The effects of pentoxifylline and caffeic acid phenethyl ester in the treatment of d -galactosamine-induced acute hepatitis in rats

Abstract

The aim of this study was to investigate histological changes in hepatic tissue and effects of pentoxifylline (PTX) and caffeic acid phenethyl ester (CAPE) on these changes using histochemical and biochemical methods in rats, in which hepatitis was established by d-galactosamine (d-GAL). Rats were divided into five groups as follows: control group, d-GAL (24 h) group, d-GAL group, d-GAL + PTX group, and d-GAL + CAPE group. In histological evaluations, the control group showed normal appearance of the liver cells. However in the d-GAL groups, focal areas consisting of inflammatory, necrotic, and apoptotic cells were detected in parenchyma. Glycogen loss was observed in the hepatocytes localized at the periphery of lobule. It was found that number of mast cells of portal areas were significantly higher in d-GAL groups compared with other groups (p = 0.0001). In addition, the number of cells with positive staining by Ki-67 and caspase-3 were significantly increased in GAL groups compared with the control group (p = 0.0001). In biochemical analysis, there was an increase in malondialdehyde and myeloperoxidase levels, while a decrease was observed in glutathione level and glutathione peroxidase activity in groups treated with d-GAL compared with the control group. On the other hand, it was seen that, in the groups treated with d-GAL, histological and biochemical injuries in the liver were reduced by administration of PTX and CAPE. In this study, we demonstrated the ameliorative effects of PTX and CAPE on d-GAL-induced liver injury.

Keywords

Hepatitis pentoxifylline caffeic acid phenethyl ester

Introduction

Acute hepatitis is a medical condition defined by the inflammation of the liver and characterized by the presence of inflammatory cells in the organ caused by several factors, such as viruses, bacterium, parasite, toxic substances, drugs, and various metabolic and autoimmune diseases.¹ Various experimental hepatitis models that have similar morphological and pathophysiological characteristics to viral hepatitis have been developed so far. d-Galactosamine (d-GAL) is one of the most commonly used hepatitis-inducing agent in rats.² d-GAL administration causes hepatocyte death resulting in an acute damage to liver parenchyma.³ d-GAL also induces the secretion of cytokines, mainly tumor necrosis factor α (TNF-α), by activating Kupffer cells.⁴ TNF-α production has been shown to induce hepatocyte apoptosis.^5,6 Oxygen-free radicals released by activated Kupffer cells are suggested to be the main reason of parencyhmal damage after the administration of d-GAL.⁷

Pentoxifylline (PTX, nonselective phosphodiesterase inhibitor) is a methyl xanthine derivative that has been used for its regulatory effect on blood flow. PTX increases the flexibility of red and white blood cells, reduces blood viscosity by decreasing plasma fibrinogen concentrations, and decreases platelet aggregation and thrombus formation. Polymorphonuclear leukocyte-mediated effects, such as superoxide production, chemotaxis, phagocytosis, and TNF production, are inhibited by PTX.^8,9

Caffeic acid phenethyl ester (CAPE), an active component of honeybee propolis, is structurally similar to flavonoids.¹⁰ CAPE is known to have antioxidant properties as well as antitoxic, anti-inflammatory, antiviral, immunomodulatory, neuroprotective, and cytostatic effects.^11,12

The aim of this study was to investigate the effects of PTX and CAPE on d-GAL-induced acute hepatitis using histochemical and biochemical methods.

Material and methods

Animals

Forty male Wistar albino rats (3–4 months old) weighing 200–250 g obtained from the Experimental Animal Research Center of Inonu University were used in the present study. The animals were housed in individual cages for 21 days in a well-ventilated room with a 12-h light/12-h dark cycle at 21°C. Animals were fed with standard rat chow and tap water ad libitum. The experiments were performed in accordance with the guidelines for animal research from National Institute of Health and were approved by the Committee on Animal Research at Inonu University, Malatya, Turkey.

Experimental induction of hepatotoxicity

Hepatotoxicity was induced in animals by an intraperitoneal (i.p.) injection of d-GAL (d-galactosamine hydrochloride, 500 mg/kg body weight; BioChemica, Germany) in a freshly prepared physiological saline as a single dose on day 0.

Experimental protocol

The animals were randomly divided into five groups each consisting of eight rats.

Group I: Control

Group II (d-GAL (24 h) group): Rats received single i.p. injection of d-GAL (500 mg/kg body weight) dissolved in 0.9% saline on day 0, and at the end of the following 24 h, they were killed.

Group III (d-GAL group): Rats received single i.p. injection of d-GAL (500 mg/kg body weight) dissolved in 0.9% saline on day 0, and at the end of 21st day, they were killed.

Group IV (d-GAL + PTX group): Rats received single i.p. injection of d-GAL on day 0 and were treated with PTX (i.p; Trental ampule) dissolved in 0.9% saline at a dose of 50 mg/kg/day for 20 days.

Group V (d-GAL + CAPE group): Rats received single i.p. injection of d-GAL on day 0 and they were treated with CAPE (i.p.; Sigma Aldrich, Steinheim, Germany) dissolved in 4% ethanol at a dose of 10 µmol/kg body weight for 20 days.

At the end of the experiment, rats were killed by ketamine anesthesia. After blood samples were collected from tail vein, livers were removed and divided into two portions. One sample was used for histopathological examination, and the other was used for evaluation of oxidative stress parameters by biochemical methods.

Histopathological evaluation

Liver tissue was fixed in 10% formalin and was embedded in paraffin. Tissue sections were cut at 5μm, mounted on slides, stained with hematoxylin–eosin (H-E) for histopathological grading on the number of local areas of inflammation consisting inflammatory, necrotic, and apoptotic cells, periodic acid–Schiff (PAS) for demonstrating hepatic glycogen content, and toluidin blue for indicating mast cells. The number of mast cells was calculated and glycogen loss was scored as follows: 0 = Normal, 1 = Mild, 2 = Moderate, and 3 = Severe. The local area was counted in 10 microscopic fields under 20× objective magnification using Leica Q Win Image Analysis System (Leica Micros Imaging Solution Ltd. Cambridge, UK). Sections were examined using a Leica DFC280 light microscope.

Immunohistochemical methods

For immunohistochemical analyses, thick sections cut at 5 μm were mounted on polylysine-coated slides. After rehydrating, the samples were transferred to citrate buffer (pH 7.6) and heated in a microwave oven at 65°C for 20 min. After cooling for 20 min at room temperature, they were washed with phosphate-buffered saline (PBS). Then the sections were kept in 0.3% hydrogen peroxide (H₂O₂) for 7 min, followed by a wash with PBS. Subsequently, the sections were incubated with primary rabbit-polyclonal cysteine aspartate specific proteinase (caspase-3; Neomarker, Fremont, California, USA) and Ki-67 (Lab Vision, Fremont, California, USA) rinsed in PBS and then incubated with biotinylated goat antipolyvalent for 10 min and streptavidin peroxidase for 10 min at room temperature. The staining procedure was completed with chromogen + substrate for 15 min and the slides were counterstained with Mayer’s hematoxylin for 1 min. Caspase-3 and Ki-67 (+) cells were counted in the 10 microscopic areas under 40× objective magnification using Leica Q Win Image Analysis System (Leica Micros Imaging Solution Ltd). Sections were examined using a Leica DFC280 light microscope.

Biochemical evaluation

Determination of AST and ALT levels

Blood samples were centrifuged for 10 min at 3000 r/min (1500g centrifugal force) and plasma samples were stored at −20°C until the measurement. Cobas Integra 800 (Roche, Nutley, New Jersey, USA) was used for measuring aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels.

Preparation of tissue homogenates

Tissues were homogenized (PCV Kinematica Status Homogenizator, Switzerland) in ice-cold PBS (pH 7.4). The homogenate was sonicated with an ultrasonicator (Branson sonicator 450, Branson Ultrasonics Corporation, Danbury, CT, USA) by three cycles (20 s sonications and 40 s pause on ice) and centrifuged (15,000g, 10 min, 4°C), and cell-free supernatant was subjected to enzyme assay immediately.

Determination of protein

Protein levels of the tissue samples were measured by the Bradford method.¹³ The absorbance measurement was taken at 595 nm using an ultraviolet–visible spectrophotometer. Bovine serum albumin was used as protein standard.

Determination of MPO levels

Tissue myeloperoxidase (MPO) activity was measured using a procedure similar to that documented by Hillegas et al.¹⁴ One unit of enzyme activity was defined as the amount of MPO present that caused a change in absorbance measured at 460 nm for 3 min. MPO activity was expressed as units per gram tissue.

Determination of MDA levels

The analysis of lipid peroxidation was carried out as described by Buege and Aust¹⁵ with a minor modification. The reaction mixture was prepared by adding 250 μL homogenate into 2 mL reaction solution (15% trichloroacetic acid:0.375% thiobarbituric acid:0.25 N hydrochloric acid, 1:1:1, w/v) and heated at 1000°C for 15 min. The mixture was cooled to room temperature, centrifuged (10,000g for 10 min), and absorbance of the supernatant was recorded at 532 nm. 1,1,3,3-Tetramethoxypropane was used as malondialdehyde (MDA) standard. MDA results were expressed as nanomoles per milligram protein in the homogenate.

Determination of GSH levels

The formation of 5-thio-2-nitrobenzoate was followed spectrophotometrically at 412 nm.¹⁶ The amount of glutathione (GSH) in the extract was determined as nanomoles per milligram protein utilizing a commercial GSH as the standard.

Determination of GPx activities

Glutathione peroxidase (GPx) activity was measured according to Lawrence and Burk.¹⁷ In brief, 1.0 mL of 50 nmol/L PBS solution (pH 7.4) including 5 mmol/L ethylenediaminetetraacetic acid, 2 µmol/L nicotinamide adenine dinucleotide phosphate (NADPH, 20 µmol/L GSH, 10 µmol/L sodium azide, and 23 mU of glutatione reductase was incubated at 37°C for 5 min. Then 20 µL of 0.25 mmol/L H₂O₂ solution and 10 µL of supernatant were added to the assay mixture. The change in absorbance at 340 nm was monitored for 1 min. A blank with all ingredients except for supernatants was also monitored. Specific activity was calculated as micromole NADPH consumed per minute per milligram of protein (i.e. U/mg protein) using an appropriate molar absorptivity coefficient (6220 M/cm). GPx levels were expressed as a micromole per milligram of protein (U/mg protein).

Determination of SOD activities

Superoxide dismutase (SOD; copper and zinc-SOD) activity was measured in the supernatant fraction using xanthine oxidase/cytochrome c method according to the method followed by McCord and Fridovich,¹⁸ where one unit of activity is the amount of enzyme needed to cause half-maximal inhibition of cytochrome c reduction. The amount of SOD in the extract was determined as nanograms of enzyme per milligram protein utilizing a commercial SOD as standard.

Statistical analysis

Statistical analysis was carried out using the Statistical Package for Social Sciences for Windows version 13.0 (SPSS Inc., Chicago, Illinois, USA) and MedCalc (SBB Consulting 10, UK) statistical programs. All data are expressed as arithmetic mean ± standard error. Normality for continued variables in groups was determined by the Shapiro–Wilk test. The variables didn’t show normal distribution (p < 0.05). So, Kruskal–Wallis and Conover tests were used for comparison of variables among the studied groups. The value of p < 0.05 was regarded as significant.

Results

Histopathology

Control group showed normal histological appearance (Figure 1(a)). In d-GAL groups, local areas that consisted of inflammatory, necrotic, and apoptotic cells were detected in parenchyma (Figure 1(b)). Apoptotic cells with their eosinophilic cytoplasm and pyknotic nuclei were separated from the surrounding cells with a halo (Figure 1(c)). In these areas, numerous eosinophils with reddish granules were noticed (Figure 1(c)). Another striking finding was the presence of necrotic cells with eosinophilic cytoplasm and pyknotic nuclei scattering among intact hepatocytes (Figure 1(d)).

Figure 1.

Control group. Histological appearance was normal (a) d-GAL (24 h) group. (b) Local areas consisted of inflammatory, necrotic, and apoptotic cells were observed (arrows; H-E, ×X20) and (c) the apoptotic cells (arrows) and eosinophils (arrowheads; H-E, ×100). (d) Necrotic cells with eosinophilic cytoplasm and pyknotic nuclei (arrows; H-E, ×40). d-GAL: d-galactosamine; H-E: hematoxylin–eosin.

The number of affected local areas in d-GAL groups was statistically higher than that of the control group (p = 0.0001; Figure 2(a)). However, damaged areas were clearly decreased statistically in d-GAL + PTX and d-GAL + CAPE groups when compared with d-GAL groups (p = 0.0001; Figure 2(b) and (c)).

Figure 2.

d-GAL group. (a) The inflammatory cells (arrows; H-E, ×20) d-GAL + PTX (b) and d-GAL + CAPE (c) groups. The appearance of hepatocytes is almost intact but mild inflammatory cell infiltration still was marked in some area (H-E, ×20). d-GAL: d-galactosamine; H-E: hematoxylin–eosin; CAPE: caffeic acid phenethyl ester; PTX: pentoxifylline.

Glycogen storage

Hepatic glycogen content was marked by the presence of a granular magenta staining called PAS-positive reaction (Figure 3(a)). The glycogen content of hepatocytes was decreased in the d-GAL groups (Figure 3(b) and (c)). Conversely, PTX and CAPE treatments reduced loss of the glycogen (Figure 3(d) and (e)).

Figure 3.

(a) Control group. The PAS-positive reaction shows a magenta staining where glycogen is present within hepatocytes, (b) d-GAL (24 h) and (c) d-GAL groups marked reduction in glycogen content especially in the periphery of classic liver lobule (asterisks) (d) d-GAL + PTX and (e) d-GAL + CAPE groups: the view of glycogen is nearly normal except mild loss of the glycogen in the some area (asterisks; PAS, ×10). d-GAL: d-galactosamine; PTX: pentoxifylline; PAS: periodic acid–Schiff; CAPE: caffeic acid phenethyl ester.

Mast cells

The number of mast cells was significantly higher in the d-GAL groups when compared with the control and treatment groups (Figure 4(a) to (c)). On the other hand, mean number of mast cells was found to be significantly decreased in d-GAL + PTX and d-GAL + CAPE groups in comparison with d-GAL groups (Figure 4(d) and (e)). Mean histopathological damage scores and mean number of mast cells are summarized in Table 1.

Figure 4.

(a) Control, (b) d-GAL (24 h), (c) d-GAL, (d) d-GAL + PTX, and (e) d-GAL + CAPE groups: the view of mast cells around portal area (arrows; toluidin blue, ×40). d-GAL: d-galactosamine; PTX: pentoxifylline; CAPE: caffeic acid phenethyl ester.

Table 1.

The results of semiquantitative histological assessment.^a

Parameters	Control	d-GAL (24 h)	d-GAL	d-GAL (PTX)	d-GAL (CAPE)
The number of local area	0.07 ± 0.025	2.25 ± 1.51^b	0.47 ± 1.54^b	0.14 ± 0.35^c,d	0.21 ± 0.41^c,d
Loss of glycogen	0.27 ± 0.44	1.94 ± 0.79^b	1.25 ± 0.75^b	1.27 ± 0.47	0.98 ± 0.67^b
The number of mast cells	4.20 ± 2.77	130.2 ± 35.42^b	108.5 ± 33.01^b	53.42 ± 13.08^c	35.42 ± 20.39^c

d-GAL: d-galactosamine; PTX: pentoxifylline; CAPE: caffeic acid phenethyl ester.

^aResults are expressed as mean ± SD.

^bSignificant increase (p = 0.0001), versus control.

^cSignificant decrease (p = 0.0001), versus d-GAL (24 h).

^dSignificant decrease (p = 0.0001), versus d-GAL.

Immunohistochemistry

Ki-67 immunoreactivity

Hepatocyte nuclei showing positive reaction by Ki-67 immunostaining method were stained brown (Figure 5(a)). Statistical significant increase in the number of Ki-67 (+) cells was detected in d-GAL groups compared with the control group (p = 0.0001; Figure 5(b) and (c)). Additionally, in d-GAL + PTX and d-GAL + CAPE groups, the number of Ki-67 (+) cells was higher than that of d-GAL groups (p = 0.0001; Figure 5(d) and (e)). Comparison of the number of cells between d-GAL + CAPE group and d-GAL + PTX group in terms of staining intensity did not exhibit any statistically significant difference (p > 0.05).

Figure 5.

(a) Control, (b) d-GAL (24 h), (c) d-GAL, (d) d-GAL + PTX, and (e) d-GAL + CAPE groups: the appearance of Ki-67 (+) cells (arrows; Ki-67, ×40). d-GAL: d-galactosamine; PTX: pentoxifylline; CAPE: caffeic acid phenethyl ester.

Caspase-3 (+) immunoreactivity

In the control group, positive cells for caspase-3 have been not detected (Figure 6(a)). However, a statistical significant increase in the number of caspase (+) cells in d-GAL group was detected in comparison with control group (p = 0.0001; Figure 6(b) and (c)). On the contrary, in d-GAL + PTX and d-GAL + CAPE groups, the number of caspase (+) cells was decreased when compared with d-GAL groups (p = 0.0001; Figure 6(d) and (e)). Mean numbers of Ki-67 and caspase-3 (+) cells are summarized in Table 2.

Figure 6.

(a) Control, (b) d-GAL (24 h), (c) d-GAL, (d) d-GAL + PTX, and (e) d-GAL + CAPE groups: the appearance of caspase (+) cells (arrows; caspase 3, ×40). d-GAL: d-galactosamine; PTX: pentoxifylline; CAPE: caffeic acid phenethyl ester.

Table 2.

The results of immunohistochemical assessment.^a

Parameters	Control	d-GAL (24 h)	d-GAL	d-GAL (PTX)	d-GAL (CAPE)
Ki-67 (+) cells	5.93 ± 4.04	6.86 ± 4.61^b	6.51 ± 5.22^b	9.65 ± 6.16^c,d	10.89 ± 6.12^c,d
Caspase (+) cell	0.00 ± 0.00	101.9 ± 22.43^b	38.66 ± 12.01^b	2.42 ± 1.90^e,f	1.71 ± 2.21^e,f

d-GAL: d-galactosamine; PTX: pentoxifylline; CAPE: caffeic acid phenethyl ester.

^aResults are expressed as mean ± SD.

^bSignificant increase (p = 0.0001), versus control.

^cSignificant increase (p = 0.0001), versus d-GAL (24 h).

^dSignificant increase (p = 0.0001), versus d-GAL.

^eSignificant decrease (p = 0.0001), versus d-GAL (24 h).

^fSignificant decrease (p = 0.0001), versus d-GAL.

Biochemical assessment

Mean AST and ALT levels were significantly increased in d-GAL groups when compared with the control group (p = 0.0001). Treatment with PTX and CAPE at all doses significantly prevented the elevation of the levels of serum enzymes compared with d-GAL groups (p = 0.0001). Mean blood AST and ALT levels of all groups are summarized in Table 3.

Table 3.

The levels of liver function tests of all groups.^a

Parameters	Control	d-GAL (24 h)	d-GAL	d-GAL (PTX)	d-GAL (CAPE)
AST	79.2 ± 6.5	1854.7 ± 76^b	102.66 ± 19^b	82.6 ± 12.3^c,d	80.6 ± 9.11^c,d
ALT	48.4 ± 8.5	1936.6 ± 83^b	54.3 ± 6.71^b	43.5 ± 6.1^c,d	39.1 ± 6.5^c,d

AST: aspartate transaminase; ALT: alanine transaminase; d-GAL: d-galactosamine; PTX: pentoxifylline; CAPE: caffeic acid phenethyl ester.

^aResults are expressed as mean ± SD.

^bSignificant increase (p = 0.0001), versus control.

^cSignificant decrease (p = 0.0001), versus d-GAL (24 h).

^dSignificant decrease (p = 0.0001), versus d-GAL.

Mean tissue MDA and MPO levels of d-GAL groups were significantly higher than that of the control group (p = 0.0002). On the contrary, mean MDA and MPO levels of d-GAL + PTX and d-GAL + CAPE groups were significantly lower than those of d-GAL groups (p = 0.0002).

Mean tissue GSH level of d-GAL groups was significantly lower than that of the control group (p = 0.0003). Mean tissue GSH level of d-GAL + PTX group was significantly higher than that of d-GAL groups (p = 0.0003).

Mean tissue SOD activities of all the groups were similar, so no significant difference was detected. GPx activity of d-GAL group was significantly lower than that of the control group, whereas that of d-GAL + PTX and d-GAL + CAPE groups was significantly increased compared with that of d-GAL group. Mean tissue MDA, MPO, and GSH levels and GPx and SOD activities of all groups are summarized in Table 4.

Table 4.

The levels of biochemical parameters of all groups.^a

Parameters	Control	d-GAL (24 h)	d-GAL	d-GAL (PTX)	d-GAL (CAPE)
MDA (nmol/mg)	0.13 ± 0.02	0.20 ± 0.04^b	0.18 ± 0.05^b	0.13 ± 0.03^c,d	0.13 ± 0.01^c,d
MPO (U/g)	17.17 ± 2.36	32.43 ± 6.46^b	29.30 ± 7.6^b	15.61 ± 2.68^c,d	17.58 ± 2.95^c,d
GSH (nmol/mg)	16.44 ± 2.85	12.40 ± 2.73^e	13.28 ± 2.99^e	20.89 ± 2.49^f	14.17 ± 2.08
GPx (U/mg)	28.19 ± 8.87	26.44 ± 4.97	22.36 ± 5.77^g	34.64 ± 3.65^h	31.43 ± 6.69^h
SOD (U/mg)	8.45 ± 3.2	8.48 ± 1.1ⁱ	7.28 ± 1.8ⁱ	9.32 ± 0.99^j	8.54 ± 1.9^j

d-GAL: d-galactosamine; PTX: pentoxifylline; CAPE: caffeic acid phenethyl ester; MDA: malondialdehyde; MPO: myeloperoxidase; GSH: glutathione; GPx: glutathione peroxidase; SOD: superoxide dismutase.

^aResults are expressed as mean ± SD.

^bSignificant increase (p = 0.0002), versus control.

^cSignificant decrease (p = 0.0002), versus d-GAL (24 h).

^dSignificant decrease (p = 0.0002), versus d-GAL.

^eSignificant decrease (p = 0.0003), versus control.

^fSignificant increase (p = 0.0003), versus d-GAL (24 h) and d-GAL.

^gSignificant decrease (p = 0.0002), versus control.

^hSignificant increase (p < 0.0002), versus d-GAL.

ⁱNo significant change (p > 0.05), versus control.

^jNo significant change (p > 0.05), versus d-GAL (24 h) and d-GAL.

Discussion

d-GAL is one of the most commonly used hepatitis-inducing agents in rats.^19,20 The aim of this study was to investigate the effects of PTX and CAPE on acute hepatitis induced by d-GAL.

Development of hepatocellular injury following the administration of D-GAL in rats has been demonstrated by several authors.^21,22 In the present study, we also observed focal parenchymal areas consisting of inflammatory, necrotic, and apoptotic cells in the sections from d-GAL groups. Takano et al. reported congestion, cell infiltration, and necrosis due to d-GAL administration.²³ We detected a significant loss of glycogen especially at the periphery of the lobules in d-GAL-administered groups. Glycogen, the main energy source of hepatocytes, is an important indicator of liver damage. Loss of cytoplasmic glycogen granules is attributed to dependence of the cell on glucose as a result of the deterioration of energy metabolism.²⁴ Mangeney et al. reported that d-GAL might inhibit the energy metabolism in hepatocytes.²⁵

d-GAL administration changed the number of periportal mast cells. The mean number of mast cells was significantly increased after d-GAL administration. Liehr et al. have reported that d-GAL directly activates mast cells resulting in the release of histamine.²⁶ We also suggest that necrotic areas are possibly related to the mast cell degranulation.

In vitro and in vivo studies show that d-GAL induces hepatocyte apoptosis^27,28 via reduction of intracellular concentration of uridine nucleotides resulting in the inhibition of protein synthesis, which is essential for cell survival.²⁹ d-GAL-induced Kupffer cell activation resulting in reactive oxygen radical production might also cause apoptosis. Apoptotic cells in the liver have been shown to appear within 18 h after injection of d-GAL.³⁰ Catal Bolkent have reported that d-GAL administration causes a significant increase in the number of caspase-3-positive cells.³¹ Although we recognized large number of apoptotic cells with H-E staining in d-GAL-administered groups, we confirmed them using caspase-3 immunostaining method. By this method, we recognized that the number of caspase-3 (+) cells was significantly higher in d-GAL groups than that of control and treatment groups. We suggest that d-GAL causes apoptosis via activation of caspases that play a central role in the execution phase of apoptosis.

Ki-67 immunostaining method is used to determine the hepatocytes undergoing mitosis when Ki-67 antibody reacts with Ki-67 epitope located in the nucleus of cells in G1, S, G2, and M phases of mitotic cycle.^32,33 We observed in our study that only in the d-GAL-administered group the number of Ki-67 (+) cells was higher than that of the control group. As we previously mentioned, d-GAL induces the secretion of cytokines, particularly TNF-α by activating Kupffer cells.⁴ TNF is an inflammatory cytokine that induces context-dependent proliferation, survival, and apoptosis responses in hepatocytes. TNF stimulates and enhances growth factor-mediated hepatocyte proliferation and survival but also acts in concert with other inflammatory cytokines to induce apoptosis in hepatocytes.³⁴ We suggest that d-GAL induces apoptosis and cell proliferation by stimulating TNF-α production.

AST and ALT are the most frequently used enzymes for determining liver damage and necrosis.^35,36 Although these enzymes normally exist in hepatocyte cytoplasm, they pass into the blood after cell injury.³⁷ Long-term application of high doses of d-GAL exacerbates liver damage and causes intracellular enzymes to pass into the blood.^38,39 Following i.p. administration of d-GAL, serum level of ALT starts to increase after 18 h and it reaches its peak level at 24 h.³⁰ We also measured the peak levels of AST and ALT at the 24th h after d-GAL administration in rats.

In order to investigate membrane damage and capacity of cellular antioxidant system, we evaluated lipid peroxidation status by analyzing tissue MDA levels and GSH (nonenzymatic) levels and GPx and (enzymatic) SOD activities. In addition, we investigated tissue MPO levels for assessing severity of inflammation. Increase in MDA levels has been reported as an indicator of lipid peroxidation in previous studies conducted with d-GAL administration.^40,41 Previous studies reported significant increases in the levels of MDA in d-GAL-administered groups compared with the control group,^42,43 as we detected. There was a statistically significant increase in MPO levels in GAL groups compared with the control group. Similarly, Ito et al. observed that MPO activity increased in the liver of d-GAL-treated rats.⁴⁴

SOD dismutates superoxide radical and provides its molecular conversion to H₂O₂ and molecular oxygen.⁴⁵ Taye et al. and Shi et al. revealed a decrease in SOD activity after administration of D-GAL in their studies.^43,46 However, we did not find any significant difference in terms of SOD activity between groups in our study.

GSH, a free radical scavenger, is one of the most important intracellular mechanisms as a substrate in GSH redox cycle to protect normal cells from oxidative damage.⁴⁷ It exists at a high concentration in the liver.⁴⁸ Shi et al. demonstrated significant reduction in hepatic GSH level in d-GAL-induced hepatitis model compared with the control group.⁴⁶ In our study, we also detected significant decrease in d-GAL group and attributed it to the oxidative stress induced by d-GAL.

We observed that histological and biochemical damage induced by d-GAL was improved with the treatment of PTX and CAPE. Liver damage was reduced significantly in PTX and CAPE-treated groups. AST and ALT levels of PTX- and CAPE-administered rats were lower than those of d-GAL-treated groups.

PTX supports tissues by accelerating blood flow and increasing tissue oxygenation.⁴³ Additionally, PTX ameliorates microvascular perfusion by protecting the integrity of the vascular system and preventing adhesion of leukocytes along the endothelium of postcapillary venules, which is thought to be the most important step of inflammation.²⁴ Moreover, PTX acts as a TNF-α inhibitor by inhibiting TNF-α release from macrophages. Thus it reduces production of chemotactic mediators including Interleukin 1 (IL-1) and IL-6.⁴⁹ Novick et al. reported that polymorphonuclear leukocytes activated by TNF-α became deactivated after exposure to PTX.⁵⁰ PTX was shown to have hepatoprotective effects in different models of hepatotoxicity.⁵¹ A research on the therapeutic effects of PTX on carbon tetrachloride (CCl₄)-induced liver damage revealed that vacuolization, necrosis, and apoptosis were significantly decreased in PTX-treated rats.⁵² Usta et al. reported significant reduction in apoptosis on kidney damage induced by adriamycine following PTX administration.⁵³ We also detected caspase-3 cell decrease in PTX-treated group in comparison with d-GAL-treated group.

Different results of the effect of CAPE on apoptosis were obtained in several studies. CAPE was shown to induce apoptosis in human colon and prostate cancer cell cultures.^54,55 On the contrary, anti-apoptotic properties of CAPE have been shown in an experimentally induced apoptosis model in neuron cultures.⁵⁶ Cagli et al. reported that CAPE inhibited apoptosis in myocardial ischemia-reperfusion injury.⁵⁷ In our study, we observed that the number of caspase-3 (+) cells was significantly reduced in CAPE-treated group compared with D-GAL-treated groups.

Total number of inflammatory areas in d-GAL + CAPE group was less than that of d-GAL group. We attribute it to anti-inflammatory effects of CAPE since CAPE specifically inhibits nuclear factor-κB (NF-κB) and oxygen radicals and inhibits the inflammatory pathway. NF-κB is a redox-sensitive transcription factor that is activated in response to oxidative stress. It has been shown that NF-κB level increases following d-GAL administration, and CAPE administration specifically inhibits NF-κB.⁵⁸ Anti-inflammatory efficiency of CAPE has been shown in many studies. Lee et al.⁵⁹ reported an anti-inflammatory effect of CAPE on tetrachloride-induced liver damage.

In the present study, both PTX and CAPE induced cell proliferation. We found that the number of KI-67 (+) cells in d-GAL + PTX and d-GAL + CAPE groups was significantly increased. Martino et al. have reported that PTX provides regeneration by reducing the amount of transforming growth factor β1 (TGF-β1).⁶⁰ TGF-β, an inhibitor of hepatocyte growth, is the most important regeneration inhibiting agent.⁶¹ The inducing effect of CAPE on cell proliferation is related to its arginine content. Arginine converts to agmatine by decarboxylation, which latter accelerates tissue regeneration by increasing mitosis and protein synthesis.⁶²

In the present study, increases in MDA levels but decreases in GSH levels and GPx activities in D-GAL-treated groups indicate oxidative stress and resulting injury. These findings were diminished by administration of PTX and CAPE. One of the most important properties of PTX⁶³ and CAPE⁶⁴ is the protection of tissues from H₂O₂ and superoxide anion produced by neutrophils. Lipid peroxidation has been reported to be reduced by administration of PTX⁶⁵ and CAPE⁶⁶ on CCl₄-induced liver damage. Studies have shown that antioxidant agents on hepatic lesions induced by d-GAL increase GSH level.⁶⁷ Zaitone et al. revealed that PTX significantly increased GSH level in an experimental model of nonalcoholic fatty liver disease.⁶⁸ Another study conducted that CAPE increased the level of GSH in cisplatin-induced hepatic damage.⁶⁹ CAPE has been reported to block all reactive oxygen species produced by xanthine dehydrogenase/xanthine oxidase system at 10 μmol/L concentration.⁷⁰ Moreover, CAPE inhibits the oxygenation of linoleic acid and arachidonic acid by 5′-lipoxygenase.⁷¹ GPx enzyme activity that metabolizes H₂O₂ has significantly increased in d-GAL + PTX and d-GAL + CAPE groups compared with d-GAL group. Catal et al. revealed that GPx enzyme activity had increased by the administration of the antioxidant combination consisting of beta carotene, ascorbic acid, α-tocopherol, and sodium selenate in renal damage induced by d-GAL.⁷² In our opinion, the significant positive effects of PTX and CAPE on oxidative stress parameters observed in this study are attributed to their antioxidant properties.

Superoxide and H₂O₂ radicals synthesized from neutrophils are the sources that increase the oxidative damage caused by d-GAL. PTX⁷³ and CAPE⁷⁴ have powerful inhibitor effects on neutrophils. Neutrophils release harmful substances including lysosomal enzymes and reactive oxygen radicals. When neutrophils are activated by various types of stimulants, MPO is also released with aforementioned harmful substances. MPO plays a major role in producing oxidants. Neutrophils are the most important cells that play a role in the tissue damage in inflammatory diseases such as rheumatoid arthritis, inflammatory bowel diseases, and reperfusion injury.⁷⁵ Administration of PTX and CAPE reduced MPO activity to control level, which had increased after d-GAL administration. These findings show us that inflammation in hepatitis model provoked by d-GAL administration can be suppressed by the administration of anti-inflammatory agents such as PTX and CAPE.

Our study demonstrated that PTX and CAPE have some healing effects on d-GAL-induced hepatitis. In conclusion, we believe that antioxidant and anti-inflammatory agents such as PTX and CAPE may have positive effects in the treatment of liver diseases.

Footnotes

Conflict of interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was financially supported by a grant from Scientific Research Fund of Inonu University (grant number: 2011/55).

References

Guicciardi

Gores

. Apoptosis: a mechanism of acute and chronic liver injury. Gut 2005; 54: 1024–1033.

Keppler

Lesch

Reutter

. Experimental hepatitis induced by d-galactosamine. Exp Mol Pathol 1968; 9: 279–290.

Decker

Keppler

Pausch

. The regulation of pyrimidine nucleotide level and its role in experimental hepatitis. Adv Enzyme Regul 1973; 11: 205–230.

Kasravi

Wang

. Bacterial translocation in acute liver injury induced by D-galactosamine. Hepatology 1996; 23: 97–103.

Keppler

Decker

. Studies on the mechanism of galactosamine hepatitis: accumulation of galactosamine-1-phosphate and its inhibition of UDP-glucose pyrophosphorylase. Eur J Biochem 1969; 10: 219–225.

Leist

Gantner

Bohlinger

. Murine hepatocyte apoptosis induced in vitro and in vivo by TNF-α requires transcriptional arrest. J Immunol 1994; 153: 1778–1788.

Sakaguchi

Yokota

. Role of Ca2+ on endotoxin-sensitivity by galactosamine challenge: lipid peroxide formation and hepatotoxicity in zymosan-primed mice. Pharmacol Toxicol 1995; 77: 81–86.

Thompson

Tuma

Young

. The effects of pentoxifylline on spinal cord blood flow after experimental spinal cord injury. J Assoc Acod Minor Phys 1999; 10: 23–26.

Ward

Clissold

. Pentoxifylline: a review of its pharmacodynamics and pharmacokinetic properties, and its therapeutic efficacy. Drugs 1987; 34: 50–97.

10.

Borrelli

Maffia

Pinto

. Phytochemical compounds involved in the anti-inflammatory effect of propolis extract. Fitoterapia 2002; 73: 53–63.

11.

Castaldo

Capasso

. Propolis, an old remedy used in modern medicine. Fitoterapia 2002; 73: 1–6.

12.

Dobrowolski

Vohora

Sharma

. Antibacterial, antifungal, antiamoebic, antiinflammatory and antipyretic studies on propolis bee products. J Ethnopharmacol 1991; 35: 77–82.

13.

Bradford

. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248–254.

14.

Hillegas

Griswold

Brickson

. Assesment of myeloperoxidase activity in whole rat kidney. J Pharmacol Methods 1990; 24: 285–295.

15.

Buege

Aust

. Microsomal lipid peroxidation. Methods Enzymol 1978; 12: 302–310.

16.

Akerboom

Sies

. Assay of glutathione, glutathione disulfideand glutathione mixed disulfides in biological samples. Methods Enzymol 1981; 77: 373–383.

17.

Lawrence

Burk

. Glutathione peroxidase activity in selenium-deficient rat liver. Biochem Biophys Res Commun 1976; 71: 952–958.

18.

McCord

Fridovich

Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem 1969; 244: 6049–6055.

19.

Medline

Schaffner

Popper

. Ultrastructural features in galactosamine-induced hepatitis. Exp Mol Pathol 1970; 12(2): 201–211.

20.

Cao

Wang

. Protective effect of prostaglandin e on hepatocytes and its value of early treatment of severe viral hepatitis. Zhonghua Nei Ke Za Zhi 1991; 30: 17–20.

21.

Tsutsui

Itagaki

Kawamura

. d-Galactosamine induced hepatocyte apoptosis is inhibited in vivo and in cell culture by a calcium calmodulin antagonist, chlorpromazine, and a calcium channel blocker, verapamil. Exp Anim 2003; 52: 43–52.

22.

Najmi

Pillai

Pal

. Hepatoprotective and behavioral effects of jigrine in galactosamine-induced hepatopathy in rats. Pharm Biol 2010; 48: 764–769.

23.

Takano

Inoue

Shimada

. Urinary trypsin inhibitor protects against liver injury and coagulation pathway dysregulation induced by lipopolysaccharide/d-galactosamine in mice. Lab Invest 2009; 89: 833–839.

24.

Abdel Salam

Baiuomy

El-Shenawy

. Effect of pentoxifylline on hepatic injury caused in the rat by the administration of carbon tetrachloride or acetaminophen. Pharmacol Rep 2005; 57: 596–603.

25.

Mangeney

Sire

Montagne

. Effect of D-galactosamine in vitro on [U-14C] palmitate oxidation, triacylglycerol synthesis and secretion in isolated hepatocytes. Biochim Biophys Acta 1985; 833: 119–127.

26.

Liehr

Grün

Seelig

. On the pathogenesis of galactosamine hepatitis. indications of extrahepatocellular mechanisms responsible for liver cell death. Virchows Arch B Cell Pathol 1978; 26: 331–344.

27.

Fortes Aiub

Bortolini

Arrieira Azambuja

. Alterations in the indexes of apoptosis and necrosis induced by galactosamine in the liver of wistar rats treated with fructose-1,6-bisphosphate. Hepatol Res 2003; 25: 83–91.

28.

Cheng

Etoh

Tanimura

. Effects of dietary gluten on the hepatotoxic action of galactosamine and/or endotoxin in rats. Biosci Biotechnol Biochem 1996; 60: 439–443.

29.

Gujral

Farhood

Jaeschke

. Oncotic necrosis and caspase-dependent apoptosis during galactosamine-induced liver injury in rats. Toxicol Appl Pharmacol 2003; 190: 37–46.

30.

Sun

Hamagawa

Tsutsui

. Evaluation of oxidative stress during apoptosis and necrosis caused by d-galactosamine in rat liver. Biochem Pharmacol 2003; 65: 101–107.

31.

Catal

Bolkent

. Combination of selenium and three naturally occurring antioxidants administration protects d-galactosamine-induced liver injury in rats. Biol Trace Elem Res 2008; 122: 127–136.

32.

Arıcı

Kıvanç

Özer

. Mide adenokarsinomlarinda CD44 ve ki 67 ekspresyonu. Turk Patoloji Dergisi 2006; 22: 26–31.

33.

Tosun

Avunduk

. Hücre çoğalma belirleyicilerinin önemi ve kullanim alanları. Türkiye Klinikleri Tıp Bilimleri Dergisi 2001; 3: 235–244.

34.

Cosgrove

Cheng

Pritchard

. An inducible autocrine cascade regulates rat hepatocyte proliferation and apoptosis responses to tumor necrosis factor-alpha. Hepatology 2008; 48: 276–288.

35.

Rosalki

Foo

. Two new methods for separating and quantifying bone and liver alkaline phosphatase isoenzymes in plasma. Clin Chem 1984; 30: 1182–1186.

36.

McIntyre

. The liver in systemic disease. Gut 1994; 35: 432.

37.

Recknagel

Glende

Jr Dolak

. Mechanisms of carbon tetrachloride toxicity. Pharmacol Ther 1989; 43: 139–154.

38.

Aristatile

Al-Numair

Veeramani

. Effect of carvacrol on hepatic marker enzymes and antioxidant status in d-galactosamine-induced hepatotoxicity in rats. Fundam Clin Pharmacol 2009; 23: 757–765.

39.

Aristatile

Al-Numair

Veeramani

. Antihyperlipidemic effect of carvacrol on d-galactosamine induced hepatotoxic rats. J Basic Clin Physiol Pharmacol 2009; 20: 15–27.

40.

Sakaguchi

Furusawa

Yokota

. Modification of tumor necrosis factor-induced acute toxicity d-galactosamine challenge by polymyxin B, an anti-endotoxin. J Immunopharmacol 2000; 22: 935–942.

41.

Yoshikawa

Furukawa

Murakami

. Effects of vitamin E on d-galactosamine-induced or carbon tetrachloride-induced hepatotoxicity. Digestion 1982; 25: 222–229.

42.

Wen

Liu

. Upregulation of heme oxygenase-1 with hemin prevents d-galactosamine and lipopolysaccharide-induced acute hepatic injury in rats. Toxicology 2007; 237: 184–193.

43.

Taye

El-Moselhy

Hassan

. Hepatoprotective effect of pentoxifylline against d-galactosamine-induced hepatotoxicity in rats. Ann Hepatol 2009; 8: 364–370.

44.

Ito

Ozasa

Noda

. Effects of free radical scavenger on acute liver injury induced by d-galactosamine and lipopolysaccharide in rats. Hepatol Res 2008; 38: 194–201.

45.

Gürdöl

Genç

Oner-Iyidogan

. Coadministration of melatonin and estradiol in rats: effects on oxidant status. Horm Metab Res 2001; 33: 608–611.

46.

Shi

Sun

. Hepatoprotective effects of ganoderma lucidum peptides against d-galactosamine-induced liver injury in mice. J Ethnopharmacol 2008; 117: 415–419.

47.

Bounous

Molson

. The antioxidant system. Anticancer Res 2003; 23(2B): 1411–1415.

48.

Victor

Rocha

De la Fuente

. N-Acetylcysteine protects mice from lethal endotoxemia by regulating the redox state of immune cells. Free Radic Res 2003; 37: 919–929.

49.

Sztrymf

Rabiller

Nunes

. Prevention of hepatopulmonary syndrome and hyperdynamic state by pentoxifylline in cirrhotic rats. Eur Respir J 2004; 23: 752–758.

50.

Novick

Sullivan

Mandell

. New pharmacological studies with pentoxifylline. Biorheolog 1990; 27: 449–454.

51.

Sneed

Buchweitz

Jean

. Pentoxifylline attenuates bacterial lipopolysaccharide-induced enhancement of allyl alcohol hepatotoxicity. Toxicol Sci 2000; 56: 203–210.

52.

Noyan

Onem

Ramazan Sekeroglu

. Effects of erythropoietin and pentoxifylline on the oxidant and antioxidant systems in the experimental short bowel syndrome. Cell Biochem Funct 2003; 21: 49–54.

53.

Usta

Ismailoglu

Bakkaloglu

. Effects of pentoxifylline in adriamycin-induced renal disease in rats. Pediatr Nephrol 2004; 19: 840–843.

54.

Wang

Xiang

. Effect of caffeic acid phenethyl ester on proliferation and apoptosis of colorectal cancer cells in vitro. World J Gastroenterol 2005; 26: 4008–4012.

55.

McEleny

Coffey

Morrissey

. Caffeic acid phenethyl ester-induced PC-3 cell apoptosis is caspase dependent and mediated through the loss of inhibitors of apoptosis proteins. BJU Int 2004; 94: 402–406.

56.

Amodio

De Ruvo

Dimatteo

. Caffeic acid phenethyl ester blocks apoptosis induced by low potassium in cerebellar granule cells. Int J Dev Neurosci 2003; 2: 379–389.

57.

Cagli

Bagci

Gulec

. In vivo effects of caffeic acid phenethyl ester on myocardial ischemia-reperfusion injury and apoptotic changes in rats. Ann Clin Lab Sci 2005; 35: 440–448.

58.

Watabe

Hishika

Takayanagi

. Caffeic acid phenethyl ester induces apoptozis by inhibition of nfkappaB and activation of fas in human breast cancer MCF-7 cells. J Biol Chem 2004; 279: 6017–6026.

59.

Lee

Choi

Khanal

. Protective effect of caffeic acid phenethyl ester against carbon tetrachloride-induced hepatotoxicity in mice. Toxicology 2008; 248: 18–24.

60.

Martino

Coelho

Kubrusly

. Pentoxifylline improves liver regeneration through down-regulation of TNF-α synthesis and TGF-β1 gene expression. World J Gastrointest Surg 2012; 4: 146–151.

61.

Michalopoulos

De Frances

. Liver regeneration. Science 1997; 276: 60–66.

62.

Gabrys

Konecki

Krol

. Free amino acids in bee hive products (propolis) as identified and quantified by gas-liquid chromatography. Pharmacol Res Comm 1986; 18: 513–518.

63.

McDonald

. Pentoxifylline reduces injury to isolated lungs perfused with human neutrophils. Am Rev Respir Dis 1991; 144: 1347–1350.

64.

Michaluart

Masferrer

Carothers

. Inhibitory effects of caffeic acid phenethyl ester on the activity and expression of cyclooxygenase-2 in human oral epithelial cells and in a rat model of inflammation. Cancer Res 1999; 59: 2347–2352.

65.

Montosi

Garuti

Iannone

. Spatial and temporal dynamics of hepatic stellate cell activation during oxidant-stress-induced fibrogenesis. Am J Pathol 1998; 152: 1319–1326.

66.

Ozen

Akyol

Iraz

. Role of caffeic acid phenethyl ester, an active component of propolis, against cisplatin-induced nephrotoxicity in rats. J Appl Toxicol 2004; 24: 27–35.

67.

Shanmugarajan

Sivaraman

Somasundaram

. Influence of alpha lipoic acid on antioxidant status in d-galactosamine-induced hepatic injury. Toxicol Ind Health 2008; 24: 635–642.

68.

Zaitone

Hassan

El-Orabi

. Pentoxifylline and melatonin in combination with pioglitazone ameliorate experimental non-alcoholic fatty liver disease. Eur J Pharmacol 2011; 662: 70–77.

69.

Kart

Cigremis

Karaman

. Caffeic acid phenethyl ester (CAPE) ameliorates cisplatin-induced hepatotoxicity in rabbit. Exp Toxicol Pathol 2010; 62: 45–52.

70.

Yılmaz

Yucel

. Protective effect of caffeic acid phenethyl ester (CAPE) on lipid peroxidation and antioxidant enzymes in diabetic rat liver. J Biochem Mol Toxicol 2004; 18: 234–238.

71.

Borrelli

Izzo

Di Carlo

. Effect of a propolis extract and caffeic acid phenethyl ester on formation of aberrant crypt foci and tumors in the rat colon. Fitoterapia 2002; 73: 38–43.

72.

Catal

Sacan

Yanardag

. Protective effects of antioxidant combination against d-galactosamine-induced kidney injury in rats. Cell Biochem Funct 2010; 28: 107–113.

73.

Schoenberg

Muhl

Sellin

. Posthypotensive generation of superoxide free radicals – possible role in the pathogenesis of the intestinal mucosal damage. Acta Chir Scand 1984; 150: 301–309.

74.

Scapagnini

Foresti

Calabrese

. Caffeic acid phenethyl ester and curcumin: a novel class of heme oxygenase-1 inducers. Mol Pharmacol 2002; 61: 554–561.

75.

Sener

Akgün

Satiroğlu

. The effect of pentoxifylline on intestinal ischemia/reperfusion injury. Fundam Clin Pharmacol 2001; 15: 19–22.