Use of sanziguichao decoction in treatment of premature ovarian insufficiency and biomarker of premature ovarian insufficiency

Abstract

The present invention relates to the technical field of biomedicine. Disclosed are the use of Sanziguichao decoction in the treatment of premature ovarian insufficiency and a biomarker of premature ovarian insufficiency. The present invention develops traditional Chinese medicine compound Sanziguichao decoction, which has a significant effect on the treatment of premature ovarian insufficiency, is verified by means of experiments to have the effect of improving the ovarian function of rats in a model group, and provides a new therapeutic drug for treating premature ovarian insufficiency. Network pharmacology analysis further identifies the specific targets of Sanziguichao decoction in the treatment of premature ovarian insufficiency. Moreover, combined proteomic and genomic analysis further finds the key genes of premature ovarian insufficiency. These targets and key genes have a plurality of application prospects in subsequent research on the individualized efficacy of Sanziguichao decoction, the preparation or development of diagnostic kits for ovarian insufficiency, the screening of drugs for treating premature ovarian insufficiency, etc.

Description

Application of Sanzi Guichao Decoction in the Treatment of Premature Ovarian Insufficiency and Biomarkers of Premature Ovarian Insufficiency Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of Sanzi Guichao Decoction in the treatment of premature ovarian insufficiency and biomarkers of premature ovarian insufficiency. Background Technology

[0002] The statements in this section provide only background information relevant to the disclosure of this application and may not constitute prior art.

[0003] Premature ovarian insufficiency (POI) is one of the most challenging gynecological diseases to address in clinical practice. POI is defined as the loss of normal ovarian function in women before the age of 40. Currently, the incidence rate in women is approximately 0.1% before age 30 and 2-4% before age 40. POI increases the risk of chronic diseases, including osteoporosis and cardiovascular disease. The causes of POI involve multiple factors, including genetics, immunity, environment, and iatrogenic factors. The gonadotoxic components of chemotherapy drugs, particularly alkylating agents commonly used during reproductive years, have been shown to potentially induce iatrogenic ovarian insufficiency. Currently, hormone replacement therapy is the primary clinical approach to alleviate menopausal symptoms caused by hypoestrogenemia in POI patients. Physiological estrogen levels can be achieved through oral administration (1-2 mg estradiol daily or 0.625-1.25 mg estrogen daily) or transdermal estrogen administration (0.1 mg daily). However, while this medication can restore menstruation, it cannot restore ovarian function. Simultaneous estrogen / progesterone therapy increases many health risks, including breast cancer, stroke, and cardiovascular disease. Women with POI are more prone to osteoporosis and fractures due to estradiol deficiency; premature menopause and chronic estradiol deficiency can lead to osteoporosis. Furthermore, young women with POI have significantly lower bone mineral density compared to women with regular menstrual cycles. For infertility in POI patients, current main solutions include oocyte donation and cryopreservation, but unfortunately, these methods have certain limitations and challenges.

[0004] Therefore, we urgently need to find a new drug that can not only treat ovarian damage, restore and protect ovarian function, but also reduce the risk of osteoporosis. Summary of the Invention

[0005] The purpose of this invention is to address the urgent need for new drugs to treat premature ovarian insufficiency by providing the application of Sanzi Guichao Decoction in the treatment of premature ovarian insufficiency and biomarkers for premature ovarian insufficiency. This invention offers a new drug compound—Sanzi Guichao Decoction—for the treatment of premature ovarian insufficiency. Furthermore, based on proteomics and genomics research, relevant gene markers for premature ovarian insufficiency have been discovered, providing new ideas and methods for the subsequent development and screening of drugs for the diagnosis or treatment of premature ovarian insufficiency.

[0006] The technical solution of the present invention is as follows:

[0007] This invention provides the application of Sanzi Guichao Decoction in the treatment of early-onset ovarian insufficiency. Sanzi Guichao Decoction comprises the following ingredients in parts by weight: 2-8 parts of raspberry, 2-8 parts of wolfberry, 2-8 parts of dodder seed, 2-4 parts of angelica, 2-8 parts of raw white peony root, 2-4 parts of prepared rehmannia root, 2-4 parts of yam, 2-4 parts of morinda root, 1-3 parts of chuanxiong rhizome, and 1-3 parts of cyperus rhizome.

[0008] The renowned Qing Dynasty physician Ye Tianshi, in his *Clinical Guide to Medical Cases*, proposed that "the liver is the innate foundation for women," emphasizing the importance of nourishing and regulating the liver for women's physiological health. This formula has achieved excellent clinical results, primarily targeting the liver meridian. The principal herbs are: Rubus idaeus, Lycium barbarum, and Cuscuta chinensis, which are sweet and warm or sweet and neutral, entering the liver, kidney, and bladder meridians. They primarily target the root causes of liver and kidney deficiency, imbalance of the Chong and Ren meridians, and malnourishment of bone marrow, nourishing the liver and kidneys, regulating the Chong and Ren meridians, and replenishing essence. The assistant herbs are: raw white peony root, prepared Rehmannia glutinosa, and Morinda officinalis, assisting the principal herbs in enhancing their nourishing effect. Raw white peony root is bitter, sour, and slightly cold, entering the liver and spleen meridians, and also astringes liver yin. Morinda officinalis is sweet and warm, and assists yang. The adjuvant herbs are: Angelica sinensis and Dioscorea opposita, which assist the principal herbs while harmonizing yin and yang, nourishing blood, regulating menstruation, and strengthening the spleen and kidneys. The guiding herbs are: Ligusticum chuanxiong and Cyperus rotundus, which invigorate blood circulation, regulate qi, soothe the liver, relieve depression, and guide the medicine to the affected area. The entire formula focuses on tonifying the liver, kidneys, and strengthening the marrow, supplemented by promoting blood circulation and regulating qi. It aims to treat early-onset ovarian insufficiency and prevent and improve osteoporosis. By harmonizing yin and yang and tonifying the Chong and Ren meridians, it achieves the goal of restoring ovarian function and preventing and improving osteoporosis.

[0009] According to a preferred embodiment, the Sanzi Guichao Decoction targets TP53, ESR1, CASP3, IL6, JUN, CCND1, AKT1, HIF1A, VEGFA, PTGS2, and CALB2.

[0010] According to a preferred embodiment, the early-onset ovarian insufficiency includes decreased estrogen levels, with hormone indicators including E2, FSH, LH, and AMH.

[0011] According to a preferred embodiment, the early-onset ovarian insufficiency disease also includes osteoporosis caused by decreased estrogen levels.

[0012] Another aspect of the present invention provides the application of Sanzi Guichao Decoction in the preparation of a drug for treating early-onset ovarian insufficiency. The Sanzi Guichao Decoction comprises the following components by weight: 2-8 parts of raspberry, 2-8 parts of wolfberry, 2-8 parts of dodder seed, 2-4 parts of angelica, 2-8 parts of raw white peony root, 2-4 parts of prepared rehmannia root, 2-4 parts of yam, 2-4 parts of morinda root, 1-3 parts of chuanxiong rhizome, and 1-3 parts of cyperus rhizome.

[0013] Another aspect of the present invention provides a sustained-release formulation, targeted formulation, or nano-formulation for treating early-onset ovarian insufficiency. The effective components of the formulation include extracts of the following substances in parts by weight: 2-8 parts of raspberry, 2-8 parts of wolfberry, 2-8 parts of dodder seed, 2-4 parts of angelica, 2-8 parts of raw white peony root, 2-4 parts of prepared rehmannia root, 2-4 parts of yam, 2-4 parts of morinda root, 1-3 parts of chuanxiong rhizome, and 1-3 parts of cyperus rhizome.

[0014] Another aspect of the present invention provides a sustained-release formulation, targeted formulation, or nano-formulation for treating early-onset ovarian insufficiency, characterized in that the extract is an aqueous extract.

[0015] In another aspect, the present invention also provides a set of genetic markers for screening and identifying early-onset ovarian insufficiency that can be treated with Sanzi Guichao Decoction, the biomarkers including TP53, ESR1, CASP3, IL6, JUN, CCND1, AKT1, HIF1A, VEGFA and PTGS2.

[0016] The Sanzi Guichao Decoction comprises the following ingredients in parts by weight: 2-8 parts of raspberry, 2-8 parts of wolfberry, 2-8 parts of dodder seed, 2-4 parts of angelica root, 2-8 parts of raw white peony root, 2-4 parts of prepared rehmannia root, 2-4 parts of yam, 2-4 parts of morinda root, 1-3 parts of chuanxiong rhizome, and 1-3 parts of cyperus rhizome.

[0017] In another aspect, the present invention also provides a set of biomarkers for early-onset ovarian insufficiency, including CALB2 gene markers or CALB2 protein markers.

[0018] In another aspect, the present invention also provides the application of the aforementioned biomarkers in the preparation, identification, and detection of products for early-onset ovarian insufficiency.

[0019] The testing products include reagents, chips, test cards, or kits.

[0020] In another aspect, this invention also provides the use of CALB2 as a biomarker in screening or developing drugs to improve early-onset ovarian insufficiency.

[0021] In another aspect, the present invention also provides the application of reagents capable of detecting and identifying CALB2 gene markers or CALB2 protein markers in the preparation of products for the identification and detection of early-onset ovarian insufficiency.

[0022] Another aspect of the present invention provides a kit for diagnosing early-onset ovarian insufficiency, comprising reagents for detecting CALB2 expression levels.

[0023] According to a preferred embodiment, the CALB2 expression level is the CALB2 mRNA expression level or the CALB2 protein expression level.

[0024] Compared with existing technologies, the advantages of this invention are:

[0025] 1. A traditional Chinese medicine compound, Sanzi Guichao Tang, which has a significant effect on the treatment of early-onset ovarian insufficiency, was developed and formulated. Animal model experiments have confirmed that it can improve ovarian function in rats in the model group.

[0026] 2. Through network pharmacology analysis, the specific targets of Sanzi Guichao Decoction in the treatment of early-onset ovarian insufficiency were further clarified, including TP53, ESR1, CASP3, IL6, JUN, CCND1, AKT1, HIF1A, VEGFA, and PTGS2. These key targets have great practical significance for subsequent research on the individualized efficacy of Sanzi Guichao Decoction, the preparation or development of diagnostic kits for ovarian insufficiency, and the screening of drugs for the treatment of early-onset ovarian insufficiency.

[0027] 3. Further analysis using a combination of proteomics and genomics revealed key genes for early-onset ovarian insufficiency. These genes are of significant importance in the diagnosis and drug development and screening processes for early-onset ovarian insufficiency, and can promote the development of treatment for this disease. Attached Figure Description

[0028] Figure 1 shows the weight change data of rats in each group in Example 1;

[0029] Figure 2 shows the ovarian and uterine indices of rats in each group in Example 1; P<0.05, P<0.01, P<0.01, P<0.01;

[0030] Figure 3 shows the organ index of rats in each group in Example 1; P<0.05, P<0.01, P<0.01, P<0.01;

[0031] Figure 4 shows the estrous cycle variation pattern of rats in each group in Example 1;

[0032] Figure 5 shows ovarian sections of rats in each group in Example 1 (HE staining, left 100×, right 400×).

[0033] Figure 6 shows the periodic changes in estrogen levels in rats of each group in Example 1; P<0.05, P<0.01, P<0.01, P<0.01;

[0034] Figure 7 shows the changes in E2, FSH, LH, and AMH hormone levels in each group of rats in Example 1; P<0.05, P<0.01, P<0.01, P<0.01;

[0035] Figure 8 shows the immunohistochemical staining of rat ovarian tissue in each group in Example 1 (200×).

[0036] Figure 9 is a comparison of femoral biomechanical parameters of rats in each group in Example 1;

[0037] Figure 10 is a comparison of femoral bone mineral density in each group of rats in Example 1;

[0038] Figure 11 shows the protein-target interaction diagram of the top 10 intersection points in Example 2;

[0039] Figure 12 shows the differential gene intersection of normal group A, POI model group B, and Sanzi Guichao Decoction administration group C in Example 3.

[0040] Figure 13 shows the PCA scores of the proteomics samples in Example 3; the triangle formed by A1, A2, and A3 in the figure is the NC group; the triangle formed by B1, B2, and B3 is the POI group; and the triangle formed by C1, C2, and C3 is the SZGC group. Embodiments of the present invention

[0041] It should be noted that, unless otherwise specified, the test methods in the following embodiments are all conventional methods. All test materials used in the following embodiments are commercially available.

[0042] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0043] Preparation of Sanzi Guichao Decoction: Weigh each component of Sanzi Guichao Decoction according to the proportion, add water and extract to obtain Sanzi Guichao Decoction suspension; The content of each component of Sanzi Guichao Decoction is as follows: The formula of Sanzi Guichao Decoction is: Raspberry 15g, Lycium barbarum 15g, Cuscuta chinensis 15g, Angelica sinensis 15g, raw white peony root 15g, prepared Rehmannia glutinosa 15g, Dioscorea opposita 15g, Morinda officinalis 15g, Ligusticum chuanxiong 10g, Cyperus rotundus 10g.

[0044] Example 1: Animal experiment verification of Sanzi Guichao Decoction in treating premature ovarian insufficiency

[0045] I. Animal Modeling

[0046] Eight female SD rats aged 8-9 weeks were randomly selected from the control group. After one week of acclimatization, the remaining animals were injected intraperitoneally with cisplatin 2 mg / kg for 7 consecutive days to establish a POI rat model. The control group was injected intraperitoneally with the same dose of 0.9% saline.

[0047] II. Experimental Grouping and Drug Treatment

[0048] Preliminary experiments determined that rats that successfully survived the cisplatin-induced model were randomly divided into a model group, a Sanzi Guichao Decoction group, and a positive drug control group. After successful model establishment, gavage treatment was initiated. The Sanzi Guichao Decoction group (converted to an adult daily dose of 140 g / 60 kg) received 14.0 g / (kg·d) via gavage. Sanzi Guichao Decoction was diluted with water and distilled to prepare a suspension with a concentration of 1.40 g / mL, which was administered at a dose of 1 mL / 100 g for ease of administration. The positive drug control group received 0.1 mg / (kg·d) of estradiol via gavage at a dose of 1 mL / 100 g. The blank control group and the model group received 1 mL / 100 g and once daily saline via gavage, respectively.

[0049] After 28 days of administration, one animal died in both the positive control group and the model group. Subsequent data analysis was conducted using random sampling in the blank control group, the model group, and the Sanzi Guichao Decoction group to ensure data reliability, with a minimum number of animals in each group (n = 6).

[0050] III. General Condition Observation

[0051] During the experiment, we comprehensively observed and recorded the rats' food intake, water consumption, fur color, activity level, and mental state. These observations aimed to assess the overall health of the rats and ensure the accuracy of the experimental conditions. The rats were weighed daily, and changes in weight before and after drug administration were recorded. The general condition of the rats is shown in the table below:

[0052]

[0053] The weight changes of rats were statistically analyzed every 7 days. The data on weight changes of rats are shown in Figure 1. The overall trend shows that the weight change in the model group was significant during the modeling process, with a large decrease in weight, indicating a strong effect of cisplatin, thus causing emaciation in the model group. After 7 days of administration, a significant trend of weight reduction was observed between the normal group and the model group (P<0.01). After the modeling process ended on the seventh day, the positive drug control group and the model group showed little recovery, while the Sanzi Guichao Decoction group showed a slight increase, which was significant (P<0.01). On the 14th day after administration, both the traditional Chinese medicine group and the positive drug control group showed an increase compared to the model group, with significant differences (P<0.01). On the 28th day after administration, both the traditional Chinese medicine group and the positive drug control group showed a significant increase compared to the model group (P<0.01).

[0054] IV. Observation of Estrogenic Cycle Changes via Vaginal Cytological Smears

[0055] After modeling, starting from day 7 of gavage, we collected vaginal exfoliated cells from rats every morning at 9:00 AM for 14 consecutive days using sterile cotton swabs dipped in physiological saline. The collected cells were evenly spread on glass slides and then observed for morphology and staining under an optical microscope. To more accurately determine the changes in the rat estrous cycle, we stained these cells with hematoxylin and eosin (HE). The criteria for differentiation referenced the morphological changes of vaginal exfoliated cells from female rats at different stages of estrus; specifically, the ratio of leukocytes, nucleated epithelial cells, and keratinized squamous epithelial cells was used to distinguish the four stages of the estrous cycle. Proestrus smears were mainly composed of nucleated epithelial cells, estrus smears were mainly composed of nucleated keratinocytes, and diaestrus smears were predominantly composed of leukocytes.

[0056] The smear data are shown in Figure 4. After modeling, the estrous period was significantly shortened in all three groups, while the proestrous and estrous phases were prolonged. After drug administration, compared with the model group, the traditional Chinese medicine group and the positive control group had a prolonged estrous period and a shortened proestrous and estrous phase, indicating that the estrous cycle was significantly improved after drug administration.

[0057] V. Periodic submandibular blood sampling to observe changes in sex hormone levels

[0058] To collect blood, this experiment used a frequency of once every 7 days, obtaining samples via submandibular vein sampling.

[0059] The results are shown in Figure 6. In the normal group, there was no significant trend in the cycle over 35 days. In the model group, estrogen levels decreased significantly after 7 days of modeling (P<0.01), with no significant changes in subsequent periods. In the drug-treated group, estrogen levels decreased significantly after 7 days of modeling (P<0.01), remained unchanged one week after drug administration compared to 7 days, and increased significantly after day 21 (P<0.01). In the positive control group, estrogen levels decreased significantly after 7 days of modeling (P<0.01), and increased significantly after day 21 (P<0.01).

[0060] VI. Sample Collection

[0061] Animals were fasted on the evening of the last day after gavage, and samples were collected the following morning. Anesthesia was administered via intraperitoneal injection of 200 μl / 100 g of 3% pentobarbital solution, ensuring complete anesthesia. Blood samples were collected from the heart using blood collection tubes and allowed to stand at room temperature for 1 hour. After standing, the blood was centrifuged at 3000 rpm for 15 minutes, and the supernatant serum was collected and stored at -20°C for later analysis. Rats were euthanized by vertebral dislocation. The abdominal cavity was opened using surgical instruments, and the heart, liver, spleen, lungs, kidneys (bilateral), and uterus were removed and immediately weighed on ice. Both ovaries were removed, weighed, and aliquoted. Half of the ovaries were placed in paraformaldehyde (4% or higher) and the other half was stored at -80°C for later use. Both femurs were removed from the rats; half were placed in 4% paraformaldehyde, and the other half on ice and stored at -80°C for later use.

[0062] VII. Organ and Gonadal Indices

[0063] Organ index = organ wet weight (g) / mouse body weight before anesthesia × 100%.

[0064] Ovarian index = bilateral ovarian wet weight (g) / mouse body weight before anesthesia × 100%.

[0065] Uterine index = uterine wet weight (g) / mouse body weight before anesthesia × 100%.

[0066] The ovarian and uterine indices of rats in each group are shown in Figure 2, and the organ indices are shown in Figure 3. As can be seen from Figures 2 and 3, the ovarian index was significantly lower in the model group compared to the normal group (P<0.01); the drug-treated group and the positive control group were significantly higher than the model group (P<0.01), but showed no significant trend compared to the normal group. The uterine index was significantly lower in the model group compared to the normal group (P<0.01); both the drug-treated group and the positive control group showed an increasing trend relative to the model group, but there was no significant difference. The heart, liver, and lung indices showed no significant trend in any group. The kidney index was significantly higher in the model group than in the normal group (P<0.01), and significantly higher in the drug-treated group and the positive control group than in the normal group (P<0.01). The drug-treated group and the positive control group showed a decreasing trend compared to the model group, but there was no significant difference. The spleen index was significantly higher in the positive control group than in the normal group (P<0.01), and also significantly higher than in the model group (P<0.01).

[0067] 8. ELISA detection of changes in serum levels of E2, FSH, LH, and AMH in experimental animals

[0068] After the administration was completed, serum samples were collected from each group of rats to measure E2. 、 The levels of four hormones, FSH, LH, and AMH, were measured. As shown in Figure 7, the E2 level was significantly lower in the model group compared to the normal group (P<0.01); however, the levels in the treatment group and the positive control group were significantly higher than those in the model group (P<0.05), and lower than those in the normal group.

[0069] Regarding FSH levels, the model group showed a significant increase compared to the normal group (P<0.01); the traditional Chinese medicine administration group showed a significant increase compared to the normal group (P<0.05), while the traditional Chinese medicine administration group showed a decrease compared to the model group; the positive control group showed a significant increase compared to the normal group (P<0.01), while the positive control group showed a decrease compared to the model group.

[0070] Regarding AMH levels, the model group showed a decreasing trend compared to the normal group; the drug-treated group and the positive control group showed an increasing trend compared to the model group, but the decrease was not significant compared to the normal group.

[0071] Regarding LH levels, the model group showed a significant increase compared to the normal group (P<0.01); the traditional Chinese medicine administration group showed no significant difference compared to the normal group, but the traditional Chinese medicine administration group showed a significant decrease compared to the model group (P<0.01); the positive control group showed a significant decrease compared to the normal group (P<0.01), and the positive control group showed a significant decrease compared to the model group (P<0.01).

[0072] IX. HE staining to detect changes in ovarian morphology, follicles, and granulosa cells.

[0073] As shown in Figure 5, the ovarian morphology and structure of the normal group were clear, and follicles of different grades were clearly visible, with normal granulosa cells. In the model group, there were fewer follicles, fewer primordial follicles, and fewer granulosa cells. In the traditional Chinese medicine treatment group, the ovaries were improved compared with the model group, with a slight increase in follicles and an increase in granulosa cells. In the positive control group, the morphology was improved compared with the model group, with a slight increase in follicles of different grades and an increase in granulosa cells.

[0074] 10. Immunohistochemical detection of changes in apoptosis-related proteins and estrogen-related proteins in ovarian tissue.

[0075] The results are shown in Figure 8. Bax: was mainly expressed in the cytoplasm of ovarian stromal cells, and was also observed in vascular smooth muscle cells. Expression in germ cells was low or not obvious. The model group showed a significant increase compared to the normal group, while the drug-treated group showed a significant decrease compared to the positive control group.

[0076] Bcl-2 is mainly expressed in the cytoplasm of ovarian stromal cells, and can also be seen in vascular smooth muscle cells. It is also significantly expressed in the cytoplasm of germ cells, and the expression is relatively diffuse. Compared with the normal group, the expression was significantly reduced in the model group and the normal group, while it was significantly increased in the drug-treated group and the positive control group.

[0077] Caspase-3 is mainly expressed in ovarian stromal cells and vascular smooth muscle cells, with little expression in germ cells. The expression was significantly elevated in the model group compared to the normal group, and significantly decreased in the drug-treated group compared to the positive control group.

[0078] VEGF is mainly expressed in ovarian vascular endothelial cells, vascular smooth muscle cells, and stromal cells, with low or minimal expression in germ cells. The expression was significantly lower in the model group compared to the normal group, and significantly higher in the drug-treated group compared to the positive control group.

[0079] ESR2: Diffusely expressed, mainly in the cytoplasm of various cell types. It was decreased in the model group compared to the normal group, and increased in the drug-treated group compared to the positive control group.

[0080] ESR1 is diffusely expressed on the cytoplasm of various cells and can also be expressed on the cell membrane. The expression level was decreased in the model group compared to the normal group, and increased in the drug-treated group compared to the positive control group.

[0081] CYP19A1 is a member of the cytochrome P450 superfamily (EC 1.14.14.1), which are monooxygenases that catalyze many reactions in steroid synthesis.

[0082] XI. Effects of Sanzi Guichao Decoction on Bone Mineral Density in Rats

[0083] Static biomechanical testing was performed using the Instron system to measure the pressure during femoral fracture under longitudinal stress. After removing surrounding tissue, the femur was placed on a test frame and subjected to a three-point bending stress test with a span of 30 mm. Load-displacement curves were generated using the instrument's built-in analysis software, and the elastic load (N), ultimate load (N), and flexural modulus (MPa) were analyzed based on the curves. The results are shown in Figure 9. Regarding elastic modulus, the model group showed a significant decrease under cisplatin induction (P<0.05), while the treated group showed an increase after administration of the Sanzi Guichao Decoction. There was little change in flexural displacement among the groups. In terms of maximum flexural load, the model group showed a significant decrease (P<0.01), while the treated group showed an increasing trend (P<0.01).

[0084] Bone mineral density (BMD) of the entire left femur in rats was measured using a multispectral small animal in vivo imaging system (model: X-RAY SYSTEM). The left femur was harvested according to previous animal experiments, and surrounding tissues were removed. The bone was fixed with 4% paraformaldehyde and stored at 4°C for in vitro BMD analysis. The bone was placed under the imaging probe of a bone densitometer, and the femur was exposed and scanned using MI software to measure BMD values. The results are shown in Figure 10. Compared with the normal group, the model group showed a significant decrease in BMD in the proximal, middle, and distal femurs, confirming that cisplatin induces a certain degree of decrease in femoral BMD in rats. Compared with the model group, the Sanzi Guichao Decoction treatment group showed an increase in BMD in the proximal, middle, and distal femurs, indicating that the administration of the decoction improved the cisplatin-induced decrease in BMD to some extent.

[0085] XII. Summary

[0086] Based on the results of animal experiments, general observations showed that the animals recovered well before and after administration, with good mental state and significant weight recovery. Vaginal smear observation of the estrous cycle showed a significant improvement in the estrous cycle compared to the model group, with a prolonged estrous period. HE staining of the ovaries revealed an increase in follicles, a clearer and more normal overall ovarian morphology, and an increase in granulosa cells compared to the model group. ELISA analysis of hormone levels showed trend changes in E2, FSH, LH, and AMH before and after administration, indicating a significant effect. Immunohistochemical observation of apoptosis and estrogen-related protein expression showed a decrease in apoptosis proteins and an increase in estrogen-related proteins in the ovaries after administration. Cisplatin induction significantly altered indicators related to pre-osteoporosis in POI model animals, while traditional Chinese medicine administration resulted in a certain degree of recovery in bone mineral density and other indicators, suggesting that Sanzi Guichao Decoction has a certain effect in improving osteoporosis. In conclusion, animal experiments have preliminarily verified the efficacy of Sanzi Guichao Decoction in treating POI.

[0087] Example 2: Detection of active ingredients in Sanzi Guichao Decoction and prediction of potential targets for the treatment of premature ovarian insufficiency.

[0088] I. Network pharmacology prediction of key active ingredients and potential targets of Sanzi Guichao Decoction in the treatment of premature ovarian insufficiency

[0089] 1.1 Searching Traditional Chinese Medicine Databases for Drug Components and Target Screening of Sanzi Guichao Decoction

[0090] Drug components were retrieved from the TCMSP database as potential active ingredients. Targets for these components were obtained from TCMSP, and the names of these targets were standardized using the Uniprot database. Results showed that, with OB≥30% and DL≥0.18, effective components from Angelica sinensis, Cuscuta chinensis, Rehmannia glutinosa, Lycium barbarum, Paeonia lactiflora, Morinda officinalis, Rubus idaeus, Dioscorea opposita, Ligusticum chuanxiong, and Cyperus rotundus were screened in the TCMSP database. After removing duplicates, potential active ingredients were obtained, and drug targets were identified through screening using the TCMSP database.

[0091] A total of 140 chemical components of Sanzi Guichao Decoction were obtained through the TCMSP database. After removing duplicates, 87 chemical components were finally obtained. The main active ingredients corresponding to the ten herbs in Sanzi Guichao Decoction are: Morinda officinalis (18), Paeonia lactiflora (9), Ligusticum chuanxiong (7), Angelica sinensis (2), Rubus idaeus (5), Lycium barbarum (39), Dioscorea opposita (13), Rehmannia glutinosa (1), Cuscuta chinensis (11), and Cyperus rotundus (16). A total of 1927 potential targets were identified for these ten herbs, of which the potential targets for each herb are: Morinda officinalis (141), Paeonia lactiflora (123), Ligusticum chuanxiong (42), Angelica sinensis (69), Rubus idaeus (154), Lycium barbarum (364), Dioscorea opposita (168), Rehmannia glutinosa (35), Cuscuta chinensis (343), and Cyperus rotundus (488). After removing duplicates, 276 targets were identified. Importing the above targets into Cytoscape 3.8.2 software and constructing a network diagram of the active ingredients and targets of Sanzi Guichao Decoction can achieve visualization.

[0092] 1.2 Retrieve genes related to premature ovarian insufficiency from disease databases

[0093] Using the OMIM and Genecards databases, a search was conducted using "Premature ovarian insufficiency" as the keyword. After identifying disease targets, duplicates were removed. The UniprotKB database was used to standardize gene IDs, resulting in a total of 823 POI-related targets.

[0094] 1.3 Network Construction and Analysis of Disease-Compound Effective Components

[0095] By drawing Venn diagrams using R software, common drug targets-diseases can be identified. A network diagram is constructed using Cytoscape 3.7.2 software, and its function as a major active ingredient in traditional Chinese medicine is analyzed. A protein-protein interaction network diagram is constructed by searching the STRING database for the common drug-disease targets mentioned above, setting the protein type as "Homo sapiens," and the minimum interaction threshold as 0.4, thus constructing a PPI network for protein-protein interactions.

[0096] Protein interactions were performed between 276 target sites of Sanzi Guichao Decoction and 823 POI target sites, and 53 common target sites were further screened out.

[0097] The top 10 targets are shown in Figure 11, namely: TP53, ESR1, CASP3, IL6, JUN, CCND1, AKT1, HIF1A, VEGFA, and PTGS2.

[0098] Example 3: Transcriptomic and proteomic study of Sanzi Guichao Decoction in the treatment of cisplatin-induced premature ovarian insufficiency.

[0099] I. Experimental Samples

[0100] Ovarian tissue was collected from mice treated with Sanzi Guichao Decoction for 4 weeks in Example 1. Three ovarian tissue samples were randomly selected from each of the normal group, model group, and traditional Chinese medicine treatment group, for a total of 9 tissue samples, for transcriptomic sequencing. Three ovarian tissue samples were also randomly selected from each of the normal group, model group, and traditional Chinese medicine treatment group, for a total of 9 tissue samples, for proteomic sequencing.

[0101] II. Transcriptomics circRNA Sequencing Experimental Methods

[0102] 2.1 RNA Extraction

[0103] Ovarian tissue was lysed using Trizol reagent to extract total RNA. The RNA from the total sample was then separated and purified according to the manufacturer's instructions.

[0104] 2.2 circRNA library construction

[0105] When performing circRNA sequencing, we first extract total RNA from the sample and then construct a strand-specific library. This library construction involves the removal of ribosomal RNA (i.e., rRNA depletion) and the selective removal of linear RNA (via RNase R, this step is optional). During this process, we remove rRNA from the total RNA while retaining mRNA and other non-coding RNAs, such as lncRNA and circRNA, for subsequent analysis.

[0106] 2.3 Screening of differentially expressed genes and analysis of gene expression time series

[0107] The microarray dataset was downloaded from the GEO database using the search term "Premature ovarian insufficiency". "Homo sapiens" was selected as the research object. The differentially expressed genes were analyzed based on the P-value using R software, and the up- and down-regulation of their expression was analyzed using a package.

[0108] Mfuzz used differentially expressed genes obtained from differential expression analysis (finding differences between any two groups and finally merging them) for time series analysis using the R package.

[0109] 2.4 GO functional enrichment and KEGG pathway enrichment analysis of differentially expressed genes

[0110] Using the Bioconductor bioinformatics software package in R, differential gene GO and KEGG functional enrichment analysis was performed with P-value < 0.05 and Q-value < 0.05, and the results were output.

[0111] III. TMT-labeled quantitative proteomics experimental methods

[0112] 1.1 Protein Extraction

[0113] 1.2 Protein Extraction Quality Control

[0114] Protein quantification was performed using the Bradford method. SDS-PAGE gel electrophoresis was then used. After electrophoresis, the gel was stained and destained, and finally, the gel was removed for photographic recording, protein separation, and visualization analysis.

[0115] 1.3 Proteolytic enzyme digestion

[0116] Extract 150 micrograms of protein. Next, add 3 micrograms of Trypsin enzyme at a protein-to-enzyme ratio of 50:1.

[0117] 1.4 TMT Marking

[0118] Each TMT tag was added to the corresponding sample to label the peptide sample.

[0119] 1.5 High pH RP separation

[0120] The sample is separated by liquid phase separation.

[0121] 1.6 Mass spectrometry detection (Nano-LC-MS / MS)

[0122] The peptides separated by liquid phase were ionized by a nanoESI source and introduced into an Orbitrap Exploris™ 480 mass spectrometer for analysis in DDA (data-dependent acquisition) mode.

[0123] 1.7 Protein identification and quantification

[0124] MaxQuant software (version 2.1.4.0) was used to perform protein search, identification, and quantification analysis on the TMT-labeled raw mass spectrometry data. The main MaxQuant parameters were as follows: MS2-based reporter group quantification mode (TMT6plex, TMT10plex, TMT16plex, or TMT18plex) was selected; trypsin / P enzyme was used; fixed modification was Carbamidomethyl (C); variable modifications were Oxidation (M) and Acetyl (protein N-term); Match between runs and secondary peptide search were also selected; all other parameters were set to default. The database used for identification was the Uniprot corresponding species database by default. In the protein filtering conditions, both the spectrum FDR and protein FDR were set to 1%, and contaminating and reverse-referenced proteins were removed.

[0125] IV. Results of Transcriptomics circRNA Sequencing Experiment

[0126] Principal component analysis (PCA), orthogonal partial least squares analysis (OPLS-DA), and partial least squares analysis (PLS-DA) were used to further classify the samples, thereby achieving detailed analysis and classification of the data. The results showed that the differences among ovarian tissue samples in the same group were small.

[0127] 4.1 Results of differential gene expression analysis

[0128] Compared with the normal group, the POI model group and the combined drug treatment group showed significant differences in 188 genes, including 76 upregulated genes and 112 downregulated genes. In the combined drug treatment group, 68 genes showed significant differences, including 40 upregulated genes and 28 downregulated genes. Comparing the POI model group and the combined drug treatment group, 123 genes showed significant differences in the combined drug treatment group, including 81 upregulated genes and 42 downregulated genes.

[0129] 4.2 Venny analysis of differentially expressed gene sets

[0130] We used Venn diagrams to analyze the differentially expressed gene intersections among the normal group A, POI model group B, and Sanzi Guichao Decoction treatment group C, and the results allowed us to screen for the target gene set. The results are shown in Figure 12.

[0131] 4.3 GO functional enrichment analysis of differentially expressed genes

[0132] We analyzed the pairwise comparisons among the three groups: normal group A, POI model group B, and Sanzi Guichao Decoction administration group C, and selected the top 25 enriched items.

[0133] The top 30 pathways enriched in POI model group B and normal group A mainly involve biological processes such as negative regulation of amyloid precursor, mononuclear cell differentiation, and wound healing. They are related to cellular components such as extracellular matrix, mitochondrial outer membrane, organelle outer membrane, and mitochondria, and mainly include molecular functions such as protease binding, oxidoreductase activity, Nad-retinol dehydrogenase activity, and amyloid-β binding.

[0134] The top 25 pathways enriched in the Sanzi Guichao Decoction treatment group C and the normal group A mainly involve biological processes such as monocyte differentiation, DNA degradation, regulation of organelle assembly, and mononuclear cell differentiation. They are related to cellular components such as the neuromuscular junction, mitochondrial matrix, MIB complex, and prosplastic body, and mainly include molecular functions such as oxidoreductase activity, galactosidase activity, carboxylase activity, and double-stranded telomeric DNA binding.

[0135] The top 25 pathways enriched in the Sanzi Guichao Decoction administration group C and the POI model group B mainly involve biological processes such as the regulation of tissue remodeling, cell junction depolymerization, synaptic pruning, and cellular immune responses. They are related to cellular components such as collagen, extracellular matrix, nucleolus, nuclear lumen, and ribonuclease Mrp complex, and mainly include molecular functions such as peptidase inhibitor activity, endopeptidase regulatory factor activity, CoA carboxylase activity, and LBD domain binding.

[0136] 4.4 KEGG enrichment analysis of differentially expressed genes

[0137] The KEGG database can enrich pathways in different protein-coding genomes. By screening the top 30 pathways through KEGG enrichment analysis, we can explore the changes in pathways in different treatment groups and further assist in the study of mechanisms.

[0138] The top 30 enriched pathways in POI model group B and normal group A mainly include cysteine ​​and methionine metabolism, ECM-receptor interaction, propionic acid metabolism, protein digestion and absorption, and the PI3K-Akt signaling pathway.

[0139] The top 30 enriched pathways in the Sanzi Guichao Decoction treatment group C and the normal group A mainly include riboflavin metabolism, pathogenic Escherichia coli infection, lipids, and atherosclerosis.

[0140] The top 30 enriched pathways in the Sanzi Guichao Decoction administration group C and the POI model group B mainly include the complement and coagulation cascade system, valine, leucine and isoleucine degradation, ovarian steroid synthesis, and systemic lupus erythematosus pathway.

[0141] V. TMT-labeled quantitative proteomics experimental methods

[0142] 5.1 Sample Relationship Analysis

[0143] Using macroscopic quantitative proteomic information, combined with hierarchical clustering, correlation analysis, and ratio distribution, the differences between samples were determined. In hierarchical clustering, the intensity of each sample was standardized, and the Euclidean distance was calculated to obtain the differences between samples, constructing the number of clusters to understand the relationships between all samples. In correlation analysis, all protein intensities were set to log10 as the observation, with the sample as the variable, and the correlation coefficient between samples was calculated. A higher correlation coefficient indicates less sample difference. Protein intensities were compared pairwise, and the pairwise ratio distribution was divided into within-group and between-group distributions to assess the differences between samples. The results showed that the differences between samples were small, and the correlation coefficients between samples were high.

[0144] 5.2 Phenome Analysis

[0145] By calculating the differences between samples using quantitative proteomic information, we can observe the differences between and within phenotype groups. Principal component analysis is used to analyze the differences between phenotype groups, and the coefficient of variation is used to calculate the differences within phenotype groups. Then, inter-group comparisons are performed, as shown in Figure 13.

[0146] 5.3 Screening of differentially expressed proteins

[0147] Compared with the normal group, the POI model group showed 167 differentially expressed proteins, with 126 downregulated and 41 upregulated. Compared with the normal group, the Sanzi Guichao Decoction group showed 118 differentially expressed proteins, with 76 downregulated and 42 upregulated. Compared with the POI model group, the Sanzi Guichao Decoction group showed 92 differentially expressed proteins, with 59 downregulated and 33 upregulated.

[0148] 5.4 GO enrichment analysis of differentially expressed proteins

[0149] Analysis of the GO database revealed that the top 20 enriched entries in the POI model group and normal group mainly involved biological processes such as thioester metabolism, phosphoribose metabolism, regulation of cell adhesion molecule production, and oxyacid metabolism. These processes were related to cellular components such as tricarboxylic acid cycle enzyme complexes, oxidoreductase complexes, organelle inner membranes, and organelle envelopes. The molecular functions included small molecule binding, oxidoreductase activity (acting on the CH-OH group donor, NAD or N), and oxidoreductase activity (acting on the aldehyde or oxygen group of the donor).

[0150] The top 20 enriched items in the Sanzi Guichao Decoction treatment group and the normal group mainly involve biological processes such as thioester metabolism, supramolecular fibrous tissue, sphingolipid metabolism, and sphingolipid-mediated signaling pathways. They are related to cellular components such as sarcomeres, organelles, myofibrils, and mitochondria, and mainly include molecular functions such as SNARE binding, SMAD binding, purine ribonucleotide binding, and purine nucleotide binding.

[0151] The top 20 enriched items in the Sanzi Guichao Decoction treatment group and model group mainly involve biological processes such as SREBP signaling pathway, regulation of SREBP signaling pathway, regulation of macrophage migration, and regulation of lipid localization. They are related to cellular components such as proteasome complex, proteasome accessory complex, peroxisomes, and peptidase complex, and mainly include molecular functions such as ubiquitin-ubiquitin ligase activity, transferase activity, and protein-lipid complex binding.

[0152] 5.5 KEGG enrichment pathway analysis of differentially expressed proteins

[0153] The main pathways were screened by KEGG enrichment analysis. The pathways enriched in the POI model group and the normal group mainly included propionic acid metabolism, valine, leucine and isoleucine degradation, citric acid cycle (TCA cycle), and fatty acid oxidation.

[0154] The main pathways enriched in the Sanzi Guichao Decoction treatment group and the normal group mainly include valine, leucine and isoleucine degradation, fatty acid elongation, citric acid cycle (TCA cycle), and propionic acid metabolism.

[0155] The main pathways enriched in the POI model group and the Sanzi Guichao Decoction administration group mainly include terpenoid skeleton biosynthesis, systemic lupus erythematosus, proteasome, phagosome, and tryptophan metabolism.

[0156] VI. Analysis of the association between transcriptomics and proteomics

[0157] By exploring the commonalities between transcriptomics and proteomics, we conducted correlation analysis on the differentially expressed genes / proteins between the drug-treated group and the model group in proteomics and transcriptomics, and obtained the intersection of differentially expressed genes / proteins in ovarian tissue transcriptomics and proteomics. The key gene is CALB2.

[0158] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. The application of Sanzi Guichao Decoction in the treatment of premature ovarian insufficiency, characterized in that, The Sanzi Guichao Decoction comprises the following ingredients in parts by weight: 2-8 parts of raspberry, 2-8 parts of wolfberry, 2-8 parts of dodder seed, 2-4 parts of angelica root, 2-8 parts of raw white peony root, 2-4 parts of prepared rehmannia root, 2-4 parts of yam, 2-4 parts of morinda root, 1-3 parts of chuanxiong rhizome, and 1-3 parts of cyperus rhizome.

2. The application of the Sanzi Guichao Decoction according to claim 1 in the treatment of premature ovarian insufficiency, characterized in that, The Sanzi Guichao Decoction targets TP53, ESR1, CASP3, IL6, JUN, CCND1, AKT1, HIF1A, VEGFA, PTGS2, and CALB2.

3. The application of the Sanzi Guichao Decoction according to claim 1 in the treatment of premature ovarian insufficiency, characterized in that, The aforementioned early-onset ovarian insufficiency includes decreased estrogen levels, with hormone indicators including E2, FSH, LH, and AMH.

4. The application of the Sanzi Guichao Decoction according to claim 3 in the treatment of premature ovarian insufficiency, characterized in that, The aforementioned early-onset ovarian insufficiency also includes osteoporosis caused by decreased estrogen levels.

5. The application of Sanzi Guichao Decoction in the preparation of drugs for treating premature ovarian insufficiency, characterized in that... The Sanzi Guichao Decoction comprises the following ingredients in parts by weight: 2-8 parts of raspberry, 2-8 parts of wolfberry, 2-8 parts of dodder seed, 2-4 parts of angelica root, 2-8 parts of raw white peony root, 2-4 parts of prepared rehmannia root, 2-4 parts of yam, 2-4 parts of morinda root, 1-3 parts of chuanxiong rhizome, and 1-3 parts of cyperus rhizome.

6. A sustained-release formulation, targeted formulation, or nano-formulation for treating early-onset ovarian insufficiency, characterized in that, The active ingredients of the preparation include extracts of the following substances in parts by weight: raspberry 2-8 parts, wolfberry 2-8 parts, dodder seed 2-8 parts, angelica 2-4 parts, raw white peony root 2-8 parts, prepared rehmannia root 2-4 parts, yam 2-4 parts, morinda root 2-4 parts, chuanxiong rhizome 1-3 parts, and cyperus rhizome 1-3 parts.

7. A sustained-release formulation, targeted formulation, or nano-formulation for treating early-onset ovarian insufficiency, characterized in that, The extract is a water extract.

8. A biomarker for early-onset ovarian insufficiency, characterized in that, This includes CALB2 gene markers or CALB2 protein markers.

9. The application of the biomarker for early-onset ovarian insufficiency as described in claim 8 in the preparation, identification, and detection of products for early-onset ovarian insufficiency.

10. The application according to claim 9, characterized in that, The products include reagents, chips, test cards, or kits.

11. Application of reagents capable of detecting and identifying CALB2 gene markers or CALB2 protein markers in the preparation, identification, and detection of products for early-onset ovarian insufficiency.

12. Application of CALB2 as a biomarker in screening or developing drugs to improve early-onset ovarian insufficiency.

13. The application of a set of gene markers in screening and identifying early-onset ovarian insufficiency that can be treated with Sanzi Guichao Decoction, characterized in that, The biomarkers include TP53, ESR1, CASP3, IL6, JUN, CCND1, AKT1, HIF1A, VEGFA, and PTGS2; The Sanzi Guichao Decoction comprises the following ingredients in parts by weight: 2-8 parts of raspberry, 2-8 parts of wolfberry, 2-8 parts of dodder seed, 2-4 parts of angelica root, 2-8 parts of raw white peony root, 2-4 parts of prepared rehmannia root, 2-4 parts of yam, 2-4 parts of morinda root, 1-3 parts of chuanxiong rhizome, and 1-3 parts of cyperus rhizome.

14. A reagent kit for diagnosing early-onset ovarian insufficiency, characterized in that, It contains reagents for detecting CALB2 expression levels.

15. A reagent kit for diagnosing early-onset ovarian insufficiency according to claim 13, characterized in that, The CALB2 expression level refers to either the CALB2 mRNA expression level or the CALB2 protein expression level.

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