Ruscogenin reduces cerebral ischemic injury via NF-κB-mediated inflammatory pathway in the mouse model of experimental stroke

European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Neuropharmacology and analgesia

Ruscogenin reduces cerebral ischemic injury via NF-κB-mediated inflammatory pathway in the mouse model of experimental stroke Teng Guan a,b, Qian Liu a, Yisong Qian c, Haopeng Yang a, Jiming Kong b, Junping Kou a,n, Boyang Yu a,n a State Key Laboratory of Natural Medicines, Department of Complex Prescription of TCM, China Pharmaceutical University, 639 Longmian Road, Nanjing 211198, PR China b Department of Human Anatomy and Cell Science, University of Manitoba, 745 Bannatyne ave, Winnipeg, MB, Canada R3E 0J9 c National Key Laboratory of Crop Genetics and Germplasm Enhancement, National Center for Soybean Improvement, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 29 January 2013 Received in revised form 10 July 2013 Accepted 16 July 2013

Transient cerebral ischemia initiates a complex series of inflammatory events, which has been associated with an increase in behavioral deficits and secondary brain damage. Ruscogenin is a major steroid sapogenin in the traditional Chinese herb Ophiopogon japonicus that have multiple bioactivities. Recent studies have demonstrated that Ruscogenin is involved in down-regulation of intercellular adhesion molecule-1 (ICAM-1) and nuclear factor-κB (NF-κB) activation in anti-inflammatory pathways. We hypothesized that Ruscogenin protects against brain ischemia by inhibiting NF-κB-mediated inflammatory pathway. To test this hypothesis, adult male mice (C57BL/6 strain) were pretreated with Ruscogenin and then subjected to transient middle cerebral artery occlusion (MCAO)/reperfusion. After 1 h MCAO and 24 h reperfusion, neurological deficit, infarct sizes, and brain water content were measured. Ruscogenin markedly decreased the infarct size, improved neurological deficits and reduced brain water content after MCAO. The activation of NF-κB Signaling pathway was observed after 1 h of ischemia and 1 h of reperfusion, and Ruscogenin significantly inhibited NF-κB p65 expression, phosphorylation and translocation from cytosol to nucleus at this time point in a dose-dependent manner. NF-κB DNA binding activity, and the expression of NF-κB target genes, including ICAM-1, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), were also suppressed by Ruscogenin pretreatment after 1 h MCAO and 24 h reperfusion. The results indicated that Ruscogenin protected the brain against ischemic damage caused by MCAO, and this effect may be through downregulation of NF-κB-mediated inflammatory responses. & 2013 Elsevier B.V. All rights reserved.

Keywords: Ruscogenin Stroke Middle cerebral artery occlusion NF-κB Inflammation

1. Introduction Stroke is one of the major public health concerns worldwide with high mortality and morbidity both in developing and developed countries. Mechanisms involved in cerebral ischemic damage are related to inflammation, excitotoxicity, mitochondrial dysfunction and oxidative stress (Lakhan et al., 2009; Martin et al., 1998; Pandya et al., 2011; Saito et al., 2005). Unfortunately, this knowledge has not yet translated into new clinical therapies. To date, tissue-plasminogen activator (t-PA) is the only FDA-approved therapy for acute ischemic stroke. However, t-PA therapy may exacerbate the cerebral injury through a series of inflammatory

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Corresponding authors. Tel./fax: +86 25 86185158. E-mail addresses: [email protected] (J. Kou), [email protected] (B. Yu).

cascades. Development of neuroprotective agents that are clinically effective remains a high priority. Researches have been focused on the effect and potential benefits of traditional Chinese medicine in stroke recently (Gong and Sucher, 2002; Kim, 2005). Herbal products show antioxidant and anti-inflammatory properties, making them an attractive option to be investigated for stroke prevention. Ruscogenin (Fig. 1), is a major effective steroidal sapogenin found in the roots of Ophiopogon japonicus (L.f) Ker-Gawl., a Chinese herb that has been applied to treat acute and chronic inflammatory and cardiovascular diseases for years (Kou et al., 2005a). It has also been widely used to treat chronic venous insufficiency and vasculitis in Europe for decades due to its ability to decrease capillary permeability and anti-elastase activity (Bouskela et al., 1994; Facino et al., 1995). Our previous studies have demonstrated that Ruscogenin had significant anti-inflammatory and anti-thrombotic activities, which might be related to the inhibition of intercellular adhesion

0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.07.036

Please cite this article as: Guan, T., et al., Ruscogenin reduces cerebral ischemic injury via NF-κB-mediated inflammatory pathway in the mouse model of experimental stroke. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.07.036i

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1-h-ischemia and 3-h-reperfusion group treated with 10 mg/kgRuscogenin. The vehicle-treated groups received equal volumes of 0.5% sodium carboxymethyl cellulose. 2.3. Focal cerebral ischemia

2. Material and methods

C57BL/6 Mice were anesthetized with chloral hydrate (400 mg/kg i.p.), supplemented as required to maintain optimal anesthesia throughout the experiment. Body temperature was kept at 37 1C by means of a thermostatically controlled heating blanket until the animals had recovered from surgery. The blood gas conditions were kept at constant levels throughout the surgery (PO2, 120 710 mm Hg; PCO2, 3573 mm Hg). Focal cerebral ischemia was induced by intraluminal middle cerebral artery occlusion (MCAO), as described previously (Li et al., 2013). Local blood flow was monitored using BLF21C Laser Doppler Flow-meter with LLM1043 fiber flow-probe (Transonic System Inc, New York). Ischemia was induced by introducing a 7-0 surgical monofilament nylon suture (Ethilon, Johnson & Johnson, Somerville, NJ, USA), pre-coated with silicone mixed with a hardener (Heraeus Kulzer, Germany) via the external carotid artery into the internal carotid artery to block the origin of the MCA. Animals that did not show a cerebral blood flow reduction of at least 70% were excluded from the experimental group. The filament was left in place for 60 min and then withdrawn. The sham-operated animals were treated identically except that the middle cerebral artery was not occluded after the neck incision.

2.1. Materials

2.4. Behavioral analysis

Unless stated otherwise, all compounds were purchased from the Sigma-Aldrich Company Ltd. (St. Louis, USA). Ruscogenin was isolated from the tubers of Ophiopogon japonicus by successive chromatographic steps and the purity analyzed by high performance liquid chromatography–evaporative light scattering detection (HPLC–ELSD) was 98.6%.

Behavioral assessment was performed 24 h after reperfusion. Neurological deficits of the experimental animals were graded on an 18-point scale as previously described (Garcia et al., 1995). The measurement of neurological deficits consisted of the following tests: spontaneous activity, symmetry of movements, symmetry of forelimbs, climbing, reaction to touch, and response to vibrissae touch. All six individual tests were graded as score 3, 2, 1 and 0. Final scoring was obtained by adding the scores recorded in each individual test. A maximum score of 18 was observed in healthy animals. In an open field test, mice were placed in the center of 100  100 cm2 arena with 45-cm-high walls and allowed to explore. The parameters measured are distance traveled and time spent in the center and peripheral zones. Anxious animals spend less time in the center zone compared with less anxious animals. Observations were recorded for 5 min.

Fig. 1. Chemical structure of Ruscogenin.

molecule-1 (ICAM-1) and nuclear factor-κB (NF-κB) pathways (Huang et al., 2008; Kou et al., 2005b; Sun et al., 2012). As in vivo and in vitro studies showed that inhibition of NF-κB pathway has beneficial effects on ischemic stroke (Jiang et al., 2011; Wang et al., 2009); regulation of NF-κB by Ruscogenin might be a promising therapeutic strategy for cerebral ischemia. In our preliminary screening, Ruscogenin significantly alleviated cerebral edema, oxidative damage and energy metabolism disorder in a mice bilateral carotid artery ligation and reperfusion model. The present study was conducted to further examine whether Ruscogenin is neuroprotective in ischemic stroke, and, if so, to assess whether these neuroprotective effects are associated with the inhibition of NF-κB and cerebral inflammation.

2.2. Animal and treatment All experimental protocols were approved by Institutional Animal Care and Use Committee of China Pharmaceutical University. C57BL/6 mice were provided by Model Animal Research Centre of Yangzhou University (Yangzhou, Jiangsu, PR China). Mice were housed in a temperature-controlled environment (24 7 2 1C) with a 12-h-light-dark cycle and allowed free access to food and water. All efforts were made to minimize animal suffering and reduce the number of animals used. In protocol 1, three different doses of Ruscogenin were administrated intragastrically 1 h before occlusion. Mice were then subjected to 1 h MCAO. At 1 h of reperfusion, mice were sacrificed for NF-κB p65 expression, phosphorylation, and translocation. At 24 h of reperfusion, infarct volume, neurological scores, brain water content, NF-κB p65 DNA binding activity, and the expression of NF-κB target genes were investigated. Mice were randomly divided into five groups (n¼ 6 for each group): (1) sham-operated group, (2) MCAO group treated with vehicle, (3–5) MCAO group treated with Ruscogenin at doses of 2.5, 5 and 10 mg/kg. In protocol 2, 10 mg/kg Ruscogenin was administrated intragastrically 1 h before occlusion and was used to observe the time response of NF-κB activation after ischemia. Mice were divided into seven groups (n ¼6 for each group): (1) sham-operated group, (2) 1-h-ischemia group treated with vehicle, (3) 1-h-ischemia group treated with 10 mg/kg-Ruscogenin, (4) 1-h-ischemia and 1-h-reperfusion group treated with vehicle, (5) 1-h-ischemia and 1-h-reperfusion group treated with 10 mg/kg-Ruscogenin, (6) 1-hischemia and 3-h-reperfusion group treated with vehicle, and (7)

2.5. Infarct volume quantification and brain water content determination Brains were quickly removed at 24 h post-ischemia and the wet weight was measured. The 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining was performed to evaluate tissue viability and measure the infarct size. The infarct area was measured in NIH Image J software (Version 1.42; National Institutes of Health, Bethesda, Md). The infarct areas on each slice were summed and multiplied by slice thickness to give the infarct volume. Infarct volume was expressed as a percentage of infarction per ipsilateral hemisphere. Brain samples of animals were dried in an oven at 100 1C for 24 h to obtain the dry weight. Brain water content was calculated with the following equation: (wet weight  dry weight)/ wet weight  100%. 2.6. Western blot analysis Sham and ischemic mice were sacrificed 0 h (immediately after 1 h MCAO), 1 h and 3 h after reperfusion, according to protocol 2.

Please cite this article as: Guan, T., et al., Ruscogenin reduces cerebral ischemic injury via NF-κB-mediated inflammatory pathway in the mouse model of experimental stroke. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.07.036i

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Dose response of Ruscogenin on NF-κB activation, including p65 expression, phosphorylation and translocation to the nucleus, were examined in another set of animals according to protocol 1. Western blots were carried out as previously described (Guan et al., 2011). The protein expression of NF-κB targeted genes, including ICAM-1, iNOS and COX-2 was detected using protocol 1. Ipsilateral cortex tissue was rapidly dissected and the cytosolic and nuclear fractions were extracted respectively. Proteins were extracted and separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinyldenedifluoride membranes. The membranes were blocked with 3% bovine serum albumin (BSA) for 1 h and were incubated with primary antibody overnight. Blots were then incubated with secondary antibodies and developed with the ECL detection system. The immunoreactive bands were visualized by autoradiography and the density of the bands was evaluated densitometrically using the ChemiDoc XRS system with Quantity One software (Bio-Rad, Richmond, CA, USA).

2.7. NF-κB binding assay NF-κB p65 DNA binding activities were assessed using the nuclear extracts mentioned in the above method, using the TransAM transcription factor assay kit (Active Motif) according to the manufacturer′s instructions. Five micrograms of brain nuclear extracts was added to 96-well plates coated with an oligonucleotide containing consensus sequences. A primary antibody that specifically recognizes activated NF-κB was added to the wells, followed by a secondary horseradish peroxidase-conjugated antibody. Developing solution (tetramethylbenzidine) was added and the colorimetric reaction was halted by a adding stop solution (0.5 mol/L H2SO4). Absorbance was measured at 450 nm with a reference wavelength of 655 nm on a spectrophotometer. The NFκB wild-type and mutated consensus oligonucleotides were used in order to monitor the specificity of the assay.

2.8. Real-time PCR Animals were decapitated and the ipsilateral cortex tissue was rapidly dissected for Real-time PCR experiments, according to protocol 1. Total RNA was isolated from the cortex tissue samples using the RNeasy kit (Qiagen, Hilden, Germany). Complementary DNA synthesis was performed using a PrimeScript 1st strand cDNA synthesis kit (Takara, Shiga, Japan). Real-time PCR was performed using the ABI 7500 sequence detection system (Applied Biosystems, Foster City, CA) with a reaction mixture that consisted of SYBR Green 2  PCR Master Mix (Applied Biosystems), cDNA template, and forward and reverse primers. Primer sequences were as follows: 5′-CCTGATGGGCAGTCAACAGCTA-3′ and 5′-ACAG CTGGCTCCCGTTTCA-3′ (ICAM-1), 5′-TGACCCCCAAGGCTCAAATAT3′ and 5′-CCCAGGTCCTCGCTTATGATC-3′ (COX-2), 5′-CAGCTGGGC TGTACAAACCTT-3′ and 5′-CATTGGAAGTGAAGCGTTTGG-3′ (iNOS), 5′-AAGCCTCGTGCTGTCGGACC-3′ and 5′-TGAGGCCCAAGGCCACAG GT-3′(IL-1β), 5′-GCTGGTGACAACCACGGCCT-3′ and 5′-AGCCTCCGA CTTGTGAAGTGGT-3′ (IL-6) 5′-CAAGGGACAAGGCTGCCCCG-3′ and 5′-GCAGGGGCTCTTGACGGCAG-3′ (TNF-α), and 5′-GCCAAGGCTGT GGGCAAGGT-3′ and 5′-TCTCCAGGCGGCACGTCAGA-3′ (glyceraldehyde-3-phosphate dehydrogenase, GAPDH, for an endogenous control). The PCR protocol consisted of 40 cycles of the following profile: 95 1C for 15 s (denaturation step) and 60 1C for 1 min (to allow extension and amplification of the target sequence). Data were analyzed using the ABI 7500 sequence detection system software. The amount of ICAM-1, COX-2, iNOS, IL-1β, IL-6 and TNF-α mRNA was normalized to GAPDH expression using the CT method.

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2.9. Enzyme-linked immunosorbent assay (ELISA) Mice in each group according to protocol 1 were sacrificed and the brain tissue homogenates were obtained from the cortexes of ischemic hemisphere. The concentration of TNF-α and IL-1β were measured using specific ELISA kits according to the manufacturers' instructions (Boster, Wuhan, China). Plates were read in an ELISA reader (Biotek Instruments, USA) at 450 nm with a correction wavelength of 570 nm. The level of these cytokines was obtained from a standard plot made using the standard provided by the kit. The total protein levels of each sample were quantified by Lowry′s method. 2.10. Statistical analyses Results were expressed as mean 7S.D. of the indicated number of experiments. Statistical analysis was performed using the oneway ANOVA analysis followed by a Tukey post-hoc test. Behavioral test between groups was analyzed by nonparametric Kruskal– Wallis test. A difference with P o0.05 was considered statistically significant.

3. Results 3.1. Ruscogenin did not affect physiologic parameters in MCAO Key physiological parameters were monitored in vehicle- or Ruscogenin-treated mice before and after induction of ischemia. No statistically significant differences were observed with respect to body weight, temperature, pO2, pCO2 or pH among groups. pO2 was 143.7 710.86 mmHg in vehicle treated animals vs 152.1 7 14.27 mmHg in Ruscogenin-treated animals, pCO2 as 40.5 7 8.3 mmHg in vehicle treated animals vs 43.6 76.7 mmHg in Ruscogenin-treated animals and pH was 7.45 70.05 in vehicle treated animals vs 7.43 70.05 in Ruscogenin-treated animals. There were no differences between the physiological parameters among groups before or at the end of protocol. The regional cerebral blood flow was reduced to approximately 20% of the baseline value immediately after MCAO and recovered nearly to baseline with reperfusion, and Ruscogenin administration had no effect on cerebrovascular blood flow when examined either immediately before or after MCAO. 3.2. Ruscogenin reduced infarct sizes, alleviated cerebral edema and improved neurological deficits following MCAO To determine whether or not Ruscogenin is neuroprotective, vehicle or various doses of Ruscogenin were administered 1 h before MCAO. Infarct volume, neurological deficit and brain water content were evaluated after 1 h of ischemia and 24 h of reperfusion. Observation of TTC-stained sections clearly showed the infarcted area, appearing as a section of unstained tissue (Fig. 2A). In the ischemic hemispheres, infarction was located mainly in the frontoparietal cortex and striatum. Ischemic mice that received 5 mg/kg or 10 mg/kg Ruscogenin had much smaller infarct size. Their infarct size decreased by 37.4% and 63% respectively (F¼15.22, Po0.05, Po0.01, Fig. 2B) compared with vehicle. Similar reductions were observed 72 h post-insult (data not shown). Effect of Ruscogenin on neuro-function was also examined. All animals in each group had normal neurological scores of 18 before experiment. Animals showed normal spontaneous activity. All four limbs extended symmetrically and walked symmetrically. Mice climbed easily and gripped tightly to the wire. They reacted by turning head and were equally startled by the stimulus on both sides. However, 24 h after MCAO, vehicle-treated mice exhibited

Please cite this article as: Guan, T., et al., Ruscogenin reduces cerebral ischemic injury via NF-κB-mediated inflammatory pathway in the mouse model of experimental stroke. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.07.036i

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Fig. 2. Ruscogenin (RUS) reduced infarct sizes, alleviated cerebral edema and improved neurological deficits following MCAO. (A) Representative TTC-stained brain sections show smaller infarcts among mice treated with Ruscogenin compared with vehicle. (B) Quantitative analysis of infarct volume. Ruscogenin also (C) improves neurological outcome and (D) alleviates cerebral edema following MCAO. Exploration, anxiety and locomotion were assessed using the open-field test. (E) Open field test indicates the average distance covered by the mice in each group. (F) Ruscogenin-treated mice spent significant more time in the center of the arena. Data are given as mean 7S.D. (n¼6).

significant neurological deficit. They hesitated to move and did not approach all sides. The limbs on left side extended less or more slowly than those on the right, and forepaw walking, climbing and reaction to stimulus on the left side were impaired. Their neurological evaluation scores decreased significantly (P o0.01, Fig. 2C). In agreement with the infarct volume measurement, mice treated with 5 mg/kg or 10 mg/kg Ruscogenin had improved neurological performances compared with the vehicle-treated group (P o0.05, P o0.01, Fig. 2C). As seen in Fig. 2D, mice treated with 5 mg/kg or 10 mg/kg Ruscogenin, the brain water content was significantly reduced (78.7271.1% and 78.371.18%, F¼ 3.69, Fig. 2D) compared with the vehicle group (80.4271.32%). No significant decrease was observed in the mice treated with 2.5 mg/kg Ruscogenin (79.4971.35%). In the open field test, 10 mg/kg Ruscogenin did not have a significant effect on the total travel distance/speed, showing that locomotor activity was equally reduced in vehicle and Ruscogenin treated mice. Another parameter that we determined in the openfield test was center/periphery ratio. The percentage of time spend in the center vs periphery area has been used as a measurement of anxiety level in rodents (Crawley, 1999). Before MCAO conduction, vehicle-treated mice and Ruscogenin-treated mice spent similar

amount of time in the center or periphery area. When tested after 1 h MCAO with 3 days reperfusion, vehicle-treated mice spent significantly less time in the center area and longer time in the periphery area, reflecting higher anxiety levels than Ruscogenintreated mice when tested 3 days after MCAO (F¼22.75, P o0.01, Fig. 2F). 3.3. Ruscogenin inhibits NF-κB activation in ischemic brain To elucidate the mechanism underlying the beneficial effect of Ruscogenin on cerebral ischemia, we determined the effect of Ruscogenin on NF-κB activation in ischemic animals. 10 mg/kg Ruscogenin showed the best protective effects in ischemic mice. Therefore, 10 mg/kg Ruscogenin was used for the time response study. The expression and phosphorylation of NF-κB p65 subunit were assessed by Western blot at different time points to confirm NF-κB activation according to protocol 2. Ischemic insult did not alter p65 expression or phosphorylation immediately after 1 h of ischemia (i.e. 1 h/ 0 h) or after 3 h of reperfusion (i.e. 1 h/3 h), but resulted in a statistically significant increase in p65 expression (1.86 folds to sham, F¼6.28, P o0.01, Fig. 3B) and phosphorylation (2.3 folds to sham, F¼18.95, Po 0.01, Fig. 3C) after 1 h of ischemia

Please cite this article as: Guan, T., et al., Ruscogenin reduces cerebral ischemic injury via NF-κB-mediated inflammatory pathway in the mouse model of experimental stroke. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.07.036i

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Fig. 3. Time response of Ruscogenin (RUS, 10 mg/kg) on NF-κB activation in ischemia/reperfusion mice. (A) Immunoblots of p65, phospho-p65 (P-p65) and GAPDH. (B) Quantitative analysis of p65 expression. (C) Quantitative analysis of P-p65 expression. The figure showed representative gels from one set of experiments. Data are given as mean 7 S.D. (n¼ 6) of three independent experiments, normalized with the corresponding GAPDH.

and 1 h of reperfusion (i.e. 1 h/1 h). In the Ruscogenin-treated group, the elevation of p65 expression after 1 h of reperfusion was strongly reversed (46.1% of vehicle, P o0.01, Fig. 3B), and the phosphorylation of p65 reversed to normal levels at the corresponding time (26.6% of vehicle, P o0.01, Fig. 3C). NF-κB activation achieved the highest level after 1 h of ischemia and 1 h of reperfusion. Therefore, we tested the dose response of Ruscogenin under this experimental condition. Ruscogenin treatment dose-dependently inhibited p65 expression (F ¼4.61, P o0.05, 5 mg/kg group vs vehicle and P o0.01, 10 mg/kg vs vehicle; Fig. 4B). Phospho-p65 was only influenced by 10 mg/kg of Ruscogenin (F¼ 2.93, P o0.01 vs vehicle, Fig. 4C). To further examine whether Ruscogenin regulates p65 translocation to the nucleus in MCAO-induced ischemia, the nucleus and cytoplasm fractions were extracted. In sham samples, immunoblotting for p65 appeared faint and was predominately located in the cytoplasm. We observed a 4.7-fold increase in nucleus cytoplasm ratio (F ¼8.65, P o0.01 vs sham, Fig. 5), indicating the remarkable translocation of p65 in the ischemic cortex. Ruscogenin profoundly prevented p65 nuclear translocation (P o0.01, 5 mg/kg group vs vehicle and P o0.01, 10 mg/kg vs vehicle; Fig. 5). These data suggested that Ruscogenin could inhibit NF-κB activation/translocation induced by cerebral ischemia/ reperfusion. We also investigated the effect of 10 mg/kg Ruscogenin on NFκB p65 subunit DNA binding capacity. The data in Fig. 6 showed that the binding activity of NF-κB p65 subunit in the cerebral cortex of ipsilateral hemisphere was found to be increased 24 h after MCAO. Treatment with Ruscogenin led to statistically significant reduction in the DNA binding capacity (F¼152.55, P o0.01 vs vehicle; Fig. 6). 3.4. Ruscogenin suppressed NF-κB-dependent gene expression of proinflammatory cytokines NF-κB is a well-known regulator for the expression of various pro-inflammatory factors. Therefore, the mRNA expression of cytokines (TNF-α, IL-1β and IL-6), as well as ICAM-1, iNOS and

COX-2, in the ischemic cortex of mice 24 h after MCAO were tested. Real-time PCR showed that after 24 h of reperfusion, all the values of inflammatory factors, except IL-6, elevated significantly in the MCAO groups. However, the mRNA levels of IL-1β (F¼ 6.50, Po 0.01 vs vehicle, Fig. 7D) and TNF-α (F¼3.98, P o0.05 vs vehicle, Fig. 7F) dropped nearly to the sham baseline in 10 mg/kg Ruscogenin-treated mice. Meanwhile, Ruscogenin also significantly decreased the mRNA levels of ICAM-1 (F¼3.88, P o0.05 5 mg/kg group vs vehicle, Po 0.01 10 mg/kg group vs vehicle, Fig. 7A), COX-2 (F¼ 10.33, Po 0.05 5 mg/kg group vs vehicle, Po 0.01 10 mg/kg group vs vehicle, Fig. 7B) and iNOS (F ¼25.14, Po 0.05 5 mg/kg group vs vehicle, P o0.01 10 mg/kg group vs vehicle, Fig. 7C). IL-6 mRNA expression increased slightly after 24 h of reperfusion, and Ruscogenin could inhibit its production to some extent.

3.5. Ruscogenin inhibits NF-κB-dependent protein expression induced by MCAO Similar results were observed in protein assay of these proinflammatory mediators. The protein levels of ICAM-1, COX-2 and iNOS were also increased after MCAO (2.4-, 13.8- and 2.8-fold increase compared with control respectively). In the presence of Ruscogenin, iNOS was reduced by 34.0% in 5 mg/kg group and 49.4% in 10 mg/kg group (F¼13.86, P o0.05 and Po0.01 vs vehicle, respectively, Fig. 8B); ICAM-1 was attenuated by 43.3% in 5 mg/kg group and 52.2% in 10 mg/kg group (F¼9.33, P o0.05 and P o0.01 vs vehicle, respectively, Fig. 8C); COX-2 was decreased by 43.4% in 5 mg/kg group and 58.7% in 10 mg/kg group (F¼15.71, Po 0.05 and P o0.01 vs vehicle, respectively, Fig. 8D). After 1 h MCAO and 24-h reperfusion both IL-1β and TNF-α expression levels were elevated. Notably, ischemia/reperfusion-induced increase in IL-1β (F¼8.55, P o0.05, Fig. 8E) and TNF-α was more prominent in vehicle-treated mice than in Ruscogenin-treated mice (F¼65.53, P o0.01, Fig. 8F). The results indicate that the protective effect of Ruscogenin is attributed to effective suppression of ischemia-induced pro-inflammatory factors.

Please cite this article as: Guan, T., et al., Ruscogenin reduces cerebral ischemic injury via NF-κB-mediated inflammatory pathway in the mouse model of experimental stroke. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.07.036i

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Fig. 4. Dose response effects of Ruscogenin (RUS) on NF-κB activation in mice after MCAO. (A) Immunoblots of p65, phospho-p65 (P-p65) and GAPDH. (B) Quantitative analysis of p65 expression. (C) Quantitative analysis of P-p65 expression. The figure showed representative gels from one set of experiments. Data are given as mean 7 S.D. (n¼6) of three independent experiments, normalized with the corresponding GAPDH.

Fig. 6. Treatment with Ruscogenin (RUS, 10 mg/kg) led to reduction in NF-κB p65 DNA binding capacity. NF-κB p65 DNA binding capacity was assessed using a TransAM transcription factor assay kit. Peri-infarct cortical regions were harvested and prepared for binding activity assay. Tissue samples from sham-operated mice were taken from the same regions and served as controls. 24 h after ischemia, binding activity increased and this increase was significantly suppressed by Ruscogenin treatment. Data are given as mean 7S.D. (n¼6) of three independent experiments.

Fig. 5. Effects of Ruscogenin (RUS) on NF-κB translocation in mice after MCAO. (A) Immunoblots of p65 in nucleus and cytoplasm fractions. (B) Quantitative analysis of nucleus/cytoplasm ratio of p65. The figure showed representative gels from one set of experiments. Data are given as mean 7 S.D. (n ¼6) of three independent experiments, normalized with the corresponding GAPDH.

4. Discussion This present study provides the first evidence that Ruscogenin exerts neuroprotection in an animal model of focal stroke. It decreases the infarct volume, improves neurological deficit and reduces the brain edema following ischemia/reperfusion. We

propose that inhibiting NF-κB-dependent proinflammatory responses is a potential mechanism for Ruscogenin intervention. Ruscogenin′s inhibitory influence on NF-κB stops the postischemic cytokine production in sub-acute phase and therefore shows a promising neuroprotection. We have reported that Plant-derived Ruscogenin could suppress zymosan A-evoked peritoneal total leukocyte migration in mice (Huang et al., 2008), inhibit TNF-α-induced over-expression of ICAM-1 and suppress NF-κB activation in human umbilical vein endothelial cells (Song et al., 2010). We performed our preliminary screening using a mice bilateral carotid artery ligation and reperfusion model. Results showed that Ruscogenin significantly alleviated cerebral edema, oxidative damage and energy metabolism disorder. In the present study, Ruscogenin attenuates the brain

Please cite this article as: Guan, T., et al., Ruscogenin reduces cerebral ischemic injury via NF-κB-mediated inflammatory pathway in the mouse model of experimental stroke. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.07.036i

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Fig. 7. Effects of Ruscogenin (RUS) on the mRNA expression of pro-inflammatory factors. Quantitative analysis of mRNA expression of (A) ICAM-1, (B) COX-2, (C) iNOS, (D) IL1β, (E) IL-6 and (F) TNF-α was determined by real-time PCR. Data are given as mean 7 S.D. (n ¼6) of three independent experiments.

Fig. 8. Effects of Ruscogenin (RUS) on the protein expression of pro-inflammatory factors. (A) Immunoblots of ICAM-1, iNOS, COX-2 and GAPDH. Quantitative analysis of (B) iNOS, (C)ICAM-1, (D) COX-2, (E) TNF-α and (F) IL-1β protein expression. The figure showed representative gels from one set of experiments. Data are given as mean 7 S.D. (n¼ 6) of three independent experiments, normalized with the corresponding GAPDH.

Please cite this article as: Guan, T., et al., Ruscogenin reduces cerebral ischemic injury via NF-κB-mediated inflammatory pathway in the mouse model of experimental stroke. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.07.036i

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injury, decreases the infarct volume, improves neurological deficit and reduces the brain edema following ischemia/reperfusion. Potential mechanisms of Ruscogenin′s beneficial effects might be related to its anti-inflammatory property and NF-κB inhibition. The NF-κB pathway is considered to play crucial roles in a variety of CNS injury such as spinal cord injury, traumatic brain injury, global ischemia, and transient focal ischemia (Williams et al., 2006). Previous studies have indicated that activation of NF-κB was sustained to at least 72 h in the ipsilateral cortex and striatum of the injured rat brain (Berti et al., 2002). Similarly, other studies have reported the activation, nuclear translocation, and DNAbinding of NF-κB at 2 h post-injury in rat MCAO model (Nurmi et al., 2004; Stephenson et al., 2000). Blockade of NF-κB pathway has shown therapeutic efficacy. For example, the novel IKK inhibitor MLN1145 exerts neuroprotective effects, which has at least a 2 h therapeutic window for treatment of MCAO rats (Williams et al., 2006). In our experiment, the expression and translocation of NF-κB p65 in the ischemic cortex peaked at 1 h after reperfusion and Ruscogenin treatment attenuated the peak rise of NF-κB, as well as thereafter DNA binding activity, reflecting the effective blockage of MCAO-induced NF-κB activation. Inhibition of NF-κB activity is neuroprotective, since the activation of multiple inflammatory genes could be reduced or prevented (Verma, 2004). NF-κB controls the expression of genes encoding the pro-inflammatory cytokines, chemokines, adhesion molecules, inducible enzymes and immune receptors (Karin et al., 2002). Cytokines formed after ischemia stimulates the expression of adhesion molecules on endothelial cells and leukocytes, leading to leukocyte adherence and extravasation into brain parenchyma. Further, two inducible isoform of enzymes iNOS and COX-2 are upregulated in inflammatory stimuli and result in the further production of large amounts of nitric oxide and prostaglandins. All of these play critical roles in controlling most inflammatory processes. The enzymes and inflammatory molecules have been implicated as causative factors. We demonstrated that Ruscogenin administration prior to ischemia inhibits the rise in mRNA level of IL-1β and TNF-α in the brain. Up-regulation of iNOS and COX-2 was not observed in Ruscogenin-treated mice. Our results showed that NF-κB occupies a prominent role in the inflammatory cascade. Ruscogenin is a therapeutic candidate for inflammation-related ischemic disease. We summarized the in vivo experimental results highlight the possible role of Ruscogenin as a neuroprotective natural product with a particular focus on stroke. Whether Ruscogenin can directly target NF-κB or act as the upstream signaling molecules for its activation requires further investigation. This is currently being tested in our laboratory.

Role of the funding source This work was supported by the Major Research Plan of the National Natural Science Foundation of China (No. 90713042), National Natural Science Foundation of China (81274131), Program for New Century Excellent Talents in University (NCET-07-849), 2011' Program for Excellent Scientific and Technological Innovation Team of Jiangsu Higher Education, the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the Project Program of the State Key Laboratory of Natural Medicines, China Pharmaceutical University (No. JKGZ201107).

Contributors All authors have participated and made substantial contributions to this paper. Teng Guan contributed to designing the study, conducting the experiments, data interpretation and the writing of

the manuscript. Qian Liu did a part of the western blot assay and analyzed the study data. Yisong Qian performed real-time PCR experiments and contributed to the collecting and analyzing the data. Jiming Kong contributed to the manuscript revisions. Haopeng Yang helped in the animal neurological functional test. Junping Kou, Boyang Yu contributed to the study design, interpreting data and manuscript revisions. All authors contributed to and have approved the final manuscript.

Acknowledgment We are very grateful to Xueping Chen for her helpful suggestions and comments. We are grateful for English language editing by Jacqueline Hogue, research assistant to Dr. Kong, in Department of Human Anatomy & Cell Science, Faculty of Medicine, University of Manitoba. References Berti, R., Williams, A.J., Moffett, J.R., Hale, S.L., Velarde, L.C., Elliott, P.J., Yao, C., Dave, J.R., Tortella, F.C., 2002. Quantitative real-time RT-PCR analysis of inflammatory gene expression associated with ischemia-reperfusion brain injury. Journal of Cerebral Blood Flow and Metabolism 22, 1068–1079. Bouskela, E., Cyrino, F.Z., Marcelon, G., 1994. Possible mechanisms for the inhibitory effect of Ruscus extract on increased microvascular permeability induced by histamine in hamster cheek pouch. Journal of Cardiovascular Pharmacology 24, 281–285. Crawley, J.N., 1999. Behavioral phenotyping of transgenic and knockout mice: experimental design and evaluation of general health, sensory functions, motor abilities, and specific behavioral tests. Brain Res 835, 18–26. Facino, R.M., Carini, M., Stefani, R., Aldini, G., Saibene, L., 1995. Anti-elastase and anti-hyaluronidase activities of saponins and sapogenins from Hedera helix, Aesculus hippocastanum, and Ruscus aculeatus: factors contributing to their efficacy in the treatment of venous insufficiency. Archiv der Pharmazie (Weinheim) 328, 720–724. Garcia, J.H., Wagner, S., Liu, K.F., Hu, X.J., 1995. Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats. Statistical validation. Stroke 26, 627–634. (discussion 635). Gong, X., Sucher, N.J., 2002. Stroke therapy in traditional Chinese medicine (TCM): prospects for drug discovery and development. Phytomedicine 9, 478–484. Guan, T., Qian, Y., Tang, X., Huang, M., Huang, L., Li, Y., Sun, H., 2011. Maslinic acid, a natural inhibitor of glycogen phosphorylase, reduces cerebral ischemic injury in hyperglycemic rats by GLT-1 up-regulation. Journal of Neuroscience Research 89, 1829–1839. Huang, Y.L., Kou, J.P., Ma, L., Song, J.X., Yu, B.Y., 2008. Possible mechanism of the antiinflammatory activity of ruscogenin: role of intercellular adhesion molecule-1 and nuclear factor-kappaB. Journal of Pharmacological Sciences 108, 198–205. Jiang, W., Fu, F., Tian, J., Zhu, H., Hou, J., 2011. Curculigoside A attenuates experimental cerebral ischemia injury in vitro and vivo. Neuroscience 192, 572–579. Karin, M., Cao, Y., Greten, F.R., Li, Z.W., 2002. NF-kappaB in cancer: from innocent bystander to major culprit. Nature Reviews Cancer 2, 301–310. Kim, H., 2005. Neuroprotective herbs for stroke therapy in traditional eastern medicine. Neurological Research 27, 287–301. Kou, J., Sun, Y., Lin, Y., Cheng, Z., Zheng, W., Yu, B., Xu, Q., 2005a. Anti-inflammatory activities of aqueous extract from Radix Ophiopogon japonicus and its two constituents. Biological and Pharmaceutical Bulletin 28, 1234–1238. Kou, J., Yu, B., Xu, Q., 2005b. Inhibitory effects of ethanol extract from Radix Ophiopogon japonicus on venous thrombosis linked with its endotheliumprotective and anti-adhesive activities. Vascular Pharmacology 43, 157–163. Lakhan, S.E., Kirchgessner, A., Hofer, M., 2009. Inflammatory mechanisms in ischemic stroke: therapeutic approaches. Journal of Translational Medicine 7, 97. Li, C., Guan, T., Chen, X., Li, W., Cai, Q., Niu, J., Xiao, L., Kong, J., 2013. BNIP3 mediates pre-myelinating oligodendrocyte cell death in hypoxia and ischemia. Journal of Neurochemistry. Martin, L.J., Al-Abdulla, N.A., Brambrink, A.M., Kirsch, J.R., Sieber, F.E., PorteraCailliau, C., 1998. Neurodegeneration in excitotoxicity, global cerebral ischemia, and target deprivation: a perspective on the contributions of apoptosis and necrosis. Brain Research Bulletin 46, 281–309. Nurmi, A., Lindsberg, P.J., Koistinaho, M., Zhang, W., Juettler, E., KarjalainenLindsberg, M.L., Weih, F., Frank, N., Schwaninger, M., Koistinaho, J., 2004. Nuclear factor-kappaB contributes to infarction after permanent focal ischemia. Stroke 35, 987–991. Pandya, J.D., Sullivan, P.G., Pettigrew, L.C., 2011. Focal cerebral ischemia and mitochondrial dysfunction in the TNFalpha-transgenic rat. Brain Research 1384, 151–160. Saito, A., Maier, C.M., Narasimhan, P., Nishi, T., Song, Y.S., Yu, F., Liu, J., Lee, Y.S., Nito, C., Kamada, H., Dodd, R.L., Hsieh, L.B., Hassid, B., Kim, E.E., Gonzalez, M., Chan, P.

Please cite this article as: Guan, T., et al., Ruscogenin reduces cerebral ischemic injury via NF-κB-mediated inflammatory pathway in the mouse model of experimental stroke. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.07.036i

T. Guan et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ H., 2005. Oxidative stress and neuronal death/survival signaling in cerebral ischemia. Molecular Neurobiology 31, 105–116. Song, J., Kou, J., Huang, Y., Yu, B., 2010. Ruscogenin mainly inhibits nuclear factorkappaB but not Akt and mitogen-activated protein kinase signaling pathways in human umbilical vein endothelial cells. Journal of Pharmacological Sciences 113, 409–413. Stephenson, D., Yin, T., Smalstig, E.B., Hsu, M.A., Panetta, J., Little, S., Clemens, J., 2000. Transcription factor nuclear factor-kappa B is activated in neurons after focal cerebral ischemia. Journal of Cerebral Blood Flow and Metabolism 20, 592–603. Sun, Q., Chen, L., Gao, M., Jiang, W., Shao, F., Li, J., Wang, J., Kou, J., Yu, B., 2012. Ruscogenin inhibits lipopolysaccharide-induced acute lung injury in mice:

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involvement of tissue factor, inducible NO synthase and nuclear factor (NF)kappaB. International Immunopharmacology 12, 88–93. Verma, I.M., 2004. Nuclear factor (NF)-kappaB proteins: therapeutic targets. Annals of the Rheumatic Diseases 63 (Suppl 2), ii57–ii61. Wang, T., Gu, J., Wu, P.F., Wang, F., Xiong, Z., Yang, Y.J., Wu, W.N., Dong, L.D., Chen, J.G., 2009. Protection by tetrahydroxystilbene glucoside against cerebral ischemia: involvement of JNK, SIRT1, and NF-kappaB pathways and inhibition of intracellular ROS/RNS generation. Free Radical Biology and Medicine 47, 229–240. Williams, A.J., Dave, J.R., Tortella, F.C., 2006. Neuroprotection with the proteasome inhibitor MLN519 in focal ischemic brain injury: relation to nuclear factor kappaB (NF-kappaB), inflammatory gene expression, and leukocyte infiltration. Neurochemistry International 49, 106–112.

Please cite this article as: Guan, T., et al., Ruscogenin reduces cerebral ischemic injury via NF-κB-mediated inflammatory pathway in the mouse model of experimental stroke. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.07.036i