WO2024207648A1 - Gpr139 receptor activation detection method, physiological function evaluation method, and use - Google Patents

Abstract

A GPR139 receptor activation detection method, a physiological function evaluation method, and a use. The method comprises the steps: (a) providing a test object; (b) providing a first detection sample in a test group, detecting a level of cortical hormone in the first detection sample, and recording the level as Y1; and providing a second detection sample in a negative control group, detecting a level of cortical hormone in the second detection sample, and recording the level as Y0; and (c) comparing the level Y1 of the cortical hormone in the first detection sample with the level Y0 of the cortical hormone in the second detection sample, and determining whether the test object is an agonist or inhibitor of the activity of a GPR139 receptor. The method is simple and fast to operate, has high repeatability, and is easy for clinical application.

Description

GPR139 receptor activation detection method, physiological function evaluation method and application Technical Field

The present invention relates to the field of biotechnology, and in particular to a GPR139 receptor activation detection method, a physiological function evaluation method and applications.

Background Art

The function of glucocorticoid (GC) is mainly achieved through glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). GR mediates the damage to emotion, cognition, and neuronal viability, plasticity, and gene expression, while MR plays a protective role. The balance of MR/GR plays a key role in neuronal excitability, stress response, and behavioral adaptation. Abnormal MR/GR mRNA ratio under stress makes neurons vulnerable and prone to stress-induced diseases such as depression. Long-term increase in GC levels can lead to degenerative changes in hippocampal neurons, causing cognitive impairment in patients with depression, and symptoms such as low mood and insomnia.

Clinically, the method of detecting glucocorticoids is often used to evaluate primary aldosteronism, secondary aldosteronism, Cushing's syndrome, pituitary ACTH tumor, etc.

When stress stimulates the body, corticosteroids rise, and after the stressful event passes, corticosteroids fall. Long-term stress stimulation is associated with hyperactivity of the HPA pathway, shortened sleep time, and reduced deep sleep and rapid eye movement sleep, leading to poor sleep quality, impaired memory, and poor emotional regulation, which in turn leads to more stress. The greater the stress, the worse the sleep quality.

Therefore, it is particularly important to develop drugs or methods that can restore the balance of the body after stress stimulation.

Therefore, there is an urgent need in the art to develop a method that is highly effective in slowing down the rise of corticosteroids or reducing corticosteroids under stress stimulation.

Summary of the invention

The present invention provides a GPR139 receptor activation detection method, a physiological function evaluation method and applications.

In a first aspect of the present invention, a method for evaluating the regulatory function of a test substance on GPR139 receptor activity is provided, comprising the steps of:

(a) providing a test object;

(b) in the test group, providing a first test sample, and detecting the level of corticosteroids in the first test sample, which is recorded as Y1; and in the negative control group, providing a second test sample, and detecting the level of corticosteroids in the second test sample, which is recorded as Y0;

Wherein, the first test sample and the second test sample are plasma samples, blood samples or tissue samples, and are samples of the same type;

Wherein, in the test group, the first test sample comes from a model animal to which the test substance is administered; and in the negative control group, the second test sample comes from a model animal to which the test substance is not administered; except for the difference between administering and not administering the test substance, the test conditions of the test group and the negative control group are the same;

(c) comparing the level Y1 of the corticosteroid in the first test sample with the level Y0 of the corticosteroid in the second test sample,

Among them, when the level of corticosteroids Y1 is significantly lower than the level of corticosteroids Y0 in the second test sample, it indicates that the test substance is an agonist of GPR139 receptor activity; when the level of corticosteroids Y1 is significantly higher than the level of corticosteroids Y0 in the second test sample, it indicates that the test substance is an inhibitor of GPR139 receptor activity.

In another preferred embodiment, when the test substance is an agonist, the method further comprises the steps of:

(d) administering the test substance to a model animal, and measuring the effect of the test substance on the sleep of the model animal.

In another preferred embodiment, when the test substance is an agonist, the method further comprises the steps of:

(e) applying the test substance in combination with a sleep-inducing drug to a model animal, and measuring the effect of the combined application on the sleep of the model animal.

In another preferred embodiment, the sleep-aiding drug includes: sodium pentobarbital.

In another preferred embodiment, the measurement includes measuring the sleep latency, sleep time, or a combination thereof of the model animal.

In another preferred embodiment, the method comprises: based on the comparison result of step (c) and/or the measurement result of step (d), evaluating the efficacy of the test substance as a GPR139 receptor-related drug.

In another preferred embodiment, the test substance includes small molecule compounds, nucleic acid drugs, antibodies, plant extracts or a combination thereof.

In another preferred embodiment, the test substance is a GPR139 ligand.

In another preferred embodiment, the test substance is an agonist of GPR139 receptor activity or an inhibitor of GPR139 receptor activity.

In another preferred embodiment, the test substance is an agonist of GPR139 receptor activity.

In another preferred embodiment, the level of corticosteroids in the second test sample Y0 is The ratio of the level of corticosteroids to the level of Y1 (Y0/Y1) is ≥120%, preferably ≥150%, and more preferably ≥200%, indicating that the test substance is an agonist of GPR139 receptor activity; or

If the ratio of the level Y1 of the corticosteroid in the first test sample to the level Y0 of the corticosteroid in the second test sample (Y1/Y0) is ≥120%, preferably ≥150%, and more preferably ≥200%, it indicates that the test substance is an inhibitor of GPR139 receptor activity.

In another preferred embodiment, the corticosteroid is selected from the group consisting of glucocorticoids, mineralocorticoids, or a combination thereof.

In another preferred embodiment, the corticosteroid is a glucocorticoid.

In another preferred embodiment, the model animals include rodents (such as mice, rats), humans and non-human primates.

In another preferred embodiment, the glucocorticoid can be detected by the following methods: enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (EIA), high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), fluorescence detection, isotope labeling, non-isotope labeling, or a combination thereof.

In another preferred embodiment, the test substance is liquid, solid, or semi-solid.

In another preferred embodiment, the test substance is administered to the model animal by the following means: oral administration, intravenous injection, or local administration.

In another preferred embodiment, the “significantly lower than” refers to Y0/Y1≥1.2, preferably, ≥1.5, and more preferably, ≥2.

In another preferred embodiment, the “significantly higher than” refers to Y1/Y0≥1.2, preferably, ≥1.5, and more preferably, ≥2.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

In another preferred embodiment, the method is an in vitro method.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the dynamic detection of corticosterone levels in acutely restrained mice when given a GPR139 agonist. V20, V40, V60, V120, and V180 represent samples of the placebo group after acute restraint for 20 minutes, 40 minutes, 60 minutes, 120 minutes, and 180 minutes, respectively; C20, C40, C60, C120, and C180 represent samples of the placebo group after acute restraint for 20 minutes, 40 minutes, 60 minutes, 120 minutes, and 180 minutes, respectively. Samples of the GPR139 agonist group after acute restraint for 20 minutes, 40 minutes, 60 minutes, 120 minutes, and 180 minutes; *: p<0.05 compared with the corresponding control group).

Figure 2 shows the detection of corticosterone levels in acutely restrained mice when given different GPR139 agonists (V: placebo group; C0-C4: different GPR139 agonist groups. **: p<0.01 compared with the control group; ***: p<0.001 compared with the control group).

FIG3 shows the synergistic effect of GPR139 agonist C4 on sodium pentobarbital hypnosis in acute restrained mice (V: placebo group; C4: GPR139 agonist group; *: p<0.05 compared with the compound group and the placebo group).

DETAILED DESCRIPTION

After extensive and in-depth research, the inventors unexpectedly discovered for the first time that there is a correlation between the level of corticosteroids and the activation or inhibition of the GPR139 receptor, so the measurement of the level of corticosteroids can be used to evaluate whether the test object has the function of regulating the GPR139 receptor. Taking the GPR139 agonist as an example, it can upregulate the function of the GPR139 receptor and cause the level of corticosteroids to decrease. In addition, the GPR139 agonist or inhibitor determined based on the method of the present invention can further evaluate its effect on physiological conditions such as sleep, and use this to evaluate the efficacy of GPR139 receptor-related drugs. On this basis, the present invention has been completed.

GPR139

G protein-coupled receptor 139 (GPR139) was first discovered by Gloriam et al. in 2005 (Biochim Biophys Acta 2005; 1722: 235-246), also known as GPCR12, PGR3, KOR3L, and GPRg1.

GPR139 is mainly expressed in the habenula, striatum, thalamus, hypothalamus and pituitary in the central nervous system (CNS). Its amino acid sequence is highly conserved in different species. For example, the homology of human GPR139 protein sequence with mouse, chicken and zebrafish is 96%, 92% and 70%, respectively.

Existing studies have shown that the GPR139 receptor has important physiological functions and is a potential drug target for anti-anxiety, anti-depression, anti-schizophrenia, treatment of Parkinson's syndrome, treatment of drug and alcohol abuse, treatment of autism, treatment of epilepsy, treatment of mania, and treatment of metabolic-related diseases.

Glucocorticoids

The function of glucocorticoid (GC) is mainly achieved through glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). GR mediates the viability, plasticity, The balance of MR/GR plays a key role in neuronal excitability, stress response and behavioral adaptation.

Abnormal MR/GR mRNA ratio under stress conditions makes neurons vulnerable and prone to stress-induced diseases such as depression. Long-term elevated GC levels can lead to degenerative changes in hippocampal neurons, causing cognitive impairment in patients with depression, and symptoms such as low mood and insomnia. Methods for detecting glucocorticoids in plasma or tissues include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (EIA), high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), fluorescence detection, isotope labeling, non-isotope labeling, etc.

Clinically, the method of detecting glucocorticoids is often used to evaluate primary aldosteronism, secondary aldosteronism, Cushing's syndrome, pituitary ACTH tumor, etc.

When the body is exposed to stressors such as stress, the body's hormonal stress response is triggered, causing the endocrine system to release adrenal hormones. These hormones increase blood pressure, muscle tension, breathing and heart rate, and blood sugar, while also increasing alertness, decreasing sensitivity to pain, and slowing digestion. Corticosteroids rise when stress is present and fall after the stressful event has passed. Prolonged stress is associated with hyperactivity of the HPA pathway, shortened sleep time, and reduced deep sleep and REM sleep, leading to poor sleep quality, impaired memory, and poorer emotional regulation, which in turn leads to more stress. The greater the stress, the worse the sleep quality.

Method of the present invention

The present invention provides a method for evaluating the regulatory function of a test substance on GPR139 receptor activity, comprising the steps of:

(a) providing a test object;

(b) in the test group, providing a first test sample, and detecting the level of corticosteroids in the first test sample, which is recorded as Y1; and in the negative control group, providing a second test sample, and detecting the level of corticosteroids in the second test sample, which is recorded as Y0;

Wherein, the first test sample and the second test sample are plasma samples, blood samples or tissue samples, and are samples of the same type;

Wherein, in the test group, the first test sample comes from a model animal to which the test substance is administered; and in the negative control group, the second test sample comes from a model animal to which the test substance is not administered; except for the difference between administering and not administering the test substance, the test conditions of the test group and the negative control group are the same (or substantially the same);

(c) comparing the level Y1 of the corticosteroid in the first test sample with the level Y0 of the corticosteroid in the second test sample,

Among them, when the level of corticosteroids Y1 is significantly lower than the level of corticosteroids Y0 in the second test sample, it indicates that the test substance is an agonist of GPR139 receptor activity; when the level of corticosteroids Y1 is significantly higher than the level of corticosteroids Y0 in the second test sample, it indicates that the test substance is an inhibitor of GPR139 receptor activity.

In another preferred embodiment, the model animal is a mammal, more preferably derived from a rodent (such as a mouse, a rat), a human, and a non-human primate.

The main advantages of the present invention include:

(1) The present invention provides a new method for evaluating the ability of a GPR139 agonist to activate the GPR139 receptor.

(2) The method of the present invention is simple to operate, rapid, highly accurate and reliable.

(3) The method of the present invention has high repeatability, good stability, and can be easily transformed into clinical application.

The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

Example 1. ELISA method for detecting corticosterone levels in acutely restrained mice when given GPR139 agonists 1.1 Experimental materials

1.1.1 Experimental animals

SPF male C57BL/6J; 8 weeks old; weight: 20-25g; quantity: 100

Feeding requirements: 5 animals/cage; 12-hour light/dark cycle in the animal room; no restrictions on food and water; animals were purchased and allowed to acclimate to the environment 1 week before the experiment.

1.1.2 Corticosterone detection kit

Abcam:ab108821

1.2 Experimental Grouping

Group 1, acute restraint placebo group, 50 mice: divided into 5 cages, 10 mice/cage, numbered V20, V40, V60, V120, V180. On the day of the experiment, the mice in this group were acutely restrained, and no food or water was allowed during the period.

The second group, acute restraint compound (GPR139 agonist) group, 50 mice: divided into 5 cages, 10 mice/cage, numbered C20, C40, C60, C120, C180. The mice in this group were acutely restrained on the day of the experiment, and were deprived of food and water during the period.

1.3 Animal Experiment Procedures

1.3.1 The placebo was prepared one day before the experiment: 0.5% sodium CMC + 1% Tween80 + normal saline.

1.3.2 The compound solution was prepared one day before the experiment: 2.5 mg/ml, and the vehicle was the placebo.

1.3.3 On the day of the experiment, all experimental mice were placed in the behavioral laboratory and allowed to adapt for 1 hour.

1.3.4 The first group of mice were gavaged with placebo, 0.1ml/10g, and the V20 group was operated first, followed by the V40, V60, V120, and V180 groups. After 30 minutes, 50 mice were placed in 50 restrained centrifuge tubes from the V20 group to the V180 group, with their tails passing through the centrifuge tube lids and leaking out. Acute dynamic restraint was performed, and no food or water was allowed during the period.

1.3.5 The second group of mice were gavaged with compound solution, 0.1 ml/10 g, first in group C20, then in groups C40, C60, C120, and C180. After 30 minutes, 50 mice were placed in 50 restrained centrifuge tubes from group C20 to group C180, with their tails passing through the caps of the centrifuge tubes and leaking out. No food or water were allowed during this period.

1.3.6 After 20 minutes of restraint, the 10 mice in the V20 group in the first group were killed and blood was collected; after 40 minutes of restraint, the 10 mice in the V40 group were killed and blood was collected; after 60 minutes of restraint, the 10 mice in the V60 group were killed and blood was collected; after 120 minutes of restraint, the 10 mice in the V120 group were killed and blood was collected; after 180 minutes of restraint, the 10 mice in the V180 group were killed and blood was collected.

1.3.7 After 20 minutes of restraint, the 10 mice in group C20 of the second group were killed and blood was collected; after 40 minutes of restraint, the 10 mice in group C40 were killed and blood was collected; after 60 minutes of restraint, the 10 mice in group C60 were killed and blood was collected; after 120 minutes of restraint, the 10 mice in group C120 were killed and blood was collected; after 180 minutes of restraint, the 10 mice in group C180 were killed and blood was collected.

1.3.8 Test the corticosterone levels of the above 100 samples.

1.4 ELISA detection of corticosterone content in plasma: The dilution multiple of plasma samples was 50 times, and the specific detection method was carried out according to the instructions of the ELISA kit (ab108821).

1.5 Experimental Results

The experimental results are shown in Figure 1. GPR139 agonist can significantly reduce the expression level of corticosterone in mice after acute restraint for 3 hours 30 minutes after administration.

Example 2. ELISA method to detect corticosterone levels in acutely restrained mice when given different GPR139 agonists

2.1 Experimental Materials

2.1.1 Experimental animals

SPF male C57BL/6J; 8 weeks old; weight: 20-25g; quantity: 50

Feeding requirements: 5 animals/cage; 12-hour light/dark cycle in the animal room; no restrictions on food and water; animals were purchased and allowed to acclimate to the environment 1 week before the experiment.

2.1.2 Corticosterone detection kit

Abcam:ab108821

2.2 Experimental Grouping

Group 1, acute restraint placebo group, 10 mice; 10 mice/cage. The mice in this group were acutely restrained in the morning of the experiment, and were deprived of food and water during the period.

The second group, acute restraint compound C0 group, 10 mice; 10 mice/cage. The mice in this group were acutely restrained in the morning of the experiment, and were deprived of food and water during the period.

The third group, acute restraint compound C2 group, 10 mice; 10 mice/cage. The mice in this group were acutely restrained in the morning of the experiment, and were deprived of food and water during the period.

The fourth group, acute restraint compound C3 group, 10 mice; 10 mice/cage. The mice in this group were acutely restrained in the morning of the experiment, and were deprived of food and water during the period.

The fifth group, acute restraint compound C4 group, 10 mice; 10 mice/cage. The mice in this group were acutely restrained in the morning of the experiment, and were deprived of food and water during the period.

2.3 Animal experiment steps:

2.3.1 Placebo was prepared one day before the experiment: 0.5% sodium CMC + 1% Tween80 + normal saline

2.3.2 The compound solutions were prepared one day before the experiment: C0, C2, C3, C4, each with a concentration of 2.5 mg/ml; the vehicle was the placebo.

2.3.3 On the morning of the experiment, all experimental mice were placed in the behavioral laboratory and allowed to adapt for 1 hour.

2.3.4 The first group of mice were gavaged with placebo, 0.1 ml/10 g. 30 minutes later, 10 mice were placed in 10 restrained centrifuge tubes in turn, with their tails passing through the caps of the centrifuge tubes and hanging out. Acute dynamic restraint was performed, during which no food or water was allowed.

2.3.5 The second group of mice were gavaged with C0 solution, 0.1 ml/10 g. After 30 minutes, 10 mice were placed in 10 restrained centrifuge tubes in turn, with their tails passing through the caps of the centrifuge tubes and leaking out. No food or water were allowed during this period.

2.3.6 Next, the third group was given C2 solution, the fourth group was given C3 solution, and the fifth group was given C4 solution by gavage in sequence, and each mouse was subjected to acute restraint operation as in step 2.3.5.

2.3.7 Three hours after the acute restraint began in each group from the first to the fifth group, the mice were killed and blood was collected to detect the content of corticosterone in each sample.

2.4. ELISA detection of corticosterone content in plasma: The dilution factor of plasma samples was 50 times, and the specific detection method was carried out according to the instructions of the ELISA kit (ab108821).

2.5 Experimental Results

The experimental results are shown in Figure 2. GPR139 agonists C0, C2, C3, and C4 can significantly reduce the expression level of corticosterone in acutely restrained mice.

Example 3. Evaluation of sleep status in acutely restrained mice when administered a GPR139 agonist

3.1 Experimental Materials

3.1.1 Experimental animals

SPF male C57BL/6J; 8 weeks old; weight: 20-25g; quantity: 20

Feeding requirements: 5 animals/cage; 12-hour light/dark cycle in the animal room; no restrictions on food and water; animals were purchased and allowed to acclimate to the environment 1 week before the experiment.

3.2 Experimental Grouping

The first group, acute restraint sleep placebo group, consisted of 10 rats, which were raised in the usual way.

The second group, the acute restraint hypnotic compound C4 (GPR139 agonist) group, consisted of 10 mice, which were deprived of food and water during the acute restraint period and were kept normally at other times.

3.3 Animal experiment steps:

3.3.1 Placebo was prepared 1 day before the experiment: 0.5% sodium CMC + 1% Tween80 + normal saline

3.3.2 One day before the experiment, compound C4 solution was prepared: 2.5 mg/ml; the vehicle was the placebo.

3.3.3 On the day of the experiment, the mice were placed in the behavioral laboratory and allowed to adapt for 1 hour.

3.3.4 The first group of mice were gavaged with placebo, 0.1 ml/10 g. After 30 minutes, 10 mice were placed in 10 restrained centrifuge tubes in turn, with their tails passing through the caps of the centrifuge tubes and leaking out. Acute restraint was performed for 3 hours, during which no food or water was allowed. After the acute restraint was completed, the mice were returned to the corresponding cages and raised normally.

3.3.5 The second group of mice was gavaged with C4 solution, 0.1 ml/10 g. 30 minutes later, 10 mice Place the mice in 10 restraint centrifuge tubes in turn, with their tails sticking out of the tube caps. Perform acute restraint for 3 hours, during which they are not allowed to eat or drink. After acute restraint, place the mice back into the corresponding cages and feed them normally.

3.3.6 One hour after the acute restraint of the first mouse in group 1, 40 mg/kg sodium pentobarbital was injected intraperitoneally. Each mouse was injected intraperitoneally with 40 mg/kg sodium pentobarbital until the last mouse in group 2 was injected with sodium pentobarbital.

3.3.7 After the administration of sodium pentobarbital, the sleep latency (the time when the righting reflex disappears) and the sleep time (the time from the disappearance of the righting reflex to the recovery) of mice in each group were observed.

3.4 Experimental Results

The experimental results are shown in Figure 3. The GPR139 agonist C4 can significantly prolong the sleep time of acutely restrained mice under the action of sodium pentobarbital.

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims (10)

A method for evaluating the regulatory function of a test substance on GPR139 receptor activity, characterized in that it comprises the steps of: (a) providing a test object; (b) in the test group, providing a first test sample, and detecting the level of corticosteroids in the first test sample, which is recorded as Y1; and in the negative control group, providing a second test sample, and detecting the level of corticosteroids in the second test sample, which is recorded as Y0; Wherein, the first test sample and the second test sample are plasma samples or blood samples or tissue samples, and are samples of the same type; Wherein, in the test group, the first test sample comes from a model animal to which the test substance is administered; and in the negative control group, the second test sample comes from a model animal to which the test substance is not administered; except for the difference between administering and not administering the test substance, the test conditions of the test group and the negative control group are the same; (c) comparing the level Y1 of the corticosteroid in the first test sample with the level Y0 of the corticosteroid in the second test sample, Among them, when the level of corticosteroids Y1 is significantly lower than the level of corticosteroids Y0 in the second test sample, it indicates that the test substance is an agonist of GPR139 receptor activity; when the level of corticosteroids Y1 is significantly higher than the level of corticosteroids Y0 in the second test sample, it indicates that the test substance is an inhibitor of GPR139 receptor activity. The method according to claim 1, characterized in that, when the test substance is an agonist, it further comprises the steps of: (d) administering the test substance to a model animal, and measuring the effect of the test substance on the sleep of the model animal. The method of claim 2, wherein the measurement comprises measuring the sleep latency, sleep time, or a combination thereof of the model animal. The method according to claim 1 or 2, characterized in that the method comprises: based on the comparison result of step (c) and/or the measurement result of step (d), evaluating the efficacy of the test substance as a GPR139 receptor-related drug. The method according to claim 1, characterized in that the test substance comprises a small molecule compound, a nucleic acid drug, an antibody, a plant extract or a combination thereof. The method of claim 1, wherein the test substance is a GPR139 ligand. The method of claim 1, wherein the ratio of the level Y0 of the corticosteroid in the second test sample to the level Y1 of the corticosteroid in the first test sample (Y0/Y1) is ≥ 120%, preferably ≥ 150%, More preferably, ≥200%, indicating that the test substance is an agonist of GPR139 receptor activity; or If the ratio of the level Y1 of the corticosteroid in the first test sample to the level Y0 of the corticosteroid in the second test sample (Y1/Y0) is ≥120%, preferably ≥150%, and more preferably ≥200%, it indicates that the test substance is an inhibitor of GPR139 receptor activity. The method of claim 1, wherein the corticosteroid is selected from the group consisting of a glucocorticoid, a mineralocorticoid, or a combination thereof. The method of claim 1, wherein the glucocorticoid is detected by an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (EIA), a high performance liquid chromatography (HPLC), a liquid chromatography-mass spectrometry (LC-MS), a fluorescence detection method, an isotope labeling method, a non-isotope labeling method, or a combination thereof. The method according to claim 1, characterized in that the “significantly lower than” means Y0/Y1≥1.2, preferably ≥1.5, and more preferably ≥2.