rutin in rat liver ischemia/reperfusion injury: effect on ddah/nos pathway

RUTIN IN RAT LIVER ISCHEMIA/REPERFUSION INJURY: EFFECTON DDAH/NOS PATHWAY

RAFFAEL LANTERI, M.D., Ph.D.,1 ROSARIA ACQUAVIVA, Ph.D.,2* CLAUDIA DI GIACOMO, Ph.D.,2

VALERIA SORRENTI, Ph.D.,2 GIOVANNI LI DESTRI, M.D.,1 MARCO SANTANGELO, M.D.,1 LUCA VANELLA, Pharm.S.,1

and ANTONIO DI CATALDO, M.D.1

Nitric oxide (NO) plays a key role in the relationship between microcirculatory disorders and I/R injuries. Our results demonstrated a signifi-cant modification in the hepatic function of I/R rats compared with the control group; treatment with rutin reported hepatic damage markersto control value. Levels of plasmatic and hepatic thiol groups decreased in the I/R untreated group, and this decrease was inhibited by rutintreatment. In addition, we observed an increase in the iNOS expression in I/R group compared with control and rutin administration attenu-ated this increase; in post-ischemic reperfused rutin-treated rats there was a significant increase in eNOS expression compared with theI/R untreated group. In the same experimental conditions an increase in DDAH 1 expression was observed in I/R group only; rutin treat-ment also counteracted this increased expression. These data suggest that rutin treatment could be useful for preventing oxidative damageassociated with hepatic post-ischemic reperfusion injury. VVC 2007 Wiley-Liss, Inc. Microsurgery 27:245–251, 2007.

Ischemia/reperfusion (I/R) injury of the liver is an impor-

tant clinical problem and a serious postoperative compli-

cation of liver surgery. I/R injury is intimately related to

the inflammatory response which results in microcircula-

tory failure followed by necrosis and cell death. Circula-

tory disorders of the liver can develop after manipulation,

mechanical compression, or occlusion of the hepatic

hylum during experimental I/R model preparation or clin-

ical liver surgery.

Reperfusion affects hepatic function adversely because

it leads to the generation of oxygen reactive species

(ROS), such as superoxide anion, hydrogen peroxide,

hydroxyl radical (OH�), lipid peroxides, or related spe-

cies. Such toxic agents can result in either transient or ir-

reversible tissue damage. It has also been shown that

ROS may lead to inactivation of some enzymes, mito-

chondrial injury, release of proinflammatory cytokines,

breakage of DNA strands and alterations of DNA bases,

apoptosis, necrosis, ultimately, to organ failure.1,2 In

response to increased oxidative stress, cells undergo spe-

cific changes in enzyme activities, cytoskeletal structure,

membrane transport, antioxidant defenses, and induction

of several proteins.3

Intensive research efforts have been focused on the

amelioration of various patho-physiological components of

I/R injury, to limit the extent of tissue injury and necrosis.

Nitric oxide (NO) plays a key role in the relationship

between microcirculatory disorders and I/R injuries.4 It

has been shown that NO, NO donors, and NO synthase

activation or transgenic over-expression exert protective

effects in a number of experimental models of I/R5–7 and

that the suppression of inducible nitric oxide synthase

(iNOS) improves I/R hepatic injury. However other stud-

ies reported harmful effects of NO over-exposure,8,9 sug-

gesting a critical role of dose and duration of NO expo-

sure and indicating a narrow therapeutic safety window

for NO in I/R pathophysiology.2,10

Recently, it was reported that asymmetric dimethylar-

ginine (ADMA), a major endogenous inhibitor of NOS,

could reduce NO production.11 There is growing evidence

that higher circulating levels of ADMA are involved in

endothelial dysfunction in some pathologies.11 In vivo

most of ADMA is degraded by dimethylarginine dime-

thylaminohydrolase (DDAH), which hydrolyzes ADMA

to l-citrulline and dimethylamine. It has been suggested

that a decrease in DDAH activity is a key factor contrib-

uting to the elevation of ADMA levels under some patho-

physiological conditions.12–14 The DDAH/ADMA system

is, therefore, now considered a pathway modulating NO

production and endothelial function.

In view of increasing interest in the protective ‘‘in

vivo’’ effects of natural dietary supplements against oxi-

dative damage, several free radical scavengers have been

evaluated in I/R injury.15,16 Our previous ‘‘in vitro’’ stud-

ies have demonstrated the antioxidant properties of rutin

and, considering the implication of NO-mediated path-

ways in post-ischemic reperfusion liver damage,4,17 the

aim of the present study was to evaluate the effect of

rutin on hepatic structural and functional parameters in

rats undergoing an experimental model of hepatic ische-

mia/reperfusion, and its involvement in the DDAH/NOS

pathway. Consequently, plasmatic alanine aminotransfer-

1Department of Surgical Sciences, Organ Transplantation and AdvancedTechnologies, University of Catania, Catania, Italy2Department of Biochemistry, Medical Chemistry and Molecular Biology, Uni-versity of Catania, Catania, Italy

Grant sponsor: MURST (Ministero dell’Universita e della Ricerca Scientifica eTecnologica), Italy.

*Correspondence to: Prof. Rosaria Acquaviva Pharm.D., Ph.D., Departmentof Biochemistry, Medical Chemistry and Molecular Biology, University ofCatania, V.le Andrea Doria 6, Catania, Italy. E-mail: [email protected]

Received 11 March 2007; Accepted 14 March 2007

Published online 3 May 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/micr.20345

VVC 2007 Wiley-Liss, Inc.

ase (ALT), aspartate aminotransferase (AST) activities,

lipid hydroperoxides (LOOH), and thiol groups (RSH)

levels were evaluated. Further, hepatic DNA fragmenta-

tion, endothelial nitric oxide synthase (eNOS), iNOS,

DDAH-1, DDAH-2 expressions, DDAH enzyme activity

were evaluated with or without the treatment with rutin.

MATERIALS AND METHODS

Chemicals

Rutin was purchased from Sigma Aldrich (St. Louis,

MO), ALT, and AST kits were Chematil and Qiamp

DNA mini kit was purchased from Qiagen.

Monoclonal eNOS antibody was purchased from

Stressgen Biotechnologies (Victoria, BC, Canada). iNOS

antibody and secondary horseradish peroxidase-conju-

gated anti-mouse antibody were Santa Cruz Biotechnol-

ogy (Santa Cruz, CA). DDAH 1 and DDAH 2 antibodies

were Calbiochem EMD Biosciences (Darmstadt, Ger-

many). The Enhanced Chemiluminescence System for

developing immunoblots and nitrocellulose membranes

was purchased from Amersham (Milano, Italy).

Animals and Experimental Protocol

All the experimental procedures met the guidelines of

Institutional Animal Care and Use Commitee of Univer-

sity of Catania, Italy. Male Wistar rats (200–220 g b.w.)

were fed with balanced diet and kept in temperature (20

8C) and humidity (50%) controlled rooms. The animals

fasted for 12 h before experiments but were allowed free

access to water.

One lot of animals was treated with rutin (30 mg/Kg

b.w. i.p. once/day for 3 days); 1 h after last rutin admin-

istration, animals were submitted to experimental surgical

procedure. Another group of rats, placed under the same

experimental conditions but treated with saline solution,

was considered as reference group.

Surgical Procedure

Both rutin and saline-treated animals underwent nor-

mothermic ischemia by selective occlusion of the portal

vein and hepatic artery for 30 min, using vascular clips

with a closing force of 0.95 N. A group of six animals

was killed immediately after 30 min ischemia (ischemic

rats). In another group of six animals, after 30 min ische-

mia the blood flow was restored for 3 h and the animals

were then killed (post-ischemic reperfused rats). In sham-

operated animals portal vein and hepatic artery were

exposed without occluding.

Sample Preparation

At the end of the reperfusion, 5 ml of blood was col-

lected from the caval vein in heparinized tubes. Samples

were centrifuged at 800g for 10 min at room temperature

to separate plasma for analysis of ALT, AST, RSH, and

LOOH levels. Livers were rapidly removed in a cold

room and processed for biochemical analysis.

Liver Biochemical Analysis

Livers for biochemical investigation were homoge-

nized in nine volumes of cold PBS. Aliquots of homoge-

nate were used to evaluate hepatic RSH and LOOH lev-

els, DNA fragmentation, DDAH enzyme activity assay

eNOS, iNOS, DDAH 1, and DDAH 2 expressions.

Determination of Plasma Alanine and Aspartate

Aminotransferase Activities

Plasmatic ALT and AST activities were evaluated, in

100 ll of plasma, using a spectrophotometric method at

k ¼ 340 nm3.

Determination of Aspartate Aminotransferase

Activity

Plasmatic AST activity was evaluated, in 100 ll of

plasma, using a spectrophotometric assay at k ¼ 340 nm3.

Determination of Lipid Hydroperoxide Levels

Both plasmatic and hepatic levels of lipid hydroperox-

ide were evaluated following the oxidation of Feþ2 to

Feþ3 in the presence of xylenol orange at k ¼ 560 nm3.

Thiol Group Determination

Both plasmatic and hepatic levels of thiol groups

were measured, in 200 ll of plasma or liver homogenate,

by using a spectophotometric assay.3

DNA Fragmentation

Genomic DNA was isolated from liver homogenate

with the Qiamp DNA micro kit (Qiagen) according to the

manufacturer’s instructions, and electrophoreses on a 2%

agarose gel stained with ethidium bromide.3

DDAH Activity Assay

Hepatic homogenate was centrifugated at 5000g for

60 min at 4 8C and supernatants were collected for

DDAH activity assay, performed by determining L-citrul-

line formation in 96-well microtirer plate, according to

Knipp’s method.18

Western Blotting

Liver homogenates were collected for Western blot

analysis and protein levels were visualized by immuno-

blotting with antibodies eNOS, iNOS, DDAH 1, or

DDAH 2.

Briefly, aliquots of homogenate containing 50 lg of

proteins were separated by sodium dodecyl sulfate/poly-

acrylamide gel electrophoresis and transferred to a nitro-

cellulose membrane. To block nonspecific binding sites,

246 Lanteri et al.

Microsurgery DOI 10.1002/micr

the membranes were incubated overnight with 5% nonfat

dry milk in 10 mM Tris-HCl (pH 7.4), 150 mM NaCl,

0.05% Tween 20 (TBST) buffer at 4 8C. After washing

with TBST, the membranes were incubated with a 1:1000

dilution of anti-DDAH 1 and DDAH 2 antibody and with

1:500 dilution of anti-eNOS and i-NOS over night at

room temperature with constant agitation. The filters

were then washed and subsequently probed with horse-

radish peroxidase-conjugated anti-goat for DDAH 1 and

DDAH 2 at a dilution of 1:10,000, anti-rabbit for eNOS

and iNOS at a dilution of 1:20,000. Chemiluminescence

detection was performed using an Enhanced Chemilumi-

nescence Detection kit according to the manufacturer’s

instructions.

Protein Assay

Protein content was evaluated according to the

method of Lowry.19

Statistical Analysis

One-way analysis of variance (ANOVA) followed by

Bonferroni’s t test was performed to estimate significant

differences among groups. Data were reported as mean

values 6 SD and differences between groups were con-

sidered to be significant at P < 0.005.

RESULTS

Data obtained in the present study demonstrated sig-

nificant alterations in hepatic functions in untreated I/R

rats when compared with sham-operated animals; in fact,

significant increases in plasmatic ALT and AST activities

were observed (ALT: percentage increment over to con-

trol of untreated I/R 380 6 5 vs. sham-operated animals;

AST: percentage increment over to control of untreated

I/R 350 6 12 vs. sham-operated animals; P < 0.001)

(Fig. 1). These enzymes are liver specific and are

Figure 1. Plasmatic ALT and AST levels. Results are expressed as percentage increment over to control. Value are the mean 6 SD of

four experiments in duplicate. P < 0.001.

Figure 2. Plasmatic and hepatic lipid hydroperoxides levels. Plasmatic LOOH levels are expressed as nanomolar per milliliter plasma; he-

patic LOOH levels are expressed as nanomolar per millgram protein. Value are the mean 6 SD of four experiments in duplicate. P <

0.001.

Rutin and DDAH/NOS in I/R Hepatic Injury 247


released from the liver during injury. Their increased ac-

tivity in plasma thus represents a marker of liver damage.

Treatment with rutin significantly reduced plasmatic ALT

and AST activity in I/R rats while it did not induce sig-

nificant modifications in treated, non ischemic rats (ALT:

rutin-treated rats and I/R 200 6 3.5 vs. untreated I/R 380

6 5; AST: I/R rutin-treated rats 190 6 7.8 vs. untreated

I/R 350 6 12; P < 0.001) (Fig. 1).

Liver damage was also confirmed by the elevated plas-

matic and hepatic LOOH levels found in I/R rats (plas-

matic LOOH: untreated I/R 1.6 6 0.04 vs. untreated con-

trol 0.21 6 0.09; P < 0.001. Hepatic LOOH untreated I/R

0.9 6 0.085 vs. untreated control 0.06 6 0.07 P < 0.001)

(Fig. 2). Treatment with rutin effectively counteracted the

increased lipoperoxidation in post-ischemic reperfused rats

(plasmatic LOOH: untreated I/R 1.6 6 0.04 vs. I/R rutin-

treated rats 0.58 6 0.05; P < 0.001. Hepatic LOOH

untreated I/R 0.9 6 0.085 vs. I/R rutin-treated rats 0.2 60.06; P < 0.001) (Fig. 2). Hepatic damage was also eval-

uated by total thiol group determination; significantly

lower plasmatic and hepatic RSH levels were found in

untreated I/R rats with respect to controls; treatment with

rutin supplemented the ��SH group defenses in I/R rats so

that these levels remained high (plasmatic RSH: untreated

I/R 0.12 6 0.03 vs. controls 0.195 6 0.04; P < 0.001.

Hepatic RSH: untreated I/R 160 6 0.04 vs. I/R rutin-

treated rats 246 6 0.05; P < 0.001) (Fig. 3).

A marked DNA laddering induced by ischemia (lane

4) is evident with respect to control (lane 2) and rutin-

treated (lane 3). This laddering is further increased by

reperfusion (lane 5). DNA ladderization in I/R rats was

significantly attenuated by treatment with rutin (lane 6)

(Fig. 4).

As shown in Figure 5, a significant increase in iNOS

expression was observed in the I/R group with respect to

control (untreated I/R 6.4 6 0.2 vs. control 3.1 6 0.5;

P < 0.001); rutin administration attenuated this increase

(untreated I/R 6.4 6 0.2 vs. I/R rutin-treated rats 2.75 60.3; P < 0.001). No significant difference in eNOS

expression was observed between I/R group and control;

in rutin-treated I/R a significant increase compared with

the I/R group was detected (Fig. 5).

Figure 3. Plasmatic and hepatic RSH levels. Plasmatic RSH levels are expressed as micromolar per milliliter plasma; hepatic RSH levels

are expressed as micromolar per milligram protein. Value are the mean 6 SD of four experiments in duplicate. P < 0.001.

Figure 4. DNA fragmentation. Lane 1: marker, lane 2: control, lane

3: rutin-treated group, lane 4: ischemic group, lane 5: ischemic and

reperfusion group, lane 6: post-ischemic reperfused and rutin

treated.

248 Lanteri et al.


Figure 6A reports the DDAH 1 expression. A signifi-

cant increase in this protein was observed in I/R group

only; rutin treatment also counteracted this increased

expression (untreated I/R 1.32 6 0.35 vs. I/R rutin-

treated rats 0.92 6 0.15; P < 0.001).

No significant difference in DDAH 2 expression was

observed among the experimental groups (Fig. 6B).

Figure 7 reports DDAH enzymatic activity; the increased

activity found in I/R rats was significantly reduced in the

I/R rutin treated group (untreated I/R 0.98 6 0.15 vs. I/R

rutin-treated rats 0.66 6 0.21; P < 0.001), while no sig-

nificant modification was observed between treated and

untreated sham operated animals.

DISCUSSION

I/R injury remains an unsolved problem in organ trans-

plantation.20 The transplantation procedure requires cold

preservation and warm reperfusion of liver grafts, result-

ing in some degree of cold I/R injury in all liver grafts.

The ischemic period occurring during organ retrieval,

preservation, and implantation is necessary in organ trans-

plantation. Although donor/recipient factors are important

in terms of organ outcome, it is well known that there is

a deterioration of organ function post-transplantation,

when cold ischemia time is extended more than 24 h.21,22

Oxidative damage is thought to play an important

role in ischemia injury and the outcome of organ trans-

plantation. In the liver, ROS may contribute significantly

to hepatic injury in the post ischemic period.23,24 It was

been suggested that lipid peroxidation, is closely related

to I/R-induced tissue damage.3,20,25 In agreement with

other authors, results obtained in the present study con-

firm that plasmatic and hepatic LOOH levels significantly

increased in post-ischemic reperfusion3,25; the pretreat-

ment with the natural antioxidant rutin, resulted in a sig-

nificant decrease in LOOH levels, suggesting that the

protective effect of this drug may be due, in part, to its

antioxidant capacity.17

The role of oxidative stress in post-ischemic reperfu-

sion liver damage is furtherly confirmed by significant

decreases in plasmatic and hepatic RSH levels found in

rats undergoing to post-ischemic reperfusion and are in

agreement with other authors reporting a significant dif-

ference in RSH levels between control and I/R groups.26

In this study, oxidative stress involvement in liver

damage was also confirmed by an increased DNA frag-

mentation observed in I/R groups compared with control

group.

In the present study, we observed that I/R-induced

increases in plasmatic levels of liver transaminases (AST

and ALT) and in both liver and plasma LOOH levels

Figure 5. Immunoblotting of iNOS (A) and eNOS (B) levels in all groups. Values are expressed as densitometric units corresponding to

signal intensity present on the autoradiographs. The values are the mean 6 SD of four experiments performed in duplicate. P < 0.001.



were significantly reduced by treatment with rutin. This

treatment was also able to inhibit plasmatic and hepatic

decreases in RSH levels and to protect against DNA frag-

mentation.

Emerging evidence suggests that also NO has an im-

portant role in ischemia injury; however there are con-

flicting reports regarding the action of NO in reperfusion

damage. Several reports suggested that moderate levels of

NO, generated by e-NOS, may be beneficial for its vaso-

dilatator action, whereas high levels of NO, produced by

iNOS, interacting with superoxide anion can produce the

peroxynitrite anion, a potent oxidant associated to patho-

logical liver conditions,27,28 which spontaneously cleaves

to OH radical and nitrogen dioxide.

Moreover, NO generated by iNOS, may induce leuko-

cyte adhesion, inflammatory cell infiltration, and paren-

chyma cell dysfunction.4 In fact, over-expression of

iNOS has been correlated with several acute and chronic

diseases. In our study, we observed a significant increase

in iNOS expression in the I/R group with respect to con-

trol and treatment with rutin attenuated this increase. No

significant difference in eNOS expression was observed in

I/R group compared with control, whereas, in post-isch-

emic reperfused, rutin-treated rats there was a significant

increase respect to the I/R group.

The present study also evaluated the dynamic inter-

play between DDAH and NOS expression and the

involvement of rutin in DDAH/NOS pathway. Data

obtained suggest that increased iNOS expression may be

attributable to DDAH undergoing modifications and,

therefore, protective effects of rutin may be due both to

its scavenger capacity and the antioxidant properties of

eNOS-derived NO.29,30

CONCLUSIONS

The majority of in vitro and in vivo studies have

attributed the protective effect of rutin to its chemical

reactivity toward free radicals and its antioxidant17; our

results confirm that rutin provide protection in post-isch-

emic reperfusion injury by participating in the cellular

Figure 6. Immunoblotting of DDAH1 (A) and DDAH2 (B) levels in all groups. Values are expressed as densitometric units corresponding to

signal intensity present on the autoradiographs. The values are the mean 6 SD of four experiments performed in duplicate. P < 0.001.

Figure 7. DDAH activity. The values are the mean 6 SD of four

experiments performed in duplicate. P < 0.001.

250 Lanteri et al.


defense systems against oxidative damage; however, we

suggest that the protective effect of rutin may be also

due to its capacity to interfere with NO/DDAH pathway.

In fact, we hypothesize that, reducing DDAH activity in

I/R rats, rutin induces an accumulation of ADMA, the en-

dogenous inhibitor of NOS isoforms.

In the present study, we demonstrated that this con-

clusion is supported by the following results: 1) DDAH 1

expression and activity undergo modification (increase)

following ischemia and reperfusion (Figs. 6 and 7); 2)

the administration of rutin to I/R rats induced an increase

in DDAH expression and a decrease in DDAH activity

compared with control and rutin-treated sham operated

animals. So rutin, although inducing an increase in

DDAH expression, reduced DDAH enzymatic activity,

favoring the accumulation of molecules able to antago-

nize over-production of NO from iNOS.

In conclusion, our data demonstrate that rutin treat-

ment might provide potent protection against I/R hepatic

injury because it acts both as antioxidant and is involved

in DDAH/NOS pathway.

ACKNOWLEDGMENT

The authors thank Dr. Mike Wilkinson for proofread-

ing the manuscript.

REFERENCES

1. Warner HR. Superoxide dismutase, aging and degenerative diseases.Free Rad Biol Med 1994;17:249–258.

2. Duranski MR, Gree JJ, Dejam A, Jaganmohan S, Hogg N, LangstonW, Patel RP, Yet SF, Wang X, Kevil CG, Gladwin MT, Lefer DJ.Cytoprotective effects of nitrite during in vivo ischemia–reperfusionof the heart and liver. J Clin Invest 2005;115:1232–1240.

3. Lanteri R, Acquaviva R, Di Giacomo C, Li Destri G, Santangelo M,Vanella L, Caltabiano R, Lanzafame S, Di Cataldo A. Heme oxy-genase 1 expression in post-ischemic reperfusion liver damage:Effect of L-Arginine. Microsurgery 2006;26:25–32.

4. Lanteri R, Greco R, Licitra E, Di Benedetto F, Li Destri G, Di Cata-ldo A. Ischemia and hepatic reperfusion: Is it possible to reduce he-patic alterations? Microsurgery 2003;23:458–460.

5. Lefer DJ. Myocardial protective actions of nitric oxide donors aftermyocardial ischemia and reperfusion. New Horiz 1995;3:105–112.

6. Jones SP, Greer JJ, Kakkar AK, Ware PD, Turnage RH, Hicks M,van Haperen R, de Crom R, Kawashima S, Yokoyama M, Lefer DJ.Endothelial nitric oxide synthase overexpression attenuates myocar-dial reperfusion injury. Am J Physiol 2004;286:H276–H282.

7. Kanno S, Lee PC, Zhang Y, Ho C, Griffith BP, Shears LL II, BiliarTR. Attenuation of myocardial ischemia/reperfusion injury by super-induction of inducible nitric oxide synthase. Circulation 2000;101:2742–2748.

8. Menezes J, Hierholzer C, Watkins SC, Lyons V, Peitzman AN, Bil-liar TR, Tweardy DJ, Harbrecht BG. A novel nitric oxide scavengerdecreases liver injury and improves survival after hemorrhagicshock. Am J Physiol 1999;277:G144–G151.

9. Woolfson RG, Patel VC, Neild GH, Yellon DM. Inhibition of nitricoxide synthesis reduces infarct size by an adenosine-dependentmechanism. Circulation 1995;91:1545–1551.

10. Bolli R. Cardioprotective function of inducible nitric oxide synthaseand role of nitric oxide in myocardial ischemia and preconditioning:An overview of a decade of research. J Mol Cell Cardiol 2001;33:1897–1918.

11. Jiang DJ, Jia SJ, Yan J, Zhou Z, Yuan Q, Li YJ. Involvement ofDDAH/ADMA/NOS pathway in nicotine-induced endothelia dys-function. Bioch Biophys Res Commun 2006;349:683–693.

12. Ito A, Tsao PS, Adimoolam S, Kimoto M, Ogawa T, Cooke JP.Novel mechanism for endothelial dysfunction:dysregulation of dime-thylarginine dimethylaminohydrolase. Circulation 1999;99:3092–3095.

13. Lin KY, Ito A, Asagami T, Tsao PS, Adimoolam S, Kimoto M,Tsuji H, Reaven GM, Cooke JP. Impaired nitric oxide synthase path-way in diabetes mellitus: Role of asymmetric dimethylarginine anddimethylarginine dimethylaminohydrolase. Circulation 2002;106:987–992.

14. Stuhlinger MC, Tsao PS, Her JH, Kimoto M, Balint RF, Cooke JP.Homocysteine impairs the nitric oxide synthase pathway: Role ofasymmetric dimethylarginine. Circulation 2001;104:2569–2575.

15. Grupta R, Singh M, Sharma A. Neuroprotective effect of antioxi-dants on ischemia and reperfusion-induced cerebral injury. Pharma-col Res 2003;48:209–211.

16. Su JF, Guo CJ, Wei JY, Yang JJ, Jiang YG, Li YF. Protectionagainst hepatic ischemia-reperfusion injury in rats by oral pre-treat-ment with quercetin. Biomed Environ Sci 2003;16:1–8.

17. Russo A, Acquaviva R, Campisi A, Sorrenti V, Di Giacomo C, Vir-gata G, Barcellona ML, Vanella A. Bioflavonoids as antiradicals,antioxidants and DNA cleavage protectors. Cell Biol Toxicol 2000;16:91–98.

18. Knipp M, Vasak M. A colorimetric 96-well microtirer plate assayfor the determination of enzymatically formed citrulline. Analyt Bio-chem 2000;286:257–264.

19. Lowry OH, Rosebrough NJ, Farr AL, Randal RJ. Protein measure-ment with the Folin phenol reagent. J Biol Chem 1951;193:265–275.

20. Jassem W, Roake JA. The molecular and cellular basis of reperfu-sion injury following organ transplantation. Transplant Rev 1998;12:14–33.

21. Ploeg RJ, Ploeg RJ, D’Alessandro AM, Knechtle SJ, Stegall MD,Pirsch JD, Hoffmann RM, Sasaki T, Sollinger HW, Belzer FO,Kalayoglu M. Risk factors for primary dysfunction after liver trans-plantation—A multivariate analysis. Transplantation 1993;55:807–813.

22. Belle SH, Belle SH, Beringer KC, Murphy JB, Plummer CC, BreenTJ, Edwards EB, Daily OP, Detre KM. Liver transplantation in theUnited States 1988 to 1990. Clin Transplant 1991;13:13–29.

23. Lemasters JJ, Thurman RG. Reperfusion injury: A role for neutro-phils. Hepatology 1991;14:954–955.

24. Caldwell-Kenkel JC, Currin RT, Tanaka Y, Thurman RG, LemastersJJ. Kupffer cell activation and endothelial cell damage after storageof rat livers: Effects of reperfusion. Hepatology 1991;13:83–95.

25. Kacmaz A, User Y, Sehirli AO, Tilki M, Ozkan S, Sener G. Protec-tive effect of melatonin against ischemia/reperfusion-induced oxida-tive remote organ injury in the rat. Surg Today 2005;35:744–750.

26. Rudiger HA, Graf R, Clavien PA. Sub-lethal oxidative stress triggersthe protective effects of ischemic preconditioning in the mouse liver.J Hepatol 2003;39:972–977.

27. Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA.Apparent hydroxyl radical production by peroxynitrite: implicationsfor endothelial injury from nitric oxide and superoxide. Proc NatlAcad Sci USA 1990;87:1620–1624.

28. Koken T, Inal M. The effect of nitric oxide on ischemia-reperfusioninjury in rat liver. Clin Chim Acta 1999;288:55–62.

29. Wink DA, Miranda KM, Espey MG, Pluta RM, Hewett SJ, ColtonC, Vitek M, Feelisch M, Grisham MB. Mechanisms of the antioxi-dant effects of nitric oxide. Antioxid Redox Signal 2001;3:203–213.

30. Lu P, Liu F, Wang CY, Chen DD, Yao Z, Tian Y, Zhang JH, WuYH. Gender differences in hepatic ischemic reperfusion injury inrats are associated with endothelial oxide synthase-derived nitric ox-ide. World J Gastroenterol 2005;14:3441–3445.



rutin in rat liver ischemia/reperfusion injury: effect on ddah/nos pathway

Documents