Animals and troxerutin administration
Postnatal 14 days female SD rats were purchased from the Breeder center of Rodents (Jinan, China).The rats were housed under controlled lighting (12h light, 12h darkness, lights were turned on at 08:00 and turned off at 20:00) and temperature (24℃) with free access to food and water for a week of acclimatization. All experimental protocols used in this study were approved by the Animal Care and Ethical Committee of Xuzhou medical University. Postnatal 21 days rats weighing approximately 50-55g, were randomly divided into four groups: sham group (each rat was implanted with a tube without DHT), PCOS group (each rat was implanted with a silastic tube filled with 5α- DHT at dose of 7.5mg under anesthesia), PCOS+150mg/kg troxerutin group and PCOS+300mg/kg troxerutin group (PCOS rat with intraperitoneal injection of 150mg/kg or 300mg/kg troxerutin daily during the 4 weeks after implantation). Due to the high water-solubility of troxerutin, the gastrointestinal system can absorb it well without toxicity. Troxerutin interacts with tRNA by external binding manner with low binding affinity, and also binds with DNA through groove binding manner. Sham and PCOS groups were injected daily with equivalent 0.9% saline which was used to dissolve troxerutin. DHT was procured from sigma, St. Louis, Mo and troxerutin (purity >95%) was from Baoji Fangsheng Biotechnology Co., Ltd, Baoji, China. The rats were housed individually after surgery.
Blood and tissue sampling
At the terminal of the study, rats were decapitated and trunk blood was collected and centrifuged at 3000 rpm for 15 minutes. Plasma was separated and stored at -80℃ until analyzed for biochemical and hormonal analysis. Ovaries were cleaned in saline and managed fat free. Ovaries were fixed in 10% buffered formalin for 48 hours and paraffin-embedded. Paraffin-embedded tissue sections were de-waxed, sectioned (6 μm) and stained with hematoxylin and eosin (H&E). Numbers of follicles were classified and counted in every 40th ovarian section for 10 ovaries per group. Follicles were counted according to the following definition. Primordial follicle was surrounded by a single layer of squamous granulosa cells. Primary follicle contained a single layer of cuboidal granulosa cells. Secondary follicle contained two or more layers of granusola cells, but no antral space in the granulosa layer. Antral follicle displayed multi-layers of granusola cells with the presence of follicular antrum.
Vagina smears and estrous cycles
Vaginal smears were taken daily at 09:00am from the 18th to the 28th day after the first day of DHT treatment,which were the terminal ten days of the study. Microscopic analysis (BX41, Olympus) was carried out to determine the stage of the estrous cycle by the predominant cell types in vaginal smears following methylene blue staining(Solarbio, Beijing). Proestrus consists of enlarged,round,nucleated epithelical cells; estrus consists of a large number of cornified squamous epithelical cells; metestrus consists of two kinds of cells: epithelial cells and leukocytes with approximately same amount; and diestrus is characterized by a small amount of cells with the predominance of leukocytes.
Body weight, biochemical and western blot analysis
Body weight was measured every week after implantation for a total of 4 weeks.
The serum concentration of LH, FSH and testosterone were measured via Enzyme Linked Immuno Sorbent Assay (ELISA) with the help of commercial kits (ELISA kit, CUSABIO, Inc, Wuhan, China)and the procedure was followed as given in the kit catalog. The concentration of LH, FSH and testosterone was estimated by the standard curve.
To identify the hypothalamic GnRH status, hypothalamus was dissected out. The rat brain was carefully taken out and temporarily put on ice with the ventral surface upward. Thalamus is the ellipsoidal part at the center of the ventral brain. With the middle of tuber cinereum and optic chiasma as the center, prechiasmal border as the anterior edge, postmammillary border as the posterior edge, hypothalamus (4x4x2mm) was carefully dissected out.Hypothalamus tissue were homogenized to obtain protein samples. Then the protein (25μg) was separated by SDS-PAGE and transferred to membranes. The membranes were incubated with mouse monoclonal anti-GnRH antibody (1:1000, MAB5456-C, Millipore, Billerica, MA) and mouse anti-GAPDH antibody (1:20000, Proteintech, Chicago , USA). After being washed with TBST three times, the membranes were incubated with IRDye-labeled secondary antibodies in TBST for 2h. The bands on the membrane were scanned with an Odyssey infrared scanner (LI-COR Biosciences, Lincoln, NE, USA) and the density of the bands was analyzed with ImageJ software.
Chromatographic analysis
Chromatographic analysis was carried out to evaluate the levels of two kinds of GnRH regulatory neurotransmitters GABA and glutamate in the brain/pituitary. Weigh the brain and add in the mix of methanol and water (1:1) at the ratio of 100mg/ml to prepare homogenate. The brain homogenate was centrifuged at 12000 rpm for 30 min at 4°C. The mix of supernate and 80μL acetonitrile were centrifuged at 12000 rpm for 30 min at 4°C. The new supernate was derivatized, mixed with 80μL borate saline buffer, 160μL water and 80μL F-moc, filtered by 0.45μm filtration membrane and immersed in 40°C bath for 5 minutes. Then, the samples were used for the measurement of GABA and glutamate using chromatographic analysis.All standard biomarkers used for identification purpose in chromatographic studies were procured from Zhongke Co, Ltd, Beijing, China. High-performance liquid chromatography (HPLC)was performed on a Waters e2695 system(Waters, USA)equipped with a hypersil ODS column(Elite, Dalian, China). The mobile phase A was a mix of natrium aceticum(pH4.8),water and tetrahydrofuran with the ratio of 410:85:5, and mobile phase B was pure acetonitrile. Flow rate was 1ml/min. The column temperature was set at 30℃. Samples were detected at the wavelength of 265nm. Injection volume was 20µL.
Immunofluorescent staining
After intracardiac perfusion with normal saline followed by fixation with 4% cold paraformaldehyde (PFA), the rat brains were separated and postfixed in 4% PFA for 6 h at 4 °C. Then the brains were incubated in 30% sucrose- 100 mM sodium phosphate buffer (pH 7.4) for 48h at 4 °C. Serial coronal sections (20 μm) were made from the bregma anterior-posterior −2.0 mm to −3.30mm. Totally 60 sections were collected per brain. At least three sections per brain were selected for NKB, Kisspeptin, GnRH/NK3R and GnRH/Kiss1r immunofluorescent staining, respectively.
For immunofluorescence, the primary antibodies rabbit anti-NKB (1:1000, Novus biologicals, NB300-201), rabbit anti-kisspeptin (1:1000, H-048-56, Phoenix Pharmaceuticals), rabbit anti-Kiss-1r (1:500, AKR-001, Alomone Labs), rabbit anti-NK3r(1:500, abx217136, Abbexa) and mouse anti-GnRH (1:1000, MAB5456-C, Millipore, Billerica, MA) were used. After incubating with the antibodies for 24h at 4 °C, the sections were washed with PBS and then were treated with goat anti-mouse IgG (H + L) Alexa Fluor ®555 or 488 -conjugated or goat anti-rabbit IgG (H + L) Alexa Fluor ®488 (Invitrogen, Eugene, OR, USA) secondary antibodies. According to the manufacturer’s instructions, DAPI (Beyotime Biotechnology, Shanghai, China) was used to label nucleus. For negative controls, sections were incubated with PBS instead of the primary antibodies. Fluorescence images were captured using a Zeiss Axioskop 40 microscope(Carl Zeiss, Oberkochen, Germany). Image Pro-Plus 6.0 software was used for semi-quantitative measurement of immunofluorescent density. Values (three slides for each brain) of integral optical density (IOD) in individual cells represented the quantity of objective protein and were calculated using the following equation: Σ IOD/Σ DAPI.
Statistical analysis
All statistical analyses were performed with SPSS software (version 16.0), and the data were analyzed using the one-way Analysis of Variance (ANOVA). The data were expressed as the mean ± s.e.m. Statistical significance was set at P<0.05 for all tests.