IJMS Vol 39, No 2, March 2014

Original Article

Mulberry Leaf Extract Attenuates Oxidative Stress-Mediated Testosterone Depletion in Streptozotocin-Induced Diabetic Rats Mohammad Reza Hajizadeh1, PhD; Ebrahim Eftekhar 2, PhD; Fatemeh Zal3, PhD; Aida Jafarian4, BSc; Zohreh Mostafavi-Pour4,5, PhD

1 Department of Biochemistry, Rafsanjan University of Medical Sciences, Rafsanjan, Iran; 2 Molecular Medicine Research Center, Department of Biochemistry, Hormozgan University of Medical Sciences, Bandar Abbas, Iran; 3 Department of Reproductive Biology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran; 4 Recombinant Protein Laboratory, Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran; 5 Maternal-Fetal Medicine Research Center, Hafez Hospital, Shiraz University of Medical Sciences, Shiraz, Iran


Background: It has been proposed that oxidative stress may contribute to the development of testicular abnormalities in diabetes. Morus alba leaf extract (MAE) has hypoglycemic and antioxidant properties. We, therefore, explored the impact of the administration of MAE on steroidogenesis in diabetic rats. Methods: To address this hypothesis, we measured the serum level of glucose, insulin, and free testosterone (Ts) as well as oxidative stress parameters (including glutathione peroxidase, glutathione reductase, total antioxidant capacity, and malondialdehyde) in the testis of control, untreated and MAE-treated (1 g/day/kg) diabetic rats. In order to determine the likely mechanism of MAE action on Ts levels, we analyzed the quantitative mRNA expression level of the two key steroidogenic proteins, namely steroid acute regulatory protein (StAR) and P450 cholesterol side-chain cleavage enzyme (P450scc), by real-time PCR. Results: The MAE-treated diabetic rats had significantly decreased glucose levels and on the other hand increased insulin and free Ts levels than the untreated diabetic rats. In addition, the administration of MAE to the diabetic rats restored the oxidative stress parameters toward control. Induction of diabetes decreased testicular StAR mRNA expression by 66% and MAE treatment enhanced mRNA expression to the same level of the control group. However, the expression of P540scc was not significantly decreased in the diabetic group as compared to the control group. Conclusion: Our findings indicated that MAE significantly increased Ts production in the diabetic rats, probably through the induction of StAR mRNA expression levels. Administration of MAE to experimental models of diabetes can effectively attenuate oxidative stress-mediated testosterone depletion. Please cite this article as: Hajizadeh MR, Eftekhar E, Zal F, Jaffarian A, Mostafavi-Pour Z. Mulberry Leaf Extract Attenuates Oxidative Stress-Mediated Testosterone Depletion in Streptozotocin-Induced Diabetic Rats. Iran J Med Sci. 2014;39(2):123-129.

Keywords ● Diabetes mellitus ● Morus alba ● Oxidative stress ● Testosterone

Correspondence: Zohreh Mostafavi-Pour, PhD; Recombinant Protein Laboratory, Department of Biochemistry, School of Medicine, Zand Street, Shiraz, Iran P. O. Box: 71345-1167 Tel/Fax: +98 711 2303029 Email: [email protected] Received: 31 July 2013 Revised: 19 October 2013 Accepted: 24 November 2013

Diabetes mellitus (DM) is one of the most common chronic diseases worldwide. It has been estimated that the prevalence of DM will increase from 275 million adults in 2010 to 439 million by 2030.1 It has been posited that oxidative stress plays a pivotal role

Iran J Med Sci March 2014; Vol 39 No 2



Hajizadeh MR, Eftekhar E, Zal F, Jaffarian A, Mostafavi-Pour Z

in the pathogeneses of DM.2 Accumulated data suggest that DM is linked with male reproductive dysfunction.3 In rats, diabetes induces apoptosis in the testicular germ cell4 and decreases sperm count and plasma testosterone (Ts) levels.5 However, the potential contribution of oxidative stress due to diabetic condition in the development of testicular abnormalities has not been fully clarified. It has been shown that reactive oxygen species (ROS) can inhibit the steroid hormone production of cultured leydig cells by directly affecting the steroidogenic enzymes.6 Be that as it may, the testicular activity of steroidogenic proteins under diabetic condition has yet to be studied. Steroid acute regulatory protein (StAR) and P450 cholesterol side-chain cleavage enzyme (P450scc) are two important proteins that catalyze the first steps in steroidogenesis.7 Diemer et al.8 demonstrated that the in vitro exposure of MA-10 tumor leydig cells to ROS decreased StAR protein expression levels. Morus alba (mulberry) is a plant rich in phytochemicals, which have an important role in diet-based therapies to cure several diseases.9 Antioxidant and antidiabetic activity of Morus alba leaf extract (MAE) in Streptozotocin-induced diabetic rats has been shown.10,11 The objective of this study was to investigate the effect of the long-term administration of MAE on oxidative stress markers and steroidogenesis in diabetic rats. The likely mechanism of MAE action on steroidogenesis was also explored. Materials and Methods Chemicals Streptozotocin, thiobarbituric acid (TBA), 1,1,3,3 -tetramethoxypropane, reduced glutathione (GSH), oxidized glutathione (GSSG), nicotine-amid-adenine-dinucleotide phosphate (NADPH), glutathione reductase (GR), tripyridyls-triazine (TPTZ), and other high-grade chemicals were purchased from Sigma Chemical Company (St. Louis, Missouri, USA). Preparation of Morus Alba Leaf Extract Leaves of Morus alba were collected from a local area (Shiraz, Iran) in April. Having been authenticated by the Botany Department of Shiraz University, dried leaves were extracted with 50% of ethanol. The mixture was filtered, concentrated in a vacuum evaporator, and then lyophilized. Using this procedure, the yield was 22% of the starting dry weight of the leaves. The final extract was kept in an air-tight container at -80°C until it was used. Induction of Diabetes Diabetes was induced 124 




intraperitoneal injection of 50 mg/kg body weight Streptozotocin (STZ) dissolved in 0.1 M citrate buffer (pH 4.5). Seven days after STZ injection, the blood was collected from the tail vein to determine the fasting blood glucose level. Rats with blood glucose >300 mg/dl were considered diabetic. The experiment was carried out 5 days after the confirmation of the existence of diabetes mellitus. Experimental Design Thirty healthy adult male Wistar rats (8 weeks old), weighting about 250±10 g, were obtained from the Animal House Unit of Shiraz University of Medical Sciences, Shiraz, Iran. They were kept under controlled conditions with a regular light cycle (12; 12 h light; dark cycle). During the experiment, the rats had free access to food and water. All the animal experiments were approved by the Ethics Committee of Shiraz University of Medical Sciences. Using a random number table, the rats were divided into three groups with 10 rats in each group and were treated through a gavage tube for a period of 2 months as follows: 1) non-diabetic rats with distilled water (control); 2) diabetic rats with distilled water; and 3) diabetic rats with MAE extract at the dose of 1 g/kg per day. At the end of the 8th week, all the rats were euthanized by an overdose of anesthetic drug. Blood samples were collected into a tube and serum was separated for the determination of glucose, insulin, and free Ts. The testes were separated and washed with ice cold 0.9% NaCl, blotted dry, decapsulated, and homogenized at 4°C in 50 mM phosphate buffer saline (pH 7.4). Homogenate was centrifuged at 10,000×g for 10 min at 4°C, and the supernatant was used to measure GR and glutathione peroxidase (GPx) activities and malondialdehyde (MDA) levels as well as total antioxidant capacity (TAC). The total protein content of the supernatant was determined using the Bradford method.12 Measurement of Serum Glucose, Insulin, and Free Ts Serum glucose level was measured using a commercial kit (Pars Azmoon Company, Tehran, Iran) based on the hexokinase method. Serum insulin and free Ts concentrations were measured using the enzyme linked immunosorbent assay (ELISA) kit (DRG instruments, GmbH, Marburg, Germany). Biochemical Analysis of Testicular Antioxidant Status MDA, an indirect index of lipid peroxidation, was assayed as TBARS (thiobarbituric reactive substance) using a colorimetric method.13 TBARS concentration was calculated using 1,1,3,3-tetramethoxypropane as a standard. The results are expressed as nanomoles per mg protein. Iran J Med Sci March 2014; Vol 39 No 2

Mulberry leaf extract attenuates testosterone depletion in diabetic rats

GPx was measured as described by a previous study.14 Briefly, the enzymatic reaction in the cuvette that contained NADPH, GSH, NaN3, and GR was initiated by adding H2O2 and the change in absorbance was monitored spectrophotometrically at 340 nm.14 GR was assayed using the method described by Carlberg and Mannervik.15 The GR assay was performed in a tube that contained Tris–HCl buffer, EDTA, GSSG, NADPH, and a sample in a final volume of 1.0 mL. The decrease in the absorbance of NADPH at 340 nm was monitored using a spectrophotometer.15 The results are expressed as units of enzyme activities per mg of protein. TAC was determined by the ferric-reducing antioxidant power assay (FRAP assay).16 RNA Extraction and Real-Time RT-PCR Analysis Total RNA was extracted from the testis using Tripure RNA Isolation Reagent according to the manufacturer’s instructions (Roche, Germany) and was quantified by spectrophotometry. The integrity of the extracted total RNA was checked by agarose gel electrophoresis and verified by the presence of 28S, 18S rRNA bands. Total RNA was reverse transcribed into the first strand of complementary DNA (cDNA) with Revert AidTM First Strand cDNA Synthesis Kit (Fermentas, EU). Real-time RT-PCR assays for the quantitative determination of StAR, P450scc, and beta actin (internal control) were performed in duplicate using the ABI system (Applied ABI Company, Foster City, CA USA). Primer sequences which were used for the beta actin, StAR, and P450scc amplification are shown in table 1. Amplifications were performed in 25-μl mixtures containing 1/20 volume of cDNA preparation (2 μl) and 1X SYBR Green PCR Master Mix (PE Applied Biosystems, CA, USA) according to the manufacturer’s instructions. Additionally, cDNA samples were amplified with pre-cycling heat activation at 95°C for 10 min, followed by 40 cycles (15 s Table 1: Sequence of the primers used for real-time PCR Genes Forward Actin CCACACCCGCCACCAGTTCG P450scc AAACACCACGCACTTCCG StAR CTGCTAGACCAGCCCATGGAC

at 95°C, 30 s at 58°C, and 30s at 60°C). The concentration of beta actin was determined in each sample, and the relative quantification of mRNA in each sample was conducted using the comparative Ct (threshold cycle) method. The results are depicted as mRNA copies/beta actin units, allowing a direct comparison of the expression levels between the different mRNA species. The quality and correct size of the PCR products were checked by electrophoresis on 1% agarose gel. Statistical Analysis The data are expressed as mean±SEM. Differences in the variables between the three groups were determined by the analysis of variance (ANOVA), followed by the Student Newman-Kelus test, to show specific group differences. The level of significance was taken as P

Mulberry leaf extract attenuates oxidative stress-mediated testosterone depletion in streptozotocin-induced diabetic rats.

It has been proposed that oxidative stress may contribute to the development of testicular abnormalities in diabetes. Morus alba leaf extract (MAE) ha...
286KB Sizes 0 Downloads 0 Views