Use of growth hormone receptor as a biomarker for depression

Abstract

The present application provides a new potential marker of depression, i.e., serum growth hormone receptor (GHR). In a mouse model of depression, the serum GHR level is significantly reduced, and increasing the serum GHR level can significantly improve the depressive symptoms. The application of acantho-side E for preparing a medicament for treating depression is also provided, wherein acantho-side E increases the serum GHR level and improves the depressive symptoms, and thus can be used for treating depression and / or relieving depressive symptoms.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of growth hormone receptor as a biomarker for depression and the use of eleutheroside E for treating depression. Background Art

[0002] Depression belongs to the category of affective disorders and is a mood disorder or affective disorder caused by various factors. Depression can be divided into three categories, namely endogenous (endogenous) depression, psychogenic depression, and symptomatic (secondary) depression. Among them, endogenous depression is the most common type, mainly manifested as low mood, slow thinking, decreased volitional activity, cognitive impairment, loss of interest and ability to move, loss of self-esteem, inappropriate guilt, thoughts of death and suicide, decreased concentration, sleep disorders, anorexia, decreased libido, and various somatic symptoms can also be accompanied. However, the pathogenesis of depression is not yet clear. Generally, it is believed that the pathogenesis of depression is related to monoamine neurotransmitters in the brain, including serotonin, norepinephrine, epinephrine, and dopamine, etc. In response to these mechanisms, monoamine oxidase inhibitor antidepressants, tricyclic and tetracyclic antidepressants, and serotonin reuptake inhibitor drugs have emerged one after another. However, these antidepressants often have toxic side effects and are likely to cause damage to the cardiovascular system and liver damage, etc. Moreover, the inducing factors of depression are very complex and the symptoms are diverse. At present, there is still no appropriate treatment method for depression clinically. Therefore, it is still necessary to deeply study the pathogenesis or potential biomarkers of depression in order to provide new ideas, targets, and drugs for the treatment of depression.

[0003] Depression belongs to the category of "depressive disorders" in traditional Chinese medicine. Most traditional Chinese medicine experts believe that the occurrence of "depressive disorders" is mostly caused by the injury of the seven emotions such as anger, worry, sadness, and sorrow, resulting in the failure of the liver to disperse qi, the failure of the spleen to transport and transform, the loss of nourishment of the heart spirit, and the imbalance of the zang-fu organs, yin and yang, qi and blood. With the in-depth research, the advantages of traditional Chinese medicine in treating depression have become increasingly prominent. A variety of single traditional Chinese medicines such as Hypericum perforatum, Morinda officinalis, Ginkgo biloba, and Acanthopanax senticosus, etc., as well as compound traditional Chinese medicines such as Chaihu Shugan Powder and Xiaoyao Powder, etc. have been reported to have effects on depression. Although it has been reported that Acanthopanax senticosus capsules, injections or extracts have antidepressant effects, there is no literature reporting that eleutheroside E has antidepressant effects. Summary of the Invention

[0004] In this study, the inventors used a high-throughput cytokine microarray to screen for potential depressive biomarkers in the serum of young men who performed a task under prolonged stress. They found that serum GHR levels decreased after the task compared to before the task. To demonstrate the relationship between GHR and depression, the inventors established a social frustration stress model to induce depression in mice and induced anxiety in mice through restrictive restraint. They then analyzed changes in serum GHR levels in both models using ELISA. The results showed no significant change in GHR levels in anxious mice, but a significant decrease in depressed mice, suggesting that serum GHR levels may be a peripheral biomarker of depression. Furthermore, the inventors discovered that eleutheroside E can increase serum GHR levels and improve depressive symptoms in a mouse depression model. This completes the present invention.

[0005] On the one hand, the present invention provides the use of growth hormone receptor (GHR) as a biomarker in the preparation of products for diagnosing depression and / or assessing the risk of developing depression.

[0006] According to the present invention, in the application described, the growth hormone receptor is used as the detection target.

[0007] According to the present invention, in the application described, the growth hormone receptor is a serum growth hormone receptor.

[0008] According to the present invention, the GHR is downregulated or its level is reduced in blood or serum samples of patients with depression or individuals at high risk of developing depression.

[0009] According to the present invention, if the expression or level of GHR of the test individual is downregulated or reduced by at least 30% or more, such as at least 40%, at least 50% or at least 60%, compared with the GHR level of the same individual when not diagnosed with depression or the average GHR level of a group of healthy individuals, it can predict that the test individual has depression or has a high risk of having depression.

[0010] According to the present invention, the product comprises a chip or a kit. The kit includes, but is not limited to, ELISA detection kits, colloidal gold detection kits, immunoprecipitation kits, chemiluminescence kits, immunofluorescence kits, etc. These kits can be prepared according to conventional methods in the art. For example, an ELISA detection kit includes: a solid-phase carrier coated with an antibody specifically binding to serum GHR, an enzyme-labeled antibody, an enzyme substrate, GHR standards, a negative control, a diluent, a washing buffer, and an enzyme reaction termination solution, etc. The antibody specifically binding to serum GHR can be a GHR monoclonal antibody.

[0011] On the other hand, the present invention provides the use of GHR in screening drugs for treating depression and / or alleviating symptoms of depression.

[0012] According to the present invention, drugs capable of increasing blood or serum GHR levels can be used to treat depression and / or alleviate symptoms of depression.

[0013] In another aspect, the present invention provides the use of Acanthopanax senticosin E in the preparation of medicaments for treating depression and / or relieving depressive symptoms.

[0014] According to the present invention, Acanthopanax senticosin E is the sole active ingredient.

[0015] According to the present invention, the depression includes mild, moderate, and severe depression, postpartum depression, adolescent depression, adult depression, and geriatric depression. The depressive symptoms include depressed mood, loss of interest, feelings of worthlessness, self-blame and guilt, slowed thinking, slowed behavior, cognitive impairment, decreased recent memory, attention deficit, sleep disturbances, decreased appetite, and decreased libido in patients diagnosed with depression.

[0016] According to the present invention, the drug can be any clinically suitable dosage form, including but not limited to tablets, capsules, granules, pills, injections, oral liquids, etc.

[0017] In another aspect, the present invention also provides the use of Acanthopanax senticosin E in the preparation of a drug that increases blood or serum GHR levels.

[0018] According to the present invention, eleutheroside E is the sole active ingredient. The drug can be any clinically suitable dosage form, including but not limited to tablets, capsules, granules, pills, injections, and oral liquids.

[0019] Beneficial effects

[0020] The inventors discovered a novel potential biomarker for depression: serum globulin level (GHR). In a mouse model of depression, serum GHR levels were significantly reduced; and increasing serum GHR levels significantly improved depressive symptoms. Targeting this potential biomarker, the inventors found that eleutheroside E can increase serum GHR levels and improve depressive symptoms in a mouse model of depression, thus it could be used to treat and / or alleviate depressive symptoms. Attached Figure Description

[0021] Figure 1 The results of the high-throughput cytokine microarray are shown.

[0022] Figure 2 The results of the elevated cross maze experiment are shown.

[0023] Figure 3 The results of the sugar water preference experiment are shown.

[0024] Figure 4 The results of the forced swimming experiment are shown.

[0025] Figure 5 The ELISA results for serum GHR levels are shown.

[0026] Figure 6 The results of ELISA for serum GHR levels in a mouse model of chronic social frustration stress, with or without drug treatment, are shown.

[0027] Figure 7 The results show the effects of drug treatment or non-treatment on depressive symptoms in a mouse model of chronic social frustration stress. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0029] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0030] Example

[0031] I. Materials and Methods (I) High-throughput Cytokine Microarray Screening

[0032] Peripheral venous blood samples were collected from young men facing significant psychological stress during a long-term task, both before and after the task. Eight samples were collected before the task, and 12 samples were collected after the task. Target proteins were screened from 507 factors using the Raybiotech AAH-BLG-1 chip. The results were read as fluorescence intensity; after standardization, higher values ​​indicated higher protein concentrations.

[0033] (II) The Chronic Social Frustration Stress Model

[0034] The method for constructing a chronic social frustration stress model was slightly modified from existing classical methods. Male CD1 mice were housed individually and rigorously selected for three consecutive days to identify highly aggressive mice (attacking C57 mice for at least two consecutive days with an attack latency of <30s) for modeling. C57 mice were divided into a frustration group and a control group. At the same time each day, the frustration group C57 mice were directly exposed to the side of the CD1 mice's enclosure as "intruders" for 10 minutes of physical contact. After the frustration period, the C57 mice were moved to the other side of the enclosure and separated by a transparent, breathable partition, maintaining visual and olfactory contact while avoiding physical contact. During the 10-day stress period, the intruder C57 mice were exposed to different CD1 mouse cages each day. Control group mice were housed individually under normal conditions.

[0035] (III) Chronic Restraint Stress Model

[0036] A restraint device was created by chiseling small ventilation holes into the walls and caps of 50ml conical centrifuge tubes. The holes should be as smooth as possible to avoid unnecessary injury to the mice. Restrained mice were placed in the centrifuge tubes, with their tails protruding through the central ventilation hole in the cap. The cap was tightened to restrict mouse movement, ensuring the centrifuge tube was tilted at a 45° angle and the mouse's head was facing upwards. Restraint stress was administered daily from 8:00 AM to 2:00 PM. After the stress period, the centrifuge tubes were washed, ensuring they were clean, free of feces and odor residue, and allowed to air dry before being reused. Restraint stress lasted for 8 days. The elevated cruciate maze test was performed the day after the stress period ended. Control mice were not restrained but were deprived of food and water during the same time periods.

[0037] (iv) Elevated Cross Maze

[0038] The elevated cross maze experimental setup consists of three parts: the cross maze itself, a camera system, and a small animal trajectory analysis system. The elevated cross maze comprises two open arms, two closed arms, and a central area. At the start of the test, mice are placed facing the closed arms through the central area and allowed to move freely within the maze for 5 minutes. The small animal trajectory analysis system records the time the mice spend freely exploring and lingering in the closed arms, open arms, and central area, as well as the number of times they enter the closed and open arms.

[0039] (V) Sugar Water Preference Experiment

[0040] Two bottles of 1% sucrose solution and pure water were placed on opposite sides of the mouse cages. Mice were allowed free access to food and water for two days, with the positions of the sucrose and pure water bottles swapped every 12 hours to prevent positional preference. On the third day, the sucrose and pure water solutions were removed, weighed, and randomly returned to the cages for the mice to drink freely. After 12 hours, the solutions were removed again and the remaining mass was weighed. The consumption of sucrose solution and pure water was calculated. The degree of sucrose preference = sucrose solution consumption / (sucrose solution consumption + pure water consumption).

[0041] (vi) Forced swimming experiment

[0042] Mice were placed in a cylindrical glass tank 50 cm high and 20 cm in diameter, with the water depth maintained at approximately 40 cm and the water temperature maintained at (25±1)℃. During the experiment, the mice were first allowed to swim in the tank for 2 minutes to acclimatize to the swimming environment. After acclimatization, the cumulative immobility time of the mice in the following 4 minutes was recorded (the mice stopped struggling in the water and floated, or only made small limb movements to maintain balance).

[0043] (vii) Drug treatment of mice

[0044] Eleutheroside E, 98% purity, was purchased from Chengdu Ruifensi Biotechnology Co., Ltd., and administered by gavage at a dose of 30 mg / kg once daily. Hydropidone hydrochloride, 99% purity, was synthesized by the Institute of Toxicology and Pharmacology, Academy of Military Medical Sciences, batch number 110116, and administered by gavage at a dose of 2.5 mg / kg once daily. Untreated mice were administered an equal volume of physiological saline by gavage.

[0045] (viii) ELISA detection of mouse serum GHR levels

[0046] 1. Experimental Materials

[0047] Mouse GHR / Growth Hormone R ELISA kit (manufacturer: Raybiotech; catalog number ELM-GHR-1); pipettes with a volume range of 2 μl to 1 ml; adjustable 1 ml to 25 ml pipettes for reagent preparation; 100 ml and 1 L graduated cylinders; absorbent paper; distilled or deionized water; computer and software for ELISA data analysis; EP tubes for preparing standard solutions or sample diluents; microplate reader capable of measuring absorbance at 450 nm.

[0048] 2. Reagent Preparation

[0049] 2.1 All reagents and samples should be brought to room temperature (18-25℃) before use.

[0050] 2.2 Diluent (E2): Dilute with deionized water or distilled water 5 times before use.

[0051] 2.3 Sample Dilution: Dilute serum, plasma, and cell culture supernatant samples with 1× diluent (E2). A dilution of 15 times the normal serum or plasma concentration is recommended. (Note: GHR levels may vary between samples. The optimal dilution factor must be determined by the laboratory for each sample.)

[0052] 2.4 Preparation of Standards: Centrifuge reagent c briefly, add 600 μl of 1× diluent E2, and prepare a 50 ng / ml standard solution. Gently mix to completely dissolve the powder. Prepare EP tubes, adding 300 μl of 1× diluent to each tube. Perform serial dilutions using 200 μl of the pre-prepared 50 ng / ml standard solution. Mix each tube thoroughly. Use the 1× diluent as a blank control (0 ng / ml). The standard concentrations are as follows: 50 ng / ml, 20 ng / ml, 8 ng / ml, 3.200 ng / ml, 1.280 ng / ml, 0.512 ng / ml, 0.205 ng / ml, and 0 ng / ml.

[0053] 2.5 If the wash concentrate (20×) (Reagent B) contains visible crystals, heat to a moderate temperature and stir until completely dissolved. Dilute 20 mL of the wash buffer concentrate with deionized or distilled water to make 400 mL of 1× wash buffer.

[0054] 2.6 Briefly centrifuge the antibody vial (F) before use. Add 100 μl of 1× diluent (E2) to prepare the antibody concentrate. Gently mix with a pipette (can be stored at 4°C for 5 days). Dilute the antibody concentrate 80 times with 1× diluent (E2) for testing.

[0055] 2.7 Instant centrifugation of streptavidin vials (G): Before use, gently mix with a pipette, as precipitation may form during storage. Dilute the HRP-Streptavidin concentrate 350 times with 1× diluent (E2). Prepare and use the diluted HRP-Streptavidin solution immediately; do not store diluted solutions.

[0056] 3. Experimental Procedure

[0057] 3.1 All reagents and samples should be brought to room temperature (18-25℃) before use.

[0058] 3.2 Select an appropriate number of 8-well strips and mark them according to the experimental requirements.

[0059] 3.3 Add 100 μl of the sample and each standard (see reagent preparation step 2.3) to the sample well. Gently shake at room temperature and incubate for 2.5 hours.

[0060] 3.4 Discard the solution and wash four times with 1× wash buffer. Wash each well with 300 μl of wash buffer using a multichannel pipette or automated plate washer. After the final wash, completely discard any remaining wash buffer. Invert the ELISA plate and blot dry with a clean paper towel.

[0061] 3.5 Add 1× diluted antibody (reagent preparation step 2.6). Gently shake at room temperature and incubate for 1 hour.

[0062] 3.6 Discard the solution and repeat the washing process as described in step 3.4.

[0063] 3.7 Add 100 μl of the prepared streptavidin solution (see reagent preparation step 2.7), gently shake well at room temperature, and incubate for 45 minutes.

[0064] 3.8 Discard the solution and repeat the washing process as described in step 3.4.

[0065] 3.9 Add 100 μl of TMB one-step substrate reagent (H) to each well. Gently shake to mix at room temperature and incubate in the dark for 30 minutes.

[0066] 3.10 Add 50 μl of stop solution (I) to each well. Immediately take a reading at 450 nm using a microplate reader.

[0067] 3.11 Calculate the average absorbance of each set of standards, controls, and samples, and then subtract the average absorbance of the blank control. Plot the best-fit line through the standard points, with the standard concentration on the x-axis and absorbance on the y-axis. Calculate the concentration of each sample using OD.

[0068] (ix) Statistical Analysis

[0069] Statistical analysis of all data was performed using GraphPad Prism 6.01 and SPSS 19.0 software. Quantitative data were expressed as mean ± standard deviation (Mean ± SD). The t-test was used to analyze the means between groups, and p < 0.05 was considered statistically significant.

[0070] II. Results

[0071] 1. Cytokine microarray results ( Figure 1 This indicates that serum GHR levels decreased in young men performing tasks under long-term stress. P = 0.018738, which is statistically significant.

[0072] 2. Results of the elevated cross-maze experiment ( Figure 2 This indicates that both the chronic social frustration stress model and the chronic restraint stress model induced anxiety in mice. *P<0.05; **P<0.01; ns: no significant difference.

[0073] 3. Results of the sugar water preference experiment ( Figure 3 This indicates that the chronic social frustration stress model induced depression in mice, while the chronic restraint stress model did not have this effect. **P<0.01; ns: no significant difference.

[0074] 4. Results of the forced swimming experiment ( Figure 4This further demonstrates that the chronic social frustration stress model leads to depression in mice. ****P<0.0001.

[0075] 5. ELISA results ( Figure 5 The study revealed that GHR levels did not change significantly in mice with chronic restraint stress-induced anxiety (i.e., the restraint group) (P = 0.3655), but decreased significantly in mice with chronic social frustration stress-induced depression (i.e., the frustration group) (P = 0.00027), suggesting that serum GHR levels may be a peripheral biomarker of depression.

[0076] 6. While inducing depression in mice using chronic social frustration stress, mice were simultaneously administered Acanthopanax senticosus glycoside E or hydroxypiperidone hydrochloride via gavage daily. ELISA results ( Figure 6 The study revealed that GHR levels were significantly decreased in stunted, untreated mice (P = 5.10985E-05), and that eleutheroside E (P = 0.00106) or hydroxypiperidone hydrochloride (P = 0.00047) could effectively increase the concentration of GHR in the serum of stunted mice.

[0077] 7. Simultaneously administering Acanthopanax senticosus glycoside E or hydroxypiperidone hydrochloride via gavage to mice induced with chronic social frustration stress effectively increased serum GHR concentration in frustrated mice, improved depressive symptoms, significantly increased sucrose preference, and significantly shortened the time spent immobile in water during forced swimming. Figure 7 This further suggests that serum GHR levels may be a peripheral biomarker for depression. Figure 7 In the above, *P<0.05, **P<0.01, and ****P<0.0001.

[0078] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of serum growth hormone receptor as a biomarker in the preparation of products for diagnosing depression and / or assessing the risk of developing depression; The serum growth hormone receptor was used as the detection target. The serum growth hormone receptor was downregulated or its level was reduced in blood or serum samples from patients with depression or individuals at high risk of developing depression.

2. The application according to claim 1, wherein the product comprises a chip or a reagent kit.

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