Th e protective eff ect of diosmin on hepatic ischemia reperfusion injury: an experimental study

Liver ischemia reperfusion injury (IRI) is an important pathologic process leading to bodily systemic eff ects and liver injury. Our study aimed to investigate the protective eff ects of diosmin, a phlebotrophic drug with antioxidant and anti-infl ammatory eff ects, in a liver IRI model. Forty rats were divided into  groups. Sham group, control group (ischemia-reperfusion), intraoperative treatment group, and preoperative treatment group. Ischemia reperfusion model was formed by clamping hepatic pedicle for a  minute of ischemia followed by liver reperfusion for another  minutes. Superoxide dismutase (SOD) and catalase (CAT) were measured as antioaxidant enzymes in the liver tissues, and malondialdehyde (MDA) as oxidative stress marker, xanthine oxidase (XO) as an oxidant enzyme and glutathione peroxidase (GSH-Px) as antioaxidant enzyme were measured in the liver tissues and the plasma samples. Hepatic function tests were lower in treatment groups than control group (p<. for ALT and AST). Plasma XO and MDA levels were lower in treatment groups than control group, but plasma GSH-Px levels were higher (p<. for all). Tissue MDA levels were lower in treatment groups than control group, but tissue GSH-Px, SOD, CAT and XO levels were higher (p<. for MDA and p<. for others). Samples in control group histopathologically showed morphologic abnormalities specifi c to ischemia reperfusion. It has been found that both preoperative and intraoperative diosmin treatment decreases cellular damage and protects cells from toxic eff ects in liver IRI. As a conclusion, diosmin may be used as a protective agent against IRI in elective and emergent liver surgical operations. ©  Association of Basic Medical Sciences of FB&H. All rights reserved


INTRODUCTION
Ischemia reperfusion injury (IRI) is an important clinical issue which is a concern to many organs including brain, heart, kidneys, and liver [,].Hepatic ischemia-reperfusion periods may take place during hepatic tumor resection, trauma surgery of liver, vessel reconstruction surgery, and liver transplantation.Furthermore, hepatic circulation is known to be affected by hemorrhagic shock, advanced sepsis, and severe trauma, independent of the surgery [].Different mechanisms such as hypoxic response, inflammatory reaction, free radical injury, and apopto-sis play role in development of cellular damage in liver during ischemic period following reperfusion [].Diosmin is a hesperidin-derivative bioflavonoid.Flavonoids have been demonstrated to exert anti-lipoperoksidant, anti-tumoral, anti-platelet, anti-ischemic, anti-allergic, and anti-inflammatory activities [].
In this study we aimed to investigate the protective eff ect of diosmin on oxidative stress and cellular damage in IRI.

Animals
Forty female Wistar-Albino rats of ± gr weight were kept in separate wire cages at constant room temperature ( ±  o C) in cycle of light and darkness of  hours.Th ey were fed with water and rat feed.Th ey were kept off food by  hours prior to the surgery.Th ey were allowed to drink water until  hours prior to the surgery.No parenteral or enteral antibiotics were administered in any stage of the study.given diosmin in the form of gavage per orogastric tube at a dose of  mg/kg/day for ten days prior to operation.Th e tube was removed after daily drug was administered.IRI was generated in operation time.All rats were sacrificed simultaneously.No rat died during the study period.At the end of these procedures blood and liver tissue samples were obtained for biochemical and histopathologic assessment.

Procedure of IRI
Th e animals were anesthetized by administering ketamine hydrochloride  mg/kg and xylasine  mg/kg.After abdomen was shaved and disinfected a midline incision was made.Ischemic period was generated by clamping hepatic pedicle with micro vascular bulldog clamp for  minutes.Following ischemic period liver was reperfused by declamping and reperfusion was continued until  minute.

Biochemical analysis
Liver tissue samples were kept at - o C for assessment of oxidative stress.Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gama glutamyl transferase (GGT) levels were measured using Olympus Au  autoanalysator to assess liver functions.To assess oxidative injury, blood malondialdehyde (MDA) levels, glutathion peroxidase (GSH-Px), and xanthine oxidase (XO) enzymatic activities were studied.MDA levels, GSH-Px, XO, superoxide dismutase (SOD), and catalase (CAT) enzymatic activities were measured in liver tissue samples.

Assessment of oxidative stress
Following sacrifi ce of animals liver samples were obtained and exposed to ice bath until homogenization.Liver samples were fi rst washed with distilled water, tissues were homogenized with physiologic saline ( w/v, approximately g in  ml for each).Later, supernatants were centrifuged at  rpm, for  minutes.All procedures were completed at +°C.Protein concentrations of supernatants obtained from tissue homogenates were measured with Lowry's method for protein measurement [].Th e obtained supernatants were separated to be studied in different measurement devices.MDA Measurement: As an end product of lipid peroxidation, MDA is used as a marker of oxidation.By measuring the absorbance level of the complex formed by MDA and thiobarbituric acid at a wave length of  nm, which is the basis of the Dahle's spectrophotometric method, the results were expressed in terms of nmol/mg tissue weight [].SOD Determination: Measurement of SOD is based on the principle of reduction of nitroblue tetrazolium (NBT) compound found in the reaction medium when superoxide radical formed by xanthine-xanthine oxidase system cannot be removed by SOD enzyme; the results were expressed in terms of U/mg.One unit of SOD was expressed as the substance amount causing  inhibition in the NBT reduction rate [].GSH-Px Determination: Glutathione Peroxidase activity was measured according to Paglia method [].GSH-Px activity was calculated with the absorbance decrease during oxidation of NADPH to NADP + read at  nm and the results were expressed in terms of milliinternational unit/milligram (mIU/mg) tissue protein.
CAT Determination: Catalase activity was measured according to Aebi method [].Measurement of catalase enzyme activity was based on the principle of spectrophotometric observation of decrease of absorbance level of hydrogen peroxide at a wave length of  nm and the results were expressed in terms of IU/mg.XO Determination: Xanthine oxidase enzyme activity was measured by spectrophotometric determination of absorbance level of uric acid formation from xanthine at a wave length of  nm and the results were expressed in terms of mIU/mg [].

Histopatologic assessment
For light microscopic analysis, liver tissue samples obtained from the animals were fi xed by keeping in  neutral buffered formalin for  days.Tissues were washed with water and dehydrated by treating with ethanol with increasing concentrations (, , , and ).Following dehydration, specimens were immersed in xylene to obtain transparency.Th ey were then immersed in paraffi n and infi ltrated.Sections with a thickness of  μm from paraffi n-immersed YUSUF TANRIKULU ET AL.: THE PROTECTIVE EFFECT OF DIOSMIN ON HEPATIC ISCHEMIA REPERFUSION INJURY: AN EXPERIMENTAL STUDY tissues were obtained using Leica RM  RT.Sections which were systematically chosen in a random manner were stained with hematoxylin-eosin (H&E) and Periodic Acid-Schiff (PAS).Histopathologic examination was performed by two histologists blinded to the study.Study photographs were taken with Nikon eclipse E  and marked.

Statistical analysis
Data were analyzed using SPSS . package program.Data were given as mean± standard deviation.Th e diff erences between the groups were compared with One-Way ANOVA or Kruskal Wallis variance analysis.When p value was signifi cant, Mann-Whitney U multi variance analysis was used to detect the group creating the diff erence.While evaluating the histopathology results, Student's t-test variance analysis was used.A p< . was considered statistically signifi cant.

RESULTS
Table  shows the liver function test results (AST, ALT and GGT) of the groups.Th ere was a signifi cant diff erence between SHAM group and controls in terms of AST and ALT levels (p<.).GGT levels were significantly lower in SHAM group compared to control group, albeit statistically non-significant (p=.).SHAM group and intraoperative treatment group were signifi cantly diff erent in terms of AST and GGT levels (p<. for AST, p<. for GGT).Th ere was a signifi cant diff erence between SHAM and preoperative groups in terms of AST and Alt levels (p<. for all).Th ere was a signifi cant diff erence between control and intraoperative treatment group in terms of all liver function tests (p<. for AST and ALT, p<. for GGT).Control and preoperative treatment group were signifi cantly diff erent in terms of AST and ALT levels (p<. for all).GGT levels were lower in preoperative treatment group, albeit statistically non-significant (p=.).There was no difference between treatment groups in terms of AST levels.However, there were significant differences between treatment groups in terms of ALT and GGT levels (p<.).ALT levels were lower in preoperative treatment group and GGT levels were lower in intraoperative treatment group.Plasma MDA, GSH-Px and XO results are summarized in Table .Control group and other groups were significantly diff erent in terms of all results (p<. for all).Plasma XO and MDA levels were lower in treatment groups than control group, but plasma GSH-Px levels were higher (p<.for all).Tissue MDA levels were lower in treatment groups than control group, but tissue GSH-Px, SOD, CAT and XO levels were higher (p<.for MDA and p<. for others).Treatment groups did not differ significantly in terms of MDA and XO levels, however, GSH-Px levels were signifi cantly higher in treatment group (p<.).Liver tissue MDA, GSH-Px, SOD, CAT and XO results are summarized in Table .SHAM group and control group diff ered signifi cantly in terms of all results (p<. for all).SHAM group and treatment groups had signifi cant diff erences in MDA, GSH-Px, CAT and XO levels (p<. for MDA and XO, p<. for GSH-Px and CAT).Control group and treatment groups were signifi cantly diff erent in terms of all results (p<. for MDA and XO, p<. for GSH-Px, SOD and CAT).MDA, SO, and XO levels were not diff erent in treatment groups, however, GSH-Px and CAT levels were sig-    nifi cantly higher in intraoperative treatment group (p<.).Tissues from the SHAM group presented no morphological alterations in the normal lobular structure of liver tissue and portal tract (Figure A).Th e sinusoids can just be seen as pale-stained spaces between the plates of liver cells (Figure A).Th e hepatic asinus is a more physiologically useful model of liver structure and lies between two or more terminal hepatic venules and blood fl ows from the portal tracts through the sinusoids to the venules.Th e asinus is divided into zones , , and .Th e glycogen in hepatocytes which, being polysaccharide, is PAS-positive was found homogenous in the hepatocyte cytoplasm of all three zones (Figure A).Th e control group, showed multiple and extensive areas of portal infl ammation with a moderate increase in the level of infl ammatory cell infi ltration (Figure B).We observed an massive congestion in the parenchyma of the liver and dilatation of the sinusoidal spaces (Figure B).The PAS stained sections showed heterogeneous distribution of glycogen in the hepatic asinus.In consequence of the glycolysis there was a signifi cant reduction in the store of glycogen.
Th is reduction was especially evident in the zone  which was the mostly affected region of the ischemia in the hepatic asinus.Th e cytoplasm of the hepatocytes of the zone  were homogeneous due to the glycogen loss (

DISCUSSION
IRI is an important clinical problem involving many organs including brain, heart, kidneys, and liver [, ].Ischemia reperfusion leads to a series of pathologic reactions resulting in cellular death and organ dysfunction.Different mechanisms take part in hepatic cellular damage during ischemia and after reperfusion.Despite it is not known which mechanism is important in the pathogenesis of ischemic cellular damage, oxygen deprivation is the most commonly accused factor.During ischemia, multiple diff erent cellular and subcellular dysfunctions such as mitochondrial dysfunction, dysfunction in cell membrane, and decreased protein synthesis arise.Recent studies have suggested that the most detrimental factor in cellular necrosis after temporary and permanent hepatic ischemia is the reperfusion injury [].
The most common reasons for IRI in liver are resection surgery for large hepatic tumors, transplantation, and trauma surgery.In addition, hepatic circulation is known to be affected by hemorrhagic shock, advanced sepsis, and severe trauma, independent of the surgery [, ].Hepatic blood fl ow has to be partially or completely interrupted during surgeries for extensive hepatic damage and large tumor resections.Various experimental studies have demonstrated that liver can tolerate ischemic periods of - minutes [].Recovery of liver function have been reported following vessel clamping up to  minutes in hepatic resections [].We designed an injury time of  minutes for ischemia and  minutes for reperfusion, consistent with the literature.
Many complex mechanisms such as lipid peroxidation, free radical injury, and increased inflammatory response play a role in ischemic cellular damage.Thus, to prevent negative effects of hepatic ischemia various agents and methods have been employed, including melatonin, L-arginine, allopurinol, and TNF-α [, ].Diosmin, is a hesperidine-derivative bioflavonoid [].Flavonoids have antibacterial, antiviral, anti-inflammatory, anti-allergic, and vasodilatory effects.Studies have shown that they also inhibit lipid peroxidation, thrombocyte aggregation, capillary permeability, and various enzymatic systems including cyclooxygenase and lipooxygenase.In addition, diosmin inhibits formation of free oxygen radicals both in vivo and in vitro [].Diosmetin is the active metabolite of diosmin and is rapidly absorbed.Its half-life is - hours.It reaches peak levels one hour following oral intake and plasma concentration starts to fall by  hours [].In studies investigating the eff ects of mikronized purified flavonoid fraction (MPFF) on microcirculation it has been shown that MPFF intercellular adhesion molecule expression, leucocyte adhesion and migration, formation of free oxygen radicals, synthesis of prostoglandin E, Fα, and tromboksan B, thrombocyte functions, and increased micro vascular permeability in ischemia-reperfusion. Furthermore, MPFF has favorable eff ects on lymphatic drainage [].Diosmin reinforces venous tonus by prolonging parietal norepinephrine activity.Th e protective eff ect of diosmin-hesperidin complex in ischemia reperfusion injury may be explained by preservation of mean arteriolar and venular diameter [, ].Diosmin also has antioxidant effect.Flavonoids inhibit oxidation of low density lipoproteins in vitro.In addition, they exert antioxidant eff ect against peroxyl and hydroxyl radicals [].In a study exploring the effect of diosmin-hesperidin on oxidative stres Unlu et al. [] showed that, in addition to antioxidant eff ect, periglomerular and perivascular leucocyte infi ltration is signifi cantly lower in rats given diosmin-hesperidin complex.Diosmetin, the main metabolite of diosmin, has been shown to exert protective eff ect on hepatocytes against cellular damage induced by erithromycine estolate and tert-butylhydroperoxide in humans [].Moreover, in another study we conducted in our clinics diosmin decreased small intestinal damage and exerted a protective eff ect in ischemia reperfusion injury [].
Liver IRI occurs in a number of clinical settings in general surgery such as trauma, transplantation, hepatic resection and is associated with increased morbidity and mortality.Reactive oxygen radicals and reactive nitrogen species play a major role in the pathophysiology of IRI.Th e antioxidant defence system is a complex and it normally controls the production their.Oxidative stress occurs when there is significant imbalance between production and removal their.Th is condition accelerates degradation of membrane phospholipids by damage of lipids, proteins, carbohydrates, and nucleic acids.Hepatocytes tend to be resistant to injury by reactive oxygen and nitrogen species, since they contain high intracellular antioxidants' concentrations such as GSH-Px, SOD, CAT and lipid soluble antioxidants [].
MDA, an interval metabolite of the lipid degradation and polyunsaturated fatty acid preoxidation, is a sensitive indicator of IRI [].Adenine nucleotides are catabolized to hypoxanthine during ischemic insult.When perfusion of ischemic organ is restored hypoxanthine is oxidized to xanthine by the enzyme xanthine oxidase, releasing free oxygen radicals which cause cell membrane damage by peroxidizing fatty acids found in the structure of phospholipid layer of cell membranes [].Th e XO pathway has been implicated as an important route in the oxidative injury to tissues, especially after ischemia-reperfusion.It scavenges reactive oxygen species and reactive nitrogen species [].
We observed a signifi cantly lower levels of MDA and XO, markers of tissue lipid peroxidation, in treatment groups.
All aerobic creatures are subject to physiologic oxidative stress during metabolism.Body glutathion is an important component of the antioxidant system and protects the cell against oxidative damage by reacting with free radicals and peroxides.Hepatic GSH concentrations have been shown to decrease during hepatic ischemia reperfusion injury.Our study revealed that GSH-Px levels which were measured to assess GSH levels, were signifi cantly high in both treatment groups, particularly so in the group given diosmin preoperatively.CAT, an endogenous antioxidant, and SOD which protects oxygen-metabolizing cells against detrimental effects such as lipid peroxidation of superoxide free radical and has a role in intracellular killing of phagocyted bacteria were also high in the treatment group.Th e degree of damage ischemia and reperfusion infl ict in liver at a cellular basis is best refl ected by serum enzyme levels such as ALT, AST, and GGT as well as histopathologic changes [].Uhlmann et al. [] found an AST level of  U/L and an ALT level of  U/L after reperfusion following a -minute partial ischemia in sham-operated rats while the same numbers following reperfusion were  U/L and  U/L, respectively.Our study demonstrated signifi cantly lower AST, ALT, and GGT levels in treatment groups, consistent with the literature.Under light microscopy hepatic ischemia reperfusion is characterized by neutrophil infi ltration, regional hemorrhage and necrosis, congestion, sinusoidal enlargement, regional hepatocellular vacuolization, hepatocyte swelling while under ultra-structural examination it is evident by distorted mitochondrial structure, swelling, staining diff erences, and neutrophil aggregation [].Crockett et al. [] observed sinusoidal congestion, cytoplasmic vacuolization, hepatocellular necrosis, neutrophil infi ltration, and a high ALT level in hepatic ischemia reperfusion group.We histopathologically investigated dilatation in vena porta branches and vena centralis, sinusoidal congestion, parenchymal congestion, sinusoidal dilatation, and portal infl ammatory cell infi ltration.We found that SHAM group and diosmin-treated groups had a normal liver and cellular structure in terms of histologic features observed in SHAM, intraoperative diosmin and preoperative diosmin administered groups whereas we found portal infl ammation, sinusoidal dilatation, congestion, and glycogen deficiency in tissue sections obtained from control group.

CONCLUSION
In conclusion, we found that diosmin administered both preoperatively and intraoperatively decreases cellular damage and protects cells against harmful eff ects during hepatic IRI.However, we also found that this eff ect is more pronounced in the group treated preoperatively.We think that the protective eff ect of diosmin may be due to its anti-infl ammatory and antioxidant eff ects.In addition, we think that diosmin treatment can decrease morbidity and mortality by preventing free radical-induced oxygen injury in hepatic ischemia reperfusion.According to fi ndings obtained from our study, we think that diosmin can be used as a protective agent against IRI in both elective and emergent liver surgeries.Nevertheless, further studies are needed to obtain better outcomes.

DECLARATION OF INTEREST
Th e authors declare no confl ict of interest.

FIGURE 1 .
FIGURE 1. Histopathological fi ndings in groups (A micrographs on the fi rst line is SHAM group, B micrographs on the second line is control group, C micrographs on the third line is intraoperative treatment group and, D micrographs on the fourth line is preoperative treatment group) Figure Legend: This panel of the rat liver is stained by hematoxylin and eosin (1 st and 2 nd micrographs of the each group on the left two columns of the panel) and Periodic Acid-Schiff reaction (3 rd micrograps of the each group on the right column of the panel).A micrographs shows the structure of the liver composed of tightly packed, pink-staining plates of hepatocytes (H).Portal tracts (P) which contain the main blood vessels, hepatic (centrilobular) venule " vena centralis" (VC), the sinusoids (S) are lined by fl at endothelial lining cells.The hepatic asinus (HA) lies between two terminal hepatic venules and divided into zones 1,2 and 3 (Z1, Z2, Z3).B micrographs, infl ammatory cell infi ltration (arrow) in the portal tract.Sinusoidal dilatation (arrow head) and congestion (*).C, D micrographs, shows the diosmin treated groups in close morphology to the regular structure of liver except the mild congestion (*) in certain regions.
Figure B).Th e marked congestion in the distended sinusoids seen in the control group was restricted to rare areas in the treatment groups (Figure C).Th e other diagnosis of the ischaemia-reperfusion group was not observed in both single and ten days dose treated groups (Figure C, D).As a conclusion; the histological features shown in Figure A, C and D suggests the livers from the sham and diosmin treated animals have a normal liver lobular architecture and cell structure.However, the liver sections obtained from the control group showed portal infl ammation, sinusoidal dilatation and congestion and glycogen depletion (Figure B).
YUSUF TANRIKULU ET AL.: THE PROTECTIVE EFFECT OF DIOSMIN ON HEPATIC ISCHEMIA REPERFUSION INJURY: AN EXPERIMENTAL STUDY study was conducted in accordance with National Guide for Care and Use of Laboratory Animals after approval of Ethical Committee of Ankara Research and Education Hospital.Rats were randomly divided into  groups each containing  rats: .SHAM group.Rats in this group, hepatic pedicule was mobilized..Control group (ischemia-reperfusion).Rats in this group, IRI was generated.Any treatment was given..Intraoperative treatment group.Rats in this group, IRI was generated.Rats in this group were given diosmin  mg/kg in in the form of gavage per orogastric tube ( Gauge feeding tube) immediately following induction of ischemia to reach of plasma concentration of diosmin at the beginning of reperfusion..Preoperative treatment group.Rats in this group were Th is  Bosn J Basic Med Sci 2013; 13 (4): 219-224

TABLE 1 .
Liver function tests