Animals
Fifteen specific-pathogen free (SPF) level female Sprague Dawley (SD) weighting 50g± 5g, were aged 21 days at the onset of experiments. They were from the Experimental Animal Science Department of Guangzhou University of Chinese Medicine, Guangzhou, China (License number SCXK-2016-0168). The experimental protocols were performed after approval and in accordance with the guidelines set by the Experimental Committee of Guangzhou Medical University (Protocol number: GY2018-021). All rats were provided with humane care in a temperature-controlled room with a 12-hr light/dark cycle (lights on 07:00–19:00) and ad libitum access to food and water in their cages (22 °C–24 °C and 60% humidity).
Study procedure
Fifteen rats were randomly divided into three groups on average. According to previous studies [16,17], from 3w age, the rats in DHT group were subcutaneously injected daily with 83μg 5α-DHT which dissolved in 0.2ml tea oil for 6 weeks to mimic the hyperandrogenic state in women with PCOS. The rats in other two groups were injected daily with the same volume of tea oil alone. Follow the experiment of Kafali H et al [10], from 6week age, the rats in letrozole (LET) group were oral administration with letrozole (1mg/kg body weight of letrozole dissolved in 1 ml of 1% carboxymethyl cellulose (CMC) for 3 weeks. At the same time, the rats in control (C) group and DHT group were daily oral administration with 1 ml of 1% CMC. All the rats were fed commercial chow (Research Diets GB 14924.3-2010) [67% carbohydrate, 21% protein, 12% fat, and total 3.45kcal/g], was provided by Guangdong Medical Laboratory Animal Center.
Vaginal smears
The major cell type in vaginal smears obtained every day from 6 weeks old to the end of the experiment were analyzed by microscope to determine the periodic stage. Rats showing regular cycles of 4–5 days complete with the proestrus, estrus, metestrus and diestrus stages were defined as normal cyclic rats, whereas animals in which the estrus cycle was found arrested in any one of the stages for 4 consecutive days were termed as acyclic rats [18].
Blood and tissue collection
Blood was withdrawn through orbital sinus in a tube and separated by 10-min centrifugation (3,000 revolutions/min) at 4°C. Supernatant containing serum was separated and immediately stored at-20°C until analyzed for biochemical and hormonal analysis.
Blood sampling for lipid profile, sex steroids and other biochemical parameters
Fasting blood glucose (FBG) was analyzed by GOD-PAP. Testosterone (T), Free testosterone (FT), Fasting insulin (INS), Luteinizing hormone (LH), Follicle stimulating hormone (FSH), Superoxide dismutase (SOD), Malondialdehyde (MDA), Lnterleukin-22(IL-22), Lipopolysaccharide (LPS), Toll-like receptors 4(TLR4) and Tumor necrosis factor-α (TNF-α) were determined using enzyme-linked immunosorbent assay (ELISA) kit (mlbio, Shanghai, China). Sensitivity of methods are 0.1 ng/ml (FT, TLR4), 1.0 U/ml (SOD), 1.0 pg/ml (TNF-α), 0.1mU/L (INS) ,0.1 pg/ml (IL-22) ,0.1 mmol/ml (MDA), 1.0 pg/ml (T) ,1.0 EU/L(LPS), and 0.1 mIU/ml (LH,FSH) at 95% confidence limit. Assays were performed according to manufacturers’ protocols. The optical density values were read at 450 nm using a microplate reader. HDL-C, LDL-C, total cholesterol and triglyceride levels were quantified by following the protocols provided with kits on UniCel DxC 600800 Synchron Clinical System. IR was appraised with the homeostasis model assessment of insulin resistance (HOMA-IR) method. HOMA-IR was calculated using the following formula:
HOMA-IR =FBG (mmol/L) * FINS (mU/L)/22.5[19].
Ovarian Histology
The left ovary of rats was fixed in 4% paraformaldehyde and embedded in paraffin. 5 μm thick sections were prepared and stained with Hematoxylin-Eosin and histo-anatomical changes were observed and photographed under light microscope (BX-51, Olympus, Tokyo, Japan atX40 magnification).
16s rDNA sequencing data analysis
The fecal microbiome for 15 fecal samples collected from 15 rats in the three groups. The 16S rDNA high through put sequencing (V3-V4 region) was performed using an Illumina MiSeq platform. After assembly, quality filtering and the random extraction of sequences at 97% similarity, the operational taxonomic units (OTUs) for species classification were obtained. The Chao1 and Shannon α-diversity indices were used to assess bacterial richness and diversity. In the aspect of β diversity, based on Bray-Curtis dissimilarity, permutational ANOVA (PERMANOVA) was used to assess bacterial community compositional difference. Linear discriminant analysis effect size (LEfSe) analysis coupled with the Kruskal-Wallis rank sum test was performed to identify the microbial differences among all groups. In LEfSe analysis, LDA effect size of >3 was used as the threshold.
Statistical analyses
Most statistical evaluations were performed with SPSS 20.0 for Windows (SPSS Inc., Chicago, IL, United States). All data were presented as mean± SEM. One-way ANOVA was used to determine the significance, p<0.05 was considered significant. When the ANOVA revealed significant differences among three groups, post hoc analysis was performed by a Tukey honest significant difference test. Kruskal–Wallis test was used for not normally distributed values.