http://informahealthcare.com/ijf ISSN: 0963-7486 (print), 1465-3478 (electronic) Int J Food Sci Nutr, 2014; 65(4): 482–488 ! 2014 Informa UK Ltd. DOI: 10.3109/09637486.2013.869796

IN VITRO AND ANIMAL STUDIES

Effects of polysaccharide from fruiting bodies of Agaricus bisporus, Agaricus brasiliensis, and Phellinus linteus on alcoholic liver injury

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Mustafa Uyanoglu1, Mediha Canbek1, Leo J. L. D. van Griensven2, Mustafa Yamac1, Hakan Senturk1, Kazım Kartkaya3, Aysegul Oglakcı3, Ozge Turgak4, and Gungor Kanbak5 1

Department of Biology, Science and Arts Faculty, Eskisehir Osmangazi University, Eskisehir, Turkey, 2Plant Research International, Wageningen University and Research Centre, Wageningen, The Netherlands, 3Graduate School of Medical Sciences, Eskisehir Osmangazi University, Eskisehir, Turkey, 4Graduate School of Sciences, Eskisehir Osmangazi University, Eskisehir, Turkey, and 5Department of Biochemistry, Medicine Faculty, Eskisehir Osmangazi University, Eskisehir, Turkey Abstract

Keywords

In the present study, the curative effects of crude polysaccharides (PSs) from mushrooms on the symptoms of alcoholic liver injury were investigated. PSs from Agaricus bisporus, Agaricus brasiliensis, and Phellinus linteus fruiting bodies were administered by gavage at levels of 100 mg per kg body weight per day for 7 d after the onset of the disease. The caspase-3 activity, mitochondrial membrane potential, mitochondrial outer membrane integrity of the liver tissues of sacrificed rats, and the serum alanine aminotransferase (ALT) levels were determined. In addition, light and transmission electron microscope (TEM) studies were performed for histopathological and cytological evaluations on liver sections. PSs from A. brasiliensis decreased ALT level and mitochondrial membrane potential and increased the outer membrane integrity; microscopic examinations also revealed normal hepatocytes and tissue. On the basis of our data, it can be argued that crude PSs from Agaricus brasiliensis have therapeutic potential for alcoholic liver injury.

Alcohol toxicity, basidiomycetes, polysaccharide, rat

Introduction The liver has extremely important functions, such as glycogen storage, plasma protein synthesis, decomposition of red blood cells, detoxification, hormone production, and synthesis of enzymes necessary for digestion. Therefore, the liver is a vital organ in vertebrates, and it is not possible to compensate for the absence of liver function (Khabiya & Joshi, 2010). Similarly to toxic and/or poisonous chemicals, a high level of alcohol consumption has damaging effects on liver tissue and also leads to many negative metabolic changes (Mochizuki et al., 1993). Fatty liver and steatohepatitis are characteristics of the early stages of alcohol damage. First, the liver mitochondria swell and extend, and the cristae are impaired; then, the mitochondrial permeability increases in parallel with the onset of steatosis (Kunitoh et al., 1997). Almost all ethanol is catabolized by oxidation. In contrast, oxidant-induced lipid peroxidation may cause necrosis and/or apoptosis (Kunitoh et al., 1997). Caspase-3 activation is responsible for the alcohol-induced apoptotic changes in hepatocytes (Wang et al., 2006). Caspases are proteases that stimulate apoptosis in damaged cells. Oxidative stress may cause an increase in the inflammatory response. Mitochondrial membrane depolarization and concurrent effects are major factors in

Correspondence: Dr. Mustafa Uyanoglu, Eskisehir Osmangazi University, Science and Arts Faculty, Department of Biology, 26480 Eskisehir, Turkey. Tel: þ90 222 239 37 57/2433. E-mail: [email protected]

History Received 25 June 2013 Revised 8 November 2013 Accepted 17 November 2013 Published online 23 December 2013

apoptotic cell death. Mitochondria-mediated apoptosis is related to the release of apoptosis-inducing factors (AIF) that stem from bioenergetic failure. Alcohol consumption for long periods of time leads to increased inflammation in the liver and an increase in polymorphonuclear leukocytes (PMNL) (Kunitoh et al., 1997). As a result of alcoholism, pro-apoptotic products stimulate the release of cytochrome-c and AIFs from mitochondria to the cytosol and activate caspase (Arthur, 2001). Thus, a natural defense mechanism begins to recover or remove cells or tissues that have been damaged by alcohol consumption. Synthetic drugs are used to prevent and/or treat liver tissue damage. However, the continuous use of synthetic drugs causes various side effects. Due to their effectiveness, limited side effects, and relatively low cost, natural drugs are increasingly used even though their biologically active compounds are unknown (Hwang et al., 2005). Edible and medicinal mushrooms have been used by mankind for hundreds of years to prevent and treat various diseases. Traditional medicine has a long history of using mushrooms as curatives not only in ancient Oriental countries but also in Eastern and Mesoamerican cultures (Wasser, 2011). The mechanisms of some of these activities have been clarified in the last decade (Chen & Seviour, 2007). Studies showed that the mushroom polysaccharides (PSs) and mycelia have many biological activities, such as antitumor, antifungal, antibacterial, antiallergic, immunomodulating, hypoglycemic, and hypolipidemic activities (Poucheret et al., 2006). Furthermore, some mushroom PSs, such as lentinan, schizophyllan, and krestin, are commercially available (Bae et al., 2005). Modern clinical practice in Far East countries, such as Japan, China, and Korea,

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uses mushroom-derived preparations (Zaidman et al., 2005). Many mushrooms, such as Grifola frondosa, Lentinus edodes, and Tricholoma lobayense, have hepatoprotective effect on liver disorders induced by different agents (Ooi, 1996). White button mushrooms (Agaricus bisporus) have also been reported to act as a protective agent against liver steatosis (Kanaya et al., 2011). Nevertheless, the published studies are limited in the diversity of macrofungi species. Therefore, in this study, the biochemical, histological, and cytological effects of PSs from the fruiting bodies of three macrofungi species on the alcohol-induced liver disease of rats were reported.

Materials and methods

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Mushroom PSs Fruiting bodies from A. bisporus (J.E. Lange) Imbach strain Sylvan strain A15, which is a commercial substrain of Horst U1 (ATCC 62462), and Agaricus blazei Murill (syn. Agaricus brasiliensis, Wasser, 2011) Mycelia strain M7700 were cultivated at the (former) Mushroom Experimental Station at Horst, The Netherlands. In short, intact cultivated fruiting bodies were harvested from the first flush and dried at 45  C under a constant stream of air. The fruiting bodies were then powdered using a hammer mill and a 2-mm screen filter. Phellinus linteus (Berk. et M.A. Curt) Teng fruiting body powder was kindly provided by Amazing Grace Health Industries of Bangkok, Thailand, and was made from fruiting bodies collected from the wild; cf. identification by Dr. Usa Klinhom of Mahasarakham University, Thailand. Dry fruiting body powder (50 g) was dispersed in 1 l of water and autoclaved at 120  C. The resulting suspension was cooled down and centrifuged at 7000 rpm for 15 min to remove solid materials. PSs were semi-purified, as described in Kozarski et al. (2011), by precipitation in 65% ethanol, followed by washing and dialysis to remove the excess small sugars and ethanol-soluble phenolic compounds. After centrifugation, the high MW semipurified PSs were ethanol precipitated and vacuum dried for later use. The yields of the dry semi-purified PSs varied in a range from 2 to 10% of the initial fruiting body powder weight. PSs from A. brasiliensis, A. bisporus, and P. linteus were dissolved in physiological saline and termed PS 1, PS 2, and PS 3, respectively. Modified liquid diet Throughout the 60-d course of the experiment, modified calorieadjusted liquid alcohol diet of Lieber et al. (1989) was given orally. The total calorie content (based on approximately 1000 kcal) of the applied liquid diet consisted of 40% fat, 36% ethanol, 20% protein, and 4% carbohydrates. The calories in the liquid diet were adjusted using dextrin, and the liquid diet was prepared fresh daily. Animals All experimental animals were obtained from Refik Saydam National Public Health Agency, Experimental Animals

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Reproduction Laboratory, The Republic of Turkey. The animals were maintained in transparent polycarbonate boxes in rooms with a 12:12 light/dark photoperiod, automatically adjusted temperature (22  2  C) and humidity (45–50%) throughout the experiment. Experimental protocols This study was conducted with approval no. 257/2012 of Eskisehir Osmangazi University, Faculty of Medicine. The rats used in the study were 3-month-old healthy male Sprague Dawleys weighing 220  20 g. The animals were randomly divided into five groups, with eight rats in each group. The experimental design of the study is presented in Table 1. Group I animals were orally administered the alcohol-free diet for 60 d, and the rest of the groups were given the same diet for only the first week. The prepared diet was put in the water bowls of the cages once a day for ad libitum use. Except for Group I, 96.5% ethyl alcohol (EtOH) (density (d)¼0.81 g/cm3) was added to the bowl of all other groups for periods of 1 week, gradually increasing the rate to 2.4–4.8% and 7.2%. During the rest of the experimental period, the diet was continued with the alcohol rate at 7.2% until dissection (Uzbay et al., 2006). The experimental animals from Group II were administered 1 ml of 0.9% NaCl each day from day 54 to 60, single doses (100 mg/kg/day) of the different PSs (PS 1, PS 2, and PS 3, respectively), which were dissolved in 1 ml of 0.9% NaCl, were administered to the experimental animals in Groups III, IV, and V by oral gavage. The consumption of the alcohol-free diet that was provided to the experimental groups every morning between 09:00 and 10:00 was measured and recorded. The daily alcohol consumption of the alcohol group animals was calculated using the formula (Uzbay et al., 2006) A ¼ dx½ðV  75Þ=W  where A is the g/kg/day alcohol consumption, V is the daily consumed fluid diet amount (ml), W is the weight of experimental animals (g), and d is the density of 96.5% EtOH (0.81 g/cm3 in 25  C). At the end of the experimental process, all experimental animals were dissected under ether anesthesia and were then sacrificed by removing all the blood from the heart. The entire liver of each experimental animal was taken out, and a piece of the liver was put in the freezer in polyethylene tubes at 80  C for biochemical analyses. Fresh liver tissue samples of standard weight were then collected for mitochondrial analyses. The rest of the liver tissue samples were fixed for histological and transmission electron microscope (TEM) studies. Biochemical analysis To measure possible function disorders in the liver cells, the serum levels of the alanine aminotransferase (ALT) enzyme were determined biochemically from serum samples by using a Roche/ Hitachi MODULAR P autoanalyzer (Mannheim, Germany) and Roche commercial kits (Montpellier, France).

Table 1. Experimental design of the study. Experimental groups Group Group Group Group Group

I (normal control) II III IV V

1–7th days

8–14th days

Diet with alcohol (2.4%)

Diet with alcohol (4.8%)

15–53th days Diet without alcohol Diet with alcohol (7.2%)

54–60th days Diet Diet Diet Diet

with with with with

alcohol alcohol alcohol alcohol

(7.2%) þ 0.9% NaCl (7.2%) þ PS 1 (7.2%) þ PS 2 (7.2%) þ PS 3

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The caspase-3 activities in the liver tissues were determined using a colorimetric caspase-3 measurement kit (Sigma, St Louis, MO), as described in Zovein et al. (2004), to form acetyl– Asp–Glu–Val–Asp–p-nitroaniline (AC-DEVD-pNA) pNA; the activity was based on a spectrophotometric measurement of the pNA absorbance at 405 nm after hydrolysis by caspase-3. The results were expressed in proportion to tissue protein amount. A Sigma mitochondria isolation kit and a Sigma cytochrome oxidase kit were used to isolate the mitochondria from the liver tissue and to determine the integrity of the mitochondrial inner and outer membranes. The mitochondrial membrane potential was determined by measuring the inner mitochondrial membrane permeability (JC-1 absorbance in mitochondria) as the fluorescence unit per mg mitochondrial protein (FLU/mgP) produced in the mitochondria suspension. To determine the mitochondrial outer membrane integrity and the separate of the mitochondrial subcellular fraction, cytochrome-c oxidase analysis was conducted using a commercial Sigma kit. Colorimetric analyses were performed based on the decrease in the absorbance at 550 nm during oxidation of ferrocytochrome-c to ferricytochrome-c. The mitochondrial outer membrane integrity was reported as the percentage of the activity difference appearing in the presence (total cytochrome-c oxidase activity) and absence (cytochrome-c oxidase activity in intact mitochondria) of n-dodecyl-b-D-maltoside, a detergent (Layne, 1957). Histological and TEM analysis For histological analyses, the liver tissue samples were embedded in paraffin blocks and were cut to 4 mm on a microtome. All sections were stained using hematoxylin & eosin. The samples were investigated by an Olympus CX31 light microscope using Spot Advanced Software (V.4.0.6; Diagnostic Instruments, Sterling Heights, MI) and an Olympus Camedia C-5060 compact digital camera (Tokyo, Japan). Histopathological examinations were conducted on 20 different section sites that were randomly selected from each histological liver section from animals in all groups. The morphological changes, such as sinusoidal congestion, enlarged sinusoids, cytoplasmic vacuolation, and PMNL infiltration, were scored (1; no evidence of injury, 2; mild, 3; moderate, 4; severe) to compare the control and experimental groups. Liver tissue samples, which were fixed with 4% glutaraldehyde and then osmium tetroxide for TEM tissue tracking, were shaped into araldite blocks following dehydration, and thin sections of 60 nm were obtained. Following the application of 2% uranyl acetate and lead acetate, thin sections were studied under a JEOL-JEM-1220 Transmission Electron Microscope (Tokyo, Japan), and their images were recorded with an Olympus MegaView G2 camera (Tokyo, Japan). Statistical analysis The data from the study were evaluated using the SPSS 12.0 for Windows software package (SPSS Inc., Chicago, IL).

The LSD method was used for inter-group comparisons. The Mann–Whitney U test was utilized to compare the scores that were used in inter-group histopathological evaluations. The data from each experimental group were expressed as ‘‘mean  standard error (SE)’’. Differences between the experimental groups were considered significant when p50.05.

Results The amounts of EtOH ingested daily by the experimental animals were statistically similar in all the groups (Table 2). Biochemistry The serum ALT levels of the experimental animals were significantly different in Group II (p50.001), Group III (p50.05), Group IV (p50.001), and Group V (p50.01) compared with Group I. The ALT values of Group III were significantly different from those of Group II (Figure 1A) at p50.05. In the present study, the caspase-3 activity in Group II was higher than that of Group I. Although the caspase-3 activity of all the groups were statistically similar, the crude PSs of A. brasiliensis led to a decrease in the caspase-3 activity compared to that in Groups I and II (Figure 1B). The mitochondrial membrane potentials (DC) in the liver tissue samples of all groups had significantly lower values than those in Group I. DC demonstrated an insignificant increase in Group III compared to that in Group II and a significant increase in Group IV (p50.05) (Figure 1C). Alcohol consumption caused a loss in the mitochondrial outer membrane integrity (Figure 1D). Although a significant difference was not observed, the outer membrane integrity in Groups III and IV was at a level closer to that of Group I. Therefore, it can be argued that the crude PSs of A. brasiliensis and A. bisporus prevent the loss of mitochondrial outer membrane integrity. However, the outer membrane integrity of the P. linteus-treated group was significantly lower than those of Group I (p50.05) and all the other groups (Figure 1D). Histological and TEM analysis The histological changes of the liver are given in Table 2. Histopathological examination showed that the liver sections of the rats in Group II (Figure 2B) had more sinusoidal widening and congestion and denser vacuoles in the hepatocyte cytoplasm than those from the animals of Group I (Figure 2A). In the sections from Group II, PMNL infiltration was observed, although it was not common in the liver parenchyma. This result was not found in the other groups. In the liver sections of Group III (Figure 2C), statistically significant decreases in vacuolization was observed in the hepatocyte cytoplasm and liver parenchyma compared to that in Group II. Widened sinusoids were not found in the liver sections of Group III; however, sinusoidal congestion was observed, although it was not dense.

Table 2. Ethanol consumption and histopathologic evaluations for each group. Group I II III IV V

EtOH consumption (g/kg/day)

Sinusoidal congestion

Enlarged sinusoids

Cytoplasmic vacuolation

PMNL infiltration

– 12.7  0.2 12.5  0.3 12.7  0.3 12.0  0.2

1.0  0.0 3.1  0.3a 1.8  0.3b 2.8  0.3a 3.0  0.3a

1.0  0.0 2.9  0.3a 1.6  0.3b 2.8  0.3a 2.9  0.3a

1.0  0.0 3.8  0.2a 2.0  0.3ab 2.4  0.2ab 3.3  0.3a

1.1  0.1 2.0  0.3a 1.5  0.2 1.6  0.3 1.5  0.2

p50.05 (a) significantly different from the Group I and (b) Group II. Data are mean  SE values (n ¼ 8).

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0,06

(B)

80 70 60 50 40 30 20 10 0

a

a Caspase-3

a ab

(µmol pNA/min) / mg pro.

ALT (U/L)

(A)

0,05 0,04 0,03 0,02 0,01 0,00

I

II

III

IV

I

V

II

IV

V

(D) 7000 6000 5000

ab

4000 3000

a

a

II

III

a

2000 1000 0 I

IV

V

GROUP

Mitochondrial outher membrane integrity (%)

(C) Potential of the inner mitochondrial membrane (ΔΨ)

III GROUP

GROUP

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90 80 70 60 50 40 30 20 10 0

a

I

II

III

IV

V

GROUP

Figure 1. (A) ALT serum levels, (B) caspase-3 activity, (C) potential of the inner mitochondrial membrane, and (D) mitochondrial outer membrane integrity. p50.05 (a) significantly different from the Group I and (b) Group II. Data are mean  SE values (n¼8).

All histopathological findings from the liver sections of Group IV were significantly different from those of Group I. The cytoplasmic vacuolization level was lower in Group IV than in Group II (Figure 2D). All the histopathological parameters of the liver sections from Group V were similar to those of Group II. In the thin sections of the liver tissues of Group I, the organelle structure, especially the mitochondria and smooth endoplasmic reticulum in the hepatocyte cytoplasm, was normal. In Group II, abnormal images were found in the form of swelling in the mitochondrial ultrastructure and impairment and melting in the cristae structures. In addition, swelling and breaks were found in the cytoplasmic fatty vacuoles and in the smooth endoplasmic reticulum (Figure 2F). Electron microscopy showed that PSs obtained from A. brasiliensis (Group III) led to a morphological structure similar to that of Group I (Figure 2E). The impairments observed in the thin liver structures of the animals that were administered PSs from A. bisporus (Group IV) and P. linteus (Group V) were similar to those of Group II.

Discussion In this study, PSs obtained from basidiocarps of A. bisporus, A. brasiliensis, and P. linteus were tested in an experimental alcoholic liver damage model. Biochemical, histopathological, and TEM examinations were used to measure the parameters in our study. The serum ALT levels of all groups were higher than that of Group I. Saravanan & Nalini (2007) reported high serum ALT levels in alcohol groups in their study investigating the effects of Hemidesmus indicus root extract on alcoholic liver damage. In our study, the hepatotoxic effect measured by the serum ALT level in Groups IV and V did not decrease; however, Group III (PS from A. brasiliensis) did experience a remission in hepatic degeneration. Kanaya et al. (2011) administered a fatty diet to ovariectomized rats and reported that the serum ALT level,

which increased due to fat accumulation in the liver, decreased when A. bisporus extract was added to that same diet for 3 months. In our study, the PSs obtained from A. bisporus did not decrease the Group IV ALT level. A possible reason for the difference might be that our experimental model was different and also that the PSs were only administered for 7 d. The fact that PSs obtained from A. brasiliensis decreased the ALT level during period was, however, a significant result. Zhou et al. (2001) studied alcohol consumption in rats and demonstrated that alcohol increased the caspase-3 level in liver tissue. In our study, the caspase-3 level of Group II showed a statistically insignificant increase compared to that of Group I due to alcohol. However, the caspase-3 level of Group III, which was administered PSs from A. brasiliensis, decreased compared to that of Group II. Alcohol induces the deterioration of the mitochondrial permeability transition (MPT). In relation to this effect, longterm alcohol consumption was reported to decrease the transmembrane potential (Yan et al., 2007). In our study, the outer membrane integrities were estimated by measuring the mitochondrial membrane potential (DC) and the cytochrome-c oxidase activity for MPT. In the groups that were administered an alcohol diet, DC was significantly reduced compared with that in the normal controls. The mitochondrial outer membrane integrity of Group II was lower than that of Group I. The DC and outer membrane integrity decreased in Group II as a result of the deterioration in MPT and the release of cytochrome-c that is necessary for caspase-mediated apoptosis. Higuchi et al. (2001) showed that MPT inhibitors, such as cyclosporin A (cys-A), in hepatocytes could hamper caspase activation and apoptosis by preventing cytochrome-c release. Jacotot et al. (2000) reported in their study that the pan-caspase inhibitor (Z-VAD.fmk) failed, although permeability transition pore complex (PTPC) inhibitors prevented the decrease in DC; the PTPC inhibitors did not have a protective effect on mitochondria. Therefore, it can be suggested

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Figure 2. (A) Liver section from Group I, (B) cytoplasmic vacuolization (arrow) and enlarged sinusoids (arrow head) in a liver section from Group II, (C) cytoplasmic vacuolization in a liver section from Group III (arrow), (D) cytoplasmic vacuolization (arrow) and enlarged sinusoids (arrow head) in a liver section from Groups IV and V, (E) normal mitochondria in a hepatocyte from Group III, and (F) swollen mitochondria and lipid vacuoles in a hepatocyte from Group II. mt, mitochondria; V, vacuole; mm, micrometer; nm, nanometer.

that the PSs exerted their effect on the mitochondria through the membrane potential gradient and that the effect was independent of the caspase-3 activation. Lipidosis is one of the most significant liver diseases, and macrovesicular adiposity has been reported to result from triglyceride accumulation in hepatocytes (Yang, 2008). Although protein malnutrition, diabetes, obesity, and various liver toxins may lead to fat accumulation in the liver, alcohol consumption is mainly responsible for the increase in free fatty acid production, the decrease in triglyceride use and fatty acid

oxidation, the blocking of lipoprotein excretion, and the hike in lipolysis in liver. In our study, fat vacuolizations and their densities also appear to histologically demonstrate alcoholic liver damage in the liver biopsies of the animals from Group II. In addition, the mean values of daily alcohol consumption of each experimental animal conform to those of similar studies. Keane & Leonard (1989) support the idea that liver damage in animals with an alcohol diet is inevitable. In our study, a substantial decrease in the fat accumulations in the PS-treated Group III was observed. The fat accumulation/cytoplasmic vacuolization in Group III was

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reversed by the PSs from A. brasiliensis (Table 1). The light and transmission electron microscopy data confirmed the decrease in lipid vacuolization in this experimental group (Figure 2C and E). Kanaya et al. (2011) also reported similar results for A. bisporus. In their study, the decreased lipid vacuolization caused by A. bisporus was explained by a suppressive effect on the expression of enzymes responsible for fatty acid biosynthesis. Our study, however, showed that the lipid vacuolization level caused by alcohol consumption in Group IV was not decreased very much by the administration of PSs from A. bisporus (Table 1); however, the PSs seemed to prevent new vacuole formation. In contrast, interestingly, chronic consumption of Shiitake (L. edodes) was reported to be associated with the development of fatty liver (Chandra et al., 2011). Thus, the effects of different types of mushroom species on liver steatosis may be different. The early effects of alcohol consumption in humans and animals reveal themselves as changes in mitochondrial morphology and functions. Structural changes, such as swelling in the mitochondria or losses in cristae, are observed. Excessive calcium and phosphate from external sources or the inability to excrete calcium as a result of alcohol-induced mitochondrial membrane damage causes mitochondrial calcification and can permanently impair mitochondrial function. Such impairments in mitochondria may lead to a decrease in fatty acid oxidation and to an accumulation of fat in cells. In parallel to an increase in the lipid storage capacity of stellate cells as a result of alcohol consumption, the lipid vacuole count was reported to be a hundred times higher than that in the normal control group (Cameron & Neuma, 1999). Alcohol stimulates enzyme systems found in the smooth endoplasmic reticulum in liver parenchyma cells (Lieber, 2004). A large amount of enzymes are required for alcohol catabolism. The widening in the lumen of the smooth endoplasmic reticulum in the alcohol groups may be associated with these phenomena. The findings obtained from TEM examinations demonstrated that the cellular damage caused by alcohol was reduced in Group III, which was administered PSs from A. brasiliensis. Fukumura et al. (2007) reported that the mitochondrial function disorder caused by chronic alcohol consumption was due to oxidative stress. It was reported that intracellular reactive oxygen species formed by alcohol decreased the membrane potential DC and increased the amount of intracellular cytochrome-c (Guan et al., 2004). Alcohol consumption for long periods of time was reported to cause an increase in PMNL infiltration and inflammation of the liver (Kunitoh et al., 1997). In their alcoholic liver study in rats, Ronis et al. (2004) reported PMNL infiltration and inflammation as a histopathological finding in addition to adiposity. Antioxidant treatments seem to gain significance for preventing alcoholic liver damage or for removing the damage. Lower activity values have been reported for the endogenous antioxidant enzymes in the liver of alcohol groups; in alcohol groups that were administered H. indicus root extract, the activity values increased and demonstrated a protective effect against oxidative damage (Saravanan & Nalini, 2007). In our study, the PMNL infiltration was low in the alcohol Group II and lower in Group III. In terms of PMNL infiltration, the PSs from A. brasiliensis may have reduced the experimentally induced alcohol damage in the liver. PS extracts of A. brasiliensis were found to be strong scavengers of reactive oxygens (Kozarski et al., 2011), and Al-Dbass et al. (2012) reported that A. brasiliensis is a natural source of antioxidant compounds and has hepatoprotective activities against CCl4-induced liver damage. TGF-b was found in hepatocytes at quite low levels or was not produced at all; the TGF-b was claimed to activate stellate cells in the liver and produce collagen. The produced collagen caused the

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development of fibrosis in alcoholic liver patients (Neuman, 2003). Yang (2008) reported that sinusoidal fibrosis development was caused by alcoholism. Moving from the findings of these studies, the sinusoidal widening observed in the liver sections of the alcohol diet group animals supported the possibility of fibrosis. In the liver sections of the experimental groups in our study, the sinusoidal widening and congestions decreased and increased in parallel to each other in terms of scoring statistics. These histopathological changes increased in the alcohol Group II and decrease in the PS-treated Groups V, IV, and III. In terms of the histopathological parameters, PS from A. brasiliensis alleviates the alcoholic damage in the widened sinusoidal regions of the liver that we assumed to have stemmed from fibrosis. The damage was alleviated/stopped by PSs from A. brasiliensis, perhaps through a mechanism that prevents stellate and Kupffer cell activation, which causes fibrosis, as noted by others (Cameron & Neuma, 1999; Neuman, 2003; Yang, 2008). Based on the biochemical and histological results of this study, it can be concluded that the PSs obtained from A. brasiliensis in particular have therapeutic activity against alcoholic liver damage. In terms of both alternative and allopathic medicines, further studies are required to clarify the mechanisms at a molecular level.

Acknowledgements The present study was supported by the Eskisehir Osmangazi University Research Fund by Project number 200819002.

Declaration of interest All authors report that there is not any actual or potential conflict of interest, including any financial, personal, or other relationships with other people or organizations, within 3 years of beginning the submitted work that could inappropriately influence (bias) their work.

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Effects of polysaccharide from fruiting bodies of Agaricus bisporus, Agaricus brasiliensis, and Phellinus linteus on alcoholic liver injury.

In the present study, the curative effects of crude polysaccharides (PSs) from mushrooms on the symptoms of alcoholic liver injury were investigated. ...
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