Application of three-seed homing decoction in treatment of premature ovarian insufficiency and premature ovarian insufficiency biomarker

Abstract

The invention relates to the technical field of biological medicines, discloses application of a three-seed homing decoction in treatment of premature ovarian insufficiency and a premature ovarian insufficiency biomarker, and researches and develops a traditional Chinese medicine compound, namely the three-seed homing decoction, which has a remarkable effect on treatment of the premature ovarian insufficiency. Experiments prove that the traditional Chinese medicine composition has the effect of improving the ovarian function of model group rats, and a new treatment medicine is provided for treating premature ovarian insufficiency. The specific target spot of the three-seed homing decoction for treating the premature ovarian insufficiency is further analyzed and defined through network pharmacology, and key genes of the premature ovarian insufficiency are further analyzed and found through combination of proteomics and genomics. The target spots and the key genes have a plurality of application prospects for the subsequent research on the individualized drug effect of the three-seed homing decoction, the preparation or development of diagnostic kits for ovarian insufficiency, the screening of drugs for treating premature ovarian insufficiency and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of Sanzi Guichao Decoction in treating premature ovarian insufficiency and biomarkers of premature ovarian insufficiency. Background Art

[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0003] Premature ovarian insufficiency (POI) is one of the most difficult diseases faced by women in clinical practice. POI is defined as the loss of normal ovarian function before the age of 40. The current incidence of this disease in women is about 0.1% before the age of 30, and 2-4% before the age of 40. POI increases the risk of chronic diseases, including osteoporosis and cardiovascular disease. The causes of POI involve multiple factors such as genetics, immunity, environment, and iatrogenicity. Gonadal toxic components in chemotherapy drugs, especially alkylating agents commonly used in childbearing age, have been shown to cause iatrogenic ovarian insufficiency. Currently, hormone replacement therapy is mainly used clinically to relieve menopausal symptoms caused by hypoestrogenemia in POI patients. Physiological estrogen levels can be achieved through oral administration (1-2 mg estradiol per day or 0.625-1.25 mg estrogen per day) or transdermal estrogen regimens (0.1 mg per day). However, the drug can restore menstruation but not ovarian function. The use of estrogen / progestin therapy at the same time increases many health risks, including breast cancer, stroke, and cardiovascular disease. Osteoporosis and fractures are more common in women due to estradiol deficiency. Premature menopause and chronic estradiol deficiency can lead to osteoporosis. At the same time, young women with POI have significantly lower bone density compared to women with regular menstruation. However, the main existing solutions to the infertility problem of POI patients include oocyte donation and deep cryopreservation technology, but unfortunately, this method has 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 the present invention is to provide the application of Sanzi Guichao Decoction in the treatment of premature ovarian insufficiency and biomarkers of premature ovarian insufficiency in response to the current urgent need for new drugs for the treatment of ovarian insufficiency, and to provide a new drug compound - Sanzi Guichao Decoction for the treatment of premature ovarian insufficiency. At the same time, based on proteomics and genomics research and analysis, related gene markers of premature ovarian insufficiency were discovered, providing new ideas and methods for subsequent development and screening for the diagnosis or treatment of premature ovarian insufficiency.

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

[0007] On one hand, the present invention provides an application of Sanzi Guichao Decoction in the treatment of premature ovarian insufficiency. The Sanzi Guichao Decoction comprises the following substances in parts by weight: 2-8 parts of raspberries, 2-8 parts of wolfberries, 2-8 parts of dodder seeds, 2-4 parts of angelica sinensis, 2-8 parts of raw white peony roots, 2-4 parts of prepared rehmannia roots, 2-4 parts of yam, 2-4 parts of Morinda officinalis, 1-3 parts of chuanxiong, and 1-3 parts of cyperus rotundus.

[0008] Ye Tianshi, a famous doctor in the Qing Dynasty, proposed in his "Clinical Guide Medical Cases" that "women are born with liver", emphasizing the importance of nourishing and soothing the liver to women's physiological health. This prescription has achieved very good results in clinical practice, and most of them enter the liver meridian. The main medicine: raspberry, wolfberry, dodder seed, sweet and warm or sweet and flat, enter the liver, kidney, and bladder meridians, mainly targeting the root causes of liver and kidney deficiency, Chong and Ren disorders, and bone marrow malnutrition, and play the role of nourishing the liver and kidney, regulating Chong and Ren, and replenishing the essence. Minister medicine: raw white peony, prepared rehmannia, Morinda officinalis, assist the main medicine to strengthen the nourishing effect, raw white peony is bitter, sour, slightly cold, enters the liver and spleen meridians, and can also restrain liver yin, Morinda officinalis is sweet and warm and helps yang. Auxiliary medicine: Angelica sinensis, yam, these medicines assist the main medicine, and at the same time reconcile yin and yang, nourish blood and regulate menstruation, and strengthen the spleen and kidney. Envoy medicine: Chuanxiong, Cyperus rotundus, these medicines activate blood circulation and qi, soothe the liver and relieve depression, and lead the medicine to the diseased area. The whole prescription is mainly aimed at nourishing the liver, kidney and marrow, supplemented by promoting blood circulation and qi movement. It aims to treat premature ovarian insufficiency and prevent and improve osteoporosis. By harmonizing yin and yang and nourishing Chong and Ren, the purpose of restoring ovarian function and preventing and improving osteoporosis can be achieved.

[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 premature ovarian insufficiency disease includes decreased estrogen levels, and the hormone indicators are E2, FSH, LH, and AMH.

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

[0012] On the other hand, the present invention also provides a group of gene markers for use in screening and identifying premature ovarian insufficiency diseases that can be treated by Sanzi Guichao Decoction, wherein the biomarkers include TP53, ESR1, CASP3, IL6, JUN, CCND1, AKT1, HIF1A, VEGFA and PTGS2;

[0013] The Sanzi Guichao Decoction comprises 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 officinalis, 1-3 parts of chuanxiong, and 1-3 parts of cyperus rotundus.

[0014] Another aspect of the present invention provides a group of premature ovarian insufficiency biomarkers, including CALB2 gene markers or CALB2 protein markers.

[0015] On the other hand, the present invention also provides the use of the aforementioned biomarkers in the preparation, identification and detection of premature ovarian insufficiency detection products.

[0016] The detection products include reagents, chips, detection cards or test kits.

[0017] Another aspect of the present invention provides the use of CALB2 as a biomarker in screening or developing drugs for improving premature ovarian insufficiency.

[0018] On the other hand, the present invention also provides the use of a reagent capable of detecting and identifying a CALB2 gene marker or a CALB2 protein marker in the preparation of a product for identifying and detecting premature ovarian insufficiency.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. We developed a Chinese medicine compound that has a significant effect on the treatment of premature ovarian insufficiency: Sanzi Guichao Decoction. Animal model experiments have shown that it can improve the ovarian function of model group rats;

[0021] 2. Through network pharmacology, the specific targets of Sanzi Guichao Decoction for the treatment of premature ovarian insufficiency were further analyzed and clarified, including TP53, ESR1, CASP3, IL6, JUN, CCND1, AKT1, HIF1A, VEGFA and PTGS2. These key targets are of great practical significance in the 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 premature ovarian insufficiency.

[0022] 3. Through the combination of proteomics and genomics, we further discovered the key genes of premature ovarian insufficiency. These genes are of great significance for the diagnosis process and drug development and screening process of premature ovarian insufficiency, and can promote the development of the treatment process of ovarian insufficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The weight change data of each group of rats in Example 1;

[0024] Figure 2 are the ovarian and uterine indexes of rats in each group in Example 1; *P<0.05, **P<0.01, ***P<0.01, ****P<0.01;

[0025] Figure 3 The organ index diagram of rats in each group in Example 1; *P<0.05, **P<0.01, ***P<0.01, ****P<0.01;

[0026] Figure 4 The diagram of the estrous cycle changes of each group of rats in Example 1;

[0027] Figure 5 The ovarian sections of rats in each group in Example 1 (HE staining, left 100×, right 400×);

[0028] Figure 6 This is a graph showing the periodic changes in the estrogen levels of rats in each group in Example 1; *P<0.05, **P<0.01, ***P<0.01, ****P<0.01;

[0029] Figure 7 The rat E in each group in Example 1 2 , FSH, LH, AMH hormone levels; *P<0.05, **P<0.01, ***P<0.01, ****P<0.01;

[0030] Figure 8 The immunohistochemical results of ovarian tissues of rats in each group in Example 1 (200×);

[0031] Fig. 9 This is a comparison chart of femoral mechanical indicators of rats in each group in Example 1;

[0032] Fig.10 This is a comparison of femoral bone density of rats in each group in Example 1;

[0033] Fig.11 The protein interaction map of the top 10 targets in the intersection in Example 2;

[0034] Fig.12The intersection of differentially expressed genes among the normal group A, the POI model group B, and the Sanzi Guichao Decoction-administered group C in Example 3;

[0035] Fig.13 This is the PCA score diagram of each group of proteomics samples in Example 3. DETAILED DESCRIPTION

[0036] It should be noted that the test methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available.

[0037] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0038] Preparation of Sanzi Guichao Decoction: Weigh the ingredients of Sanzi Guichao Decoction in proportion, add water to extract to obtain Sanzi Guichao Decoction suspension; the content of each ingredient of Sanzi Guichao Decoction is as follows: The formula of Sanzi Guichao Decoction is: 15g of raspberry, 15g of wolfberry, 15g of dodder seed, 15g of angelica, 15g of raw white peony root, 15g of rehmannia root, 15g of yam, 15g of Morinda officinalis, 10g of Chuanxiong, and 10g of Cyperus rotundus.

[0039] Example 1 Animal experiment verifies that Sanzi Guichao Decoction treats premature ovarian insufficiency

[0040] 1. Experimental Animal Modeling

[0041] Eight SD female rats aged 8-9 weeks were randomly selected from the blank group. After 1 week of adaptive feeding, the remaining animals were injected intraperitoneally with 2 mg / kg cisplatin for 7 consecutive days to establish the POI rat model. The blank group was injected intraperitoneally with the same dose of 0.9% saline.

[0042] 2. Experimental Grouping and Drug Treatment

[0043] According to the preliminary experiment, after the administration of cisplatin, the rats that survived the successful model were randomly divided into a model group, a Sanzi Guichao Decoction group, and a positive drug control group. After the model was successful, gavage treatment was started, and the rats in the Sanzi Guichao Decoction group (converted to 140g / 60kg per day for adults) were gavaged with 14.0g / (kg·d). Sanzi Guichao Decoction was added with water and then rotary evaporated to prepare a Sanzi Guichao Decoction suspension with a concentration of 1.40g / mL, that is, for the convenience of feeding during gavage, it was administered at 1mL / 100g; the positive drug control group was gavaged with 0.1mg / (kg·d), 1mL / 100g of Progynova, and the blank group and model group were gavaged with normal saline at 1mL / 100g and once a day, respectively.

[0044] After 28 days of drug administration, one animal died in each of the positive drug control group and the model group. Later, data analysis was performed in the blank group, model group and Sanzi Guichao Decoction group by random sampling to ensure the reliability of the data, with the minimum number of animals in each group (n=6).

[0045] 3. General status observation

[0046] During the experiment, we conducted comprehensive observations and records of the rats' food intake, water intake, fur color, activity status, and mental state. These observations were intended 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 were recorded before and after drug administration. The general condition of the rats is shown in the following table:

[0047]

[0048]

[0049] The weight changes of rats were statistically analyzed every 7 days. Figure 1 As shown in the figure, from the overall trend, during the modeling process, the overall weight of the modeling group changed greatly, and the weight loss was large, indicating that cisplatin had a greater effect, causing the modeling group to be thin. After 7 days of administration, the comparison between the normal group and the modeling group clearly showed a trend of weight loss, and there was a significant difference (P<0.01). After the modeling was completed on the seventh day after administration, the positive drug control group and the model group recovered less, and the Sanzi Guichao Decoction administration group had a slight increase, and there was a significant difference (P<0.01). On the 14th day after administration, both the Chinese medicine administration group and the positive drug control group were higher than the model group, and there was a significant difference (P<0.01). On the 28th day after administration, the drug administration group and the positive drug control group had a significant increase and difference compared with the model group (P<0.01). 4. Observation of estrous cycle changes by vaginal detachment cytology smear

[0050] After the modeling was completed, starting from the 7th day of gavage, we used sterile cotton swabs dipped in saline to collect vaginal exfoliated cells from rats at 9 am every day for 14 consecutive days. The collected cells were evenly spread on a glass slide, and then their morphology and coloration were observed under an optical microscope. In order to more accurately judge the changes in the estrous cycle of rats, we performed HE staining on these cells. The judgment criteria refer to the morphological changes of vaginal exfoliated cells of female rats at various stages of estrus, that is, the proportion of white blood cells, nucleated epithelial cells and keratinized squamous epithelial cells is used to distinguish the four stages of the estrous cycle. The proestrus smear is mainly composed of nucleated epithelial cells, the estrus smear is mainly composed of nucleated keratinocytes, and the diestrus smear is mainly composed of white blood cells.

[0051] Smear data such as Figure 4As shown, the estrus period was significantly shortened and the proestrus and interestrus were prolonged in the three groups after modeling; after drug administration, the estrus period was prolonged and the proestrus and interestrus were shortened in the Chinese medicine group and the positive control group compared with the model group, indicating that the estrus cycle was significantly improved after drug administration.

[0052] 5. Periodic submandibular blood sampling to observe changes in sex hormone indicators

[0053] For blood collection, this experiment adopted a frequency of once every 7 days, and samples were obtained through submandibular vein blood collection.

[0054] The results are as follows Figure 6 As shown in the figure, there was no trend change in the cycle of the normal group within 35 days, and there was no significant difference. After the 7-day modeling, the estrogen level of the model group decreased significantly (P<0.01), and there was no significant change in the subsequent period. After the 7-day modeling, the estrogen level of the drug-treated group decreased significantly (P<0.01). One week after the drug administration, the estrogen level did not change compared with the 7th day, and the estrogen level increased significantly after the 21st day (P<0.01). After the 7-day modeling, the estrogen level of the positive control group decreased significantly (P<0.01), and the estrogen level increased significantly after the 21st day (P<0.01).

[0055] 6. Sample Collection

[0056] The animals were fasted on the evening of the end of gavage. The animals were taken the next morning and anesthetized with 3% pentobarbital solution, 200μl / 100g intraperitoneal injection, and then anesthesia was confirmed to be complete. Blood samples were collected by heart bleeding, and blood was collected and kept still using a blood collection tube. After standing at room temperature for 1h, it was centrifuged at 3000rmp for 15min, and the upper serum was collected and stored in a -20℃ refrigerator for subsequent testing. After the rats were killed by spinal dislocation, surgical instruments were used to open the abdominal cavity of the animals, and the heart, liver, spleen, lung, kidney (bilateral), and uterine tissues were taken out and placed on the ice surface for immediate weighing and recording. The bilateral ovaries were taken out, weighed and then packaged, half of the ovaries were placed in more than 4% paraformaldehyde, and half were placed in a -80℃ refrigerator for standby use. The bilateral femurs of the rats were taken out, half of the femurs were placed in 4% paraformaldehyde, and the other half were placed on the ice surface and stored in a -80℃ refrigerator for standby use.

[0057] VII. Organ and gonad index

[0058] Organ index = organ wet weight (g) / mouse body weight before anesthesia × 100%.

[0059] Ovarian index = wet weight of bilateral ovaries (g) / body weight of mouse before anesthesia × 100%.

[0060] Uterine index = uterine wet weight (g) / mouse body weight before anesthesia × 100%.

[0061] The ovarian and uterine indexes of rats in each group were as follows Figure 2 As shown in Figure 3 As shown, from Figure 2 and Figure 3 It can be seen that in the ovarian index, the model group was significantly lower than the normal group (P<0.01); the drug group and the positive control group were significantly higher than the model group (P<0.01), and there was no obvious trend compared with the normal group. In the uterine index, the model group was significantly lower than the normal group (P<0.01); both the drug group and the positive control group showed an upward trend compared with the model group, but there was no significant difference. In the heart index, liver index, and lung index, there was no trend change in each group. In the kidney index, the model group was significantly higher than the normal group (P<0.01), the drug group and the positive control group were significantly higher than the normal group (P<0.01), and the drug group and the positive control group were both lower than the model group, with no significant difference. In the spleen index, the positive control group was significantly higher than the normal group (P<0.01), and at the same time, it was significantly higher than the model group (P<0.01).

[0062] 8. ELISA detection of E in experimental animal serum 2 , FSH, LH, AMH level changes

[0063] After the administration, the serum of rats in each group was taken to measure E 2、 The results are as follows: Figure 7 Visible: E 2 In terms of levels, the model group was significantly lower than the normal group (P<0.01); the drug-treated group and the positive control group were significantly higher than the model group (P<0.05), and lower than the normal group.

[0064] In terms of FSH level, the model group had a significant increase compared with the normal group (P<0.01); the Chinese medicine group had a significant increase compared with the normal group (P<0.05), and the Chinese medicine group had a decrease compared with the model group; the positive control group had a significant increase compared with the normal group (P<0.01), and the positive control group had a decrease compared with the model group.

[0065] In terms of AMH levels, the model group showed a downward trend compared with the normal group; the drug-treated group and the positive control group showed an upward trend compared with the model group, but the decrease was not obvious compared with the normal group.

[0066] In terms of LH level, the model group had a significant increase compared with the normal group (P<0.01); there was no significant difference between the Chinese medicine group and the normal group, and the Chinese medicine group had a significant decrease compared with the model group (P<0.01); the positive control group had a significant decrease compared with the normal group (P<0.01), and the positive control group had a significant decrease compared with the model group (P<0.01). IX. HE staining to detect changes in ovarian morphology, follicles and granulosa cells

[0067] like Figure 5 The ovarian morphology and structure of the normal group were clear, the follicles of different grades were clearly visible, and the granulosa cells were normal; in the model group, there were fewer follicles, fewer primordial follicles, and fewer granulosa cells; in the Chinese medicine group, the ovaries were improved compared with the model group, the follicles increased slightly, and the granulosa cells increased compared with the model group; in the positive control group, the morphology was improved compared with the model group, the follicles of different grades increased slightly, and the granulosa cells increased compared with the model group. X. Immunohistochemical detection of changes in apoptosis-related proteins and estrogen-related proteins in ovarian tissue

[0068] The results are as follows Figure 8 As shown in the figure, Bax: is mainly expressed in the cytoplasm of ovarian interstitial cells, and can also be seen in vascular smooth muscle cells, and the expression in germ cells is low or not obvious. The model group is significantly increased compared with the normal group, and the drug group is significantly decreased compared with the positive control group.

[0069] Bcl-2: Mainly expressed in the cytoplasm of ovarian interstitial cells, also expressed in vascular smooth muscle cells, obviously expressed in the cytoplasm of germ cells, and the expressing cells are more diffuse; the model group was significantly lower than the normal group, and the drug group was significantly higher than the positive control group.

[0070] Caspase-3: Mainly expressed in ovarian interstitial cells, vascular smooth muscle cells, etc., and not significantly expressed in germ cells. The model group was significantly higher than the normal group, and the drug group was significantly lower than the positive control group.

[0071] VEGF: It is mainly expressed in ovarian vascular endothelial cells, vascular smooth muscle, and interstitial cells, and is lowly expressed or expressed in small amounts in germ cells; the model group was significantly lower than the normal group, and the drug-treated group was significantly higher than the positive control group.

[0072] ESR2: diffusely expressed in the cytoplasm of various cells. The model group was lower than the normal group, and the drug group was higher than the positive control group.

[0073] ESR1: It is diffusely expressed on the cytoplasm of various cells and can also be expressed on the capsule. The model group was lower than the normal group, and the drug group was higher than the positive control group.

[0074] CYP19A1: is a member of the cytochrome P450 superfamily (EC 1.14.14.1), which are monooxygenases that catalyze many reactions in steroidogenesis.

[0075] 11. Effect of Sanzi Guichao Decoction on Bone Density in Rats

[0076] Use the instron system to perform static biomechanical tests to test the pressure of the femur fracture under longitudinal pressure. Remove the tissue around the bone, place the femur on the test frame, and use a three-point bending pressure test with a span of 30mm. Use the analysis software configured with the instrument to obtain the load-displacement curve, and then analyze the elastic load (N), limit load (N) and bending modulus (MPa) based on the curve graph. The results are as follows Fig. 9 As shown in the figure, in terms of elastic modulus, the model group was significantly reduced under the induction of cisplatin (P<0.05), and the Sanzi Guichao Decoction group increased after drug treatment. In terms of bending displacement, there was no significant change in each group. In the maximum bending load, the model group was significantly reduced (P<0.01), and the drug group had an increasing trend (P<0.01).

[0077] The multispectral small animal in vivo imaging system (model: X-RAY SYSTEM) was used to measure the bone density of the entire left femur of rats. According to the previous animal experiment, the left femur was taken, the tissue around the bone was removed, fixed with 4% paraformaldehyde, and stored in a 4°C refrigerator for in vitro bone density examination. The bone was placed under the camera probe of the bone densitometer, and the MI software was used to perform an exposure scan of the femur to measure the bone density value. The results are as follows Fig.10 As shown, compared with the normal group, the model group had an obvious trend of decreased bone density in the proximal, middle and distal femur, confirming that cisplatin would cause a decrease in femoral bone density in rats to a certain extent; compared with the model group, the Sanzi Guichao Decoction-treated group had an upward trend in bone density in the proximal, middle and distal femur, indicating that the drug administration would improve the decrease in bone density caused by cisplatin to a certain extent.

[0078] 12. Summary

[0079] According to the results of animal experiments, the animals recovered before and after administration through general observation, their mental state recovered well, and their weight recovered significantly after administration. Through vaginal smear observation of the estrous cycle, the estrous period of the model group was significantly improved after administration, and the estrous period was prolonged compared with the model group. In the HE staining observation of the ovarian morphology, the number of ovarian follicles increased after administration, the overall morphology of the ovary recovered to normal, and the number of granulosa cells increased compared with the model group. Through Elisa experiments to detect changes in hormone levels, it was found that E 2, FSH, LH, and AMH all showed trend changes before and after administration, indicating that the effect of administration was more obvious. The expression of apoptosis and estrogen-related proteins was observed by immunohistochemistry, and it was found that the apoptosis protein in the ovary decreased and the estrogen-related protein increased after administration. Cisplatin induction caused significant changes in the related indicators of osteoporosis precursors in POI model animals, while the administration of traditional Chinese medicine can restore indicators such as bone density to a certain extent, indicating that Sanzi Guichao Decoction has a certain effect in improving osteoporosis. In summary, the efficacy of Sanzi Guichao Decoction in the treatment of POI has been preliminarily verified through animal experiments.

[0080] Example 2 Detection of active ingredients in Sanzi Guichao Decoction and prediction of potential targets for the treatment of premature ovarian insufficiency

[0081] 1. Network pharmacology prediction of key active ingredients and potential targets of Sanzi Guichao Decoction for the treatment of premature ovarian insufficiency

[0082] 1.1 Searching the Chinese medicine database for drug components and target screening of Sanzi Guichao Decoction

[0083] The TCMSP database was used to search for Chinese medicinal ingredients as potential active ingredients, and the targets of related ingredients were obtained from TCMSP, and the target names were standardized using the Uniprot database. Results In the TCMSP database, OB≥30% and DL≥0.18 were set, and the effective ingredients such as Angelica sinensis, Cuscuta australis, Rehmannia glutinosa, Lycium barbarum, White Peony Root, Morinda officinalis, Rubus idaeus, Dioscorea opposita, Ligusticum chuanxiong, and Cyperus rotundus were screened. After removing duplicates, the potential active ingredients were obtained, and the drug targets were screened out through the TCMSP database.

[0084] A total of 140 chemical components of Sanzi Guichao Decoction were obtained through the TCMSP database, and 87 chemical components were finally obtained after removing duplicates. The main active ingredients corresponding to the ten herbs in Sanzi Guichao Decoction were 18 from Morinda officinalis, 9 from white peony root, 7 from Chuanxiong, 2 from Angelica sinensis, 5 from Rubus idaeus, 39 from Lycium barbarum, 13 from Dioscorea opposita, 1 from Rehmanniae glutinosae, 11 from Cuscuta chinensis, and 16 from Cyperus rotundus. A total of 1927 potential targets were retrieved from these ten herbs, including 141 from Morinda officinalis, 123 from white peony root, 42 from Chuanxiong, 69 from Angelica sinensis, 154 from Rubus idaeus, 364 from Lycium barbarum, 168 from Dioscorea opposita, 35 from Rehmanniae glutinosae, 343 from Cuscuta chinensis, and 488 from Cyperus rotundus, of which 276 were found after removing duplicates. The above targets were imported into Cytoscape3.8.2 software to construct the active ingredient-target network diagram of Sanzi Guichao Decoction for visualization purposes.

[0085] 1.2 Searching for genes related to premature ovarian insufficiency in disease databases

[0086] The OMIM and Genecards databases were used to search for "Premature ovarian insufficiency" as the keyword, and after obtaining the disease targets, duplicates were removed. The UniprotKB database was used to standardize gene IDs, and a total of 823 POI-related targets were obtained.

[0087] 1.3 Construction and analysis of disease-compound active ingredient network

[0088] By drawing a Venn diagram using R software, common drug target-disease relationships can be obtained. Using Cytoscape 3.7.2 software, a network diagram was constructed, and the functions of the main active ingredients of traditional Chinese medicine were analyzed using the network. A protein-related interaction network diagram was constructed, and the above drug-disease common targets were entered into the STRING database for retrieval. The protein type was set to "Homosapiens" and the minimum interaction threshold was set to 0.4 to construct a PPI network of protein interactions.

[0089] The 276 Sanzi Guichao Decoction targets were interacted with the 823 POI targets by protein interaction, and 53 common targets of the two were further screened out.

[0090] The top 10 targets are: Fig.11 As shown, they are: TP53, ESR1, CASP3, IL6, JUN, CCND1, AKT1, HIF1A, VEGFA, and PTGS2.

[0091] Example 3 Transcriptomics and proteomics study of Sanzi Guichao decoction in the treatment of cisplatin-induced premature ovarian insufficiency

[0092] 1. Experimental Sample

[0093] Ovarian tissue was obtained 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 Chinese medicine administration group, for a total of 9 tissue samples for transcriptomic sequencing. Three ovarian tissue samples were randomly selected from each of the normal group, model group, and Chinese medicine administration group, for a total of 9 tissue samples for proteomic sequencing.

[0094] 2. Transcriptomics circRNA sequencing experimental methods

[0095] 2.1 RNA extraction

[0096] Ovarian tissue was lysed using Trizol reagent to extract total RNA from tissues. RNA from total samples was isolated and purified according to the protocol provided by the manufacturer.

[0097] 2.2 circRNA library construction

[0098] When performing circRNA sequencing, we first extract Total RNA from the sample and then construct a specific strand-specific library. The construction of this library involves the removal of ribosomal RNA (i.e., rRNA depletion) and the selective removal of linear RNA (through RNase R treatment, this step is optional). In this process, we remove rRNA from total RNA while retaining mRNA and other non-coding RNAs, such as lncRNA and circRNA, for subsequent analysis.

[0099] 2.3 Screening of differentially expressed genes and temporal analysis of gene expression

[0100] The microarray data set was downloaded using the GEO database, the search term was “Premature ovarianinsufficiency”, “homo sapiens” was selected as the research object, and the differentially expressed genes were analyzed according to the P value using R software, and the up- and down-regulation of their expression was analyzed using the program package.

[0101] The gene expression time series analysis Mfuzz used the differentially expressed genes obtained by differential expression analysis (find the difference between any two groups and merge them) to perform time series analysis through the R program package.

[0102] 2.4 Analysis of GO function enrichment and KEGG pathway enrichment of differentially expressed genes

[0103] The Bioconductor bioinformatics software package was used based on R software to perform GO and KEGG functional enrichment analysis of differentially expressed genes with Pvalue<0.05 and Qvalue<0.05, and the results were output.

[0104] 3. TMT labeling quantitative proteomics experimental method

[0105] 1.1 Protein extraction

[0106] 1.2 Protein extraction quality control

[0107] Bradford method was used for protein quantification. After SDS-PAGE gel electrophoresis analysis, we stained and destained the gel, and finally took out the gel to take pictures and record, and separated and visualized the proteins.

[0108] 1.3 Proteolysis

[0109] 150 micrograms of protein were extracted. Next, 3 micrograms of Trypsin enzyme were added at a protein to enzyme ratio of 50:1.

[0110] 1.4 TMT Marking

[0111] Each TMT label was added to the corresponding sample to label the peptide sample.

[0112] 1.5High pH RP separation

[0113] The samples were separated by liquid phase separation.

[0114] 1.6 Mass spectrometry detection (Nano-LC-MS / MS)

[0115] The peptides separated by liquid phase are ionized by nanoESI source and introduced into OrbitrapExploris TM The analysis was performed in DDA (data dependent acquisition) mode in a 480 mass spectrometer.

[0116] 1.7 Protein identification and quantification

[0117] MaxQuant software (version 2.1.4.0) was used to search and identify proteins and conduct quantitative analysis on the raw data of TMT-labeled mass spectrometry. The main parameters of MaxQuant were as follows: check the reporter group quantitative mode based on MS2 (TMT6plex, TMT10plex, TMT16plex or TMT18plex), trypsin was Trypsin / P enzyme, fixed modification was Carbamidomethyl (C), variable modification was Oxidation (M) and Acetyl (protein N-term), Matchbetween runs and secondary peptide search were also checked, and the rest of the parameters were default parameters. The database used for identification was the Uniprot corresponding species database by default, and the spectrum FDR and protein FDR in the protein filtering conditions were both set to 1%, and the contamination library and anti-library proteins were removed.

[0118] 4. Transcriptomics circRNA sequencing results

[0119] The samples were further classified using principal component analysis (PCA), orthogonal partial least squares method (OPLS-DA) and partial least squares method (PLS-DA), thereby achieving detailed analysis and classification of the data. The results showed that the differences in the ovarian tissue samples in the same group were small.

[0120] 4.1 Results of differential gene expression analysis

[0121] The POI model group and the compound drug group were compared with the normal group. The POI model group found 188 genes with significant differences, of which 76 genes were upregulated and 112 genes were downregulated. The drug group found 68 genes with significant differences, of which 40 genes were upregulated and 28 genes were downregulated. The POI model group and the compound drug group were compared. In the compound drug group, 123 genes were found to have significant differences, of which 81 genes were upregulated and 42 genes were downregulated.

[0122] 4.2 Venny analysis of differentially expressed gene sets

[0123] We used the Venn diagram to analyze the intersection of differentially expressed genes among the normal group A, POI model group B, and Sanzi Guichao Decoction-administered group C, and the target gene set was screened out through the results. Fig.12 shown.

[0124] 4.3 GO functional enrichment analysis of differentially expressed genes

[0125] We analyzed the normal group A, POI model group B, and Sanzi Guichao Decoction-treated group C and compared them pairwise, and took the top 25 enriched entries.

[0126] The top 30 pathways enriched in POI model group B and normal group A were mainly involved in biological processes such as negative regulation of amyloid precursor, mononuclear cell differentiation, and wound healing, and were related to cellular components such as extracellular matrix, mitochondrial outer membrane, organelle outer membrane, and mitochondria, mainly including molecular functions such as protease binding, oxidoreductase activity, Nad-retinol dehydrogenase activity, and amyloid-β binding.

[0127] The top 25 pathways enriched in the Sanzi Guichao Decoction-treated group C and the normal group A were mainly involved in biological processes such as monocyte differentiation, DNA decomposition process, regulation of organelle assembly, and mononuclear cell differentiation, and were related to cellular components such as neuromuscular junction, mitochondrial matrix, MIB complex, and pre-spliceosome, mainly including molecular functions such as oxidoreductase activity, galactosidase activity, carboxylase activity, and double-stranded telomere DNA binding.

[0128] The top 25 pathways enriched in Sanzi Guichao Decoction-administered group C and POI model group B were mainly involved in biological processes such as the regulation of tissue remodeling, cell junction dissociation, synaptic pruning, and cellular immune response, and were related to cellular components such as collagen-containing extracellular matrix, nucleolus, nuclear cavity, and ribonuclease Mrp complex, mainly including molecular functions such as peptidase inhibitor activity, endopeptidase regulatory factor activity, CoA carboxylase activity, and LBD domain binding.

[0129] 4.4 KEGG enrichment analysis of differentially expressed genes

[0130] The KEGG database can be enriched with pathways of different protein-coding genomes. The top 30 pathways can be screened out through KEGG enrichment analysis to explore the changes in pathways of different treatment groups and further provide assistance for mechanism research.

[0131] The top 30 pathways enriched in POI model group B and normal group A mainly included cysteine ​​and methionine metabolism, ECM-receptor interaction, propionate metabolism, protein digestion and absorption, PI3K-Akt signaling pathway and other pathways.

[0132] The top 30 pathways enriched in the Sanzi Guichao Decoction-treated group C and the normal group A mainly included riboflavin metabolism, pathogenic Escherichia coli infection, lipids, and atherosclerosis.

[0133] The top 30 pathways enriched in Sanzi Guichao Decoction-administered group C and POI model group B mainly included complement and coagulation cascade system, valine, leucine and isoleucine degradation, ovarian steroid synthesis, systemic lupus erythematosus and other pathways.

[0134] 5. TMT labeling quantitative proteomics experimental method

[0135] 5.1 Sample relationship analysis

[0136] Using macro-quantitative information of the proteome, combined with hierarchical clustering, correlation analysis, and ratio distribution, the difference relationship between samples is judged. In hierarchical clustering, the intensity of each sample is standardized, the Euclidean distance is calculated to obtain the difference between samples, the number of clusters is constructed, and the relationship between all samples is understood. In the correlation analysis, all protein intensities are taken as log10 as the observation quantity, and the sample is used as the variable to calculate the correlation coefficient between samples. The higher the correlation coefficient, the less the difference between samples. The protein intensity is compared in pairs of samples, and the pairwise ratio distribution is divided into intra-group and inter-group display of phenotypic groups to evaluate the difference between samples. It is concluded that the sample differences between samples are small, and the correlation coefficients between samples are high.

[0137] 5.2 Phenotype group analysis

[0138] By calculating the differences between samples through the quantitative information of the proteome, we can observe the differences between and within each phenotypic group. The differences between phenotypic groups are analyzed using principal components, and the differences within phenotypic groups are calculated using the coefficient of variation, and then inter-group comparisons are performed, such as Fig.13 shown.

[0139] 5.3 Screening of differentially expressed proteins

[0140] Compared with the normal group, the POI model group had 167 differentially expressed proteins, 126 down-regulated proteins, and 41 up-regulated proteins. Compared with the normal group, the Sanzi Guichao Decoction group had 118 differentially expressed proteins, 76 down-regulated proteins, and 42 up-regulated proteins. Compared with the POI model group, the Sanzi Guichao Decoction group had 92 differentially expressed proteins, 59 down-regulated proteins, and 33 up-regulated proteins.

[0141] 5.4 GO enrichment analysis of differentially expressed proteins

[0142] Through GO database analysis, the top 20 enriched entries in the POI model group and the normal group were mainly involved in biological processes such as thioester metabolism, ribose phosphorylation metabolism, the production of regulatory cell adhesion molecules, and oxoacid metabolism, and were related to cellular components such as tricarboxylic acid cycle enzyme complexes, oxidoreductase complexes, organelle inner membranes, and organelle envelopes, mainly including molecular functions such as small molecule binding, oxidoreductase activity (acting on donors of CH-OH groups, NAD or N), and oxidoreductase activity (acting on the aldehyde or oxygen group of the donor).

[0143] The top 20 enriched entries in the Sanzi Guichao Decoction-treated group and the normal group mainly involved biological processes such as thioester metabolism, supramolecular fiber organization, sphingolipid metabolism, and sphingolipid-mediated signaling pathways, which were related to cellular components such as sarcomeres, organelles, myofibrils, and mitochondria, and mainly included molecular functions such as SNARE binding, SMAD binding, purine ribonucleotide knots, and purine nucleotide binding.

[0144] The top 20 items enriched in the Sanzi Guichao Decoction-treated group and the model group mainly involved biological processes such as the SREBP signaling pathway, regulation of the SREBP signaling pathway, regulation of macrophage migration, and regulation of lipid localization, and were related to cellular components such as the proteasome complex, proteasome-affiliated complex, peroxisome, and peptidase complex, mainly including molecular functions such as ubiquitin-ubiquitin ligase activity, transferase activity, and binding of protein-lipid complexes.

[0145] 5.5 KEGG enrichment pathway analysis of differentially expressed proteins

[0146] The main pathways were screened out through KEGG enrichment analysis. The pathways enriched in the POI model group and the normal group mainly included propionate metabolism, valine, leucine and isoleucine degradation, citric acid cycle (TCA cycle), fatty acid oxidation and other pathways.

[0147] The main pathways enriched in the Sanzi Guichao Decoction-treated group and the normal group included valine, leucine and isoleucine degradation, fatty acid elongation, citric acid cycle (TCA cycle), propionic acid metabolism, etc.

[0148] The main pathways enriched in the POI model group and the Sanzi Guichao Decoction-treated group included terpene skeleton biosynthesis, systemic lupus erythematosus, proteasome, phagosome, tryptophan metabolism, etc.

[0149] VI. Correlation analysis between transcriptomics and proteomics

[0150] By deeply exploring the similarities between transcriptomics and proteomics, the differential genes / proteins between the drug-treated group and the model group in proteomics and transcriptomics were correlated and analyzed, and the intersection of differential genes / proteins in ovarian tissue transcriptomics and proteomics was obtained, and the key gene was CALB2.

[0151] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. The application of Sanzi Guichao Decoction in the treatment of premature ovarian insufficiency is characterized by: The Sanzi Guichao Decoction comprises 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 officinalis, 1-3 parts of chuanxiong, and 1-3 parts of cyperus rotundus.

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

3. The use of Sanzi Guichao Decoction in the treatment of premature ovarian insufficiency according to claim 1, characterized in that: The premature ovarian insufficiency disease includes decreased estrogen levels, and the hormone indicators are E2, FSH, LH, and AMH.

4. The use of Sanzi Guichao Decoction in the treatment of premature ovarian insufficiency according to claim 3, characterized in that: The premature ovarian insufficiency disease also includes osteoporosis caused by decreased estrogen levels.

5. A biomarker for premature ovarian insufficiency, characterized in that: Including CALB2 gene markers or CALB2 protein markers.

6. Use of a premature ovarian insufficiency biomarker as claimed in claim 5 in the preparation of a product for identifying and detecting premature ovarian insufficiency.

7. The use according to claim 6, characterized in that: The products include reagents, chips, test cards or test kits.

8. Use of reagents capable of detecting and identifying CALB2 gene markers or CALB2 protein markers in the preparation of products for identification and detection of premature ovarian insufficiency.

9. Application of CALB2 as a biomarker in screening or developing drugs to improve premature ovarian insufficiency.

10. The use of a group of gene markers in screening and identifying premature ovarian insufficiency diseases that can be treated by 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 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 officinalis, 1-3 parts of chuanxiong, and 1-3 parts of cyperus rotundus.

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