阅读说明：本技术 佛手柑内酯在治疗抑郁症中的应用 (Application of bergapten in treating depression ) 是由 程金波 闫美辰 薄雪娜 于 2021-09-15 设计创作，主要内容包括：本发明公开佛手柑内酯在治疗抑郁症中的应用,属于医药技术领域。本发明首次提出了天然药物佛手柑内酯可通过调节炎症反应防治抑郁症,显著改善抑郁症状。佛手柑内酯使脂多糖(LPS)诱导的抗抑郁小鼠悬尾测试和强迫游泳测试中不动时间明显下降；糖水偏好百分比升高,自主活动增强。其作用机制与调节抑郁小鼠小胶质细胞活化介导的神经炎症反应有关。本发明为改进目前抑郁症的治疗方法提供新的途径。本发明的方案可实现“老药新用”,可大大缩短药物发现到临床转化的时间。(The invention discloses an application of bergapten in treating depression, belonging to the technical field of medicines. The invention firstly provides a natural medicine bergapten which can prevent and treat depression by adjusting inflammatory reaction and obviously improve depression symptoms. Bergapten can obviously reduce the immobility time in a Lipopolysaccharide (LPS) -induced antidepressant mouse tail suspension test and a forced swimming test; the percentage of sugar water preference is increased and the autonomic activity is enhanced. The action mechanism is related to the regulation of nerve inflammation reaction mediated by the activation of the microglia of depressed mice. The invention provides a new way for improving the existing treatment method of depression. The scheme of the invention can realize the new application of the old medicine and can greatly shorten the time from the discovery of the medicine to the clinical conversion.)

1. Application of bergapten in preparing medicine for preventing and/or treating depression symptom diseases is provided.

2. The use according to claim 1, wherein the depressive symptom class of diseases includes unidirectional depressive disorder, bidirectional depressive disorder, secondary depression, dysthymic disorder, postpartum depression, psychotic depression, seasonal affective disorder, and juvenile depression.

3. Use according to claim 1 or 2, characterized in that bergapten according to claim 1 is used in combination with other active ingredients with similar pharmacological activity.

4. A medicament for preventing and/or treating depression symptom diseases, which is characterized by comprising the bergapten and/or pharmaceutical excipients in claim 1.

5. The pharmaceutical composition of claim 4, wherein the pharmaceutical excipients comprise solvents, propellants, solubilizers, solubilizing agents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, tonicity adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, permeation enhancers, pH adjusting agents, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, encapsulation agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, and release retardants.

6. The drug of claim 4, wherein the dosage form of the drug comprises injection, lyophilized powder for injection, controlled release injection, liposome injection, suspension, implant, suppository, capsule, tablet, pill and oral liquid.

7. The medicament of claim 4, further comprising a medicament carrier.

8. The drug of claim 7, wherein the drug carrier comprises microcapsules, microspheres, nanoparticles, and liposomes.

9. The medicament according to any one of claims 4 to 9, wherein the medicament further comprises the administration of a combination of other active ingredients with similar pharmacological activities.

10. Application of bergapten in preparing food for preventing and/or treating depression symptom diseases is provided.

Technical Field

The invention relates to application of bergapten in treating depression, and belongs to the technical field of medicines.

Background

Depression is a major mental disorder that afflicts human health worldwide, with about 3.5 million people diagnosed as depression worldwide, and the incidence of depression is rising year by year. The pathogenesis of depression is relatively complex, a clear and unified theoretical mechanism is not formed at present, and researches show that the inflammatory reaction generated by stress is an important way for inducing depression. At present, most of the clinically used antidepressant drugs have difficult side effects and slow effect, so that safe and effective new antidepressant drugs need to be further developed urgently.

In recent years, traditional Chinese medicine has achieved remarkable clinical efficacy in treating depression, and more reports focus on the research of natural medicine anti-depression. As a huge drug molecule treasure house, the traditional Chinese medicine has unique advantages in the aspects of side effects, drug resistance, dependence and the like.

Bergapten (BG) is a coumarin compound, widely exists in natural plants such as bergamot, notopterygium, radix glehniae, radix angelicae, fructus cnidii and the like, and researches show that the bergamot lactone has various pharmacological activities of resisting tumors, diminishing inflammation, improving insomnia, resisting allergy and the like. However, the effect and efficacy of bergapten in depression resistance are not reported in the invention.

Disclosure of Invention

The invention provides a product which can relieve inflammation induced by Lipopolysaccharide (LPS) and reduce depression symptoms caused by neuroinflammation and can be used for preventing and/or treating depression.

The invention provides an application of bergapten in preparing a medicament for preventing and/or treating depression symptom diseases, wherein the structure of the bergapten is shown as follows:

in one embodiment, the depressive symptom class of disease includes unidirectional depressive disorder, bidirectional depressive disorder, secondary depression, dysthymic disorder, postpartum depression, psychotic depression, seasonal affective disorder, and juvenile depression.

In one embodiment, bergapten as described above is used in combination with other active ingredients of similar pharmacological activity.

The invention also provides a medicine for preventing and/or treating depression symptom diseases, which comprises the bergapten and/or pharmaceutical auxiliary materials.

In one embodiment, the pharmaceutical excipients comprise solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, tonicity adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, permeation enhancers, pH adjusting agents, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickeners, encapsulation agents, humectants, absorbents, flocculants and deflocculants, filter aids, and release retardants.

In one embodiment, the dosage form of the medicament comprises injection, freeze-dried powder for injection, controlled release injection, liposome injection, suspension, implant, suppository, capsule, tablet, pill and oral liquid.

In one embodiment, the medicament may further comprise a medicament carrier.

In one embodiment, the drug carrier includes microcapsules, microspheres, nanoparticles, and liposomes.

In one embodiment, the above drugs may also be administered in combination with other active ingredients of similar pharmacological activity.

The invention also protects the application of the bergapten in preparing the food for preventing depression symptom diseases.

Has the advantages that:

(1) the invention discloses a new application of bergapten in preparing a medicine for treating and/or preventing depression symptom diseases. According to the invention, the results of animal efficacy evaluation show that bergapten obviously reduces the immobility time of LPS-induced antidepressant mouse tail suspension test and forced swimming test, increases the percentage of sweet water preference and enhances the autonomous activity.

(2) According to the invention, the observation of mouse brain coronal section shows that bergapten can reverse the abnormal shape change of microglia caused by LPS, so that the shape of the microglia is recovered, and the quantity of the microglia and the astrocyte is obviously reduced.

(3) The bergapten can effectively inhibit the expression quantity of inflammatory factors IL-6, TNF-alpha and IL-1 beta induced by LPS on mRNA level and NF-kappa B and MAPK signal channels in microglia through in vitro BV2 cell verification. Namely, bergapten has good anti-inflammatory effect and good prevention and treatment effect on depression symptoms caused by neuroinflammation.

Drawings

The N.S appearing in the figures of the present invention indicate no significance, P <0.05, P <0.01, P < 0.001.

FIG. 1 shows: a bergapten action depression model mouse anxiety behavior result graph; FIG. 1A: a mouse moving path diagram in an open field test; FIG. 1B: the duration of time for a mouse to enter the central area in an open field test; FIG. 1C: total distance of movement of the mouse in the open field test; FIG. 1D: a mouse moving path diagram in the elevated plus maze test; FIG. 1E: the number of times of entering an open arm in the elevated plus maze test; FIG. 1F: open arm time was entered in the elevated plus maze test.

FIG. 2 shows a plot of the results of depressive-like behavior in bergapten-acting depressive model mice; FIG. 2A: immobility time of mice in tail suspension test; FIG. 2B: immobility time of mice in forced swim test; FIG. 2C: percentage of sugar water preference in the sugar water preference experiment.

FIG. 3 shows: a result graph of the shapes and the quantity of microglia in different brain areas of a model mouse with depressed bergapten effect; FIG. 3A: microglial cell morphology in hippocampal CA1 region; FIG. 3B: microglial cell morphology in hippocampal DG region; FIG. 3C: cortical microglial cell morphology; FIG. 3D: the number of microglia in the hippocampal CA1 region; FIG. 3E: the number of microglia in the hippocampal DG region; FIG. 3F: number of cortical microglia.

FIG. 4 shows: a result graph of the shapes and the quantity of astrocytes in different brain regions of a model mouse with depressed bergapten effect; FIG. 4A: astrocytic morphology in the hippocampal CA1 region; FIG. 4B: astrocytic morphology in hippocampal DG area; FIG. 4C: cortical astrocyte morphology; FIG. 4D: astrocyte number in the hippocampal CA1 region; FIG. 4E: number of astrocytes in the hippocampal DG region; FIG. 4F: number of cortical astrocytes.

FIG. 5 shows: graph of bergapten's action in LPS-stimulated BV2 cell inflammatory response; FIG. 5A: mRNA expression level of IL-6; FIG. 5B: mRNA expression level of IL-1 beta; FIG. 5C: mRNA expression level of TNF-alpha.

FIG. 6 shows: the effect of bergapten on NF-kB and MAPK signal paths of BV2 cells is shown in the figure; FIG. 6A: NF- κ B signal path; FIG. 6B: MAPK signaling pathway.

Detailed Description

The present invention will now be described in further detail with reference to specific embodiments thereof, which are given by way of illustration only and are not intended to limit the scope of the invention. Those skilled in the art can, in light of the present disclosure, modify the present invention without in any way constituting a limitation thereof.

Bergapten, referred to in the examples below, was purchased from Designt Bio Inc., LPS was purchased from Sigma, mice were purchased from Wintonlifa laboratory animals Co., Ltd, Beijing, and microglia (BV2 cells) were purchased from ATCC.

Example 1 animal feeding and grouping

(1) Laboratory animal

40 male C57BL/6J mice of 6 weeks of age were purchased from Wintonlifa laboratory animals Co., Ltd, Beijing, and the average body weight was between 21 and 25 g. Mice were housed in clean or SPF animal rooms at room temperature of 23 ℃ for 12 hours day and night, acclimatized for one week before the start of the experiment, and water was freely available during the period. Mice were housed in groups until the start of the behavioural test.

All animal experiments were reviewed and approved by the central national university committee on biology and medical ethics.

(2) Pharmaceutical preparation and administration

1) Dissolving Bergapten (BG) in dimethyl sulfoxide (DMSO), and performing intraperitoneal injection according to 10mg/kg mouse

2) LPS was dissolved in physiological saline and administered by intraperitoneal injection to mice at 1 mg/kg.

(3) Establishment and grouping of experimental models

The mice after one week of pre-feeding were randomly divided into 4 groups of 10 animals, i.e., a normal group (n ═ 10), a single administration group (n ═ 10), an LPS group (n ═ 10), and a BG treatment group (n ═ 10), administered for 4 days, all animals were allowed to drink water freely, fed with normal daily diet, and after completion of the behavior test, the mice were anesthetized with 70mg/kg of sodium pentobarbital, and after cardiac perfusion, brain tissues were fixed.

TABLE 1 animal experiment grouping and processing method

Example 2 Effect of bergapten on Depression-like behavior in mice

1. Behavioural test

(1) Open field test

The open field test is a method for evaluating the autonomous behavior of a mouse in a new and different environment and exploring behavior and tensity. Mice were pre-acclimated to the experimental environment 2 hours prior to the experiment. During the experiment, mice were placed in a test chamber of size 32 × 32 cm. The test procedure was initiated, spontaneous activity of the mice was recorded over 5 minutes, and the total distance of activity and the time spent in the central zone were counted. Each mouse was tested and cleaned with 75% ethanol solution and placed into the next mouse after drying. The experimental results are shown in fig. 1A-C, the total distance of movement and the residence time in the central area of four groups of mice have no significant difference, and bergapten has no significant effect on the mouse anxiety behavior induced by LPS.

(2) Elevated plus maze test

The elevated plus maze test explores anxiety states by conflicting studies of the mouse's exploratory properties in a new and different environment and the fear of highly suspended open arms. Mice were pre-acclimated to the experimental environment 2 hours prior to the start of the experiment. During the test, the mice were gently placed in the central area towards the open arms, the test software was started, and the time of activity and the number of entries of the mice in the open arms were recorded within 5 minutes. Open arm entry times (two front melons must enter the arm), open arm dwell time, closed arm entry times, and closed arm dwell time. Each mouse was tested and cleaned with 75% ethanol solution and placed into the next mouse after drying. The experimental results are shown in fig. 1D-F, the times and residence times of the four groups of mice entering the open arms were not significantly different, and bergapten had no significant effect on the LPS-induced anxiety-like behavior of the mice.

(3) Tail overhang test

The tail suspension test is a classical method capable of rapidly evaluating the drug effects of antidepressant drugs, stimulants and sedatives. The experiment was taped down 2.5 cm from the end of the mouse tail and the head 15 cm from the ground. The experiment was carried out for 6 minutes and recorded with video. The initial 1 minute is the adaptation period of the mouse, and the total immobility time of the mouse (i.e., the mouse stopped struggling, the body was in a vertical suspended state, and the mouse was still) was counted within 5 minutes. The experimental result is shown in fig. 2A, the immobility time of the mice in the LPS group in the tail suspension experiment is remarkably increased compared with that of the normal group and the single administration group, while the immobility time of the mice in the BG group is reduced by 54.7% compared with that of the mice in the LPS group, and the injection of bergapten can remarkably reduce the immobility time of depressed mice, relieve the depressed state and remarkably improve the depression-like behavior of the mice induced by LPS.

(4) Forced swimming test

The forced swim test is widely used for antidepressant drug screening and basic research, and is one of the most commonly used experiments to evaluate depressive-type behavior in rodent animal models. In the experiment, a mouse is independently placed in a cylindrical glass beaker, the diameter of the beaker is 12 cm, the height of the beaker is 25 cm, the water temperature is 25 +/-2 ℃, the time is measured for 6 minutes after the mouse is placed in the water, and a camera records the specific details of the swimming of the mouse from the side surface of the beaker. The initial 2 minutes was the mouse acclimation period, and the cumulative swimming immobility time (meaning the mice stopped struggling in water, or showed a floating state with only minor limb movements to keep the head floating) was recorded within 4 minutes after each mouse experiment, the water in the glass beaker was replaced immediately. The experimental result is shown in fig. 2B, the immobility time of the depressed mice in the forced swimming experiment is significantly increased compared with the normal group and the single-dose group, while the immobility time of the BG group mice is reduced by 53.8% compared with the LPS group, so the injection of bergapten can significantly reduce the immobility time of the mice, and significantly improve the depression-like behavior of the mice induced by LPS.

(5) Sweet water preference experiment

Before testing, a bottle of 1% sucrose water and a bottle of plain drinking water were placed in the cages of all mice, and the mice were acclimated for 2 days. On the test day, one bottle of 1% sucrose water and 1 bottle of common drinking water were placed in all the mouse cages, the positions of the two bottles were randomized, the liquid consumption within 23 hours was observed, the consumption of water leaking from the water bottle was excluded, and the recording was performed.

The percentage of sugar water preference is equal to the sucrose water consumption/(sucrose water consumption + common drinking water consumption) × 100%.

The experimental results are shown in fig. 2C, the percentage of sugar preference of the mice in the LPS group is significantly reduced compared to the normal group and the single-dose group, the percentage of sugar preference of the mice in the BG group is significantly increased by 40.7% compared to the LPS group, and the mice in the BG group are basically restored to the normal sugar preference degree, which indicates that the bergapten relieves the anhedonia caused by depression.

2. Determination of biochemical index

(1) Sample collection and tissue fixation

Immediately after the behavioral testing was completed, the mice were anesthetized (pentobarbital; intraperitoneal injection; 70 mg/kg). Blood was collected by cardiac puncture, 5 mice per group were perfused with sterile normal saline and then the hippocampal tissue and cerebral cortex were separated on ice, the remaining 5 mice per group were perfused with 4% Paraformaldehyde (PFA) and then the brain tissue of the mice was taken on ice, fixed overnight in 4% paraformaldehyde and then dehydrated with 15-30% sucrose water gradient.

(2) Effect of bergapten on inflammatory factors

2×10⁵Each BV2 cell was plated in 12-well plates containing 1mL complete medium (DMEM, 10% fetal bovine serum, 1% diabody), 37 ℃ 5% CO₂After culturing in an incubator with saturated humidity for 24 hours, adding 20 mu g/ml bergapten for pretreatment for 30min, adding 1 mu g/ml LPS to stimulate for 6 hours and 12 hours respectively, washing with PBS, and collecting cells. Addition of T to cellsCells were lysed by rizol, total RNA was isolated, reverse transcribed into cDNA using a reverse transcription kit, and real-time fluorescent quantitative PCR was performed using the following primers.

TABLE 2 qPCR primer Table

As shown in FIG. 5, in vitro experiments, bergapten can significantly inhibit the expression level of mRNA of inflammatory factors IL-6, TNF-alpha and IL-1 beta induced by LPS, and has good anti-inflammatory effect.

(3) Effect of bergapten on inflammatory pathways

2×10⁵Each BV2 cell was plated in 12-well plates containing 1mL complete medium (DMEM, 10% fetal bovine serum, 1% diabody), 37 ℃ 5% CO₂After culturing in an incubator with saturated humidity for 24 hours, adding 20 mu g/ml bergapten for pre-protection for 30 minutes, adding 1 mu g/ml LPS to stimulate for 6 hours and 12 hours respectively, washing with PBS, extracting total protein and determining the protein concentration by using a BCA protein concentration detection kit. Proteins were separated by 10% SDS-PAGE gel electrophoresis (120V, 1h), and after NC membrane transfer (250mA, 2h), the NC membrane was blocked with 10% skim milk for 1 h. The NC membrane was then incubated overnight with different primary antibodies (p-I κ B, I κ B, iNOS, p-JNK, p-p38, p38, p-ERK, ERK), respectively, using β -tubulin as the internal control. After washing with TBST, incubating with horseradish peroxidase-labeled secondary antibody at room temperature for 1 hour, washing the membrane with TBST for 3 times, and finally developing with ECL developing solution.

The result is shown in figure 6, bergapten can effectively inhibit NF-kB and MAPK signal channels in microglia, and has good anti-inflammatory effect.

(4) Effect of bergapten on microglia and astrocytes

The brain coronary sections were prepared using the mice fixed with 4% paraformaldehyde in step (1), and 40 μm brain coronary sections were cut with a Leica CM1950 cryostat, followed by rinsing and blocking the sections with 0.3% Triton and 5% bovine serum at room temperature for 1.5 hours. The blocked sections were incubated with Iba-1(1:200, WAKO, Japan), GFAP (1:200, Millipore, Germany), and DAPI, respectively, overnight in the dark at 4 ℃ followed by incubation of fluorescent secondary antibodies for 1 hour at room temperature. Images were taken using a Nikon confocal fluorescence microscope.

The morphological changes of the microglia were observed, and the experimental results are shown in FIGS. 3A-C and 4A-B. Microglia of the LPS group mice were debranched and presented with an amoeba-like morphology, and the morphologies of microglia of the hippocampal CA1 region and DG region of the BG group mice were significantly restored, compared to the normal group and the single-dose group. It can be seen that bergapten can reverse abnormal morphological changes of microglia caused by LPS.

To quantify the staining of microglia and astrocytes in the hippocampal and cortical regions, the areas of Iba-1 and GFAP labeling were analyzed in proportion, and the results are shown in FIGS. 3D-F and FIGS. 4C-D. Compared with the normal group and the single administration group, the proportional area of the microglia and the astrocyte in the CA1 region, the DG region and the cerebral cortex of the hippocampus of the mice in the LPS group is obviously increased, and the proportional area of the mice in the BG group is reduced by 10 to 26 percent compared with that of the LPS group and is basically recovered to the normal level. Therefore, bergapten can reverse the abnormal morphological change of microglia caused by LPS, and the number of the microglia and the astrocyte is obviously reduced.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.