Use of an inhibitor of hepatic 20-hete synthesis in the manufacture of a medicament for the treatment of perimenopausal mood disorders

Abstract

The application discloses application of a liver 20-HETE synthesis inhibitor in preparation of a medicine for treating perimenstrual mood disorders. The application constructs a perimenstrual mouse model through ovariectomy, and it is observed through behavior test that the perimenstrual model mouse shows obvious depressive and anxious behaviors. The expression of 20-HETE synthesis enzyme Cyp4a12a in the liver of the ovariectomy model mouse is significantly increased, and the 20-HETE level in the plasma is significantly increased. Through intervention by using a liver 20-HETE synthesis enzyme inhibitor TS-011, the 20-HETE content in the plasma of the ovariectomy mouse is significantly reduced, and the depressive and anxious behaviors of the mouse are obviously improved. It is found by the application that the liver 20-HETE synthesis inhibitor can be applied to development or preparation of a perimenstrual mood disorder treatment medicine with low side effects and high clinical acceptance, and has high clinical transformation value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of hepatic 20-HETE synthesis inhibitors in the preparation of drugs for treating perimenopausal mood disorders. Background Technology

[0002] Perimenopause is a transitional period for women from peak fertility to decline, and also a high-risk period for mood disorders. 45-68% of perimenopausal women report significant depressive symptoms, and 20.2% report significant anxiety symptoms. Currently, first-line antidepressants (such as serotonin and norepinephrine reuptake inhibitors and selective serotonin reuptake inhibitors) are commonly used to treat perimenopausal mood disorders. However, long-term use of these drugs significantly increases the incidence of nausea, headache, dizziness, and changes in appetite. Furthermore, the blood-brain barrier limits the efficiency of antidepressant entry into the brain, resulting in approximately 50% of patients not responding to antidepressants. In addition, estrogen replacement therapy is widely used to improve perimenopausal symptoms and mood disorders. However, long-term estrogen use increases the risk of various diseases, including endometrial and breast cancer, and mood disorders often worsen dramatically after estrogen withdrawal, leading to withdrawal symptoms. Therefore, perimenopausal mood disorders have become a pressing clinical challenge, and finding new treatments for perimenopausal mood disorders is of paramount importance.

[0003] Brain homeostasis is regulated by other organs, particularly through mechanisms such as the liver-brain axis, gut-brain axis, and fat-brain axis. These mechanisms play a crucial role in regulating key physiological processes such as brain inflammation, neuronal survival, and synaptic plasticity, as well as higher brain functions like emotion, learning, and memory. The liver, as the core of energy metabolism, significantly influences the integrity of the central nervous system and processes like neuroinflammation. Cyp4a12a, an important member of the cytochrome P450 enzyme system in the liver and belonging to the Cyp4a subfamily, is primarily responsible for catalyzing the metabolism of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE is a lipid mediator with diverse biological activities, playing a vital role in regulating cell proliferation, inflammatory responses, sodium transport, oxidative stress, and blood-brain barrier integrity. Furthermore, 20-HETE acts as a potent vasoconstrictor, inhibiting KCa channel activation of protein kinase C and depolarizing vascular smooth muscle, and facilitating L-type Ca2+ metabolism. 2+ Channel increases Ca 2+ internal flow. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to develop or prepare a drug for treating or adjuvant treatment of perimenopausal mood disorders and / or how to develop and / or prepare a drug for improving or adjuvant improvement of perimenopausal mood disorders.

[0005] To address the aforementioned technical problems, this invention first provides the application of 20-hydroxyeicosatetraenoic acid synthesis inhibitors in the development and / or preparation of medicaments for the treatment and / or prevention of perimenopausal mood disorders.

[0006] In the above applications, the term "prevention" generally refers to methods implemented to prevent or delay the occurrence of a disease, condition, or symptom in a subject.

[0007] The term "treatment" generally refers to a method implemented to achieve a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, reduction of symptoms, lessening of disease severity, reduction of disease extent, stabilization of disease (i.e., cessation of disease progression), delay or slowing of disease progression, improvement or relief of disease status (whether partial or complete remission), whether detectable or undetectable. Furthermore, treatment can also refer to an extension of survival compared to the expected survival of a subject without treatment.

[0008] In the above applications, the drug can be used in animals with ovaries, such as mammals.

[0009] In the above applications, the symptoms of perimenopausal mood disorders may include at least one of the following:

[0010] 1) Depression

[0011] 2) Anxiety;

[0012] 3) Hair removal;

[0013] 4) The content of 20-hydroxyeicosatetraenoic acid in body fluids increases.

[0014] In the above applications, the 20-hydroxyeicosatetraenoic acid synthesis inhibitor can be a substance that inhibits the expression of the enzyme that catalyzes the formation of 20-hydroxyeicosatetraenoic acid. In one specific embodiment of the present invention, the enzyme is Cyp4a12a.

[0015] In one specific embodiment of the present invention, the 20-hydroxyeicosatetraenoic acid synthesis inhibitor is N-(3-chloro-4-morpholinophenyl)-N'-hydroxyformamidinium (TS-011).

[0016] In one specific embodiment of the present invention, the body fluid is blood plasma.

[0017] To address the aforementioned technical problems, this invention also provides a 20-hydroxyeicosatetraenoic acid synthesis inhibitor for the development and / or preparation of a medicament for treating or preventing diseases caused by ovarian dysfunction or loss.

[0018] In the above applications, the symptoms of the disease may include at least one of the following:

[0019] 1) Depression

[0020] 2) Anxiety;

[0021] 3) Hair removal;

[0022] 4) The content of 20-hydroxyeicosatetraenoic acid in body fluids increases.

[0023] In one specific embodiment of the present invention, the body fluid is blood plasma.

[0024] In the above applications, the 20-hydroxyeicosatetraenoic acid synthesis inhibitor can be a substance that inhibits the expression of the enzyme that catalyzes the formation of 20-hydroxyeicosatetraenoic acid. In one specific embodiment of the present invention, the enzyme is Cyp4a12a.

[0025] In one specific embodiment of the present invention, the 20-hydroxyeicosatetraenoic acid synthesis inhibitor is N-(3-chloro-4-morpholinophenyl)-N'-hydroxyformamidinium (TS-011).

[0026] In one specific embodiment of the present invention, the ovarian dysfunction or loss is caused by removal of the ovary.

[0027] In the above applications, the term "prevention" generally refers to methods implemented to prevent or delay the occurrence of a disease, condition, or symptom in a subject.

[0028] The term "treatment" generally refers to a method implemented to achieve a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, reduction of symptoms, lessening of disease severity, reduction of disease extent, stabilization of disease (i.e., cessation of disease progression), delay or slowing of disease progression, improvement or relief of disease status (whether partial or complete remission), whether detectable or undetectable. Furthermore, treatment can also refer to an extension of survival compared to the expected survival of a subject without treatment.

[0029] In the above applications, the drug can be used in animals with ovaries, such as mammals.

[0030] The mammals mentioned above can be mice or humans.

[0031] To address the aforementioned technical problems, the present invention also provides a medicine, which may be a medicine for treating or adjuvant treatment of perimenopausal mood disorders or a medicine for treating or adjuvant treatment of diseases caused by ovarian dysfunction or loss. The medicine may contain a 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis inhibitor.

[0032] In the aforementioned drug, the 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis inhibitor can be a substance that inhibits the expression of a catalytic enzyme, wherein the catalytic enzyme is an enzyme that catalyzes the production of 20-hydroxyeicosatetraenoic acid. In a specific embodiment of the present invention, the catalytic enzyme is Cyp4a12a, and the 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis inhibitor is TS-011.

[0033] In one specific embodiment of the present invention, the ovarian dysfunction or loss is caused by removal of the ovary.

[0034] In the context of the aforementioned medications, the term "prevention" typically refers to methods implemented to prevent or delay the occurrence of a disease, condition, or symptom in a subject.

[0035] The term "treatment" generally refers to a method implemented to achieve a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, reduction of symptoms, lessening of disease severity, reduction of disease extent, stabilization of disease (i.e., cessation of disease progression), delay or slowing of disease progression, improvement or relief of disease status (whether partial or complete remission), whether detectable or undetectable. Furthermore, treatment can also refer to an extension of survival compared to the expected survival of a subject without treatment.

[0036] The above-mentioned drugs can be used in animals with ovaries, such as mammals.

[0037] The mammals mentioned above can be mice or humans.

[0038] The behavioral experiments described above may include sucrose preference tests, open field tests, elevated cross maze tests, elevated zero maze tests, and / or forced swimming and tail suspension tests. The mammal may be a mouse.

[0039] The applications or methods described above are not for disease diagnosis. They are not intended to directly obtain disease diagnoses or health status results from living humans or animals.

[0040] The above applications or methods are for non-disease treatment purposes. They are not intended to restore or restore health or reduce suffering in living human or animal bodies.

[0041] This invention establishes a perimenopausal mouse model through ovariectomy, and conducts behavioral tests to observe whether the perimenopausal model mice develop emotional disorders. The expression of Cyp4a12a in the liver of ovariectomized mice was detected by Western blotting and Real-time PCR, and the level of 20-HETE in mouse plasma was detected by high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS). Ovariectomized mice were treated with TS-011 (a liver 20-HETE synthase inhibitor) via subcutaneous implantation of a slow-release pump. After four weeks, changes in 20-HETE levels in mouse plasma were detected by HPLC-MS / MS, and behavioral changes in the mice were observed. Attached Figure Description

[0042] Figure 1 The results are as follows: (A) In the saccharide preference test, the percentage of mice in the sham-operated group and those that had their ovariectomized prefer saccharide; the vertical axis represents the percentage of mice that preferred saccharide. (B) In the open field test, the total distance traveled by mice in the sham-operated group and those that had their ovariectomized within 30 minutes; the vertical axis represents the total distance traveled by mice in the open field test. (C) In the open field test, the number of times mice in the sham-operated group and those that had their ovariectomized entered the central area within 10 minutes; the vertical axis represents the number of times mice entered the central area in the open field test within 10 minutes. (D) In ​​the elevated cross maze test, the time taken for mice in the sham-operated group and those that had their ovariectomized to enter the open arm; the vertical axis represents the percentage of time mice spent in the open arm within 10 minutes. (E) In the elevated cross maze test, the number of times mice in the sham-operated group and those that had their ovariectomized entered the open arm; the vertical axis represents the number of times mice spent in the open arm within 10 minutes. (F) In the elevated zero maze test, the time taken for mice in the sham-operated group and those that had their ovariectomized to enter the open arm; the vertical axis represents the percentage of time mice spent in the open arm within 10 minutes. (G) In the elevated zero-maze test, the number of times the sham-operated group and ovariectomized mice entered the open arm; the vertical axis represents the number of times the mice entered the open arm within 10 minutes. (H) In the tail suspension test, the percentage of immobile time for the sham-operated group and ovariectomized mice; the vertical axis represents the percentage of immobile time for the mice. (I) In the forced swimming test, the percentage of immobile time for the sham-operated group and ovariectomized mice; the vertical axis represents the percentage of immobile time for the mice. (AC) Blue represents sham-operated mice, n=8; pink represents ovariectomized mice, n=8. (DI) Blue represents sham-operated mice, n=16; pink represents ovariectomized mice, n=8. An independent samples t-test was performed, and p < 0.05 was considered statistically significant compared to the sham-operated group.

[0043] Figure 2 This image shows hair loss in mice after ovariectomy. Sham: sham-operated group; OVX: ovariectomy-operated group.

[0044] Figure 3The results show the detection of Cyp4a family members in mouse liver. (A) Expression level of Cyp4a12a mRNA in mouse liver; the vertical axis represents the expression level of Cyp4a12a mRNA in mouse liver. Blue represents sham-operated mice, n=8; pink represents ovariectomized mice, n=8. (B) Western Blot results of CYP4A12 and CYP4A-pan in mouse liver. (C) Statistical graph of Western Blot results; the vertical axis represents the relative expression level of the CYP4A family in mouse liver. Blue represents sham-operated mice, n=3; pink represents ovariectomized mice, n=3. Independent samples t-test, p<0.05 was considered statistically significant compared with the sham-operated group.

[0045] Figure 4 The vertical axis represents the relative expression level of 20-HETE in the plasma of mice after ovariectomy; the vertical axis represents the expression level of 20-HETE in mouse plasma. Blue represents sham-operated mice (n=21); pink represents ovariectomized mice (n=18). Independent samples t-test, p<0.05 was considered statistically significant compared to the sham-operated group.

[0046] Figure 5The results are as follows: (A) Changes in plasma 20-HETE levels in mice in the TS-011 intervention group compared to the ovariectomy group; the vertical axis represents the relative change in plasma 20-HETE levels in mice. (B) Hair loss in mice after TS-011 intervention. (C) Sugar water preference test: Sugar water preference in mice in the TS-011 intervention group and the ovariectomy group; the vertical axis represents the percentage of mice that prefer sugar water. (D) Open field test: Number of times mice in the TS-011 intervention group and the ovariectomy group entered the central area within 10 minutes; the vertical axis represents the number of times mice entered the central area within 10 minutes in the open field test. (E) Open field test: Time taken for mice in the TS-011 intervention group and the ovariectomy group to enter the central area within 10 minutes; the vertical axis represents the percentage of time mice spent entering the central area within 10 minutes in the open field test. (F) Elevated cross maze test: Number of times mice in the TS-011 intervention group and the ovariectomy group entered the open arm; the vertical axis represents the number of times mice entered the open arm within 10 minutes. (G) In the elevated cross maze test, the time it took for mice in the TS-011 intervention group and the ovariectomy group to enter the open arm; the vertical axis represents the percentage of time mice spent entering the open arm within 10 minutes. (H) In the elevated zero maze test, the number of times mice in the TS-011 intervention group and the ovariectomy group entered the open arm; the vertical axis represents the number of times mice spent entering the open arm within 10 minutes. (I) In the elevated zero maze test, the time it took for mice in the TS-011 intervention group and the ovariectomy group to enter the open arm; the vertical axis represents the percentage of time mice spent entering the open arm within 10 minutes. (J) In the tail suspension test, the percentage of immobile time for mice in the TS-011 intervention group and the ovariectomy group; the vertical axis represents the percentage of immobile time for mice. Blue represents the solvent group, n=6; pink represents the TS-011 intervention group, n=6 (one outlier was removed using the ROUT method in the open field test, elevated cross maze, and elevated zero maze); independent samples t-test, p<0.05 was considered statistically significant compared to the solvent group. Detailed Implementation

[0047] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0049] TS-011 in the following examples is a MedChemExpress (MCE) product, catalog number HY-15603. Its scientific name is N-(3-chloro-4-morpholinophenyl)-N'-hydroxymethylamidine, CAS number: 339071-18-0, and it belongs to the inhibitors of 20-hydroxyeicosatetraenoic acid synthesis.

[0050] The behavioral tests in this embodiment of the invention include the following:

[0051] All animal behavioral tests were recorded using an infrared camera (with supplemental infrared lighting) and analyzed using SMART software. One hour before each test, the mice were placed in the testing room to acclimatize, and the room was kept quiet during the test. The equipment was thoroughly cleaned and dried with 75% ethanol before each test.

[0052] 1) Open field test: Each mouse was placed in the center of an open field, and its activity was observed 10 minutes and 30 minutes after entering the open field. The statistical indicators were: the total distance the mouse moved within 30 minutes, the number of times the mouse entered the central area in the first 10 minutes, and the time the mouse stayed there.

[0053] 2) Elevated Zero Maze Experiment: The elevated zero maze consists of a circular ring, with two quarter-circular closed arms surrounded by 20cm high walls on both sides. The apparatus is 60cm above the ground. During the experiment, mice are placed at the junction of the open and closed arms, facing the closed arms, and allowed to explore freely for 10 minutes. The mice's activity trajectory is analyzed, and the number of times the mice enter the open arms and the time spent in the open arms are recorded.

[0054] 3) Elevated Cross Maze Experiment: The elevated cross maze is a cross-shaped device with each arm 30cm long. The closed arms are surrounded by 20cm high walls on both sides, and the device is 60cm above the ground. During the experiment, mice are placed facing the closed arms and allowed to explore freely for 10 minutes. The mice's activity trajectories are analyzed, and the number of times a mouse enters an open arm and the time spent in an open arm are recorded.

[0055] 4) Tail suspension test: The mouse's tail (approximately 2 cm from the tip) was secured with medical tape to maintain a suspended state, with the mouse's head approximately 15 cm above the table. The experiment lasted for 5 minutes. The immobility time of the mice was analyzed and recorded.

[0056] 5) Forced Swimming Experiment: The forced swimming apparatus consisted of a cylindrical, transparent plastic container filled with water approximately 20 cm deep at around 22°C. The experiment was divided into two phases: adaptation and testing. Adaptation Phase: Mice were placed in the water container and allowed to swim for 15 minutes. Testing Phase: After 24 hours of adaptation swimming, mice were placed in the water container again and allowed to swim for 5 minutes. The immobility time of the mice was analyzed and recorded.

[0057] In this embodiment of the invention, the CYP4A12 primary antibody was custom-made by Huaan Bio (species: rabbit); the CYP4A-pan primary antibody was from Aifang Bio (catalog number: AFRM82001); and the β-actin antibody was from Zhongshan Jinqiao (catalog number: TA-09).

[0058] The following examples used Graphpad Prism 9 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. The t-test was used, and P < 0.05 indicated a significant difference.

[0059] Example 1. Preparation of perimenopausal mouse model

[0060] 1. Ovariectomy in mice

[0061] Ovariectomized mice – perimenopausal model mice (OVX) – were obtained using female C57BL / 6J mice:

[0062] 1.1 Experimental grouping: Sixteen 8-week-old female C57BL / 6J mice, weighing 19±1g, were randomly divided into two groups: sham group and ovariectomy group (OVX), with 8 mice in each group.

[0063] 1.2 Surgical plan:

[0064] Sham surgery group: Mice were anesthetized with isoflurane (5% for induction, 1% for maintenance). During the operation, the mice were placed in a prone position, and the skin was prepared according to the surface location of the ovary. The surgical procedure was performed according to aseptic principles. The skin incision was made with scissors to expose the peritoneum. After opening the abdominal cavity with an incision of about 0.5-1.0 cm in the peritoneum, the adipose tissue located below and outside the kidneys could be seen. After removing part of the adipose tissue, the peritoneum was closed layer by layer, and the skin incision was sutured with 5-0 suture needles. After the operation, an appropriate amount of penicillin solution was dripped into the skin incision to prevent bacterial infection.

[0065] Ovariectomy group (OVX): Mice were anesthetized with isoflurane (5% for induction, 1% for maintenance). During surgery, the mice were placed in a prone position, and the skin was prepared according to the surface location of the ovary. The surgical procedure was performed under aseptic conditions. A skin incision was made using scissors to expose the peritoneum. After opening the abdominal cavity with an incision of approximately 0.5-1.0 cm, the adipose tissue located below and outside the kidneys could be seen. Slight retraction revealed the ovary and fallopian tubes, which were encased in fat. The ovary and fallopian tubes were ligated using 5-0 surgical sutures. After ovary removal, the peritoneum was closed layer by layer, and the skin incision was sutured using 5-0 suture needles. After surgery, an appropriate amount of penicillin solution was instilled into the skin incision to prevent bacterial infection, resulting in ovariectomy mice (OVX).

[0066] 2. Ovariectomized mice (OVX) exhibited mood disorders.

[0067] Behavioral tests were performed on ovariectomized mice 4 weeks post-operation. Results showed a decreasing preference for sucrose in ovariectomized mice (OVX) compared to the sham group. Figure 1 (A) and exhibited significant depressive and anxiety-like behaviors;

[0068] In the mouse sucrose preference experiment, mice were first acclimatized to the experimental environment and two bottles containing purified water and a 1% sucrose solution, respectively. Before the formal experiment, the mice were fasted for 12 hours, followed by 30 ml of purified water and 30 ml of 1% sucrose solution, which they were allowed to drink freely for 6 hours. To eliminate the influence of positional preference on the experimental results, the positions of the two bottles were exchanged at the 3rd hour of the experiment. After the experiment, the consumption of purified water and sucrose solution per mouse was accurately estimated by weighing the bottles, and the data was analyzed using the sucrose preference calculation formula: Sucrose preference = [Sucrose consumption / (Sucrose consumption + Purified water consumption)] × 100%.

[0069] In the open field experiment, compared with the sham group, ovariectomized mice (OVX) showed a significantly reduced total distance traveled into the open field within 30 minutes, and a decreasing trend in the number of times they entered the center (central region) of the open field in the first 10 minutes. Figure 1 (B and C);

[0070] In the elevated cross maze experiment, compared with the sham group, ovariectomized mice (OVX) showed a significantly shorter time to enter the open arm and a decreasing trend in the number of times they entered the open arm. Figure 1 middle DE);

[0071] In the elevated zero maze experiment, compared with the sham group, ovariectomized mice (OVX) had significantly reduced time to enter the open arm and number of times they entered the open arm (FG in Figure 1).

[0072] In the forced swimming and tail suspension tests, the immobility time of ovariectomized mice (OVX) was significantly increased compared with the sham group (HI in Figure 1).

[0073] The above behavioral results suggest that ovariectomized mice (OVX) exhibited significantly depressive and anxiety-like behaviors compared to the sham group.

[0074] 3. Ovariectomized mice (OVX) exhibited significant hair loss.

[0075] Excessive stress and anxiety in mice can cause changes in hormone levels, ultimately leading to hair loss. This invention found that compared to control mice (…),… Figure 2Compared to the Sham model mice, the ovariectomized mice showed significant hair loss. Figure 2 The presence of OVX (represented by Ovarian Virus X) also indicates that ovariectomized mice exhibited emotional disturbances.

[0076] 4. Cyp4a12a expression was significantly increased in the liver of ovariectomized mice (OVX).

[0077] Previous studies have found that Cyp4a12a expression was significantly upregulated in the liver of perimenopausal model mice, and the level of 20-HETE in plasma was significantly increased. Simultaneously, the mice exhibited pronounced depressive and anxiety-like behaviors. This suggests that the Cyp4a12a-20-HETE axis may be a key factor influencing mood disorders in perimenopausal model mice.

[0078] 4.1 Real-time PCR detection of Cyp4a12a expression

[0079] After the behavioral testing experiment, real-time PCR was performed to detect Cyp4a12a mRNA (NCBI Entrez ID: 277753) in the liver of mice. The results showed that compared with sham mice, the expression level of Cyp4a12a mRNA in the liver of ovariectomized mice (OVX) was significantly increased. Figure 3 (A)

[0080] The primers for detecting Cyp4a12a and the internal reference gene (β-actin) are as follows:

[0081] Cyp4a12a-F: 5'-TCCTCTAATGGCTGCAAGGCTA-3';

[0082] Cyp4a12a-R: 5'-CCAGGTGATAGAAGTCCCATCT-3';

[0083] Actb-F: 5'-CAGCCTTCCTTCTTGGGTATG-3';

[0084] Actb-R: 5'-GGCATAGAGGTCTTTACGGATG.

[0085] The real-time PCR detection method is as follows:

[0086] Total RNA was extracted from 50 mg of liver tissue using 1 mL of Trizol, and the purity and concentration of the RNA were determined. cDNA was synthesized using a reverse transcription kit and stored at -20°C. Amplification was then performed using a Real-time PCR kit, with a reaction volume of 20 μL. The amplification conditions were: 95°C pre-denaturation for 5 min; 95°C denaturation for 10 s, 55-60°C annealing for 20 s, and 72°C extension for 20 s, for 40 cycles. Results were presented as follows: -ΔΔCt Data analysis methods are used to perform relative quantitative calculations of gene expression.

[0087] 4.2 Western Blot Detection

[0088] Western blot analysis of CYP4A12 (an antibody that specifically recognizes two CYP4A12 isoforms, CYP4A12a and CYP4A12b) and CYP4A-pan (a broad-spectrum antibody against CYP4A family proteins) proteins in mouse liver showed that, compared with sham-operated mice, the expression levels of CYP4A12 and CYP4A-pan in the liver of ovariectomized mice were significantly increased. Figure 3 (B and C in the middle).

[0089] The Western Blot detection method is as follows:

[0090] 50 mg of liver tissue was added to lysis buffer and protease inhibitor, and lysed thoroughly on a shaker at 4°C. The supernatant was collected by centrifugation, and protein quantification was performed using the BCA method. The samples were diluted according to their protein concentration to ensure consistent final protein concentration across different samples. After boiling, the samples were rapidly cooled on ice. SDS-PAGE gels were prepared, loaded, and subjected to electrophoresis. When the loading reached the bottom of the gel, the membrane was transferred and blocked with 5% skim milk for 1 hour. The membrane was then incubated overnight with primary antibody (CYP4A12 or CYP4A-pan). After washing with TBST, the membrane was incubated with secondary antibody (Donkey Anti-Rabbit IgG (H+L) HRP Conjugated, Jakson, 715-035-151) for 2 hours. After ECL staining, the membrane was exposed and developed using an imaging system. Data analysis was performed using ImageJ.

[0091] 5. Plasma levels of 20-HETE were significantly elevated in ovariectomized mice (OVX).

[0092] Following behavioral tests, high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) was used to detect 20-HETE levels in mouse plasma. The results showed that, compared to the sham-operated group, ovariectomy (OVX) significantly increased 20-HETE levels in mouse plasma. Figure 4Meanwhile, considering the significant depression and anxiety-like behaviors exhibited by the ovariectomized mice (OVX) in step 2, and the emotional disorders observed in the ovariectomized mice in step 3, it is evident that the present invention successfully constructed a perimenopausal model mouse by removing the ovaries of mice, and the ovariectomized mice (OVX) are the perimenopausal model mice.

[0093] The 20-HETE detection method is as follows:

[0094] Plasma samples underwent protein precipitation and liquid-liquid extraction for sample pretreatment to remove interfering substances. The pretreated samples were then injected into a high-performance liquid chromatography-mass spectrometry (HPLC-MS) system, where 20-HETE was separated using an appropriate mobile phase (e.g., gradient elution of methanol and water). After separation, the samples were detected by mass spectrometry, typically using electrospray ionization and multiple reaction monitoring (MRM) modes to improve specificity and sensitivity. Finally, the concentration of 20-HETE was quantified using the internal standard method (20-HETE-D6, CAYMAN CHEMISTRY) to ensure the accuracy and repeatability of the results.

[0095] Specifically, gradient elution was used, the ESI ionization source was in positive mode, and the data acquisition mode was data-dependent acquisition mode. Detailed chromatographic and mass spectrometry parameters are as follows:

[0096] Ultra-high performance liquid chromatograph: ExionLC TM AD (HPLC): SCIEX (USA);

[0097] Ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometer Qtrap 7500: SCIEX (USA);

[0098] Liquid chromatography conditions: XBridge BEH C18 column (100 mm × 2.1 mm, 2.5 μm, Waters, USA), column temperature 40 °C, injection volume 10 μL;

[0099] Mobile phase: A is ultrapure water, B is acetonitrile, both containing 0.1% formic acid (v / v), flow rate is 0.4 mL / min. -1 ;

[0100] Gradient elution conditions: 0-0.5 min, 30% B; 0.5-2 min, 30%-99% B; 2.0-4.0 min, 99% B; 4.0-4.1 min, 99%-30% B; 4.1-6.0 min, 30% B;

[0101] Mass spectrometry conditions: Heated electrospray ionization source (HESI) temperature 300℃; capillary voltage 1.5kV; ion transmission tube temperature 320℃; sheath gas 35 units; auxiliary gas 10 units. Full scan / ddms2 scan mode: acquisition range 50-1000Da, negative ion acquisition.

[0102] Example 2. TS-011 Treatment of Perimenopausal Mood Disorders

[0103] Following the method in Example 1, 12 eight-week-old female C57BL / 6J mice, weighing 19±1g, were used to establish a perimenopausal mouse model.

[0104] Dissolve TS-011 in DMSO (5%) and PEG300 (95%) to prepare a 50 mg / mL solution.

[0105] Twelve perimenopausal model mice were randomly divided into two groups: a control group (OVX+Vehicle) and a TS-011 intervention group (OVX+TS-011), with six mice in each group.

[0106] TS-011 intervention group: Each perimenopausal model mouse was administered TS-011 via a subcutaneous implanted slow-release pump. The administration volume for each mouse was 3.5 uL / day and the dosage was 5 mg / kg / day.

[0107] Control group (OVX+Vehicle): Each perimenopausal model mouse was administered the drug to the control group via a subcutaneous implanted slow-release pump using a solvent (DMSO (5%) and PEG300 (95%)), with a dosage volume of 3.5 uL / day per mouse.

[0108] The specific method is as follows: Mice were anesthetized with isoflurane (5% induction, 1% maintenance). During the operation, the mice were placed in a prone position, and the hair on their backs or between their shoulder blades was shaved for skin preparation. The surgical procedure was performed under aseptic conditions. The skin incision was made with scissors, and the subcutaneous tissue was bluntly dissected to form a pouch to accommodate the pump. The sustained-release pump (Reward Biotech, catalog number: 140W, catheter facing down) was gently implanted into the pouch, ensuring complete embedding of the pump without twisting or compression. The skin incision was sutured with a 5-0 suture needle. After the operation, an appropriate amount of penicillin solution was instilled into the skin incision to prevent bacterial infection. Four weeks later, the changes in 20-HETE levels in mouse plasma were detected using high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS), with the 20-HETE level detection method being the same as in step 5.

[0109] The results showed that compared with the control group ( Figure 5 Compared to OVX+Vehicle mice with type A, OVX model mice after TS-011 intervention ( Figure 5The level of 20-HETE in plasma was significantly reduced in OVX-+TS-011-represented mice. Figure 5 Compared to OVX+Vehicle (B), hair loss in mice was significantly improved. Figure 5 The OVX+TS-011 in the middle B group represents this. Behavioral test results showed that, compared to the control group ( Figure 5 Compared to OVX+Vehicle mice, TS-011 intervention significantly increased the sucrose preference in OVX model mice. Figure 5 In the open field experiment, compared with the control group (OVX+TS-011 represents C); Figure 5 Compared with OVX+Vehicles in D and E models, TS-011 intervention showed an increasing trend in the number of times OVX model mice entered the central region, and the time spent entering the central region was significantly increased. Figure 5 OVX+TS-011 represented by D and E mice; in the elevated cross maze and elevated zero maze experiments, compared with the control group, the number of times OVX model mice entered the open arm was significantly increased after TS-011 intervention ( Figure 5 In the tail suspension test, compared with the control group, the immobility time of OVX model mice was significantly reduced after TS-011 intervention (F and H); Figure 5 The results showed that intervention with the liver 20-HETE inhibitor TS-011 significantly improved depression and anxiety-like behaviors in perimenopausal model mice.

[0110] In summary, perimenopausal model mice exhibited significant depressive and anxiety-like behaviors. The expression of the 20-HETE synthase Cyp4a12a in the liver of OVX model mice was significantly increased, and the plasma 20-HETE level was also significantly elevated. Intervention with the hepatic 20-HETE synthase inhibitor TS-011 significantly improved the depressive and anxiety-like behaviors in the perimenopausal model mice. This invention demonstrates that intervention with hepatic 20-HETE synthase inhibitors can be applied to the development or preparation of drugs for the treatment of perimenopausal mood disorders with low side effects and high clinical acceptance, possessing high clinical translational value.

[0111] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The use of a 20-hydroxyeicosatetraenoic acid synthesis inhibitor in the preparation of a medicament for treating perimenopausal mood disorders, wherein the 20-hydroxyeicosatetraenoic acid synthesis inhibitor is N-(3-chloro-4-morpholinophenyl)-N'-hydroxyformamidin.

2. The application according to claim 1, characterized in that: The symptoms of perimenopausal mood disorders include at least one of the following: 1) Depression; 2) Anxiety; 3) Hair removal; 4) The content of 20-hydroxyeicosatetraenoic acid in body fluids increases.

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