Materials and reagents
BSTJF consists of seven herbs, including H. glutinosa Libosch. (Dihuang), 20 g; Ligustrum lucidum Ait. (Nvzhenzi), 20 g; Rubus chingii Hu (Fupenzi), 10 g; Cwscwia australis R. Br. (Tusizi), 15 g; Psoralea corylifolia L. (Buguzhi), 15 g; Astragalus membranaceus (Fisch.); Bge. var. mongholicus (Bge.); Hsiao (Huangqi), 10 g; and Salvia m iltiorrhiza Bge. (Danshen), 10 g, which were purchased from Huadong Medicine Co., Ltd. The reference standards, including catalpol, sodium danshensu, 3,4-dihydroxybenzaldehyde, neochlorogenic acid, salidroside, chlorogenic acid, p-coumaric acid, calycosin-7-O-beta-d-glucoside, rutin, rosmarinic acid, salvianolic acid B, and salvianolic acid A, were purchased from Shanghai Winherb Medical Technology Co., Ltd. (Shanghai, China). Adenosine, rehmannioside D, nuezhenidic acid, psoralenoside, isopsoralenoside, hyperoside, acteoside, salvianolic acid D, specneuzhenide, ononin, salvianolic acid E, quercetin, calycosin, and isopsoralen were provided by Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Schaftoside and astragalin were obtained from Chengdu Must Biotechnology Co., Ltd. (Chengdu, China). Sucrose was purchased from Aladdin Industrial Corporation (Shanghai, China). Psoralen was purchased from the National Institutes for Food and Drug Control (Beijing, China). HPLC-grade acetonitrile, methanol, and formic acid were obtained from Merck (Darmstadt, Germany). The samples were stored at 4 °C and the reference standards were stored at − 20 °C at the Pharmaceutical Informatics Institute of Zhejiang University. Deionized water was prepared using an Elga PURELAB flex system (ELGA LabWater, UK). All other chemicals and reagents used were of analytical grade.
Testosterone propionate was purchased from Beijing Solaibao Technology Co., Ltd. (Beijing, China). Sesame oil (S3547) was purchased from Sigma–Aldrich (Steinheim, Germany). Pregnant mare serum (PMSG) and human chorionic gonadotropin (HCG) were obtained from Ningbo Second Hormone Factory (Ningbo, China). Sump dye was purchased from Haoke Biotechnology Co., Ltd. (Hangzhou, China). Primary antibodies against Foxl2, ERK1/2, Grin2b, and Adra1b were procured from Proteintech Group, Inc. (Rosemont, USA). Primary antibodies against c-FOS and Gabrb1 were obtained from Affinity Biosciences, Ltd. (Jiangsu, China). The secondary antibody goat anti-rabbit IgG H&L (HRP) was provided by Abcam (Cambridge, UK). TSA-FITC and TSA-CY3 were purchased from Pinuofei Biotechnology Co., Ltd. (Wuhan, China). Ziehl and Golgi dye kits were obtained from ServiceBio (Wuhan, China).
Pharmacological research on BSTJF
LC–MS analysis of BSTJF
BSTJF was prepared by soaking all the raw materials in water for 30 min and then decocting it for 60 min. Standard stock solutions of 30 chemical compounds were prepared at a concentration of 1 mg/ml by dissolving them in 70% methanol solutions and then mixing. All samples were centrifuged at 10,000 rpm for 20 min. The supernatants were collected and subjected to LC–MS analysis.
An Acquity UPLC system (Waters, Milford, MA, USA) coupled with a Triple TOF 5600plus MS (AB SCIEX, Framingham, MA, USA) was employed for chemical identification. Chromatographic separation was performed on a Waters ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm i.d. 1.8 μm) at 35 °C with mobile phase A (0.1% formic acid–water) and mobile phase B (0.1% formic acid-40% acetonitrile–water). The flow rate was 0.25 ml/min and a linear gradient elution was programmed: 0–7 min, 0–5%B; 7–14 min, 5–12%B; 14–25 min, 12–16%B; 25–40 min, 16–25%B; 40–50 min, 25–35%B; 50–60 min, 35–55%B; 60–62 min, 55–65%B; 62–65 min, 65–75%B; 65–68 min, 75–100%B; 68–73 min, 100–100%B. The injection volume was 3μL. Mass spectrometry analysis was performed in positive and negative mode with the following parameters: scan range, m/z 90–1500; ion source GS1, 50 psi; ion source GS2, 50 psi; curation gas (CUR), 35 psi; temperature, 600 °C for ESI+ and 550 °C for ESI-; ion spray voltage (IS), -4.5 kV for ESI-; and 5.0 kV for ESI+.
Molecular networking and network pharmacology analyses
The molecular network was constructed as previously reported [33]. MS Convert and MZ mine 2.53 were used for LC–MS/MS data processing.
Mass detection was performed by fixing the noise level at 100 for MS1 and 0 for MS2. Chromatogram building was achieved using the ADAP chromatogram builder with a minimum group size of 5 scans, a group intensity threshold of 100, a minimum highest intensity of 100 and a m/z tolerance of 0.01 Da (or 10 ppm). For chromatogram deconvolution, the wavelet (ADAP) method was selected with the following settings: S/N threshold = 10, minimum feature height = 100, coefficient/area threshold = 50, peak duration range 0.00–10.00 min, and retention time (RT) wavelet range 0.0–0.50. MS/MS scans were paired using a m/z tolerance range of 0.02 Da and RT tolerance range of 1 min. The isotopic peak grouper algorithm was used with a m/z tolerance of 0.01 Da (or 10 ppm) and an RT tolerance of 0.1 min. Thereafter, the peak list was filtered using a peak list row filter to keep only peaks with the MS2 scan and reset the peak number ID. Finally, the data file, including the retention time, peak area, and other information of the compounds, were exported.
The corresponding molecular networking was created according to the online workflow at GNPS (http://gnps.ucsd.edu) [34]. In this analysis, the parent mass tolerance and fragment ion mass tolerance were set at 0.02 Da. The minimum cluster size was set at 1, the network topology was 20, and the maximum connected component size was 200. In addition, the MS cluster and filter precursor window tools were turned off. The network was created with a cosine score above 0.7 and more than six matched peaks. The molecular networking data were visualized using Cytoscape (version 3.8.0).
The ingredients identified by UPLC-Q/TOF–MS analysis of BSTJF were considered candidate compounds. Symmap database (https://www.symmap.org/) [35], BATMAN database (http://bionet.ncpsb.org/) [36] and TargetNet database (http://targetnet.scbdd.com) [37] was used to predict the putative targets. The selection criteria of the target were as follows: for the Target-Net database, targets with area under the receiver operating characteristic curve ≥ 0.7 and probability > 0.9 and targets with score cutoff > 20 were retained. Then, all targets were converted to official gene symbols using the UniProt database (https://www.UniProt.org/). KEGG pathway enrichment analysis was performed using the DAVID database (https://david.ncifcrf.gov/) [38].
The effects of BSTJF on the PCOS model rats
Establishment of the PCOS rat model
All animal studies were approved by the Ethical Review Committee of the Experimental Animal Welfare, Zhejiang University (Zhejiang, China), according to the European Community guidelines (EEC Directive of 1986; 86/609/EEC). Forty-five neonatal female Sprague–Dawley rats, provided by Vital River (Beijing, China), were randomly divided into the PCOS model and control groups (n = 30 in the PCOS group and n = 15 in the control group). All animals were fed a standard laboratory diet and housed under a 12:12 h light/dark cycle at 25 °C.
Nine days after birth, the PCOS group was induced by subcutaneous injection of testosterone propionate at a dose of 0.1 mg/0.004 ml sesame oil per g of animal [39], and the controls only received sesame oil. When the rats were 8 weeks old, the estrous cycle of the rats was measured for 14 consecutive days by analyzing the vaginal smears cell types [40]. For the PCOS group, we retained only rats with irregular vaginal smears for the subsequent experiments. In addition, two rats in each group were randomly sacrificed to obtain ovarian tissue for model verification. Then, the PCOS model rats were randomly divided into the PCOS and BSTJF groups.
Intervention with BSTJF on PCOS rat
The BSTJF decoction was as follows: A total of 100 g of raw herb (the composition of BSTJF is described in 2.1. of the Materials and reagents section), a daily dose for adults, were decocted with water 10 times for each decoction with three total decoctions (1 h each). The decoction solutions were then combined, filtered, and concentrated into a solution with a concentration of 2.00 g/ml, which was hermetically stored at 4 °C.
The BSTJF group received a BSTJF decoction infusion once a day at a dose of 333 mg/200 g for 4 consecutive weeks, whereas the control and PCOS groups only received the same volume of distilled water. The estrous cycle was observed during the last 2 weeks of infusion, and the ovaries were removed from the sacrificed rats (4 rats in each group) and fixed in 4% paraformaldehyde for morphological observation by hematoxylin and eosin (H&E) staining.
Double-labeling immunofluorescence
Double-labeling immunofluorescence was used to detect the expression of target proteins in ovarian granulosa cells according to the manufacturer's instructions. Ovarian granulosa cells were specifically labeled with anti-Foxl2 antibody [41], and the target proteins were labeled with the corresponding antibodies.
Two slides were prepared for double-labeling immunofluorescence, and the samples on the slides were cut from each ovary sample. The slides were blocked with 3% bovine serum albumin (BSA) for 30 min at room temperature (RT) (20–25 °C). The slides were then incubated with the primary antibodies overnight at 4 °C. The following antibodies were used: anti-ERK1/2 (1:200; Proteintech Group, Inc., Rosemont, USA; no. 11257–1-AP) and anti-FOS (1:200; Affinity Biosciences, Ltd., Jiangsu, China; no. AF0132). The slides were then incubated with goat anti-rabbit IgG H&L (HRP) (1:2000; Abcam, Cambridge, UK; no. Ab205718) secondary antibody for 50 min at RT. Then, anti-Foxl2 (1:200; Proteintech Group, Inc., Rosemont, USA; no. 19672–1-AP) was added to the slide and incubated overnight at 4 °C. Thereafter, goat anti-rabbit IgG H&L (HRP) (1:2000; Abcam, Cambridge, UK; no. Ab205718) secondary antibody was incubated with the sample for 50 min at RT. Nuclei were stained with 40,6-diamidino-2-phenylindole (DAPI). Fluorescence signals were visualized using a confocal laser microscope. The value of Foxl2 and target protein double-positive cell number/FoxL2 positive cell number were used to describe the expression rate of ovarian granulosa cells to the target protein.
Observation of the neurobehavioral manifestations and morphology of the dentate gyrus of female offspring born to PCOS rats administered by BSTJF
Offspring acquisition
The three groups of rats were injected intraperitoneally with 20 IU pregnant mare serum (PMSG) at 5:00 pm, followed by 20 IU of human chorionic gonadotropin (HCG) after 48 h [42] and were immediately mated with male rats at a scale of 1:1. Once vaginal sperm plugs were observed the following day, the female rats were raised separately, and their weights were monitored twice a week to determine if they were pregnant. The dams were allowed to deliver the pups naturally, and the birth date of progeny was considered postnatal day (PND) 0. The pups were weaned on PND 21, and then the female and male pups were fed separately.
Learning and memory testing with the Morris water maze in the female offspring
The Morris water maze test was performed on female offspring at 5 weeks (puberty) and 13 weeks (adulthood) old. Three progeny females were randomly selected from the progeny of each female parent to participate in the experiment. The water maze apparatus consisted of a circular water tank 1.5 m in diameter and was filled to a depth of 60 cm with water (22 ± 2 °C). The tank was divided into four quadrants. A circular platform was placed in one of the quadrants (target quadrant), 1 cm below the water surface. The rats were subjected to three learning sessions per day for 5 days and a memory retention test (probe test) on the fifth day. Subsequently, an estrous cycle was observed after the probe testing. In the learning sessions, the rats were placed at the same starting positions per quadrant and allowed to freely swim and find the hidden platform (target) for 60 s. The time it took a rat to reach the platform after it was placed in the water was recorded as the latency to the target. If the rat did not locate the platform within 60 s, it was guided to the platform and remained there for 10 s, and its latency to target was recorded as 60 s. On the fifth day of the probe test, the platform was removed, and the rats were allowed to swim freely for 60 s. One female offspring rat at the diestrus stage was randomly taken from each family. The distance to the target, latency to the target in the training sessions, and the distance, time, entries in the target zone and in the target quadrant during the probe test were recorded using a camera linked to a computerized video tracking system (DMS-2MORRIS water maze system, Institute of Medicine, Chinese Academy of Medical Sciences. Beijing, China).
Brain tissue collection of the female offspring rats
After completing the Morris water maze test, the female offspring were anesthetized with sodium pentobarbital and transcardially perfused with 100 ml of saline followed by 200 ml of a fixative containing 2% glutaraldehyde and 2% paraformaldehyde in 0.01 mol/l phosphate buffer (PB, pH 7.4). Then, the brains were removed from the skulls and cut sagittally in half. The left hemispheres were stored in a fixative solution at 4 °C for subsequent paraffin embedding. For the right hemispheres, the dentate gyrus of the hippocampus in some of the samples from each group was exfoliated completely and cut into 1 × 3 mm pieces to be preserved in glutaraldehyde for subsequent electron microscope observation. The remaining samples of each group were fixed for Golgi staining or the hippocampus was removed and stored at − 80 °C.
Nissl staining
The left hemisphere samples were paraffin-embedded and sectioned coronally into 4-μm slices in the dentate gyrus area. Nissl staining was used to observe the morphology of the dentate gyrus of female offspring. The results were examined under a light microscope. (DM2500, Leica Microsystems, Germany).
Golgi staining
Golgi staining was used to observe the morphology of dendrites and analyze the density of dendritic spines in the dentate gyrus. The hemisphere tissue of the female offspring was stained with Golgi dye according to the manufacturer's instructions and then sliced. Image J software was used to analyze the Golgi-stained images, and dendritic spines were observed under a 400-fold microscope. Starting from the first branch of the dendritic cells from the cell body, the number of dendritic spines in the 30–60 μm length range was calculated, and the density was obtained.
Transmission electron microscopy
Transmission electron microscopy was used to observe the ultrastructure of neurons and synapses in the dentate gyrus of female offspring. First, the dentate gyrus tissue was cut into 1 × 3 mm blocks and stored in glutaraldehyde for 2 h at room temperature. Thereafter, the samples were rinsed with BP buffer, postfixed with 2% osmium tetroxide for 1–2 h, rinsed again with BP buffer, dehydrated in an ethanol series, infiltrated in a mixture of Spurr and acetone, embedded in 100% Spurr embedding agent overnight and polymerized for 24 h, and then the samples were cut into 70 nm thick slices for transmission electron microscopy (Hitachi H-7650, Japan) observation using a side-mounted CCD camera (Model 830 SC200, Gatan, USA) at 8000×, 25000×, and 50,000× magnification.
Immunohistochemistry
To further investigate the mechanism of action of BSTJF on female offspring born to PCOS, based on our previous work [27], immunohistochemistry was used to observe the expression of Gabrb1, Grin2b, and Adra1b in the dentate gyrus of female offspring rats. Anti-Gabrb1 (1:100; Affinity Biosciences, LTD., Jiangsu, China; no. AF6207), anti-Grin2b (1:50; Proteintech Group, Inc., Rosemont, USA; no. 21920–1-AP), and anti-Adra1b (1:100; Proteintech Group, Inc., Rosemont, USA; no. 22419–1-AP) were used as primary antibodies at RT for 1 h, followed by incubation with hypersensitive rabbit/mouse universal secondary antibody (Tuling Hangzhou Biomedical Co., Ltd; Hangzhou, China; no. I20012C) for 30 min at RT. Digital images of the dentate gyrus were captured using a light microscope (ECLIPSE E100, NIKON, Japan). Gabrb1-, Grin2b-, and Adra1b-immunoreactive neurons were counted, and the positive rate was described by integrated optical density (IOD).
Western blotting
The hippocampal tissue was fully ground with a mortar and pestle, followed by treatment with lysate. Next, ultrasound was used to turn the tissue into a homogenate, which was centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatants were collected. The protein concentration was measured by bicinchoninic acid (BCA) assay. Protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE 8%) and transferred to polyvinylidene difluoride membranes (soaked in methanol approximately 10 s before use) in transfer buffer. The membranes were incubated for 2 h in blocking solution (5% skim milk). Then, the membranes were washed with TBST and incubated with primary antibodies at 4 °C overnight. The membranes were washed three times in TBST for 10 min each time and incubated with secondary antibody for 1 h at room temperature. β-actin was used as a loading control. The immune complexes were detected using enhanced chemiluminescence. The density of specific bands was analyzed using Image J software.
The dilution ratios of primary antibodies were as follows: anti-Gabrb1 (1:1000; Affinity Biosciences, LTD., Jiangsu, China; no. AF6207); anti-Grin2b (1:1000; Proteintech Group, Inc., Rosemont, USA; no. 21920–1-AP); and anti-Adra1b (1:1000; Proteintech Group, Inc., Rosemont, USA; no. 22419–1-AP).
Statistics analysis
Data are presented as the mean ± SD. Statistical significance was determined using one-way ANOVA or non-parametric test for multiple comparisons and LSD test for pairwise comparisons between groups. Fisher’s exact test was used for comparisons among categorical variables. Statistical significance was set at p < 0.05.
