Effects of Nourishing “Yin”-Removing “Fire” Chinese Herb Mixture on the Differential Expression of GABAA Receptor α Subunits in Hypothalamus of Precocious Puberty Female Rats ()
1. Introduction
Precocious puberty is defined as the onset of puberty before age of 8 years in girls and 9 years in boys. Precocious puberty in general is more frequent in girls, of which the majority have idiopathic precocious puberty whose etiology is unknown [1]. It has been well established that pulsatile GnRH release played a crucial role in triggering the onset of puberty [2]. GABA is one of the primary inhibitory neurotransmitters in the regulatory network of GnRH and contributes a great deal modulating pubertal progress via its receptors in the hypothalamus [3,4]. GABA receptors are widely distributed throughout the central nervous system where they predominantly mediate inhibitory neurotransmission. Specific to the hypothalamus, studies supported that GABA, particularly through its action on GABAA receptor, regulated firing of pubertal GnRH neurons [5].
GABAA receptor includes six α, three β, three γ, one δ, one ε, one π, one θ and three ρ subunits, of which each composition form involves at least one α subunit [6]. It is revealed that the pattern of each GABAA receptor subunit is complex and differs from phase to phase and region to region in mammal species, which ensures the functional heterogeneity of GABA input throughout the life span [7]. There was a normal switch from some GABAA receptor α subunits highly expressed during development (α2, α3, α4) to those subunits highly expressed in adulthood (α1) in rats model [8]. And compared with adult subunits mRNA expression in GnRH neurons of female mice, juvenile GnRH neurons expressed a much more heterogenous population of GABAA receptors that α5, β1, and γ2 were the most frequently co-expressed except α4 and γ1 [9]. In addition, it has been reported that α2 subunit expression is very high before birth to shortly after birth, and decreases gradually to adult levels, whereas α1 expression is minimal before birth and increases after birth until adulthood [10,11]. Examination of GABAA receptor subunit expression in GnRH neurons of hypothalamus supports this trend of age-associated going on during pubertal development [5].
Nourishing “Yin”-Removing “Fire” Chinese herb mixture has been successfully used for the management of idiopathic precocious puberty for a long time. It is reported that it could significantly alleviate the symptoms on precocious patients whose serum gonadotropins and estrogen reduce and secondary sexual characteristics subside. And a series of experiments on female precocious puberty rats model supported that Chinese herb mixture could regulate the function of hypothalamus-pituitarygonadal axis (HPGA) through modulating the gene and protein expressions of GnRH, kisspeptin, GABA and glutamate [12,13]. However, the mechanism of GABA receptor subunits participates in the Chinese herb mixture curative effects of precocious puberty is merely mentioned. We hypothesized that GABAA receptor subunit composition might involve the onset of precocious puberty. The present work was to observe the effects of Nourishing “Yin”-Removing “Fire” Chinese herb mixture on the expression of GABAA receptor α subunit mRNA in Danazol induced female precocious puberty rats and to explore the possibility of GABAA receptor participating in advance GnRH release in precocious puberty.
2. Methods
2.1. Animal
Female Sprague-Dawley rats at 3 days of age were purchased from Medical Experimental Animals Center of Chinese Academy of Sciences (Shanghai, China). Animals were housed under laminar flow in an isolated room with controlled temperature of about 22˚C under a 12-h light/dark cycle with lights on from 7:00 am to 7:00 pm. The model litters at P5 were given a single subcutaneous injection of 300 µg of danazol (Hualian Pharm Ltd., Shanghai, China) dissolved in 25 µl of propylene glycolethanol (1:1, v/v), and allowed to grow without further treatment, the vehicles were administered with the same volume of propylene glycol-ethanol (1:1, v/v). Animals were weaned on P21, and then vaginal opening was examined daily afterwards, and the daily vaginal smears were examined. All experiments procedures involving the use of animals were conducted in accordance with NIH Guidelines and were reviewed and approved by Animal Use and Care Committee for the Fudan University.
All rats were randomly divided into normal (N), precocious puberty model (M), model exposed to saline (MS) and model exposed to Nourishing “Yin”-Removing “Fire” Chinese herb mixture (CHM) four groups. From P15, rats in CHM and MS were fed with CHM (including crude drug 3.3 g per ml) or the same volume normal saline every day. The intragastric dose increased by about 0.1 ml per day as the rat grew.
At the day of vaginal opening in M, rats of all four groups were decapitated with blood and hypothalamus tissue collected, the uterus and ovaries were dissected out of the surrounding fat, and the organ coefficients (mg/100g) were evaluated.
2.2. Hormone Measurement by RIA
At the time of decapitate, the blood of all rats was collected. The serum was separated by centrifugation and stored at −80˚C until assayed. The gonadotropins and oestradiol levels were measured by double-antibody RIA kits purchased from the Beijing Sinouk Institute of Biological Technology (Beijing, China). The samples were assayed in duplicate, and all the subjects’ samples were assayed together. The sensitivity for the E2 was less than 5 pg/ml; the intraand inter-assay coefficients were less than 10% and 15.2%, respectively. For LH, the assay sensitivity was 0.2mIU/ml, and the intraand inter-assay coefficient of variation was 2.0% - 2.4% and 4.2% - 7.5%, respectively. For FSH, the assay sensitivity was 0.25 mIU/ml, and the intraand inter-assay coefficient of variation was 2.2% - 2.5% and 3.7% - 8.7%, respectively.
2.3. Tissue Collection and Total RNA Preparation
The rat brains in every group (n = 5) were rapidly removed and hypothalamus were separated immediately and frozen in liquid nitrogen. The target regions, including mediobasal hypothalamus and the suprachiasmaticpreoptic areas were dissected. Total hypothalamic RNA was extracted by “Trizol Regent” (Invitrogen Inc., America) according to the manufacturer’s instructions. The concentration of RNA was estimated by spectrophotometry with UV absorbance at 260 nm and 280 nm. The purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis before carrying out the analytical procedures.
2.4. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
The total RNA from each tissue was reverse transcribed using M-MuLV reverse transcriptase (Superscript II, Promega) and 2.5 mM oligo dT (BioDev-Tech) in the presence of 250 mM of deoxynucleotides triphosphate in a final volume of 12.5 ml at 37˚C for 1 h. The reaction mixture was denatured for 5 min at 95˚C and stored at −70˚C. The transcribed DNA for each subunit was amplified by PCR in a thermal cycler using specific primers for each subunit. The primers were designed in order to amplify DNA sequences (ERα, sense: 5’-AATTCTGACAATCGACGCCAG-3’; antisense: 5’-GTGCTTCAACATTCTCCCTCCTC-3’. β-actin, sense: 5’-AAGCAGGAGTATGACGAGTCCG-3’, Antisens: 5’-GCCTTCATACATCTCAGGTTGG-3’). The amplification mixture contained 1.5 U of Taq PCR Master Mix (Shanghai Lifefeng Biotechnology) and 20 μM of specific primer pairs in a final volume of 20 ml. The mixture was amplified for 30 cycles. Each amplification cycle consisted of 5 min of denaturation at 95˚C to activate the enzyme and 1 min denaturation at 95˚C then 1 min at 58˚C for annealing and 1 min at 72˚C. The final extension step was 15 min at 72˚C. The relative abundance of each product was estimated by the visual intensity of ethidium bromide stained bands under UV light.
2.5. Real-Time Reverse Transcriptase-PCR (qRT-PCR)
Prior to conducting real-time reverse transcriptase-PCR (qRT-PCR), the total RNA was digested with RNase-free DNase I (Invitrogen, Carlsbad, CA), by which possible contamination of genomic DNA. The SuperScrip III reverse transcription system (Invitrogen Corp., Carlsbad, CA, USA) was used for reverse transcription with 2 μg of total RNA according to the manufacturer’s specifi- cations. The primers used for GABAA receptor α subunits and GnRH were designed and synthesized by Invitrogen with standard purity. To determine the sensitivity and efficiency of the amplification, PCR assay linearity ranges were previously established for each gene cDNA. Quantitative Real-Time PCR was carried out in IQ5 Real-time PCR system (Bio-Rad). The amplification protocol was as follows: an initial denaturing step at 95˚C for 2 min followed by 40 cycles of a 95˚C for 10 sec, 60˚C for 30 sec, and 72˚C for 30 sec. Following amplification, a dissociation curve analysis was performed to insure purity of PCR products. All real-time experiments were run in triplicate and a mean value was used for the determination of mRNA levels. The relative linear quantity of the target gene was calculated using the formula 2−ΔΔCt. Therefore, the data were expressed an n-fold change in gene expression normalized to a reference gene (β-actin) and relative to a calibrator sample. The primer sequences and realtime PCR conditions for GABAA receptor α subunits and GnRH mRNA were listed in Table 1.
2.6. Western Blot
Each of six rats in four groups was used to investigate GABAA receptor α1 and α3 subunits protein expression by western blot with a standard procedure. For total protein extraction, hypothalamus was homogenized in lysate (BioDev-Tech. Co., Ltd) and protease inhibitors using a polytron homogenizer. After homogenization, samples were centrifuged at 10,000 rpm for 15 min at 4˚C. The supernatant fraction was the total tissue lysate. Protein concentration in the lysate was determined by a total protein measurement kit from BioDev-Tech. Protein samples were denatured in loading buffer and separated on a sodium dodecyl sulfate-polyacrylamide gel after loading equal amount of protein in each lane. Separated proteins were transferred to PVDF membrane at 100 V for 110 mins in 25 mM Tris-glycine buffer, pH 8.3, 10% methanol using a Transblot apparatus (Bio-Rad Laboratories, Inc.). Following the transfer, the membranes were rinsed five times with TTBS (20 mM Tris, 0.1% Tween- 20) for 10 min each rinse and then incubated with 5% BSA for 4 h at 4˚C to block non specific/unbound surface. The membrane was incubated overnight with polyclonal rabbit anti-rat (1:200; Millipore). The membrane was then washed with TTBS to remove unbound antibody, followed by incubation with secondary HRP-conjugated sheep anti-rabbit IgG (1:2000, Millipore) for 1 h at room temperature. The signal was detected using an ECL detection kit (GE Healthcare) and the membranes were exposed to ImageQuant LAS 4000 mini (GE Healthcare).
2.7. Statistics Analysis
All data are presented as means ± SEM. Statistical analysis was performed on raw data using one-way analysis of variance (ANOVA), with the significance concentrations of P < 0.05 in two-tailed testing chosen. Comparisons among groups were made using the Student’s t-test.
3. Results
3.1. Effects of Nourishing “Yin”-Removing “Fire” Chinese Herb Mixture on the Timing of Vaginal Opening
On P22 at the beginning of observation, one third of the
Table 1. Primer sequences of GABAA receptor α1-6 subunits and GnRH in realtime PCR.
rats in M and MS had showed vaginal opening, while few in N and CHM did. The discrepancy in the proportion of vaginal opening rats in groups sustained from then on till P44 when all rats had steady-state sexual cycles (Figure 1.)
3.2. Effects of Nourishing “Yin”-Removing “Fire” Chinese Herb Mixture on Serum E2, LH and FSH Levels
The concentration of serum E2, LH and FSH in M were significantly higher than those of N (p < 0.01). And the serum E2 and LH levels were significantly lower in CHM than those in M (p < 0.01, respectively), and FSH level were also lower in CHM than that in M (p < 0.05). There were not statistically significant differences observed between the M and MS (Figure 2.).
3.3. Effects of Nourishing “Yin”-Removing “Fire” Chinese Herb Mixture on Reproductive Organs’ Coefficients
Table 2 shows changes in reproductive organs’ coefficients of the different rat groups. On P22 the uterus coefficient of M were 80.18 ± 2.19 mg/100g which was significant increased than those of N (59.78 ± 1.30 mg/100g), and the uterus coefficient of CHM were significantly decreased to 58.62 ± 1.72 mg/100g (p < 0.01, respectively). Meanwhile the ovaries coefficient of M were 5.87 ± 0.38 mg/100g which were also increased significantly compared to those of N, and the coefficients of CHM were decreased to 4.36 ± 0.22 mg/100g (p < 0.05, respectively). These reproductive organ coefficients on P26 were similar to those at P22 (p < 0.05, respectively).