Biomarker for potential treatment needing Qingshu granules and application of biomarker

Abstract

The invention belongs to the technical field of biology, and particularly relates to a biomarker for potential treatment needing Qingshu granules and application of the biomarker. The biomarkers comprise a series of differentially expressed genes (such as Egr3, Gpr88 and Adora2a) and proteins (such as GAD67, ER beta and the like), the markers are highly associated with occurrence and development of hypertension, especially pathological mechanisms of estrogen deficiency related hypertension, so that the biomarkers are applied to preparation of novel antihypertensive drugs, hypertension is reduced through a multi-channel synergistic effect, and the antihypertensive effect is achieved. Comprising the following steps: regulating hypothalamic functions, recovering estrogen level, improving neurotransmitter balance, inhibiting oxidative stress and inflammatory response and the like; the Qingshu granules disclosed by the invention can be used as a corresponding functional medicine. The key effects of the Egr3 and the GAD67 in a central nervous system, especially in function connection of an emotion regulation brain region, are defined for the first time.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a biomarker for potentially requiring treatment with Qingshu Granules and its application. Background Art

[0002] As a disease that requires long-term medication maintenance, modern medical treatment of menopausal hypertension still mainly focuses on symptomatic treatment, including: RAAS inhibitors, including β-blockers, ACEI drugs, ARB drugs, and aldosterone receptor antagonists. Although its antihypertensive effect is rapid, the long-term efficacy is not ideal, and it cannot improve the central nervous system symptoms during menopause. Long-term use will also cause adverse drug reactions such as dry cough, edema, gastrointestinal discomfort, and rash.

[0003] Hormone replacement therapy (HRT) can lower blood pressure, dilate blood vessels, and relieve related symptoms of the central nervous system, but there is a risk of inducing thrombotic diseases and sex hormone-related cancers.

[0004] In recent years, the complex pathogenesis of menopausal hypertension has made traditional Chinese medicine, which currently has the characteristics of multi-component, multi-target, and multi-pathway treatment, attract much attention in the treatment of complex diseases such as menopausal hypertension. Compounds or traditional Chinese medicine monomers such as Qingxuan Jiangya Decoction, Erxian Decoction, Erzhi Pills, Liuwei Dihuang Decoction, icariin, Xuzao Gengping Pills, and Sangdi Gengping Pills can lower blood pressure, improve menopausal symptoms, and improve the quality of life through the effects of vasodilation and estrogen-like substances.

[0005] Based on the clinical manifestations of menopausal hypertension, it belongs to the categories of "zangzao", "syndrome of pre- and post-menopausal disorders", "depressive syndrome", "lily disease", and "palpitation" in traditional Chinese medicine. There are many causes of this disease. Deficiency of the liver and kidney in the constitution, yin deficiency and yang hyperactivity, emotional disorders, improper diet, and excessive consumption of fatty and sweet foods can all cause this disease. The relevant etiology and pathogenesis have been discussed by medical experts of past dynasties for a long time, and it is believed that it is mostly related to "deficiency", "wind", "phlegm", and "fire". Most traditional Chinese medicine experts believe that after menopause, the kidney yin essence is insufficient, the water fails to nourish the wood, the liver qi is not smooth, stagnates and transforms into fire, resulting in liver fire flaring up, presenting symptoms such as elevated blood pressure, irritability, anxiety, and tension; excessive heat injures yin, the liver yin deficiency is aggravated, yin fails to control yang, resulting in liver yang hyperactivity, presenting symptoms such as headache, dizziness, restlessness, and insomnia; when yin deficiency is more severe, virtual fire flares up, yin damages yang, and there is yin-yang deficiency, presenting symptoms such as anxiety, depression, hot flashes, and night sweats, and forgetfulness. Therefore, hypertension after menopause is often accompanied by mental and neurological symptoms.

[0006] Clinical traditional Chinese medicine experts have achieved good curative effects in treating the discomfort caused by female menopausal hypertension by using the method of nourishing the liver and kidney and suppressing liver yang. It has definite curative effects in lowering blood pressure and improving central nervous system symptoms during menopause such as dizziness, anxiety, and depression, and has the effect of increasing estrogen levels.

[0007] The Qingxuan Jiangya Decoction is a prescription for hypertension formulated based on this traditional Chinese medicine theory, and it has definite clinical efficacy for menopausal hypertension, but its mechanism of action is not yet clear. Summary of the Invention

[0008] (1) Technical problems to be solved

[0009] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a biomarker for potentially needing Qingshu Granule treatment, which provides new biomarkers that can be used to indicate hypertension and can indicate the discovery of new targets for hypertension treatment drugs;

[0010] Correspondingly, the present invention also provides the application of a biomarker for potentially needing Qingshu Granule treatment, which can be used to prepare hypertension treatment drugs.

[0011] (2) Technical solutions To achieve the above object, the main technical solutions adopted by the present invention include:

[0012] In the first aspect, the present invention provides a biomarker for potentially needing Qingshu Granule treatment, which includes: a combination of one or two of the following genes:

[0013] Dsp, LOC108351703, Otop3, Ccdc77, LOC120097335, Gpr88, Egr3, Atp2a1, Nxnl2, LOC120101770, LOC120099591, RGD1561730, Chrm1, Myo5c, Adora2a, Lbp, Ca12, Arhgef16, Cfi, Krtap11-1;

[0014] Or / and

[0015] A combination of one or more of the following bioactive substances:

[0016] GAD67 protein, Egr3 protein, ERβ protein, ERα protein.

[0017] The new biomarker provided by the present invention includes gene types and protein types,

[0018] These markers can distinguish the molecular characteristics of different causes of hypertension, especially showing specificity in the hypertension model caused by ovariectomy.

[0019] In the second aspect, the present invention also provides the application of the above biomarker in the preparation of products for preventing, diagnosing or treating hypertension or hypertension-related analysis methods.

[0020] The present invention also provides an application in the preparation of products for preventing, diagnosing or treating hypertension developed for biomarkers, which can significantly improve problems such as poor efficacy and strong non-specificity of traditional drugs for castration-induced hypertension. Among them, by regulating Egr3, ERβ and Glu / GABA balance, blood pressure is significantly stabilized, and the problem of drug resistance that may occur in traditional treatments is prevented.

[0021] Optionally, the application includes a gene detection method for hypertension risk assessment, comprising the following steps:

[0022] S1 Extract genomic RNA from a subject sample;

[0023] S2 Detect the transcript expression levels of one or both of the following genes in the genomic RNA;

[0024] Dsp, LOC108351703, Otop3, Ccdc77, LOC120097335, Gpr88, Egr3, Atp2a1, Nxnl2, LOC120101770, LOC120099591, RGD1561730, Chrm1, Myo5c, Adora2a, Lbp, Ca12, Arhgef16, Cfi, Krtap11-1;

[0025] S3 Generate a personalized hypertension risk assessment report based on the correlation between the detection results of the transcript expression levels in step S2 and the systolic or diastolic blood pressure of the subject.

[0026] By screening the expression levels of specific genes and performing correlation analysis with systolic or diastolic blood pressure, the present invention can generate a personalized hypertension risk assessment report. This method significantly improves the sensitivity and specificity of prediction compared with traditional risk assessment models, and at the same time simplifies the operation steps, providing technical support for large-scale clinical promotion.

[0027] Optionally, the hypertension-related product is a drug for reducing blood pressure, and the drug includes a combination of the following one or more actions to reduce blood pressure:

[0028] Regulate the estrogen receptor expression in the paraventricular nucleus of the hypothalamus;

[0029] Regulate the expression of the Egr3 gene in the paraventricular nucleus of the hypothalamus;

[0030] Improve the Glu / GABA balance; improve the functions of brain regions related to emotion regulation;

[0031] Regulate the sympathetic nerve activity of the paraventricular nucleus of the hypothalamus; restore estrogen levels;

[0032] Regulate the RAS system; inhibit oxidative stress; anti-inflammatory effect.

[0033] In this solution, it is further defined that the drug has the above-mentioned effects, which should be understood that any drug with the above-mentioned effects can be used to reduce blood pressure.

[0034] Optionally, the drug includes at least one of the following bioactive substances: ERβ agonist, ERα agonist, Egr3 regulator, GAD67 expression regulator, Glu and GABA regulator, mood regulation brain region functional connectivity regulator, metabolic regulator that regulates pentose and gluconate metabolic pathways, anti-inflammatory factor, bile secretion promoter, antibacterial agent against Staphylococcus aureus infection, immune regulator against Staphylococcus aureus infection;

[0035] The mood regulation brain region functional connectivity includes one or a combination of two or more of the following connections: the connection between the right primary cingulate cortex, the left hypothalamus and the right primary cingulate gyrus, the connection between the right hypothalamus and the left striatum, the connection between the right perirhinal cortex area 35 and the right cerebellar granule cell layer.

[0036] In this solution, it is defined that the drug can significantly improve the hypertension and related mood disorder problems caused by castration through a combination including but not limited to the above bioactive substances. By targeting and regulating the functions of brain regions related to mood regulation (such as the right primary cingulate cortex, the connection between the hypothalamus and the striatum, etc.), the effect of reducing blood pressure can be achieved.

[0037] Optionally, the drug is Qingshu Granules.

[0038] Optionally, the application includes a multi-marker detection kit for hypertension diagnosis.

[0039] The kit in this solution, through biomarkers, including gene types (such as Egr3, Atp2a1) and protein types (such as GAD67, ERβ), significantly improves the sensitivity and specificity of hypertension diagnosis, especially the early recognition ability for castration-related hypertension.

[0040] In the third aspect, the present invention also provides an application of an Egr3-expressed protein as a Glu / GABA regulator.

[0041] The present invention discovers that the Egr3-expressed protein can significantly improve the Glu / GABA imbalance, which is one of the core causes of hypertension caused by the decline of estrogen levels. Compared with traditional antihypertensive drugs, the Egr3-expressed protein realizes the function of fundamentally restoring balance by directly acting on the neurotransmitter regulation network. This molecular mechanism has not been publicly reported in the prior art and has a significant technological breakthrough.

[0042] The present invention also provides a screening method for biomarkers, including the following steps:

[0043] S1 Establish an experimental group: Use castrated animals to establish a model group, an estrogen group, and a treatment drug group, and use normal animals to establish a normal group;

[0044] Estrogen group: Intervene with E2 drug;

[0045] Treatment drug group: Intervene and treat with treatment drugs;

[0046] S2 Screening of gene-based biomarkers:

[0047] S21 Collect differentially expressed genes between the normal group and the model group, between the model group and the treatment drug group, and between the model group and the estrogen group;

[0048] S22 Analyze and screen the differentially expressed genes that are significantly correlated with the animals in the corresponding experimental groups;

[0049] S3 The screening method of the protein includes:

[0050] Regularly detect the expression of estrogen receptors and related regulatory proteins in the experimental group animals, and through analysis and screening, obtain: ERβ, ERα, Egr3, GAD67.

[0051] Fourthly, the present invention also provides an application of Qingshu Granules, and the application includes being used for preparing the following drugs: ERβ agonist, ERα agonist, Egr3 regulator, GAD67 expression regulator, Glu and GABA regulator, mood regulation brain region functional connectivity regulator, metabolic regulator for regulating pentose and gluconate metabolic pathways, anti-inflammatory factor, bile secretion promoter, antibacterial agent against Staphylococcus aureus infection, immune regulator against Staphylococcus aureus infection;

[0052] The mood regulation brain region functional connectivity includes one or a combination of two or more of the following connections: right primary cingulate cortex, connection between the left hypothalamus and the right primary cingulate cortex, connection between the right hypothalamus and the left striatum, connection between the right 35 area of the perirhinal cortex and the right cerebellar granular cell layer.

[0053] Qingshu Granules have the following functions:

[0054] Regulate the expression of estrogen receptors in the paraventricular nucleus of the hypothalamus; regulate the expression of the Egr3 gene in the paraventricular nucleus of the hypothalamus; improve the Glu / GABA balance; improve the functions of brain regions related to mood regulation; regulation of sympathetic nerve activity by the paraventricular nucleus of the hypothalamus; restore estrogen levels; regulation of the RAS system; inhibit oxidative stress; anti-inflammatory effect. Therefore, it can be used as the corresponding functional drug.

[0055] (III) Beneficial effects

[0056] The beneficial effects of the present invention are:

[0057] The biomarkers of the present invention include a series of differentially expressed genes (such as Egr3, Gpr88, Adora2a) and proteins (such as GAD67, ERβ, etc.). These biomarkers are highly associated with the occurrence and development of hypertension, especially the pathological mechanism of estrogen deficiency-related hypertension. Thus, they are applied to the preparation of new antihypertensive drugs, achieving multi-pathway synergistic effects in reducing hypertension, including regulating hypothalamic function, restoring estrogen levels, improving neurotransmitter balance, inhibiting oxidative stress and inflammatory responses, etc.;

[0058] Among them, through the biomarkers, the present invention discovers that bioactive substances with corresponding functions can be used to reduce blood pressure; such bioactive substances can be used as antihypertensive drugs, thus providing new and effective antihypertensive drugs;

[0059] Among them, through the biomarkers, the present invention discovers the key role of Egr3 in regulating the Glu / GABA balance and the functional connectivity of emotion-regulating brain regions, combined with the regulation of sympathetic nerve activity by the hypothalamus; thus, Egr3 can be used as a new Glu / GABA regulatory drug;

[0060] Among them, through the biomarkers, the present invention determines Egr3 and GAD67 as antihypertensive drug targets;

[0061] Among them, the Qingshu Granule of the present invention can be used as a corresponding functional drug

[0062] The present invention for the first time clarifies the key role of Egr3 and GAD67 in the central nervous system, especially the participation mechanism in the functional connectivity of emotion-regulating brain regions. This discovery not only explains the emotion regulation disorder in hypertensive patients, but also provides a new target for the future development of treatment plans for emotion-related hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 Shows the effect of Qingshu Granule on the levels of serum blood pressure regulatory factors in ovariectomized female rats in Example 3;

[0064] Figure 2 Shows the effect of Qingshu Granule on the serum oxidative stress level in ovariectomized female rats in Example 3, and the statistical chart of the levels of reactive oxygen species (ROS) in the serum of rats in each group;

[0065] Figure 3 Shows the statistical analysis chart of the different brain regions with ALFF in rats in each group in Example 3;

[0066] Figure 4 Shows the statistical analysis chart of the brain functional connectivity with the right hypothalamus as the seed point in rats in each group in Example 3;

[0067] Figure 5Statistical analysis diagram of brain functional connectivity with the left hypothalamus as the seed point for each group of rats in Example 3;

[0068] Figure 6 Statistical analysis diagram of brain functional connectivity with the left amygdala as the seed point for each group of rats in Example 3;

[0069] Figure 7 Statistical analysis diagram of brain functional connectivity with the right amygdala as the seed point for each group of rats in Example 3;

[0070] Figure 8 Nissl staining results of the paraventricular nucleus of the hypothalamus in Example 3;

[0071] Figure 9 (OVX / Ctrl) vs (QSG-H / OVX) differentially expressed gene statistical chart;

[0072] Figure 10 Cluster heat map of differentially expressed genes related to Qingshu Granule regulating blood pressure in Example 3;

[0073] Figure 11 ERα immunohistochemical results of the paraventricular nucleus of the hypothalamus in Example 3;

[0074] Figure 12 ERβ immunohistochemical results of the paraventricular nucleus of the hypothalamus in Example 3;

[0075] Figure 13 Egr3 immunohistochemical results of the paraventricular nucleus of the hypothalamus in Example 3. Detailed implementation manners

[0076] To illustrate in detail the possible application scenarios, technical principles, specific implementable solutions, achievable purposes and effects of this application, etc., the following will be described in detail with reference to the specific examples listed. The examples recorded in this article are only used to more clearly illustrate the technical solutions of this application, so they are only used as examples and cannot be used to limit the protection scope of this application.

[0077] Referring to "Example" in this article means that the specific features, structures or characteristics described in combination with the example can be included in at least one embodiment of this application. The word "Example" appearing in various positions in the specification does not necessarily refer to the same example, nor does it particularly limit its independence or relevance to other examples. In principle, in this application, as long as there is no technical contradiction or conflict, the technical features mentioned in each example can be combined in any way to form the corresponding implementable technical solutions.

[0078] Unless otherwise defined, the meanings of the technical terms used herein are the same as those commonly understood by those skilled in the technical field to which this application belongs; the use of relevant terms herein is only for describing specific embodiments and is not intended to limit this application.

[0079] In the description of this application, the term "and / or" is an expression used to describe the logical relationship between objects, indicating that three relationships can exist. For example, A and / or B means: there is A, there is B, and there is both A and B at the same time. In addition, the character " / " in this text generally represents an "or" logical relationship between the associated objects before and after.

[0080] In this application, terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual quantitative, primary-secondary or sequential relationships between these entities or operations.

[0081] Without further limitation, in this application, the expressions "including", "comprising", "having" or other similar expressions used in a statement are intended to cover non-exclusive inclusion. These expressions do not exclude that there may be additional elements in the process, method or product including the said elements, so that the process, method or product including a series of elements may not only include those defined elements, but also include other elements not explicitly listed, or also include elements inherent to such process, method or product.

[0082] Similar to the understanding in the "Examination Guidelines", in this application, expressions such as "greater than", "less than", "exceeding" are understood not to include the number itself; expressions such as "above", "below", "within" are understood to include the number itself. In addition, in the description of the embodiments of this application, the meaning of "a plurality of" is two or more (including two), and similar expressions related to "many", such as "multiple groups", "multiple times", etc., are understood in this way unless otherwise specifically defined.

[0083] In the description of the embodiments of this application, the spatially related expressions used, such as "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "perpendicular", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc., the indicated orientation or positional relationship is based on the orientation or positional relationship shown in the specific embodiment or the drawing, and is only for the convenience of describing the specific embodiment of this application or for the reader's understanding, rather than indicating or implying that the device or component referred to must have a specific position, a specific orientation, or be constructed or operated in a specific orientation, and therefore cannot be understood as a limitation on the embodiments of this application.

[0084] Unless otherwise clearly specified or defined, in the description of the embodiments of the present application, the terms such as "installation", "connection", "attachment", "fixation", "setting" and the like shall be understood in a broad sense. For example, the "connection" may be a fixed connection, a detachable connection, or an integral setting; it may be a mechanical connection, an electrical connection, or a communication connection; it may be a direct connection, or an indirect connection through an intermediate medium; it may be the communication inside two components or the interaction relationship between two components. For those skilled in the art to which the present application pertains, the specific meanings of the above terms in the embodiments of the present application can be understood according to specific circumstances.

[0085] In the following specific embodiments,

[0086] The animal model experimental method is as follows:

[0087] Fifty 8-week-old female Sprague-Dawley rats were randomly divided into a normal group (Ctrl), a model group (OVX), an estrogen group (E2) (0.1 mg / kg / d), a low-dose Qingshu Granule group (QSG-L, 5.8725 g / kg / d), and a high-dose Qingshu Granule group (QSG-H, 11.745 g / kg / d). Except for the normal control group, ovariectomy was performed on the rats in the other groups to establish an ovariectomized female rat (OVX) model. Five days after the operation, the estrous cycle was continuously detected by vaginal smear, and blood pressure and body weight were monitored weekly; small animal magnetic resonance fMRI was used to analyze the functional changes and differences in the brain regions of the rats; blood was collected after 8 weeks of intervention, and Nissl staining was used to observe the neuronal activity in the paraventricular nucleus of the hypothalamus; enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of serum E2, RAS-related factors, neurotransmitters, and oxidative stress, and immunohistochemistry (IHC) was used to detect the expression of estrogen receptors, ER-α, and ER-β in the paraventricular nucleus of the hypothalamus.

[0088] Example 1

[0089] The present invention provides a biomarker, which includes the following genes:

[0090] Dsp, LOC108351703, Otop3, Ccdc77, LOC120097335, Gpr88, Egr3, Atp2a1, Nxn12, LOC120101770, LOC120099591, RGD1561730, Chrm1, Myo5c, Adora2a, Lbp, Ca12, Arhgef16, Cfi, Krtap11-1;

[0091] Or

[0092] GAD67 protein, Egr3 protein, ERβ protein, ERα protein.

[0093] In this example, differential genes intervened by Qingshu Granule were screened out through RNA sequencing of the paraventricular nucleus of the hypothalamus, and correlation analysis was performed between these genes and blood pressure. A total of 20 differential genes significantly related to blood pressure were screened out.

[0094] Among them, Egr3, i.e., early growth response 3, is a member of the early growth response gene family. Its coding gene is located on human chromosome 8p21-23, and the encoded protein contains 387 amino acids.

[0095] ERα and ERβ are estrogen nuclear receptors located in the cell nucleus, mediating the genotypic effects of estrogen, that is, exerting "genotypic" regulatory effects by regulating the transcription of specific target genes.

[0096] GAD67, i.e., glutamate decarboxylase 67, is a key enzyme for synthesizing the inhibitory neurotransmitter γ-aminobutyric acid (GABA).

[0097] Egr3, i.e., early growth response 3, is a member of the early growth response gene family. Its coding gene is located on human chromosome 8p21-23, and the encoded protein contains 387 amino acids.

[0098] ERα and ERβ are estrogen nuclear receptors located in the cell nucleus, mediating the genotypic effects of estrogen, that is, exerting "genotypic" regulatory effects by regulating the transcription of specific target genes.

[0099] GAD67, i.e., glutamate decarboxylase 67, is a key enzyme for synthesizing the inhibitory neurotransmitter γ-aminobutyric acid (GABA).

[0100] In some other specific examples, the specific method for the above gene screening is as follows:

[0101] Using a Venn diagram to aggregate the differential genes of (OVX / Ctrl) vs (QSG-H / OVX) based on the results of RNA sequencing analysis, 170 common differential expression genes were screened out. As Figure 9 shown, KEGG enrichment was performed on the 170 differential expression genes. The top 5 pathways include cell adhesion molecules; bile secretion; Staphylococcus aureus infection; ascorbate and aldarate metabolism; pentose and glucuronate interconversions, which mainly focus on immune response, inflammatory response, and energy metabolism.

[0102] Figure 9 Among them, A. Venn diagram of (OVX / Ctrl) vs (QSG-H / OVX); B. Bubble chart of KEGG enrichment pathways of differential expression genes of (OVX / Ctrl) vs (QSG-H / IVX).

[0103] The Spearman correlation analysis was used to analyze the correlation between the TPM values of these 170 gene transcripts and the systolic blood pressure of the sequenced rats at the 8th week. As shown in Table 13, a total of 20 differentially expressed genes significantly related to blood pressure changes were screened out: Dsp, LOC108351703, Otop3, Ccdc77, LOC120097335, Gpr88, Egr3, Atp2a1, Nxnl2, LOC120101770, LOC120099591, RGD1561730, Chrm1, Myo5c, Adora2a, Lbp, Ca12, Arhgef16, Cfi, Krtap11-1. The pairwise correlation analysis of the TPM values of these 20 differentially expressed genes was performed, and the results are as Figure 11 shown. Through relevant cluster analysis, a strong correlation was found among the three genes Gpr88, Egr3, and Chrm1 related to inflammatory response and central mood regulation. Among them, Egr3 is regulated by estrogen receptor and is related to emotional cognition and inflammatory response. Whether this gene is the key gene for QXJYD to intervene in the central blood pressure elevation of ovariectomized female rats regulated by estrogen receptor remains to be further verified.

[0104] Table 13 Results of Spearman correlation analysis between differentially expressed genes and blood pressure changes

[0105]

[0106]

[0107] The new biomarkers provided by the present invention include gene-based and protein-based ones.

[0108] These biomarkers can distinguish the molecular characteristics of different etiologies of hypertension, especially showing specificity in the hypertension model caused by ovariectomy.

[0109] Example 2

[0110] This example provides an application of the above-mentioned biomarker in the preparation of products for preventing, diagnosing or treating hypertension or analysis methods related to hypertension.

[0111] The present invention also provides an application in the preparation of products for preventing, diagnosing or treating hypertension developed for biomarkers, which can significantly improve problems such as poor efficacy and strong non-specificity of traditional drugs for ovariectomized hypertension. Among them, by regulating the balance of Egr3, ERβ and Glu / GABA, the blood pressure is significantly stabilized, and the problem of drug resistance that may occur in traditional treatments is prevented.

[0112] Specific applications also include:

[0113] Evaluating and identifying individuals at potential risk of hypertension: One of the methods includes identifying individuals at potential risk of hypertension among high-risk populations with a family history of hypertension, castration, or decreased estrogen levels by detecting the gene expression and protein levels of Egr3 and GAD67.

[0114] By using selected specific biomarkers (such as Egr3, GAD67, ERβ protein, etc.), early warning of hypertension can be achieved, guiding personalized intervention strategies and realizing disease prevention. Screening of high-risk populations, non-pharmacological intervention guidance, and according to the expression levels of biomarkers, specific lifestyle interventions (such as diet regulation, psychological counseling, etc.) are recommended to delay the disease process. Dynamic health management: By regularly monitoring the change trends of these biomarkers, data support is provided for individualized health management, reducing the incidence of hypertension.

[0115] Developing gene detection and biochemical detection methods using biomarkers to provide new technical means for the precise diagnosis of hypertension. Gene detection technology: Detecting the expression levels of genes including but not limited to Dsp, Egr3, Nxnl2, etc. in blood, urine or other samples, and combining with the patient's blood pressure situation to determine whether it belongs to hormone-related hypertension.

[0116] Multi-biomarker diagnostic kit: Developing a kit for combined detection of multiple biomarkers, and realizing the precise classification of hypertension (such as estrogen-related hypertension, neuromodulation-related hypertension) by detecting bioactive substances such as Egr3, GAD67, ERβ, etc.

[0117] Imaging-assisted diagnosis: Combining the biomarker detection results and using them in conjunction with brain region functional imaging (such as fMRI) to analyze the functional changes in brain regions such as the hypothalamus and primary cingulate cortex, providing multi-dimensional support for the classification diagnosis of hypertension.

[0118] Developing targeted treatment plans based on the change rules of biomarkers (such as Egr3, ERβ, Glu / GABA), including the preparation of targeted drugs and biological agents, and the optimization of adjuvant treatment methods.

[0119] Developing ERβ agonists or Egr3 regulators. For patients with estrogen-related hypertension, reducing blood pressure by improving hypothalamic function or regulating the Glu / GABA balance.

[0120] Designing drugs that target the regulation of GAD67 expression to improve abnormal blood pressure from the perspective of neuromodulation by balancing neurotransmitter activities.

[0121] Taking Egr3, GAD67, ERβ as targets, further optimizing the formula ratio or dosage form of Qingshu Granules to achieve stronger antihypertensive effects and minimize side effects.

[0122] Individualized treatment plan: Based on the test results of the patient's markers, customize the drug selection and dosage (such as adjusting the proportion and components of Qingshu Granule) adjustment strategy to avoid unnecessary drug interventions and improve the treatment effect.

[0123] Application of hypertension-related analysis methods: Develop marker-based hypertension-related analysis techniques to explore the pathological mechanisms of the disease and provide a scientific basis for future hypertension prevention and treatment. Utilize the expression changes of Egr3 and GAD67 to deeply study the relationship between estrogen level decline and blood pressure regulation mechanism, and promote the progress of basic research on hypertension.

[0124] Quantify the efficacy of drugs or treatment plans by detecting the changes of markers such as Egr3, GAD67, and ERβ before and after treatment in patients.

[0125] Example 3

[0126] A verification method of Qingshu Granule as a corresponding functional drug provided in this example

[0127] 1. Effects of Qingshu Granule on the levels of serum blood pressure regulation-related factors in ovariectomized female rats

[0128] 1.1 Effects of Qingshu Granule on the levels of serum NE and AngII in ovariectomized female rats

[0129] As Figure 1 shown, in the animal experiment, after 8 weeks of intervention, the levels of NE and AngII in the serum of rats in each group were detected. Compared with the Ctrl group, the levels of NE and AngII in the OVX group increased. Compared with the OVX group, the levels of NE and AngII in the Qingshu Granule intervention group decreased. The above results indicate that the levels of NE and AngII increase after ovariectomy in female rats, and Qingshu Granule can effectively reduce the levels of serum NE and AngII in ovariectomized female rats. Therefore, the increase in blood pressure in ovariectomized female rats may be related to the abnormal levels of peripheral blood pressure regulation-related factors, and Qingshu Granule can regulate the balance of blood pressure regulation-related factor levels.

[0130] Figure 1 In, *P < 0.05 compared with the Ctrl group, #P < 0.05 compared with the OVX group.

[0131] 1.2 Regulation of the Glu / GABA balance in the serum of ovariectomized female rats by Qingshu Granule

[0132] In the brain, Glu and GABA are an important pair of neurotransmitters that are interrelated and complementary. Glutamatergic neurons (excitatory) and GABAergic neurons (inhibitory) work together to maintain the stability of neural networks. The balance between them is crucial for maintaining normal neural function. There is a metabolic conversion between Glu and GABA, so the stability of neural function regulation cannot be simply measured by the level of Glu or the level of GABA. Therefore, Pearson correlation analysis was used to study the correlation between Glu and GABA in each group of rats to determine the balance relationship between the two.

[0133] As shown in Table 1, there was a highly positive correlation between serum Glu and GABA levels in the Ctrl group of rats, a highly negative correlation in the OVX group, a highly positive correlation in the QSG-L group, a highly positive correlation in the QSG-H group, and a highly positive correlation in the E2 group.

[0134] The above results indicate that after ovariectomy in female rats, the regulation of Glu and GABA is disordered. When Glu increases, GABA decreases instead, and the balance between excitation and inhibition is lost. Qingshu Granule and E 2 can effectively improve the disordered balance regulation of Glu / GABA after ovariectomy.

[0135] Table 1 Pearson correlation study on the contents of serum Glu and GABA in each group of rats

[0136]

[0137] 2. Effects of Qingshu Granule on the serum oxidative stress level in ovariectomized female rats

[0138] As Figure 2 shown, compared with the Ctrl group, the serum ROS level in the OVX group increased significantly; compared with the OVX group, each intervention group could significantly reduce the serum ROS level after ovariectomy, and among them, the QSG-H group had the most significant reduction effect.

[0139] The above results indicate that after ovariectomy in female rats, the increase in blood pressure may be related to the increase in peripheral oxidative stress level, and Qingshu Granule can significantly improve the oxidative stress level.

[0140] Figure 2 Among them, *P < 0.05 compared with the Ctrl group, #P < 0.05 compared with the OVX group.

[0141] 3. Effects of Qingshu Granule on the amplitude of low-frequency oscillation (ALFF) of resting-state brain function in ovariectomized female rats

[0142] To further investigate the central mechanism of elevated blood pressure in ovariectomized female rats, fMRI technology was used to perform resting-state brain function scans on rats in each group, and the scan data was processed to analyze the low-frequency amplitude of the brain to study the activity pattern of the brain in the resting state. The results are shown in Table 2 and Figure 3 as follows. In the resting state, the active brain regions included the left periaqueductal gray (PAG), the right primary cingular cortex (PCC), and the right medio medial secondary visual cortex (MMV). Periaqueductal gray (PAG): PAG plays a key role in pain regulation, emotional response, respiratory control, and stress response. It affects pain perception at the spinal level through descending pathways and is connected to brain regions related to emotion and stress, such as the amygdala. Primary cingulate (PCC): PCC is part of the cingulate cortex and is related to functions such as emotional regulation, attention, decision-making, and social cognition. It interacts with multiple brain regions when processing complex emotions and cognitive tasks, including its connection with PAG. Medio medial secondary visual cortex (MMV): MMV is part of the visual cortex and is involved in the processing of visual information, such as the perception of shape, color, and motion. It receives information from the primary visual cortex and may be related to visual memory and attention. These three brain regions are interconnected in terms of emotional and pain regulation functions.

[0143] To further clarify the activity differences in the three brain regions of rats in each group at rest, multiple comparisons of the ALFF values of the three brain regions of rats in each group were performed. The results are as Figure 4 follows: Compared with the Ctrl group, the ALFF values of the three different brain regions in the OVX group showed statistically significant differences. Among them, PAG showed a functional activation state, PCC showed overactivation, and MMV showed decreased activity; there was no significant difference in the ALFF values of PAG and PCC in the Qingshu Granule intervention group compared with the Ctrl group, while the ALFF value of the MMV brain region was significantly decreased in each group after modeling compared with the Ctrl group, and the ALFF value of the PAG brain region in the estrogen group showed overactivation.

[0144] The above results indicate that after ovariectomy of female rats, in the resting state, the abnormally active brain regions are mainly concentrated in the brain regions responsible for emotional and pain regulation, and Qingshu Granule can effectively improve the abnormal activity in the primary cingulate cortex region at rest.

[0145] Table 2 Statistical table of ALFF-different brain regions (voxel P < 0.001, cluster P < 0.05, FWE corrected)

[0146]

[0147] 4. Effects of Qingshu Granule on Brain Functional Connectivity with the Hypothalamus as the Seed Point in Ovariectomized Female Rats

[0148] The main brain regions for central emotion regulation are the hypothalamus and the amygdala. However, the changes in brain function of the hypothalamus and the amygdala after ovariectomy in female rats and their relationship with blood pressure regulation are still unclear. To further clarify the changes in the functional connectivity of the hypothalamus with the whole brain after ovariectomy in female rats, in this study, brain functional connectivity analysis with the hypothalamus as the seed point was performed by fMRI technology, and the results are as follows.

[0149] 4.1 Effects of Qingshu Granule on Brain Functional Connectivity of the Right Hypothalamus in Ovariectomized Female Rats

[0150] As shown in Table 3 and Figure 4 as indicated, there were differences in brain functional connectivity between the right hypothalamus and the left striatum. Compared with the Ctrl group, the connection functions of the two differential connection networks in the OVX group were opposite, showing a connection activation state with the left striatum; compared with the OVX group, the functional connectivity of this brain region in the high-dose Qingshu Granule group was significantly different.

[0151] The above results indicate that after ovariectomy in female rats, the connection between the right hypothalamus and the left striatum (Striatum L) is abnormal. This neural network connection is closely related to emotion regulation and output. After intervention with high-dose Qingshu Granule, the abnormal functions of these two neural network connections can be improved.

[0152] Table 3 ROI values of brain functional difference regions based on the right hypothalamus (Hypothalamus R) as the seed point (voxel P < 0.05, cluster size >= 625)

[0153]

[0154] 4.2 Effects of Qingshu Granule on Brain Functional Connectivity of the Left Hypothalamus in Ovariectomized Female Rats

[0155] As shown in Table 4 and Figure 5As shown in the figure, the brain regions with different functional connections to the left hypothalamus are the molecular layer of the left cerebellum (Molecular Layer of the Cerebellum L), the right primary cingulate cortex (Primary Cingular Cortex R), and the agranular insular cortex of the left insula (Agranular Insular Cortex L). Among them, compared with the Ctrl group, the connection function between the left hypothalamus and the three different brain regions in the OVX group is opposite. The connection between the molecular layer of the left cerebellum and the agranular insular cortex of the left insula shows an inhibitory regulation state, and the connection in the right primary cingulate cortex shows an activation state. Compared with the OVX group, the QSG-H group has a better effect on improving the functional connection of the right primary cingulate cortex. In terms of the connection between the granular cell layer of the left cerebellum and the agranular insular cortex of the left insula, the low-dose Qingshu Granule group has a better improvement effect, with significant differences.

[0156] The above results indicate that after ovariectomy in female rats, the increase in blood pressure may be related to the abnormal functional connections between the left hypothalamus and the molecular layer of the left cerebellum, the right primary cingulate cortex, and the agranular insular cortex of the left insula. These three neural network connections are closely related to emotion regulation and output. After intervention with Qingshu Granule, the abnormal functions of these three different brain connections can be improved.

[0157] Table 4 ROI values of brain functional difference regions based on the left hypothalamus (Hypothalamus L) as the seed point (voxel P < 0.05, cluster size >= 580)

[0158]

[0159] 5 Effects of Qingshu Granule on the brain functional connections of ovariectomized female rats with the amygdala as the seed point

[0160] 5.1 Effects of Qingshu Granule on the brain functional connections of the left amygdala in ovariectomized female rats

[0161] As shown in Table 5 and Figure 6 As shown, the brain region with different functional connections to the left amygdala is the granular cell layer of the right cerebellum (Granule Cell Level of the Cerebellum R). Among them, compared with the Ctrl group, the functional connection between the left amygdala and the granular cell layer of the right cerebellum in the OVX group is opposite, and the connection shows an inhibitory regulation state (P < 0.05). Compared with the OVX group, the difference in brain functional connection signals in the QSG-H group is significant (P < 0.05). The above results indicate that after ovariectomy in female rats, the functional connection between the left amygdala and the granular cell layer of the left cerebellum is abnormal. This neural network connection is closely related to emotion regulation output. After intervention with high-dose Qingshu Granule, the abnormal function of this neural network connection can be improved.

[0162] Table 5 ROI values of brain functional difference regions with the left amygdala (amygdalaL) as the seed point (voxel P < 0.005, cluster size >= 77)

[0163]

[0164]

[0165] 5.2 Effects of Qingshu Granule on the brain functional connectivity of the right amygdala in ovariectomized female rats

[0166] As shown in Table 6 and Figure 7 as follows, the brain regions with different functional connectivity with the right amygdala are the right primary cingulate gyrus (PrimaryCingular Cortex R). Among them, compared with the Ctrl group, the functional connectivity between the right amygdala and the right primary cingulate gyrus in the OVX group and the E 2 group is opposite, and the connection shows an inhibitory regulation state (P < 0.05); compared with the OVX group, the functional connectivity signal of this brain region in the QSG-L group is enhanced (P < 0.05).

[0167] The above results indicate that after ovariectomy of female rats, the brain regions with different functional connectivity of the right amygdala show abnormal functional connectivity of the right primary cingulate gyrus. This neural network connection is closely related to the output of emotional regulation. After intervention with low-dose Qingshu Granule, the abnormal function of this neural network connection can be improved.

[0168] Table 6 ROI values of brain functional difference regions with the right amygdala (amygdalaR) as the seed point (voxel P < 0.01, cluster size >= 106)

[0169]

[0170] 6 Effects of Qingshu Granule on the whole-brain functional connectivity of ovariectomized female rats

[0171] To further clarify the changes in whole-brain functional connectivity after ovariectomy in female rats, this study analyzed the whole-brain functional connectivity using the results of fMRI. The results are shown in Table 7. There were a total of 5 different brain connections in the whole-brain functional connectivity analysis: the right entorhinal cortex (Ectorhinal Cortex R) and the right perirhinal cortex area 35 (Perirhinal Cortex Area35 R), the left perirhinal cortex area 36 (Perirhinal Area 36L) and the right commissure of the inferior colliculus (Commissure of the Inferior Colliculus R), the right commissure of the inferior colliculus and the left globus pallidus (Globus Pallidum L), the right perirhinal cortex and the right commissure of the superior colliculus, and the right cerebellar granular cell layer and the right perirhinal cortex area 35. Among them, compared with the Ctrl group, all five different brain connections in the OVX group were abnormal (P<0.05); compared with the OVX group, the high-dose Qingshu Granule group could effectively improve the abnormal functional connectivity of this brain region (P>0.05).

[0172] The above results indicate that after ovariectomy in female rats, there are five abnormal brain functional connections in total on the right. These five neural network connections are closely related to the output of emotional regulation. After the intervention of Qingshu Granule, the abnormal function of this neural network connection can be improved. However, the differential brain regions related to blood pressure need further analysis.

[0173] Table 7 Differential brain regions of whole-brain functional connectivity in rats of each group

[0174]

[0175] 7 Correlation analysis of signal changes in differential brain regions and blood pressure changes

[0176] To identify the differential brain regions closely related to blood pressure changes, correlation statistical analysis was used. Since the data did not conform to the normal distribution, Spearman correlation analysis was adopted. The statistical results are shown in Table 8. The differential brain regions related to blood pressure changes are: the right primary cingulate gyrus, the brain connection between the left hypothalamus and the right primary cingulate gyrus, the brain connection between the right hypothalamus and the left striatum, and the brain connection between the right cerebellar granular cell layer and the right perirhinal cortex.

[0177] The above results show that the blood pressure changes in ovariectomized female rats are closely related to brain regions such as the right primary cingulate gyrus, hypothalamus, left striatum, cerebellar granular cell layer, and right perirhinal cortex. These brain regions are mainly responsible for emotional regulation, suggesting that the increase in blood pressure in ovariectomized female rats is mainly related to the central emotional regulation mechanism.

[0178] Table 8 Results of Spearman correlation analysis between differential brain regions and blood pressure changes

[0179]

[0180] Effect of Qingshu Granule on the metabolic level and quantity of neurons in the paraventricular nucleus of the hypothalamus in ovariectomized female rats

[0181] Effect of Qingshu Granule on the quantity of neurons in the paraventricular nucleus of the hypothalamus in ovariectomized female rats

[0182] To further explore the neuronal activity in the paraventricular nucleus of the hypothalamus after ovariectomy in female rats and the intervention effect of Qingshu Granule, through neuronal Nissl staining analysis, the results are as Figure 8 shown. Compared with the Ctrl group, the number of neuronal cell bodies in the paraventricular nucleus of the hypothalamus in the OVX group decreased significantly (P<0.05), the arrangement was disorderly, the cytoplasmic staining became significantly lighter, the number of Nissl bodies decreased, and the structure became loose; compared with the OVX group, the number of neuronal cell bodies in the E 2 group and the QSG-H group increased significantly (P<0.05), the damage of Nissl bodies was significantly alleviated, the staining gradually became deeper, and the number of neuronal cells recovered somewhat.

[0183] It shows that after ovariectomy in female rats, due to the decrease of estrogen, the number of neuronal cell bodies in the paraventricular nucleus of the hypothalamus decreases significantly, and Qingshu Granule can increase the number of neuronal cell bodies in the paraventricular nucleus of the hypothalamus after ovariectomy.

[0184] 9 Effect of Qingshu Granule intervention on differentially expressed genes in the paraventricular nucleus of the hypothalamus in ovariectomized female rats

[0185] The cardiovascular regulation center in the hypothalamus is the paraventricular nucleus of the hypothalamus. To further study the molecular mechanism of the hypothalamus regulating blood pressure in ovariectomized female rats and the intervention effect of Qingshu Granule, by means of transcriptome high-throughput sequencing, the paraventricular nucleus tissues of the Ctrl group, OVX group, QSG-H group and E 2 group were subjected to high-throughput sequencing. In this study, by comparing the differentially expressed genes, enriched pathways and intersections between groups among the Ctrl group and the OVX group, OVX and the QSG-H group, OVX and the E 2 group, it is expected to predict the potential molecular mechanism and signaling pathway of the hypothalamus in reducing blood pressure in ovariectomized female rats.

[0186] To further clarify the results of the enriched pathways of differentially expressed genes between groups: The top 5 pathways with the most significant enrichment of differentially expressed genes in the hypothalamus tissues between the Ctrl group and the OVX group include Pentose and glucuronate interconversions, Ascorbate and aldarate metabolism, Gastric acid secretion, Cell adhesion molecules, Bile secretion.

[0187] The OVX group and group E 2 The top 5 pathways with the most significant enrichment of differentially expressed genes between the OVX group and group E include ECM-receptor interaction, Cell adhesion molecules, Antigen processing and presentation, Phagosome, and Allograft rejection.

[0188] The top 5 pathways with the most significant enrichment of differentially expressed genes between the OVX group and the QSG-H group include Cell adhesion molecules, Bile secretion, Staphylococcus aureus infection, Graft-versus-host disease, and Type l diabetes mellitus.

[0189] The above results indicate that the differential pathways in the paraventricular nucleus of the hypothalamus of ovariectomized female rats are mainly metabolism, immune response, and inflammatory response.

[0190] 10. Effect of Qingshu Granule on the expression of estrogen receptors in the paraventricular nucleus of the hypothalamus of ovariectomized female rats

[0191] To clarify the effect of Qingshu Granule on the expression of estrogen receptors in the paraventricular nucleus of the hypothalamus of ovariectomized female rats, the immunohistochemical results are as Figure 11 and Figure 12 shown. Compared with the Ctrl group, the expressions of ERα and ERβ in the paraventricular nucleus of the hypothalamus in the OVX group were significantly decreased (P<0.05); compared with the OVX group, Qingshu Granule could significantly up-regulate the expression levels of ERα and ERβ (P<0.05), indicating that after ovariectomy of female rats, the decrease in estrogen levels led to a decrease in the estrogen receptor levels in the paraventricular nucleus of the hypothalamus, and ERp was more significant. Qingshu Granule could increase the expression levels of estrogen receptors in the paraventricular nucleus of the hypothalamus of ovariectomized female rats.

[0192] 11. Effect of Qingshu Granule on the expression of Egr3 in the paraventricular nucleus of the hypothalamus of ovariectomized female rats

[0193] To verify the expression of the differentially expressed gene Egr3 related to the intervention of Qingshu Granule on the blood pressure of ovariectomized female rats screened by RNA sequencing, immunohistochemical staining was used, and the results are as Figure 13 shown. Compared with the Ctrl group, the expression of Egr3 in the paraventricular nucleus of the hypothalamus in the OVX group was significantly decreased (P<0.05); compared with the OVX group, E2 The Hehe Qingshu Granule group could significantly up-regulate the expression level of Egr3 (P<0.05).

[0194] The above results indicate that after ovariectomy of female rats, the estrogen level decreases, and the expression level of Egr3 in the paraventricular nucleus of the hypothalamus decreases. Qingshu Granule can increase the expression level of Egr3 in the paraventricular nucleus of the hypothalamus of ovariectomized female rats.

[0195] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A biomarker for potential treatment with Qingshu granules, characterized in that: It includes: one or a combination of two of the following genes: Dsp, LOC108351703, Otop3, Ccdc77, LOC120097335, Gpr88, Egr3, Atp2a1, Nxnl2, LOC12010177 0, LOC120099591, RGD1561730, Chrm1, Myo5c, Adora2a, Lbp, Ca12, Arhgef16, Cfi, Krtap11-1; or / and One or a combination of two or more of the following biologically active substances: GAD67 protein, Egr3 protein, ERβ protein, and ERα protein.

2. Use of the biomarker as claimed in claim 1 in the preparation of a product for preventing, diagnosing or treating hypertension or a hypertension-related analysis method.

3. The use according to claim 2, characterized in that It includes a genetic testing method for hypertension risk assessment, comprising the following steps: S1 extracts genomic RNA from subject samples; S2 detects the transcript expression levels of one or two of the following genes in genomic RNA; Dsp, LOC108351703, Otop3, Ccdc77, LOC120097335, Gpr88, Egr3, Atp2a1, Nxn12, LOC12010177 0, LOC120099591, RGD1561730, Chrm1, Myo5c, Adora2a, Lbp, Ca12, Arhgef16, Cfi, Krtap11-1; S3 generates a personalized hypertension risk assessment report by correlating the test results of the transcript expression level in step S2 with the systolic blood pressure or diastolic blood pressure of the subject.

4. The use according to claim 2, characterized in that The hypertension-related product is a drug for lowering blood pressure, which has one or a combination of two or more of the following effects to lower blood pressure: Regulates estrogen receptor expression in the paraventricular nucleus of the hypothalamus; Regulates the expression of Egr3 gene in the paraventricular nucleus of the hypothalamus; Improve Glu / GABA balance; improve brain function related to emotion regulation; The paraventricular nucleus of the hypothalamus regulates sympathetic nerve activity and restores estrogen levels; Regulation of RAS system; inhibition of oxidative stress; anti-inflammatory effects.

5. The use according to claim 2, characterized in that It includes at least one of the following biologically active substances: ERβ agonist, ERα agonist, Egr3 regulator, GAD67 expression regulator, Glu and GABA regulator, emotion regulation brain region functional connectivity regulator, metabolic regulator regulating pentose and gluconate metabolic pathways, anti-inflammatory factor, bile secretion promoter, antibacterial agent for Staphylococcus aureus infection, immunomodulator for Staphylococcus aureus infection; The functional connections of the emotion regulation brain regions include one or a combination of two or more of the following connections: right primary cingulate cortex, left hypothalamus connected to right primary cingulate cortex, right hypothalamus connected to left striatum, right perirhinal cortex area 35 connected to right cerebellar granule cell layer.

6. The use according to claim 2, characterized in that: It includes a multi-marker detection kit for the diagnosis of hypertension.

7. The biomarker for potential need of Qingshu granule treatment according to claim 1, characterized in that: The biomarker screening method comprises the following steps: S1 Establishment of experimental groups: Model group, estrogen group and treatment drug group were established with castrated animals, and normal group was established with normal animals; Estrogen group: intervention with E2 drugs; Therapeutic drug group: intervention with therapeutic drugs; Screening of S2 gene biomarkers: S21 collects differentially expressed genes between the normal group and the model group, between the model group and the treatment drug group, and between the model group and the estrogen group; S22 analyzing and screening the differentially expressed genes that are significantly correlated with the corresponding experimental group animals; The method for screening the protein described in S3 includes: The expression of estrogen receptors and related regulatory proteins in the experimental group animals were regularly tested, and after analysis and screening, the following proteins were obtained: ERβ, ERα, Egr3, and GAD67.

8. Use of Egr3 expression protein as a Glu / GABA regulator.

9. An application of Qingshu granules, characterized in that: The application includes the preparation of the following drugs: ERβ agonists, ERα agonists, Egr3 regulators, GAD67 expression regulators, Glu and GABA regulators, mood regulating brain region functional connectivity regulators, metabolic regulators regulating pentose and gluconate metabolic pathways, anti-inflammatory factors, bile secretion promoters, antibacterial agents for Staphylococcus aureus infection, and immunomodulators for Staphylococcus aureus infection; The functional connections of the emotion regulation brain regions include one or a combination of two or more of the following connections: right primary cingulate cortex, left hypothalamus connected to right primary cingulate cortex, right hypothalamus connected to left striatum, right perirhinal cortex area 35 connected to right cerebellar granule cell layer.

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