Animal assays
The 8 weeks old female rats was obtained from laboratory animal center of Guangdong province (Guangdong, China). These experimental rats were bred normally for one week. After that, the vaginal smear was performed and the estrous cycle was observed. Then, 24 rats with two normal estrous cycles were selected for the next experiments. These rats were divided into three groups (Control, Model and EBM treatment groups). Next, the gavage was performed to give the tripterysium glycosides (50 mg/kg.d) to the rats of model and EBM treatment groups for two weeks. And the estrous cycle was observed from the eighth day after the gavage.
When the estrous cycle disorder occurs in these rats, the establishment of DOR model is successful. When the DOR models was constructed, the extractive of EBM (110 mg/kg.d) was dissolved in the saline solution and given to the rats of EBM treatment group by gavage. The rats of control and model groups were given with the equal volume of normal saline solution. These rats were fasted for 24 hours after the last treatment, then these rats were anesthetized and the blood was collected for the next research. After that, the ovaries were quickly gathered and weighed. At last, half of the ovarian tissues was fixed in 4% paraformaldehyde solution for HE staining. Granulosa cells were extracted from the other half of the ovarian tissues for subsequent analysis.
Elisa Assays
The blood was centrifuged using a high-speed centrifuge (Beckman Coulter, USA) and then isolated the serum. The serum was collected into the sterilized tube for the ELISA assays. The ovary granulosa cells were separated from the ovarian tissue and lysed with the RIPA buffer (Beyotime, China). After that, the FSH Rat ELISA kit (Senbeijia Bio, SBJ-R0712), LH Rat ELISA kit (Runyu Bio, China), E2 ELISA kit (Baiaolaibo Bio,ARB12674), AMG antibody (Abcam, ab54507) and INHB Rat ELISA kit (Jianglai Bio, China) were used for the detection of the levels of corresponding indicators in serum of rats from diverse groups. Next, the androgen receptor (AR) Rat ELISA kit (Tongwei Bio, China), estrogen receptor (ESR1) Rat ELISA kit (Jianglai Bio, China), CYP19A1 Rat ELISA kit (Boshikang Bio, China) were used for the detection of the levels of the corresponding indicators in the granulosa cells from these rats in different groups. All the operations in these assays was followed the guidelines.
He Staining
The procedures of HE staining followed the routine protocols [8]. Firstly, the deparaffinization and rehydration were performed for the staining of the ovarian tissue. After that, 5 µm tissue sections was stained with hematoxylin solution for 6 minutes. Then these sections were steeped in the 1% acid ethanol and washed with the distilled water. Next, these sections were stained with the eosin solution for 5 minutes. At last, these sections were dehydrated with the graded alcohol and washed with the xylene. The photographs were taken with the fluorescence microscope (Olympus, Tokyo, Japan).
Chemical Composition Database And Active Ingredient Screening
The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, http://lsp.nwu.edu.cn/) and the Traditional Chinese Medicines Integrated Database (TCMID, http://119.3.41.228:8000/) were used to search the information of active ingredients in EBM. Because the composition of traditional Chinese medicine is complicated, not all ingredients will play the role in human bodies. Therefore, the active ingredients of EBM were selected according to the pharmacokinetic parameters including the oral bioavailability (OB) ≥ 30%, drug-likeness (DL) ≥ 0.18 and permeability of Caco2 > 0 [9, 10]. Oral bioavailability (OB) is the percentage of orally drugs that entered the systemic circulation, which is one of the most commonly used pharmacokinetic properties in drug screening. DL value is mainly used to indicate the similarity of related active ingredients with known drugs in the compound [11]. The absorption of oral drugs is mainly in the small intestine. The presence of villi and microvilli greatly increases the absorption rate. The absorption rate of drugs in the intestine is the crucial factor which could affect the bioavailability of drugs. Furthermore, gastrointestinal epithelial cells are the only way for the absorption of oral drugs. The Caco-2 cell model is an intestinal transport model and an epithelial transport model which were widely used in laboratories to evaluate the absorption of drug in vitro. Compounds are impermeable when the values of Caco-2 less than 0, so the threshold of Caco-2 permeability is set to 0.
Disease-related Targets
The 19 compounds screened in the previous step were imported into PharmaMapper (http://lilab-ecust.cn/pharmmapper/) to predict the potential therapeutic targets of EBM. According to the keywords “diminished ovarian reserve”, we searched the DOR related genes in the GeneCards database (https://www.genecards.org/). At last, the VENNY2.1 (https://bioinfogp.cnb.csic.es/tools/venny/index.html) was used for the screening of the potential targets which was affected by EBM active ingredients during the development of DOR.
Construction Of Target Proteins Network
The String database (http://string-db.org) was used for the analysis of target proteins network. The species of these proteins are limited to “Homo sapiens”. In the network, the green elements represent active ingredients in EBM, while the yellow elements represent disease-related proteins. The red line represents the targeted effect of the active ingredients in the EBM for these proteins, and the blue line represents the interaction between these proteins. We used Network Analyzer to analyze the connectivity of each protein to other proteins. The higher values of connectivity implied that numerous proteins interacted with this protein. It also showed that this protein played the critical role in the occurrence and development of disease.
Enrichment Analysis For Target Proteins
The Gene Ontology (GO) enrichment analysis was used for the analysis of the function of target genes. Therefore, we further predicted that EBM affected the occurrence and development of DOR by affecting the biological function of the corresponding proteins and signaling pathways. And the lower of P values implied that the close relationship between the targeted protein and DOR. And values of P less than 0.05 is considered statistically significant.
Statistical analysis
All the experiments in this study was repeated for three times. And the data was presented as mean ± SD. All the data was analyzed with the Graphpad Prism7.0 (GraphPad Software, La Jolla, CA, USA). And the comparison between diverse groups in histograms was performed with the student’s t test. When the P value is less than 0.05, the difference is considered statistically significant.