Ingredients of SXF
Every 100 g of SXF was composed of the following ingredients: Chinese Thorowax Root (10 g), Angelica sinensis (10 g), White paeony root (10 g), Poriacocos Wolf (10 g), Rhizomaatractylodismacrocephalae (10 g), Peppermint (10 g), Herbaecliptae (10 g), Ligustrumlucidum (10 g), Spica prunellae (10 g), Radix glycyrrhizaepreparata (10 g). All Chinese medicine ingredients were purchased from JiangyinTianjiang Pharmaceutical Co., Ltd. (Jiangsu, China). SXF was diluted in distilled water to a concentration of 2 g/mL and then placed in a 4°C refrigerator for storage.
A total of 60 female Sprague-Dawley (SD) rats (71.15 ± 8.22 g) were commercially provided by Shanghai Laboratory Animal Co. Ltd. (Shanghai, China). Rats were kept separately in cages in an SPF laboratory animal room at (22 ± 1) ℃ and (55 ± 5)% humidity with free access to water and food. The light/dark cycle was 12 h. All animal experiments in this study have been approved by the Animal Ethics Committee (No.: R-082018033).
Construction and treatment of female rat model with CPP
Rats were randomly divided into 6 groups (10 rats per group) and named as follows: Control group, CPP group, CPP-LP group, CPP-HD-SXF group, CPP-MD-SXF group and CPP-LD-SXF group. Rats of Control group were injected subcutaneously with 0.9% sodium chloride injection (0.2 mL/time) at 14:00 and 16:00 daily. Notably, for rats of the other 5 groups, they were subjected to the construction of CPP model through subcutaneous injection of NMA (40 mg/kg) (Solarbio, Beijing, China) at 14:00 and 16:00 daily. Meanwhile, at 9:00 every day, rats in Control group and CPP group were given gavage of 0.9% sodium chloride injection (1.0 mL per rats). However, rats in CPP-LP group were injected intramuscularly with leuprolide (LP) acetate microspheres (100 μg/kg). At the same time point, rats of CPP-LD-SXF group, CPP-MD-SXF group and CPP-HD-SXF group were subjected to SXF gavage with a dose of 2.13 g/kg, 4.25 g/kg and 8.50 g/kg, respectively.
The vaginal opening of rats in each group was observed at 8:30 every day. When the vaginal opening of rats in CPP group was observed to be open, the injection of NMA was stopped. At the same time, rats of Control group were stopped the injection of 0.9% sodium chloride injection. Meanwhile, the injection of NMA for rats of CPP-LP group, CPP-LD-SXF group, CPP-MD-SXF group and CPP-HD-SXF group was also stopped. In CPP group, rats with open vaginal opening were subjected to vaginal smears. The vaginal smears were observed under a microscope in order to determine the sexual cycle of the rats. Then rats of CPP group were sacrificed at the first pre-estrus stage, and rats in the other 5 groups were also sacrificed at the same time. It should be noted that, before being sacrificed, the body weight was measured and the peripheral blood was obtained. After being sacrificed, the bilateral ovaries, uterus, hypophysis and hypothalamus of each rat was obtained, weighted and stored at -80°C.
Determination of estrogen concentration in peripheral blood
The peripheral blood of each rat was obtained and concentrated for 5 min at 3000 × g, 4°C to collect the serum. The concentration of follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL) and estradiol (E2)in the serum was determined by radioimmunoassay testing kits (Hengyuan biotechnology Co., Ltd., Shanghai, China).
Measurement of sex organ index
Before being sacrificed, the body weight of each rat was measured. The weight of ovary, uterus, hypophysis and hypothalamus was also measured after rat being sacrificed. The ratio of ovary/body weight, uterus/body weight, hypophysis/body weight and hypothalamus/body weight was subsequently calculated.
Hematoxylin-eosin (HE) staining
The ovary and uterus of rats were fixed with 4% paraformaldehyde for 24 h at 4°C. Gradient alcohol was used to dehydrate. After treatment with xylene, the ovary and uterus was embedded in the paraffin, followed by being prepared into sections with a thickness of 5 μm. Sections were dried in an oven at 45°C. Thereafter, xylene was used to dewax the sections and gradient alcohol was applied to hydrate the sections. The sections were stained with hematoxylin and eosin subsequently. After being dehydrated with alcohol and transparentized by xylene, sections were sealed in neutral resin and placed under a microscope to observe the growth of follicles and thickness of the uterine wall.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Sex hormone receptor related genes expression in hypophysis, including GnRH, GnRHR, estrogen receptor alpha (ERα) and G protein-coupled receptor 30 (GPR30), was investigated by qRT-PCR. In general, hypophysis tissues were lysed on ice with the addition of TRIZOL reagent (Thermo Fisher Scientific, Waltham, MA, USA) to extract total RNA. PrimeScriptTMRT kit (TaKaRa, Shiga, Japan) was used for the reverse transcription reaction with 5 μg total RNA sample to synthesis cDNA template. Thereafter, a total of 2 μL cDNA template was subjected to the PCR amplification reaction in a 20 μL reaction system. The amplification reaction conditions were 40 cycles of 95℃ for 30s, 95℃ for 15s and 62 ℃ for 20 s. β-actin was used as the control. Primers were as follows: GnRH, forward: 5'-CCGCTGTTGTTCTGTTGACT-3', reverse: 5'-GCAGATCCCTAAGAGGTGAA-3'. GnRHR, forward: 5'-TCACTCAGCCCTTAGCTGTCC-3', reverse: 5'-GAAGGCTTCATGCCACCATTG-3'. ERα, forward: 5'-TCAGGCTACCATTACGGAGT-3', reverse: 5'-CGCTTGTGCTTCAACATTCT-3'. GPR30, forward: 5'-TCATTTCTGCCATGCACCCA-3', reverse: 5'-GTGGACAGGGTGTCTGATGT-3'. β-actin, forward: 5'-GGAGATTACTGCCCTGGCTCCTA-3', reverse: 5'-GACTCATCGTACTCCTGCTTGCTG-3'. At last, 2-ΔΔCt method was used for the calculation of relative mRNA expression.
Hypophysis tissues were ground into powder in liquid nitrogen. Cell lysate was added into the hypophysis tissues powder to collected total proteins on ice. The concentration of total proteins was detected using BCA assay kit (Pierce Chemical Company, Rockford, IL, USA). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used for the separation of proteins in each sample. After being transferred onto a polyvinylidene fluoride (PVDF) membrane, total proteins were experienced blocking with 5% skim milk for 1 h. Rabbit anti-GnRH, anti-GnRHR, anti-ERα and anti-GPR30 primary antibodies (1:1000, Cell Signaling, Danvers, MA, USA) were used to incubate the membrane at 4℃ overnight. The membrane was then washed with Tris-buffered saline/0.1% Tween (TBST) for 3 times with 10 min per time. Thereafter, horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody (1:5000, Solarbio, Beijing, China) was added onto the membrane for 1 h incubation at room temperature. The membrane was washed with TBST for 3 times again. The proteins bands were visualized using enhanced chemiluminescence (ECL) regent. β-actin was used as the control.
The expression of GnRH in the hypothalamus and GnRHR in the ovary was detected by immunohistochemistry. In short, the hypothalamus and ovary were subjected to fix with 4% paraformaldehyde and then were prepared into sections with a thickness of 5 μm. The sections were blocked with 5% bovine serum albumin for 1 h. Rabbit anti-GnRH and anti-GnRHR (1:250, Lichen Biotechnology Co., Ltd., Shanghai, China) was used to treat the sections overnight at 4 °C. Subsequently, biotin-labeled anti-rabbit IgG antibody (1: 200, Solarbio, Beijing, China) was added onto the sections for 2 h incubation at room temperature. Sections were then stained with 3, 3-diaminobenzidine (DAB) and counterstained with hematoxylin. After being dehydrated and transparentized, the sections were sealed in neutral resin and observed under a microscope. Brown-yellow particles were considered to be positive GnRH and GnRHR expression signals.
In this research, all data were expressed as mean ± standard deviation based on three independent repeated trials. SPSS 19 software (SPSS Inc., Chicago, IL, USA) was used to process the data. GraphPad Prism (version 5, La Jolla, CA, USA) was used for the making of statistical graphs. Student's t-test was responsible for the comparison between two groups. One-way analysis of variance was applied for the comparison among at least three groups. P < 0.05 indicated the statistically significant differences.