Pectinase-treated Panax ginseng extract (GINST) rescues testicular dysfunction in aged rats via redox-modulating proteins

Experimental Gerontology 53 (2014) 57–66

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Pectinase-treated Panax ginseng extract (GINST) rescues testicular dysfunction in aged rats via redox-modulating proteins Yu-Jin Won a,1, Bo-kyung Kim b,1, Yong-Kyu Shin a, Seung-Hyo Jung b, Sung-Kwang Yoo a, Seock-Yeon Hwang c, Jong-Hwan Sung d, Si-Kwan Kim a,⁎ a

Department of Life Science, College of Biomedical & Health Science, Konkuk University, Chungju 380-701, Republic of Korea Department of Physiology, College of Medicine, Konkuk University, Chungju 380-701, Republic of Korea Department of Biomedical Laboratory Science, College of Applied Science and Industry, Daejeon University, Daejeon 300-716, Republic of Korea d Il Hwa Co., Ltd., Ginseng Research Institute, Guri 471-711, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 26 August 2013 Received in revised form 12 February 2014 Accepted 20 February 2014 Available online 1 March 2014 Section Editor: Holly M Brown-Borg Keywords: Panax ginseng Testicular dysfunction Glutathione peroxidase Glutathione-S-transferase Glutathione

a b s t r a c t The root of Panax ginseng improves testicular function both in humans and animals. However, the molecular mechanism by which ginseng exerts this effect has not been elucidated. Changes in protein expression in the rat testis in response to a pectinase-treated P. ginseng extract (GINST) were identified using 2-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF MS. Number of sperm, Sertoli cells and germ cells, and the Sertoli Cell Index decrease in the testis of aged rats (AR) relative to young control rats (YCR). However, those parameters were completely restored in GINST-treated AR (GINST-AR). A proteomic analysis identified 14 proteins that were differentially expressed between vehicle-treated AR (V-AR) and GINST-AR. Out of these, the expression of glutathione-S-transferase (GST) mu5 and phospholipid hydroperoxide (PH) glutathione peroxidase (GPx) was significantly up-regulated in GINST-AR compared to V-AR. The activity of GPx and GST, as well as the expression of glutathione, in the testis of GINST-AR was higher than that in V-AR. The levels of lipid peroxidation (LPO) increased in AR compared with YCR, but this change was reversed by GINST-AR. These results suggest that the administration of GINST enhances testicular function by elevating GPx and GST activity, thus resulting in increased glutathione, which prevents LPO in the testis. © 2014 Elsevier Inc. All rights reserved.

1. Introduction The root of Panax ginseng C.A. Meyer (P. ginseng) is one of the most frequently used traditional medicines in Korea, China, Japan, and other Asian countries. P. ginseng has been used for the treatment of a broad spectrum of ailments, and previous reports have shown that extracts of P. ginseng have many biological activities including antiinflammatory (Yayeh et al., 2012), anti-diabetic (Xiong et al., 2010), anti-tumor (Du et al., 2011), neuroprotective (Zheng et al., 2011), cardioprotective (Wang et al., 2010), and hepatoprotective (Lee et al., 2005) effects. In addition, recent studies show that P. ginseng can result in improved hair regeneration in Alopecia Areata (Oh and Son, 2012) and protected on UVB-irradiated human keratinocytes and dermal fibroblasts (Lee et al., 2012). Moreover, P. ginseng has potent effects on sexual function, and can relieve erectile dysfunction (Choi et al., 1995), senile testicular dysfunction (Hwang et al., 2010), and dioxin-induced testicular damage (Hwang et al., 2004). ⁎ Corresponding author. Tel.: +82 43 840 3574; fax: +82 43 840 3872. E-mail address: [email protected] (S.-K. Kim). 1 Contributed equally to the manuscript.

http://dx.doi.org/10.1016/j.exger.2014.02.012 0531-5565/© 2014 Elsevier Inc. All rights reserved.

P. ginseng saponins, commonly referred to as ginsenosides, are regarded to be the main active components responsible for the pharmacological activities of P. ginseng. To date, 38 different ginsenosides have been characterized, and most of these are protopanaxdiol and protopanaxtriol dammarane-type triterpenoids (Fuzzati, 2004). The pharmacological actions of ginsenosides are the result of their biotransformation in the human intestine (Hasegawa et al., 1997). Ginsenosides are hydrolyzed by intestinal bacteria before absorption. The microbes transform ginsenosides to other forms that contain fewer sugar moieties. For example, P. ginseng fermentation by lactic acid bacteria generates compound K (CK), which is transformed from the ginsenosides Rb1, Rb2 and Rc. Interestingly, CK has a potent cytotoxic effect on tumor cells much stronger than found in the ginsenosides. Pectinase is commonly produced by lactic acid bacteria in the intestine (Hasegawa and Uchiyama, 1998), and recent reports have shown that pectinase-treated extracts of P. ginseng (GINST) contain large amounts of CK as well as several other forms of ginsenosides, including Rg3, Rg5, Rk1, Rh1, F2, and Rg2 (Chen et al., 2008). These results suggest that extracts of GINST may have higher bioactivities in many physiological contexts, including testicular function in aged animals. However, the function and its mechanism by which GINST modulates male sexual function have not been elucidated. In

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the present study, the effects of GINST on various testicular functions related to spermatogenesis and on the composition of the testis proteins in aged rats (AR) were investigated using a 2-dimensional electrophoresis (2-DE)-based proteomic analysis. Furthermore, we examined the roles of glutathione-S-transferase (GST) mu5 and phospholipid hydroperoxide (PH) glutathione peroxidase (GPx), two redox-related proteins which were differentially expressed between AR and GINSTtreated AR (GINST-AR).

2.5. Measurement of serum biochemical parameters To measure biochemical parameters, blood was taken from the abdominal vein and collected in s SST® gel & clot activator tube (Becton and Dickinson, Franklin Lakes, New Jersey). Serum was separated by centrifugation at 1500 ×g for 10 min at room temperature. Serum testosterone, Luteinizing Hormone (LH) and Follicle-stimulating Hormone (FSH) contents were analyzed using radioimmunoassay kit (Diagnostic Product Corporation, Los Angeles, USA).

2. Materials and methods 2.1. Preparation of GINST The GINST used in this study was produced from an extract of P. ginseng treated with pectinase (Hu et al., 2008). Briefly, dried ginseng (1 kg) was extracted with 5 L of 50% aqueous ethanol at 85 °C and concentrated in vacuo to obtain a dark-brown, viscous solution. The extract was then dissolved in water containing 2.4% pectinase (Sigma, St. Louis, MO) and incubated at 55 °C for 24 h. The fermented P. ginseng extract was then subjected to vacuum concentration, and ginsenosides in the extract were analyzed. 2.2. HPLC analysis of ginsenosides The ginsenosides were analyzed by High-performance liquid chromatography (Agilent, Santa Clara, CA, US) with a diode array detector. An HS C18 (25 cm × 4.6 mm, i.d., 5 μm; SUPELCO, St. Louis, MO, USA) was used for all separations at room temperature. The binary gradient elution system consisted of water (solvent A) and acetonitrile (solvent B). The separation was achieved using the following gradient program: 0–10 min (20% B), 40 min (32% B), 48 min (42% B), 60 min (45% B), 78 min (75% B), and 80 min (100% B). The column temperature was kept constant at room temperature; the flow rate was 1.6 mL/min. 2.3. Experimental animals Male Sprague–Dawley rats [AR = 12-month-old (750 ± 20 g, n = 18), YCR = 7-week-old (280 ± 10 g, n = 9)] were purchased from Samtako Korea (Osan, Korea) and acclimatized to the facility for at least 1 week prior to experimental treatment. Treatment groups consisted of young control rats (n = 9, YCR), vehicle (distilled water)treated AR (n = 9, V-AR), and GINST-treated AR (n = 9, GINST-AR). The GINST was administered at daily dose of 200 mg/kg body weight for 4 months. The GINST was mixed evenly with a sterilized standard powder type diet and then pelletized. Animals were provided with the standard pellet diet and water ad libitum and kept at constant temperature (21 ± 2 °C) and relative humidity (55 ± 10%) on a 12/12-h light/ dark cycle. Rats were maintained in accordance with the Institutional Animal Care and Use Committee Guidelines of Konkuk University. The study was approved by the Animal Ethics Committee in accordance with the 14th article of the Korean Animal Protection Law. 2.4. Histology Testes were removed under ethyl ether anesthesia and weighed after removing the surrounding adipose tissue. The left testis was cut into small pieces (≈5 mm3), and fixed in Bouin's solution containing 25% formaldehyde, 5% glacial acetic acid and 75% picric acid. The fixed testicular tissues were dehydrated, paraffin-embedded using an automatic tissue processor (ASP300, Leica, Wetzlar, Germany), and sectioned to a thickness of 4–6 μm using a microtome (RM2245, Leica). Sections were stained with hematoxylin and eosin (H&E), and examined using light microscopy (CX31, Olympus, Tokyo, Japan). More than 100 longitudinal sections of seminiferous tubules per group were measured to determine the spermatogenesis-related parameters.

2.6. Measurement of sperm kinematic values Sperm samples were extracted from the left caudal epididymis by cutting it with scissors; one drop of caudal fluid was immediately placed in a culture dish containing 5 mL of Hanks' balanced salt solution prewarmed to 37 °C and supplemented with 10 mg/mL BSA (bovine serum albumin). After incubation for 5 min at 37 °C, an aliquot of the suspension was collected with a micropipette and diluted to contain 40 ± 10 sperm under the defined microscopic field (×100 magnification); 10 μL of the suspension was then added to a 2X-CEL slide (depth: 80 μm, thickness: 0.15 mm, Hamilton Thorne Res., Massachusetts, USA) that had been prewarmed in a CO2 incubator (Sanyo Electric Co., Osaka, Japan) at 37 °C. Sperm motility was recorded using a computer-assisted sperm analyzer (CASA, Hamilton Thorne Res., Massachusetts, USA) with a ×4 objective lens and a charge-coupled device (CCD) camera. At least 200 sperm in each sample were monitored for motility pattern analysis. 2.7. Analysis of spermatogenesis-related parameters The germinal cells in the seminiferous epithelium were classified into three morphological categories according to the developmental stage: spermatogonia, spermatocytes and spermatids. For the identification of each stage: testis sections were stained with H&E and examined under a light microscope and classified according to the criteria proposed by Hankinson (2005). The ratio of the total number of germinal cells to the total number of Sertoli cells (Sertoli Cell Index, SCI) was calculated, and the numbers of germ cells at different stages of maturation in the seminiferous epithelium were analyzed as recommended by Hankinson (2005).

2.8. Assay of antioxidant status The testis was thoroughly rinsed in saline, and approximately 100 mg of tissue was then homogenized at 4 °C in Tris–HCl buffer (0.1 mol; pH 7.4). The homogenates were centrifuged at 2500 rpm for 30 min, and the resultant supernatants were refrigerated until further biochemical analysis was conducted. All the assay procedures were carried out within 48 h after sample collection. The reduced glutathione (GSH) content of the tissue homogenate was measured at 412 nm using the method of Beutler et al. (1963). Results were expressed as μmol/mg protein. The GPx activity of the tissue was quantified spectrophotometrically at 412 nm according to Rotruck et al. (1973). Values were expressed as μmol/min/mg protein, enzyme activity being the amount of activity that oxidizes 1 μmol of glutathione (GSH) to GSSG in the presence of H2O2/min. GST activity was assayed in tissue homogenates by the method of Habig et al. (1974) and quantified by measuring the increase in absorbance at 340 nm. Results were expressed in μmol/min/mg protein. The level of lipid peroxidation (LPO) was estimated according to the method of Ohkawa et al. (1979). Malondialdehyde (MDA), one of the major secondary products of LPO, was quantified spectrophotometrically by measuring the increase in absorbance at 532 nm, and results were expressed as nmol/min/mg protein in tissue samples.

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2.9. Western blot analysis

2.12. In-gel digestion and MS of protein spots

Equal amounts of testis protein from each sample were separated by 10% SDS-PAGE and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). Each membrane was incubated for 1 h in TBS containing 0.1% Tween-20 and 5% skim milk to block non-specific binding. Membranes were then incubated with primary antibodies that recognized GSTmu5, PH-GPx, or actin (1:2000, Santa Cruz Biotech, Santa Cruz, CA). Each protein was detected using horseradish peroxidase conjugated secondary antibodies and a chemiluminescence detection system (GE Healthcare Life Sciences, Little Chalfont, U.K.).

Silver-stained protein spots were excised from the stained gel and destained with freshly prepared 15 mmol K3[Fe(CN)6] and 50 mmol Na2S2O3 for 10 min. The gel pieces were washed with 50% ACN and 100 mmol NH4HCO3 three times for 15 min. After dehydrating the gel spot with ACN for 15 min, each gel spot was dried in a SpeedVac centrifuge for 10 min. Samples were then reduced with DTT and subsequently alkylated with iodoacetamide. Finally, samples were digested in 20 μL of digestion buffer (12.5 ng/mL trypsin in 20 mmol NH4HCO3) at 37 °C for at least 16 h. The peptide samples were extracted with 50 μL of 50% ACN/0.1% formic acid and dried in a SpeedVac centrifuge. Extracts were resuspended in 10 μL of 0.1% TFA, and desalted with ZipTip C18 columns (Millipore) according to the manufacturer's instructions. The peptide samples were then mixed (1:1) with a matrix consisting of a saturated solution of CHCA prepared in 50% ACN/0.5% TFA. Aliquots of samples (1 μL) were spotted onto stainless–steel sample target plates. Peptide mass spectra were obtained by MALDI-TOF/TOF mass spectrometer (AB4700, AB SCIEX, Foster City, CA, USA) in the positive ion reflector mode. For precursor ion selection, all fractions were measured in single MS before MS/MS was performed. For MS/MS spectra, the peaks were calibrated by default. The 10 most abundant precursor ions per sample were selected for subsequent fragmentation by high-energy collision-induced dissociation (CID). The collision energy was set to 1 keV, and air was used as the collision gas. The criterion for precursor selection was a minimum signal-to-noise ratio of 20. The mass accuracy was within 100 ppm for the mass measurement and within 0.2 Da for CID experiments. The other parameters for searching were: use of trypsin, 1 missed cleavage, variable modification of carbamidomethyl, oxidation, propionamide and pyro-glu (N-term), peptide charge of 1+, and monoisotopic. For database searches, known contamination peaks such as keratin and autoproteolysis peaks were removed before searching. Spectra were processed and analyzed using the Global Protein Server Explorer 3.0 software (AB SCIEX). This software uses an internal MASCOT (Matrix Science, London, U.K.) program for matching MS and MS/MS data against database information. The data obtained were screened against rat databases downloaded from both NCBI (http://www.ncbi.nlm.nih.gov) and Swiss-Prot/TrEMBL (http://www. expasy.ch/sprot). Further confirmations of protein identification were obtained using the MS-Fit (http://prospector.ucsf.edu) and ProFound (http://www.prowl.rockefeller.edu) programs.

2.10. Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted from testis tissue using the RNA-Bee reagent (AMS Bio, Abingdon, U.K.) according to the manufacturer's instructions. RNA (1 μg) was reverse-transcribed for 50 min at 37 °C in a mixture containing 1 μL oligo (dT), 10 mmol dNTP, 0.1 mol DTT, 5× PCR buffer and 1 μL M-MLV RT (Invitrogen, Carlsbad, CA). An aliquot (200 ng) of the RT product was amplified in a 25-μL reaction using GoTaq® Green Master Mix (Promega, Madison, WA, USA) in the presence of 10 pmol oligonucleotide primer. The following primers were used: GSTmu5 (forward, 5′- TAT GCT CCT GGA GTT TAC TGA TAC C -3′; reverse, 5′- GA CGT CAT AAG TGA GAA AAT CCA C -3′); PH-GPx (forward, 5′- GCA AAA CCG ACG TAA ACT ACA CT -3′; reverse, 5′- CGT TCT TAT CAA TGA GAA ACT TGG T -3′); and GAPDH (forward, 5′-AAC TTT GGC ATT GTG GAA GGG C -3′; reverse, 5′- ACA CAT TGG GGG TAG GAA CAC G -3′). PCR was performed for 20 cycles at 95 °C for 30 s, 56 °C for 40 s, and 72 °C for 40 s. After the amplification, PCR products were separated by electrophoresis on a 2.0% agarose gel containing ethidium bromide, and the bands were visualized by fluorescence. 2.11. Two-dimensional electrophoresis (2-DE) Testicular tissues were homogenized in 2-DE buffer containing 8 mol urea, 2 mol thiourea, 65 mmol DTT, 2% CHAPS, and complete protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany). The homogenate was centrifuged at 12,000 × g for 10 min at 10 °C and the supernatant was collected. The protein concentrations were determined using BioRad protein assay reagents with BSA as the standard. The protein homogenates were diluted with rehydration buffer containing 8 mol urea, 0.28% DTT, 0.5% CHAPS, 10% glycerol, 0.5% appropriate ampholyte, and 0.002% bromophenol blue. The IPG strips (pH 3–10 nonlinear) were rehydrated at 50 V for 12 h at 20 °C in 200 mg of whole cell protein extract. IEF was carried out sequentially with a BioRad Protein IEF Cell at 100 V for 2 h, 250 V for 1 h, 500 V for 1 h, 1000 V for 1 h, 3000 V for 1 h, 5000 V for 1 h, and 8000 V for 9 h at 20 °C. After IEF, individual strips were equilibrated for 20 min in 10 mL of equilibration solution containing 50 mmol Tris–HCl (pH 6.8), 6 mol urea, 20% glycerol, 2% SDS, 0.01% bromophenol blue, and 5 mmol tributylphosphine. The equilibrated strips were transferred onto 12% acrylamide SDS-PAGE gels, and a potential of 30 V was applied for 1 h followed by a potential of 80 V until the bromophenol blue marker reached the bottom of the gel. To visualize proteins, gels were incubated for 2 h in fixing solution containing 50% methanol, 12% acetic acid, and 0.05% formaldehyde, and then washed twice for 20 min, each time in 50% ethanol. Sensitization was carried out for 1 min in 0.02% Na2S2O3. After three consecutive washes with distilled water, each for 20 s, gels were impregnated with 0.1% silver nitrate solution containing 0.075% formaldehyde for 20 min. Excess silver nitrate was washed with distilled water for a few seconds, and gels were developed with a solution of 0.0002% Na2S2O3, 0.025% formaldehyde and 3% Na2CO3 for 10 min. The development was stopped by the addition of 1.5% EDTA. The density of silverstained spots from four different experiments was determined by both automation and manual spot detection, and statistically analyzed using the PDQuest software (Version 7.1.1, BioRad).

2.13. Statistical analysis Data are expressed as the mean ± SD. Statistical evaluation of the data was performed using Student's t-test for comparisons between two groups and ANOVA for multiple comparisons using GraphPad Prism version 4.0 (GraphPad Software, San Diego, CA). A value of p b 0.05 was considered to indicate a significant difference.

3. Results 3.1. HPLC analysis HPLC chromatograms of ginseng radix and pectinase-treated ginseng radix extracts are shown in Fig. 1. The saponin peaks that include ginsenosides Rg1, Rg2 R, Rb1, Rb2 and Rd in ginseng radix were decreased during the enzyme treatment. On the other hand, compound K peak in GINST was higher than in ginseng radix. The ginsenoside content of GINST determined on a dry weight basis was as follows: Rg1 (46.72 mg/g), Rf (62.18 mg/g), Rh1 (33.68 mg/g), Rg2 S (10.02 mg/g), Rg2 R (10.34 mg/g), Rb1 (18.99 mg/g), Rb2 (79.98 mg/g), Rd (103.77 mg/g), Rg3 S (21.65 mg/g), Rg3 R (28.85 mg/g), compound K (280.48 mg/g), Rh2 S (35.68 mg/g) and Rh2 R (53.94 mg/g).

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Fig. 1. HPLC profiles of ginseng radix (A) and pectinase-processed ginseng radix (GINST) (B). Instrument; Agilent HPLC system, Column; HS C18 (4.6 × 250 mm, 5 μm; SUPELCO, St. Louis, Missouri, USA), Mobile-phase; water (solvent A) and acetonitrile (solvent B): 0–10 min (20% B), 40 min (32% B), 48 min (42% B), 60 min (45% B), 78 min (75% B), 80 min (100% B), Flow rate; 1.6 mL/min.

3.2. Effect of GINST on testis morphology in aged rats The morphological characteristics of testicular tissues from YCR, V-AR, and GINST-AR were examined. Cross-sections of seminiferous tubules of the YCR testis showed the typical arrangement of cells at different stages (Fig. 2). Spermatogonia and Sertoli cells rest on the basement membrane, which is surrounded by a concentric myofibroblast layer similar to that of the human testis (Ling et al., 1996). In the YCR testis, the tubules were densely packed with Sertoli cells and post-meiotic cells at various stages of differentiation (Fig. 2A). By contrast, cells in the seminiferous tubules of the AR testis were loosely packed (Fig. 2B), and the number of spermatozoa at the center of the tubules was low. The administration of GINST significantly improved the cell numbers and spermatogenesis in AR testis (Fig. 2C). Moreover, degraded Leydig cells were rejuvenated by treatment with GINST and were uniformly scattered through the interstitial tissue.

3.3. Effect on sperm kinetic values related to sperm quality The ratio of motile sperm in YCR was 74.7 ± 8.0% and the ratio in V-AR decreased to 34.0 ± 7.8% (p b 0.001, Table 1). On the other hand, the

motile sperm ratio in the GINST group increased significantly to 52.0 ± 10.4% (p b 0.01) when compared with that in the old control. The ratio of sperm with straightforward movement in the young control group was 33.7 ± 3.8%, and the ratio in the old control group decreased markedly to 12.7 ± 3.5% (p b 0.001). The ratio of sperm with straightforward movement in GINST group was significantly higher, 22.8 ± 4.0% (p b 0.001), when compared with old control.

3.4. Effect on serum sex hormone levels There was not a marked variation in testosterone level between the YCR (4.63 ± 0.74 ng/mL) and V-AR (4.81 ± 0.86 ng/mL) groups (Fig. 3). However, the GINST-AR group revealed markedly elevated level of testosterone (5.48 ± 0.73 ng/mL, p b 0.05) compared with that of V-AR. The FSH level of V-AR (23.54 ± 2.46 mIU/mL, p b 0.01) was significantly higher than that of YCR (17.24 ± 3.26 mIU/mL). On the other hand, GINST-AR demonstrated significantly lower value (19.64 ± 2.15 mIU/mL) compared with that of V-AR. The LH level of the YCR, V-AR and GINST-AR groups demonstrated 20.56 ± 1.31, 23.61 ± 1.98 and 21.09 ± 1.41 mIU/mL, respectively: a significant increase in V-AR

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A. YCR

B. V-AR

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C. GINST-AR

Fig. 2. Histological analysis of seminiferous tubules in the rat testis. Representative images of tubular cross-sections of testis from young control rats (YCR, A), vehicle-treated aged rats (V-AR, B), and GINST-treated aged rats (GINST-AR, C). Sections were stained with H&E. The images are typical of those obtained in five independent experiments. Scale bar = 45 μm. LC, Leydig cell; PS, primary spermatocyte; SG, spermatogonium; SP, spermatozoa; SR, Sertoli cell; ST, spermatid.

(p b 0.05) compared with YCR and a significant decrease in GINST-AR (p b 0.05) compared with V-AR. 3.5. Effect of GINST on spermatogenesis-related parameters in aged rats Sperm number was significantly lower in the V-AR testis compared to the YCR testis (Fig. 4A). A significant increase in sperm number in the GINST-AR testis relative to V-AR testis was measured. Sertoli cell and germ cell counts per tubule and the SCI were compared in YCR, V-AR, and GINST-AR testes. These parameters were significantly decreased in V-AR testis compared to YCR testis, but the GINST-AR testis parameters were significantly greater than those of the V-AR testis (Fig. 4B, C, D). 3.6. Isolation and characterization of the testis proteome of aged rats Proteins were extracted from V-AR and GINST-AR testes, separated by 2-DE and visualized with silver-staining. The average matching rate on 4 gels was 82.5% in V-AR, and that in GINST-AR was 76.6%. Fig. 5 shows typical images of 2-DE gels of total testicular proteins from V-AR and GINST-AR. More than 900 proteins can be seen in each gel, with isoelectric point values of pH 3–10 and masses of 15–180 kDa. Among these proteins, 14 proteins were significantly increased in the GINST-AR testis. Table 2 shows the properties of the altered proteins, including representative peptide sequences, sequence coverage, theoretical and experimental pI and MW values, and accession numbers from both the Swiss-Prot and NCBI databases. Among these proteins, similar to capping protein beta subunit, triosephosphate isomerase 1, and peroxiredoxin 4 (Prx4) were up-regulated in the GINST-AR testis compared to the V-AR testis (Fig. 6). Carbonyl reductase (NADPH) was down-regulated in the GINST-AR testis. In particular, as shown in Fig. 7A, GSTmu5 (spot 3217) and PH-GPx (spot 8106) were clearly increased in the GINST-AR testis. To validate the differential protein expression, we selected the two antioxidant enzymes, GSTmu5 and PH-GPx, and examined their distribution

in the AR testis by immunoblotting and PCR analysis. The protein levels of GSTmu5 and PH-GPx were higher in testis from GINST-AR than those of V-AR (Fig. 7B). Similar results were observed in the mRNA analysis (Fig. 7C).

3.7. Effect of GINST on enzymatic and non-enzymatic antioxidants in aged rats Proteomics analysis shows that the expression of antioxidant enzymes was strongly altered in the GINST-AR testis. Therefore, we determined the total activities of lipid peroxidases, GST, and GPx, as well as the levels of GSH in testis from YCR, V-AR, and GINST-AR. The GST activity was significantly diminished in the AR testis compared with that of YCR, and GINST administration significantly increased the GST activity in the AR testis (Fig. 8A). In addition, the GPx activity was reduced in AR testis relative to YCR, and activity was restored in GINST-AR (Fig. 8B). GSH levels were significantly lower in V-AR compared to YCR. The administration of GINST restored the concentrations of GSH in AR compared to V-AR testis (Fig. 8C). Elevated levels of reactive oxygen species (ROS) contribute to oxidation of macromolecules, and PH-GPx is involved in reducing oxidized phospholipids in the membranes of testicular cells (Wolin, 2000). Therefore, the effect of GINST on the extent of lipid peroxidation in the YCR and AR testes was examined. The level of MDA, an indicator of lipid peroxidation, was significantly elevated in the AR testis compared to the YCR testis (Fig. 8D). The level of lipid peroxidation was reduced near to YCR levels in the GINST-AR testis.

Table 1 Effect of GINST on kinematic values related to sperm quality. VAP; average path velocity, VSL; straight line velocity, VCL; curvilinear velocity, LIN; linearity = VSL / VCL × 100, STR; straightness = VSL / VAP × 100. Statistical comparisons: YCR vs V-AR, *p b 0.05, **p b 0.01; V-AR vs. GINST-AR, #p b 0.05, ##p b 0.01. ###p b 0.001. Values are mean ± SD (n = 6). Parameter

YCR

Motility (%) Progressive (%) VAP (mm/s) VSL (mm/s) VCL (mm/s) LIN (%) STR (%)

74.7 33.7 220.3 158 268.3 58.7 71.5

V-AR ± ± ± ± ± ± ±

8.0 3.8 30.0 25.1 5.6 5.6 3.9

34.0 ± 12.7 ± 165.3 ± 125 ± 254.7 ± 49.4 ± 74.2 ±

GINST-AR 7.8** 3.5** 66.7* 48.8* 95.5 4.8** 5.0

52.0 22.8 207.0 140.4 263.0 53.6 68.3

± ± ± ± ± ± ±

10.4### 4.0### 41.6# 24.6# 47.9 5.0### 4.3##

Fig. 3. Effect of GINST on sex hormone levels in aged rats. FSH; follicle stimulating hormone, LH; luteinizing hormone. Statistical comparisons: YCR vs V-AR, ⁎p b 0.05; V-AR vs. GINST-AR, #p b 0.05, ##p b 0.01. Values are mean ± SD (n = 6).

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Fig. 4. Effects of GINST on testis parameters related to spermatogenesis in aged rats. (A) Sperm number per tubule, (B) Sertoli cells per tubule, (C) Germ cells per tubule, and (D) Sertoli cell index. Statistical comparisons: YCR vs V-AR, ⁎p b 0.05, ⁎⁎p b 0.01; V-AR vs. GINST-AR, #p b 0.05, ##p b 0.01. Values are mean ± SD (n = 6).

4. Discussion In this study, we analyzed the rat testis proteome and resolved more than 900 different protein spots using 2-DE; the resulting pattern shares some similarities with previous analyses of various testicular tissue or cells (Com et al., 2003; Huang et al., 2005; Huo et al., 2008; Li et al., 2011). The administration of GINST to AR differentially affected the expression of 14 proteins, which were subsequently identified using MALDI-TOF/TOF MS. These differentially expressed proteins are included in common antioxidant, metabolic, or mitochondrial functions. For example, we were able to identify Prx4, an antioxidant protein that is widely expressed in mammalian cells (Com et al., 2003; Rolland et al., 2007), including rat testis (Huo et al., 2008; Iuchi et al., 2009). There are six Prx proteins, which are classified into three groups according

A. V-AR

to the number of cysteine residues: two-Cys, one-Cys, and atypical two-Cys groups (Lee et al., 2007). The cysteine residues of Prx participate in reducing H2O2 levels in cells. Prx is involved in a number of cellular responses, including proliferation and apoptosis (Zhang et al., 1997). Previously, we demonstrated that the expression of Prx isoforms in vascular cells during hypertension was significantly altered in response to treatment with ROS (Lee et al., 2007). These results imply that Prx may be involved in testicular dysfunction that results from oxidative stress. A key finding of this study is that several proteins showed increases in expression in response to GINST administration. The expression of GSTmu5 and PH-GPx significantly increased in testis from GINST-AR compared with V-AR. These changes in expression were verified by Western blot and PCR analyses, indicating that altered expression of

B. GINST-AR

Fig. 5. Representative images of silver-stained 2-DE gels showing the rat testis proteome. Aged rats were administered 200 mg/kg body weight GINST (GINST-AR, B) or vehicle (V-AR, A) daily for 4 months. Proteins were subjected to IEF on IPG pH 3–10 NL strips and then separated by 12% SDS-PAGE. Arrows show protein spots in which expression is altered in GINST-AR relative to V-AR. The numbers indicated in the gels correspond to those in Table 2. The images are typical of those obtained in four independent experiments.

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Table 2 Altered proteins in rat testis tissue identified by MALDI-TOF/TOF. Protein spots were quantified in testis from AR treated with vehicle or GINST. Increases or decreases of at least 1.5-fold in three independent experiments were considered significant (p b 0.05). No.

Δ

Protein name

Peptide sequences

SC(%)/score

pI/Mr (kDa) theoretical (experimental)

Accession no./database

RLEAAYTDLR QILESEKDLEEAEEYKEAR RLPPQQIEK DYLLCDYNR KLEVEANNAFDQYR FFVGGNWK DLGATWVVLGHSER HIFGESDELIGQK CNVSEGVAQCTR DYGVYLEDSGHTLR GLFIIDDKGVLR QITLNDLPVGR SMVLGYWDIR QYTCGEAPDYDR FTWFAGEK IAAFLQSDR SWNETFHTR LASLSEKPPAIDWAYYR NCAQFVTGSQAR KYPYWPHQPIENL ENECHFYAGGQVYPGEVSR DYGVYLEDSGHTLR QITLNDLPVGR LVQAFQYTDK HITIFSPEGR AINQGGLTSVAVR ITENIGCVMTGMTADSR CDPAGYYCGFK LALQLHPDRNPDDPQAQEK FQMTQEVVCDECPNVK TLEVEIEPGVR DGMEYPFIGEGEPHVDGEPGDLR NGYELSPTAAANFTR FILNLPTFSVR FHQLDIDNPQSIR ELLPIIKPQGR ILLNACCPGWVR DETNYGIPQR DVLSVAFSSDNR YTVQDESHSEWVSCVR VWQVTIGTR IGGHGGEYGEEALQR TYFSHIDVSPGSAQVK AADHVEDLPGALSTLSDLHAHK TDVNYTQLVDLHAR ILAFPCNQFGR QEPGSNQEIKEFAAGYNVR EFAAGYNVR

26/87

5.43/12.7 (5.46/17.8)

gi|37194701/NC

11/88

5.69/30.6 (5.59/33.3)

gi|54400732/NC P10111/SP

18/219

6.45/26.9 (5.54/34.0)

gi|12621074/NC P48500/SP

13/170

6.18/30.9 (5.64/32.0)

gi|16758274/NC Q63716/SP

17/186

6.33/26.6 (5.75/29.1)

gi|25282395/NC

31/167

5.78/18.7 (5.81/25.5)

gi|220904/NC

2002

2.4

Unknown (protein for MGC:72998)

2211

2.8

Similar to capping protein beta subunit, isoform 2

2308

2.3

Triosephosphate isomerase 1

3203

1.9

Peroxiredoxin 4

3217

2.9

Glutathione-S-transferase, mu 5

4103

1.9

Subunit d of mitochondrial H-ATP synthase

4202

2.0

Peroxiredoxin 4

5207

1.7

Similar to proteasome subunit alpha type 6

5401

2.0

LRRGT00084

7104

2.0

Proteasome subunit, beta type 2

7216

−4.3

7318

4.3

Guanine nucleotide binding protein, beta polypeptide 2-like 1

8003

2.4

Similar to hemoglobin alpha chain

8106

1.5

Phospholipid hydroperoxide glutathione peroxidase

Carbonyl Reductase (NADPH)

antioxidant proteins is a prominent event in aging in the testis. There are several studies that tested the effects of ginsenosides on expression of proteins using proteomic analyses in cancer and vascular cells and platelets (Lee et al., 2009; Ma et al., 2006; Yao et al., 2008). To the best of our knowledge, the present study is the first report to clarify the roles of P. ginseng, especially GINST, on the expression of proteins in the testis. In this study, GST activity was decreased in the AR testis; this may be due to excessive generation of H2O2 and diminished GSH concentrations in aged animals. These observations are consistent with earlier reports (Luo et al., 2006; Rao and Shaha, 2000). Fulcher et al. (1995) identified a major protein component of mouse fibrous sheath as a GSTmu5 and showed that its mRNA was enriched in testis, as well as in isolated spermatogenic cells. In addition, the diminished GST activity in the testis during aging may reduce the conversion of GSSG to GSH (Bray and Taylor, 1993). In cellular systems, there are three classes of peroxidases that scavenge cellular H2O2: catalase, GPx, and Prx (Wolin, 2000). This study showed that the expression of PHGPx was elevated in the GINST-AR testis compared with the V-AR testis. PH-GPx is the major GPx in testis, and total GPx activity may reflect primarily PH-GPx activity (Roveri et al., 1992). In rat testis, PH-GPx is

P31399/SP 19/167

6.18/30.9 (5.81/29.1)

gi|16758274/NC

20/148

6.34/27.3 (6.24/29.4)

Q63716/SP gi|8394076/NC P60901/SP

19/163

5.92/40.4 (5.92/40.4)

gi|37361854/NC

12/47

6.96/22.8 (7.64/26.3)

12/194

8.22/30.5 (8.02/32.9)

gi|34849630/NC P40307/SP gi|1217651/NC P47727/SP

14/250

7.60/35.0 (8.03/33.4)

gi|18543331/NC P63245/SP

37/252

8.45/15.2 (8.45/15.2)

gi|34875788/NC P01946/SP

25/337

8.30/19.4 (8.44/21.7)

gi|1041645/NC P36970/SP

greater than 10-fold more active than in liver, kidney, or lung, and PHGPx mRNA levels in testis reflect the activity level (Lei et al., 1995). GPx expression and activity are diminished in the testis in animals treated with male reproductive toxicants (Anjum et al., 2011). Moreover, the activity of GPx is highly dependent on the GSH concentration (Kumaran et al., 2009). The decreased GSH concentration in the AR testis reveals a serious impairment in the GSH circuitry essential for the optimal functioning of the catalytic activities of GSH-dependent enzymes (Mates, 2000). Here, a decrease in GSH coincided with a reduction in the activity of GSH-dependent enzymes. GSH plays an essential role in the cellular antioxidant defenses by removing H2O2 and organic peroxides (Bray and Taylor, 1993). Therefore, increases in the expression and activity of antioxidant enzymes may be considered to be a defense mechanism in response to the formation of ROS in the testis during aging. Therefore, the decreased levels of GST and PH-GPx may lead to ROS increases and may participate in altering the characteristics of the testis during aging. Aging is correlated with increased oxidative stresses and oxidative damage in all the vital organs, including the testis. Oxidative stresses play a major role in the etiology of male infertility (Bindhu and Annamalai, 2004). Therefore, age-induced oxidative stress in the testis

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Fig. 6. Silver-stained 2-DE images showing up- and down-regulated proteins in V-AR versus GINST-AR testis. The arrows indicate differentially expressed proteins. The numbers indicated in the gels correspond to those in Table 1. Statistical analysis was performed on gels from four independent experiments using PDQuest. White bars, V-AR; black bars, GINST-AR (⁎p b 0.05).

can cause physiological changes and biochemical perturbations that are likely to culminate in testicular dysfunction, leading to the development of male infertility (Aitken et al., 1989; Rajeswary et al., 2007). Previously, GPx activity and expression were shown to be reduced in the AR testis (Luo et al., 2006). In this study, we confirmed that the number of fully matured spermatids in the seminiferous tubules was low, the periphery of the tubules appeared like degenerated, and number of cells lining in the tubular membrane was decreased in the AR testis. Furthermore, the level of testis GSH was restored near the level of YCR in the GINST-treated AR testis. Moreover, degenerated Leydig cells were rejuvenated by treatment with GINST and were uniformly spread throughout the interstitial tissue. Histological analysis showed that GINST restored the healthy appearance of the seminiferous tubules. In addition, GINST ameliorated various parameters related to spermatogenesis, such as the SCI and the number of sperm, Sertoli cells, and germ cells per tubule. In our experiment, there was not a significant change in the serum testosterone level between YCR and V-AR. However, the level was markedly increased by GINST (p b 0.05). LH and FSH levels in V-AR were significantly higher when compared with those in YCR (p b 0.05). It can be postulated that those 2 hormones increased in aged rats to compensate for the lowered function of Leydig and Sertoli cells. It was interesting to note that LH and FSH levels were decreased by GINST. Also, inhibin-α is an important adhesion molecule in Leydig cells and promote to synthesize testosterone. From this, it can be supposed that GINST elevated sensitivity of Leydig and Sertoli cells to LH and

testosterone, respectively. However, we need further study on the expression levels of androgen, LH and FSH receptors, and inhibin-α to verify the hypothesis. Moreover, the activities of GPx and GST and the level of GSH in the GINST-AR testis were higher than those in the V-AR testis. These results suggested that the administration of GINST enhances testicular function via the elevation of GPx and GST, with a corresponding increase in the overall levels of GSH in the testis. Besides, our results suggested that GINST prevented the ROS-mediated damage in AR, thus leading to an improvement in the steroidogenic function of the testis. P. ginseng contains a variety of active ingredients such as ginsenosides, polysaccharides and polyacetylenes (Park, 1996). Therefore, the action of GINST in testicular tissue may not be solely dependent on the antioxidant system. However, ginseng clearly showed the ability to rejuvenate senile testicular dysfunction in part by stimulating the testicular antioxidant system. Moreover, GINST is a pectinase-treated extract of P. ginseng, and pectinase is an enzyme commonly produced by lactic acid bacteria in the intestine (Hu et al., 2008). P. ginseng fermented by lactic acid bacteria generates CK from ginsenosides. CK is potently cytotoxic to tumor cells, more than ginsenosides (Bae et al., 2000). Hence, GINST may be a valuable therapeutic agent for testing aging-related sexual dysfunction in men. In this study, we confirmed the age-related alterations of the rat testis by measuring spermatogenesis-related parameters, and showed that testicular function was significantly restored following the administration of GINST. Moreover, we identified 14 proteins, including GST,

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Fig. 7. Protein expression and confirmation of changes in expression in AR and GINST-AR. (A) Arrows indicate proteins that were differentially expressed in V-AR relative to GINST-AR. Protein levels were quantified from four independent experiments (⁎p b 0.05). The numbers indicated in the gels correspond to the numbers in Table 1. (B) Protein expression of GSTmu5 and PH-GPx in testis tissues. Tissue lysates from V-AR and GINST-AR testis were immunoblotted with anti-GSTmu5 and -PH-GPx antibodies. The images are typical of those obtained in blots from three independent experiments. (C) GSTmu5 and PH-GPx mRNA levels in testis from V-AR and GINST-AR. GAPDH served as a loading control. The images are representative of three independent experiments.

Fig. 8. Effects of GINST on testicular antioxidant enzymes in aged rats. (A) GST activity (μmol/min/mg of protein). (B) GPx activity (μmol/min/mg of protein). (C) GSH level, expressed as μg/mg protein. (D) MDA level, expressed as nmol/min/mg protein. Statistical comparisons: YCR vs. V-AR, ⁎⁎p b 0.01; V-AR vs. GINST-AR, ##p b 0.01. n = 6.

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PH-GPx, and GSH, that were differentially expressed in the GINST-AR testis relative to V-AR testis. The activities of GPx and GST and the level of GSH in the GINST-AR testis were higher than in the V-AR testis. These results suggest that the administration of GINST enhances testicular function via the elevation of GPx and GST activities, which may be associated with an increase in GSH levels in the testis. Conflict of interest The authors have no conflicts of interests. Acknowledgement This study was supported by a grant from the ILHWA Co., (ILHWA2011-003) Guri-si, Gyeonggi-do, Republic of Korea. References Aitken, R.J., Clarkson, J.S., Fishel, S., 1989. Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol. Reprod. 41, 183–197. Anjum, S., Rahman, S., Kaur, M., Ahmad, F., Rashid, H., Ansari, R.A., Raisuddin, S., 2011. Melatonin ameliorates bisphenol A-induced biochemical toxicity in testicular mitochondria of mouse. Food Chem. Toxicol. 49, 2849–2854. Bae, E.A., Park, S.Y., Kim, D.H., 2000. Constitutive β-glucosidases hydrolyzing ginsenoside Rb1 and Rb2 from human intestinal bacteria. Biol. Pharm. Bull. 23, 1481–1485. Beutler, E., Duron, O., Kelly, B.M., 1963. Improved method for the determination of blood glutathione. J. Lab. Clin. Med. 61, 882–888. Bindhu, M.P., Annamalai, P.T., 2004. Combined effect of alcohol and cigarette smoke on lipid peroxidation and antioxidant status in rats. Indian J. Biochem. Biophys. 41, 40–44. Bray, T.M., Taylor, C.G., 1993. Tissue glutathione, nutrition, and oxidative stress. Can. J. Physiol. Pharmacol. 71, 746–751. Chen, G.T., Yang, M., Song, Y., Lu, Z.Q., Zhang, J.Q., Huang, H.L., Wu, L.J., Guo, D.A., 2008. Microbial transformation of ginsenoside Rb1 by Acremonium strictum. Appl. Microbiol. Biotechnol. 77, 1345–1350. Choi, H.K., Seong, D.H., Rha, K.H., 1995. Clinical efficacy of Korean red ginseng for erectile dysfunction. Int. J. Impot. Res. 7, 181–186. Com, E., Evrard, B., Roepstorff, P., Aubry, F., Pineau, C., 2003. New insights into the rat spermatogonial proteome: identification of 156 additional proteins. Mol. Cell. Proteomics 2, 248–261. Du, G.J., Dai, Q., Williams, S., Wang, C.Z., Yuan, C.S., 2011. Synthesis of protopanaxadiol derivatives and evaluation of their anticancer activities. Anti-Cancer Drugs 22, 35–45. Fulcher, K.D., Welch, J.E., Klapper, D.G., O'Brien, D.A., Eddy, E.M., 1995. Identification of a unique mu-class glutathione S-transferase in mouse spermatogenic cells. Mol. Reprod. Dev. 42, 415–424. Fuzzati, N., 2004. Analysis methods of ginsenosides. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 812, 119–133. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione s-transferases. The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139. Hankinson, O., 2005. Role of coactivators in transcriptional activation by the aryl hydrocarbon receptor. Arch. Biochem. Biophys. 433, 379–386. Hasegawa, H., Uchiyama, M., 1998. Antimetastatic efficacy of orally administered ginsenoside Rb1 in dependence on intestinal bacterial hydrolyzing potential and significance of treatment with an active bacterial metabolite. Planta Med. 64, 696–700. Hasegawa, H., Sung, J.H., Benno, Y., 1997. Role of human intestinal Prevotella oris in hydrolyzing ginseng saponins. Planta Med. 63, 436–440. Hu, J.N., Zhu, X.M., Lee, K.T., Zheng, Y.N., Li, W., Han, L.K., Fang, Z.M., Gu, L.J., Sun, B.S., Wang, C.Y., Sung, C.K., 2008. Optimization of ginsenosides hydrolyzing betaglucosidase production from Aspergillus niger using response surface methodology. Biol. Pharm. Bull. 31, 1870–1874. Huang, S.Y., Lin, J.H., Chen, Y.H., Chuang, C.K., Lin, E.C., Huang, M.C., Sunny Sun, H.F., Lee, W.C., 2005. A reference map and identification of porcine testis proteins using 2-DE and MS. Proteomics 5, 4205–4212. Huo, R., He, Y., Zhao, C., Guo, X.J., Lin, M., Sha, J.H., 2008. Identification of human spermatogenesis-related proteins by comparative proteomic analysis: a preliminary study. Fertil. Steril. 90, 1109–1118. Hwang, S.Y., Kim, W.J., Wee, J.J., Choi, J.S., Kim, S.K., 2004. Panax ginseng improves survival and sperm quality in guinea pigs exposed to 2,3,7,8-tetrachlorodibenzop-dioxin. BJ. Int. 94, 663–668. Hwang, S.Y., Sohn, S.H., Wee, J.J., Yang, J.B., Kyung, J.S., Kwak, Y.S., Kim, S.W., Kim, S.K., 2010. Panax ginseng improves senile testicular function in rats. J. Ginseng Res. 34, 327–335.

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