nebivolol and chrysin protect the liver against ischemia/reperfusion-induced injury … · 2017. 1....

w.sciencedirect.com

b e n i - s u e f un i v e r s i t y j o u rn a l o f b a s i c a n d a p p l i e d s c i e n c e s 4 ( 2 0 1 5 ) 8 6e9 2

HOSTED BY Available online at ww

ScienceDirect

journal homepage: www.elsevier .com/locate/bjbas

Full Length Article

Nebivolol and chrysin protect the liver againstischemia/reperfusion-induced injury in rats

Sayed M. Mizar a, Hany A. Omar a,b,*, Gamal A. El Sherbiny c,Mohammed A. El-moselhy d,e

a Department of Pharmacology and Toxicology, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, 62514, Egyptb Sharjah Institute for Medical Research, Department of Pharmacology, College of Pharmacy, University of Sharjah,

Sharjah, 27272, United Arab Emiratesc Department of Pharmacology and Toxicology, Faculty of Pharmacy, Kafr El-Sheikh University, Egyptd Department of Pharmacology and Toxicology, Ibn sina National College for Medical Studies, Jeddah, Saudi Arabiae Department of Pharmacology and Toxicology, Faculty of Pharmacy, El-minia University, Egypt

a r t i c l e i n f o

Article history:

Received 10 January 2015

Accepted 17 February 2015

Available online 6 March 2015

Keywords:

Ischemia-reperfusion

Nitric oxide synthetase

Nebivolol

Chrysin

Liver injury

Oxidative stress

* Corresponding author. Department of PhaEgypt. Tel.: þ20 01000882828; fax: þ20 082 23

E-mail address: [email protected]

Peer review under the responsibility of Benhttp://dx.doi.org/10.1016/j.bjbas.2015.02.0122314-8535/Copyright 2015, Beni-Suef UniversNC-ND license (http://creativecommons.org/

a b s t r a c t

Oxidative stress plays a key role in the pathogenesis of hepatic ischemia/reperfusion (I/R)-

induced injury, one of the leading causes of liver damage post-surgical intervention,

trauma and transplantation. This study aimed to evaluate the protective effect of nebivolol

and chrysin against I/R-induced liver injury via their vasodilator and antioxidant effects,

respectively. Adult male Wister rats received nebivolol (5 mg/kg) and/or chrysin (25 mg/kg)

by oral gavage daily for one week then subjected to ischemia via clamping the portal triad

for 30 min then reperfusion for 30 min. Liver function enzymes, alanine transaminase

(ALT) and aspartate transaminase (AST), as well as hepatic Myeloperoxidase (MPO), total

nitrate (NOx), glutathione (GSH) and liver malondialdehyde (MDA) were measured at the

end of the experiment. Liver tissue damage was examined by histopathology. In addition,

the expression levels of nitric oxide synthase (NOS) subtypes, endothelial (eNOS) and

inducible (iNOS) in liver samples were assessed by Western blotting and confirmed by

immunohistochemical analysis. Both chrysin and nebivolol significantly counteracted I/R-

induced oxidative stress and tissue damage biomarkers. The combination of these agents

caused additive liver protective effect against I/R-induced damage via the up regulation of

nitric oxide expression and the suppression of oxidative stress. Chrysin and nebivolol

combination showed a promising protective effect against I/R-induced liver injury, at least

in part, via decreasing oxidative stress and increasing nitric oxide levels.

Copyright 2015, Beni-Suef University. Production and hosting by Elsevier B.V. This is an open

access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/

4.0/).

rmacology and Toxicolo17958.u.edu (H.A. Omar).

i-Suef University.

ity. Production and hostilicenses/by-nc-nd/4.0/).

gy, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514,

ng by Elsevier B.V. This is an open access article under the CC BY-

http://creativecommons.org/licenses/by-nc-nd/4.�0/http://creativecommons.org/licenses/by-nc-nd/4.�0/mailto:[email protected]://crossmark.crossref.org/dialog/?doi=10.1016/j.bjbas.2015.02.012&domain=pdfwww.sciencedirect.com/science/journal/23148535www.elsevier.com/locate/bjbashttp://dx.doi.org/10.1016/j.bjbas.2015.02.012http://dx.doi.org/10.1016/j.bjbas.2015.02.012http://dx.doi.org/10.1016/j.bjbas.2015.02.012http://creativecommons.org/licenses/by-nc-nd/4.�0/

b e n i - s u e f u n i v e r s i t y j o u r n a l o f b a s i c a n d a p p l i e d s c i e n c e s 4 ( 2 0 1 5 ) 8 6e9 2 87

1. Introduction

Hepatic ischemia/reperfusion (I/R) injury is a major hepatic

problem that arises in many situations such as liver trans-

plantation, resection, circulatory shock, and post traumatic

interruption of blood supply to liver (Kim et al., 2013). The

release of reactive oxygen species (ROS) underlies the patho-

physiology of the injury during the reperfusion stage which

provokes inflammatory response in the form of activation of

kuppfer cells, inflammatory cytokines and infiltration of leu-

cocytes which, in turn, leads to liver cells damage (Jaeschke,

2006; Schwartz et al., 2013). The activation of Kuppfer cells

leads to the release of ROS such as superoxide, hydroxyl, and

hydrogen peroxide radicals which are the main causes of

tissue damage especially after the release of nitric oxide (NO)

from endothelium upon reperfusion forming peroxynitrite

with oxygen free radicals (Taniai et al., 2004).

In normal liver, there is a delicate balance between NO

vasodilating effect and endothellin (ET) vasoconstricting ef-

fect which controls the vascular rhythm in liver (Jaeschke,

2003; Nakamura et al., 1995). NO is produced in liver by two

subtypes of nitric oxide synthetases; endothelial nitric oxide

synthetase (eNOS) and inducible nitric oxide synthetase

(iNOS) (Tian et al., 2010). eNOS is produced naturally by

endothelium and it is responsible for maintaining blood sup-

ply to the liver. While the production of iNOS is induced in the

endothelial, hepatocyte and other hepatic cells under special

conditions such as decreased blood supply or increased

vasoconstriction injury (Serracino-Inglott et al., 2001). iNOS is

responsible for the vasodilatation of liver sinusoid and

consequent hypotension and stroke following the hepatic

injury (Serracino-Inglott et al., 2001). Thus, we hypothesized

that the use of some agents with vasodilating effects before

the ischemic stage would increase the amount of NO pro-

duced from eNOS and decrease the induction of iNOS during

the reperfusion stage. In addition, the use of agents with anti-

oxidant activities should be beneficial in the treatment of

hepatic injury following I/R.

In the current study, we assessed the potential protective

effect of an NO producing drug such as nebivolol and an

antioxidant such as chrysin against I/R-induced liver injury in

rats. Nebivolol is a long acting vasodilating b1-blocker with

some b3 agonist activity (Chlopicki et al., 2002; Feng et al., 2012;

Gupta andWright, 2008; Maffei and Lembo, 2009). It acts as NO

producer by activating soluble guanylate cyclase which in-

creases cGMP. cGMP acts as a smooth muscle relaxant in

endothelium and may alleviate I/R-induced injury (Parenti

et al., 2000; Sobocanec et al., 2006). Chrysin is a natural

flavone which is used as a dietary supplement found in many

plants, honey, and propolis (Williams et al., 1997). It has many

reported biological activities such as anti-oxidant (Lapidot

et al., 2002; Pushpavalli et al., 2010a), anti-inflammatory

(Gresa-Arribas et al., 2010), Antidiabetic (Luka�cı́nov�a et al.,

2008), anxiolytic properties (Wolfman et al., 1994) as well as

vasorelaxant effects (Duarte et al., 2001).

In an attempt to elaborate the mechanism of the potential

hepatoprotective effects of nebivolol and chrysin, we inves-

tigated its effect on the redox status of liver rats by assessing

the levels of lipid peroxides, NO and protein thiols. In addition,

the expression levels of nitric oxide synthetase subtypes

(eNOS and iNOS) in liver samples were assessed by Western

blotting.

2. Materials and methods

2.1. Animals

Adult male Wister rats weighing 220e250 gm were used for

performing this experiment and were obtained from national

research center (Giza, Egypt), they were housed in cages, 10

animal per each cage and maintained at room temperature

and 12/12 h lightedark cycle and free access ofwater and food.

All animal use and handling were done in accordance with

protocols approved by the Faculty of Pharmacy, Beni-Suef

University Animal Care and Use Committee.

2.2. Surgical procedure and experimental design

Fifty rats were used for performing this study divided into 5

groups, the first one receiving dimethyl sulphoxide (DMSO) in

saline and kept as control, second group is kept as ischemic

control given DMSO in saline, third group was given chrysin

(25fmg/kg/day) (Pushpavalli et al., 2010b), fourth group was

given nebivolol (5 mg/kg/day) (Heeba and El-Hanafy, 2012),

fifth group was given combination (chrysin plus nebivolol). All

rats were pretreated with drugs by oral gavage once daily for 7

consecutive days,after the 30 min of the last dose and over-

night fasting animals were anaesthetized with 70 mg/kg

thiopental sodium (Fouad & Jresat, 2011) and middle laparot-

omy was incised and portal triad (portal vein, hepatic artery,

bile duct) clamped for 30 min (ischemic period) and releasing

the clips for allow reperfusion for another 30 min then the

animal was sacrificed and blood sample collected as well as

liver was excised and median and left lobe was removed for

further biochemical investigations (Dulundu et al., 2007).

2.3. Determination of serum alanine aminotransferase(ALT), aspartate aminotransferase (AST)

Determination of ALT and AST was performed using the

commercially available diagnostic kits (Randox Laboratories

Ltd., UK) according to the manufacturer instructions. The

assay is based on the measurement of hydrazone derivatives

which are produced by the interaction of alpha oxoglutarate

with alanine and aspartate respectively. The amount of

hydrazone derivative is proportional to the quantity of

released enzyme.

2.4. Determination of oxidative stress biomarkers

Hepatic glutathione (GSH) was measured according the

method described before and expressed as mmol/gm wet tis-

sue (Beutler and West, 1977), hepatic malondialdehyde (MDA)

was measured according to the method of Mihara and

Uchiyama (1978)and expressed as nmol/gm wet tissue

(Mihara and Uchiyama, 1978), hepatic Myeloperoxidase (MPO)

was measured according to the method of Harada et al. (1999)

and expressed as U/gm wet tissue (Harada et al., 1999), and

http://dx.doi.org/10.1016/j.bjbas.2015.02.012http://dx.doi.org/10.1016/j.bjbas.2015.02.012

Table 1 e Effects of nebivolol and/or chrysin on serumalanine aminotransferase (ALT) and aspartateaminotransferase (AST) in rats subjected to I/R.

GROUPS ALT(u/l) AST (u/l)

CONTROL 21.7 ± 0.7 103.4 ± 11.2ISCHEMIC 122.4 ± 4.9a 205.0 ± 13.2a

CHRYSIN 70.4 ± 5.2a,b 126.0 ± 8.1b

NEBIVOLOL 81.4 ± 6.7a,b 166.7 ± 17.1a

NEBIVOLOL & CHRYSIN 61.7 ± 9.4a,b 116.8 ± 11.2b

Each group consist of 6e10 animals.

Values are mean ± SEM.a Significant from normal at P < 0.05.b Significant from I/R at P < 0.05.


total nitrate/nitrite was measured according to method of

Miranda et al. (2001) and expressed as mmol/gm wet tissue

(Miranda et al., 2001).

2.5. Histopathology

Autopsy samples were taken from the liver of rats in different

groups and fixed in 10% formol saline for 24 h. Washing was

done in tap water then serial dilutions of alcohol (methyl,

ethyl and absolute ethyl) were used for dehydration. Speci-

mens were cleared in xylene and embedded in paraffin at 56�

in hot air oven for 24 h. Paraffin bees wax tissue blocks were

prepared for sectioning at 4 microns thickness by slidge

microtome. The obtained tissue sections were collected on

glass slides, deparaffinized, stained by hematoxylin &eosin

stain for routine examination then examination was done

through the light electric microscope.

2.6. Western blotting

Western blottingwas performed essentially as reported before

(Omar et al., 2011). Briefly, proteins were extracted from liver

tissues which were subjected to ischemia, and their concen-

trations were determined by the Bradford assay (Bio-Rad, CA,

USA). Equivalent amounts of proteins were subjected to 10%

SDS polyacrylamide gel electrophoresis. Blots were trans-

ferred onto a nitrocellulose membrane using a semidry

transfer cell. After blocking, the membranes were then incu-

bated with the appropriate primary and then secondary an-

tibodies. The immunoblots were detected using enhanced

chemiluminescence.

Table 2 e Effects of nebivolol and/or chrysin on oxidative stres

GROUPS GSH(mg/g wet tissue) MDA(nmol/g we

CONTROL 1.1 ± 0.10 57.5 ± 2.5ISCHEMIC 0.4 ± 0.02a 123.3 ± 5.8CHRYSIN 0.9 ± 0.10b 63.9 ± 5.0NEBIVOLOL 0.7 ± 0.40a,b 56.4 ± 2.6NEBIVOLOL & CHRYSIN 0.9 ± 0.08b 47.9 ± 3.4



2.7. Immunohistochemistry

Liver Samples fixed in paraffin were cutted into 4 mm sections

then deparrafinezed with citrate buffer (pH ¼ 6) (Omar et al.,2009). Samples were rehydrated in methanol and washed

with phosphate buffered saline bath,samples were treated

with primary antibody for iNOS or eNOS for 1 h then washed

and treated with biotinylated secondary antibody for

20 min,washed,incubated with streptividin peroxidase then

with diaminobenzidine with chromogen,finally slides were

counterstained with hematoxylin and visualized under light

microscope,immunopositive patches colored with red brown

color in the cytoplasm and the nucleus is stained with dark

blue color.

2.8. Statistical analysis

All values of results are expressed as Mean ± SE of means. Theresults were analyzed by one way analysis of variance

(ANOVA), followed by Tukey HSD test for multiple compari-

sons using SPSS (version 16). The Differences were considered

significant at P < 0.05.

3. Results

3.1. Effect of nebivolol and/or chrysin on liver enzymes

I/R markedly increased serum ALT and AST by 5- and 2-folds

respectively. The rise of these enzymes was inhibited by

prior administration of chrysin, nebivolol or their combina-

tion to 57%, 66% and 50% for ALT and 61%, 80% and 56% for

AST respectively (Table 1).

3.2. Effect of nebivolol and/or chrysin on oxidative stressbiomarkers

I/R injury markedly increased oxidative stress by decreasing

GSH to 33% and increasing MDA, total NITRATE, and MPO to

227%, 225% and 286% respectively as compared to normal

control. Chrysin effectively antagonized I/R-induced depletion

of glutathione by increasing its level to 226%. It decreased

MDA, total NITRATE and MPO by 52%, 75% and 52% respec-

tively as compared to ischemic group. Nebivolol increased

GSH and total NITRATE by 196% and 111% respectively while

decreased MDA and MPO by 45% and 60% respectively as

s biomarkers in rats subjected to I/R.

t tissue) NITRATE(mM/g wet tissue) MPO(U/g wet tissue)

1.0 ± 0.04 1.2 ± 0.03a 2.1 ± 0.10a 3.3 ± 0.12ab 1.6 ± 0.12a 1.7 ± 0.05a,bb 2.4 ± 0.15a 2.0 ± 0.03a,bb 2.3 ± 0.17a 1.5 ± 0.04a,b


Fig. 1 e Photomicrographs (x40) of rat hepatic tissue stained with hematoxylin & eosin (H&E) stain. A) Section from hepatic

tissue of normal rats with normal histological structure of the central vein and surrounding hepatocytes. B) Section of

hepatic tissue of I/R showing Fatty change in diffuse manner all over the hepatocytes. C) Section of hepatic tissue of group

treated with chrysin followed by I/R injury showing fatty liver changes in some of the hepatocyte. D) Section of hepatic

tissue of group treated with nebivolol followed by I/R injury showing congestion in the central and portal vein as well as

sinusoids in association with diffuse kuppfer cells proliferation in between the hepatocytes. E) Section of hepatic tissue of

group treated with combination of chrysin and nebivolol followed by I/R showing very low fatty changes in hepatocyte.


compared to ischemic group. The combination of chrysin and

nebivolol markedly increased GSH content and total NITRATE

by 226% and 111% respectively while decreasedMDA andMPO

by 38% and 46%respectively as compared to ischemic group

(Table 2).

3.3. Histopathological evaluation

I/R caused some fatty changes noticed in diffusive manner all

over the hepatocyte with degeneration and vacuolization

(ballooning degeneration) which decreasedwith pretreatment

with chrysin and nebivolol. Congestion in central and portal

Table 3 e Effects of nebivolol and/or chrysin on I/R-induced iNOS and eNOS expression in rat hepatic tissuesmeasured by Western blotting.

GROUP eNOS(mg/g wet tissue) iNOS(mg/gm wet tissue)

CONTROL 1.4 ± 0.15 0.1 ± 0.01ISCHEMIC 0.3 ± 0.06a 0.8 ± 0.09a

CHRYSIN 0.7 ± 0.05a 0.7 ± 0.06a

NEBIVOLOL 0.7 ± 0.07a 0.7 ± 0.06a

NEBIVOLOL

&CHRYSIN

1.0 ± 0.15a,b 0.3 ± 0.04a,b



vein as well as sinusoids with diffusive kuppfer cell prolifer-

ation between the hepatocyte was observed in nebivolol

group. Both fatty changes and congestion were nearly absent

in the combination group (Fig. 1).

3.4. Western blotting of iNOS and eNOS

I/R increased the expression of iNOS by about 6-fold as

compared to control group. Pretreatment with chrysin, nebi-

volol or their combination antagonized I/R-induced expres-

sion of iNOS by 83%, 85% and 35% respectively as compared to

ischemic group. With regard to eNOS, I/R group showed a

decrease in the expression of eNOS by 19% as compared to

normal control. Pretreatment with chrysin, nebivolol or their

combination markedly increased the expression of eNOS by

242%, 235% and 350% respectively as compared to ischemic

group (Table 3, Fig. 2).

3.5. Immunohistochemistry

Immunohistochemical examination showed that I/R

decreased the immunoreactivity to eNOS which increased in

the groups pretreated with nebivolol or chrysin or their com-

bination. eNOS is expressed as brown color in the hepatocyte

cytoplasm as compared to control group (Fig. 3). On the other

hand, I/R increased the immunoreactivity to iNOS which was

counteracted by the nebivolol or chrysin or their combination

as compared to control group (Fig. 4).


Fig. 2 e Western blots showing the effect of nebivolol and/or chrysin on the expression of iNOS and eNOS in rat liver tissues

subjected to I/R. N ¼ normal control, I/R ¼ ischemia reperfusion, CHR ¼ chrysin, Neb ¼ nebivolol, Mix ¼ combination ofnebivolol and chrysin


4. Discussion

Hepatic ischemiaereperfusion (I/R) is amajor clinical problem

associated with numerous conditions such as liver trans-

plantation, liver resection, hypovolemic shock and cardiac

failure (Gracia-Sancho et al., 2010). Many pharmacological and

clinical research and trials have been done to protect against I/

R-induced liver injury. However, there is a paradox about the

optimal measures and treatments to protect against the post

ischemic hepatocyte damage.

Because of the complicated pathophysiology of I/R-

induced liver injury, it is generally difficult to achieve

optimal treatment or protection using individual medication.

The goal of the current study was to analyze the single and

combined effects of chrysin and nebivolol pretreatment in

preventing liver damage during hepatic I/R injury. Chrysin is

known to counteract oxidative stress and replenish GSH levels

in vitro and in vivo (Lapidot et al., 2002; Pushpavalli et al.,

2010a). On the other hand, nebivolol is a long acting vaso-

dilating b1-blocker which acts as NO producer by activating

Fig. 3 e Immunohistochemical staining of the inducible nitric ox

of control group (brown color). B) Ischemic group showing incre

Chrysin group showing decreased intensity of staining relative

decreased intensity of staining compared to ischemic or chrysi

showing decreased intensity of staining compared to ischemic

soluble guanylate cyclase which increases cGMP in vascular

smooth muscles (Parenti et al., 2000; Sobocanec et al., 2006).

Our results indicated that decreased NO during the

ischemic stage and increased ROS during reperfusion stage

are themain causes of I/R-induced injury. Nebivolol decreased

hepatocyte damage and partially restored its functions and

morphology. The protective effect of nebivolol was attributed

to its vasodilator effect via the induction of NO production and

release. These results are in accordance with the reported

activity of nebivolol as a vasodilator b1-blocker (Gao et al.,

1991; Wehland et al., 2012). In addition, the observed antiox-

idant effect of nebivolol in the present study by increasing

hepatic glutathione level and the decreasing MDA, MPO and

total nitrate was also reported by Munzel and Gori (2009).

Many studies have shown that nebivolol acts as anti-oxidant

via its ability to scavenge oxygen free radicals (de Groot

et al., 2004; Erdamar et al., 2009).

Kuppfer cell edema and damaged hepatocyte release ROS

and pro-inflammatory cytokines such as TNF-alpha IL-1, and

IL-8 (Mehany et al., 2013; Witthaut et al., 1994). These media-

tors activate the chemotaxis of neutrophils which, in-turn,

ide synthase (iNOS) in rat hepatic tissue (x160). A) Staining

ased intensity of staining and the expression of iNOS. C)

to the ischemic group. D) Nebivolol group showing

n group. E) Combination of nebivolol and chrysin group

group.


Fig. 4 e Immunohistochemical staining of constitutive nitric oxide synthase (eNOS) of rat hepatic tissue subjected to I/R

(x160). A) Staining of control group (brown color). B) Ischemic group showing the lowest color intensity and the lowest

expression of eNOS in the hepatic tissue. C) Chrysin group showing increased intensity of staining relative to ischemic

group. D) Nebivolol group showing increased intensity of staining relative to ischemic group. E) Combination of chrysin and

nebivolol treated group showing increased intensity of staining relative ischemic group.


produce more ROS and increase the production of TNF-alpha

from Kuppfer cells and consequently cause more hepatocyte

damage (Mehany et al., 2013; Perry et al., 2011; Witthaut et al.,

1994).

In the current study, chrysin protected hepatocyte from I/

R-induced injury via its antioxidant activity which was evi-

denced by decreased lipid peroxidation and nitrate production

as well as increased protein thiols in the treated animals. The

antioxidant effect of chrysin is attributed to the hydroxyl

group in carbon 5 which give chrysin complexing activities for

free radicals (Torel et al., 1986). In addition, chrysin decreased

neutrophils adhesion to endothelial cells which was evi-

denced by decreased MPO activity. These results match those

reported by other groups considering chrysin antioxidant ac-

tivity both in vitro and in vivo (Anand et al., 2012; Vollmar et al.,

1994). Furthermore, chrysin inhibited pro-inflammatory cy-

tokines and prevented inflammation by inhibiting TNF-alpha

and NF-kB. These results are in accordance with the re-

ported anti-inflammatory activity of chrysin and its ability to

release NO from the endothelial cells (Ha et al., 2010;

Harasstani et al., 2010; Lee et al., 2009; Parenti et al., 2000).

The combination of nebivolol and chrysin showed more

significant hepatocyte protective effect than the single treat-

ment. The additive effect could be due to the ability of nebi-

volol to decrease the injury during the ischemic stage and

chrysin to decrease the damage during the reperfusion stage.

In conclusion, preconditioning with nebivolol and/or

chrysin had a beneficial effect in protecting the rat liver

against I/R-induced injury. These protective effects could be

beneficial in certain clinical conditions such as cardiovascular

disorders associated with liver failure and waiting for trans-

plantation. Themajor limitation for the current study could be

the possible decrease of nebivolol metabolism in chronic liver

disorders (Hilas and Ezzo, 2009). This combination may

provide a new option for the prevention of I/R-induced liver

injury.

r e f e r e n c e s

Anand KV, Mohamed Jaabir MS, Thomas PA, Geraldine P.Protective role of chrysin against oxidative stress in d-galactose-induced aging in an experimental rat model. GeriatrGerontol Int 2012;12:741e50.

Beutler E, West C. Comment concerning a fluorometric assay forglutathione. Anal Biochem 1977;81:458e60.

Chlopicki S, Kozlovski VI, Gryglewski RJ. No-dependentvasodilation induced by nebivolol in coronary circulation isnot mediated by beta-adrenoceptors or by 5 HT1A-receptors. JPhysiol Pharmacol 2002;53:615e24.

de Groot AA, Mathy MJ, van Zwieten PA, Peters SL. Antioxidantactivity of nebivolol in the rat aorta. J Cardiovasc Pharmacol2004;43:148e53.

Duarte J, Jimenez R, Villar IC, Perez-Vizcaino F, Jimenez J,Tamargo J. Vasorelaxant effects of the bioflavonoid chrysin inisolated rat aorta. Planta Med 2001;67:567e9.

Dulundu E, Ozel Y, Topaloglu U, Toklu H, Ercan F, Gedik N, et al.Grape seed extract reduces oxidative stress and fibrosis inexperimental biliary obstruction. J Gastroenterol Hepatol2007;22:885e92.

Erdamar H, Sen N, Tavil Y, Yazici HU, Turfan M, Poyraz F, et al.The effect of nebivolol treatment on oxidative stress andantioxidant status in patients with cardiac syndrome-X.Coron Artery Dis 2009;20:238e44.

Feng MG, Prieto MC, Navar LG. Nebivolol-induced vasodilation ofrenal afferent arterioles involves beta3-adrenergic receptorand nitric oxide synthase activation. Am J Physiol RenalPhysiol 2012;303:F775e82.

Fouad AA, Jresat I. Therapeutic potential of cannabidiol againstischemia/reperfusion liver injury in rats. Eur J Pharmacol2011;670:216e23.

http://refhub.elsevier.com/S2314-8535(15)00013-X/sref1http://refhub.elsevier.com/S2314-8535(15)00013-X/sref1http://refhub.elsevier.com/S2314-8535(15)00013-X/sref1http://refhub.elsevier.com/S2314-8535(15)00013-X/sref1http://refhub.elsevier.com/S2314-8535(15)00013-X/sref1http://refhub.elsevier.com/S2314-8535(15)00013-X/sref2http://refhub.elsevier.com/S2314-8535(15)00013-X/sref2http://refhub.elsevier.com/S2314-8535(15)00013-X/sref2http://refhub.elsevier.com/S2314-8535(15)00013-X/sref3http://refhub.elsevier.com/S2314-8535(15)00013-X/sref3http://refhub.elsevier.com/S2314-8535(15)00013-X/sref3http://refhub.elsevier.com/S2314-8535(15)00013-X/sref3http://refhub.elsevier.com/S2314-8535(15)00013-X/sref3http://refhub.elsevier.com/S2314-8535(15)00013-X/sref4http://refhub.elsevier.com/S2314-8535(15)00013-X/sref4http://refhub.elsevier.com/S2314-8535(15)00013-X/sref4http://refhub.elsevier.com/S2314-8535(15)00013-X/sref4http://refhub.elsevier.com/S2314-8535(15)00013-X/sref5http://refhub.elsevier.com/S2314-8535(15)00013-X/sref5http://refhub.elsevier.com/S2314-8535(15)00013-X/sref5http://refhub.elsevier.com/S2314-8535(15)00013-X/sref5http://refhub.elsevier.com/S2314-8535(15)00013-X/sref6http://refhub.elsevier.com/S2314-8535(15)00013-X/sref6http://refhub.elsevier.com/S2314-8535(15)00013-X/sref6http://refhub.elsevier.com/S2314-8535(15)00013-X/sref6http://refhub.elsevier.com/S2314-8535(15)00013-X/sref6http://refhub.elsevier.com/S2314-8535(15)00013-X/sref7http://refhub.elsevier.com/S2314-8535(15)00013-X/sref7http://refhub.elsevier.com/S2314-8535(15)00013-X/sref7http://refhub.elsevier.com/S2314-8535(15)00013-X/sref7http://refhub.elsevier.com/S2314-8535(15)00013-X/sref7http://refhub.elsevier.com/S2314-8535(15)00013-X/sref8http://refhub.elsevier.com/S2314-8535(15)00013-X/sref8http://refhub.elsevier.com/S2314-8535(15)00013-X/sref8http://refhub.elsevier.com/S2314-8535(15)00013-X/sref8http://refhub.elsevier.com/S2314-8535(15)00013-X/sref8http://refhub.elsevier.com/S2314-8535(15)00013-X/sref9http://refhub.elsevier.com/S2314-8535(15)00013-X/sref9http://refhub.elsevier.com/S2314-8535(15)00013-X/sref9http://refhub.elsevier.com/S2314-8535(15)00013-X/sref9http://dx.doi.org/10.1016/j.bjbas.2015.02.012http://dx.doi.org/10.1016/j.bjbas.2015.02.012


Gao YS, Nagao T, Bond RA, Janssens WJ, Vanhoutte PM. Nebivololinduces endothelium-dependent relaxations of caninecoronary arteries. J Cardiovasc Pharmacol 1991;17:964e9.

Gracia-Sancho J, Villarreal Jr G, Zhang Y, Yu JX, Liu Y, Tullius SG,et al. Flow cessation triggers endothelial dysfunction duringorgan cold storage conditions: strategies for pharmacologicintervention. Transplantation 2010;90:142e9.

Gresa-Arribas N, Serratosa J, Saura J, Sola C. Inhibition of CCAAT/enhancer binding protein delta expression by chrysin inmicroglial cells results in anti-inflammatory andneuroprotective effects. J Neurochem 2010;115:526e36.

Gupta S, Wright HM. Nebivolol: a highly selective beta1-adrenergic receptor blocker that causes vasodilation byincreasing nitric oxide. Cardiovasc Ther 2008;26:189e202.

Ha SK, Moon E, Kim SY. Chrysin suppresses LPS-stimulatedproinflammatory responses by blocking NF-kappaB and JNKactivations in microglia cells. Neurosci Lett 2010;485:143e7.

Harada N, Okajima K, Kushimoto S, Isobe H, Tanaka K.Antithrombin reduces ischemia/reperfusion injury of rat liverby increasing the hepatic level of prostacyclin. Blood1999;93:157e64.

Harasstani OA, Moin S, Tham CL, Liew CY, Ismail N,Rajajendram R, et al. Flavonoid combinations causesynergistic inhibition of proinflammatory mediator secretionfrom lipopolysaccharide-induced RAW 264.7 cells. InflammRes 2010;59:711e21.

Heeba GH, El-Hanafy AA. Nebivolol regulates eNOS and iNOSexpressions and alleviates oxidative stress in cerebralischemia/reperfusion injury in rats. Life Sci 2012;90:388e95.

Hilas O, Ezzo D. Nebivolol (bystolic), a novel Beta blocker forhypertension. Pharmacol Ther 2009;34:188e92.

Jaeschke H. Molecular mechanisms of hepatic ischemia-reperfusion injury and preconditioning. Am J PhysiolGastrointest Liver Physiol 2003;284:G15e26.

Jaeschke H. Mechanisms of Liver Injury. II. Mechanisms ofneutrophil-induced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions. Am JPhysiol Gastrointest Liver Physiol 2006;290:G1083e8.

Kim JS, Wang JH, Biel TG, Kim DS, Flores-Toro JA, Vijayvargiya R,et al. Carbamazepine suppresses calpain-mediated autophagyimpairment after ischemia/reperfusion in mouse livers.Toxicol Appl Pharmacol 2013;273:600e10.

Lapidot T, Walker MD, Kanner J. Antioxidant and prooxidanteffects of phenolics on pancreatic beta-cells in vitro. J AgricFood Chem 2002;50:7220e5.

Lee S, Kim YJ, Kwon S, Lee Y, Choi SY, Park J, et al. Inhibitoryeffects of flavonoids on TNF-alpha-induced IL-8 geneexpression in HEK 293 cells. BMB Rep 2009;42:265e70.

Luka�cı́nov�a A, Moj�zi�s J, Be�na�cka R, Keller J, Maguth T, Kurila P,et al. Preventive effects of flavonoids on alloxan-induceddiabetes mellitus in rats. Acta Veterinaria Brno2008;77:175e82.

Maffei A, Lembo G. Nitric oxide mechanisms of nebivolol. TherAdv Cardiovasc Dis 2009;3:317e27.

Mehany HA, Abo-youssef AM, Ahmed LA, Arafa E-SA, Abd El-Latif HA. Protective effect of vitamin E and atorvastatinagainst potassium dichromate-induced nephrotoxicity in rats.Beni-Suef Univ J Basic Appl Sci 2013;2:96e102.

MiharaM, UchiyamaM. Determination ofmalonaldehyde precursorin tissuesbythiobarbituricacid test.AnalBiochem1978;86:271e8.

Miranda KM, Espey MG, Wink DA. A rapid, simplespectrophotometric method for simultaneous detection ofnitrate and nitrite. Nitric Oxide 2001;5:62e71.

Munzel T, Gori T. Nebivolol: the somewhat-different beta-adrenergic receptor blocker. J Am Coll Cardiol 2009;54:1491e9.

Nakamura S, Nishiyama R, Serizawa A, Yokoi Y, Suzuki S,Konno H, et al. Hepatic release of endothelin-1 after warm

ischemia. Reperfusion injury and its hemodynamic effect.Transplantation 1995;59:679e84.

Omar HA, Chou CC, Berman-Booty LD, Ma Y, Hung JH, Wang D,et al. Antitumor effects of OSU-2S, a nonimmunosuppressiveanalogue of FTY720, in hepatocellular carcinoma. Hepatology2011;53:1943e58.

Omar HA, Sargeant AM, Weng JR, Wang D, Kulp SK, Patel T, et al.Targeting of the Akt-nuclear factor-kappa B signaling networkby [1-(4-chloro-3-nitrobenzenesulfonyl)-1H-indol-3-yl]-methanol (OSU-A9), a novel indole-3-carbinol derivative, in amouse model of hepatocellular carcinoma. Mol Pharmacol2009;76:957e68.

Parenti A, Filippi S, Amerini S, Granger HJ, Fazzini A, Ledda F.Inositol phosphate metabolism and nitric-oxide synthaseactivity in endothelial cells are involved in the vasorelaxantactivity of nebivolol. J Pharmacol Exp Ther 2000;292:698e703.

Perry BC, Soltys D, Toledo AH, Toledo-Pereyra LH. Tumor necrosisfactor-alpha in liver ischemia/reperfusion injury. J Invest Surg2011;24:178e88.

Pushpavalli G, Kalaiarasi P, Veeramani C, Pugalendi KV. Effect ofchrysin on hepatoprotective and antioxidant status in D-galactosamine-induced hepatitis in rats. Eur J Pharmacol2010a;631:36e41.

Pushpavalli G, Veeramani C, Pugalendi KV. Influence of chrysinon hepatic marker enzymes and lipid profile against D-galactosamine-induced hepatotoxicity rats. Food ChemToxicol 2010b;48:1654e9.

Schwartz J, Holmuhamedov E, Zhang X, Lovelace GL, Smith CD,Lemasters JJ. Minocycline and doxycycline, but not othertetracycline-derived compounds, protect liver cells fromchemical hypoxia and ischemia/reperfusion injury byinhibition of the mitochondrial calcium uniporter. ToxicolAppl Pharmacol 2013;273:172e9.

Serracino-Inglott F, Habib NA, Mathie RT. Hepatic ischemia-reperfusion injury. Am J Surg 2001;181:160e6.

Sobocanec S, Sverko V, Balog T, Saric A, Rusak G, Likic S, et al.Oxidant/antioxidant properties of Croatian native propolis. JAgric Food Chem 2006;54:8018e26.

Taniai H, Hines IN, Bharwani S, Maloney RE, Nimura Y, Gao B,et al. Susceptibility of murine periportal hepatocytes tohypoxia-reoxygenation: role for NO and Kupffer cell-derivedoxidants. Hepatology 2004;39:1544e52.

Tian J, Yan Z, Wu Y, Zhang SL, Wang K, Ma XR, et al. Inhibition ofiNOS protects endothelial-dependent vasodilation in agedrats. Acta Pharmacol Sin 2010;31:1324e8.

Torel J, Cillard J, Cillard P. Antioxidant activity of flavonoids andreactivity with peroxy radical. Phytochmistry 1986;25:383e5.

Vollmar B, Glasz J, Leiderer R, Post S, Menger MD. Hepaticmicrocirculatory perfusion failure is a determinant of liverdysfunction in warm ischemia-reperfusion. Am J Pathol1994;145:1421e31.

Wehland M, Grosse J, Simonsen U, Infanger M, Bauer J, Grimm D.The effects of newer beta-adrenoceptor antagonists onvascular function in cardiovascular disease. Curr VascPharmacol 2012;10:378e90.

Williams CA, Harborne JB, Newman M, Greenham J, Eagles J.Chrysin and other leaf exudate flavonoids in the genusPelargonium. Phytochmistry 1997;46:1349e53.

Witthaut R, Farhood A, Smith CW, Jaeschke H. Complement andtumor necrosis factor-alpha contribute to Mac-1 (CD11b/CD18)up-regulation and systemic neutrophil activation duringendotoxemia in vivo. J Leukoc Biol 1994;55:105e11.

Wolfman C, Viola H, Paladini A, Dajas F, Medina JH. Possibleanxiolytic effects of chrysin, a central benzodiazepinereceptor ligand isolated from Passiflora coerulea. PharmacolBiochem Behav 1994;47:1e4.

http://refhub.elsevier.com/S2314-8535(15)00013-X/sref10http://refhub.elsevier.com/S2314-8535(15)00013-X/sref10http://refhub.elsevier.com/S2314-8535(15)00013-X/sref10http://refhub.elsevier.com/S2314-8535(15)00013-X/sref10http://refhub.elsevier.com/S2314-8535(15)00013-X/sref11http://refhub.elsevier.com/S2314-8535(15)00013-X/sref11http://refhub.elsevier.com/S2314-8535(15)00013-X/sref11http://refhub.elsevier.com/S2314-8535(15)00013-X/sref11http://refhub.elsevier.com/S2314-8535(15)00013-X/sref11http://refhub.elsevier.com/S2314-8535(15)00013-X/sref12http://refhub.elsevier.com/S2314-8535(15)00013-X/sref12http://refhub.elsevier.com/S2314-8535(15)00013-X/sref12http://refhub.elsevier.com/S2314-8535(15)00013-X/sref12http://refhub.elsevier.com/S2314-8535(15)00013-X/sref12http://refhub.elsevier.com/S2314-8535(15)00013-X/sref13http://refhub.elsevier.com/S2314-8535(15)00013-X/sref13http://refhub.elsevier.com/S2314-8535(15)00013-X/sref13http://refhub.elsevier.com/S2314-8535(15)00013-X/sref13http://refhub.elsevier.com/S2314-8535(15)00013-X/sref14http://refhub.elsevier.com/S2314-8535(15)00013-X/sref14http://refhub.elsevier.com/S2314-8535(15)00013-X/sref14http://refhub.elsevier.com/S2314-8535(15)00013-X/sref14http://refhub.elsevier.com/S2314-8535(15)00013-X/sref15http://refhub.elsevier.com/S2314-8535(15)00013-X/sref15http://refhub.elsevier.com/S2314-8535(15)00013-X/sref15http://refhub.elsevier.com/S2314-8535(15)00013-X/sref15http://refhub.elsevier.com/S2314-8535(15)00013-X/sref15http://refhub.elsevier.com/S2314-8535(15)00013-X/sref16http://refhub.elsevier.com/S2314-8535(15)00013-X/sref16http://refhub.elsevier.com/S2314-8535(15)00013-X/sref16http://refhub.elsevier.com/S2314-8535(15)00013-X/sref16http://refhub.elsevier.com/S2314-8535(15)00013-X/sref16http://refhub.elsevier.com/S2314-8535(15)00013-X/sref16http://refhub.elsevier.com/S2314-8535(15)00013-X/sref17http://refhub.elsevier.com/S2314-8535(15)00013-X/sref17http://refhub.elsevier.com/S2314-8535(15)00013-X/sref17http://refhub.elsevier.com/S2314-8535(15)00013-X/sref17http://refhub.elsevier.com/S2314-8535(15)00013-X/sref18http://refhub.elsevier.com/S2314-8535(15)00013-X/sref18http://refhub.elsevier.com/S2314-8535(15)00013-X/sref18http://refhub.elsevier.com/S2314-8535(15)00013-X/sref19http://refhub.elsevier.com/S2314-8535(15)00013-X/sref19http://refhub.elsevier.com/S2314-8535(15)00013-X/sref19http://refhub.elsevier.com/S2314-8535(15)00013-X/sref19http://refhub.elsevier.com/S2314-8535(15)00013-X/sref20http://refhub.elsevier.com/S2314-8535(15)00013-X/sref20http://refhub.elsevier.com/S2314-8535(15)00013-X/sref20http://refhub.elsevier.com/S2314-8535(15)00013-X/sref20http://refhub.elsevier.com/S2314-8535(15)00013-X/sref20http://refhub.elsevier.com/S2314-8535(15)00013-X/sref21http://refhub.elsevier.com/S2314-8535(15)00013-X/sref21http://refhub.elsevier.com/S2314-8535(15)00013-X/sref21http://refhub.elsevier.com/S2314-8535(15)00013-X/sref21http://refhub.elsevier.com/S2314-8535(15)00013-X/sref21http://refhub.elsevier.com/S2314-8535(15)00013-X/sref22http://refhub.elsevier.com/S2314-8535(15)00013-X/sref22http://refhub.elsevier.com/S2314-8535(15)00013-X/sref22http://refhub.elsevier.com/S2314-8535(15)00013-X/sref22http://refhub.elsevier.com/S2314-8535(15)00013-X/sref23http://refhub.elsevier.com/S2314-8535(15)00013-X/sref23http://refhub.elsevier.com/S2314-8535(15)00013-X/sref23http://refhub.elsevier.com/S2314-8535(15)00013-X/sref23http://refhub.elsevier.com/S2314-8535(15)00013-X/sref24http://refhub.elsevier.com/S2314-8535(15)00013-X/sref24http://refhub.elsevier.com/S2314-8535(15)00013-X/sref24http://refhub.elsevier.com/S2314-8535(15)00013-X/sref24http://refhub.elsevier.com/S2314-8535(15)00013-X/sref24http://refhub.elsevier.com/S2314-8535(15)00013-X/sref24http://refhub.elsevier.com/S2314-8535(15)00013-X/sref24http://refhub.elsevier.com/S2314-8535(15)00013-X/sref24http://refhub.elsevier.com/S2314-8535(15)00013-X/sref24http://refhub.elsevier.com/S2314-8535(15)00013-X/sref24http://refhub.elsevier.com/S2314-8535(15)00013-X/sref24http://refhub.elsevier.com/S2314-8535(15)00013-X/sref25http://refhub.elsevier.com/S2314-8535(15)00013-X/sref25http://refhub.elsevier.com/S2314-8535(15)00013-X/sref25http://refhub.elsevier.com/S2314-8535(15)00013-X/sref26http://refhub.elsevier.com/S2314-8535(15)00013-X/sref26http://refhub.elsevier.com/S2314-8535(15)00013-X/sref26http://refhub.elsevier.com/S2314-8535(15)00013-X/sref26http://refhub.elsevier.com/S2314-8535(15)00013-X/sref26http://refhub.elsevier.com/S2314-8535(15)00013-X/sref27http://refhub.elsevier.com/S2314-8535(15)00013-X/sref27http://refhub.elsevier.com/S2314-8535(15)00013-X/sref27http://refhub.elsevier.com/S2314-8535(15)00013-X/sref28http://refhub.elsevier.com/S2314-8535(15)00013-X/sref28http://refhub.elsevier.com/S2314-8535(15)00013-X/sref28http://refhub.elsevier.com/S2314-8535(15)00013-X/sref28http://refhub.elsevier.com/S2314-8535(15)00013-X/sref29http://refhub.elsevier.com/S2314-8535(15)00013-X/sref29http://refhub.elsevier.com/S2314-8535(15)00013-X/sref29http://refhub.elsevier.com/S2314-8535(15)00013-X/sref30http://refhub.elsevier.com/S2314-8535(15)00013-X/sref30http://refhub.elsevier.com/S2314-8535(15)00013-X/sref30http://refhub.elsevier.com/S2314-8535(15)00013-X/sref30http://refhub.elsevier.com/S2314-8535(15)00013-X/sref30http://refhub.elsevier.com/S2314-8535(15)00013-X/sref31http://refhub.elsevier.com/S2314-8535(15)00013-X/sref31http://refhub.elsevier.com/S2314-8535(15)00013-X/sref31http://refhub.elsevier.com/S2314-8535(15)00013-X/sref31http://refhub.elsevier.com/S2314-8535(15)00013-X/sref31http://refhub.elsevier.com/S2314-8535(15)00013-X/sref32http://refhub.elsevier.com/S2314-8535(15)00013-X/sref32http://refhub.elsevier.com/S2314-8535(15)00013-X/sref32http://refhub.elsevier.com/S2314-8535(15)00013-X/sref32http://refhub.elsevier.com/S2314-8535(15)00013-X/sref32http://refhub.elsevier.com/S2314-8535(15)00013-X/sref32http://refhub.elsevier.com/S2314-8535(15)00013-X/sref32http://refhub.elsevier.com/S2314-8535(15)00013-X/sref33http://refhub.elsevier.com/S2314-8535(15)00013-X/sref33http://refhub.elsevier.com/S2314-8535(15)00013-X/sref33http://refhub.elsevier.com/S2314-8535(15)00013-X/sref33http://refhub.elsevier.com/S2314-8535(15)00013-X/sref33http://refhub.elsevier.com/S2314-8535(15)00013-X/sref34http://refhub.elsevier.com/S2314-8535(15)00013-X/sref34http://refhub.elsevier.com/S2314-8535(15)00013-X/sref34http://refhub.elsevier.com/S2314-8535(15)00013-X/sref34http://refhub.elsevier.com/S2314-8535(15)00013-X/sref35http://refhub.elsevier.com/S2314-8535(15)00013-X/sref35http://refhub.elsevier.com/S2314-8535(15)00013-X/sref35http://refhub.elsevier.com/S2314-8535(15)00013-X/sref35http://refhub.elsevier.com/S2314-8535(15)00013-X/sref35http://refhub.elsevier.com/S2314-8535(15)00013-X/sref36http://refhub.elsevier.com/S2314-8535(15)00013-X/sref36http://refhub.elsevier.com/S2314-8535(15)00013-X/sref36http://refhub.elsevier.com/S2314-8535(15)00013-X/sref36http://refhub.elsevier.com/S2314-8535(15)00013-X/sref36http://refhub.elsevier.com/S2314-8535(15)00013-X/sref37http://refhub.elsevier.com/S2314-8535(15)00013-X/sref37http://refhub.elsevier.com/S2314-8535(15)00013-X/sref37http://refhub.elsevier.com/S2314-8535(15)00013-X/sref37http://refhub.elsevier.com/S2314-8535(15)00013-X/sref37http://refhub.elsevier.com/S2314-8535(15)00013-X/sref37http://refhub.elsevier.com/S2314-8535(15)00013-X/sref37http://refhub.elsevier.com/S2314-8535(15)00013-X/sref38http://refhub.elsevier.com/S2314-8535(15)00013-X/sref38http://refhub.elsevier.com/S2314-8535(15)00013-X/sref38http://refhub.elsevier.com/S2314-8535(15)00013-X/sref39http://refhub.elsevier.com/S2314-8535(15)00013-X/sref39http://refhub.elsevier.com/S2314-8535(15)00013-X/sref39http://refhub.elsevier.com/S2314-8535(15)00013-X/sref39http://refhub.elsevier.com/S2314-8535(15)00013-X/sref40http://refhub.elsevier.com/S2314-8535(15)00013-X/sref40http://refhub.elsevier.com/S2314-8535(15)00013-X/sref40http://refhub.elsevier.com/S2314-8535(15)00013-X/sref40http://refhub.elsevier.com/S2314-8535(15)00013-X/sref40http://refhub.elsevier.com/S2314-8535(15)00013-X/sref41http://refhub.elsevier.com/S2314-8535(15)00013-X/sref41http://refhub.elsevier.com/S2314-8535(15)00013-X/sref41http://refhub.elsevier.com/S2314-8535(15)00013-X/sref41http://refhub.elsevier.com/S2314-8535(15)00013-X/sref42http://refhub.elsevier.com/S2314-8535(15)00013-X/sref42http://refhub.elsevier.com/S2314-8535(15)00013-X/sref42http://refhub.elsevier.com/S2314-8535(15)00013-X/sref43http://refhub.elsevier.com/S2314-8535(15)00013-X/sref43http://refhub.elsevier.com/S2314-8535(15)00013-X/sref43http://refhub.elsevier.com/S2314-8535(15)00013-X/sref43http://refhub.elsevier.com/S2314-8535(15)00013-X/sref43http://refhub.elsevier.com/S2314-8535(15)00013-X/sref44http://refhub.elsevier.com/S2314-8535(15)00013-X/sref44http://refhub.elsevier.com/S2314-8535(15)00013-X/sref44http://refhub.elsevier.com/S2314-8535(15)00013-X/sref44http://refhub.elsevier.com/S2314-8535(15)00013-X/sref44http://refhub.elsevier.com/S2314-8535(15)00013-X/sref45http://refhub.elsevier.com/S2314-8535(15)00013-X/sref45http://refhub.elsevier.com/S2314-8535(15)00013-X/sref45http://refhub.elsevier.com/S2314-8535(15)00013-X/sref45http://refhub.elsevier.com/S2314-8535(15)00013-X/sref46http://refhub.elsevier.com/S2314-8535(15)00013-X/sref46http://refhub.elsevier.com/S2314-8535(15)00013-X/sref46http://refhub.elsevier.com/S2314-8535(15)00013-X/sref46http://refhub.elsevier.com/S2314-8535(15)00013-X/sref46http://refhub.elsevier.com/S2314-8535(15)00013-X/sref47http://refhub.elsevier.com/S2314-8535(15)00013-X/sref47http://refhub.elsevier.com/S2314-8535(15)00013-X/sref47http://refhub.elsevier.com/S2314-8535(15)00013-X/sref47http://refhub.elsevier.com/S2314-8535(15)00013-X/sref47http://dx.doi.org/10.1016/j.bjbas.2015.02.012http://dx.doi.org/10.1016/j.bjbas.2015.02.012

Nebivolol and chrysin protect the liver against ischemia/reperfusion-induced injury in rats1. Introduction2. Materials and methods2.1. Animals2.2. Surgical procedure and experimental design2.3. Determination of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST)2.4. Determination of oxidative stress biomarkers2.5. Histopathology2.6. Western blotting2.7. Immunohistochemistry2.8. Statistical analysis

3. Results3.1. Effect of nebivolol and/or chrysin on liver enzymes3.2. Effect of nebivolol and/or chrysin on oxidative stress biomarkers3.3. Histopathological evaluation3.4. Western blotting of iNOS and eNOS3.5. Immunohistochemistry

4. DiscussionReferences

nebivolol and chrysin protect the liver against ischemia/reperfusion-induced injury … · 2017. 1....

Documents