The mushroom Lactarius deterrimus (Ld) was collected in the Istra region in Croatia, in the summer of 2008. Fruiting bodies were gently cleansed of any residual compost, air-dried and stored in airtight plastic bags at room temperature. Samples of spiny burrs of the sweet chestnut (Cs) (C. sativa Mill.) were collected in western Bosnia and Herzegovina. The chestnut samples were collected during the chestnut-ripening season, from the middle of September to the end of October 2006. The collected samples were kept at -20°C and protected from light before further use. The dried mushroom samples and chestnut samples (spiny-burrs) was obtained from Dr. Senka Vidović (Faculty of Technology, University of Novi Sad) The dried samples were milled in a blender before extraction with 50% ethanol, at a sample:solvent ratio of 1:10 (w/v) for the mushroom extract, and 1:5 (w/v) for the chestnut extract. The extraction process was carried out using an ultrasonic bath (B-220; Branson and SmithKline Company) at 45°C for 40 min for the mushroom extract, and at room temperature for 30 min for the chestnut extract. After filtration, the extraction solvent was removed using a rotary evaporator (Devarot; Elektromedicina) under vacuum. The obtained extracts were dried at 60°C to a constant mass and stored in glass bottles at -80°C to prevent oxidative damage.

The contents of total phenolic compounds in the dry mushroom and chestnut extracts were determined by the Folin–Ciocalteu procedure at 765 nm (Singleton and Rossi, 1965). The values are expressed as mg of gallic acid equivalents (GAE) per 1 g of dry extract. The Cs and Ld extracts contained 252 and 14.8 mg gallic acid per gram of dry material, respectively, in total phenolics. The total flavonoid contents were established by the aluminum-chloride colorimetric assay at 510 nm (Markham, 1989). The values are expressed as mg of quercetin equivalents (QE) per 1 g of dry extract. The flavonoid contents in the Cs and Ld extracts were 192 and 5.07 mg quercetin per gram of dry material, respectively. Previous detailed qualitative and quantitative analyses of the extract by High-Performance Liquid Chromatography with Diode-Array Detection (HPLC/DAD) revealed in Cs extract the highest content of ellagic acid structures, followed by gallic acid derivatives and flavonoid structures (Grdović et al., 2012). In the Ld extract, compounds containing a phenolic group were tryptophan and p-hydroxybenzoic acid, while other detected compounds were unsaturated and oxy (hydroxy- or epoxy-) fatty acids (Grdović et al., 2012).

Experiments were performed on 2.5-month-old adult albino Wistar rats weighing 220–250 g. All animal procedures were approved by the Committee for Ethical Animal Care and Use of the Institute for Biological Research “Siniša Stanković,” University of Belgrade, which acts in accordance with the EEC Directive (86/609/EEC) on the protection of animals used for experimental and other scientific purposes. The experimental model of multiple low-dose STZ-induced diabetes was used. Diabetes was induced by intraperitoneal (i.p.) injections of 40 mg STZ/kg to Wistar rats for 5 consecutive days. STZ was dissolved immediately before use in sodium citrate buffer (0.1 M, pH 4.5). Blood glucose was measured 24 h after the last STZ injection. Blood samples were obtained from the tail vein of overnight-fasted rats, and glucose was measured with a blood glucometer (Accu-Chek Active). Rats were considered to have diabetes when the fasting blood glucose level exceeded 20 mmol/l. Male Wistar rats were randomly divided into five groups: (i) ND – the non-diabetic control group (n = 7) which received an equivalent volume of citrate buffer i.p. for 5 consecutive days; (ii) D – the diabetic group (n = 8) that received STZ (40 mg/kg/5 days, i.p.) and was left untreated throughout the 4-week period; (iii) D + Cs – the diabetic group treated with the chestnut extract (Cs; 60 mg/kg, i.p.) daily for 4 weeks (n = 8), starting from the last day of STZ administration; (iv) D + Ld – the diabetic group treated with the mushroom extract (Ld; 60 mg/kg, i.p.) daily for 4 weeks (n = 8), starting from the last day of STZ administration; (v) D + Cs/Ld – the diabetic group treated with the combination of chestnut and mushroom extract (Mix Cs/Ld; 60 + 60 mg/kg, i.p.) daily for 4 weeks (n = 8), starting from the last day of STZ administration. The mode of administration was chosen to be i.p. because rats refused to drink the extract due to its bitterness. Based on our previous experimental experience, we estimated that i.p. administration is less stressful for the rats than force-feeding by gavage and it ensures that animal receive the desired dose. Even dough, the i.p. delivery is considered a parenteral route of administration, the pharmacokinetics of substances administered intraperitoneally are more similar to those seen after oral administration (Turner et al., 2011) as well as the bioavailability of substances is higher. The rats were killed 4 weeks after diabetes was established. Following blood collection, an abdominal incision was made and the liver and both kidney were removed and processed further as indicated. The remaining liver and kidney tissue was frozen in liquid nitrogen, kept at -80°C and used for cytoplasmic and nuclear protein preparation according to the instructions described in ProteoJET cytoplasmic and nuclear protein extraction kit (Fermentas).

After 4 weeks of diabetes, the rats were fasted overnight and their blood was collected in heparinized tubes (1,000 IU heparin). Blood plasma was obtained after centrifugation at 2,000 × g for 10 min and used for determination of glucose, total cholesterol, triglycerides, aspartate and alanine aminotransferases (AST and ALT, respectively), and blood urea nitrogen (BUN). After centrifugation and collection of plasma fractions, the remaining pellets were used for the preparation of red blood cell (RBC) hemolysates. RBC were washed twice with 0.9% NaCl and lysed in three volumes of cold water for 30 min on ice. Hemolysates were used for the determination of glycated (Gly) hemoglobin (Hb).

Blood glucose concentrations were measured with a commercial kit (Gluco-quant Glucose/HK, Boehringer Mannheim, Germany), which is based on the hexokinase/G6P-DH enzymatic method. Concentrations of GlyHb were determined by the colorimetric assay according to Parker et al. (1981). Serum triglyceride concentrations were determined by the glycerol-3-phosphate (GPO) oxidase-p-aminiphenazone (PAP) method with an enzymatic kit (Randox Laboratories, United Kingdom). Total cholesterol concentrations were measured by the enzymatic cholesterol esterase-cholesterol oxidase method using the CHOD-PAP test kit (Randox Laboratories, Crumlin, United Kingdom). Activities of ALT and AST were estimated by measuring the produced oxaloacetate and pyruvate, respectively, using an optimized standard UV kinetic method kit [GPT (ALAT) IFCC mod.; GOT (ASAT) IFCC mod.]. Blood urea nitrogen level was estimated according to GLDH method (Human, Wiesbaden, Germany).

The liver and kidney tissue from all experimental groups were removed and fixed in 10% buffered formalin for histological examination. Liver and kidney tissues from all examined groups were embedded in paraffin wax and sectioned at 5 mm thickness for histological examination. For histological analysis tissue sections were stained with Hematoxylin (H) and Eosin (E), mounted and observed under a light microscope (Leica DMLB; objective magnification 20×).

The level of oxidative stress in liver and kidney tissue was estimated by using 2′,7′-dichlorofluorescin diacetate (DCFDA), a fluorogenic dye that measures hydroxyl, peroxyl, and other reactive oxygen species (ROS) activity. Briefly, an aliquot of the liver and kidney homogenates (0.01 ml) were mixed with 0.15 ml ethanol solution of DCFDA to the final concentration of 10 mM. After incubation for 30 min at RT and in dark the fluorescence was measured using Tecan microplate reader Safire II (excitation and emission wavelengths of 488 and 520 nm, respectively).

Lipid peroxidation was examined with the TBARS assay according to Ohkawa et al. (1979). Briefly, an aliquot of the liver and kidney homogenates (0.1 ml) prepared in 1.15 M KCl were mixed with 0.2 ml of 8.1% SDS, 1.5 ml of 20% acetic acid (pH 3.5), 1.5 ml of 0.8% TBA, and 0.7 ml water and heated at 95°C for 60 min. After cooling to room temperature, 1 ml of water and 5 ml of n-butanol/pyridine (15:1, v/v) were added, mixed and centrifuged at 3,000 × g for 10 min. The red pigment in the supernatant fractions was estimated by absorbance at 532 nm. A calibration curve was prepared with 1,1,3,3-tetramethoxypropan (concentrations ranged from 25 nmol/ml to 1 μmol/ml).

The level of DNA damage was assessed by the alkaline Comet assay (Singh et al., 1988). Liver and kidney samples from all experimental groups were minced in ice-cold HBSS buffer (0.14 g/l CaCl₂, 0.4 g/l KCl, 0.06 g/l KH₂PO₄, 0.1 g/l MgCl₂ × 6H₂O, 0.1 g/l MgSO₄ × 7H₂O, 8.0 g/l NaCl, 0.35 g/l NaHCO₃, 0.09 g/l Na₂HPO₄ × 7H₂O, 1.0 g/l D-glucose) containing 20 mM EDTA and 10% DMSO. Ten microliters of a liver or kidney cell suspension was mixed with low-melting agarose (0.75%) and applied to a microscope slide. Cells were lysed for 2 h at 4°C in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10, 1% Triton X-100). After lysis, the slides were incubated for 30 min at 4°C in electrophoresis solution (300 mM NaOH, 1 mM EDTA, pH 13.0) and subjected to electrophoresis in order to separate the damaged DNA fragments. The slides were placed in neutralization buffer (0.4 M Tris-HCl, pH 7.4) and stained with SYBR Green I (1:10,000 dilution; Sigma–Aldrich). Fluorescence microscopy was performed with a Zeiss Axiovert 135TV microscope equipped for epifluorescence. DNA damage was quantified by measuring the displacement between the genetic material contained inside the nuclear sphere or comet ‘head’ and the resulting comet ‘tail.’ Images were analyzed with TriTekCometScore Freeware v1.5.¹

Reaction mixtures containing bovine serum albumin (BSA) (20 mg/ml)/glucose (1 M)/fructose (1 M) system (1 ml), dissolved in 20 mM Tris-HCl (pH 7.4), in the presence or absence of examined extracts were incubated at 37°C for 7 days (Brownlee et al., 1986). The incubated solution was mixed with 0.5 mM NBT (0.5 ml) dissolved in 100 mM carbonate puffer pH 10.8. The reduction of NBT was measured at 595 nm according to the formula: %Inhibition = ((A_blank - A_test)/A_blank) × 100, where A_blank is the absorbance of the BSA/glucose system without the test sample, and A_test is the absorbance of BSA/glucose system in the solution with the test sample.

Bovine serum albumin (20 mg/ml) was incubated with glucose (1 M) and fructose (1 M) in Tris-HCl pH 7.4 containing 0.02% sodium azide. All of the reagents and the examined extracts were passed through filter paper and each of the mixtures was incubated at 37°C for 7 days. The formation of AGE was measured by the fluorescence intensity at an excitation wavelength of 330 nm and an emission wave-length of 410 nm with a Luminescence spectrometer LS50B (PerkinElmer Ltd., Buckinghamshire, England) (Lee et al., 1998).

For determination of antioxidant enzyme activities, a homogenate of fresh liver and renal tissue was prepared in sucrose buffer (0.25 M sucrose, 1 mM EDTA, 0.05 M Tris-HCl, pH 7.4). Firstly, 1 g of liver and kidney tissue was homogenized in 10 ml of sucrose buffer, sonicated, and at the end centrifuged at 100,000 × g in a Beckmann rotor Ti 50 for 90 min. Total SOD activity was measured by the epinephrine method (Misra and Fridovich, 1972). SOD activity was expressed as U/mg of protein. MnSOD activity was performed after preincubation with 8 mmol/l KCN. CuZnSOD activity was calculated from the difference between total SOD and MnSOD activities. CAT activity was measured by the rate of hydrogen peroxide decomposition and expressed as l M H₂O₂/min/mg protein (U/mg of protein) (Claiborne, 1984). Protein concentrations were determined according to Lowry et al. (1951).

Total GSH and GSSG levels were determined by the standard recycling method using the Glutathione Assay Kit (Cayman Chemicals Company, Ann Arbor, MI, United States). A 20% homogenate of fresh liver and renal tissue was prepared in phosphate buffer (100 mM NaH₂PO₄, 1 mM EDTA, pH 7.5). Samples for GSSG determination were incubated at room temperature with 2 μl of 0.5 M 2-vinyl pyridine solution per 50 μl of deproteinized sample for 1 h. Samples were deproteinized with 5% 5-Sulfosalicylic acid solution. Incubation with 2-vinyl pyridine conjugates any GSH present in the sample so that only GSSG is recycled to GSH. The GSSG was then subtracted from the total GSH to determine the actual GSH level and GSH/GSSG ratio.

For immunoblot analysis, homogenates of fresh liver and renal tissue prepared in sucrose buffer were used. Twenty micrograms of liver and kidney protein fractions were separated onto 4% stacking/12% separating slab gels. After electrophoresis, proteins were transferred to PVDF membranes (Hybond-P, Amersham Pharmacia Biotech) and immunoblot analysis was performed using rabbit polyclonal antibodies to O-linked β-N-acetylglucosamine (O-GlcNAc), N-(carboxymethyl)lysine (CML), RAGE, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65, β-actin; goat polyclonal anti-bodies to lamin B (Santa Cruz Biotechnology, Santa Cruz, CA, United States). Appropriate horseradish peroxidase-conjugated anti-rabbit, anti-goat (Santa Cruz Biotechnology, Santa Cruz, CA, United States), and anti-mouse (Cell Signaling) immunoglobulins were used as a secondary antibody. Each PVDF membrane was reprobed according to the supplier’s protocol for reprobing. Immunoreactive bands were identified by an enhanced chemiluminescence (ECL) detection system (Santa Cruz Biotechnology) according to the manufacturer’s instructions. The blots were scanned, and the intensities of the signals were quantified using TotalLab (Phoretix, Newcastle Upon Tyne, England) electrophoresis software (ver. 1.10).

The data were expressed as the mean ± SEM (standard error of mean). Before statistical analysis we tested the obtained data for normality using a Kolmogorov–Smirnov test. For intergroup comparison between two means, first a one-way Analysis of Variance (ANOVA) was used and in case of significance a Duncan’s multiple range test (DMRT) was applied as a post hoc test. The difference was considered statistically significant at p < 0.05. Mann–Whitney test was used in one particular case (for total amount of Nf-kB) to compare differences between two independent groups (p < 0.05).

The biochemical markers of diabetes in different experimental groups are presented in Table 1. Compared to the control non-diabetic rats, the STZ-treated animals displayed typical signs of diabetes: elevated fasting blood glucose and total glycated hemoglobin level (5- and 2-fold, respectively), as well as increased total cholesterol and triglyceride concentrations (1.6- and 1.9-fold, respectively). The activation of detrimental processes in the liver and kidney of diabetic rats was observed as increased enzymatic activities of ALT and AST, markers of hepatocellular injury, and increased BUN, an indicator of renal functionality. The treatment of diabetic rats with Cs extract lowered fasting blood glucose, GlyHb, cholesterol, triglyceride and ALT levels to the control values and significantly lowered the concentration of BUN. In contrast, the treatment of diabetic rats with the Cs extract did not cause any significant change in AST activity. Administration of the Ld extract to diabetic rats alleviated hyperglycemia, decreased GlyHb and triglyceride levels and restored the total cholesterol concentration to the control values. While the treatment of diabetic rats with the Ld extract improved the enzymatic activity of AST, the level of ALT activity and BUN concentration remained elevated. Diabetic rats treated with the combination of the mushroom and chestnut extracts MIX Ld/Cs displayed improvements in all measured parameters, indicating a better glycemic and lipid status and improved liver and kidney functioning.

Figure 1 depicts the photomicrographs of hematoxylin and eosin staining of liver and kidney tissues of control and experimental groups of rats. ND control rats showed liver parenchyma with general structures preserved, including hepatic lobules with normal hepatocytes surrounded by sinusoids and distributed radially toward the centrilobular veins with no observed inflammatory infiltration and fatty degeneration. In contrast, untreated D rats presented morphological changes in the liver that were characterized by hepatocytes that contained focal or generalized fatty vacuoles and micro- or macro vesicular features that were associated with the presence of dilated sinusoids and a progressive loss of general organ structure (disorganization of the lobular architecture) together with mild inflammatory cell infiltration. Histological examination of the livers of diabetic rats treated with Cs extract and MixCs/Ld maintained normal lobular architecture and had mild fatty change, feathery degeneration, and only slight neutrophil infiltration almost comparable to the normal. Also, Ld treatment protected liver tissue from diabetes-induced damage, but to a lesser extent.

The sections of kidney tissue of control rats demonstrated normal architecture with normal glomeruli and tubules. On the other hand, section of kidney tissues of diabetic group of rats revealed visible distortion in the architecture of the kidney tissue showing features of glomerulopathy such as compression of the glomerular tuft into nodules with decreased cellularity of the glomeruli, dilated tubules lined by cuboidal epithelium without brush borders and marked inflammatory cells infiltration. However, the treatment of diabetic rats with Cs extract and MixCs/Ld attenuated the histological changes in the kidney of diabetic rats showing better patterned renal architecture with fairly normal glomeruli and tubules and mild inflammatory cells infiltration. Also, Ld treatment attenuated kidney damage, but to a lesser extent.

To estimate the level of oxidative stress we used a non-fluorescent substrate 2′,7′-dichlorofluorescein (DCFDA) which undergoes oxidation to green fluorescent product in the presence of ROS. The highest level of oxidative stress, based on the measured fluorescence, was detected in the liver and kidney of diabetic animals (Figure 2A). On the other hand, in the liver and kidney tissue of diabetic animals treated with the extracts, separately and in combination, the extent of ROS production was significantly lower. The strongest effect on the ROS production in both examined tissues was displayed by mushroom extract (Figure 2A).

The end products of lipid peroxidation are reactive aldehydes, such as MDA, which served as an indicator of lipid peroxidation in liver and kidney in each experimental group (Figure 2B). Highest concentration of MDA equivalents (MDAeq), measured using the thiobarbituric acid-reactive substance assay, were detected in the liver and kidney of diabetic rats. Comparison of MDAeq levels in the liver and kidney of diabetic animals and the diabetic animals treated with the extracts revealed that both extracts, as well as MIX Ld/Cs, exhibited statistically significant inhibition of lipid peroxidation. The mushroom extract showed the highest potential for inhibition of lipid peroxidation, based on the MDAeq level that was 2-fold lower than in the liver of diabetic rats and 1.6-fold lower than in the kidney of diabetic rats. The chestnut extract and the combination of extracts were less effective, as they both induced a 1.4-fold decrease of MDAeq in both liver and kidney when compared to diabetic rats (Figure 2B).

Oxidative damage to DNA in the liver and kidney in each experimental group was evaluated by the alkaline Comet assay (Figures 2C,D). The extent of DNA damage was expressed as % of DNA in comet’s tail (Figure 2D). Representative images of liver and kidney cells whose DNA was visualized by staining with SYBR Green I are shown in Figure 2C. In the control, non-diabetic group, the majority of analyzed liver and kidney cells did not exhibit DNA damage (Figure 2C, ND panels). In STZ-induced diabetic rats, extensive DNA damage was observed in liver and kidney cells. This was revealed by the large amount of DNA in the comet tails (Figures 2C,D panels). Treatments of diabetic animals with the extracts, separately and in combination, led to a pronounced decrease in DNA damage in liver and kidney cells (panels D + Cs, D + Ld, and D + Cs/Ld). Analysis of % of DNA in comet’s tail revealed that the mushroom extract and the combination of the extracts provided the best protection against diabetes-induced DNA damage in liver cells (Figure 2D). On the other hand, in kidney cells, the chestnut extract and the combination of the extracts caused the greatest decrease in DNA damage (Figure 2D). Taken together, these results indicate that both extracts and their combination significantly suppressed oxidative stress and thereby oxidative damage of lipids and DNA in liver and kidney tissue of diabetic animals.

To determine whether the observed suppression of oxidative stress after the treatment with extracts was caused by changes in activities of antioxidant enzymes, we examined the activities of MnSOD, CuZnSOD, and CAT. As can be seen in Figure 3, STZ-induced diabetes was associated with significantly decreased MnSOD, CuZnSOD, and CAT activities in liver and kidney tissue. The treatment of diabetic rats with both extracts separately and in combination promoted increased activity of MnSOD to nearly control level in both examined tissues, except in the case of Cs-treated animals where the level of liver MnSOD activity remained below its control level (Figure 3A). Treatment of STZ-diabetic rats with the Ld extract and MIX Ld/Cs restored the activity of liver and kidney CuZnSOD to the control levels (Figure 3B). However, the chestnut extract caused a significant increase of CuZnSOD activity which was 18 and 14% below its control values in the liver and kidney, respectively. In contrast to the restoration of SOD activities, the treatments of diabetic rats with Cs, Ld, and MIX Ld/Cs did not produce any effect on CAT activity in the liver and kidney of diabetic rats (Figure 3C).

During oxidative stress, the concentration of GSH decreases and is accompanied by an increase in its oxidized form (GSSG). A significant decrease in the GSH/GSSG ratio was detected in the liver and kidney of diabetic rats as compared to non-diabetic controls (Figure 3D). The treatment of diabetic rats with either extract or their combination shifted the redox balance of glutathione in favor of its reduced form in both tissues, except in kidney tissue of Ld-treated rats. The strongest effect on the GSH/GSSG ratio in both tissues was displayed by MIX Ld/Cs (Figure 3D).

We examined the potential inhibitory effects of the separate extracts and their combination on the formation of fructosamines during the process of protein glycation. This was studied by monitoring the reduction of NTB on the 7th day of incubation in the BSA/glucose/fructose system in the presence or absence of the examined extracts. The concentration ranges differed for the two extracts and were previously defined (Grdović et al., 2012). As can be seen on Figure 4A, the inhibition of NBT reduction, which is directly related to the ability of the examined extracts to suppress glycation, was dose-dependent in the case of both extracts, while the combination of the two extracts exhibited the strongest anti-glycation activity, providing nearly 80% inhibition of NBT reduction (Figure 4A). The formation of AGE was monitored by measuring the fluorescence intensity of the BSA/glucose/fructose solution attributed to the formation of glycophore groups. As shown in Figure 4B, when the extracts were added to the reaction media containing the BSA/glucose/fructose system, a significant concentration-dependent reduction in fluorescence intensity was observed at the end of study period. Again, the combination of different extracts displayed the greatest effect, inhibiting AGE formation by 90%.

After establishing that both extracts inhibit protein glycation and AGE formation in vitro, we investigated in vivo protein glycation in the liver and kidney. We observed increased protein glycation in both the liver and kidney of diabetic rats in comparison to the control glycation levels (Figure 5A). Treatments of diabetic rats with the Cs, Ld, and MIX Ld/Cs significantly diminished the amount of glycated proteins in both examined tissue. In liver tissue, the combination of the Cs and Ld extracts provided the best protection against diabetes-induced protein glycation, while in kidney tissue all three treatments decreased glycated proteins to a similar level.

Reactive derivatives of non-enzymatic glucose-protein condensation reactions form a heterogeneous group of irreversible adducts called AGE, which refers to a broad range of advanced products of the Maillard reaction (Reinauer, 1993), including stable AGE compounds such as CML. As expected, immunostaining for CML revealed that diabetic rats exhibited significantly elevated AGE levels in the liver and kidney as compared to non-diabetic controls (Figure 5B). Densitometric analysis of the electrophoretic protein profiles showed that the treatment of diabetic rats with Ld, Cs, and MIX Ld/Cs reduced CML immunostaining in both tissues to significantly lower levels than in diabetic rats. The greatest reduction of CML was detected in liver and kidney tissues of diabetic animals that were treated with the Cs extract.

To examine whether the detected suppression of AGE formation after treatment of diabetic rats with extracts affected the RAGE-NF-κB axis, we performed immunoblot analysis of liver and kidney tissues with anti-RAGE and anti-NF-κB antibodies. Densitometric analysis of the bands obtained after immunoblot analysis (Figure 6A) shows increased levels of RAGE in liver and kidney homogenates of diabetic rats. This increase was effectively prevented by treatments of diabetic rats with the Ld and Cs extracts separately, as well as their combination MIX Ld/Cs, in both examined tissue. Treatments with either with Cs or MIX Ld/Cs manage to retain the level of RAGE in both examined tissues at the control level (Figure 6A). Since RAGE activation perpetuates NF-κB activation, we examined the translocation of NF-κB p65 from the cytosol to the nucleus (Figure 6B). Western blot analysis revealed an overall increase in NF-κB p65 in liver and kidney nuclear fractions of diabetic rats (observed as an approximate nuclear:cytoplasmic p65 ratio >1), as compared to non-diabetic controls (where the nuclear:cytoplasmic p65 ratio < 1). While the cellular distribution of p65 changed, the amount of total p65 was not significantly altered in the livers of diabetic rats. However, in the kidney of diabetic rats, the amount of total p65 was higher than in control kidney. Administration of the extracts to diabetic rats produced varied responses. In the liver, their administration lowered the amount of total p65 significantly below the control level, with Cs producing the most pronounced decrease. Also, administration of extracts led to significant decrease of p65 in the liver nuclear fraction of diabetic rats, In the kidney, Cs administration in diabetic animal effectively lowered the relative amount of total p65 to the control level, bringing the nuclear:cytoplasmic p65 ratio <1. While Ld administration was less effective in lowering the relative amount of total p65, it also shifted the nuclear:cytoplasmic p65 ratio <1. The administration of MIX Ld/Cs had no effect in lowering the relative amount of total p65, but it shifted the nuclear:cytoplasmic p65 ratio <1.

In addition to the AGE/RAGE pathway, we examined weather changes in AGE formation influenced enzymatic protein O-GlcNAcylation. To that end, we performed immunoblot analysis of liver and kidney homogenates with anti-O-GlcNAc antibody. Representative immunoblots for liver and kidney tissue are shown in Figures 7A,B, respectively. Densitometric analysis showed an increased level of total protein O-glycosylation in both tissue of diabetic rats (Figures 7A,B). The treatments of diabetic rats with the extracts significantly reduced the amount of protein O-glycosylation in both organs. The greatest reduction of O-GlcNAc proteins was detected in diabetic rats treated with the combination of mushroom and chestnut extracts. The observed changes in the level of O-GlcNAc proteins were most apparent in the band indicated as p135, which has been identified as O-N-acetylglucosamine transferase (Konrad et al., 2001; Zraika et al., 2006). Densitometric analysis of the protein band at 135 kDa showed a similar pattern of change as total O-GlcNAc proteins. However, the reduction of p135 glycosylation in both the liver and kidney of diabetic rats treated with Ld the extracts was more pronounced than the reduction of total O-GlcNAc proteins with regard to their levels in untreated, diabetic rats.