Animals
Twenty adult female rats (Sprague–Dawley), purchased from the Department of Laboratory Animal Science of Peking University Health Science Center, which were 8–10 weeks old and weighed 240–280 g, were used for the study. The rats were maintained under controlled conditions (25 ± 1 °C, room temperature, with a light/dark cycle of 12 h and 40–50% humidity), and fed with soy-free forage and given free access to water under indirect light for 2 weeks. The study was approved by the local Ethics Committee, and all experiments were conducted in conformity with the National Institutes of Health Guidance for the Care and Use of Laboratory Animals. The study was also approved by the Biomedical Ethics Committee of Peking University (approval number: LA2012–82).
Reagents and instruments
Rabbit anti-rat caspase-3 polyclonal antibodies (#ZS-7148) and a streptavidin/peroxidase staining kit (Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd. Beijing, China) were used for immunohistochemistry. Commercially available estradiol valerate (Bujiale; 1 mg per tablet, Bayer Health Care Co. Ltd, Batch number 256A 2; Guangzhou, China) and C. racemosa (Remifemin tablets; 20 mg per tablet, Schaper & Brümmer Ltd & Co KG, Batch number 123813; Germany) extracted using 40% isopropyl alcohol were used in this study.
Establishment of the ovariectomized rat model
Experimental groups were established as follows: a sham-operated group (SHAM, n = 5), an ovariectomized group (OVX, n = 5), an OVX group treated with estradiol (E2) valerate (OVX + E2, n = 5), and an OVX group treated with the iCR (OVX + iCR, n = 5). Firstly, ovariectomy was performed on rats under general anesthesia. Following appropriate asepsis and antisepsis, the abdominal cavity was exposed using a scalpel. Instead of the ovaries, an equal quantity of fat from around the ovaries was resected from the SHAM group. In the other groups, bilateral ovaries were totally removed. Finally, the abdominal cavity was closed with a 3-0 seam interrupted suture. The exfoliated vaginal cells, changing in cell morphology and types with fluctuations in estrogen levels, were observed for 7 consecutive days from the third day after surgery to verify successful OVX [13]. Two weeks were allowed after the operation for wound healing, and then rats were given daily gavage for 4 weeks [14, 15].
Dosage and administration
The preparation of experimental medicines was as follows: the estradiol valerate and C. racemosa tablets were dissolved in distilled water by sonication to form a uniform suspension. The concentration of estradiol was 0.2 g/l and of iCR, 12 g/l. Gavage was administered to all rats from 8:30 to 9:30 a.m. Doses were as follows: the SHAM group received distilled water (10 ml/kg); the OVX group received distilled water (10 ml/kg); the OVX + E2 group received estradiol valerate (0.8 mg/kg) and the OVX + iCR group received C. racemosa (60 mg/kg) [13, 16]. Rats were weighted on alternate days and the dose was adjusted according to any change in body weight.
Sample collection
To avoid the normal fluctuation of estrogen affects the results, the sample collection of SHAM group was performed in estrus used the vaginal smear method. Following sodium pentobarbital (40 mg/kg intraperitoneal injection) anesthesia, tissues were first collected for electron microscopy. The neck skin and fascia were cut with scissors, revealing the sublingual glands. A small part of the left gland was cut away with a sharp blade and cut into pieces of approximately 1 mm3. These were fixed in 3% v/v glutaraldehyde for 2 h. Tissues were washed with 0.1 mol/l phosphate-buffered (PB) buffer, fixed in osmium tetroxide for 2 h and then, following dehydration in ethanol solutions of increasing concentration, embedded in Epok 812. Ultrathin sections (70 nm thick) were taken and stained with uranyl acetate and lead citrate. These were then examined by electron microscopy (JEM-2100). Each image was viewed at the same brightness and contrast for production of photomicrographs, in order to compare changes in the rough endoplasmic reticulum, mitochondria and secretory granules.
After collection of tissues for electron microscopy, the heart was exposed, a perfusion catheter was inserted into the ascending aorta and the right atrium was incised. Perfusion was carried out using 200 ml normal saline (0.9% g/ml sodium chloride aqueous solution) and 300 ml 4% g/ml paraformaldehyde solution (paraformaldehyde dissolved in 0.1 mol/l PB). After perfusion, the right sublingual gland was immediately removed and post-fixed in 4% g/ml paraformaldehyde solution for 24 h. After dehydrated, the tissue was embedded in paraffin. Three serial sections of a sample, 5.0 μm in thickness, were mounted on each microscope slide, and 20 slides were collected of per animal. After drying, the paraffin sections were stained with hematoxylin and eosin (HE).
Immunohistochemical staining
Paraffin-embedded sections were heated to 60 °C to melt the paraffin, then washed in xylene and rehydrated by graded washing in 100, 95, 80, 70, 50% ethanol in distilled water (each concentration for 20 min). Sections were then incubated in 0.3% Triton X-100 at 37 °C for 30 min, and, for antigen retrieval, placed in a boiling water bath for 15 min. They were next placed in 0.3% hydrogen peroxide for 15 min. The sections were washed with 0.01 mol/l phosphate-buffered saline (PBS; pH 7.4, 4 °C) (3 × 5 min) between each step. Sections were then incubated in biotinylated goat anti-rabbit IgG serum (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd. Beijing, China) for 2 h, and then with rabbit anti-rat caspase-3 polyclonal antibody (1:100 dilution in 0.01 mol/l PBS; Beijing Zhongshan Golden Bridge Biotechnology Co, Ltd. Beijing, China) at room temperature for 1 h and then at 4 °C overnight. The following day, the sections were placed at room temperature for 1 h, and then avidin–biotin complex (ABC) staining kit (Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd. Beijing, China) was used. Finally, each section was stained with DAB, using a staining kit (Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd. Beijing, China), for approximately 5 min, and the immunoreactive products were visualized as a brown stain. PBS (0.01 mol/l) was used for washing between each step (3 × 5 min). Then, hematoxylin was used to stain the cell nuclear in order to facilitate observation. After dehydration and mounting of neutral resins, sections were observed under a light microscope (Leica; BX51). The same process was used for negative controls, except that 0.01 mol/l PBS was used instead of rabbit anti-rat caspase-3 polyclonal antibodies.
Data collection and statistics
The percentage of the gap area
Under the same conditions of brightness and contrast (at 200× magnification), five fields of vision were randomly selected from each HE film. Image-Pro Plus 6.0 software was used. Firstly, the non-staining acini was filled with red color manually in order to distinct with the gap, and then the area of the gap and the total area of the lobule were calculated used Count/Size instrument of Image-Pro Plus 6.0 software. The percentage of the gap area was then obtained by dividing the area of the gap by the total area (Additional file 1).
Percentage of caspase-3 positive cells
At a magnification of 400×, five fields of vision were randomly selected from each immunohistochemical film: “a”, represents the number of positive acinar cells in each photo; “b” is the total number of acinar cells in the image; “a/b” is the percentage of positive cells in each film. A mean of the data for the five fields of vision was taken to obtain the ratio of positively stained cells to the total cells in each rat (Additional file 2).
Average optical density (mean absorbance, MA) of caspase-3 immunoreactivity in the striated ducts
Under the same conditions of brightness and contrast (at 400× magnification), five fields of vision were randomly selected from each immunohistochemical film. Image-Pro Plus 6.0 software was used to analyze the MA. Firstly, the area of striated ducts was measured manually (area of interest, AOI), and then the total area and the integral absorbance (IA) of AOI were calculated used Count/Size instrument of Image-Pro Plus 6.0 software; MA was then obtained by dividing IA by the total area (Additional file 3).
All the data were analyzed using SPSS17.0 and recorded as means ± standard deviations (x ± SD). To analyse caspase-3 expression, a one-way analysis of variance (one-way ANOVA) was performed, followed by an LSD post hoc test.