Application of SSK-9-1 in preparation of medicine for preventing or treating depression and anxiety

Abstract

The invention belongs to the technical field of application of natural medicines, and particularly discloses application of SSK-9-1 in preparation of medicines for preventing or treating depression and anxiety. Animal behavioral experiments prove that SSK-9-1 shows a positive improvement effect on depression and anxiety behaviors of a depression model mouse, and the exploration behavior and interest level of the mouse in a new environment can be improved. Specifically, in an open field experiment, SSK-9-1 can significantly prolong the residence time of a depression mouse in a central area and increase the number of times of entering the central area; in an elevated cross maze experiment, the SSK-9-1 can obviously increase the number of times that a depression mouse enters an open arm and the staying time of the depression mouse in a target area. SSK-9-1 provides a new thought and basis for developing natural medicines to prevent or treat depression and anxiety, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of natural medicine application technology, specifically relating to the application of SSK-9-1 in the preparation of drugs for the prevention or treatment of depression and anxiety. Background Technology

[0002] Depression (DEP), a serious mental disorder that severely impacts human health and quality of life, is characterized by symptoms such as anhedonia, depressed mood, fatigue, sleep disturbances, loss of appetite, and cognitive decline. The etiology of depression is complex, involving multiple factors, including neuronal atrophy, reduced synaptic connections, and decreased neurotransmitter activity in the central nervous system (CNS) due to inflammatory responses. In research on the pathogenesis of depression, the monoamine neurotransmitter system theory (norepinephrine, serotonin, and dopamine, etc.) has become dominant. Currently, based on this pathogenesis, various antidepressants have been developed, including selective serotonin reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors (SNRIs), tricyclic antidepressants, and monoamine oxidase inhibitors. However, these drugs suffer from slow efficacy, numerous side effects, and even poor efficacy in some patients.

[0003] Anxiety disorders (AD) are mental disorders characterized by excessive and persistent worry and tension. Patients with anxiety disorders often experience symptoms of autonomic hyperactivity, such as palpitations, chest tightness, and restlessness, leading to impaired social functioning, often manifesting as inattention, memory loss, and other cognitive impairments. It is generally believed that anxiety disorders result from the interaction of genetic and environmental factors. This interaction can trigger neurotransmitter metabolism disorders and dysfunction of the endocrine and immune systems. Clinical treatment of anxiety disorders primarily employs psychotherapy, physical therapy, and pharmacology. Among these, pharmacology is considered the first-line treatment due to its low cost and rapid effectiveness. However, existing medications have drawbacks such as tolerance, high addictive potential, and potential side effects, which significantly weaken treatment efficacy and affect patient recovery. Therefore, exploring safe, effective, and low-side-effect natural drugs to improve depressive and anxious behaviors has become a major goal in this research field.

[0004] SSK-9-1 is a natural compound isolated from plants in the Cupressaceae, Araucariaceae, and Pinaceae families. Currently, there are no published studies reporting on the effects of SSK-9-1 on depression and anxiety disorders. Therefore, the potential role and mechanism of SSK-9-1 in the field of depression and anxiety disorders remain to be explored and studied. Summary of the Invention

[0005] In view of the problems in the technical background, the present invention aims to provide the application of SSK-9-1 (3β-(equatorial)-hydroxy-labdane-type diterpene) in the preparation of drugs for the prevention or treatment of depression and anxiety.

[0006] One objective of this invention is to provide the use of SSK-9-1 in the preparation of drugs for the prevention or treatment of depression and anxiety. Another objective is to provide a pharmaceutical composition for the prevention or treatment of depression and anxiety. A third objective is to provide a pharmaceutical formulation containing SSK-9-1.

[0007] This invention first provides the application of SSK-9-1 in the preparation of drugs for the prevention or treatment of depression and anxiety, wherein the structural formula of SSK-9-1 is: .

[0008] The SSK-9-1 described in this invention can be isolated from plants of the Cupressaceae family, Araucariaceae family, and Pinaceae family, or it can be chemically synthesized.

[0009] Pharmacological experiments have confirmed that SSK-9-1 has significant effects in preventing and treating depressive and anxious behaviors, and significantly increases the number of times and the duration of depressed mice entering the open arms of the elevated cross maze, as well as the number of times and the duration of their stay in the central area of ​​the open field.

[0010] Based on network pharmacology analysis and in vitro cell experiments, SSK-9-1 can increase the expression level of Bcl-2 protein, thereby improving anxiety-depression-like behaviors.

[0011] The aforementioned depression and anxiety include, but are not limited to, depression-anxiety-like behaviors caused by damage to the nervous system, neuroinflammation, endocrine abnormalities, or stress.

[0012] The depression and anxiety mentioned include, but are not limited to, depression-anxiety-like behaviors caused by neuropsychiatric disorders such as chronic pain, neurodegenerative diseases, cerebrovascular diseases, metabolic syndrome, autoimmune diseases, post-infectious sequelae, perinatal physiological changes, or drug / substance withdrawal.

[0013] The aforementioned depressive and anxiety-like behaviors include any one or more of the following: self-isolation, decreased social activity, low exploratory ability, and reduced willpower.

[0014] The present invention also provides a pharmaceutical composition for the prevention or treatment of depression and anxiety, the pharmaceutical composition comprising SSK-9-1, its pharmaceutically acceptable salts, stereoisomers, solvates, etc.

[0015] The present invention also provides a pharmaceutical formulation comprising SSK-9-1, a pharmaceutically acceptable salt thereunder, a stereoisomer thereunder, a solvate thereunder, or the above-described pharmaceutical composition, and a pharmaceutically acceptable carrier.

[0016] The formulations of the pharmaceutical preparations include, but are not limited to, injections, capsules, tablets, granules, powders, oral liquids, and pills.

[0017] This invention rationally utilizes medicinal chemistry, network pharmacology prediction, and in vivo and in vitro biological verification to confirm that SSK-9-1 exerts its preventive and therapeutic effects through multiple signaling pathways closely related to the pathology of depression and anxiety, and possesses clear antidepressant and anti-anxiety activity, providing multi-level scientific evidence for its role as a novel substance for the prevention and treatment of depression and anxiety behaviors. Attached Figure Description

[0018] Figure 1 The effect of SSK-9-1 on the number of times a depressed mouse model enters the open arm of an elevated cross maze.

[0019] Figure 2 The effect of SSK-9-1 on the time spent in the target area of ​​an elevated cross maze in a mouse model of depression.

[0020] Figure 3 The effect of SSK-9-1 on the number of times a depressed mouse model enters the central region of an open field.

[0021] Figure 4 The effect of SSK-9-1 on the time spent in the central region of an open field in a mouse model of depression.

[0022] Figure 5 Venn diagram showing the predicted targets of SSK-9-1 active ingredients and targets of depression.

[0023] Figure 6 Venn diagram showing the predicted targets of SSK-9-1 active ingredients and targets for anxiety disorders.

[0024] Figure 7 SSK-9-1 is the core PPI target for treating depression.

[0025] Figure 8 SSK-9-1 is the core PPI target for the action of anxiety disorders.

[0026] Figure 9 To detect the effect of SSK-9-1 on Bcl-2 protein expression levels using Western blotting. Detailed Implementation

[0027] The present invention is further described in detail through the following embodiments, but it should be noted that the scope of the present invention is not limited by these embodiments.

[0028] Example 1: Elevated Cross Maze Experiment to investigate the effect of SSK-9-1 on depressive-anxiety-like behaviors in a corticosterone-induced depression model mouse.

[0029] The elevated cruciform maze experiment utilizes mice's innate exploratory nature towards novel environments and their fear of suspended open spaces to construct a conflicting scenario for assessing the animals' anxiety levels. Mice, due to their dark-loving nature, tend to seek security in closed arms, but their instinct to explore new environments makes them crave to enter open arms. This conflict between their exploratory impulse and their fear of open spaces triggers anxiety, manifested as altered time allocation between open and closed arms or a reduced frequency of entering open arms. By quantitatively analyzing indicators such as the time mice spend in open and closed arms and the number of entries, their anxiety levels can be objectively assessed.

[0030] The mice used in the experiment were 7-week-old SPF-grade male C57BL / 6 mice, weighing 20-25 g, provided by the Experimental Animal Center of Nantong University. After 7 days of acclimatization, all mice were randomly divided into four groups: a normal control group (Control); a corticosterone-induced depression model group (Model, 20 mg / kg, sc); a corticosterone-induced group treated with the positive control drug fluoxetine (Fluoxetine, 20 mg / kg, ig); and a corticosterone-induced group treated with SSK-9-1 (SSK-9-1, 10 mg / kg, ig). Drug administration was performed simultaneously with model establishment, and both the modeling agent and the drugs were administered for 21 days. After completing the model establishment and drug administration procedures, behavioral assessment experiments were conducted on the mice in each group.

[0031] The elevated cross maze (EPM) experimental setup is designed as follows: The setup consists of a pair of open arms and a pair of closed arms, each arm measuring 25 × 5 cm, extending outward from a central cross platform of 5 × 5 cm. The two opposing arms are enclosed by an opaque wall 20 cm high, forming two closed arm regions. The entire cross maze is 40 cm above the ground.

[0032] Mice were placed in the central intersection area of ​​an elevated cross maze, with their heads facing the open arms. The mice's exploration trajectories were then recorded freely over 5 minutes using the ANY-maze analysis system. Anxiety levels were quantified by analyzing the number of times mice entered and exited the open and closed arms, as well as the duration of their stay within each arm. Data were statistically analyzed using GraphPad Prism 10.0 software and are expressed as mean ± standard deviation (SD). P < 0.05 indicates a statistically significant difference.

[0033] Open arm dwell time percentage (%) = Open arm dwell time / (Open arm dwell time + Closed arm dwell time) × 100%

[0034] Percentage of open arm dwell times (%) = Number of open arm dwell times / (Number of open arm dwell times + Number of closed arm dwell times) × 100%.

[0035] In model mice, depressive and anxious behaviors were negatively correlated with open arm exploration behavior; that is, fewer entries into the open arm and shorter dwell times reflected more severe depressive and anxious symptoms. If drug intervention significantly increased the number of times mice entered the open arm and the dwell time, it indicated that the drug had activity in improving depressive and anxious behaviors.

[0036] Experimental results are as follows Figure 1 , Figure 2 As shown, in the elevated cross maze experiment, the model group of depressed mice exhibited significantly abnormal exploration behavior in the elevated cross maze; that is, compared with the normal control group, the model group mice entered the open arms a significantly fewer time. P < 0.001), and the dwell time within the open arm was significantly shortened ( P < 0.001), indicating significant depression and anxiety-like mood disorders. Further analysis revealed that after treatment with SSK-9-1, the number of times the depressed model mice entered the open arm was significantly increased compared to the model group ( P < 0.001), and its efficacy is superior to that of the positive control drug fluoxetine at the same dose ( P < 0.001). Meanwhile, SSK-9-1 significantly prolonged the time depressed mice remained in the open arm ( P <0.001), bringing it close to the level of the normal group.

[0037] The above results confirm that SSK-9-1 can effectively enhance the autonomous exploration behavior of depressed mice in novel environments, reduce their anxiety levels, and its anti-anxiety effect has significant advantages.

[0038] Example 2: Open field experiment to investigate the effect of SSK-9-1 on depressive-anxiety-like behavior in a corticosterone-induced depression model mouse.

[0039] The open field test (also known as the open box test) is a commonly used behavioral testing method in mental illness research. It explores the behavior of rodents by assessing their spontaneous movements in an unfamiliar open environment in order to indirectly reflect their anxiety levels.

[0040] The grouping and administration regimens of the experimental animals were the same as in Example 1.

[0041] The Open Field Experiment (OFT) setup consists of four 50 cm high white acrylic panels forming a 50 cm × 50 cm square open box, with a camera mounted on top to record the animals' activity trajectory throughout.

[0042] After removing the mice from their cages, the experimenters placed them gently in the center of a pre-installed open space test chamber, ensuring the mice faced away from the experimenters to avoid human interference. Once placed, the experimenters immediately withdrew, allowing the mice 15 minutes of free exploration without external disturbance.

[0043] Throughout the experiment, data was collected using the ANY-maze video tracking and analysis system. This system automatically recorded and analyzed the mice's spontaneous activity, dwell time in the central and peripheral areas, and number of entries within 15 minutes to evaluate the amount of spontaneous activity and anxiety behavior. After each monitoring session, the following cleaning procedure was strictly followed: After returning the mice to their original cages, the experimenter thoroughly wiped and disinfected the bottom, interior items, and walls of the enclosure with a 75% ethanol solution to eliminate the influence of odor on the next mouse. Subsequent behavioral tests were only conducted after the ethanol had completely evaporated and there was no irritating odor inside the enclosure to ensure the reliability of the experimental results.

[0044] Experimental results are as follows Figure 3 , Figure 4 As shown, in the behavioral assessment of the open field experiment, the model group of depressed mice exhibited significant anxiety-like behavioral characteristics, namely, compared with the normal control group, the model group mice spent a significantly shorter time in the central region of the open field. P < 0.001), and the number of times it entered the central region also decreased significantly ( P < 0.001 indicates a decreased interest in exploring novel environments and severe anxiety. Further analysis revealed that after SSK-9-1 treatment, the time spent in the central region of the depressed model mice was significantly longer than that in the model group ( P < 0.001), the number of entries increased significantly ( P < 0.001). This confirms that SSK-9-1 can not only effectively alleviate anxiety in depressed mice, but also enhance their autonomous exploration behavior in novel environments.

[0045] Example 3: Network pharmacology analysis of key targets of SSK-9-1 in depression and anxiety disorders

[0046] The two-dimensional structure diagram of SSK-9-1 was retrieved from the PubChem database and imported into the SwissTargetPrediction database, with the species limited to "Homo sapiens," to query potential targets. The protein names of the targets were converted to gene names using the UniProt database, and duplicates were removed to construct a dataset of SSK-9-1 active ingredient targets. Targets for depression and anxiety were retrieved from the GeneCards, DrugBank, OMIM, and DisGeNet databases, and duplicate values ​​were removed to construct datasets for depression and anxiety disease targets. The intersections of the active ingredient targets with the targets for depression and anxiety were calculated, and Venn diagrams were generated using the Venny 2.1 platform. The resulting intersection targets were the genes at which SSK-9-1 acts on depression and anxiety, respectively. These intersections were then plotted as protein-protein interaction (PPI) diagrams using Cytoscape 3.10.1 software.

[0047] like Figure 5 , Figure 6 As shown, a total of 100 predicted targets for the active ingredient of SSK-9-1 were obtained, including 17,411 disease targets for depression and 9,872 disease targets for anxiety. SSK-9-1 targets 90 and 80 intersection genes for depression and anxiety, respectively.

[0048] Figure 7 , Figure 8 As shown, a PPI network diagram was constructed based on the intersection genes of SSK-9-1 acting on depression and anxiety disorders, with core intersection targets presented according to degree values. Analysis of the PPI network diagram suggests that Bcl-2 may be a key target gene of SSK-9-1. Bcl-2 can inhibit apoptosis by preventing the activation of pro-apoptotic proteins and maintaining mitochondrial integrity. In the brain, Bcl-2 can protect neurons from stress factors, producing an antidepressant-like effect and improving behavioral performance in animals.

[0049] Example 4

[0050] To verify the above predictions, this embodiment used Western blotting to detect the expression level of Bcl-2 protein.

[0051] Depression is currently considered closely related to microglia and can be defined as a microglia-related disease. Studies have shown that microglia-induced neuroinflammatory activation accelerates the development of depression and increases an individual's susceptibility to it. Further research has found a significant increase in microglia density in the brain tissue of depressed patients, and the expression level of microglia density is associated with depressive and anxious behaviors.

[0052] This embodiment uses the mouse microglial cell line BV2. After resuscitation, BV2 cells were passaged and used in the experiment. BV2 cells (1.5 × 10⁻⁶) 6 The samples were seeded into 6-well plates and incubated for 12 hours. According to experimental requirements, 500 ng / mL LPS and SSK-9-1 (2.5, 5, 10 μM) were prepared using complete culture medium. After drug administration, the plates were incubated for another 24 hours. All experimental samples were set up in triplicate to calculate bias. Western blotting was used to detect BCL-2 protein expression. The bands were exposed in a chemiluminescence analyzer using ECL chemiluminescence developing solution, and the protein blots were quantified and analyzed using ImageJ software.

[0053] The results are as follows Figure 9 As shown, Western blotting analysis revealed that the expression level of Bcl-2 protein in the model group (Model) BV2 cells was significantly lower than that in the control group (Control). P < 0.01). Treatment with SSK-9-1 (2.5, 5, 10 μM) significantly increased Bcl-2 protein expression in BV2 cells. P < 0.001). The above results confirm that the antidepressant effect of SSK-9-1 may be related to the activation of Bcl-2, thereby inhibiting apoptosis.

[0054] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of SSK-9-1 in the preparation of drugs for the prevention or treatment of depression and anxiety, characterized in that, The structural formula of the SSK-9-1 is as follows: 。 2. The application as described in claim 1, characterized in that, The depression and anxiety mentioned include depressive and anxiety-like behaviors caused by damage to the nervous system, neuroinflammation, endocrine abnormalities, or stress.

3. The application as described in claim 1, characterized in that, The depression and anxiety mentioned include depressive and anxiety-like behaviors caused by chronic pain, neurodegenerative diseases, cerebrovascular diseases, metabolic syndrome, autoimmune diseases, post-infectious sequelae, perinatal physiological changes, or drug / substance withdrawal.

4. The application as described in claim 2 or 3, characterized in that, The aforementioned depressive and anxiety-like behaviors include any one or more of the following: self-isolation, decreased social activity, low exploratory ability, and reduced willpower.

5. A pharmaceutical composition for the prevention or treatment of depression and anxiety, characterized in that, The pharmaceutical composition comprises SSK-9-1, its pharmaceutically acceptable salt, stereoisomer, and solvate.

6. A pharmaceutical preparation, characterized in that, The pharmaceutical composition comprises SSK-9-1, its pharmaceutically acceptable salt, stereoisomer, solvate, or the pharmaceutical composition of claim 5, and a pharmaceutically acceptable carrier.

7. The pharmaceutical preparation according to claim 6, characterized in that, The pharmaceutical preparations can be in the form of injections, capsules, tablets, granules, powders, oral liquids, and pills.

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