz), PhosphoERK (1:30 0 0, polyclonal antibody; Cell Signaling Technology) and β -actin (1:50 0 0, monoclonal antibody; Sigma-Aldrich). After incubation for 12 h, membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody. Dimer and monomer forms of eNOS were separated by the method described previously by our group and others (Han et al., 2011; Zhao et al., 2015). Immunoreactivity was visualized by enhanced chemiluminescence (Amersham Life Science). 18 F-FDG-

micro-PET /CT images and quantitative analysis

At 22–25 h after reperfusion, mice were anesthetized with 2% isofluorane in O2 gas and intravenously injected with approximately 0.2mCi of [18 F]-fluoro-2-deoxy-d-glucose (18 F-FDG). Following 1 h of radiotracer uptake under continuous anesthesia inhalation, mice were placed prone in the center of Siemens Inveon combined micro positron emission tomography (micro-PET)- computed tomography (CT) scanner (Siemens Preclinical Solution USA, Inc., Knoxville, TN, USA). MicroCT scans were performed with an X-ray tube voltage of 80 kV, a current of 500 μA, an exposure time of 150 ms, and 120 rotation steps. A 10 min PET static acquisition was performed and the images were reconstructed using OSEM (ordered set expectation maximization) algorithm for 3D PET reconstruction. Images were analyzed with the Inveon Research Workplace 4.1 (Siemens, Erlangen, Germany). The standardized uptake value (SUV, in g/ml) was obtained with the formula SUV = [(RTA/cm3 )/RID] × BW, where RTA is the measured radiotracer tissue activity (in mCi), RID is the radiotracer injected dose (in mCi), and BW is the mice body weight (in grams). A coronal section (or frame) in which the right side is most severe ischemic observed by eyes was chosen (actually the frame number is close in all the mice), and the right and left brain were symmetrically manually drawn as regions of the interest (ROIs). The ratio of right to left SUVs was used for semiquantitative analysis (Gao et al., 2010).

=

F − FDG activity in the ipsilateral cortex 18 F − FDG activity in the contralateral cortex 18

Statistical analysis t-tests were used to analyze the data when means between two groups were compared. For multigroup comparisons, statistical significance was determined using one-way ANOVA followed by a post hoc Tukey’s test or Dunnett’s comparison to control. All data are expressed as the mean ± SEM. A value of p < 0.05 was considered to be significant. Results

Fig. 1. HPLC chromatogram and chemical structure of SAA. (A) Column: Altima C18 (250 × 4.6 mm id, 5 μm); temperature: 30 ◦ C, mobile phase: A-acetonitrile, B- 0.67% formic acid, with a gradient of A:B 15:85 in 0 ∼ 10 min, 25:75 in 10 ∼ 25 min, 35:65 in 25 ∼ 40 min; flow rate:1 ml/min; wavelength: 286 nm. (B) Chemical structure of SAA.

compared with that of the vehicle group (Fig. 2A). In rotarod test latency to fall was increased in vehicle-treated animals which was significantly reduced (P < 0.05) by SAA pretreatment (Fig. 2B). Representative samples of Nissl-stained sections showed large infarct volume in the vehicle group (Fig. 2C). Infarct areas were significantly reduced (P < 0.01) in SAA pretreatment groups (1 mg/kg and 5 mg/kg) compared with vehicle treatment (Fig. 2D). Effects of SAA on brain glucose metabolism A metabolism reflected by 18 F-FDG-uptake was measured 24 h after stroke and representative 18 F-FDG-microPET images are presented in Fig. 3A. Mice were studied for glucose metabolism in sham; vehicle and SAA treated (5 mg/kg) groups. 18 F-FDG-uptake was reduced in the ischemic hemisphere; the reduced region inversely related to the infarction stained by crysl violet. There was no metabolic asymmetry between the hemispheres in sham mice but MCAO decreased the metabolic activity in the affected hemisphere which was protected with SAA pretreatment. The SUVs ratio of contralateral to the ipsilateral region was significant decreased (P < 0.01) in vehicle treated group compared to sham (Fig. 3B). SAA pretreatment preserve significantly (P < 0.05) contralateral to ipsilateral part SUVs ratio and showed the increased glucose metabolism (Fig. 3B).

SAA ameliorated neurological dysfunctions and reduced brain infarction at 24 h after reperfusion

SAA inhibited calpain activation in middle cerebral artery occlusion

The chromatographic analysis of the crude product on a C18 reversed-phase silica gel column eluted with 30% MeOH finally yielded 0.015% SAA with purity ≥ 97% shown in Fig. 1A and B. The neurological deficit score was determined in SAA or vehicle group. SAA pretreatment (1 mg/kg and 5 mg/kg) significantly decreased neurological deficit score (P < 0.01) 24 h after reperfusion

In ischemic neuronal death, one of the calpain substrates, spectrin is cleaved to 145 or 150 kDa, and the amount of cleaved spectrin reflects calpain activity (Meyer et al., 2014). In western blot analysis cleaved spectrin was increased significantly (P < 0.001) in the ischemic brain hemisphere compare to sham. The breakdown products of spectrin were significantly preserved (P < 0.001) by

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Fig. 2. The effects of SAA on neurological function and infarct size. (A and B) The neurological scores and rotarod test were examined, data are expressed as the average of the values observed in vehicle-treated animals (mean ± SEM, n = 6). ∗ p < 0.05; ∗ ∗ p < 0.01 versus vehicle-treated mice. (C) The infarct area was quantified 24 h later by Nissl-staining of brain sections. (D) The data are expressed as the percentage of the infarct area/total area of each brain section (mean ± SEM, n = 6). ∗ ∗ p < 0.01 versus vehicle-treated mice.

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Fig. 4. The SAA pretreatment protects neurovascular damage after brain ischemia. (A) The proteins from penumbra brain region of mice were immunoblotted with antibodies for neurovascular damage against spectrin and calcineurin. (B) The quantitative analysis is shown in the bar graph of SBP, spectrin breakdown products as the percentage values of sham-operated animals (mean ± SEM, n = 4). ∗ ∗ ∗ p < 0.001, versus sham mice; ### p < 0.001 versus vehicle treated mice. (C) The quantitative analysis is shown in the bar graph of CBP, calcineurin breakdown products as the percentage values of sham-operated animals (mean ± SEM, n = 4). ∗ ∗ ∗ p < 0.001, versus sham mice; ### p < 0.001 versus vehicle treated mice. Immunoblotting with an anti-β -actin antibody demonstrated equal protein loading in each lane.

Fig. 3. Representative 18 FDG PET or PET/CT images of sham, vehicle, and SAA 5 mg/kg group. (A) Images are shown for coronal (i), and transverse (ii) planes. PET image in coronal plane is used for semiquantitative analysis of SUV ischemia to contralateral hemispheres ratio, under the direction of skull contour in CT image. The arrow indicates the ischemic zone after reperfusion. (B) The quantitative analysis shown in the bar graph as contralateral to ipsilateral SUVs ratio (mean ± SEM, n = 8), ∗ ∗ p < 0.01 versus sham mice; # p < 0.05 versus vehicle-treated mice.

the pretreatment with SAA (1 mg/kg and 5 mg/kg) (Fig. 4A and B). The cerebral stroke enhanced calpain-mediated activation of calcineurin (CaN) in the striatum and prefrontal region (Meyer et al., 2014; Shioda et al., 2006). We also assessed calpain-mediated CaN breakdown. SAA pretreatment with (1 mg/kg and 5 mg/kg) significantly inhibited (P < 0.001) calpain induced breakdown of 48 kDa CaN subunit (Fig. 4A and C). SAA attenuates the ischemia-induced eNOS uncoupling and inhibited peroxynitrite formation It was demonstrated that the biochemically active dimeric form of eNOS appeared to be able to generate O2 ·− , whereas the monomeric form served as a marker for eNOS uncoupling (Zhao et al., 2015). The cerebral ischemia result in ONOO− and resultant oxidative/nitrative stress production caused dysfunction of microvascular endothelial cells by enhancing the generation of O2 ·−

Fig. 5. The eNOS dimer-monomer ratio in mice after tMCAO and peroxynitrite generation. (A) In ischemic brain decreased eNOS dimer-monomer ratio in vehicle treated group. (B) The quantitative analysis of dimer-monomer ratio are shown in the bar graph as the percentage values of sham-operated animals (mean ± SEM, n = 4). Immunoblotting with an anti-β -actin antibody demonstrated equal protein loading in each lane. ∗ ∗ ∗ p < 0.001 versus sham mice; # p < 0.05, ## p < 0.01 versus vehicle-treated mice. (C) SAA decreases mice brain 3-nitrotyrosine content 24 h after reperfusion. (D) The quantitative analysis of 3-nitrotyrosine are shown in the bar graph as the percentage values of sham-operated animals (mean ± SEM, n = 4). Immunoblotting with an anti-β -actin antibody demonstrated equal protein loading in each lane. ∗ ∗ p < 0.01 versus sham mice; # p < 0.05, ## p < 0.01 versus vehicle-treated mice.

(Tao et al., 2014). The western blot analysis of penumbra brain region indicates decreased dimer-to-monomer ratio after tMCAO (Fig. 5A). The SAA with (1 mg/kg and 5 mg/kg) pretreatment significantly restored this ratio as the dose increased (P < 0.01) and enhanced the eNOS coupling (Fig. 5A and B). Peroxynitrite is a powerful oxidant exhibiting a wide array of tissue-damaging effects, ONOO− production was increased 24 h after MCAO in the injured

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Fig. 6. SAA enhanced phosphorylation of AKT, ERK, and FKHR in ischemic brain. (A) The protein extracts from penumbra brain region of mice were processed for western blotting to detect total and phosphorylated levels of AKT, ERK, and FKHR. (B-D) Densitometry values were normalized to the average of all sham values (mean ± SEM, n = 4). ∗ ∗ p < 0.01, ∗ ∗ ∗ p < 0.001 versus sham mice; # p < 0.05; ## p < 0.01 versus vehicle-treated mice. Immunoblotting with an anti-β -actin antibody demonstrated equal protein loading in each lane.

ipsilateral cortex. In our western blot analysis, 3-nitrotyrosine levels were significantly increased in penumbra brain region in vehicle treated group compared to sham (P < 0.01) (Fig. 5C and D), the footprint of ONOO- production in vivo. The SAA pretreatment significantly reduced (P < 0.01) nitrotyrosine levels in ischemic ipsilateral (Fig. 5C and D). SAA prevents the dephosphorylation of AKT, ERK, and FKHR AKT is a serine/threonine kinase and plays an important role in the regulation of cell growth and survival in ischemic vascular diseases (Gao et al., 2015). In western blot analysis, we observed dephosphorylating of AKT, ERK, and FKHR in the vehicletreated group but no change in total protein levels (P < 0.001; Fig. 6A). The decreased phosphorylation of AKT may be attribution of increased O2 ·− production as a result of eNOS uncoupling. The SAA pretreatment (1 mg/kg and 5 mg/kg) significantly enhanced in a dose-dependent manner (P < 0.01) phosphorylation of AKT, ERK and FKHR compared to vehicle treated group (Fig. 6A–D). Discussion Nitrosative stress and down-stream signaling during ischemia underlays mechanism of ischemic cerebral cell death (Tao et al., 2014). The uncoupling of eNOS exacerbated oxidative stress and enhanced NO depletion resulted in reducing endothelial-dependent vasorelaxation of vessels (Bauersachs and Schäfer, 2005). In the present study, we demonstrated that SAA pretreatment significantly protects the eNOS dimer-monomer ratio, suggested that SAA could prevent eNOS uncoupling and decreased the production of ONOO− . The peroxynitrite might be severing as a marker of neurovascular damage and neurological impairment in ischemic stroke (Tao et al., 2014). Previous work from our laboratory has demonstrated that Ca2+ /CaM dependent nitrosative stress promotes the generation of ONOO− in the vascular endothelium and this effect is associated with aberrant eNOS production (Han et al., 2006). NO is a potent vasodilator and regulates regional blood flow and endothelium-derived NO serves as a protectant in the vascular wall (Iadecola, 1997; Samdani et al., 1997). The physiological concentration of vascular NO plays a prominent role in maintaining cerebral blood flow and preventing neuronal injury by increasing col-

lateral flow to the ischemic area (Dalkara et al., 1994; Iadecola, 1997; Samdani et al., 1997). eNOS maintains homeostasis in vasculature by inducing vasodilatation, antioxidant activity, regulate glucose uptake and insulin sensitivity (Srivastava et al., 2012). In the endothelium, AKT phosphorylates eNOS at S1177 and regulates the activity of uncoupled eNOS (Brown, 2010). AKT suppress eNOS oxidase activity under conditions of reduced nitrotyrosine content in the ischemic hemisphere (Karuppiah et al., 2011). Protein kinase B (PKB) also known as AKT, regulates a variety of cellular processes that include cell proliferation, differentiation, survival, and apoptosis (Datta et al., 1999; Datta et al., 1997). It has been reported that cerebral ischemia and reperfusion induce a reduction of phospho-AKT, phospho-ERK and phospho-FKHR without changing in the total level of protein (Cho et al., 2009; Zhu et al., 2016). Consistently, our results demonstrated that a decreased phosphorylation of AKT, ERK, and FKHR after brain ischemia in the vehicle group. Ischemia injury increased generation of NO and O2 ·− results in the formation of ONOO− (Wilcox and Pearlman, 2008). This is a short-lived highly reactive oxidant that attacks many proteins specifically, prostacyclin synthase and oxidizes BH4 to BH2, thereby causes uncoupling of eNOS and directing it to generate O2 ·− in place of NO. Indeed, nitrosative stress targets primarily to endothelial cells in cardiovascular and neurovascular diseases (Han et al., 2011; Tao et al., 2012). The increased ONOO− generation was associated with MMP activation, and trigger NF-kB-mediated proinflammatory signaling (Han et al., 2006; Matata and Galiñanes, 2002; Suofu et al., 2010). ONOO− -mediated tyrosine nitration has been detected during endothelial cell injury or BBB breakdown (Han et al., 2006; Han et al., 2011; Suofu et al., 2010). We found that SAA reduced eNOS uncoupling and generation of ONOO− in the ipsilateral region of the ischemic brain, which might be lead to dephosphorylation of AKT, ERK, and FKHR. The present study suggests that SAA plays a key role against brain injury through restore the phosphorylation of AKT, FKHR, and ERK phosphorylation. To prevent extensive damage after a stroke, it is most important to restore the blood supply during therapeutic window (Wang et al., 2015b). However, reperfusion induces a massive increase in ROS/RNS production, and consequently, causes further damage and neuronal death. Therefore, strategies to reduce the ROS/RNS production are of great therapeutic interest (Margaill et al., 2005). SAA is a polyphenolic metabolically unstable compound that undergoes rapid metabolism to generate active monophenolic metabolites has strong antioxidant properties (Liu et al., 1992; Zhang et al., 2011). In the present study, SAA significantly reduced infarct volumes and against the functional deficit. Calpain activation is increased after stroke and involved in degradation of Ca2+ -regulating proteins (Bevers and Neumar, 2008; Clinkinbeard et al., 2013). The massive Ca2+ influx into neuronal cells has been implicated in NMDA receptor-mediated neuronal death (Sun et al., 2008). Our results suggested that inhibition of spectrin and CaN breakdown might be associated with neurovascular protective effects of SAA against ischemic neuronal damage. This suggestion is consistent with results indicating that inhibitory effect on calpain activation and eNOS uncoupling is highly correlated with the improvement of glucose metabolism and neurological dysfunction. In addition, microglia and astrocytes induced neurotoxicity may also contribute to the pathological process of brain ischemia, which may trigger the production of a wide array of inflammatory cytokines. Therefore, further investigation is required to evaluate whether SAA elicits its neuronal protective effect through modulate inflammatory response during brain ischemia. Conclusion These findings demonstrated that SAA pretreatment is a promising strategy for protecting the neurovascular from damage

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during cerebral ischemia through improves the glucose metabolism. eNOS uncoupling-dependent nitrosative stress and calpain signaling play a critical role during cerebral ischemia which were prevented by SAA pretreatment. Conflict of interest The authors have no conflicts in interest to disclose. Author contributions Q.M., G.F.W., H.W., and G.W. performed the majority of the experiments; C.X.Z., H.Y.Y. and Z.R.L. participate in research design and perform data analysis; F.H. and K.Z. participate in research design and wrote the manuscript. Acknowledgments This work was supported in part by National Natural Science Foundations of China (81300991, 81302765). The Science and Technology Planning Project of Zhejiang Province (2014C33182). References Alfieri, A., Srivastava, S., Siow, R.C., Cash, D., Modo, M., Duchen, M.R., Fraser, P.A., Williams, S.C., Mann, G.E., 2013. Sulforaphane preconditioning of the Nrf2/HO-1 defense pathway protects the cerebral vasculature against blood–brain barrier disruption and neurological deficits in stroke. Free Radical Biol. Med. 65, 1012–1022. Bano, D., Nicotera, P., 2007. Ca2+ signals and neuronal death in brain ischemia. Stroke. 38, 674–676. Bauersachs, J., Schäfer, A., 2005. Tetrahydrobiopterin and eNOS dimer/monomer ratio–a clue to eNOS uncoupling in diabetes. Cardiovascular Res. 65, 768–769. Bevers, M.B., Neumar, R.W., 2008. Mechanistic role of calpains in postischemic neurodegeneration. J. Cerebral Blood Flow Metab. 28, 655–673. Braeuninger, S., Kleinschnitz, C., 2009. Rodent models of focal cerebral ischemia: procedural pitfalls and translational problems. Exp. Transl. Stroke Med. 1, 8. Brown, G.C., 2010. Nitric oxide and neuronal death. Nitric oxide. 23, 153–165. Carmichael, S.T., 2005. Rodent models of focal stroke: size, mechanism, and purpose. NeuroRx. 2, 396–409. Chien, M.-Y., Chuang, C.H., Chern, C.-M., Liou, K.-T., Liu, D.-Z., Hou, Y.-C., Shen, Y.-C., 2016. Salvianolic acid A alleviates ischemic brain injury through the inhibition of inflammation and apoptosis and the promotion of neurogenesis in mice. Free Radical Biol. Med. 99, 508–519. Cho, J.H., Sung, J.H., Cho, E.H., Won, C.-K., Lee, H.-J., Kim, M.-O., Koh, P.-O., 2009. Gingko biloba Extract (EGb 761) prevents ischemic brain injury by activation of the Akt signaling pathway. Am. J. Chin. Med. 37, 547–555. Clinkinbeard, T., Ghoshal, S., Craddock, S., Pettigrew, L.C., Guttmann, R.P., 2013. Calpain cleaves methionine aminopeptidase-2 in a rat model of ischemia/reperfusion. Brain Res. 1499, 129–135. Dalkara, T., Morikawa, E., Panahian, N., Moskowitz, M., 1994. Blood flow-dependent functional recovery in a rat model of focal cerebral ischemia. Am. J. Physiol. Heart Circ. Physiol. 267 H678 h83. Datta, S.R., Brunet, A., Greenberg, M.E., 1999. Cellular survival: a play in three Akts. Genes Dev. 13, 2905–2927. Datta, S.R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y., Greenberg, M.E., 1997. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell. 91, 231–241. Du, G., Zhang, J., 1997. Protective effects of salvianolic acid A against impairment of memory induced by cerebral ischemia-reperfusion in mice. Chin. Med. J. 110, 65–68. Du, W., Huang, J., Yao, H., Zhou, K., Duan, B., Wang, Y., 2010. Inhibition of TRPC6 degradation suppresses ischemic brain damage in rats. J. Clin. Invest. 120, 3480–3492. Fan, H.-Y., Yang, M.-Y., Qi, D., Zhang, Z.-K., Zhu, L., Shang-Guan, X.-X., Liu, K., Xu, H., Che, X., 2015. Salvianolic acid A as a multifunctional agent ameliorates doxorubicin-induced nephropathy in rats. Sci. Rep. 5. Förstermann, U., Sessa, W.C., 2012. Nitric oxide synthases: regulation and function. Eur. Heart J. 33, 829–837. Fukunaga, K., Sato, H., Takatsu, K., Tominaga, A., Miyamoto, E., 1986. Monoclonal antibody against a multifunctional calmodulin-dependent protein kinase from rat brain and the tissue distribution of the enzyme. Biomed. Res. 7, 405–413. Gao, F., Wang, S., Guo, Y., Wang, J., Lou, M., Wu, J., Ding, M., Tian, M., Zhang, H., 2010. Protective effects of repetitive transcranial magnetic stimulation in a rat model of transient cerebral ischaemia: a microPET study. Eur. J. Nucl. Med. Molecular Imaging. 37, 954–961.

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