Application of DACA in development of medicine for preventing and treating depression

Abstract

The invention relates to the technical field of depression drug development, and particularly discloses application of DACA in depression prevention and treatment drug development, the molecular formula of DACA is C24H30O6, DACA is applied to anti-depression activity, DACA is used as an active component to form a depression prevention and treatment drug together with a drug carrier, and the drug carrier is used as a drug carrier. The invention provides a new thought and approach for clinical prevention and treatment of depression, can be used for preparing medicines or health-care foods for preventing and treating depression, and has remarkable popularization and application values.

Description

Technical Field

[0001] The present invention belongs to the technical field of the development of drugs for treating depression, and particularly relates to the application of DACA in the development of drugs for preventing and treating depression. Background Art

[0002] Major depressive disorder (MDD) is one of the most common mental illnesses in current society and ranks as the fourth most prevalent disease in the world. The main clinical symptoms of depression include persistent low mood, loss of appetite, decreased interest in favorite activities, despair, sleep disorders, decreased energy, feeling useless or guilty; difficulty in thinking, concentrating or making decisions, etc. Currently, there is no effective means to cure it completely.

[0003] Due to the complexity of the pathological mechanism of MDD, several hypotheses have been proposed in recent years to explain the pathogenicity of the MDD pathogenesis mechanism, including (1) the hypothalamic-pituitary-adrenal (HPA) axis dysfunction hypothesis, (2) the monoamine hypothesis, (3) the inflammation hypothesis, (4) the genetic and epigenetic abnormality hypothesis, (5) the structural and functional brain remodeling hypothesis, and (6) the social psychology hypothesis, etc.

[0004] DACA structurally belongs to abietane-type diterpenoids. Since its structure was discovered and determined, its pharmacological activities have been widely concerned. Previous studies have found that it has good antioxidant and anti-inflammatory activities and has a good protective effect in neurodegenerative diseases.

[0005] Currently, there are no relevant patents and literature reports on the use of DACA for preventing and treating depressive-like conditions. Summary of the Invention

[0006] The purpose of the present invention is to provide the application of DACA in the development of drugs for preventing and treating depression, so as to solve the problems raised in the above background art.

[0007] To achieve the above purpose, the present invention provides the following technical solutions:

[0008] The application of DACA in the development of drugs for preventing and treating depression, wherein the molecular formula of DACA is C 24 H 30 O 6 , and the structural formula is

[0009]

[0010] Preferably, the DACA is applied in anti-depressant activity.

[0011] Preferably, the DACA is applied in neuroprotection, and the neuroprotection is for BV2 cells damaged by LPS.

[0012] Preferably, the DACA is applied to improving the depressive-like behaviors of C57BL / 6 male mice induced by chronic unpredictable mild stress.

[0013] Preferably, the DACA is applied to the preparation of anti-depressant drugs or health foods.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] Through experiments on DACA, the present invention discovers new uses of DACA. By using DACA as an active ingredient together with a drug carrier to form a drug for preventing and treating depression, it provides new ideas and approaches for the prevention and treatment of clinical depression, and can be used to prepare drugs or health foods for preventing and treating depression, having significant popularization and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a schematic diagram of the structural formula of DACA of the present invention;

[0017] Figure 2 It is a schematic diagram of the cell survival rate of DACA of the present invention acting alone on BV2 cells;

[0018] Figure 3 It is a schematic diagram of the content of NO in the cell supernatant after DACA of the present invention acts on LPS-induced BV2 cells;

[0019] Figure 4 It is a trajectory diagram of DACA of the present invention improving the depressive-like behaviors of mice induced by CUMS;

[0020] Figure 5 It is a result diagram of DACA of the present invention improving the depressive-like behaviors of mice induced by CUMS. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0022] Embodiment:

[0023] Application of DACA in the research and development of drugs for preventing and treating depression. The molecular formula of DACA is C 24 H 30 O 6 , and the structural formula is

[0024]

[0025] Among them, DACA can be applied in anti - depressive activity, can be applied in neuroprotection, the neuroprotection is for BV2 cells damaged by LPS, can be applied in improving the depressive - like behavior of C57BL / 6 male mice induced by chronic unpredictable mild stress, and can be applied in the preparation of anti - depressive drugs or health foods.

[0026] Test columns:

[0027] 1. Determine the anti - depressive activity of DACA on LPS - damaged BV2 cells

[0028] Source of materials: BV2 cells were purchased from Wuhan Ponsai Life Science Co., Ltd.

[0029] Source of instruments

[0030] Multifunctional microplate reader (Thermo Scientific Multiskan FC, USA); CO 2 Thermostatic incubator (ThermoForma 3310, USA).

[0031] Test methods

[0032] Experiment:

[0033] (1) Divide the cells into a control group and a drug group, with 5 replicate wells in each group. Inoculate BV2 cells in the logarithmic growth phase onto a 96 - well cell culture plate at a density of 1×10 4 cells / ml.

[0034] Control group: BV2 cells cultured normally;

[0035] Blank control group: A group without cells;

[0036] Drug group: Treat with drugs at different concentrations (5, 10, 20, and 40 μM) for 24 h;

[0037] Replace the cell culture medium with a medium containing 10% CCK - 8 in the wells. After incubating for 2 hours, use a microplate reader to read the absorbance of each well at 450 nm, and calculate the cell survival rate of each group. The experiment was repeated 3 times.

[0038] Among them, the formula for calculating the cell survival rate: Cell survival rate (%) = [A (drug - added) - A (blank)] / [A (control) - A (blank)] × 100%

[0039] Divide the cells into a control group and a drug group, with 5 replicate wells in each group.

[0040] Inoculate BV2 cells in the logarithmic growth phase onto a 96 - well cell culture plate at a density of 5×10 5 cells / ml.

[0041] Control group: BV2 cells cultured normally;

[0042] Blank control group: cell-free group;

[0043] LPS group: After culturing the cells for another 24 h, discard the old medium and treat with 1 μg / ml LPS for 24 h;

[0044] Drug group: Treat with drugs at different concentrations (5, 10, and 20 μM) for 24 h;

[0045] Take out 50 μl of the supernatant from each group and add it to a new 96-well plate. According to 50 μl / well, add Griess Reagent I in the room temperature nitric oxide kit to each well, and then according to 50 μl / well, add Griess Reagent II to each well. Use an enzyme-linked immunosorbent assay (ELISA) reader to read the absorbance of each well at 540 nm, and calculate the nitric oxide content in the cell supernatant of each group. The experiment was repeated 3 times.

[0046] The cytotoxicity of DACA on BV2 cells and the anti-depressant activity of cells against LPS damage are shown as follows. Data are expressed as mean ± SEM. Static analysis was performed using SPSS 22.0 software. One-way analysis of variance was used for the data, and p < 0.05 was considered statistically significant.

[0047] Results:

[0048] The DACA toxicity evaluation showed that. As Figure 2 shown, DACA did not cause damage to BV2 cells, indicating that DACA is basically non-toxic to BV2 cells.

[0049] Next, we evaluated the neuroprotective activity of DACA on LPS-induced BV2 cells. As Figure 3 shown, the release of nitric oxide in the cell supernatant of the LPS group was significantly increased compared with that of the control group, indicating that the LPS-induced BV2 cell model was successfully established. The release of nitric oxide in the cell supernatant of the DACA group was significantly decreased compared with that of the LPS group, indicating that DACA has a protective effect on the nerve damage caused by LPS. In summary, DACA can play an anti-depressant role through anti-inflammation, can be used as a potential anti-depressant protective agent, and can be used to prepare anti-depressant drugs or health foods.

[0050] Figure 1 , The chemical structure formula of DACA. Figure 2 . The cell viability of DACA acting alone on BV2 cells. Figure 3 . The content of NO in the cell supernatant after DACA acts on LPS-induced BV2 cells. (Mean ± SEM, n = 5; ## p < 0.01,#### p < 0.0001 vs control group; ****p < 0.0001 vs LPS group).

[0051] 2. Measurement of the improvement of DACA on CUMS-induced depressive-like behaviors in mice

[0052] Source of materials: C57BL / 6 male mice were purchased from the Animal Department of Kunming Medical University

[0053] Source of instruments: Animal open field behavior image acquisition card (Xinruan, Shanghai), VisuTrack animal open field behavior analysis software (Xinruan, Shanghai), animal pole climbing behavior acquisition card ((Xinruan, Shanghai), VisuTrack animal pole climbing behavior analysis software (Xinruan, Shanghai).

[0054] Test method:

[0055] Experiment: After the mice were placed in standard conditions and adapted for 1 week, the animals were divided into 5 groups (n = 10). Control group: normal control group, given 0.5% CMC-Na; CUMS group: given 1 - 2 mild stimuli every day for 42 consecutive days, including; Low-concentration drug group: CUMS + DACA (10 mg / kg, i.g.); Medium-concentration drug group: CUMS + DACA (20 mg / kg, i.g.); High-concentration drug group: CUMS + DACA (40 mg / kg, i.g.). The trial lasted for 42 days. CUMS for 42 consecutive days. DACA (10, 20, and 40 mg / kg / day) was administered by gavage from day 21 to day 42. Behavioral experiments were conducted on day 42 of the experiment.

[0056] Sucrose preference experiment

[0057] Depression is accompanied by multiple behavioral phenotypes, and one important manifestation is anhedonia, that is, individuals cannot experience pleasure from rewarding or pleasant activities. In animal model experiments, the sucrose preference test (SPT) is usually used to test for anhedonia symptoms. On the first day after the end of stress, the mice were housed individually, and two bottles of ordinary drinking water were placed in each cage to allow the mice to adapt to the situation of two bottles of water. On the second day, one of the bottles was replaced with 1% sucrose water, and the positions of the two bottles were exchanged every 6 h. On the third day, the mice were fasted and water-deprived. On the fourth day, one bottle of weighed sugar water and one bottle of drinking water were placed in each cage, and the positions were exchanged every 6 h. The test lasted for 12 h. After the test, the mass of the remaining liquid was weighed again. The calculation formula is sucrose preference percentage = sucrose water consumption / (drinking water consumption + sucrose water consumption) × 100%.

[0058] Open field experiment

[0059] The open field test, also known as the open box test, is a method for evaluating the autonomous behavior, exploratory behavior, and tension of laboratory animals in a novel environment. Based on the occurrence frequency and duration of certain behaviors of laboratory animals in a novel environment, etc., it is simple to operate and the method is reliable. The experimental device consists of an open field reaction chamber and a data automatic acquisition and processing system. The mouse open field reaction chamber is 50 cm × 50 cm × 50 cm with the inner wall painted black, and the bottom surface is evenly divided into 16 small squares of 12.5 cm × 12.5 cm (see Figure 1 ). A digital camera is mounted 2 m directly above, and its field of view can cover the entire interior of the open field. Depending on the computer software design, different parameters can be observed, such as the time the animal stays in the central square within a unit time, the number of grids crossed by a certain limb as the horizontal score (crossing), the number of times of hind limb standing as the vertical score (rearing), the number of grooming times, the number of urination and defecation times; movement speed, movement distance, rest time, movement distance along the edge, movement distance in the center, etc. This experiment mainly explores the movement ability of mice by detecting the movement trajectory and movement distance of mice within 5 minutes. The experiment is carried out in a quiet environment. The animal is placed at the center of the bottom surface of the box, and at the same time, video recording and timing are carried out. After observing for 5 minutes, stop video recording, and clean the inner wall and bottom surface of the box with 75% alcohol to prevent the information left by the previous animal (such as the urine, feces, and odor of the animal) from affecting the results of the next test. Replace the animal and continue the experiment.

[0060] Tail suspension test

[0061] In the tail suspension test (TST), the tail of the experimental animal is fixed so that its head hangs down. The animal struggles in this environment, attempting to get out of this predicament. After failing to get out after efforts, it shows intermittent immobility, indicating a state of "behavioral despair". On the day after the stress ends, gently take the mouse out of the cage, quickly fix its tail with medical tape 1 cm away from the tip of the tail, and the height between the tip of the animal's tail and the ground is about 30 cm. Then record the immobility time of the mouse within 6 minutes with a camera. After the recording duration is completed, tear off the medical tape and carefully take down the experimental animal, and put it back into the original cage and make a record.

[0062] Forced swimming test

[0063] Forced Swimming Test (FST) uses the immobility time during swimming as the main indicator to detect the despair behavior of animals. It is a commonly used test for the primary screening of antidepressant drugs and for detecting whether model animals exhibit "depressive-like" behaviors. The forced swimming test was conducted on the third day after the stress ended. Before the experiment, the water temperature in the test chamber was adjusted to 23 - 25 °C. The experimental container was 50 cm high, 20 cm in diameter, and the water depth was 20 cm. At the start of the experiment, the mice were gently placed into the water, and the immobility time within 6 minutes was recorded. After the recording period was completed, the medical tape was torn off, and the experimental animals were carefully removed and placed back into the original cage for recording. After the recording was completed, the mice were taken out, dried in a timely manner, and returned to the original cage.

[0064] Morris water maze experiment

[0065] The Morris water maze (MWM) is the most widely used behavioral experiment for studying and evaluating the spatial learning and memory ability of animals, and is one of the most commonly used experimental methods for studying learning and memory. The pre-experiment learning started on the fourth day after the stress ended. Four hours before the start of the experiment, the experimental mice were transferred to the experimental environment to adapt to it. The MWM experiment mainly includes three stages: adaptive training, learning and memory training, and learning and memory testing. One day before the formal start of the MWM learning and memory training, the mice were given adaptive training for 90 s per mouse. During this period, the mice could swim freely in the water maze to adapt to the water maze environment. The learning and memory training lasted for 4 days. During this period, each mouse was trained at two random sites every day, and the movement trajectories of the mice were monitored and recorded. During the learning and memory training, the mice could freely search for the platform in the water maze. If the mouse successfully found the platform and stayed on it for 3 s within 90 s, it was taken out of the water maze, and the time required for it to find the platform was recorded; if the mouse did not find the platform within 90 s, it was guided to find the platform and made to stay on it for 30 s. After 30 s, the mouse was taken out of the water maze, and the time for it to find the platform was recorded as 90 s. The learning and memory test was conducted on the second day after the learning and memory training ended. Before the start of the test, the platform was removed, and the movement trajectory of the mouse within 90 s was recorded and analyzed.

[0066] Results

[0067] The CUMS-induced depression model is a well-known model for exploring molecular changes related to depression, which can cause behavioral changes in mice.

[0068] To explore the effects of DACA on behavioral changes, we conducted some basic behavioral studies, such as the sucrose preference test, open field test, tail suspension test, forced swimming test, and water maze test.

[0069] Such as Figure 5As shown, in the sucrose preference test, compared with the control group, the percentage of sucrose preference in the CUMS treatment group was significantly reduced. After treatment with DACA, the percentage of sucrose preference in mice was significantly increased. In the behavioral test on the 42nd day of the experiment, compared with the control group, CUMS significantly reduced the total moving distance of mice within the open area frame, while each dose group of DACA had a certain improvement effect on it. In the tail suspension test and forced swimming test, compared with the control group, the immobility time in the CUMS treatment group was significantly increased. After treatment with DACA, the immobility time was significantly reduced. In the water maze test, compared with the control group, the number of times the CUMS treatment group passed through the platform area was significantly reduced. After treatment with DACA, the number of times mice passed through the platform area was significantly increased. These data indicate that DACA can improve CUMS-induced depressive-like behaviors in mice.

[0070] Figure 4 It is a trajectory diagram of DACA improving CUMS-induced depressive-like behaviors in mice, including the trajectory of the open field test and the water maze test. Figure 5 It is a result diagram of DACA improving CUMS-induced depressive-like behaviors in mice, including the percentage of sucrose preference in the sucrose preference test of mice, the total distance of mice within the open area frame, the immobility time of mice in the tail suspension test and forced swimming test, and the number of times and escape latency of mice passing through the platform area in the water maze test. (Mean ± SEM, n = 12; ##p < 0.01, p < 0.001, #p < 0.0001 vs control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs CUMS group).

[0071] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Application of DACA in the development of drugs for the prevention and treatment of depression, wherein the molecular formula of DACA is C 24 H 30 O6, structural formula is 2. The use of DACA according to claim 1 in the development of drugs for preventing and treating depression, characterized in that: The DACA is used in antidepressant activity.

3. The use of DACA according to claim 1 in the development of drugs for preventing and treating depression, characterized in that: The DACA is used in neuroprotection, and the neuroprotection is against LPS-damaged BV2 cells.

4. The use of DACA in the development of drugs for the prevention and treatment of depression according to claim 1, characterized in that: The DACA is used to improve the depressive-like behavior of C57BL / 6 male mice induced by chronic unpredictable mild stimulation.

5. The use of DACA in the development of drugs for preventing and treating depression according to claim 1, characterized in that: The DACA is used in the preparation of antidepressant drugs or health foods.

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