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Taxifolin ameliorates cerebral ischemia-reperfusion injury in rats through its anti-oxidative effect and modulation of NF-kappa B activation Yea-Hwey Wang 1 , Wen-Yen Wang 2 , Chia-Che Chang 5 , Kuo-Tong Liou 6 , Yen-Jen Sung 4 , Jyh-Fei Liao 1 , Chieh-Fu Chen 1 , Shiou Chang 2 , Yu-Chang Hou 2 , Yueh-Ching Chou 7,8 & Yuh-Chiang Shen 3,5, * 1 Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan; 2 Departments of Surgery and Chinese Medicine, Tao-yuan and Hsin-chu General Hospitals, Department of Health, Taipei, Taiwan; 3 National Research Institute of Chinese Medicine, Taipei, Taiwan; 4 Anatomy and Cell Biology, School of Medicine, National Yang-Ming University, Taipei, Taiwan; 5 Institute of Biomedical Sciences, National Chung-Hsing University, Taichung, Taiwan; 6 Department of Chinese Martial Arts, Chinese Culture University, Taipei, Taiwan; 7 Department of Pharmacy, Veterans General Hospital, Taipei, Taiwan; 8 School of Pharmacy, Taipei Medical University, Taipei, Taiwan Received 20 June 2005; accepted 8 September 2005 ȑ 2005 National Science Council, Taipei Key words: cerebral ischemic-reperfusion, COX-2, ICAM-1 (CD54), iNOS, Mac-1 (CD11b/CD18), nuclear factor kappa B (NF-jB), reactive oxygen species, taxifolin Summary Infarction in adult rat brain was induced by middle cerebral arterial occlusion (MCAO) followed by reperfusion to examine whether taxifolin could reduce cerebral ischemic reperfusion (CI/R) injury. Taxifolin administration (0.1 and 1.0 lg/kg, i.v.) 60 min after MCAO ameliorated infarction (by 42%±7% and 62%±6%, respectively), which was accompanied by a dramatic reduction in malondial- dehyde and nitrotyrosine adduct formation, two markers for oxidative tissue damage. Overproduction of reactive oxygen species (ROS) and nitric oxide (NO) via oxidative enzymes (e.g., COX-2 and iNOS) was responsible for this oxidative damage. Taxifolin inhibited leukocyte infiltration, and COX-2 and iNOS expressions in CI/R-injured brain. Taxifolin also prevented Mac-1 and ICAM-1 expression, two key counter-receptors involved in firm adhesion/transmigration of leukocytes to the endothelium, which par- tially accounted for the limited leukocyte infiltration. ROS, generated by leukocytes and microglial cells, activated nuclear factor-kappa B (NF-jB) that in turn signaled up-regulation of inflammatory proteins. NF-jB activity in CI/R was enhanced 2.5-fold over that of sham group and was inhibited by taxifolin. Production of both ROS and NO by leukocytes and microglial cells was significantly antagonized by taxifolin. These data suggest that amelioration of CI/R injury by taxifolin may be attributed to its anti- oxidative effect, which in turn modulates NF-jB activation that mediates CI/R injury. Introduction Acute ischemic stroke is commonly disabling and also an important cause of death in industrialized countries with a high incidence affecting up to 0.2% of the population each year [1]. Ischemic stroke- induced brain injury is closely correlated with oxidative/nitrosative damage induced by overpro- duction of reactive oxygen species (ROS) [e.g., hydroxyl radicals (OH ) ), superoxide anions (O 2 .) ), and hydrogen peroxide (H 2 O 2 )] and reactive nitro- gen species (RNS) [e.g., nitric oxide (NO . ) and *To whom correspondence should be addressed. Tel: 886-2-2820 1999 ext. 9101; Fax: 886-2-28264266, E-mail: [email protected] Present address: Yea-Hwey Wang, Wen-Yen Wang, Chia-Che Chang, and Kuo-Tong Liou contributed equally to this work. Journal of Biomedical Science (2006) 13:127–141 127 DOI 10.1007/s11373-005-9031-0

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Page 1: Taxifolin ameliorates cerebral ischemia-reperfusion injury in rats … · 2017. 8. 26. · Taxifolin ameliorates cerebral ischemia-reperfusion injury in rats through its anti-oxidative

Taxifolin ameliorates cerebral ischemia-reperfusion injury in rats throughits anti-oxidative effect and modulation of NF-kappa B activation

Yea-Hwey Wang1, Wen-Yen Wang2, Chia-Che Chang5, Kuo-Tong Liou6,Yen-Jen Sung4, Jyh-Fei Liao1, Chieh-Fu Chen1, Shiou Chang2, Yu-Chang Hou2,Yueh-Ching Chou7,8 & Yuh-Chiang Shen3,5,*1Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan; 2Departments of Surgery andChinese Medicine, Tao-yuan and Hsin-chu General Hospitals, Department of Health, Taipei, Taiwan;3National Research Institute of Chinese Medicine, Taipei, Taiwan; 4Anatomy and Cell Biology, School ofMedicine, National Yang-Ming University, Taipei, Taiwan; 5Institute of Biomedical Sciences, NationalChung-Hsing University, Taichung, Taiwan; 6Department of Chinese Martial Arts, Chinese CultureUniversity, Taipei, Taiwan; 7Department of Pharmacy, Veterans General Hospital, Taipei, Taiwan; 8Schoolof Pharmacy, Taipei Medical University, Taipei, Taiwan

Received 20 June 2005; accepted 8 September 2005

� 2005 National Science Council, Taipei

Key words: cerebral ischemic-reperfusion, COX-2, ICAM-1 (CD54), iNOS, Mac-1 (CD11b/CD18),nuclear factor kappa B (NF-jB), reactive oxygen species, taxifolin

Summary

Infarction in adult rat brain was induced by middle cerebral arterial occlusion (MCAO) followed byreperfusion to examine whether taxifolin could reduce cerebral ischemic reperfusion (CI/R) injury.Taxifolin administration (0.1 and 1.0 lg/kg, i.v.) 60 min after MCAO ameliorated infarction (by42%±7% and 62%±6%, respectively), which was accompanied by a dramatic reduction in malondial-dehyde and nitrotyrosine adduct formation, two markers for oxidative tissue damage. Overproduction ofreactive oxygen species (ROS) and nitric oxide (NO) via oxidative enzymes (e.g., COX-2 and iNOS) wasresponsible for this oxidative damage. Taxifolin inhibited leukocyte infiltration, and COX-2 and iNOSexpressions in CI/R-injured brain. Taxifolin also prevented Mac-1 and ICAM-1 expression, two keycounter-receptors involved in firm adhesion/transmigration of leukocytes to the endothelium, which par-tially accounted for the limited leukocyte infiltration. ROS, generated by leukocytes and microglial cells,activated nuclear factor-kappa B (NF-jB) that in turn signaled up-regulation of inflammatory proteins.NF-jB activity in CI/R was enhanced 2.5-fold over that of sham group and was inhibited by taxifolin.Production of both ROS and NO by leukocytes and microglial cells was significantly antagonized bytaxifolin. These data suggest that amelioration of CI/R injury by taxifolin may be attributed to its anti-oxidative effect, which in turn modulates NF-jB activation that mediates CI/R injury.

Introduction

Acute ischemic stroke is commonly disabling andalso an important cause of death in industrialized

countries with a high incidence affecting up to 0.2%of the population each year [1]. Ischemic stroke-induced brain injury is closely correlated withoxidative/nitrosative damage induced by overpro-duction of reactive oxygen species (ROS) [e.g.,hydroxyl radicals (OH)), superoxide anions (O2

.)),and hydrogen peroxide (H2O2)] and reactive nitro-gen species (RNS) [e.g., nitric oxide (NO.) and

*Towhom correspondence should be addressed. Tel: 886-2-28201999 ext. 9101; Fax: 886-2-28264266, E-mail: [email protected] address: Yea-Hwey Wang, Wen-Yen Wang, Chia-CheChang, and Kuo-Tong Liou contributed equally to this work.

Journal of Biomedical Science (2006) 13:127–141 127DOI 10.1007/s11373-005-9031-0

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peroxynitrite (OONO))] in response to activation ofinflammatory mediators produced by recruitedleukocytes (e.g., neutrophils), active microglia cells,and/or neurons and astrocytes in injured tissue [2–4].

Although pathologic mechanisms leading tocerebral ischemic/reperfusion (CI/R) injury arecomplicated, it has been emphasized that multiplefundamental cell injury mechanisms are involved,including excitotoxicity, overproduction of ROS/RNS, inflammation, and apoptosis [5–7]. After theonset of ischemia, energy deficits lead to ionicdisruption and mitochondrial dysfunction thatserve as activators for free radical producing-enzyme systems including mitochondria, cycloox-ygenase (COX), xanthine oxidase, NADPHoxidase, and neuronal- or inducible-nitric oxidesynthase (nNOS or iNOS) which overproduceROS and RNS. ROS and RNS damage tissue byattacking DNA or inducing lipid peroxidation orprotein nitrosylation of the cell membrane andorganelles [6]. In tissue damaged by ROS/RNS, aninflammatory cascade is initiated leading to com-plement activation (e.g. C5a), firm adhesion mol-ecule upregulation (e.g., ICAM-1 and Mac-1),leukocyte infiltration, activation/expression ofNOS and COX, and release of cytokines (e.g.,IL-1b and TNF-a) which amplify inflammation[8]. Many reports have indicated that activation ofnuclear factor-kappa B (NF-jB) plays a pivotalrole in mediating oxidative stress-induced cellinjury and in the regulation of post-ischemicinflammation, possibly through upregulation ofinflammatory genes/proteins that contribute to celldeath in cerebral ischemia [9–11]. Multiple path-ological mechanisms as mentioned above areinvolved in CI/R-induced tissue injury, and thefact that many drugs work in animals but fail inhumans [5] suggests that combination therapies ordrug(s) that can target multiple sites of thesemechanisms can provide the potential to meet thetreatment challenges of ischemic stroke.

Flavonoids structurally similar to quercetinhave been reported to exhibit multiple pharmaco-logical effects and display beneficial actions againstcardiovascular diseases [12–13] but their potentialfor CNS protection and the exact mechanisms ofaction remain unclear. In addition, taxifolin(dihydroquercetin, Figure 1) has been shown toexhibit anti-inflammatory effects and protectagainst oxidative cellular injury in rat peritoneal

macrophages [14] and human endothelial cells [15],possibly by inhibiting the expression of intercellu-lar adhesion molecule-1 (ICAM-1) [16] and Mac-1(CD11b/CD18), a dominant b2 integrin in acti-vated neutrophils [17]. Expression of ICAM-1 oninflammatory target cells (e.g., endothelial cells)mediates activation and firm adhesion of circulat-ing neutrophils to injured tissue through interac-tion with b2 integrin expressed on neutrophils [18].Drugs with anti-oxidative or anti-inflammatoryeffects can possibly modulate the upregulation ofMac-1 [19]. These observations suggest thatimpairment of interactions between peripheralneutrophils and their target cells might preventfurther activation of these inflammatory cells toinfiltrate injured tissue (e.g., the brain) and conferon drugs potential benefits for the prevention ortreatment of cardiovascular disorders, e.g., stroke.

In our previous study, we reported that taxif-olin exhibits an inhibitory effect on surface Mac-1expression and Mac-1-dependent firm adhesion byhuman neutrophils, as well as the possible mech-anism(s) underlying its anti-inflammatory effect[17]. In this current work we set up an animalmodel of ischemic stroke to elucidate whethertaxifolin, with anti-inflammatory potential, canprotect animals from CI/R injury. In particular,some key molecular mechanisms involved in theoxidative/nitrosative damage by CI/R injury andthe possible targets of taxifolin were elucidated inin vivo and in vitro systems.

Materials and methods

Animals and induction of cerebralischemic-reperfusion injury

All animal procedures and protocols were per-formed in accordance with the Guide For TheCare and Use of Laboratory Animals (NIHpublication, 85–23, revised 1996) and werereviewed and approved by the Animal ResearchCommittee in our institute. Male adult Long-Evans rats (National Laboratory Animal Breedingand Research Center, Taipei, Taiwan) (250–350 g)were used. Rats were anesthetized with chloralhydrate (300 mg/kg, i.p.). The right femoral arterywas cannulated for continuous monitoring of theheart rate and blood pressure. The right middlecerebral artery (RMCA) was ligated. Immediately

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Figure 1. Effect of taxifolin on CI/R-induced cerebral infarction and lipid peroxidation in the rats. Whole brains from sham-operated rats (sham) or CI/R-injured rats receiving vehicle control (CI/R), 0.1 or 1.0 lg/kg (i.v.) taxifolin (CI/R+0.1T or CI/R+1.0T), 1.0 lg/kg (i.v.) quercetin (CI/R+1.0Q), or 1 lg/kg NS398 (CI/R+NS398) were removed for the determination of in-farct volume by TTC staining (upper panel) or lipid peroxidation by MDA formation (lower panel). Data were calculated asthe percent of infarct volume of the right hemisphere brain (cerebral infarction) or nmol/mg protein (MDA formation), and areexpressed as the mean±SEM (n=10 for each data point). *p<0.05 as compared with the CI/R group by one-way ANOVA fol-lowed by Dunnett’s test. The inset (top) shows the chemical structure of taxifolin.

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after occlusion of the RMCA, both commoncarotid arteries (CCAs) were clipped for 1 h toproduce cortical infarction. Twenty-four hoursafter the surgery, the rats were anesthetized andsacrificed. During the entire experiment, the bloodgas data and physiological data were monitoredas in our previous report [20]. The experimen-tal grouping included one sham-operated group(sham, n=10), two (0.1 and 1.0 lg/kg) taxifolin-treated groups (n=10, for each dose), and one CI/R group (CI/R only, n=10). Sham-operated ratsunderwent the same surgical procedures withoutRMCA occlusion or CCA clipping. Additionalanimals from the groups described above wereused for other assays (leukocyte infiltration,lipid peroxidation, immuno-blotting, and semi-quantitative RT-PCT).

Drug administration

Drug solutions (0.3 ml) were administered intra-venously (i.v.) via the right femoral vein 60 minafter RMCAO at two different doses (0.1 or1.0 lg/kg). Taxifolin (Sigma-Aldrich, USA) wasfirst dissolved in 10 ll of ethanol and furthersuspended in normal saline to make a clear stocksolution of 1.0 mg/ml, then this was diluted withnormal saline to the desired concentrations. Thefinal concentration of ethanol in the injectedtaxifolin solution was less than 0.001% (v/v). Ratsinjected with 0.3 ml of normal saline containing0.001% (v/v) ethanol were used as vehicle controlin CI/R only and sham-operated groups.

Evaluation of infarct volume

Twenty-four hours after reperfusion, rats weresacrificed by rapid decapitation under deep anes-thesia. Whole brains were rapidly removed. Imme-diately after being weighed, the brain was slicedinto 2-mm-thick coronal sections and stainedwith 2% 2,3,5-triphenyltetrazoliumchloride (TTC,Sigma-Aldrich) for 30 min at 37 �C in the dark,followed by fixation with 10% of formalin at roomtemperature overnight. Brain slices lacking redstaining defined the infarct area. The slices wererecorded by digital photography and analyzed byan image processing system (AIS software, Imag-ing Research, Brock Univ., Ontario, Canada).Infarct volume was obtained according to theindirect method proposed by Swanson et al. [21]

and corrected for edema by comparing the volumeof ischemic and nonischemic hemispheres asdescribed [22]. The infarct volume was expressedas a percentage of the hemispheric volume (% ofhemisphere).

Measurement of leukocyte infiltration

Myeloperoxidase (MPO) activity is used to quan-titatively assess leukocyte infiltration into ischemictissue [23]. Briefly, brain tissues were homogenizedin 1.5 ml of potassium phosphate buffer (PPB,50 mM, pH 6). One milliliter of the homogenatewas pelleted and suspended in PPB containing0.5% hexadecyltrimethylammonium bromide.After three cycles of freezing and thawing, samplewas sonicated on ice for 10 s, and centrifuged at12,000�g for 10 min. MPO activity was deter-mined in the supernatants by mixing supernatantwith PPB containing o-dianisidine chloride andhydrogen peroxide (H2O2). The absorbance at awavelength of 460 nm (OD460) was determined byspectrophotometry over 2 min and normalizedusing protein concentration. Values of the tissueMPO activity (infiltration index) are expressed asOD460�100/mg protein. The protein concentra-tion was determined with a commercial kit (Bio-Rad Laboratory, USA).

Measurement of lipid peroxidation

Lipid peroxidation of the frozen cerebral cortexwas determined by the tissue malondialdehyde(MDA) level as measured by the formation ofthiobarbituric acid (TBA)-reactive substances(TBARSs) [24]. Briefly, brain tissues were homog-enized with 10� (w/v) of cold 1.5% KCl. Thehomogenate was mixed with a 1% phosphoric acidand 6% TBA (Sigma-Aldrich) aqueous solution.The mixture was heated for 45 min in a boilingwater bath. After cooling, n-butanol was addedand mixed vigorously. The absorbance of thebutanol phase was measured at 525 nm. A seriallydiluted MDA (Sigma-Aldrich) solution was pre-pared and used as a standard. Data (MDA) wereexpressed as nmol/mg protein.

Western immunoblot analysis

Equal amounts of protein were subjectedto sodium dodecyl sulfate-polyacrylamide gel

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electrophoresis (SDS-PAGE) and electro-transferredto a hydrophobic polyvinylidene difluoride (PVDF)membrane. After blocking with 5% nonfat milk inPBS containing 0.05% Tween 20 (PBST) at 4 �C for1 h, the membrane was washed 3 times with PBSTand incubated overnight at 4 �C with an antibodyagainst nitrotyrosine (rabbit-anti-rat, isotype: IgG;Upstate, USA), COX-2 (goat-anti-rat, isotype:IgG; Santa Cruz Biotechnology, USA), iNOS(mouse-anti-rat, isotype: IgG; BD BiosciencePharmingen, USA) or ICAM).1 (mouse-anti-rat,isotype: IgG; Serotec, UK) at a proper dilution(1:100 or 1:1000). After additional washes withPBST, the membrane was incubated with a secondantibody (anti-rabbit, anti-goat, or anti-mouse,respectively, isotype: IgG conjugated with horse-radish peroxidase; Santa Cruz Biotechnology) for1 h at room temperature. The immunoblot on themembrane was visible after developing withan enhanced chemiluminescence (ECL) system(Perkin-Elmer).

Semiquantitative revers-transcriptase-polymerasechain reaction (RT-PCR)

Total RNA was prepared from frozen cerebralcortex using a NucleoSpin RNA II kit (BDBiosciences Clontech). RNA (300 ng) was usedto perform RT-PCR with Titanium One-step RT-PCR kit (BD Biosciences Clontech). Primers usedwere specific for COX-2 (upstream: 5¢-ACA CTCTAT CAC TGG CAT CC-3¢, downstream: 5¢-GAA GGG ACA CCC TTT CAC AT-3¢;expected PCR product size: 584 bp), iNOS (up-stream: 5¢-CCA ACA ACA CAG GAT GACC-3¢,downstream: 5¢-CCT GAT GTT GCC ACT GTTAG-3¢; expected PCR product size: 602 bp), andICAM-1 (upstream: 5¢-AGA CAC AAG CAAGAG AAG AA-3¢, downstream: 5¢-GAG AAGCCC AAA CCC GTA TG-3¢; expected PCRproduct size: 233 bp). Each PCR product wasnormalized to a housekeeping gene, glyceraldehy-des 3-phosphate dehydrogenase (GAPDH; up-stream: 5¢-CCC TCA AGA TTG TCA GCAATG C-3¢, downstream: 5¢-GTC CTC AGTGTA GCC CAG GAT-3¢; expected PCR productsize: 410 bp). Generally the following conditionwere used: 94 �C for 5 min followed by 25–30cycles of 94 �C for 30 s, 58 �C for 30 s, 68 �C for1 min. PCR products were electrophoresed andanalyzed with a Molecular Imager FX (Bio-Rad,

USA). The data were processed with Quantity Onesoftware (Bio-Rad), and RNA levels were calcu-lated as specific RNA-to-GAPDH ratios to deter-mine the relative amounts of the genes of interest.

Neutrophil preparation

Peripheral whole blood was collected for theneutrophil preparation [25]. The leukocyte-rich(upper) layer was collected and subjected to cen-trifugation. Neutrophils were separated by a Ficollgradient centrifugation method followed by lysis ofcontaminating erythrocytes and washed threetimes with cold phosphate-buffered saline (PBS).The purified sample, containing more than 95%neutrophils, was used for the in vitro assay includ-ing ROS production and Mac-1 (CD11b/CD18)expression. Cell viability was measured by trypanblue exclusion assay at the end of the experiments.

Measurement of reactive oxygen species (ROS)production by neutrophils

Production of ROS was evaluated accordingto our previous report [17]. PMA (phorbol-12-myristate-13-acetate)-induced ROS production inneutrophils was determined by luminol-amplifiedchemiluminescence in the presence or absence oftaxifolin. Briefly, taxifolin-pretreated neutrophilswere incubated with luminol. Cells were thenimmediately stimulated with PMA in whichchemiluminescence was monitored every 1 minfor 30 min with a microplate luminometer reader(Orion�, Germany), and results are represented asrelative light units (RLU). Peak levels wererecoded to calculate the activity of the test drugs.Data are expressed as RLU% of the vehiclecontrol. The 50% inhibitory concentration (IC50)of PMA-triggered chemiluminescence by testdrugs was calculated using a semilog-plot trans-formation of the data.

Measurement of Mac-1 (CD11b/CD18)up-regulation by neutrophils

Up-regulation of Mac-1 was analyzed as in ourprevious study [17]. In brief, taxifolin-pretreatedcells were stimulated with PMA. Cells were thenpelleted and resuspended in PBS containing 10%FBS and 10 mM sodium azide. Cells wereincubated in the dark for 60 min with a

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proper aliquot of fluorescein isothiocyanate(FITC)-conjugated anti-Mac-1 antibody (mouseanti-rat CD11b, class IgG1; BD BiosciencesPharmingen) or a non-specific mouse antibody(class IgG1, Sigma-Aldrich) as a negative control.After two washes with PBS containing 5% FBS,stained-cells were resuspended in flow cytometricsheath fluid (Becton Dickinson) containing 1%paraformaldehyde, and Mac-1 expression wasanalyzed by a flow cytometer (FACSCaliburTM).Data were expressed as the mean channel fluores-cence for each sample as calculated by the Cell-Quest�software (Becton Dickinson).

Determination of NF-jB activation

Frozen cerebral cortex was homogenized and wasextracted using a nuclear extraction reagent(Pierce NE-PER; USA). An NF-jB p65 Tran-scription Factor Assay Kit (Pierce) consisting of a96-well plate onto which the oligonucleotideduplex containing the NF-jB consensus site (5¢-GGGACTTTCC-3¢) was pre-coated was used tomonitor NF-jB activation. The active formof NF-jB in the nuclear extract specifically bindsto this consensus site and is recognized bythe primary antibody. A horseradish peroxidase-conjugated secondary antibody provided thechemiluminescent quantification. Results wereexpressed as relative light units (RLU). TNF-a-activated HeLa whole-cell extract provided bythe manufacturer was used as a positive control forNF-jB activity. Wild-type (5¢-CACAGTTGAGGGGACTTTCCCAGGC-3¢) and mutant (5¢-CACAGTTGAGGCCACTTTCCCAGGC-3¢) NF-jBcompetitor duplexes were included to contrastthe specific binding of NF-jB.

Cell culture and measurements of nitric oxide (NO)and intracellular reactive oxygen species (ROS)production by microglial cells

The murine microglia cell line, BV2, was gener-ously provided by Dr. Greer M. Murphy, Jr. fromthe Department of Psychiatry (Stanford Univer-sity School of Medicine, Stanford, CA). These cellswere originally isolated from neonatal mousebrain and immortalized by infection with a v-raf/v-myc-carrying retrovirus (J2) [26]. These cellsexhibit normal macrophage functions and expressproperties of activated microglia cells. BV2 cells

were cultured in Dulbecco’s modified Eaglemedium (Gibco, USA) supplemented with 5%fetal bovine serum (Hyclone). Production of NOwas measured by the accumulation of nitrite in theculture medium 24 h after stimulation with LPS(0.5 lg/ml) or IFN (20 ng/ml) by the Griessreagent. Intracellular ROS accumulation wasmeasured as in our previous report [25].

Statistical analysis

All values in the text and figures are presented as themean±SEM Data were analyzed by one-way ortwo-way analysis of variance (ANOVA) dependingon the number of parameters for comparisonfollowed by post–hoc Dunnett’s t-test for multiplecomparisons. Values of p<0.05 were consideredsignificant.

Results

Protective effect of taxifolin against CI/R-inducedcerebral infarction

The infarct volume induced by CI/R injury in thisstudy (around 20% of the right brain hemisphere)was comparable with that of our previousreports [20, 27]. Taxifolin (0.1–1.0 lg/kg) dose-dependently ameliorated CI/R-induced braininfarction by 40–65% (Figure 1 upper panel,one-way ANOVA, p<0.05). Treatment with both0.1 and 1.0 lg/kg (i.v.) taxifolin effectively reducedCI/R-induced brain infarction (Figure 1 upperpanel; one-way ANOVA followed by Dunnett’stest, p<0.05, n=10 for each group). Quercetin(1.0 lg/kg, i.v.), a flavonoid used as a referencedrug, also showed a 45% protective effect againstCI/R-induced brain infarction.

Protective effects of taxifolin against CI/R-inducedoxidative and nitrosative tissue damage

Lipid peroxidation and protein tyrosine nitrosylationare two markers for oxidative and nitrosative tissuedamage in the brain [28, 29]. In this study, CI/R-induced cerebral lipid peroxidation, as assayed byMDA formation, occurred in a time-dependent man-ner that peaked at 24 h as in our previous report [27].Taxifolin dose-dependently prevented CI/R-inducedMDA formation (Figure 1 lower panel, one-way

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ANOVA,p<0.05,n=10foreachgroup). Inaddition,NS398, a COX-2 inhibitor included as a positivecontrol for its anti-ischemicproperty [30], significantlysuppressed MDA formation.

CI/R injury also induced time-dependent ni-trosative cerebral damage as evidenced by theincrements in immunoreactivity for protein tyro-sine nitrosylation (Figure 2a). Treatment with1.0 lg/kg (i.v.) taxifolin significantly reduced theimmunoreactivity for nitrotyrosine at 24 h (Fig-ure 2b), indicating that taxifolin prevented cere-bral nitrosative damage.

Effects of taxifolin on CI/R-induced COX-2and iNOS expression, and leukocyte infiltration

Many prooxidative enzymes, including COX-2,NOS, NADPH oxidase (NOX), and myeloper-oxidase (MPO), are known to participate in theoxidative and nitrosative stress-induced signalingand brain damage in cerebral ischemia [2]. Inthis study, CI/R induced significant upregulationof COX-2 and iNOS on both the protein(Figure 3a) and gene (Figure 3b) levels at 24 h.Treatment with 0.1 and 1.0 lg/kg (i.v.) taxifolinsignificantly reduced the immunoreactivity ofCOX-2 and iNOS (Figure 3a), and their geneexpressions (Figure 3b) indicating that the upreg-ulation of prooxidative enzymes, especiallyCOX-2 and iNOS, was limited by taxifolintreatment.

Infiltration of leukocytes to CI/R-injured tissueprovides predominant sources for MPO activity,another important prooxidative enzyme responsi-ble for oxidative stress in CI/R-injured brain. Wepreviously reported that enhancement of MPOactivity in CI/R-injured brain closely paralleledcerebral MDA formation in infarct tissue in atime-dependent manner [27]. In this study, CI/Rinduced-leukocyte infiltration, as measured byMPO activity in the cerebral infarct area, peakedat 24 h, which was comparable to the result of ourprevious report [27]. MPO activity in the CI/R-injured only group was 4.2-fold higher than that inthe sham-operated group (Table 1). Treatmentwith 0.1 and 1.0 lg/kg (i.v.) taxifolin or a COX-2inhibitor (NS398, 1.0 lg/kg, i.v.) all significantlyreduced MPO activity in the CI/R-injured cerebralcortex (Table 1, one-way ANOVA followed byDunnett’s test, p<0.05, n=6 for each group)

indicating that leukocyte infiltration was inhibitedby treatment with taxifolin and NS398.

Effect of taxifolin on ROS and NO productionin neutrophils and microglial cells

During CI/R injury, activation of microglial cellsand infiltration of peripheral leukocytes maydamage brain tissue by producing enormousamounts of ROS and/or RNS (when superoxideanion combines with NO) [6]. ROS and RNS alsosignal the transcription of many inflammatory-related genes and proteins (e.g., cytokines, adhe-sion molecules, COX-2, iNOS, etc.) possibly byactivation of NF-jb [31–32]. To examine whethertaxifolin can prevent ROS or NO production, anin vitro study was performed on oxidative bursts ofrat peripheral blood neutrophils and a murinemicroglial cell line (BV2).

PMA-induced ROS production in rat neutroph-ils, as assayed by luminol-amplified chemilumines-cence at �25,879±261 RLU, was 208-fold higherthan that of the vehicle control (124±30 RLU).Pretreatment with taxifolin concentration-depen-dently inhibited the PMA-induced luminol-ampli-fied chemiluminescence (one-way ANOVA,p<0.05, n=5) with an IC50 of �28 lM (Table 2).NS-398 (a COX-2 inhibitor), quercetin (an ana-logue of taxifolin), and apocynin (an inhibitor ofNADPH oxidase) [33] used in this test as referencedrugs, also significantly inhibited PMA-inducedROS production (Table 2). Ibuprofen, a COX-1inhibitor, did not significantly inhibit ROS produc-tion in rat leukocytes (IC50>100 lM). In microg-lial cells, both LPS and IFN-c enhanced theproduction of ROS (FI) and NO (lM) to around3-fold higher than resting cells (Table 3). Pretreat-ment with taxifolin (10 lM) significantly inhibitedthe production of ROS and NO (Table 3;one-way ANOVA, p<0.05, n=5–6). Trolox(an anti-oxidant) and L-NAME (anNOS inhibitor)were included to contrast taxifolin’s effect, and bothsignificantly antagonized ROS and NO production(Table 3; one-way ANOVA, p<0.05, n=5–6).

Effect of taxifolin on CI/R-induced ICAM-1expression in the cerebral cortex and PMA-inducedMac-1 upregulation in rat peripheral neutrophils

Infiltration of peripheral leukocytes to ischemia-injured tissue primarily depends on the upregula-

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Figure 2. Effect of taxifolin on CI/R-induced protein nitrosylation in the rat cerebral cortex. (a) Time-dependent (0–24 h) analysisof protein nitrosylation after CI/R injury. The cerebral cortex was removed for determination of protein nitrosylation by immuno-blotting. Three major protein nitrosylation bands as indicated (arrow) were used for the statistical analysis (lower panel). Datawere calculated as multiples of the control (0 h) after normalization to b-actin, respectively, and are expressed as the mean±SEM(n=5 for each data point). (b) Formation of protein nitrosylation from sham-operated rats (S) or rats receiving vehicle control(CI/R-24 h) or 0.1 and 1.0 lg/kg (i.v.) taxifolin followed by CI/R injury for 24 h (CI/R+0.1T or CI/R+1.0T). Data were calcu-lated as multiples of the control (sham) after normalization to b-actin, respectively, and are expressed as the mean±SEM (n=5 foreach data point). *p<0.05 as compared with the CI/R only group by one-way ANOVA followed by Dunnett’s test.

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tion of firm adhesion molecules, especially Mac-1(CD11b/CD18) in neutrophils and its counterligands in endothelial cells, i.e., intercellular

adhesion molecule 1 (ICAM-1) [34]. In this study,CI/R induced a significant upregulation of ICAM-1 in CI/R-injured cerebral cortex at 24 h

Figure 3. Effect of taxifolin on CI/R-induced COX-2 and iNOS expression in the rat cerebral cortex. (a) COX-2 and iNOSproteins or (b) genes were determined by immunoblotting or semiquantitative RT-PCR. The cerebral cortex was removed fromsham-operated rats (S) or CI/R-injured rats receiving the vehicle control (CI/R) or 0.1 or 1.0 lg/kg (i.v.) taxifolin (CI/R+0.1T orCI/R+1.0T). b-actin or the GAPDH gene was included as reference for normalization. The lower panel of each figure shows thestatistical results (mean±SEM, n=5) of the densitometric measurements after normalization to b-actin or GAPDH. �;yp < 0:05 ascompared with the CI/R only group for COX-2 and iNOS, respectively, by one-way ANOVA followed by Dunnett’s test.

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(Figure 4). Taxifolin treatment (1.0 lg/kg, i.v.)significantly inhibited ICAM-1 protein (Figure 4a)and gene (Figure 4b) expressions in the infarctarea. In vitro examination of taxifolin’s effect onMac-1 upregulation in peripheral rat neutrophilsrevealed that taxifolin concentration-dependentlyinhibited PMA-induced Mac-1 upregulation(Figure 5, one-way ANOVA, p<0.05, n=5).

Effect of taxifolin on CI/R-induced NF-jBactivation

NF-jB has been reported to be a biomarker foroxidative stress associated with ischemic reperfu-sion injury. Activation of NF-jB may be thedown-stream signaling pathway of ROS or RNSfor the transcription of many inflammatory-related genes and proteins (e.g., cytokines,adhesion molecules, COX-2, iNOS) [31–32]. Inthis study, CI/R enhanced NF-jB activation to

around 2.5-fold over that of sham group (Table 4).Treatment with both 0.1 and 1.0 lg/kg (i.v.)taxifolin significantly reduced NF-jB activationin the CI/R-injured cerebral cortex (Table 4, one-way ANOVA followed by Dunnett’s test, p<0.05,n=4 for each data point) indicating that activationof NF-jB was modulated by taxifolin treatment.

Discussion

Taxifolin is a potent anti-inflammatory drugagainst leukocyte activation [17]. Herein we dem-onstrated for the first time that taxifolin alsoreduced cerebral ischemic reperfusion (CI/R)injury. Rats subjected to CI/R revealed typicalmarkers of cerebral inflammation and oxidative/nitrosative injury including infarction (increasedTTC-staining), oxidative and nitrosative tissuedamage (increased cerebral MDA formation andprotein tyrosine nitrosylation), leukocyte infiltra-tion into the infarct area (enhanced MPO activity),upregulation of adhesion molecules (ICAM-1),and induction of prooxidative enzymes (COX-2and iNOS). These data established a typicalpattern of CI/R injury that was characterized byprotein modifications of oxidative and nitrosativepathways in brain tissue in relation to theactivation of inflammatory mediators. Taxifolintreatment showed protective effects in the reduc-tion of CI/R-induced oxidative and nitrosativetissue damage by blocking inflammation-relatedevents and expression of prooxidative enzymes.

It has been reported that flavonoids structur-ally related to quercetin exhibit neuroprotectiveactions against oxidative injuries induced in cor-

Table 1. Decrease in CI/R-induced leukocyte infiltration(MPO activity) by taxifolin in the rat cerebral cortex 24 hafter middle cerebral arterial occlusion and reperfusion.

Groups MPO activity (10,000

�OD460/mg protein)

Sham 112±25*

CI/R only 473±30

CI/R+TAXI (lg/kg, i.v.) –

0.1 260±36*

1.0 125±17*

CI/R+NS-398 149±49*

Data are expressed as the mean±SEM (n=6 for each group).*p<0.05 as compared with the CI/R only group by one-wayANOVA followed by Dunnett’s test.

Table 2. Summary of the IC50 and maximum inhibitionvalues for the prevention of ROS production by taxifolin andsome reference drugs in rat peripheral leukocytes.

Drugs IC50 (lM) for

ROS production

Maximum

inhibition (%)

Taxifolin 28.3±3.7 95.4±0.7

Quercetin 30.2±4.2 92.5±1.4

Apocynin 21.1±7.5 72.7±3.64

Ibuprofen >100 10.0±2.6

NS-398 30.5±5.1 58.0±3.6

Data are expressed as the 50% inhibitory concentration (IC50).Maximum inhibition was determined at 50 lM for each drug.Values represent the mean±SEM of five experiments per-formed on different days using cells from different rats.

Table 3. Inhibition of ROS (fluorescence intensity) and NO(lM) production by taxifolin in murine microglial cells.

Inducer LPS IFN-c

ROS NO ROS NO

control (drug free) 10±0* 0 10±0* 0

Inducer alone 27±3 27±3 33±3 29±1

+ Taxifolin 10 lM 20±2* 13±0* 19±4* 19±2*

+ Trolox 50 lM 10±1* N.D. 15±1* N.D.

+ L-NAME 20 lM N.D. 19±1* N.D. 17±0*

Data are expressed as the mean±SEM (n=5–6 for each datapoint). *p<0.05 as compared with the inducer (0.5 lg/ml LPSor 20 ng/ml IFN-c) alone by one-way ANOVA followed byDunnett’s test. N.D., data not determined.

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tical cell cultures, but their effects in animal models(in vivo) and the molecular mechanisms of actionremain unclear [35]. In this study, we found thatboth taxifolin (dihydroquercetin) and quercetinexhibited neuroprotective effects against CI/Rinjury in rats, and taxifolin was more potent thanquercetin in the prevention of CI/R-induced braininfarction. Although quercetin has been demon-strated to be better than taxifolin in protectingagainst oxidative injuries in cortical cell cultures[35], in this study we used taxifolin in the followingexperiments to further elucidate its mechanisms ofaction in the amelioration of CI/R-induced braininjury.

Taxifolin dose-dependently (0.1 and 1.0 lg/kg)inhibited TTC staining by 40–65%, and almostcompletely blocked cerebral MDA formation (anoxidative damage marker) indicating that multiplepathological mechanisms more than lipid peroxi-dation can mediate ischemic stroke-induced cere-bral damage which was modulated by taxifolin.Therefore, other mechanisms of action of taxifolinin mediating its anti-stroke effect were furtherexplored and illustrated in this study.

Activation of inducible nitric oxide synthase(iNOS) in microglial cells and infiltration ofleukocytes (predominantly neutrophils) providean important source for NO and ROS productionduring CI/R injury [36], although different obser-vations have been reported [37]. The present studytogether with our previous results [38] confirmedthat CI/R-induced infarction developed along withleukocyte infiltration into the infarct area that alsoparalleled cerebral lipid peroxidation in the infarctarea, indicating that oxidative stress elicited byleukocytes did contribute to the infarction. Taxif-olin dose-dependently prevented CI/R-inducedleukocyte accumulation (Table 1) and effectivelydecreased the ROS produced by leukocytes(Table 2), and ROS and NO produced by microg-lial cells (Table 3), revealing that the anti-oxidativeeffect is responsible for taxifolin’s protectiveeffects, and taxifolin could exert its protectiveeffects by modulating inflammatory cells includingmicroglial cells and leukocytes. An inhibitor forCOX-2 (NS398), but not COX-1 (ibuprofen), anda direct NADPH oxidase inhibitor (apocynin)both significantly prevented ROS production byneutrophils (Table 2), indicating that direct COX-2 and/or NADPH oxidase inhibition may also bepotential targets for anti-stroke drug design.

In addition to oxidative stress, nitrosativestress-mediated activation of inflammation medi-ators is currently being emphasized as an impor-tant factor involved in post-traumatic stressdisorder, neurodegeneration, and I/R injury [3,39]. Nitrosative stress during the above-mentionedconditions arises primarily from the huge accu-mulation of NO by over-expression of iNOS ornNOS in the brains of CI/R-injured animal whichform peroxynitrite (ONOO)) by reaction with thesuperoxide anion (O2

).) [40]. Peroxynitrite is atissue-damaging agent that acts through initiationof lipid peroxidation, oxidation of sulfhydrylgroups, and nitrosylation of tyrosine-containingmolecules. In this study, we found that CI/Rinduced time-dependent protein tyrosine nitrosy-lation. Upregulation of iNOS but not nNOS (datanot shown) in the brain was responsible for thenitrosative brain damage induced by CI/R. Asimilar observation was also shown in another’report [41]. Taxifolin treatment significantly pre-vented protein tyrosine nitrosylation and iNOSexpression indicating that iNOS-induced nitrosa-tive stress was diminished by taxifolin.

Inflammatory cascades triggered by CI/R in-jury further amplify tissue damage [4]. Forexample, leukocytes, macrophages, and activemicroglial cells are recruited into ischemic tissuewhere inflammatory mediators are generated bythese cells or by neurons and astrocytes. Amongthese mediators, cyclooxygenase 2 (COX-2) has acrucial role in mediating CI/R injury. Notably,administration of a COX-2 inhibitor (NS-398) wasshown to reduce infarct volume and improveneurological deficits [30]. In this study, weobserved that rats pretreated with NS-398(1.0 lg/kg, i.v.) were protected against CI/R-induced infarction, cerebral MDA formation,and leukocyte infiltration (MPO activity) withcomparable efficacies as those of taxifolin. We alsoobserved that COX-2 was upregulated after CI/R.Taxifolin significantly reduced COX-2 gene andprotein expressions, but whether COX-2 activitywas also suppressed by taxifolin awaits furtherinvestigation.

Moreover, infiltration of leukocytes (primarilyneutrophils) into the infarct areas following CI/Rbegan with enhanced firm adhesion of peripheralleukocytes through Mac-1, a beta-2 integrin, toendothelium adhesion molecules (e.g., ICAM-1).Our data demonstrated that CI/R-induced infarc-

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Figure 4. Effect of taxifolin on CI/R-induced ICAM-1 expression in the rat cerebral cortex. (a) ICAM-1 protein and (b) gene weredetermined by immunoblotting or RT-PCR, respectively. The cerebral cortex was removed from sham-operated rats (S) or CI/R-injured rats receiving vehicle control (CI/R) or 0.1 or 1.0 lg/kg taxifolin (CI/R+0.1T or CI/R+1.0T). b-actin or the GAPDHgene was included as a reference for normalization. The lower panel of each figure shows the statistical results (mean±SEM) ofthe densitometric measurements after normalization. *p<0.05 as compared with the CI/R only group by one-way ANOVA fol-lowed by Dunnett’s test.

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tion developed along with leukocyte infiltrationinto the infarcted tissue. Expression of ICAM-1, acounter-receptor for Mac-1, was significantlyenhanced in the infarct area. Rats treated withtaxifolin significantly inhibited ICAM-1 expres-sion. In vitro examination of Mac-1 upregulationin peripheral leukocyte was prevented by taxifolin,possibly via impairment of ROS generation pro-duced by NADPH oxidase through interferingwith the p38 MAPK- and/or PKC-dependent

signals, and antagonizing the G protein-mediatedcalcium influx which in turn inhibited Mac-1-dependent neutrophil firm adhesion, a prerequisitestep before leukocyte transmigration [17]. Fur-thermore, TNF-a and IL-1b were reported to up-regulate ICAM-1 expression during CI/R injury[42–43]. We also observed that both TNF-a andIL-1b genes were up-regulated 6-fold higher thanthat in sham-operated rats in our CI/R modelat 24 h, and treatment of taxifolin significantlyreduced both genes expressions to 2–3-fold (datanot showed). These results may partially accountfor the suppression of ICAM-1 upregulation bytaxifolin. Therefore, the cerebral protective effectof taxifolin paralleled the impediment of leukocyteinfiltration into the cerebral foci by reducingadhesion molecule upregulation by CI/R. Theseresults confirmed that leukocyte activation andinfiltration play pivotal roles in mediating CI/Rinjury. Taxifolin, as a potent antioxidant and aninhibitor of leukocyte activation/infiltration, maybe a novel CI/R injury protective agent.

Furthermore, we found that taxifolin acted notonly as an anti-inflammatory and antioxidativeagent but also displayed inhibitory effects on theexpression of proinflammatory proteins (iNOSand COX-2) and cytokines (IL-1b and TNF-a),indicating that transcription factor(s) may be

Figure 5. Effect of taxifolin on Mac-1 upregulation in peripheral rat neutrophils. Surface levels of Mac-1 upregulation induced byPMA (0.2 lM) were measured by staining with FITC-conjugated anti-rat CD11b and analyzed on a flow cytometer (FACSCali-burTM) in the absence or presence of 1–50 lM taxifolin (TAXI). Staurosporine (Stau; 20 nM), a protein kinase c (PKC) inhibitor,was included as a control to block PKC activity. Samples receiving neither PMA nor TAXI were used as the background control(control). Data were represented as the mean channel fluorescence intensity and are expressed as the mean±SEM from five inde-pendent trials in each group. *p<0.05 as compared with PMA group by one-way ANOVA followed by Dunnett’s test.

Table 4. Decrease in CI/R-induced NF-jB activation bytaxifolin in the rat cerebral cortex 4 h after middle cerebralarterial occlusion and reperfusion.

Groups NF-jB activity

(RLU/mg protein)

Sham 10,444±252*

CI/R only 24,623±376

+ Wild-type competitor 7,279±340*

+ Mutant competitor 24,712±325

CI/R+TAXI (lg/kg; i.v.) –

0.1 18,264±366*

1.0 12,012±178*

TNF-a-stimulated HeLa 49,490±185*

Data are expressed as the mean±SEM (n=4 for each datapoint). *p<0.05 as compared with the CI/R only group by one-way ANOVA followed by Dunnett’s test.

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modulated by taxifolin. Since most of theseinflammatory-related proteins are down streamgene products of transcription factor NF-jB thatwas activated during oxidative and/or inflamma-tory stress, it is reasonable that taxifolin might alsomodulate the signaling pathways leading to acti-vation of NF-jB. Our data revealed that taxifolindecreased NF-jB activation in CI/R-injured rats,and our unpublished data showed that taxifolininhibited NF-jB transcriptional activation in thetransient transfected macrophage cell line with aplasmid containing a triple NF-jB binding site in aluciferase reporter gene, indicating that taxifolinsuppressed NO or inflammatory-related genes viaa NF-jB-dependent mechanism. However, wecould not rule out the possibility that suppressionof NF-jB activation by taxifolin might be asecondary event through its antioxidative effect.More precise mechanism(s) needs further investi-gations.

In conclusion, our study demonstrated thattaxifolin significantly protected rat brain againstCI/R injury through diminishing cerebral lipidperoxidation (MDA formation) and protein nit-rosylation by limiting leukocyte infiltration andupregulation of adhesion molecules (Mac-1 andICAM-1), and inflammatory-related prooxidativeenzymes (iNOS and COX-2) in injured tissue via,at least in part, reducing the activation of NF-jB.As a potent antioxidative and antinitrosative drug,taxifolin could be beneficial for the preventionand/or amelioration of CI/R injury.

Acknowledgements

This study was supported, in part, by grants fromthe National Science Council, R.O.C. (NSC-93-2320-B-077-004), the National Research Instituteof Chinese Medicine (NRICM93-DBCMR-09),and Tao-yuan and Hsin-chu General Hospitals,Department of Health, Taiwan, to Y.C. Shen,S. Chang, and W.Y. Wang, respectively.

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