ARTICLE

Vinpocetine protects liver against ischemia–reperfusion injuryHala Fahmy Zaki and Rania Mohsen Abdelsalam

Abstract: Hepatic ischemia–reperfusion (IR) injury is a clinical problem that leads to cellular damage and organ dysfunctionmediated mainly via production of reactive oxygen species and inflammatory cytokines. Vinpocetine has long been used incerebrovascular disorders. This study aimed to explore the protective effect of vinpocetine in IR injury to the liver. Ischemia wasinduced in rats by clamping the common hepatic artery and portal vein for 30 min followed by 30 min of reperfusion. Serumtransaminases and liver lactate dehydrogenase (LDH) activities, liver inflammatory cytokines, oxidative stress biomarkers, andliver histopathology were assessed. IR resulted in marked histopathology changes in liver tissues coupled with elevations inserum transaminases and liver LDH activities. IR also increased the production of liver lipid peroxides, nitric oxide, andinflammatory cytokines interleukin-1� and interleukin-6, in parallel with a reduction in reduced glutathione and interleukin-10in the liver. Pretreatment with vinpocetine protected against liver IR-induced injury, in a dose-dependent manner, as evidencedby the attenuation of oxidative stress as well as inflammatory and liver injury biomarkers. The effects of vinpocetine werecomparable with that of curcumin, a natural antioxidant, and could be attributed to its antioxidant and anti-inflammatoryproperties.

Key words: liver ischemia–reperfusion, vinpocetine, curcumin, cytokines, oxidative stress.

Résumé : Le dommage d'ischémie–reperfusion (IR) hépatique constitue un problème clinique qui mène a un dommage cellulaireet a un dysfonctionnement de l'organe, principalement a la suite de la production d'espèces réactives d'oxygène et de cytokinesinflammatoires. La vinpocétine a longtemps été utilisée dans les cas de maladies cérébrovasculaires. La présente étude visait aexplorer les effets protecteurs de la vinpocétine sur le dommage d'IR hépatique. L'ischémie a été provoquée chez le rat par leclampage de l'artère hépatique et de la veine porte pendant 30 minutes, suivi d'une reperfusion de 30 minutes. L'activité destransaminases sériques et de la lactate déshydrogénase (LDH) hépatique, les cytokines inflammatoires et les biomarqueurs dustress oxydant ont été examinés, et l'examen histopathologique du foie a été réalisé. L'IR provoquait d'importants changementshistopathologiques dans les tissus hépatiques, accompagnés d'une élévation de l'activité des transaminases sériques et de la LDHhépatique. L'IR accroissait aussi la production de peroxydes lipidiques, d'oxyde nitrique et de cytokines inflammatoires commel'interleukine-1 bêta et l'interleukine-8, parallèlement a une réduction des contenus en glutathion réduit et en interleukine-10dans le foie. Un prétraitement a la vinpocétine protégeait le foie du dommage induit par l'IR en fonction de la concentration, telque démontré par une diminution du stress oxydant et des marqueurs inflammatoires et du dommage hépatique. Les effets dela vinpocétine étaient comparables a ceux de la curcumine, un antioxydant naturel, et ils pourraient être attribuables a sespropriétés anti-oxydantes et anti-inflammatoires. [Traduit par la Rédaction]

Mots-clés : ischémie–reperfusion hépatique, vinpocétine, curcumine, cytokines, stress oxytant.

IntroductionHepatic ischemia–reperfusion (IR) injury is an important clini-

cal problem that complicates liver surgery and transplantation.The pathophysiology underlying hepatic IR injury is complicated,involving oxidative stress as well as inflammatory and apoptoticmechanisms (Vardanian et al. 2008). Interruption of blood flow tothe liver and the subsequent reperfusion lead to an acute oxida-tive stress response that may cause significant cellular damageand organ dysfunction (Lanteri et al. 2006).

Hepatocellular injury during both the initial and later phases ofreperfusion is partially caused by reactive oxygen species (ROS)(Jaeschke and Farhood 1991; Jaeschke 2003). Accordingly, antioxi-dant therapy can be used to limit IR injury (Glantzounis et al.2005).

Curcumin (CR; 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5 dione), a yellow-orange dye extracted from the Indian spiceturmeric, has been reported to possess multiple effects includingantioxidant, anticarcinogenic, antimicrobial, and anti-inflammatory

activities (Aggarwal et al. 2007). CR administration has been shownto ameliorate IR injury in rat intestine, liver, and kidney (Shen et al.2007; Awad and El-Sharif 2011; Lin et al. 2012; Onder et al. 2012).

Vinpocetine, a derivative of the alkaloid vincamine, has longbeen used for cerebrovascular disorders and cognitive impair-ment. The neuroprotective effects of vinpocetine are mediated viathe blockade of sodium channels, reducing calcium influx intoneuronal cells, as well as acting as an antioxidant (Zelles et al.2001; Mendoza et al. 2007; Sitges et al. 2007). Vinpocetine has beenfound to interfere with various stages of the ischemic cascade:ATP depletion, activation of voltage-sensitive Na2+ and Ca2+-channels, release of glutamate and free radicals (Hadjiev 2003;Sitges et al. 2007).

The neuroprotective effect of vinpocetine has been reportedwith respect to IR injury in hippocampal neuronal cells, both invivo and in vitro, (Rischke and Krieglstein 1991; Solanki et al.2011). Its role in protecting against IR injury in the liver, however,remains unexplored. This study aimed to investigate the protec-tive effect of vinpocetine in IR injury in the liver. CR was chosen as

Received 12 March 2013. Accepted 9 July 2013.

H.F. Zaki and R.M. Abdelsalam. Pharmacology and Toxicology Department, Faculty of Pharmacy, Cairo University, Faculty of Pharmacy, CairoUniversity, El-Kasr El-Eini Street, Cairo, Egypt.Corresponding author: Rania Mohsen Abdelsalam (e-mail: ranmohsen@hotmail.com).

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Can. J. Physiol. Pharmacol. 91: 1064–1070 (2013) dx.doi.org/10.1139/cjpp-2013-0097 Published at www.nrcresearchpress.com/cjpp on 19 August 2013.

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a reference standard. The parameters chosen to assess IR damageincluded estimation of serum transaminases in addition to liverlactate dehydrogenase (LDH) activity as markers of liver injuryand ischemia, respectively. Moreover, the levels of inflammatorycytokines and oxidative stress biomarkers were assessed in theliver, as well as histopathologic examination of liver tissues.

Material and methods

Drugs and chemicalsCR and vinpocetine were obtained from Sigma–Aldrich, USA.

They were suspended in 1% Tween 80 shortly before admini-stration to the animals. The concentrations of the drugs wereadjusted so that each animal received 1 mL of either the CR orvinpocetine suspension (containing the appropriate dose) per100 g of body mass. All of the other chemicals were of the highestgrade commercially available.

AnimalsMale, albino, Wistar rats, 150–200 g body mass, were purchased

from the National Cancer Institute, Cairo, Egypt, and left to accli-matise in the animal facility of the Faculty of Pharmacy, CairoUniversity, for 1 week before being subjected to experimentation.All animals were allowed free access to feed and tap water. Thestudy was conducted in accordance with the ethical proceduresand policies approved by the Ethics Committee of Faculty ofPharmacy, Cairo University.

Induction of hepatic IR injuryAnimals were anesthetized with thiopental sodium (70 mg/kg

body mass; intraperitoneal injection (i.p.)). Ischemia was inducedin rats by clamping the common hepatic artery and portal vein,according to the method described by Colletti et al. (1990), for30 min followed by 30 min of reperfusion. Throughout the oper-ation the animal’s abdomen was covered with plastic wrap toprevent dehydration.

Experimental designAnimals were randomly allocated to 5 groups, each consisting

of 8 rats. Groups I and II received 1% Tween 80 daily (per oral (p.o.))and served as the sham operated and the control IR groups, re-spectively. The remaining 3 groups were treated daily, p.o., with100 mg CR/kg body mass), or 10 mg vinpocetine/kg, or 20 mgvinpocetine/kg, respectively for 2 weeks. The doses of the testagents used were chosen from the published literature (Rischkeand Krieglstein 1991; Banji et al. 2001).

Twenty-four hours after the last treatment, rats in groups 2–5were subjected to IR injury. Thereafter, animals were sacrificed bydecapitation; blood samples were collected from the neck veinand used for the serum separation that was used for the estima-tion of serum activities of alanine transaminase (ALT) and aspar-tate transaminase (AST). The livers were then excised, washedwith saline, and weighed. Half of the liver was homogenized inice-cold phosphate buffer (pH 7.4) to prepare the 10% homogenatethat was used for estimation of hepatic LDH activity as well ashepatic contents of nitric oxide (NO), tumor necrosis factor-�(TNF-�), interleukin-1� (IL-1�), interleukin-6 (IL-6), interleukin-10(IL-10), reduced glutathione (GSH), and thiobarbituric acid reactivesubstances (TBARS). The other half was preserved in 10% formalinprepared in saline and used for histopathologic examination.

Biochemical and histological assaysSerum ALT and AST activities were estimated using commercial

kits and expressed in units per litre (U/L). Liver LDH activity wasassessed using commercial kits from Stanbio (USA) and expressedin U/L.

Liver content of GSH was determined using Ellman’s reagentaccording to the method described by Ahmed et al. (1991) andexpressed in milligrams per gram of wet tissue. NO metabolites

were measured as total nitrate/nitrite (NOx) using Griess reagentaccording to the method described by Miranda et al. (2001) andexpressed in milligrams per gram of wet tissue. Liver lipid perox-ides were determined as thiobarbituric acid reactive substances(TBARS) using malondialdehyde (MDA) as a standard according tothe method described by Mihara and Uchiyama (1978) and ex-pressed in nanomoles per gram of wet tissue.

Estimation of liver contents of TNF-�, IL-1�, IL-6, and IL10 wascarried out using ELISA reagent kits (Quantikine, R&D Systems,USA) and expressed in picograms per gram of wet tissue.

Tissue samples preserved for histopathology were fixed in10% formalin. The samples were washed with tap water, thenserial dilutions of alcohol were used for dehydration. Specimenswere cleared in xylene and embedded in paraffin at 56 °C in hot airoven for 24 h. Paraffin blocks were prepared for sectioning bySlidge microtome (slices 4 �m thick). The obtained tissue sectionswere collected on glass slides, deparaffinized, and stained withhematoxylin and eosin for histopathological examination usingan electron microscope (Banchroft et al. 1996). Images were cap-tured and processed using Adobe Photoshop (version 8).

Statistical analysisValues are the mean ± SE from 8 rats. Results were analyzed

using one-way analysis of variance (ANOVA) followed with a Tukey–Kramer multiple comparisons test. Values for p < 0.05 were consid-ered statistically significant. Graph Pad Software InStat (version 2)was used to carry out these statistical tests.

ResultsRats subjected to liver IR showed a significant increase in both

serum ALT (155.58 ± 3.01 U/L) and AST (111.31 ± 4.93 U/L) activities,reaching 129% and 149% of that in the sham-operated group, re-spectively. Pretreatment with CR (100 mg/kg) reduced the activityof AST to 83% of that in the IR treatment group. Treatment withvinpocetine (20 mg/kg) controlled the elevation in both ALT andAST to 88.3% and 80.6% of that in IR group, respectively (Fig. 1Aand 2B).

Untreated rats subjected to IR suffered from a 2-fold increase inTBARS (51.70 ± 1.79 mmol/g wet tissue) as compared with thesham-operated group (21.20 ± 1.48 mmol/g wet tissue); meanwhilethe GSH content in the IR treatment group (158.75 ± 11.43 mg/g)was reduced to almost half of the value in the sham-operatedgroup (259.43 ± 8.53 mg/g). This effect was prevented by the ad-ministration of CR and vinpocetine: the level of TBARS was re-duced to 68% of the rats in the IR treatment group, 71% of the ratstreated with CR, and 64.5% of that in the rats treated with vinpo-cetine (10 or 20 mg/kg). In addition, GSH levels were elevated to163% of that in the IR treatment group, 122% of that for the ratstreated with CR, and 145.5% of that in the rats treated with vinpo-cetine (10 or 20 mg/kg) (Fig. 2A and 2B).

Hepatic LDH activity was increased following induction of IR to1043.73 ± 119.72 U/g wet tissue, reaching 435% of that in the sham-operated group (204.14 ± 22.7 U/g wet tissue). Pretreatment withCR or vinpocetine (10 or 20 mg/kg) daily for 2 weeks protectedagainst increased LDH activity: enzyme activity was almost nor-malized in all 3 groups to be 22%, 25%, and 21% of that for rats inthe IR group treatment group, respectively (Fig. 2C).

Hepatic NOx content was elevated following induction of liverIR to 555.63 ± 17.23 mg/g wet tissue, and reached 134.5% of that inthe sham-operated group (413.06 ± 21.78 mg/g wet tissue). Pretreat-ment with CR, 10 mg vinpocetine/kg, or 20 mg vinpocetine/kg, for2 weeks, almost normalized hepatic NOx content, reducing it to73.5%, 83%, and 67.5% of that in IR group, respectively (Fig. 2D).

Induction of IR increased levels of both IL-1� (3152.73 ±325.00 pg/g wet tissue) and IL-6 (1551.48 ± 61.95 pg/g wet tissue) to264% and 147% of that in the sham-operated group, accompaniedwith a reduction in IL-10 (743.89 ± 47.79 pg/g wet tissue) to 76% ofthat in the sham-operated group. Pretreatment with CR reducedboth IL-6 and IL-1� levels to 70% and 35%, respectively, of that in IR

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treatment group, and increased IL-10 content to 116% of that in IRgroup. Similarly, 10 mg vinpocetine/kg reduced both IL-1� and IL-6levels to 55.5% and 71%, respectively, of that in IR treatment group,and increased IL-10 content to 109% of that in IR group; mean-while, 20 mg vinpocetine/kg reduced both IL-1� and IL-6 contents

to 37% and 66%, repsectively, of that in IR group and increasedIL-10 content to 141% of that in IR group (Figs. 3A–3C). On the otherhand, TNF-� wasn’t significantly altered in the IR treatment groupor in any of the treated groups, compared with the sham-operatedgroup (Fig. 3D).

Fig. 1. The effect of pretreatment with curcumin (100 mg/kg body mass) or vinpocetine (10 or 20 mg/kg) on (A) serum aspartate transaminase(AST), and (B) serum alanine transaminase (ALT) activities in male albino Wistar rats subjected to ischemia–reperfusion (IR) injury. Curcuminand vinpocetine were administered daily, per oral, for 2 weeks. Twenty-four hours after the last dose, rats were subjected to 30 min ofischemia followed by 30 min of reperfusion. Statistical analysis was carried out with one-way ANOVA followed by the Tukey–Kramer multiplecomparisons test. Value are the mean ± SE (n = 8 rats); *, p < 0.05 compared with the normal control group; @, p < 0.05 compared with the IRgroup. SO, sham operated; CR, curcumin; V10, vinpocetine (10 mg); V20, vinpocetine (20 mg).

Fig. 2. The effect of pretreatment with curcumin (100 mg/kg body mass) or vinpocetine (10 or 20 mg/kg) on (A) liver content of reducedglutathione (GSH), (B) liver content of thiobarbituric acid reactive substances (TBARS), (C) lactate dehydrogenase (LDH) activity in the liver, and(D) total nitrate/nitrite (NOx) content in the liver of male, albino, Wistar rats subjected to ischemia–reperfusion (IR) injury. Curcumin andvinpocetine were administered daily, per oral, for 2 weeks. Twenty-four hours after the last dose, rats were subjected to 30 min of ischemiafollowed by 30 min of reperfusion. Statistical analysis was carried out using one-way ANOVA followed by the Tukey–Kramer multiplecomparisons test. Values are the mean ± SE (n = 8 rats); *, p < 0.05 compared with the normal control group; @, p < 0.05 compared with the IRgroup. SO, sham operated; CR, curcumin; V10, vinpocetine (10 mg); V20, vinpocetine (20 mg).

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Histological examination of tissues of sham-operated group re-vealed that the histologic structure of the liver was normal(Fig. 4A), whereas in the IR treatment group the hepatocytes sur-rounding the central vein showed coagulative necrosis associatedwith diffuse Kupffer cell proliferation and inflammatory cell in-filtration in between the hepatocytes, while the central vein andsinusoids were dilated (Fig. 4B).

In the CR-pretreated group, some of the hepatocytes surround-ing the congested central vein showed fatty changes associatedwith slight Kupffer cell proliferation and inflammatory cell infil-tration (Fig. 4C). Pretreatment with 10 mg vinpocetine/kg pro-duced dilation and congestion in the portal vein and dilation inboth the central vein and sinusoids that was associated with infil-tration by a few inflammatory cells (Fig. 4D). Meanwhile, pretreat-ment with 20 mg vinpocetine/kg revealed dilation in both centralvein and sinusoids associated with infiltration by a few inflamma-tory cells (Fig. 4E). The severity of histopathological alterations inthe liver tissues is also presented in Table 1, with the highestseverity recorded following IR (++++). CR offered moderate protec-tion against histological alterations (++), whereas treatment withboth dose levels of vinpocetine showed better protection againstthese alterations than CR (+).

DiscussionIn this study, clamping the common hepatic artery and portal

vein of rats affected 70% of the liver mass. It has been reportedthat the resultant hepatic insult is similar to the clinical situation

in which the liver is rendered ischemic during total vascular ex-clusion for liver resection(Arab et al. 2009). Indeed, in the presentstudy, 30 min of hepatic ischemia followed by 30 min of reperfu-sion caused severe liver injury, as demonstrated by the structuraldamage to the liver and the increased serum activities of AST andALT in addition to liver LDH activity. Similar results were reportedby Taha et al. (2012).

Gao and Li (2012) concluded that oxidant stress and inflamma-tion are the most critical mechanisms contributing to the organpathophysiology after warm hepatic IR. This was evidenced in thisstudy by the depletion in hepatic GSH and the parallel increase inMDA.

It has been indicated that there are 2 distinct phases of liverinjury after warm IR. The initial phase of injury (<2 h after reper-fusion) is characterized by activation of Kupffer cells, the primarysource of the oxygen-derived free radicals that disturb cellularhomeostasis (Jaeschke and Farhood 1991; Jaeschke et al. 1992).

In addition to Kupffer-cell-induced oxidative stress, with in-creasing length of the ischemic episode, intracellular generationof ROS by xanthine oxidase may also lead to impaired adenosinetriphosphate (ATP) production and acidosis resulting in liver dys-function and cell injury during reperfusion (Kumamoto et al.1999).

IR injury in the current investigation resulted in a significantincrease in levels of pro-inflammatory cytokines as IL-1� and IL-6in the liver, with a parallel decrease in IL-10, an anti-inflammatorycytokine. Increased expression of pro-inflammatory cytokines as

Fig. 3. The effect of pretreatment with curcumin (100 mg/kg body mass) or vinpocetine (10 or 20 mg/kg) on liver content of (A) interleukin-10(IL-10), (B) interleukin-6 (IL-6), (C) interleukin-1� (IL-1�), and (D) tumor necrosis factor-� (TNF-�) in male albino Wistar rats subjected toischemia–reperfusion (IR) injury. Curcumin and vinpocetine were administered daily, per oral, for 2 weeks. Twenty-four hours after the lastdose, rats were subjected to 30 min of ischemia followed by 30 min of reperfusion. Statistical analysis was carried out using one-way ANOVAfollowed by the Tukey–Kramer multiple comparisons test. Values are the mean ± SE (n = 8 rats). *, p < 0.05 compared with the normal controlgroup; @, p < 0.05 compared with the IR group. SO, sham operated; CR, curcumin; V10, vinpocetine (10 mg); V20, vinpocetine (20 mg).

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TNF-� and IL-6 during hepatic IR was previously reported (Tahaet al. 2012). The activation of inflammatory cytokines is mediatedby ROS and is associated with the induction of protective genesthat are essential for the maintenance of liver functions (Llacunaet al. 2009). Kupffer cells and liver-infiltrating monocyte-derivedmacrophages are the primary sources of cytokines (Tacke et al.2009). Moreover, Beraza et al. (2007) have suggested that the he-patic inflammatory response to IR is driven largely by the activa-tion of NF-�B in hepatocytes.

Induction of hepatic IR injury was coupled with an increase inNO production. The role of NO in IR injury remains controversial.NO produced by endothelial nitric oxide synthase (eNOS) servesmany physiological purposes, such as the promotion of vasodila-tation and protection against IR injury (Jeyabalan et al. 2008).However, excessive levels of NO via inducible nitric oxide syn-thase (iNOS) in macrophages may cause cellular damage via aphenomenon known as nitrosative stress (Kimura et al. 2003;Vardanian et al. 2008). This process leads to the production ofperoxynitrite, which is a potent oxidant that induces cell death(Schwentker and Billiar 2002).

Indeed, reperfusion of the ischemic liver was shown to result inthe generation of oxidative and nitrosative stresses as well as in

the production of peroxynitrite, which induces rapid cytotoxicityand liver injury (Lin et al. 2012).

In this study, pretreatment with CR prevented IR-induced liverinjury, as evidenced from the observed decrease in liver lipidperoxides and NOx levels, coupled with an increase in GSH andIL-10. Similarly, pretreatment with CR prevented IR-induced in-creases in IL-1� and IL-6 in the liver. Improvements in the struc-ture of the liver correlated with a reduction in serum AST andliver LDH activities.

In a study performed by Lin et al. (2012), liver ischemia for30 min followed by 80 min of reperfusion resulted in significantelevations in serum transaminases and LDH activity, as well asincreases in blood levels of NO and TNF-�. All such changes wereattenuated by pretreatment with CR (25 mg/kg). Moreover, CR inthe same dose was shown to prevent methotrexate-induced hep-atotoxicity (Banji et al. 2001).

In a study performed by Onder et al. (2012), CR (200 mg/kg) asa single dose protected against histopathologic damage andoxidative stress in the intestine and distant organs includingliver, kidney, and lungs, and also protected against mesentericIR injury.

Fig. 4. Photomicrographs of livers from the male albino Wistar rat groups: (A) sham-operated control; (B) ischemia–reperfusion control;(C) pre-treated with curcumin (100 mg/kg body mass); (D) pre-treated with vinpocetine (10 mg/kg), or (E) vinpocetine (20 mg/kg). (A) Nohistopathological alteration was detected in the central vein (cv) or surrounding the hepatocytes (h). (B) Hepatocytes showed coagulativenecrosis (c) surrounding the central vein (cv) associated with diffused Kupffer cell proliferation (arrow) and inflammatory cells infiltration (m)in between the hepatocytes while the central vein (cv) and sinusoids (s) were dilated. (C) Fatty changes (arrow) were observed in some of thehepatocytes surrounding the congested central vein (cv) associated with the proliferation of few diffused Kupffer cells (k) as well as theinfiltration of few inflammatory cells (m) throughout the hepatic parenchyma. (D) Dilated central vein (cv) and sinusoids (s) with someinfiltration of inflammatory cells (m). (E) Dilated central vein (cv) and sinusoids (s) were observed, together with the infiltration offew inflammatory cells (m) (hematoxylin and eosin staining, magnification × 200).

Table 1. The severity of histopathological alterations in the liver tissues from male albino Wistar rats.

GroupsShamoperated

IRcontrol

Curcumin(100 mg/kg)

Vinpocetine(10 mg/kg)

Vinpocetine(20 mg/kg)

Coagulative necrosis − +++ − − −Fatty changes − ++ ++ ++ +Inflammatory cell infiltration

between hepatocytes− +++ ++ + +

Diffuse Kupffer cell proliferation − ++ ++ − −Total inflammatory reaction − − − + −Congestion − ++ + − −Total − ++++ ++ + +

Note: ++++, Very severe; +++, severe; ++, moderate; +, mild; −, nil.

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Pretreatment with CR protected the liver and kidney against IRinjury through multiple pathways involving immune-mediatedand anti-apoptotic mechanisms, mediated via inhibition of tumorgrowth factor-beta (TGF-�) and caspase-3 (Shen et al. 2007; Awadand El-Sharif 2011). CR was shown to increase antioxidant enzymeexpression and activity in tissue, inhibit neutrophil infiltration,and protect cell function under different stress conditions (Araujoand Leon 2001). Moreover, CR has been reported to be an effectivescavenger for ROS and RNS in vitro. These effects are mediatedthrough the regulation of various transcription factors, growthfactors, and inflammatory cytokines, as well as protein kinasesand other enzymes (Aggarwal et al. 2007).

Pretreatment with vinpocetine protected against IR injury tothe liver in a dose-dependent manner. Vinpocetine in the largedose (20 mg/kg) prevented all IR-induced biochemical and histo-logical changes. Vinpocetine is readily absorbed from the smallintestine following oral administration. It undergoes extensivemetabolism yielding its main metabolite, apovincaminic acid,which is absorbed from the stomach (Pudleiner and Vereczkey1993).

Zhao et al. (2011) demonstrated that by reducing the expressionof inflammation-related molecules as nuclear factor-�B (NF-�B)and activator protein-1 (AP-1), vinpocetine can inhibit the produc-tion of NO and pro-inflammatory cytokines in microglia exposedto oxygen, and glucose deprivation. Hence, the observed anti-inflammatory effects of vinpocetine could be mediated via inhibi-tion of pro-inflammatory cytokine induced NF-�B activation, aswell as inhibition of monocyte adhesion and chemotaxis, whichare critical processes during inflammation (Jeon et al. 2010). Phos-phodiesterase inhibitors such as vinpocetine are regarded as use-ful anti-inflammatory agents that down-regulate inflammatorycytokines and up-regulate inhibitory cytokines such as IL-10 in thecentral nervous system (Yoshikawa et al. 1999).

The effect of vinpocetine on acute hepatic injury caused by theadministration of CCl4 in rats has been investigated (Abdel Salamet al. 2007). Vinpocetine reduced CCl4-induced elevation in serumtransaminases and reduced associated hepatic necrosis. The abil-ity of vinpocetine to block calcium channels (Sitges et al 2007)may explain its hepatoprotective effect. In this context, limitingthe influx of Ca2+ into the hepatocytes by calcium channel antag-onists ameliorated hepatic injury in a number of experimentalmodels (Sippel et al. 1993; Romero et al. 1994).

Another proposed mechanism for the protective effect ofvinpocetine is its marked antioxidant activity from the scaveng-ing of hydroxyl radicals and other ROS (Santos et al. 2000;Herrera-Mundo and Sitges 2012). Solanki et al. (2011) showed thatvinpocetine prevented ROS generation and reduced antioxidantlevels associated with IR injury in primary hippocampal cell cul-tures. Moreover, vinpocetine was reported to possess cytoprotec-tive activity and to prevent apoptosis in hypoxia (Gabryel et al.2002).

ConclusionsIn conclusion, the experimental IR model used herein was reli-

able with respect to the promotion of parenchymal and func-tional liver damage as evidenced by the observed biochemical andhistopathological changes in liver tissues. Pretreatment of ratswith CR or vinpocetine protected against IR-induced liver injuryby virtue of their antioxidant and anti-inflammatory properties.

AcknowledgementsThe authors are very grateful to Dr. Adel M. Bakeer, professor of

pathology, Faculty of Veterinary Medicine, Cairo University, forexamining and interpreting histopathological aspects of thisstudy.

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