Attenuation of reproductive dysfunction in diabetic male rats with timber cultured Antrodia cinnamomea ethanol extract

Biomedicine & Pharmacotherapy 112 (2019) 108684

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Attenuation of reproductive dysfunction in diabetic male rats with timber cultured Antrodia cinnamomea ethanol extract

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Athira Johnson, Shu-Chun Cheng, David Tsou, Zwe-Ling Kong Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan

A R T I C LE I N FO

A B S T R A C T

Keywords: Diabetes Reproductive dysfunction Oxidative stress Anti-inflammation Antrodia cinnamomea

Diabetes mellitus together with the oxidative stress affects the process of spermatogenesis and leads to male infertility. Antrodia cinnamomea (AC) is a mushroom found unique in Taiwan and commonly used for the treatment of several types of cancers and inflammatory disorders. This study was aimed to investigate the antioxidative and the ameliorative effects of Antrodia cinnamomea ethanol extract (ACEE) on reproduction dysfunction in male diabetic rats. The diabetic condition was induced by administrating the combination of streptozotocin (STZ) (65 mg/kg) and nicotinamide (NA) (230 mg/kg). Three different doses of ACEE were tested (385, 770, 1540 mg/kg) for 5 weeks. The results indicated that the ACEE improved STZ-NA induced hyperglycemia, oxidative stress, and insulin resistance. In addition to this, ACEE reduced the degree of lipid peroxidation, recovered the abnormal structure of the seminiferous tubules, and improved sperm parameters. Moreover, the DNA damages and mitochondrial membrane potential were improved in sperm. Our study confirmed that the ACEE has anti-inflammatory and ameliorative effects to prevent diabetes-induced male reproductive dysfunction.

1. Introduction Diabetes mellitus (DM) is a metabolic disorder characterized by prolonged hyperglycemia and account for about 8.3% prevalence in worldwide [1]. It is associated with defects in insulin secretion, insulin action, or both. Diabetes is mainly categorized into type 1 (due to the destruction of β cells of the pancreas), type 2 (increases the demand for insulin due to insulin resistance and obesity), and gestational diabetes (hyperglycemia occurs during pregnancy) [2]. Increased production of free radicals or impaired antioxidant defenses cause oxidative stress and results in diabetic condition [3]. It causes long-term issues including renal failure, foot ulcers, amputations, loss of vision, and sexual dysfunctions etc. [1]. The impaired spermatogenesis and sperm deoxyribonucleic acid (DNA) damage are linked to obesity-associated insulin resistance [4]. The hypothalamic-pituitary-gonadal (HPG) axis control the whole process of spermatogenesis. Under normal conditions, the gonadotropin-releasing hormone (GnRH) released by the hypothalamus stimulates the anterior pituitary to produce luteinizing hormone (LH) and follicle stimulating hormone (FSH). LH stimulates the Leydig cells to produce testosterone and FSH stimulate the Sertoli cells to assist the process of spermatogenesis. In spermatozoa, energy is obtained from glycolysis and oxidative phosphorylation. Therefore, the deficiency of ⁎

insulin will alter the endocrine pathway and results in male reproductive dysfunction [5]. Streptozotocin (2-deoxy-2(3-methyl-3-nitrosoureido)-D-glucopyranose) is a chemical agent, exerts a toxic effect on the β cells of the pancreas. The destruction is happening via the transport of streptozotocin into the cells through the glucose transporter 2 (GLUT2). The administration of nicotinamide (pyridine-3carboxamide) protect the β cells from complete damage [6]. Antrodia cinnamomea (AC) (Syn. Antrodia camphorata) is a wellknown medicinal fungus, very expensive, and endemic to Taiwan. It was first reported as a new species in 1990 [7]. AC is found only on the rotting heartwood of Cinnamomum kanehirai (Hay tree), a large evergreen broad tree grow only at an altitude between 450 and 1200 m [8]. The main components of this fungi are polysaccharides, triterpenoids, steroids, phenolic components, cordycepin, sesquiterpene, adenosine, ergosterol, and maleic/succinic acid derivatives etc. Antrodia cinnamomea contains polysaccharides such as sugar, glucose, galactose, mannose, and galactosamine [9]. This fungus possesses anticancer activity, anti-inflammatory/ immunomodulatory effects, anti-Hepatitis B virus replication, anti-oxidant activities, hepatoprotective activity, neuroprotective effect, antihypertensive effect, and vasorelaxation effect [10]. Previous study reported that the Antrodia comphorata protected mouse beta cell line MIN6 from endoplasmic reticulum (ER) stress-induced apoptosis and increased insulin secretion in a dose-

Corresponding author at: Department of Food Science, National Taiwan Ocean University, Pei-Ning Road, Keelung City, 20224, Taiwan. E-mail address: [email protected] (Z.-L. Kong).

https://doi.org/10.1016/j.biopha.2019.108684 Received 18 December 2018; Received in revised form 28 January 2019; Accepted 12 February 2019 0753-3322/ © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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+4 times dose of Antrodia cinnamomea extract (4X ACEE) (1540 mg/kg BW). Metformin was taken as the positive control. Three doses of CTE (100, 385, and 770 mg/kg) were used according to the recommendation of the Taiwan Food and Drug Administration (TFDA) health food functional evaluation guidelines. The body weight measured in every week and the OGTT test was performed after 5 weeks. All rats were sacrificed after 5 weeks. Centrifuged (HITACHI, CF7D2, Tokyo, Japan) the blood at 3000 × g for 15 min at 4 °C to obtain the plasma for further analysis. The tissues were weighed and stored at - 80 °C for future analysis.

dependent manner through Peroxisome proliferator-activated receptor (PPAR)-γ [11]. This study was aimed to investigate the anti-inflammatory and the ameliorative effects of Antrodia cinnamomea ethanol extract (ACEE) on reproduction dysfunction in male diabetic rats. 2. Materials and methods 2.1. Materials Timber cultured Antrodia cinnamomea was supplied by Xuzhi Biotech, Taichung, Taiwan. Five-week-old male Sprague-Dawley (SD) rats were purchased from Lesco Biotech Co., Ltd., Taipei, Taiwan. Laboratory Rodent Diet 5001 were obtained from PMI® LabDiet®, St. Louis, MO., USA. Glucose enzymatic kit was purchased from EugeneChen, Taipei, Taiwan. Insulin ELISA kit was obtained from Mercodia AB Inc., Sylveniusgatan 8A, Uppsala, Sweden. Triglyceride and cholesterol enzymatic kits were purchased from Audit Diagnostics, Cork, Ireland. Superoxidase dismutase (SOD) and glutathione peroxidase (GPx) were purchased from Randox, Co. Ltd., Antrim, UK. Catalase was purchased from Cayman Chemical Company, Michigan, USA. 1, 1, 3, 3-tetramethoxypropane and thiobarbituric acid were obtained from Sigma, St. Louis, USA. Dichloro-dihydro-fluorescein diacetate (DCFH-DA), Nitroblue tetrazolium (NBT), dimethyl sulfoxide (DMSO), acridine orange (AO), rhodamine 123, and trypan blue were supplied by Sigma, MO, USA. Testosterone ELISA Kit, follicle stimulating hormone (FSH) ELISA Kit, and luteinizing hormone (LH) ELISA Kit were purchased from Medical, Taipei, Taiwan. Bradford reagent, 40% acrylamide solution, and sodium dodecyl sulfate (SDS) were supplied by Sigma, Missouri, USA. β- Actin monoclonal antibodies were obtained from Santa Cruz, California, USA. Tween 20 was purchased from Sigma, Missouri, USA. Tissue Protein Extraction Reagent (TPER) was obtained from Thermo scientific, Illinois, USA.

2.2.3. Plasma sample analysis 2.2.3.1. Determination of glucose and insulin content. 20 μl of plasma was added to 1 ml of glucose enzymatic kit reagent and allowed to react for 5 min at 37 °C. The absorbance was measured at 500 nm. For insulin content determination, 25 μl of plasma was taken and analyzed by rat insulin ELISA kit and the absorbance was measured at 450 nm. The total glucose content was calculated by the following formula:Plasma total glucose (mg/dl) = (A sample-A blank)/ (A standard-A blank) × 200 A sample: Absorbance of blood samples, A blank: Absorbance value of kits reagents without a sample, A standard: absorbance value of the standard reagent, 200: standard reagents at a concentration of 200 mg/ dl. 2.2.3.2. Determination of total triglyceride and cholesterol content in plasma. 10 μl of plasma was added to 1 ml of triglyceride enzymatic kit reagent and allowed to react for 5 min at 37 °C. The absorbance was measured at 510 nm. Total cholesterol content was determined adding10 μl of plasma to 1 ml of cholesterol enzymatic kit reagent and allowed to react for 5 min at 37 °C. The absorbance was measured at 500. The total triglyceride and cholesterol contents were calculated by the following formula:Plasma total triglyceride (mg/dl) = (A sample-A blank)/ (A standard-A blank) ×200. Plasma total cholesterol (mg/dl) = (A sample-A blank)/ (A standard-A blank) ×200.

2.2. Methods 2.2.1. Preparation of Antrodia cinnamonea ethanol extract (ACEE) Antrodia cinnamomea fruiting bodies were treated with 95% ethanol for 3 h. The obtained solution was centrifuged (10,000 × g, 5 min), filtered, and freeze dried. Later, different concentrations of samples (385, 770, 1540 mg/mL) were prepared by dissolved in 95% ethanol. The major content of ACEE was triterpenoids (6.53%). The principle triterpenoids in ACEE were given in HPLC profile (Supplementary data).

2.2.3.3. Determination of lipid peroxidation in plasma. 1 ml of reagent (Trichloroacetic acid (15% w/v) in 0.25 N hydrochloric acid (HCl) and thiobarbituric acid (0.375% w/v) in 0.25 N HCl) was added to 0.5 ml of plasma or 0.5 ml of phosphate-buffered saline (PBS) (blank). Mixed the solution well and placed in a water bath at 100 °C for 15 min. After cooling, 1 ml of n-butanol was added. The solution was shaken vigorously and centrifuged at 1500 × g for 10 min. The supernatant was collected and the absorbance was measured at 532 nm [12]. The lipid peroxidation was calculated by the following formula:Malondialdehyde (MDA) concentration (nM/ml) = (A sample-A blank)/ (A standard-A blank) ×5 A sample: Absorbance of blood samples, A blank: Absorbance value without a sample, A standard: absorbance value of the standard reagent

2.2.2. Animal model design The experimental rats (Sprague Dawley, 4 weeks old) were individually kept in a stainless steel cage with temperature control of 23 ± 1 °C, humidity of 40–60%, and an atmosphere of 12 h light/dark cycle. The rats were fed with laboratory rodent diet 5001. Food and water were provided ad libitum. All procedures followed the standard of Institutional Animal Care and Use Committee (IACUC Approval No. 103015) of National Taiwan Ocean University, Taiwan. The experiment was conducted after one week of domestication. 60 Sprague-Dawley (SD) rats were divided into 2 groups such as control (normal diet) group and diabetic group [high-fat diet (40%)]. Later, 230 mg/Kg (BW) of nicotinamide (NA) injected intraperitoneally. After 15 min, streptozotocin (STZ) (65 mg/Kg BW in citrate buffer, pH 4.6) was injected while the same volume of vehicle (0.1 mol/L citrate buffer, pH 4.5) was given to the control group. After a week, the oral glucose tolerance test (OGTT) was performed to confirm the diabetic condition. After the complete induction of diabetes, the rats were divided into 6 experimental groups such as control (Control), diabetes (DM), diabetes + metformin (100 mg/kg BW), diabetes+ 1 times dose of Antrodia cinnamomea extract (1X ACEE) (385 mg/kg BW), diabetes + 2 times dose of Antrodia cinnamomea extract (2X ACEE) (770 mg/kg BW), Diabetes

2.2.3.4. Determination of plasma testosterone and LH contents. The testosterone ELISA Kit was used to analyze the concentration of testosterone in the plasma. 50 μl of plasma was added to 50 μl of acetylcholinesterase (AChE) tracer and allowed to react. After centrifugation, the supernatant was removed and washed 5 times with wash buffer. Later, Ellman's reagent (substrate for AChE) was added and the solution was shaken under the dark condition for 80 min. The absorbance was measured at 412 nm. Radioimmunoassay (RIA) was used to determine the LH concentration.50 μl of plasma was analyzed using RIA kits. Rat LH-I-9 was calibrated with 125I using Iodo-gen tubes and the hormone concentration calculated as LH-RP-3. The coefficient of variation is less than 8˜10% and the sensitivity was 5 pg/tube. 2

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2.2.4. Sperm sample analysis 2.2.4.1. Sperm sample collection. The swim-up method was used to collect the sperm. Epididymis (cut into 3 pieces) were placed in a beaker containing 8 ml of Roswell Park Memorial Institute (RPMI) medium and shaken for 10 min at 100 rpm. After centrifugation (190 × g, 5 min), incubated the sample at 37 °C for 30 min in 5% CO2 incubator. After incubation, the sperm was collected for further analysis [13].

washed with cooled PBS. 0.5 g of testicular tissues were added to 4.5 ml of potassium chloride solution (KCl) (1.15%) to homogenize the tissues. Centrifuged the solution at 10,000 × g for 30 min (4 °C). Later, 1 ml of testicular homogenate (1 ml of PBS was taken as a blank group) was added to 2 ml of the reagent (trichloroacetic acid (15% w/v) in 0.25 N HCl and Thiobarbituric acid (0.375% w/v) in 0.25 N HCl). The further procedures were same with the Section 2.2.3.3.

2.2.4.2. Determination of sperm count, motility, and abnormal sperm count. 100 μl of sperm liquid was mixed with trypan blue and the number of sperms, motile sperms, and abnormal sperms were counted using a haematocytometer and microscope. The sperm dilution factor is the average of the total number of sperm [14]. Sperm concentration (sperms/ml) = cell number × 2 × 104× dilution factor Proportion of active sperm (%) = (number of active sperm / total sperm) × 100 The proportion of abnormal sperm (%) = (number of sperm with the abnormal shape / total number of sperm) × 100

2.2.5.2. Determination of Superoxide dismutase (SOD) and Catalase activity in testis. The SOD activity was analyzed by RANSOD kit. 0.05 ml of testis homogenate was added to 1.7 ml of the reagent (0.05 mM Xanthine, 0.025 mM 2-(4-iodophenyl)-3-(4-nitrophenol)-5phenyltetrazolium chloride (INT). After mixing, 0.25 ml of xanthine oxidase (80 U/L) was added and reacted at room temperature for 30 s. The absorbance was measured at 505 nm. The protein concentration of the testicular homogenate was determined by the Bio-Rad DC protein assay kit, and its specific activity (U/mg protein) was calculated [16]. 0.6 ml of the testis homogenate added to 0.3 ml of 30 mM H2O2 (dissolved in 50 mM PBS, pH 7.0). The absorbance was measured at 240 nm [17].

2.2.4.3. Sperm lipid peroxidation. The sperm number was adjusted to 1 × 106 cells/ml. 1 ml of sperm liquid (1 ml of PBS as a blank group) added to 2 ml of the reagent (15%, w/v trichloroacetic acid (15% w/v) in 0.25 N HCl and 0.375%, w/v thiobarbituric acid (0.375%w/v) in 0.25 N HCl). The further procedures were same with the Section 2.2.3.3.

2.2.5.3. Histology analysis of testis. Soaked the testis in 10% of formalin and transferred to a 50 ml centrifuge tube containing PBS and stored at 4 °C. Before slicing, the temperature of the cryostat was adjusted to −20 °C. Optimal cutting temperature (OCT) compound was added to the surface of the testis until the OCT completely covers the testis. Sliced the tissues into 10 micro sizes. Then quickly attached the sheet to the slide. The slides were fixed in fixative (95% methanol + 5% acetic acid) for 10 s, washed with PBS for one minute, and air dried for one hour. Later, soaked them with 50%, 70% and 90% alcohol for one minute and embedded in paraffin wax. For Hematoxylin and Eosin (H& E) staining, the slides were immersed in hematoxylin for 3 min and washed with running water for 5 min. Stained with Iraqi Eosin for 10 s and soaked in 100% alcohol for one minute until it fades. Finally, soaked the slide in xylene for one minute, air-dried, and sealed. Place the stained slide on an inverted phase contrast microscope (Inverted Phase Difference Microscope, Olympus CK-2, Japan) to observe the morphology.

2.2.4.4. Determination of ROS content in sperm. The sperm number was adjusted to 1 × 106 sperm/ml in RPMI solution. 1 ml of dichlorodihydro-fluorescein diacetate (DCFH-DA) was added and allowed to react at 37 °C. After 30 min, the solution was centrifuged at 760 × g for 5 min. The supernatant was removed and washed with PBS. After centrifugation, 1 ml of PBS was added and the ROS contents were analyzed by flow cytometry. 2.2.4.5. Nitroblue tetrazolium chloride (NBT) analysis. The sperm count was adjusted to 1 × 106 cells/ml. 0.3 ml NBT solution (0.1 mg/ml NBT, 5% fetal calf serum (FCS), and 3% dimethyl sulfoxide (DMSO) dissolved in 10 ml RPMI) was added and incubated at 37 °C for 45 min. After centrifugation (500 × g, 10 min), the supernatant was removed and washed with PBS. Later, 200 μl of DMSO was added to dissolve the purple crystals in the cells. 200 μl of the cell suspensions were transferred to a 96-well plate and the absorbance was measured at 630 nm.

2.2.5.4. Identification and quantification of protein. Bradford protein assay was used to quantify the protein. 20 μl of standard or diluted cell solution was added to 96-well plate containing 200 μL of Bradford reagent. After 15 min, the absorbance was measured at 595 nm. The standard concentration curve was obtained by serial dilution with 2.0 mg/ml bovine serum albumin. After regression, the linear equation obtained and the protein concentration (mg/mL) was converted. SDS- polyacrylamide gel electrophoresis (SDS-PAGE) was performed to identify the protein. Solution A (30% acrylamide-bisacrylamide solution), solution B (1.5 M Tris buffer, pH 8.80), solution C (0.5 M Tris buffer, pH 6.8), and solution D [10% ammonium persulfate (APS)] were the stock solutions. Samples were prepared by adding protein lysate to protein loading buffer and western blot analysis was performed to detect the specific protein.

2.2.4.6. Evaluation of sperm mitochondrial membrane potential. Rhodamine 123 (final concentration 10 μM) was added to 1 × 106 sperm/ ml cells. Mixed the solution well and incubated for 30 min at 37 °C in 5% CO2 incubator. Centrifuged the obtained solution at 760 × g for 5 min. The supernatant was removed and washed once with PBS. 1 ml of PBS was added to the cells and analyzed by flow cytometry [15]. 2.2.4.7. Acridine orange (AO) staining. Adjusted the sperm number to 1 × 106 sperm/ml. 5 μl of AO dye (dissolved in PBS containing 1% Tween-20, final concentration 6 μg/ml) was added to 95 μl of cell suspension. After 10 min in dark condition, the solution was centrifuged (300 x g, 5 min) and the supernatant was removed. Later, mixed the sperm with 1 ml PBS to avoid damage. The supernatant was removed again after centrifugation. Re-suspended the cells in 100 μl PBS, pipetted a drop on the slide, and cover with a lid. The slides were mounted and observed under a fluorescent microscope at 536 nm.

2.2.6. Statistical analysis Statistical product & service solutions (SPSS) 17.0 software was used to analyze the statistical data. The results were expressed as the mean ± standard deviation (mean ± SD). Significant differences were analyzed by one-way analysis of variance (ANOVA). Multiple comparisons of different groups were analyzed by Duncan’s test at the value of p < 0.05 as significant level.

2.2.5. Analysis of testis 2.2.5.1. Determination of lipid peroxidation in testis. The tissues were 3

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Table 1 The weight of organs and abdominal adipose tissue (% of body weight) in diabetic rats after fed with different concentration of ACEE. % of body weight

Control

DM

Metformin

1 X ACEE

2 X ACEE

4 X ACEE

Liver Kidney Heart Abdominal adipose Body weight

3.31 ± 0.34 0.70 ± 0.08 0.34 ± 0.04 1.93 ± 1.04 506.6 ± 81.2

3.96 ± 0.57 0.88 ± 0.26 0.35 ± 0.05 1.61 ± 0.69 456.5 ± 45.1

3.38 ± 0.44 0.78 ± 0.03 0.34 ± 0.03 1.81 ± 0.73 458.7 ± 45.1

3.70 ± 0.44 0.85 ± 0.21 0.32 ± 0.02 1.65 ± 0.64 477.1 ± 57.0

3.60 ± 0.48 0.80 ± 0.13 0.35 ± 0.05 1.81 ± 0.53 497.1 ± 64.4

3.34 ± 0.27 0.77 ± 0.85 0.33 ± 0.02 1.29 ± 0.80 492.7 ± 39.3

Data were shown as the mean ± S.D. (n = 10). DM: Diabetes; ACEE: Antrodia cinnamomea ethanol extract; 1X ACEE: 385 mg/kg BW; 2X ACEE: 770 mg/kg BW; 4X ACEE: 1540 mg/kg BW.

3. Results

hyperglycemia caused by diabetes.

3.1. Effect of ACEE on organ, abdominal fat, and body weights in male diabetic rats

3.2.3. Determination of plasma total cholesterol and triglyceride content The total cholesterol (Fig. 2a) and triglyceride (Fig. 2b) contents were observed in Fig. 2. The higher level of total cholesterol and triglyceride contents in the DM group was indicated the abnormal regulation of the lipolytic enzymes. As compared to metformin group, the 4X ACEE groups shown a better reduction in the level of total cholesterol and triglycerides. There was a gradual reduction in the level of total cholesterol was observed in ACEE treated groups. The triglyceride content was significantly reduced in the higher concentration of ACEE (4X ACEE).

After fed with different doses of ACEE for five weeks, the weight of each organ was measured. The results showed that the weights of liver, kidney, heart, and abdominal fat of experimental rats were not significantly different between the groups (Table 1). But the abdominal adipose tissue weight was slightly reduced in 4X ACEE groups. 3.2. Analysis of plasma biochemical parameters in diabetic rats 3.2.1. Effect of ACEE on glucose tolerance test (OGTT) and the area under the curve (AUC) To understand the glucose uptake, OGTT was performed. The results show that the blood glucose level in the DM group was significantly higher than the control group at 0, 30, 60, 90 and 120 min. The glucose uptake was significantly improved in both 4X ACEE and metformintreated groups. Glucose uptake in ACEE treated groups was proportional to their concentration (Fig. 1a). The area under the curve (AUC) of glucose uptake was shown in Fig. 1b. The AUC of diabetic rats were significantly higher and indicated the formation of the diabetic condition. The AUC of ACEE treated groups were decreased with an increase in the concentration. Metformin and 4X ACEE showed almost similar values in the case of AUC.

3.2.4. Determination of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, and creatinine level A large amount of ALT activity was observed in the DM group and a significant recovery was detected in the ACEE and metformin groups (Table 3). 2X and 4X dosages of ACEE groups showed a significant decrease in ALT activity and the level of ALT was decreased with increase in the concentration of ACEE. The AST activity was also increased in the DM group and a significant reduction was observed in 2X ACEE group. Urea is a waste, produced as the result of liver metabolism. In the DM group, the level of urea and creatinine were increased and a notable reduction was observed in the metformin and ACEE treated groups. The level of urea was almost similar in both ACEE and metformin treated groups. Highest reduction in the creatinine level was observed in the metformin group.

3.2.2. Effect of ACEE on plasma glucose level, insulin level, and homeostasis model assessment- Insulin resistance (HOMA-IR) in diabetic rats The plasma glucose, insulin, and HOMA-IR values were further determined in diabetic rats. It was found that the fasting blood glucose level of 4X ACEE group was significantly lower than the DM group. Like ACEE, metformin also improved glucose uptake. The DM group had higher insulin content and HOMA- IR value than the control group. The insulin level was decreased with increase in the concentration of ACEE. The metformin and 4X ACEE significantly reduced the insulin level and HOMA- IR value indicated the recovery from the diabetic condition (Table 2). So, it was clearly visible that the ACEE can regulate the fasting blood glucose level and can improve the symptoms of

3.2.5. Determination of lipid peroxidation in the diabetic rat after treated with ACEE The majority of diabetic patients have an abnormal reproductive function [18]. The degree of lipid peroxidation is correlated with diabetes and reproductive dysfunction. As shown in Fig. 3, lipid peroxidation was significantly higher in the DM group and it was moderately reduced after fed with 4 X dosage of ACEE.

Fig. 1. Effects of ACEE on (a) oral glucose tolerance test (OGTT) (after oral glucose load of 2 g/kg) and (b) area under the curve (AUC) of plasma concentration in diabetic rats after 5 weeks of treatment. Data were shown as the mean ± S.D. (n = 10). Differences were considered significant at p < 0.05 and denoted by letters (a–c) analyzed by Duncan’s multiple range test. DM: Diabetes; ACEE: Antrodia cinnamomea ethanol extract; 1X ACEE: 385 mg/kg BW; 2X ACEE: 770 mg/kg BW; 4X ACEE: 1540 mg/kg BW.

4

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Table 2 Plasma fasting blood glucose, insulin levels and homeostasis model assessment equation (HOMA-IR) in diabetic rats fed different concentration of ACEE after 5 weeks.

Glucose (mg/dL) Insulin (ng/mL) HOMA-IR

Control

DM

Metformin

1X ACEE

2X ACEE

4X ACEE

83.70 ± 21.14a 0.32 ± 0.05a 3.02 ± 2.10a

133.33 ± 18.85b 0.63 ± 0.06b 10.08 ± 3.84c

93.33 ± 20.95a 0.37 ± 0.06a 4.66 ± 2.05a

122.96 ± 33.52b 0.46 ± 0.18a 7.38 ± 2.22b

131.85 ± 8.38b 0.41 ± 0.07a 6.77 ± 1.87b

94.81 ± 24.20a 0.39 ± 0.10a 4.83 ± 1.83a

Data were shown as the mean ± S.D. (n = 10). Differences were considered significant at p < 0.05 and denoted by letters (a–c) analyzed by Duncan’s multiple range test. DM: Diabetes; ACEE: Antrodia cinnamomea ethanol extract; 1X ACEE: 385 mg/kg BW; 2X ACEE: 770 mg/kg BW; 4X ACEE: 1540 mg/kg BW. HOMAIR = Fasting plasma insulin concentration (mmol/L) × fasting plasma glucose concentration (ug/L) / 22.5. Fig. 2. Effects of ACEE on plasma (a) total cholesterol (b) triglyceride concentration in diabetic rats after 6 weeks of the experiment. Data were shown as the mean ± S.D. (n = 10). Differences were considered significant at p < 0.05 and denoted by letters (a–b) analyzed by Duncan’s multiple range tests. DM: Diabetes; ACEE: Antrodia cinnamomea ethanol extract; 1X ACEE: 385 mg/kg BW; 2X ACEE: 770 mg/kg BW; 4X ACEE: 1540 mg/kg BW.

testicular cells were also observed in DM group. Significant improvements were observed after treated with ACEE and metformin. The structure and cavities were restored in both groups. The recovery in ACEE groups was proportional to their concentration. 3.3.3. Effect of ACEE on sperm parameters in diabetic rats From Fig. 5, it was founded that the spermatozoa of the DM group had a lower sperm count and abnormal morphology (curled tail). An opposite result was detected in ACEE treated groups. It was observed that the number of sperm within the images were reduced in DM group and a dramatic increase was seen in ACEE treated groups. The total sperm count, motility, and sperm abnormality were further observed (Table 5). The results showed that the total sperm count and motility rate were significantly reduced in DM group and increased in both ACEE and metformin-treated groups. As compared to others, the sperm count was higher in the 4X ACEE group and the number was slightly increased with increase in the concentration. The number of abnormal sperm was significantly higher in the DM group and notable reductions were observed in ACEE groups. The abnormality was inversely proportional to ACEE concentration. Thus, it was confirmed that the ACEE improved sperm count, motility, and abnormalities under diabetic condition.

Fig. 3. Effects of ACEE on malondialdehyde level of plasma in diabetic rats after 5 weeks of treatment. Data were shown as the mean ± S.D. (n = 10). Differences were considered significant at p < 0.05 and denoted by letters (a–c) analyzed by Duncan’s multiple range test. DM: Diabetes; ACEE: Antrodia cinnamomea ethanol extract; 1X ACEE: 385 mg/kg BW; 2X ACEE: 770 mg/kg BW; 4X ACEE: 1540 mg/kg BW.

3.3. Effect of ACEE on reproductive parameters 3.3.1. Effect ACEE on the weight of testis, epididymis, and epididymal adipose tissue As shown in Table 4, the weights of the testis, epididymis, and epididymal adipose tissues were not significantly different between the groups. Since, the effect of ACEE on the reproductive system was further explored by analyzing the morphology, sperm parameters, and hormone level etc.

3.3.4. Determination of plasma FSH, and testosterone level in the diabetic rat after fed with ACEE As shown in Table 6, the level of LH was significantly decreased in the DM group and increased in both metformin and ACEE treated groups. As compared to metformin, the level of LH has increased in ACEE treated groups. There were no significant differences in FSH level between the groups. The testosterone level was decreased in the DM groups and increased in 4X ACEE treated group, which was significantly higher than the metformin group.

3.3.2. Histological evaluation of the testis after fed with ACEE The sections of testicular tissues were observed by H&E staining. From Fig. 4, it was observed that both Leydig cells and Sertoli cells in the DM group showed significant atrophy and a cavity in the lumen. The seminiferous tubules became loose and atrophic (black arrows). In DM groups, the interstitial space between the seminiferous tubules increased and the germinal cell layer thickness decreased. The loss of

3.3.5. Effect of ACEE on testosterone synthesis pathway The protein expressions of StAR, CYP11A1, and 17βHSD were shown in Fig. 6. The StAR protein expression (Fig. 7a) was reduced in the DM group. The expression level was higher in 2X ACEE treated group, which was much greater than the metformin group. The 5

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Fig. 4. The testis sections of experimental rats in each group were stained with hematoxylin and eosin (H&E) (Magnification 20x).

hyperglycemic and oxidative stress condition [20]. Therefore, GRP78 can be used as an indicator of ER stress. As shown in Fig. 8, the level of GRP 78 expression was increased in DM group indicated the development of ER stress under diabetic condition. The level of GRP 78 expression was decreased with increase in the concentration of ACEE. Metformin was also showed a significant reduction of GRP 78 expression.

CYP11A1 (Fig. 7b) and 17βHSD (Fig. 7c) protein expression were also decreased in DM groups. The expression of CYP11A1 in ACEE treated group was significantly higher than the metformin group. The level of 17βHSD protein expression was slightly increased with increase in the concentration of ACEE. 3.3.6. RAGE protein expression on the testicular tissue of diabetic rats after treated with ACEE The long-term hyperglycemic condition increases the production of advanced glycation end products (AGEs) and stimulates inflammatory reactions [19]. AGEs can cause oxidative stress by binding to RAGE on the cell membrane. The results show that the expression of RAGE was increased in the DM group and a significant reduction was observed in both ACEE and metformin groups. As compared to the metformin groups, the RAGE expressions were significantly decreased in ACEE treated groups. A notable reduction of RAGE expression was observed in both 2X and 4X ACEE treated groups (Fig. 7).

3.3.8. Determination of antioxidant enzyme activity and lipid peroxidation in diabetic rats after treated with ACEE The superoxide dismutase (SOD) (Fig. 9a) and catalase (Fig. 9b) activities were shown in Fig. 9. It was observed that the SOD and catalase activities were decreased in the DM groups. The metformin group did not show any improvements in SOD activity. In ACEE treated groups, the SOD activity was increased with increase in the concentration. The catalase activity was increased in both ACEE and metformin-treated groups. A significant increase was observed in both 2X and 4X ACEE treated groups. The effects of ACEE on lipid peroxidation of testicular tissues were shown in Fig. 10. The degree of lipid peroxidation was increased in the DM group and a moderate improvement was observed in both

3.3.7. Evaluation of endoplasmic reticulum (ER) stress on the testicular tissue of diabetic rats after fed with ACEE The endoplasmic reticulum (ER) activates GRP 78 under

Fig. 5. Effects of ACEE on the morphology of sperm in diabetic rats after 5 weeks of treatment (Magnification 40x). 6

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Fig. 6. Expression of (a) StAR, (b) CYP11A1, and (c) 17βHSD protein expression in testosterone synthesis pathway in testis of rats. Data were shown as the mean ± S.D. (n = 10). Differences were considered significant at p < 0.05 and denoted by letters (a–b) analyzed by Duncan’s multiple range test. DM: Diabetes; ACEE: Antrodia cinnamomea ethanol extract; 1X ACEE: 385 mg/kg BW; 2X ACEE: 770 mg/kg BW; 4X ACEE: 1540 mg/kg BW.

2.5

Fig. 7. Expression of RAGEs protein in testis of rats after 5 weeks of the treatment. Data were shown as the mean ± S.D. (n = 10). Data were shown as the mean ± S.D. (n = 10). Differences were considered significant at p < 0.05 and denoted by letters (a–b) analyzed by Duncan’s multiple range test. DM: Diabetes; ACEE: Antrodia cinnamomea ethanol extract; 1X ACEE: 385 mg/kg BW; 2X ACEE: 770 mg/kg BW; 4X ACEE: 1540 mg/kg BW.

Fig. 8. Expression of GRP78 protein in testis of rats after 5 weeks of the treatment. Data were shown as the mean ± S.D. (n = 10). Differences were considered significant at p < 0.05 and denoted by letters (a–b) analyzed by Duncan’s multiple range test. DM: Diabetes; ACEE: Antrodia cinnamomea ethanol extract; 1X ACEE: 385 mg/kg BW; 2X ACEE: 770 mg/kg BW; 4X ACEE: 1540 mg/kg BW.

metformin and ACEE treated groups. Metformin and 1X ACEE were showed almost similar rate. But a significant decrease in the degree of lipid peroxidation was observed only in 4X ACEE treated group.

3.3.9. Evaluation of oxidative stress in spermatozoa of diabetic rats after fed with ACEE The H2O2 production in the sperm was determined to understand the status of oxidative stress. Fig. 11 shows that the production of H2O2 was significantly higher in the DM group and a significant reduction 7

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Fig. 9. Effects of ACEE on (a) superoxide dismutase and (b) catalase activity of testis in diabetic rats after 5 weeks of the treatment. Data were shown as the mean ± S.D. (n = 10). Differences were considered significant at p < 0.05 and denoted by letters (a–b) analyzed by Duncan’s multiple range test. DM: Diabetes; ACEE: Antrodia cinnamomea ethanol extract; 1X ACEE: 385 mg/kg BW; 2X ACEE: 770 mg/kg BW; 4X ACEE: 1540 mg/kg BW.

Fig. 10. Effects of ACEE on malondialdehyde level of the testis in diabetic rats after 5 weeks of treatment. Data were shown as the mean ± S.D. (n = 10). Differences were considered significant at < 0.05 and denoted by letters (a–b) analyzed by Duncan’s multiple range test. DM: Diabetes; ACEE: Antrodia cinnamomea ethanol extract; 1X ACEE: 385 mg/kg BW; 2X ACEE: 770 mg/kg BW; 4X ACEE: 1540 mg/kg BW.

Fig. 12. The level of superoxide production of diabetic rat sperm after 5 weeks of treatment. Data were shown as the mean ± S.D. (n = 10). Differences were considered significant at p < 0.05 and denoted by letters (a–b) analyzed by Duncan’s multiple range test. DM: Diabetes; ACEE: Antrodia cinnamomea ethanol extract; 1X ACEE: 385 mg/kg BW; 2X ACEE: 770 mg/kg BW; 4X ACEE: 1540 mg/kg BW.

was observed in both metformin and ACEE treated groups. As compared to metformin, the H2O2 production was slightly decreased in ACEE treated groups. The superoxide production in each group was shown in Fig. 12. It was observed that the superoxide production was higher in the DM group and a significant reduction was observed after treated with ACEE. There was no any significant difference in superoxide production between the ACEE treated groups but the improvements were slightly higher than the metformin group.

In addition to this, the lipid peroxidation (Fig. 13a) and nitric oxide (NO) (Fig. 14b) productions in the spermatozoa were determined. In the DM group, the sperm lipid peroxidation was very heavy and serious. But after treated with metformin and ACEE, the degree of lipid peroxidation was significantly reduced and reached almost control group’ values. The NO production was higher in the DM group and the level was significantly decreased with increase in the concentration of ACEE.

Fig. 11. Effect of ACEE on the H2O2 production of sperm in rats fed different concentration after 5 weeks of treatment. Data were shown as the mean ± S.D. (n = 10). Differences were considered significant at p < 0.05 and denoted by letters (a–c) analyzed by Duncan’s multiple range test. DM: Diabetes; ACEE: Antrodia cinnamomea ethanol extract; 1X ACEE: 385 mg/kg BW; 2X ACEE: 770 mg/kg BW; 4X ACEE: 1540 mg/kg BW. 8

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Fig. 13. The level of (a) malondialdehyde and (b) NO level of diabetic rat sperm after 5 weeks of treatment. Data were shown as the mean ± S.D. (n = 10). Differences were considered significant at p < 0.05 and denoted by letters (a–b) analyzed by Duncan’s multiple range tests. DM: Diabetes; ACEE: Antrodia cinnamomea ethanol extract; 1X ACEE: 385 mg/kg BW; 2X ACEE: 770 mg/kg BW; 4X ACEE: 1540 mg/kg BW.

Fig. 14. Effect of ACEE on mitochondria membrane potential of sperm in rats fed different concentration after 5 weeks of treatment. Data were shown as the mean ± S.D. (n = 10). Differences were considered significant at p < 0.05 and denoted by letters (a–b) analyzed by Duncan’s multiple range tests. DM: Diabetes; ACEE: Antrodia cinnamomea ethanol extract; 1X ACEE: 385 mg/kg BW; 2X ACEE: 770 mg/kg BW; 4X ACEE: 1540 mg/kg BW.

diabetics. Metformin was selected as a positive control for the experiment. Metformin increases the binding of insulin to its receptor and facilitating insulin action [24]. Diabetes is a metabolic disorder associated with a defect in insulin secretion or action. Glucose absorption is carried out by insulin action. Under the diabetic condition, an increased level of glucose was observed due to the imbalance of insulin level [25]. Our results confirm this statement. The blood glucose level, insulin level, and HOMA- IR value of DM group were significantly higher than the control and ACEE treated groups (Table 2). The literature showed that the resistance to the normal signaling of both leptin and insulin is occured as the results of inflammation in hypothalamus caused by excess calories or saturated fats [26]. The amelioration of the hyperglycemic condition was observed in ACEE treated groups. Another study revealed that the dehydroeburicoic acid from AC prevented the dyslipidemic and diabetic condition by regulating glucose transporter type 4 (GLUT4), peroxisome proliferator-activated receptor alpha (PPARα), FAS, and phosphorylation of 5′ AMP-activated protein kinase (AMPK) [27]. An elevated level of cholesterol and triglycerides indicate an increased abnormality in lipid metabolism. The free radicals formed as the results of oxidative stress interacts with the lipoproteins and leads to lipid peroxidation [28]. Recent literature indicated that the accumulation of esterified cholesterol in macrophages is an indicator of the progression of atherosclerosis [29]. The level of triglycerides and cholesterols were increased in diabetic patients [30]. The conversion of fatty acids into triglycerides is increased in the liver during stress condition [26]. In this present study, we calculated the total cholesterol and triglyceride contents in plasma. The level of cholesterol and triglycerides were higher in DM groups and decreased significantly in the 4X ACEE group (Fig.2.). Previous studies reported that the antroquinonol from AC lowered the level of low-density lipoprotein (LDL) cholesterol by increasing the expression of LDL receptor genes [31]. The hypolipidemic effect of water extract of AC has also reported by Lai

2X and 4X groups showed a better reduction in the production of NO in sperm. 3.3.10. Mitochondrial membrane potential of spermatozoa The flow cytometry results show that the fluorescence intensity of Rhodamine 123 in the DM group was significantly lower than other groups. Both metformin and ACEE treated groups shown an increase in the fluorescent intensity indicated the increased mitochondrial membrane potential in the sperm. The highest activity was observed in 1X ACEE group and which was slightly higher than the metformin group (Fig. 14). 3.3.11. Effect of ACEE on DNA damage of spermatozoa The extent of DNA damage in sperm was observed by AO staining. In DM groups, the orange color in the sperm head denoted the DNA damage. The intensity of orange was significantly reduced after treated with metformin and ACEE. From the results, it could be understood that the ACEE can reduce the DNA damage in the sperm (Fig. 15). 4. Discussion Diabetes mellitus (DM) is a chronic metabolic disease leads to secondary complications such as atherosclerosis, neuropathy, and cardiovascular disease etc. The biochemical changes that happen during the diabetic condition results in an impairment of spermatogenesis process [21]. Decreased sperm motility, sperm count, semen volume, disruption of the seminiferous tubule, histological damage of epididymis and testis, and low hormone level (FSH, LH) were observed under diabetic condition [22]. Antrodia cinnamomea is a medicinal mushroom endemic to Taiwan have various activities such as anticancer, anti-hypertension, anti-oxidation, anti-inflammation, neuroprotection, immunomodulation, and hepatoprotection [23]. The present study was focused on the ameliorative effect of ACEE on reproductive dysfunction arises due to 9

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Fig. 15. Effect of ACEE on DNA of sperm in rats fed different concentration after 5 weeks of treatment (Magnification 40x).

understood that the ACEE can reduce the degree of lipid peroxidation at higher doses. Another study revealed that the polyphenols and triterpenoids from Antrodia cinnamomea play an important role in the reduction of lipid peroxidation [40]. Sexual dysfunctions such as infertility, retrograde ejaculation, and impotence are known as the consequences of diabetes mellitus in men [41]. The overall process of spermatogenesis is regulated by the HPG axis. Studies show that during diabetes, oxidative stress affects the process of spermatogenesis, damage testicular DNA, and impaired reproductive cells [42]. During diabetic condition, lower testicular weight, abnormal spermatogenesis, decreased sperm number and motility, and low testosterone level was observed. The testis is known as the male reproductive organ and epididymis is a duct seen behind the testis. From our results, the weights of the testis, epididymis, and epididymal adipose weight were not significantly different between the groups. So, further analysis such as testicular tissue morphology, sperm parameters, and hormone levels etc. were determined. The testis is composed of tightly coiled tubules called seminiferous tubules. Sertoli cells are seen in the walls of the seminiferous tubule and provide nourishment to the immature sperm. Leydig cells are found adjacent to the seminiferous tubule and produce testosterone in presence of LH. The H&E staining illustrated that significant atrophy observed in the DM group. The size of the seminiferous tubule was reduced and the interstitial space between the tubules was larger in the DM group. Previous studies reported that the STZ- induced diabetic condition caused seminiferous tubule atrophy with reduction of Sertoli and spermatogenic cells [43,44]. The ACEE and metformin-treated group show better improvements in the structure (Fig.4.). The abnormal function of Sertoli cells and Leydig cells may affect the sperm count and fertility [45]. The number of sperms and motility decreased and abnormal sperms count increased in DM group. ACEE groups improved the condition by reducing the abnormal sperm count and

et al., 2012 [32]. The liver maintains the normal glucose fasting level and is known as a site for insulin clearance. An abnormal hepatocellular function is associated with obesity, insulin resistance, and type 2 diabetes [33]. The aminotransferases such as alanine aminotransferases (ALT) and aspartate aminotransferases (AST) were mostly found in the liver and also seen in serum and other organs. Increase in the level of ALT is known as a better marker for fat accumulation and type 2 diabetes [34]. When the body tissues are damaged (especially liver), ALT and AST were released into the bloodstream [35]. Creatinine is identified as the breakdown product of creatine phosphate in muscles and excreted by the kidneys. The higher level of urea and creatinine are the sign of renal dysfunction [36]. Our results show that the level of ALT, AST, creatinine, and urea were increased in DM groups and decreased upon treated with different doses of ACEE (Table 3). The production of peroxides and free radicals leads to cell death by damaging the components of the cell such as proteins, DNA, and lipids etc. Oxidative stress is a term indicating the increased production of oxidizing species and decreases the effectiveness of antioxidants [37]. Superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) are the major antioxidant enzymes that reduce the level of ROS in the blood. SOD neutralize the superoxide radicals in the blood [38]. From our investigation, it was revealed that in the DM group, the level of SOD and catalase were decreased and a significant increase was observed in ACEE treated groups (Fig.9). Oxidation of lipids in cell membranes and plasma lipoproteins are associated with the development of vascular disease in diabetes. The cytotoxic effect of ROS on membrane phospholipids resulting in the formation of malondialdehyde (MDA) [39]. The MDA level was determined in plasma, testis, and sperm. The current study shows that the MDA level decreased in the ACEE and metformin-treated groups. But there were no improvements observed in the DM group. It could be

Table 3 Effects of ACEE on plasma ALT, AST, urea, and creatinine in diabetic rats after 5 weeks of treatment. Control ALT (U/L) AST (U/L) Urea(mg/dL) Creatinine (mg/dL)

DM a

26.19 ± 6.21 35.50 ± 5.61 28.26 ± 2.67a 0.25 ± 0.08a

Metformin b

55.87 ± 7.40 37.83 ± 7.04 43.33 ± 9.28b 0.5. ± 0.01b

1X ACEE a

31.43 ± 9.13 30.18 ± 5.40 28.10 ± 4.58a 0.16 ± 0.02a

2X ACEE ab

36.32 ± 8.94 34.22 ± 5.32 27.78 ± 9.58a 0.31 ± 0.04ab

4X ACEE a

30.68 ± 7.11 27.44 ± 3.30 28.33 ± 3.97a 0.37 ± 0.17ab

29.39 ± 4.87a 35.21 ± 5.34 26.94 ± 4.88a 0.31 ± 0.21a

Data were shown as the mean ± S.D. (n = 10). Differences were considered significant at p < 0.05 and denoted by letters (a–b) analyzed by Duncan’s multiple range test. DM: Diabetes; AST: aspartate transaminase; ALT: alanine transaminase; ACEE: Antrodia cinnamomea ethanol extract; 1X ACEE: 385 mg/kg BW; 2X ACEE: 770 mg/kg BW; 4X ACEE: 1540 mg/kg BW. 10

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Table 4 The weight of testis, epididymis, and epididymal adipose after fed with different concentration of ACEE. % of body weight

Control

DM

Metformin

1X ACEE

2X ACEE

4X ACEE

Testis Epididymis Epididymal adipose Body weight

0.72 ± 0.07 0.28 ± 0.03 1.64 ± 0.52 506.6 ± 81.2

0.80 ± 0.10 0.31 ± 0.03 1.64 ± 0.48 456.5 ± 45.1

0.76 ± 0.08 0.30 ± 0.29 1.66 ± 0.36 458.7 ± 45.1

0.74 ± 0.09 0.27 ± 0.02 1.69 ± 0.52 477.1 ± 57.0

0.74 ± 0.14 0.28 ± 0.03 1.51 ± 0.35 497.1 ± 64.4

0.75 ± 0.06 0.28 ± 0.01 1.52 ± 2.13 492.7 ± 39.3

Data were shown as the mean ± S.D. (n = 10). DM: diabetes; ACEE: Antrodia cinnamomea ethanol extract; 1X ACEE: 385 mg/kg BW; 2X ACEE: 770 mg/kg BW; 4X ACEE: 1540 mg/kg BW.

among others [53]. The oxidative stress damages the mitochondria and leads to a decrease in mitochondrial membrane potential (MMP). It resulted in reduced sperm survival and swimming ability [54]. The flow cytometry analysis revealed that the MMP increased in ACEE treated groups (Fig.14). So, it could be understood that the oxidative stress in mitochondria improved. Literature indicated that the intact sperm emits green fluorescence while DNA damaged sperm emits red fluorescence after AO staining [55]. Orange to red fluorescence of sperm DNA after treated with STZ was also mentioned in previously reported work [56]. From Fig. 15, it was understood that the ACEE reduced the DNA damage in sperm and thereby protected the sperm from stress condition.

increasing the motility (Table 5). The role of hormones such as follicle stimulating hormone (FSH) and luteinizing hormone (LH) is to regulate the process of spermatogenesis and control the Leydig cells function respectively. Previous studies revealed that the low serum level of FSH and LH was observed under diabetic condition [46]. Testosterone is a steroid hormone plays a crucial role in maintaining male fertility. Our studies revealed that the reduction in the level of LH, FSH, and testosterone in the DM group indicating the male reproductive dysfunction. But, after treated with ACEE, the level increased significantly in all ACEE groups, especially in 4X dosage (Table 6). The biosynthesis of testosterone started with the conversion of pregnenolone by CYP11 A. 3β-hydroxysteroid dehydrogenase (3β-HSD) convert the pregnenolone into progesterone [47]. StAR is another gene plays an important role in steroid synthesis by enhancing the conversion of pregnenolone into progesterone. The current investigation showed that the protein expression of StAR, CYP11A1, and 17βHSD decreased in the DM group and a significant increase in the expression was observed in ACEE treated groups (Fig.6). Advanced glycation end products (AGEs) are formed by the nonenzymatic reaction between reducing sugars and amino groups of proteins, lipids, and nucleic acids. Accumulation of AGEs induces the diabetic condition by binding to the receptor for the advanced glycation end product (RAGE) [48]. Previous studies indicated that AGEs can accelerate hyperglycemia and accumulation of AGEs enhanced in diabetic condition [49]. Our results show that the different doses of ACEE reduced the expression of RAGE and moderated the impact of diabetes (Fig.7). Endoplasmic reticulum (ER) is a large membrane-enclosed cellular compartment. The protein folding in the lumen of ER was facilitated by a number of compounds including GRP 78. It is known as the main modulator of the unfolded protein response [50]. The GRP78 expression was increased in DM group and a significant reduction was observed in ACEE treated groups (Fig. 8). The occurrence of β- cell dysfunction, insulin resistance, impaired glucose tolerance, and type 2 diabetes are connected with oxidative stress [51]. Some of the reactive oxygen species such as superoxide anion, nitric oxide (NO), and hydrogen peroxide (H2O2) etc. are responsible for the development of oxidative stress [52]. Our results show that the H2O2, superoxide, and NO production increased in DM group and reduced in ACEE and metformin-treated groups. A gradual reduction in the degree of lipid peroxidation was observed in the ACEE group. Previous study examined the antioxidant activity of n-hexane, ethyl acetate, and n-Butanol extracts of AC and they concluded that ethyl acetate extract of the AC showed better antioxidant activity

5. Conclusion Antrodia cinnamomea is a unique fungus in Taiwan and is well known for its pharmacological activities. Different doses of ACEE were tested for 5 weeks in diabetic rats. After treated with ACEE, the hyperglycemia, insulin resistance, and hyperlipidemia were improved. The ACEE reduced the degree of lipid peroxidation, improved sperm parameters, and maintained the structural integrity of testis. Amelioration of oxidative stress in ER and mitochondria were observed in ACEE treated groups. In addition to this, ACEE restored reproductive dysfunction by increasing the level of LH, FSH, and testosterone. So, it was understood that the ACEE ameliorated the reproductive dysfunction in STZ- induced male diabetic rats. Funding This work was financially supported by the Center of Excellence for the Oceans, National Taiwan Ocean University from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan and MOST 106-2320-B-019-006. Conflict of interest The authors declared no conflict of interest. Acknowledgments This work was financially supported by the Center of Excellence for the Oceans, National Taiwan Ocean University from The Featured Areas Research Center Program within the framework of the Higher

Table 5 Effect of ACEE on sperm parameters in rats fed different concentration after 5 weeks of treatment. Control 6

Counts (10 ) Mobility (%) Abnormality (%)

DM b

20.32 ± 7.05 23.22 ± 3.49b 2.89 ± 1.05a

Metformin a

13.4 ± 6.86 10.26 ± 2.87a 6.71 ± 2.77b

1X ACEE b

17.09 ± 5.38 23.76 ± 5.92b 2.58 ± 0.96a

2X ACEE b

18.78 ± 6.21 19.66 ± 7.18b 3.14 ± 1.82a

4X ACEE b

19.52 ± 6.02 22.51 ± 7.98b 2.99 ± 1.57a

20.47 ± 7.97b 21.17 ± 1.35b 2.62 ± 0.66a

Data were shown as the mean ± S.D. (n = 10). Differences were considered significant at p < 0.05 and denoted by letters (a–b) analyzed by Duncan’s multiple range test. DM: diabetes; ACEE: Antrodia cinnamomea ethanol extract; 1X ACEE: 385 mg/kg BW; 2X ACEE: 770 mg/kg BW; 4X ACEE: 1540 mg/kg BW. 11

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Table 6 Effects of ACEE on plasma hormone value in diabetic rats after 5 weeks of treatment. (ng/mL) LH FSH Testosterone

C

DM b

4.34 ± 1.10 0.84 ± 0.04a 8.14 ± 3.36c

Metformin a

2.72 ± 1.07 0.88 ± 0.05a 1.33 ± 1.07a

1X ACEE b

4.10 ± 1.03 0.88 ± 0.04a 3.51 ± 1.03b

2X ACEE b

4.32 ± 1.44 0.88 ± 0.03a 3.58 ± 1.70b

4X ACEE b

4.02 ± 1.23 0.87 ± 0.02a 3.17 ± 0.32b

4.60 ± 1.08b 0.88 ± 0.04a 6.08 ± 3.36c

Data were shown as the mean ± S.D. (n = 10). Differences were considered significant at p < 0.05 and denoted by letters (a–c) analyzed by Duncan’s multiple range test. DM: diabetes; ACEE: Antrodia cinnamomea ethanol extract; 1X ACEE: 385 mg/kg BW; 2X ACEE: 770 mg/kg BW; 4X ACEE: 1540 mg/kg BW.

Education Sprout Project by the Ministry of Education (MOE) in Taiwan and MOST 106-2320-B-019-006. [21]

Appendix A. Supplementary data [22]

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2019.108684. References

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