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Effects of Apocynin on Oxidative Stress and Expression of Apoptosis-Related Genes in Testes of Diabetic Rats

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Effects of Apocynin on Oxidative Stress and Expression of Apoptosis-Related Genes in Testes of Diabetic Rats MOLECULAR MEDICINE REPORTS 7: 47-52, 2013 Effects of apocynin on oxidative stress and expression of apoptosis-related genes in testes of diabetic rats MINGCHAO LI, ZHUO LIU, LI ZHUAN, TAO WANG, SHUIMING GUO, SHAOGANG WANG, JIHONG LIU and ZHANGQUN YE Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, P.R. China Received March 15, 2012; Accepted July 13, 2012 DOI: 10.3892/mmr.2012.1132 Abstract. Reactive oxygen species (ROS) are important in the associated with male reproductive dysfunction. Compared development of diabetic testicular dysfunction. Overproduction with non-diabetic individuals, male diabetic patients showed of ROS promotes the process of apoptosis, which shows that an increased incidence of hypogonadism and infertility (2). there is a crosstalk between oxidative stress and apoptosis. Recent Oxidative stress damage is regarded as the most influential harm- research has suggested that NADPH oxidase is the main source of causing factor affecting testicular function (2-4). The increase ROS. In this study, we investigated whether the NADPH oxidase in reactive oxygen species (ROS) causes non-specific changes inhibitor, apocynin, can improve diabetes-induced testicular in nucleic acid, protein and phospholipid levels, resulting in dysfunction by suppressing oxidative stress. The streptozocin DNA, RNA and protein damage and alterations in antioxidant (STZ)-induced diabetic rats were administered apocynin, and the enzyme levels, which lead to cellular and tissue damage (1). mRNA and protein expression of Bax, Bcl-2, p47phox and p67phox Diabetes inhibits reproductive activity in experimental animals; was examined by real-time PCR (RT-PCR) and western blot for instance, the testicular function of diabetic rats is impaired, analysis. Production of ROS was measured by thiobarbituric acid including reduced testicular weight, sperm count and sperm reactive substances (TBARS) assay. Terminal-deoxynucleoitidyl motility (5). Antioxidant vitamin E is capable of improving transferase mediated nick end-labeling (TUNEL) assay was diabetes-induced free radical damage in testicular tissue (1). used to detect apoptosis and ELISA was used to detect total A previous study showed that the apoptosis of testicular testosterone levels. The mRNA and protein expression of Bcl-2 germ cells increases in diabetic mice, leading to the disrup- was significantly reduced, and that of Bax, p47phox and p67phox tion of spermatogenesis (6). Apoptosis occurs in the testes was significantly increased in the diabetic rats compared to the of diabetic animals, but the mechanism of apoptosis has not control group. Apocynin significantly increased the expression yet been clarified (7). Apoptotic cell death is mediated by the of Bcl-2 and decreased the expression of Bax, p47phox and p67phox activation of apoptotic signaling pathways, including the Bcl-2 at both the mRNA and protein levels. The production of ROS family proteins. Bcl-2 and Bcl-xL suppress apoptotic cell death and apoptotic cells significantly increased in the diabetic group through anti-apoptotic function, whereas Bax and Bad promote compared to the control group. Apocynin significantly reduced apoptotic death through pro-apoptotic function (7). the production of ROS and apoptotic cells and increased the total The overproduction of ROS promotes the process of testosterone level. In conclusion, apocynin is capable of amelio- apoptosis by increasing caspase-3 activity and inhibiting Bcl-2 rating testicular dysfunction. expression, which demonstrates that a crosstalk exists between oxidative stress and apoptosis (2). Introduction Previous studies have indicated that NADPH oxidase is the main source of ROS, and that activated NADPH oxidase Diabetes mellitus (DM) is the most common metabolic results in increased ROS production (8-10). ROS derived from disease (1). There is increasing evidence that diabetes is closely NADPH oxidase play a potent role in initiating and acceler- ating the development of diabetic complications (11). NADPH oxidase is composed of the regulatory subunits, p67phox (phox, phagocyte oxidase), p47phox, p22phox and p40phox, and the cata- lytic subunit, gp91phox. The active oxidase generates superoxide Correspondence to: Dr Tao Wang or Dr Jihong Liu, Department by transferring the electron from NADPH to oxygen (12). of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, Apocynin has been previously shown to effectively P. R. Ch i na inhibit the increased NADPH oxidase activity in diabetic E-mail: [email protected] aortas and to restore changes in nitric oxide synthase (NOS) E-mail: [email protected] expression, thereby blocking the vicious cycle which results in diabetes-associated endothelial dysfunction (13). A recent Key words: reactive oxygen species, apocynin, testicular dysfunction study also showed that elevated activity of NADPH oxidase led to significant testicular damage, and that the antioxidant, 48 LI et al: EFFECTS OF APOCYNIN ON OXIDATIVE STRESS strontium fructose 1,6-diphosphate (FDP-Sr), restored the Table Ⅰ. Primer sequences for real-time quantitative polymerase testicular function (14). chain reaction. The aim of this study was to examine whether testicular dysfunction and apoptosis under diabetic conditions can be Gene Primers ameliorated by the NADPH oxidase inhibitor, apocynin, by phox suppressing oxidative stress. p47 F: 5'-GAGACATACCTGACGGCCAAAGA-3' p47phox R: 5'-AGTCAGCGATGGCCCGATAG-3' Materials and methods p67phox F: 5'-GAAAGCATGAAGGATGCCTGG-3' p67phox R: 5'-ATAGCACCAAGATCACATCTCC-3' Experimental animals. Tongji Hospital, Tongji Medical Bax F: 5'-GTTACAGGGTTTCATCCAGG-3' College, Huazhong University of Science (Wuhan, China) and Bax R: 5'-CGTGTCCACGTCAGCAAT-3' the Technology Animal Care and Use Committee approved all Bcl-2 F: 5'-CGGGAGAACAGGGTATGA-3' procedures undertaken in the current study. Seven-week-old Bcl-2 R: 5'-CAGGCTGGAAGGAGAAGAT-3' male Sprague-Dawley (SD) rats were obtained from Tongji β-actin F: 5'-AAGAGCTATGAGCTGCCTGA-3' Medical College, Huazhong University of Science and β-actin R: 5'-TACGGATGTCAACGTCACAC-3' Technology. Diabetes in rats was induced by a single intraperi- toneal injection of streptozocin (STZ) at a dose of 60 mg/kg in F, forward; R, reverse. citrate buffer (50 mM sodium citrate, pH 4.5), blood glucose levels in serum samples obtained from the tail vein of all rats were detected prior to diabetes induction and 72 h after intraperitoneal injection of STZ with a blood glucose meter (Johnson and Johnson, New Brunswick, NJ, USA). The rats β-actin was measured. Relative quantification of the expres- with blood glucose concentrations >11.1 mmol/l were accepted sion levels of each transcript for each group was calculated as being diabetic. using the 2-ΔΔCt method. Using the non-diabetic control as the Eight weeks after the induction of diabetes, diabetic rats calibrator, the data of other groups were presented as the fold were randomly divided into 2 groups: the untreated group change in gene expression normalized to β-actin and relative and the apocynin-treatment group (16 mg/kg). The treatment to the non-diabetic control. The sequences of the PCR primers period was 4 weeks. Age-matched male SD rats were used as used are shown in Table I. the control group. At the end of the treatment period, the blood collected Measurement of testicular protein expression. Testicular via cardiac puncture was allowed to clot and the serum was tissues were homogenized in lysis buffer on ice. Protein was obtained by centrifugation at 1,500 x g for 15 min. Serum loaded on SDS-PAGE gel. The resolved proteins were trans- samples were stored at -80˚C until analysis. The testes were ferred onto 0.2-µm nitrocellulose membranes, and blots were harvested after rats were sacrificed with an intraperitoneal blocked in 5% non-fat dried milk for 1 h at 37˚C to saturate overdose of pentobarbital. non-specific protein binding. The membranes were incubated overnight at a 1:500 dilution of polyclonal primary antibody Measurement of serum testosterone level. The serum testos- (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The terone level was detected using a rat testosterone ELISA kit membranes were then incubated with appropriate alkaline (R&D Systems, Inc., Minneapolis, MN, USA) according to the phosphatase-linked secondary antibodies (Proteintech Group, manufacturer's instructions. Inc., Chicago, IL, USA) for 1 h at 37˚C and visualized using the BCIP/NBT Chromogen system (Covance Inc., Princeton, NJ, Measurement of testicular mRNA expression. Total RNA USA). β-actin was used as the internal control to standardize was isolated from testicular tissue using TRIzol reagent each sample with equal protein quantity. Protein expression (Invitrogen, Carlsbad, CA, USA) according to the manufac- was quantified by computer-assisted densitometry with the turer's instructions. cDNA was synthesized from 500 ng of use of the Gel Pro version 4.0 software (Media Cybernetics, RNA using the PrimeScriptTM RT reagent kit (Takara, Dalian, Georgia, MD, USA). China) according to the manufacturer's instructions. Real- time PCR was carried out with a Stratagene real-time PCR Thiobarbituric acid reactive substances (TBARS) assay. system (Agilent Technologies, Inc., Santa Clara, CA, USA). TBARS measures a family of lipid peroxidation products and The real-time PCR reactions were performed using SYBR is a major indicator of oxidative stress (8).
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