Application of CREB1, ATF2 and FOS as drug target genes in screening and preparing climacteric syndrome drugs

Abstract

The invention provides application of CREB1, ATF2 and FOS as drug target genes in screening and preparing climacteric syndrome drugs, and belongs to the technical field of biological pharmacy. The target gene of the climacteric syndrome drug is screened through omics research, and plays a role by adjusting a dopamine synaptic pathway. According to the invention, the obtained key gene targets correspond to related pathways, the potential molecular mechanism is determined, and reference is provided for subsequent development and application.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and specifically relates to the application of CREB1, ATF2 and FOS as drug target genes in screening and preparing drugs for menopausal syndrome. Background Art

[0002] Perimenopausal syndrome is often referred to as menopausal syndrome, which refers to a series of physical and mental symptoms caused by fluctuations or decreases in sex hormones before and after menopause in women. Perimenopausal is the stage when women transition from regular menstruation in the reproductive period to menopause, including the onset of endocrine, biological and clinical characteristics related to decreased ovarian function to one year after the last menstrual period. The symptoms of perimenopausal syndrome mainly include menstrual changes, vasomotor symptoms, and psychoneurological symptoms. It is more common in women aged 45 to 55. The clinical manifestations of perimenopausal women vary greatly due to their different mental states, living environments, etc. The treatment goal is to relieve recent symptoms, improve the quality of life of patients, and detect them early to reduce the risk of osteoporosis and cardiovascular diseases.

[0003] Traditional treatments are mainly based on psychological counseling and oral medication. However, the side effects associated with these drugs, such as dizziness, drowsiness, and constipation, often limit their use, and some patients may not respond well to currently available drug treatments. At the same time, multiple studies have shown that the use of traditional Chinese medicine can effectively and economically relieve menopausal symptoms and improve overall quality of life. However, there is little research on the treatment and relief mechanisms of traditional Chinese medicine.

[0004] Extensive research has been conducted in the prior art to explore the causes of the increased mood disorders experienced during the menopausal transition. In addition to the potential impact of VMS, research suggests that there appears to be a potential correlation between the presence of mood disorders and decreased ovarian function, as it has been observed that decreased estradiol levels lead to increased degradation and metabolic inactivation of dopamine, norepinephrine, and serotonin, along with downregulation of monoamine neurotransmitter activity. In other words, the involvement of dopamine in regulating anxiety may be related to regulating estrogen levels.

[0005] Furthermore, it has been shown that anxiety-inducing effects can be observed by inhibiting dopamine pathways. It has been found that a decrease in dopamine release in the medial prefrontal cortex (PFC) of rodents leads to an increase in anxiety-related behaviors. This finding is consistent with previous studies showing a correlation between dopamine and anxiety, as evidenced by the increased incidence of anxiety symptoms in patients with Parkinson’s disease. In this neurodegenerative disease, a decrease in dopaminergic prominence has been observed. These empirical observations support the hypothesis that anxiety may be attributed to a deficiency in dopamine levels. The role of dopamine in the pathophysiology of anxiety has been highlighted in earlier studies and has recently been re-emphasized. For example, the prefrontal cortex (PFC), hippocampus, and extended amygdala are brain regions responsible for processing emotions, including basal and stress-induced anxiety states. These regions are influenced by dopamine-rich midbrain regions that are thought to play a role in the formation of anxiety-like behaviors. Dopamine neurons in these target regions are important for emotion processing. Studies by Zweifel et al. have shown that interference with dopamine-dependent components can lead to the emergence of a generalized anxiety-like phenotype, and that activation of the mesolimbic dopamine system plays a crucial role in avoiding generalized anxiety. The dopaminergic system can also exert anxiolytic effects through its interaction with corticotropin-releasing hormone (CRH) and its receptor CRHR1.

[0006] This study starts from the mechanism of drugs for the treatment of perimenopausal syndrome in women, in order to achieve the development of safe and effective drugs for treatment. Summary of the invention

[0007] To this end, the technical problem to be solved by the present invention is to provide the application of CREB1, ATF2 and FOS as drug target genes in the screening and preparation of menopausal syndrome drugs; the application of reagents for regulating the expression of CREB1, ATF2 and FOS genes in the preparation of drugs for treating menopause; the application of CREB1, ATF2 and FOS genes in the analysis of the mechanism of action of menopausal syndrome drugs; and a pharmaceutical composition.

[0008] The present invention provides a technical solution: application of CREB1, ATF2 and FOS as drug target genes in screening and preparing drugs for menopausal syndrome.

[0009] Preferably, the CREB1, ATF2 and FOS genes act in the dopaminergic synaptic pathway.

[0010] Preferably, the screening method for CREB1, ATF2 and FOS genes comprises the following steps: S1. Experimental grouping: participants who meet the inclusion criteria are randomly divided into a drug group and a placebo group; S2. Experimental process and sample collection: serum samples were collected from the drug group and the placebo group at three time points: before medication, during the experiment, and at the end of the experiment; S3. Sample analysis, including: S3.1, transcriptome library preparation and sequencing; S3.2. Data analysis: FC and P values ​​were used to screen the differentially expressed genes between the drug group and the placebo group; S3.3. KEGG pathway enrichment analysis and network pharmacology analysis obtained CREB1, ATF2 and FOS target genes.

[0011] Preferably, after step S3.2, the method further includes performing a combined metabolomics and transcriptomics analysis on the drug group and the placebo group to screen metabolic and transcriptional co-enriched pathways.

[0012] Preferably, the drug group adopts a traditional Chinese medicine preparation with Cyperus rotundus and White Peony Root as main ingredients.

[0013] The present invention also provides another technical solution: use of a reagent for regulating the expression of CREB1, ATF2 and FOS genes in the preparation of a drug for treating menopause.

[0014] Preferably, the application includes: S1', experimental grouping, participants who met the inclusion criteria were randomly divided into a drug group and a placebo group; S2', experimental process and sample collection, serum samples were collected from the drug group and the placebo group at three time points: before medication, during the experiment, and at the end of the experiment; S3', sample analysis, including: S3.1', transcriptome library preparation and sequencing; S3.2', data analysis, FC and P value were used to screen the differentially expressed genes between the drug group and the placebo group; S3.3', KEGG pathway enrichment analysis and network pharmacology analysis revealed that CREB1, ATF2 and FOS target genes act in the dopaminergic synaptic pathway.

[0015] The present invention also provides a technical solution: a pharmaceutical composition comprising drugs for regulating the effects of CREB1, ATF2 and FOS genes on dopaminergic synaptic pathways.

[0016] Beneficial effects: The present invention provides the use of CREB1, ATF2 and FOS as drug target genes in screening and preparing drugs for menopausal syndrome. The drug target genes for menopausal syndrome are screened out through omics research, and they play a role by regulating the dopaminergic synaptic pathway. The key gene targets obtained by the present invention correspond to the related pathways, and their potential molecular mechanisms are determined, providing a reference for subsequent development and application.

[0017] The application of CREB1, ATF2 and FOS genes provided by the present invention in analyzing the mechanism of action of menopausal syndrome drugs provides a research direction for the in-depth development of subsequent treatment plans for menopausal syndrome. The combination of network pharmacology not only strengthens the validity of the research results, but also helps to determine the expected target genes for future therapeutic interventions. Secondly, in the clinical research part, a standardized scale is used to assess the anxiety level, rather than relying on subjective judgment methods, which enhances the accuracy of the research results. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to make the contents of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings.

[0019] Figure 1a This is a schematic diagram of the comprehensive distribution of differentially expressed genes in the drug group before and after medication in Example 1 of the present invention; Figure 1b This is a schematic diagram of the comprehensive distribution of differentially expressed genes in the placebo group before and after medication in Example 1 of the present invention; Figure 2 This is a schematic diagram of comparative analysis of differentially expressed genes between two experimental groups in Example 1 of the present invention; Figure 3 This is a schematic diagram of comparative analysis of differential metabolites between the two experimental groups and before and after medication in Example 1 of the present invention; Figure 4 A comprehensive network diagram of the interactions between differentially expressed genes and differential metabolites of the drug group of the present invention before and after medication; Figure 5 A schematic diagram of the relationship between the target genes related to the drug components of the drug analyzed by the present invention and the differentially expressed genes before and after the drug group is administered; Figure 6 The figure is a schematic diagram of the enrichment pathway based on the significant sorting of overlapping genes in the present invention; Figure 7 A schematic diagram of the relationship between the various drug components and signal pathways of the drug analyzed by the present invention; Figure 8 A schematic diagram of the relationship between the target genes related to the dopaminergic synaptic pathway screened for the present invention and the various drug components of the analyzed drugs. DETAILED DESCRIPTION

[0020] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments. The principles and features of the present invention are described below in combination with the accompanying drawings. It should be noted that the embodiments in this application and the features in the embodiments can be combined with each other without conflict. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention.

[0021] The therapeutic drug used in this manual is Xiangshao Granules, which is produced by Sichuan Hairong Pharmaceutical Co., Ltd., China; other experimental reagents, unless otherwise specified, are commercially available products.

[0022] In the present invention, a double-blind, randomized, placebo-controlled, multicenter design was used to study the effects of drugs on perimenopausal and postmenopausal Chinese women and their mechanisms of action based on transcriptomic analysis. 360 participants were randomly divided into two groups: one group took therapeutic drugs (n=180) and the other group took placebo (n=180) for 8 weeks. The main outcome measure was the Self-Rating Anxiety Scale (SAS) score, which was evaluated at baseline, 4 weeks and 8 weeks. The main focus was on the changes in SAS scores observed after the 8-week intervention period. In addition, in a subset of 96 patients, blood samples were collected at enrollment and after 8 weeks of treatment for metabolomics and transcriptomics analysis, as well as network pharmacology analysis, to study the mechanism of influence on anxiety symptoms.

[0023] In the present invention, the therapeutic drug is taken as an example of Xiangshao Granules, which is a pure herbal medicine with Cyperus rotundus and White Peony Root as the main drugs, and Bupleurum Root, Chuanxiong Rhizoma and Toosendan Fructus as auxiliary drugs. In animal experiments and randomized controlled trials, it has been observed that it has a strong safety and significant therapeutic effect in the treatment of perimenopausal symptoms, especially symptoms related to emotional symptoms. Existing studies believe that the regulation of drug symptoms of perimenopausal depression in women, the potential mechanism may involve the increase of regulating serum neurotransmitter levels and hippocampal brain-derived neurotrophic factor (BDNF), which has been identified as an antidepressant factor. Bupleurum saponin A is considered to be an active ingredient related to these mechanisms. However, the mechanism by which drugs relieve anxiety symptoms during perimenopause needs further study.

[0024] The present invention uses transcriptomics to analyze the therapeutic mechanism, and can also use metabolomics to analyze the therapeutic mechanism, as well as a combination of the two to perform mechanism analysis. "Omics" encompasses a comprehensive approach to examining the complete molecular spectrum of cells and tissues, including various disciplines such as genomics, transcriptomics, proteomics, metabolomics, epigenomics, and epitranscriptomics. Transcriptomics is the study of the transcriptome, including gene structure, expression, and regulation, while metabolomics is downstream of genomics, transcriptomics, and proteomics, and aims to characterize a large number of metabolites in biological systems that are related to pathophysiological interventions or genetic modifications. Through the method of the present invention, the potential mechanism of drug therapy is analyzed based on genomics, thereby providing a basis and ideas for future drug improvement or the development of new drugs.

[0025] 1. Screening of Experimental Participants The experimental participants in the present invention came from 18 clinical centers, and a total of 360 generally healthy perimenopausal or postmenopausal women with anxiety symptoms were recruited from December 2021 to June 2023. These participants were randomly assigned to a drug group or a placebo group. Assessments were performed at three different time intervals: baseline, 4 weeks later, and 8 weeks later. A baseline medical review was conducted using a standardized questionnaire and repeated at subsequent visits. In addition, in order to more deeply study the mechanism of the drug's effect on anxiety symptoms, blood samples were extracted from selected participants for analysis at enrollment and after 8 weeks of treatment. The study was approved by the Human Research Ethics Committee of Peking Union Medical College Hospital (HS-2958D) and registered at ClinicalTrials.gov (NCT05003336) on August 10, 2021. All participants provided written informed consent before the trial.

[0026] Participants included individuals aged 40 to 65 years with an intact uterus and at least one ovary. Eligible participants were required to be in the menopausal transition stage, characterized by a persistent difference in menstrual length of at least 7 days or amenorrhea lasting more than 60 days, or the postmenopausal stage, defined as amenorrhea lasting at least 12 consecutive months. The determination of these stages was based on the use of the 2011 Reproductive Aging Stages Workshop +10 criteria. In addition, participants had to exhibit mild to moderate anxiety symptoms, as demonstrated by a Self-Rating Anxiety Scale (SAS) score of 69 or lower and 50 or higher. In addition, participants were required to exhibit significant menopausal symptoms, as evidenced by a modified Kupperman score of 16 or higher.

[0027] Participants were excluded if they had a history of prior use of exogenous reproductive hormones, psychotropic medications, or other treatments for anxiety, or if they had used health products in the past four weeks. In addition, women with anxiety or other psychiatric disorders not related to menopause, as well as women with a Self-Rating Depression Scale (SDS) score of 70 or higher during screening, were also excluded. Women with a well-documented history of severe systemic illness, including but not limited to hypothyroidism / hyperthyroidism, unstable coronary artery disease, severe hypertension, pheochromocytoma, severe liver or kidney disease, gynecological or breast malignancies, and women with known allergies or sensitivities to study products, or women with a history of alcohol or drug abuse, were also not eligible for study participation.

[0028] 2. Experimental Grouping A randomized design was used to divide participants into the pegylated shao group and the placebo group. The randomization schedule was generated using computer software. The randomization sequence was generated using SAS software (SAS Institute, Cary, North Carolina, USA). Throughout the study, participants and investigators were unaware of the group assignment.

[0029] 3. Experimental Process In the drug group of the present invention, participants took Xiangshao granules, three times a day, 4 grams of Xiangshao granules each time, brewed with warm water, and taken within 30 minutes for 8 weeks. In the placebo group of the present invention, participants took Xiangshao granule simulators under the same scheme. Xiangshao granule simulators are mainly composed of corn dextrin, soluble starch, food coloring, flavor correctors, and 150g of clear paste extracted from every 1000g of Xiangshao granules. Compared with real drugs, the potential therapeutic effect of Xiangshao granule simulators is 9.3%. In addition, it has the same visual and taste characteristics as Xiangshao granule drugs, as well as similar packaging and labeling.

[0030] The experiment was marked as follows: "in A" refers to the entry group of the drug group, and "out A" refers to the exit group of the drug group. Similarly, "in B" refers to the entry group of the placebo group, and "out B" refers to the exit group of the placebo group. Self-administered questionnaires were used, including a general situation questionnaire and a self-rating anxiety scale (SAS) questionnaire. Anxiety symptoms were assessed using the SAS questionnaire (a total of 20 items). Each item was assigned a score from 1 to 4, with 1 indicating no or rarely, 2 indicating sometimes, 3 indicating often, and 4 indicating always. The questionnaire consisted of 15 negative statements scored in order (1-4) and 5 positive statements scored in reverse order (4-1). The total SAS score was calculated by the sum of the scores of each question. The standardized total score of the SAS was obtained by applying a multiplication factor of 1.25 to the total score and then rounding it off. Individuals with a standardized total SAS score of less than 50 were classified as having no anxiety disorder. A standardized total score of 50-59 was scored as mild, 60-69 was scored as moderate, and 70 or more was scored as severe.

[0031] Clinical outcomes were determined by analyzing the change in SAS scores from baseline scores after the 8-week treatment period. The SAS reduction rate was calculated as: (SAS score at baseline - SAS score after 8 weeks of treatment) / (SAS score at baseline - 25) x 100%. Categorical variables were used to examine clinical improvement based on the degree of change in the SAS. The classification criteria were as follows: a reduction of less than 30% was classified as "ineffective", a reduction of 30% to 50% was classified as "relief", a reduction of 50% to 80% was classified as "moderately effective", and a reduction of more than 80% was classified as "highly effective". In addition, a SAS score of less than 50 at the 8-week time point was considered an indication of symptom cure.

[0032] 4. Sample Analysis (A) Metabolic analysis using metabolite identification technology.

[0033] Serum samples were diluted with LC-MS grade water to a final concentration of 53% methanol. The samples were then centrifuged at 15,000 g and 4°C for 20 min. Finally, the supernatant was injected into the LCMS / MS system for analysis. Metabolites were quantitatively analyzed using an ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) system. The raw data files generated by UPLC-MS were processed using Compound Discoverer3.1 (CD3.1, ThermoFisher), and peak alignment, peak picking, and quantification were performed for each metabolite. Afterwards, the peak intensity was normalized to the total spectral intensity. It was then compared with the mzCloud, mzVault, and Masslist databases to obtain accurate qualitative and relative quantitative results.

[0034] Metabolites were annotated using the KEGG database (https: / / www.genome.jp / kegg / pathway.html), HMDB database (https: / / hmdb.ca / metabolites), and LIPIDMaps database (http: / / www.lipidmaps.org / ). Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed on metabolic data processing software, such as metax software. Univariate analysis (t-test) was used to calculate statistical significance (p value). Metabolites with VIP>1 and p value <0.05 and fold change22 or FCS0.5 were considered differential metabolites. Volcano plots were screened for enriched metabolites based on the log2 (fold change) and -log10 (p value) of metabolites using ggplot2 in R language.

[0035] (B) Transcriptome sequencing and analysis.

[0036] First, library preparation and sequencing were performed. RNA integrity was assessed using the RNA kit (RNA Nano 6000 Assay Kit) of the Bioanalyzer2100 system (Agilent Technologies, CA, USA). Total RNA was used as input material for RNA sample preparation. Library preparation was performed using the TruSeg PE Cluster Kit v3 (Illumina). After evaluating the quality of the library, sequencing was performed on the Illumina Novaseg platform to generate 150 bp double-end sequences.

[0037] Transcriptome data were analyzed. The raw data were processed by fastp software, that is, the sequences containing adapters, sequences containing ploy-N, and low-quality sequences were removed from the raw data to obtain high-quality data. Then, the cleaned double-end sequences were aligned with the reference genome GRCh38 / hg38 using the transcriptome alignment tool (Hisat2 v2.0.5). The number of sequences for each gene was counted using feature counting v1.5.0-p3. Then, the FPKM (Reads Per Kilobase of exon model per Million mapped reads) of each gene was calculated based on the length of each gene and the number of sequences of the gene. The data of the drug group and the placebo group were differentially expressed using the DESeq2 R (1.20.0) software package. The obtained p-values ​​were adjusted using the Benjamini and Hochberg method to control the false discovery rate. Genes with adjusted p-values ​​≤ 0.05 were classified as differentially expressed by DESeq2 R (1.20.0). Common gene enrichment analysis tools (such as ClusterProfiler package) were used to perform enrichment analysis of differentially expressed genes, including Gene Ontology (GO) and KEGG enrichment analysis of differentially expressed genes, in which gene length bias was corrected. GO or KEGG terms with a corrected p value of < 0.05 were considered to be significantly enriched in differentially expressed genes.

[0038] (III) Statistical analysis

[0039] Statistical analysis Continuous variables with normal distribution were expressed as mean ± standard deviation. Categorical variables were expressed as numbers and percentages. Comparisons of continuous variables between groups were assessed by analysis of variance. Depending on the sample size, Pearson chi-square test or Fisher's exact test were used to compare categorical data frequencies. For comparisons involving non-normally distributed data sets, the Wilcoxon rank sum test was used. Statistical analyses were performed using SAS software, version 9.4, and P < 0.05 was considered statistically significant.

[0040] Example 1 This embodiment is the use of CREB1, ATF2 and FOS as drug target genes in screening and preparing drugs for menopausal syndrome. Wherein, screening and preparing drugs for menopausal syndrome includes screening drugs for preventing, alleviating and / or treating menopausal syndrome.

[0041] The process is as follows: S1. Experimental grouping: participants who meet the inclusion criteria are randomly divided into a drug group and a placebo group; S2. Experimental process and sample collection: serum samples were collected from the drug group and the placebo group at three time points: before medication, during the experiment, and at the end of the experiment; S3. Sample analysis, including: S3.1, transcriptome library preparation and sequencing; S3.2. Data analysis: FC and P values ​​were used to screen the differentially expressed genes between the drug group and the placebo group; S3.3. KEGG pathway enrichment analysis and network pharmacology analysis obtained CREB1, ATF2 and FOS target genes.

[0042] Specific: Based on 360 participants, 180 of whom were assigned to the drug group and 180 to the placebo group. Participants in the two groups showed similar baseline characteristics, including age, menopausal status, and SAS scores. No statistically significant differences were presented in these variables. In the clinical analysis, the completion rate of the drug group was 165 / 180 (91.7%) and the completion rate of the placebo group was 162 / 180 (90.0%), of which 166 participants in the drug group and 165 participants in the placebo group completed at least 4 weeks of the study. A total of 96 patients were included in the omics study, providing comprehensive omics data for entry (in) and exit (out) samples. Each patient provided a transcriptome and a metabolome sample at two different time points before and after medication. A total of 384 omics samples were obtained, including 192 transcriptome samples and 192 metabolome samples. The entry group of the drug group was marked as "in A" and the exit group was marked as "out A". Similarly, the entry group of the placebo group was marked as "in B" and the exit group was called "out B". Through quality inspection, 4 transcriptome samples and 7 metabolome samples were found to be unqualified, and a total of 373 samples were included in the omics analysis.

[0043] After the two groups of participants were treated with medication according to the above experimental process, the changes in anxiety symptoms in both groups were significantly lower than before treatment in SAS scores (P<0.0001). The scores of the medication group were 47.7±8.78 at 4 weeks and 44.7±7.94 at 8 weeks; the scores of the placebo group were 47.8±8.27 at 4 weeks and 47.2±8.79 at 8 weeks. After 8 weeks, the SAS score of the medication group was significantly lower than that of the placebo group (P=0.0086). The proportion of participants in the medication group who were classified as "cured" increased from 58.6% at 4 weeks to 69.8% at 8 weeks. At the 4th and 8th week time points, the cure rates of the placebo group were 54.0% and 56.9%, respectively. The clinical improvement of the two groups after 8 weeks was statistically significant (P=0.0151).

[0044] The differential gene enrichment in the dopaminergic synaptic pathway between the drug group and the placebo group before and after medication was compared and analyzed. Differentially expressed genes were identified by FC (Fold Change) and values. The threshold was set as |log2FC] ≥ 1, p value ≤ 0.05, and the number of differentially expressed genes screened is shown in Table 1.

[0045] Table 1 Number of differentially expressed genes between the drug group and the placebo group See also Figure 1a and Figure 1b , shows the comprehensive distribution of differentially expressed genes in the two groups before and after medication. The left side is the drug group and the right side is the placebo group. The horizontal axis represents the fold change and logarithmic transformation of the differentially expressed genes in each group, and the vertical axis represents the significance level of these differences. Each data point in the figure corresponds to a specific gene, where significantly upregulated genes are represented in red and significantly downregulated genes are represented in green. Compared with the placebo group, the number of differentially expressed genes in the drug group increased significantly. See Figure 2 and Figure 3 , showing the comparative analysis of differentially expressed genes in the two groups, intuitively seeing the overlapping and differentially expressed genes between the two groups, and thus understanding the relationship between the differentially expressed genes in the two experimental groups. After excluding 101 overlapping genes in the two groups, there were 1136 differentially expressed genes in the drug group and 602 differentially expressed genes in the placebo group.

[0046] KEGG pathway enrichment analysis was used to determine the main biological functions performed by differentially expressed genes. The enrichment results were derived from the KEGG pathways, and the hypergeometric test method was used to identify pathways that were enriched for differentially expressed genes relative to all identified gene backgrounds. This was used to determine the main biochemical, metabolic, and signal transduction pathways associated with the differentially expressed genes. The peony group showed a total of 11 enriched pathways corresponding to specific biological processes, while the placebo group showed only 2 enriched pathways. The KEGG pathway enrichment analysis of differentially expressed genes is shown in Table 2.

[0047] Table 2 KEGG pathway enrichment analysis of differentially expressed genes Metabolomics and transcriptomics were jointly analyzed for the drug group and the placebo group. Among them, there were two enriched pathways in the drug group, namely the dopaminergic synaptic pathway and the vitamin digestion and absorption pathway. Before and after medication, the dopaminergic synaptic pathway showed significant changes in both transcriptomics and metabolomics analysis (P value < 0.05), while the vitamin digestion and absorption pathway only showed significant differences in metabolomics analysis (P value < 0.05), and there was no statistical difference in transcriptomics analysis (P value < 0.75). No transcriptional metabolic co-enrichment pathways were shown in the placebo group. See Figure 4 , showing the comprehensive network diagram of the differentially expressed genes and differential metabolite changes in the drug group before and after medication. Among them, the genes are represented by ovals, namely LMBRD1, PLCB4, PPP2R5C, GNG2, CREB1, GNB4, PLCB1, ATF2, FOS, GSK3A, PPP2R2A, PPP2CA, PPP1CB, PRKACB, GNAI3, GNG3, and DRD4. The darker the background color of the ellipse, the smaller the P value in the transcriptome difference analysis, that is, the higher the significance; the signal pathway is represented by a purple rectangle, which is the dopaminergic synaptic pathway, the vitamin digestion and absorption pathway, and the tyrosine pathway; the metabolites are represented by yellow hexagons, which are homovanillic acid and mephenone. The dark connecting line represents the correlation between the signal pathway and the gene, and the light connecting line represents the correlation between the metabolite and the gene.

[0048] The network pharmacology analysis of drugs includes: (1) screening target genes related to the main components of the drugs from the network pharmacology database (Traditional Chinese Medicine Systems Pharmacology, TCMSP), and based on the drug group of this example, screening and identifying 590 genes corresponding to the active small molecules of the main components of the drugs; (2) after intersecting these 590 genes with the 1237 differentially expressed genes in the drug group, screening and obtaining 24 overlapping genes, see Figure 5 , showing the overlap and differential relationship between target genes related to drug ingredients and differentially expressed genes before and after drug administration. The drug ingredients include white peony root, cyperus rotundus, Chuanlianzi, bupleurum, ligusticum chuanxiong, fructus aurantii, pinellia tuber, cardamom, costusroot, and liquorice. The 24 overlapping genes are TERF1, PTGS2, CHUK, PPARD, KCNH2, ATF2, GLYATL1, METAP2, DMPK, CDKN2AIP, RELA, PPARG, NOS3, PCNA, ACSL4, ALB, CXCL10, FASN, JAK2, HIF1A, CREB1, CXCL8, AHR, and FOS. (3) Pathway enrichment analysis of these 24 overlapping genes was performed using the KEGG database, and 15 significantly different pathways were obtained. Figure 6 The top 15 enriched pathways of the overlapping genes are shown, arranged by the difference value. One of the important pathways is the dopaminergic synaptic pathway; (4) Comprehensive analysis of the network relationship formed by the 24 overlapping genes, the main components of the drug and the dopaminergic synaptic pathway. Figure 7As shown in the figure, the ingredients of the drug are represented by green diamonds, including white peony root, cyperus rotundus, Chuanlianzi, bupleurum, chuanxiong, fructus aurantii, pinellia tuber, cardamom, costusroot, and liquorice; the dopaminergic synaptic pathway is represented by a purple rectangle, and the genes are represented by ovals, including CXCL10, CXCL8, CHUK, FASN, NOS3, AHR, PPARD, HIF1A, PPARG, FOS, PTGS2, KCNH2, RELA, CDKN2AIP, METAP2, DMPK, ALB, ATF2, ACSL4, JAK2, TERF1, GLYATL1, PCNA, and CREB1; the size and font of the graphic represent the degree value, and the larger the size, the higher the degree value. As can be seen in the figure, the genes that play a role in the dopaminergic synaptic pathway, namely FOS, ATF2, and CREB1, are highlighted in yellow. See Figure 8 , showing the correlation between the three target genes in the dopaminergic synaptic pathway and the various components of the drug. See Table 3, showing the correlation between the three target genes in the dopaminergic synaptic pathway and the various components of the drug.

[0049] Table 3 Relationship between target genes and drug components involved in dopaminergic synaptic pathways Example 2 This embodiment provides the use of a reagent for regulating the expression of CREB1, ATF2, and FOS genes in the preparation of a drug for treating menopause. The reagent includes a reagent for regulating the expression of CREB1, ATF2, and FOS genes in the dopaminergic synaptic pathway, and is used to prepare a drug for treating menopause to achieve the treatment of female menopausal syndrome.

[0050] Example 3 This example provides the application of CREB1, ATF2 and FOS genes in analyzing the mechanism of action of menopausal syndrome drugs.

[0051] Based on the experimental process and results of Example 1, after 8 weeks of medication, the cure rate of the drug group was 69.8%, and the cure rate of the placebo group was 56.9%, and the clinical improvement was statistically significant (P=0.0151).

[0052] Combined analysis of gene transcriptomics and metabolomics showed that the dopaminergic synaptic pathway showed significant changes in transcriptomics and metabolomics analysis before and after drug administration (P value < 0.05). Figure 7 As shown, the drug components are represented by green diamonds, the pathways are represented by purple rectangles, and the genes are represented by ovals. The size and font of the graphics represent the degree value. The larger the size, the higher the degree value. As can be seen in the figure, the genes that play a role in the dopaminergic synaptic pathway, namely FOS, ATF2 and CREB1, are highlighted in yellow. Figure 8, showing the correlation between three target genes in the dopaminergic synaptic pathway and the various components of the drug.

[0053] The embodiments of the present invention provide a research direction for the further development of treatment plans for menopausal syndrome. Among them, the combination of network pharmacology not only strengthens the validity of the research results, but also helps to determine the expected target genes for future therapeutic interventions. Secondly, in the clinical research part, a standardized scale is used to assess the anxiety level, rather than relying on subjective judgment methods, which enhances the accuracy of the research results.

[0054] Example 4 This embodiment provides a pharmaceutical composition, which contains a drug that regulates the effects of CREB1, ATF2, and FOS genes in the dopaminergic synaptic pathway to achieve the treatment of female menopausal syndrome. In addition to the active ingredients, the drug may also include one or more pharmaceutically acceptable carriers or excipients according to the needs of different dosage forms.

[0055] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Application of CREB1, ATF2 and FOS as drug target genes in screening and preparing drugs for menopausal syndrome.

2. The use according to claim 1, characterized in that: The CREB1, ATF2 and FOS genes act in the dopaminergic synaptic pathway.

3. The use according to claim 1 or 2, characterized in that: The screening method of the CREB1, ATF2 and FOS genes comprises the following steps: S1. Experimental grouping: Participants who meet the inclusion criteria are randomly divided into a drug group and a placebo group; S2. Experimental process and sample collection: serum samples were collected from the drug group and the placebo group at three time points: before medication, during the experiment, and at the end of the experiment; S3. Sample analysis, including: S3.1, transcriptome library preparation and sequencing; S3.

2. Data analysis: FC and P values ​​were used to screen the differentially expressed genes between the drug group and the placebo group; S3.

3. KEGG pathway enrichment analysis and network pharmacology analysis obtained CREB1, ATF2 and FOS target genes.

4. The use according to claim 3, characterized in that: After step S3.2, the method further includes performing a combined metabolomics and transcriptomics analysis on the drug group and the placebo group to screen for metabolic and transcriptional co-enriched pathways.

5. The use according to claim 3, characterized in that: The medicine group adopts a traditional Chinese medicine preparation with Cyperus rotundus and White Peony Root as main ingredients.

6. Application of reagents that regulate the expression of CREB1, ATF2, and FOS genes in the preparation of drugs for the treatment of menopause.

7. Application of CREB1, ATF2 and FOS genes in analyzing the mechanism of action of drugs for menopausal syndrome.

8. The use according to claim 7, characterized in that: include: S1', experimental grouping, participants who met the inclusion criteria were randomly divided into a drug group and a placebo group; S2', experimental process and sample collection, serum samples were collected from the drug group and the placebo group at three time points: before medication, during the experiment, and at the end of the experiment; S3', sample analysis, including: S3.1', transcriptome library preparation and sequencing; S3.2', data analysis, FC and P value were used to screen the differentially expressed genes between the drug group and the placebo group; S3.3', KEGG pathway enrichment analysis and network pharmacology analysis revealed that CREB1, ATF2 and FOS target genes act in the dopaminergic synaptic pathway.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition contains drugs for regulating the effects of CREB1, ATF2 and FOS genes on the dopaminergic synaptic pathway.

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