ORIGINAL ARTICLE
The effects of tadalafil and pentoxifylline on
apoptosis and nitric oxide synthase in liver
ischemia/reperfusion injury
Sibel Bektas
a,*
, Kemal Karakaya
b, Murat Can
c, Burak Bahadir
d,
Berrak Guven
c, Nilsen Erdogan
a, Sukru Oguz Ozdamar
da
Department of Pathology, Gaziosmanpasa Taksim Training and Research Hospital, Gaziosmanpasa, Istanbul, Turkey
b
Department of General Surgery, Bulent Ecevit University, School of Medicine, Kozlu, Zonguldak, Turkey
c
Department of Biochemistry, Bulent Ecevit University, School of Medicine, Kozlu, Zonguldak, Turkey
d
Department of Pathology, Bulent Ecevit University, School of Medicine, Kozlu, Zonguldak, Turkey
Received 2 February 2016; accepted 8 May 2016
Available online 2 June 2016
KEYWORDS Ischemia/ reperfusion; Liver; Pentoxifylline; Tadalafil
Abstract The aim of this study was to investigate the effects of tadalafil (TDF) and pentox-ifylline (PTX) on hepatic apoptosis and the expressions of endothelial and inducible nitric oxide synthases (eNOS and iNOS) after liver ischemia/reperfusion (IR). Forty Wistar albino rats were randomly divided into five groups (nZ 8) as follows: sham group; IR group with ischemia/re-perfusion alone; low-dose and high-dose TDF groups received 2.5 mg/kg and 10 mg/kg TDF, respectively; and PTX group received 40 mg/kg PTX. Blood was collected for the analysis of serum alanine aminotransferase, aspartate aminotransferase, g-glutamyl transferase, uric acid, malondialdehyde (MDA), and total antioxidant capacity (TAC). MDA and TAC also were measured in liver tissue. Histopathological examination was performed to assess the severity of hepatic injury. Apoptosis was evaluated using the apoptosis protease-activating factor 1 (APAF-1) antibody; the expressions of eNOS and iNOS were also assessed by immunohistochem-istry in all groups. Serum alanine aminotransferase, aspartate aminotransferase, g-glutamyl transferase, uric acid, MDA, and TAC, tissue MDA and TAC levels, hepatic injury, and score for extent and for intensity of eNOS, iNOS, and apoptosis protease-activating factor 1 were significantly different in TDF and PTX groups compared to the IR group. High dose-TDF and PTX have the best protective effect on IR-induced liver tissue damage. This study showed that
Conflicts of interest: All authors declare no conflicts of interest.
* Corresponding author. Gaziosmanpas‚a Taksim Training and Research Hospital, Department of Pathology, Karayolları Gaziosmanpas‚a, _Istanbul 34255, Turkey.
E-mail address:sibel_bektas@yahoo.com(S. Bektas).
http://dx.doi.org/10.1016/j.kjms.2016.05.005
1607-551X/Copyrightª 2016, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Available online atwww.sciencedirect.com
ScienceDirect
TDF and PTX supplementation may be helpful in preventing free oxygen radical damage, lipid peroxidation, hepatocyte necrosis, and apoptosis in liver IR injury and minimizing liver damage.
Copyrightª 2016, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/
by-nc-nd/4.0/).
Introduction
Ischemia/reperfusion (IR) injury results from prolonged ischemic damage followed by restoration of blood perfusion. IR injury is a serious complication of liver surgery, transplantation, and various forms of circulatory shock. Liver IR injury is a complex cascade of events mediated by numerous inflammatory cells and molecular mediators, resulting in hepatocyte death and systemic inflammatory response. The degree of inflammatory response and organ dysfunction is dependent on the duration of liver ischemia and underlying liver disease
[1e3]. A large number of pharmacological agents have been shown to confer protection against IR injury in the liver. These agents include antioxidants, ozone, adeno-sine agonists, nitric oxide donors, sildenafil, and varde-nafil[3e5]. Nevertheless, only a few drugs are currently introduced into clinical practice.
Tadalafil (TDF) is a potent and selective inhibitor of phosphodiesterase type-5 (PDE5), which was originally studied as a potential antianginal agent, but became popular for its use in treatment of erectile dysfunction and pulmonary arterial hypertension. Hepatic vascular resistance is regulated by contraction or relaxation of smooth muscle cells in terminal arterioles. By contrast, perisinusoidal stellate cells regulate sinusoidal tonus depending on concentration of nitric oxide (NO) synthe-sized by sinusoidal endothelial cells. TDF is a specific in-hibitor of the NO/cyclic guanosine monophosphate (cGMP) pathway in vascular smooth muscle and platelets, which results in vasodilation of peripheral arteries and veins and inhibition of platelet aggregation respectively. NO gen-erates cGMP, which induces cellular response such as vasodilation. cGMP is inactivated to GMP by phosphodi-esterases. Inhibition of PDE augments and prolongs the cellular responses of vasodilation induced by NO and its products. [6,7]. NO is synthesized by one of three NO synthase (NOS) isoforms. The expression of neuronal NOS is mostly limited to neural tissue. Inducible NOS (iNOS) is not expressed under normal circumstances but is upre-gulated during inflammatory conditions in hepatocytes, endothelial cells, biliary cells, Kupffer cells, neutrophils, and T lymphocytes. Endothelial NOS (eNOS) is constitu-tively expressed in many cell types, including liver endo-thelial cells and hepatocytes. Several studies have revealed the protective effect of TDF on renal, myocar-dial and testicular IR injury[8e10].
Pentoxifylline (PTX) is a nonspecific type-4 PDE inhibi-tor that displays vasodilainhibi-tory effects on peripheral blood vessels, particularly in the liver [11]. Some studies have indicated that PTX treatment restores depressed cardiac
output and improves hepatic perfusion and intestinal blood flow after hemorrhage and resuscitation[3,12,13]. PTX has been reported to suppress the production of tumor necrosis factor-a, interleukin (IL)-1, IL-6, and IL-12 and to reduce oxidative stress and inflammatory indices
[14,15]. Although TDF and PTX have demonstrated bene-ficial effects, the mechanisms by which these drugs exert protective effects are not fully understood in liver IR injury.
Several mechanisms exist to inhibit IR injury and many drugs have also shown protective effects. The protection mechanisms against IR-induced injury are multifactorial. A number of mechanisms have been proposed, including the elimination of free radicals, inhibition of free radical pro-duction, neutrophilic inhibition, and reduction of lipid peroxidation [6,7,12e14]. Although the basic pathological mechanism underlying hepatic injury is not completely understood, it has been shown that reactive oxygen spe-cies, formed during IR and apoptosis play an important role in this process. This is the first experimental study in which the effect of two different PDE inhibitors on liver IR injury was investigated. The purpose of this study was to establish the impact of TDF and PTX on hepatic apoptosis and the expressions of eNOS and iNOS in IR-induced liver tissue damage.
Methods
The experimental protocols were conducted with the approval of the Animal Research Committee at Bulent Ecevit University, Kozlu, Zonguldak. All animals were maintained in accordance with the recommendations of the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
Animal model
Forty female Wistar rats weighing 250e300 g were housed individually in cages, and were allowed free access to standard rat chow and water before the experiments. The animal rooms were windowless with temperature (22 2C) and lighting controls. The animals were fasted overnight before the experiments but were given free access to water. They were anesthetized by 100 mg/kg ketamine intraperitoneally. All surgical interventions were performed under sterile conditions by the same surgical team at the same period and environment. After midline laparotomy, the left and median liver lobes were rendered ischemic with a microvascular clamp. The successful occlusion of the hepatic artery and portal triad branch in question was
confirmed by a change in color in the affected lobes. The hepatic ischemia lasted 90 minutes and was followed by 2 hours of reperfusion. At the end of reperfusion period, the animals were sacrificed by exsanguination through cardiac puncture and blood collection. The blood was immediately centrifuged, and the plasma supernatant was stored at 80C until it was assayed for biochemical studies. The left
and median liver lobes were harvested at the end of each procedure for histopathology, immunohistochemistry, and biochemical analysis.
Experimental groups
Sham group (nZ 8): Animals subjected to anesthesia, only laparotomy and mobilization of liver (without induction of hepatic ischemia); IR group (nZ 8): Animals subjected to IR procedures alone; Low-dose TDF (nZ 8) and high-dose TDF (nZ 8) groups: Animals received 2.5 mg/kg and 10 mg/kg TDF by gastric gavage, respectively and underwent IR pro-cedures after 45 minutes; PTX group (nZ 8): Animals received 40 mg/kg PTX by gastric gavage and underwent IR procedures after 45 minutes.
Biochemical analysis
Blood
Blood was collected into tubes at the time of death. Blood samples were centrifuged at 1000 g for 10 minutes at 4C to remove plasma. Aliquots of the samples were transferred into polyethylene tubes to be used in the assay of biochemical parameters and were stored at 80C until
analysis. Tissue
All tissues were washed twice with cold saline solution, placed into glass bottles, labeled, and stored in a deep freezer (80C) until processing. Liver tissues were
ho-mogenized in 10 volumes of 150mM ice-cold KCl using a glass Teflon Homogenizer (Ultra Turrax IKA T18 Basic; IKA, Wilmington, NC, USA) after cutting the tissues into small pieces with scissors (for 2 minutes at 5000 rpm). The ho-mogenate was then centrifuged at 5000 g for 15 minutes. The supernatant was used for analysis.
Assay of biochemical parameters
Malondialdehyde (MDA) was assayed with a commercial kit (Immundiagnostik AG, Bensheim, Germany) that is based on the high-performance liquid chromatography method using an Agilent 1200 HPLC system (Agilent, San Jose, CA, USA). Antioxidant capacity was analyzed using a TAS Kit (Randox, Crumlin, Antrim, UK) according to the manu-facturer’s instructions. Measurements were performed using a Shimadzu UV-1601 (Shimadzu, Kyoto, Japan) spectrophotometer. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), uric acid (UA), and g-glutamyl transferase (GGT) levels were measured with commercially available kits on an Advia 2400 automated analyzer (Siemens Healthcare Diagnostics, Tarrytown, NY, USA). Protein concentrations of the supernatants were determined by the method described by Lowry et al.[16].
Histopathological evaluation
Liver tissues were fixed in 10% buffered formaldehyde and prepared for routine paraffin embedding. Tissue sections (5 mm) were then stained with hematoxylin and eosin and examined under a light microscope (Olympus- BH-2, Wendenstrasse, Hamburg, Germany) by an experienced pathologist, who was unaware of the treatment conditions. A semiquantitative histopathological evaluation of the severity of the hepatic injury was graded on a scale as follows: Grade 0, minimal or no evidence of injury; Grade 1, mild injury consisting of cytoplasmic vacuolation and focal nuclear pyknosis; Grade 2, moderate to severe injury with extensive nuclear pyknosis, cytoplasmic hypereosinophilia, and loss of intercellular borders; and Grade 3, severe ne-crosis with disintegration of hepatic cords, hemorrhage, and neutrophil infiltration[5].
Immunohistochemical staining
Sections (5 mm) were deparaffinized in xylene, and then dehydrated. 3% hydrogen peroxide was used for blocking endogenous peroxidase activity. Antigen retrieval was car-ried out by incubation in citrate buffer (pH 6.0) for 10 mi-nutes in a pressure cooker. The sections were exposed to the primary antibodies to eNOS [rabbit polyclonal anti-human immunoglobulin (IgG), clone: C-20, 1:100 dilution; Santa Cruz Biotechnology, Inc., Heidelberg, Germany], iNOS (rab-bit polyclonal anti-human IgG, clone: N-20, 1:100 dilution; Santa Cruz Biotechnology), and APAF-1 (mouse monoclonal anti-human IgG, clone: 2E10, 1:100 dilution; Santa Cruz Biotechnology) for 60 minutes at room temperature. The standard streptavidin-biotin-peroxidase complex method was used for primary antibodies and by employing dia-minobenzidine as the chromogen. Intense brown staining of the cytoplasm was considered as a positive reaction for eNOS, iNOS, and APAF-1. Samples of colorectal carcinoma and placenta were used as a positive control for APAF-1 and eNOSeiNOS expressions, respectively. Negative controls were obtained by omitting the primary antibody.
Evaluation of immunohistochemical staining
We calculated the immunohistochemical score for extent and for intensity of eNOS, iNOS, and APAF-1 for each liver tissue according to the previously published methods[10]. The intensity of the immune expression was categorized into four groups: no staining (0), weak (þ1), moderate (þ2), and strong (þ3). The extent of epithelial cells with positive cytoplasmic staining was estimated and classified on a four-point scale: no stainingZ 0; 1 Z 1e9%; 2Z 10e49%; and 3 Z 50e100%.
Statistical analysis
Statistical analysis was performed with SPSS version 18.0 software (SPSS Inc., Chicago, IL, USA). All values were expressed as the mean standard error of mean. Differ-ences between the groups were evaluated by one-way analysis of variance test with posthoc multiple compari-sons, ManneWhitney U test, and KruskaleWallis test.
A p value< 0.05 was considered statistically significant for all tests.
Results
Biochemical results
Serum ALT, AST, and GGT levels, which were used as indices of hepatic injury, showed a significant increase (p< 0.05) in the IR group, while high-dose TDF and PTX administration prevented this effect (p< 0.05); there was no significant difference between low-dose TDF and IR groups (p> 0.05). ALT, AST, and GGT levels were similar in the high-dose TDF and PTX groups (p> 0.05). Each of the serum AST, ALT, and GGT levels were statistically higher in low-dose and high-dose TDF and PTX groups than the sham group (p< 0.001). MDA is an index of hepatic damage associated with lipid peroxidation and total antioxidant capacity (TAC) is used to evaluate oxidative stress. Serum and tissue MDA and TAC levels were significantly different in the IR group compared with high-dose TDF and PTX groups (p< 0.05), whereas there was no significant difference compared with low-dose TDF group (p> 0.05). Serum and tissue MDA and TAC levels were similar in high-dose TDF and PTX groups (p> 0.05). UA is released in hypoxic conditions and is a potent antioxi-dant. Serum UA levels were significantly different in the IR group compared with low- and high-dose TDF and PTX groups (p< 0.05), whereas no significant difference was observed between low- and high-dose TDF and PTX groups (p> 0.05). The biochemical characteristics of the all groups are shown inTable 1.
Histopathological results
In the sham group, the livers showed a normal lobular ar-chitecture with central veins and radiating hepatic cords with a mean overall grade of 0 in each. In low- and high-dose TDF and PTX groups, the severity of hepatic injury showed a significant decrease when compared to IR group
(p< 0.05). Hepatic injury grades of low-dose TDF and PTX groups were similar and were not significantly different (p> 0.05; Figure 1). High-dose TDF exerted the best his-topathological protective effect on IR-induced liver tissue damage (p< 0.05;Table 2). The histopathological results of the all groups are shown inTable 2.
Immunohistochemical results
Hepatocyte apoptosis was significantly higher in the IR group versus the high-dose TDF, PTX, and sham groups (p< 0.05), whereas no significant difference was evident with low-dose TDF (p> 0.05; Figure 2). The rats treated with high-dose TDF and PTX showed a significantly lower number of apoptotic cells. Scores for extent and intensity of eNOS (Figure 3) and iNOS (Figure 4) were significantly higher in IR group compared with high-dose TDF and PTX groups (p< 0.05) but not significantly different from low-dose TDF group (p> 0.05). eNOS and iNOS expressions were similar in the high-dose TDF and PTX groups (p> 0.05). The immunohistochemical results of the all groups are shown inTable 2.
Discussion
IR is a major component of injury in vascular occlusion during both liver transplantation and liver tumor resec-tion. There are other conditions that decrease hepatic blood flow and cause hepatic ischemia, such as hemor-rhagic shock, sepsis, hepatic artery ligation, trauma, and vascular lesions. The pathophysiology of liver IR includes a number of mechanisms including oxidant stress formed during IR that contribute to various degrees to organ damage. Liver IR injury is divided into two phases. The early phase covers the first 2 hours following reperfusion and is dominated by Kupffer cell activation and release of various mediators such as reactive oxygen species and cytokines. The late phase, which commences at about 6 hours of reperfusion and continues until 48 hours post-reperfusion, and is characterized by neutrophil infiltration
Table 1 Biochemical parameters of all groups.
Sham (nZ 8) IR (nZ 8) TDF (nZ 8) 2.5 mg/kg TDF (nZ 8) 10 mg/kg PTX (nZ 8) 40 mg/kg Serum ALT (u/L) 660.4 220 3020.7 1249 2254.5 939 1018.6 137a,b 1125.5 171a,b AST (u/L) 692.8 250 3904.7 1914 1823 549 1123.2 246a,b 1259.1 477a,b GGT (u/L) 3.24 1.01 6.75 2.12 6.12 0.83 3.33 1.22a,b 3.68 1.77a,b TAC (mM Trolox) 1.45 0.32 0.81 0.32 0.89 0.34 1.68 0.39a,b 1.87 1.75a,b MDA (mM) 2.41 0.78 5.25 1.02 3.75 0.66 2.62 0.56a,b 2.56 1.09a,b UA (mg/dL) 1.27 0.38 1.83 0.21 1.21 0.16a 1.10 0.10a 1.22 0.16a Tissue
TAC (nmol trolox/mg protein) 130 38.4 257.3 80.2 190.1 64.1 134 25.5a,b 139.7 19.5a,b MDA (nmol/mg protein) 2.16 0.86 4.37 1.6 3.82 0.64 2.33 1.02a,b 2.67 0.68a,b Data are presented as mean standard deviation.
ALTZ alanine aminotransferase; AST Z aspartate aminotransferase; GGT Z g-glutamyl transferase; IR Z ischemia/reperfusion; MDAZ malondialdehyde; PTX Z pentoxifylline; TAC Z total antioxidant capacity; TDF Z tadalafil; UA Z uric acid.
a p< 0.05 compared with IR group.
in the liver and the progression of hepatocyte and sinu-soidal endothelial cell damage [1,2,17]. During the ischemia as a result of glycogen consumption and lack of oxygen supply, Kupffer cells, sinusoidal endothelial cells, and hepatocytes suffer with lack of ATP production. The lack of ATP leads to failure of the sodium/potassium ATP-dependent plasma membrane pump and subsequent
intracellular Naþ accumulation, edema, and swelling. Kupffer cells and sinusoidal endothelial cells swelling combined with an increase in the vasoconstrictors endo-thelin and thromboxane A2 and a decrease in the vaso-dilator nitric oxide lead to sinusoidal narrowing. The end result is a significant reduction of microcirculatory blood flow on reperfusion[1,2,18].
Figure 1. (A 20) Ischemia/reperfusion group: Hepatocytes cells with severe necrosis, disintegration of hepatic cords and hemorrhage. (B20) Low-dose tadalafil and (C 20) pentoxifylline groups: Hepatocytes cells with moderate injury, cytoplasmic hypereosinophilia, and loss of intercellular borders. (D 20) High-dose tadalafil group: mild injury consisting of cytoplasmic vacuolation and focal nuclear pyknosis. (Hematoxylin and eosin).
Table 2 Hepatic injury grades and immunohistochemical expressions of apoptosis protease-activating factor 1, endothelial and inducible nitric oxide synthases of all groups.
Sham (nZ 8) IR (nZ 8) TDF (nZ 8) 2.5 mg/kg TDF (nZ 8) 10 mg/kg PTX (nZ 8) 40 mg/kg Hepatic injury grade 0.0 0.0 2.8 0.4 1.9 0.3a 0.6 0.5a,b 1.8 0.4a,c
APAF-1 Extent 0.6 0.51 2.5 0.50 1.8 0.51 1.0 0.0a,d 1.0 0.0a,d Intensity 0.6 0.51 2.4 0.51 1.9 0.56 1.3 0.52a,d 1.3 0.48a,d eNOS Extent 0.4 0.51 1.4 0.51 1.3 0.48 0.8 0.42a,d 1.0 0.0a,d Intensity 0.4 0.51 2.3 0.67 1.6 0.51 1.0 0.47a,d 1.5 0.52a,d iNOS Extent 0.3 0.48 1.6 0.51 1.5 0.52 0.8 0.42a,d 1.2 0.42a,d Intensity 0.3 0.48 2.3 0.48 2.1 0.31 1.1 0.56a,d 1.2 0.42a,d Data are presented as mean standard deviation.
APAF-1Z apoptosis protease-activating factor 1; eNOS Z endothelial nitric oxide synthases; iNOS Z inducible nitric oxide synthases; IRZ ischemia/reperfusion; PTX Z pentoxifylline; TDF Z tadalafil.
ap< 0.05 compared with IR group.
bp< 0.05 compared with low-dose TDF and PTX groups. c p> 0.05 compared with low-dose TDF group.
Further, reperfusion injury is caused by the generation of toxic oxygen free radicals with the return of blood flow following ischemia. These free radicals can react with lipids in the cell membranes and initiate lipid peroxidation, which
is responsible for the IR injury. During postischemic reper-fusion, liver tissue can counteract oxidative stress by upregulating antioxidant defenses with the help of the antioxidant enzymes[1,2,18].
Figure 2. APAF-1 immune expression of liver cells (A20) strong cytoplasmic staining in ischemia/reperfusion group, (B 20) moderate cytoplasmic staining in low-dose tadalafil group, weak cytoplasmic staining in (C20) pentoxifylline and (D 20) high-dose tadalafil groups.
Figure 3. eNOS immune expression of liver cells (A20) strong cytoplasmic staining in ischemia/reperfusion group, (B 20) moderate cytoplasmic staining in low-dose tadalafil group, weak cytoplasmic staining in (C20) pentoxifylline and (D 20) high-dose tadalafil groups.
NO is an important molecule in IR injury and produces cGMP. The vasodilator effect of NO is normally limited by the PDE-mediated degradation of its second messenger cGMP. TDF is a long-acting PDE5 inhibitor and could reduce the degradation of cGMP in tissues. PDE5 inhibitors have been shown to potentiate the vasodilator effect of NO, improving vascular function in patients with erectile dysfunction, pulmonary arterial hypertension, portal hy-pertension, and in cirrhotic rat liver [17,19e21]. Further-more, it has been shown that the administration of TDF may also be useful against ischemic injury in other organs and tissues such as neurons, skin island flaps, ovary, myocar-dium, and kidney[10,22e25]. Baek et al.[26]showed that 10 mg/kg of TDF suppressed apoptotic neuronal cell death and enhanced cell proliferation in the hippocampus of maternal-separated rat pups.
PTX, a methylxanthine derivative and a nonspecific type-4 PDE inhibitor, is clinically used in the treatment of lower extremity claudication. PTX has been reported to exert vasodilator effect in addition to significantly sup-pressing inflammation and to reduce hepatocyte apoptosis in experimental models [27]. Takhtfooladi et al. [28]
showed that 40 mg/kg of PTX reduced inflammatory cell infiltration, hemorrhage, and edema in lung IR injury. So¨nmez and Du¨ndar [29] reported that 50 mg/kg of PTX ameliorated eNOS, iNOS, and neuronal NOS protein levels and apoptotic cells in the rat kidney. The beneficial effects of PTX are believed to occur through various mechanisms such as inhibition of PDE, increased cAMP levels and downregulation of tumor necrosis factor-alpha, IL-1, IL-6, IL-12, transforming growth factor-b, interferon-g, stellate cell activation, and procollagen-I mRNA expression[30,31]. Recent studies have implied therapeutic effects of PTX on
alcoholic hepatitis, nonalcoholic fatty liver, and liver fibrosis; however, its effect on liver IR injury remains un-clear[30,32,33]. The results of the present study showed that, histopathologically, high-dose TDF (10 mg/kg) and PTX (40 mg/kg) improves hepatic architecture, hepatocyte necrosis, hemorrhage, and neutrophil infiltration in liver IR injury.
eNOS is constitutively expressed in liver endothelial cells and hepatocytes. iNOS is not expressed under normal circumstances, but it is upregulated in various cell types including hepatocytes, neutrophils, T-lymphocytes, endothelial, biliary, and Kupffer cells in liver IR injury. Abu-Amara et al.[34] reported that eNOS is an essential enzyme for the protective effects in liver IR injury. Another study reported that iNOS had protective effects against IR injury in heart [35]. By contrast, nitrosative stress has also been recognized as a contributor to cellular damage associated with IR injury. When iNOS and eNOS are upregulated in IR injury, the excessive NO produced will react with superoxide anion, creating peroxynitrite. Per-oxynitrite then aggravates the injury through lipid perox-idation, apoptosis, and necrosis by nitration of tyrosine residues on tissue proteins [36]. In our study, immune scores for extent and eNOS and iNOS intensities were significantly decreased in high-dose TDF and PTX groups compared with the IR group. This result can be explained by reduced nitrosative stress and inhibition of hepatocytes and endothelial cell injury subsequent to high dose TDF and PTX administration in liver IR.
In IR injury, the mitochondrion participates in various pathophysiological processes. There is failure of ATP pro-duction as a consequence of disruption of oxidative phosphorylation caused by the generation of reactive
Figure 4. iNOS immune expression of liver cells (A 20) strong cytoplasmic staining in ischemia/reperfusion group, (B 20) moderate cytoplasmic staining in low-dose tadalafil group, weak cytoplasmic staining in (C20) pentoxifylline and (D 20) high-dose tadalafil groups.
oxygen and nitrogen species. Cytosolic ionic disturbances lead to mitochondrial ionic disturbances, and vice versa, with consequent plasma and mitochondrial membrane damage, including the formation of mitochondrial permeability transition pores. Reactive oxygen and nitro-gen species can cause oxidative damage to the enzyme complexes of respiratory chain and release of cytochrome C into the cytosol, triggering both apoptotic and necrotic cell death. The protective effects of PDE5 inhibition were recently shown to be mediated by increased intracellular cGMP levels and the activation of mitochondrial K-ATP channels either directly or indirectly through a variety of signaling pathways, such as activation of protein kinase C and protein kinase G. Mitochondrial K-ATP channel open-ing may prevent apoptosis, presumably by inhibitopen-ing mitochondrial Ca2þaccumulation during ischemia and by preserving mitochondrial inner membrane potential[37]. Antiapoptotic properties of PDE5 inhibitors have been re-ported. Sildenafil, another PDE inhibitor, inhibits apoptosis by increasing cytochrome-c secretion and the Bcl-2/Bax ratio through NO[38]. These mechanisms may explain the lower APAF1 levels in high-dose TDF group and also PTX group in this study. In addition, high-dose TDF and PTX administration in liver may prevent apoptosis and necrosis by reducing the formation of reactive oxygen and nitrogen species.
TAC is used to evaluate oxidative stress. In this study, higher serum and lower tissue levels of TAC in high-dose TDF and PTX groups compared with the IR group could be due to upregulation of enzymatic antioxidant defense of liver tissue. MDA is the end product of lipid peroxidation and is a well-known marker for free radical formation in postischemic tissue. In our study, hepatic and serum MDA levels were significantly decreased in high-dose TDF and PTX groups compared to IR group. This condition indicates that high dose TDF and PTX administration decreased free radical formation and lipid peroxidation in liver IR.
Serum ALT, AST, and GGT levels are widely used as markers of liver cell damage. In this study, high-dose TDF and PTX promoted a protective effect against IR injury in liver. UA is released in hypoxic conditions and acts as a strong reducing agent and a potent antioxidant. We believe that reduced UA levels by TDF and PTX administrations could minimize liver damage.
Effective dose of PDE inhibitors are different in rats and humans due to their different metabolic rate and half-life. Previous studies reported that the half-life of PDE inhibitors in rats was at least three times less than in humans (0.4e1.3 hours in rat vs. 4 hours in humans)[39]. Therefore, the drug doses used in rats is much higher than doses commonly used therapeutically in humans. For example, Kovanecz et al.[40]indicated that a 20 mg/kg daily dose of sildenafil (PDE5 inhibitor) in rats was approximately equivalent to a 200 mg oral daily dose in humans when corrected for differences in total body surface area.
The cellular results of liver IR injury are necrosis and apoptosis of hepatocytes and sinusoidal endothelial cells. Clinically, IR injury leads to liver dysfunction, which makes a major contribution to the morbidity and mortality of liver surgery. Although considerable research efforts have been made to find and eliminate underlying causes of hepatic IR injury, many points still remain unclear.
In conclusion, we have demonstrated that high-dose TDF and PTX have beneficial histopathological and biochemical effects that are protective against liver IR injury. This an-imal model implies that TDF and PTX supplementation may be helpful in preventing free oxygen radical damage, lipid peroxidation, hepatocyte necrosis, and apoptosis in liver IR injury and minimizing liver damage. We believe that studies on the protective effects of TDF and PTX in liver IR damage will lead to the proper application of type-4 and -5 PDE inhibitors in clinical practice.
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