Pharmaceutical composition based on neural stem cells and application of pharmaceutical composition in treatment of depression

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a medicine composition based on neural stem cells and application of the medicine composition to treatment of depression. The invention finds that compared with a blank group, the movement time of a C57 mouse depression model group in an open field central region is remarkably reduced (Plt; 0.05) of the substrate (1); the time of tail suspension immobility and forced swimming immobility is significantly increased (Plt; 0.05) of the method. Compared with the model group, the motion time of the stem cell treatment group in the central area of the open field is obviously increased (Plt; 0.05) of the substrate (1); compared with the model group, the tail suspension immobility and forced swimming quiescence time of the stem cell treatment group is obviously reduced (Plt; a pathological result shows that the nerve cells in the hippocampus CA1 region of the mouse in the model group are obviously necrotic, and the number of necrotic cells after NSCs treatment is obviously reduced. And the NSCs are combined with the melatonin, so that the treatment effect can be improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a drug composition based on neural stem cells and its application in the treatment of depression. Background Art

[0002] Depression is a common neurological disease worldwide, characterized by high incidence, complex etiology, and heavy social and economic burden. Although there are currently various antidepressant drugs and psychotherapy methods, these methods have problems such as limited efficacy, significant side effects, and drug resistance in some patients, and there is an urgent need to explore new treatment strategies to meet clinical needs.

[0003] Neural Stem Cells (NSCs) can not only differentiate into various neurons and glial cells, but also have multiple functions such as secreting neurotrophic factors, regulating inflammatory responses, and antioxidant stress. These characteristics make them show broad prospects in the treatment of neurological diseases. However, there are still many unresolved scientific problems and technical challenges regarding the therapeutic effect and mechanism of NSCs on depression. First, the exact mechanism of action of NSCs in depression models has not been fully elucidated, especially their roles in anti-inflammatory, antioxidant, and neuronal repair need to be further studied. Second, current research mainly focuses on the establishment and evaluation of single depression models, and the therapeutic effects and adaptability of NSCs under different stress conditions still need to be systematically explored. In addition, how to improve the survival rate, migration ability, and targeting of NSCs in vivo remains a key technical problem restricting their clinical application.

[0004] Therefore, it is necessary to develop an application of neural stem cells in the treatment of depression and a drug composition to improve its therapeutic effect. Summary of the Invention

[0005] The purpose of the first aspect of the present invention is to provide the application of the combined use of neural stem cells and melatonin in the preparation of products for the treatment of depression.

[0006] In some embodiments of the present invention, the neural stem cells are derived from umbilical cord blood stem cells and induced pluripotent stem cells.

[0007] In some embodiments of the present invention, the product includes drugs.

[0008] In some embodiments of the present invention, the drug includes pharmaceutically acceptable excipients.

[0009] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickening agents, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, carriers.

[0010] The above-mentioned pharmaceutically acceptable excipients are generally recognized for this purpose and serve as inactive ingredients of pharmaceutical agents. Compilations of pharmaceutically acceptable excipients can be found in reference books such as Handbook of Pharmaceutical Excipients (2nd Edition, edited by A. Wade and P. J. Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994); and the list of names of pharmaceutical excipients in the Pharmacopoeia of the People's Republic of China.

[0011] In some embodiments of the present invention, the dosage forms of the product include gastrointestinal dosage forms or parenteral dosage forms.

[0012] In some embodiments of the present invention, the gastrointestinal dosage forms include at least one of powders, tablets, granules, capsules, sustained-release formulations, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, syrups, drops, chewable tablets.

[0013] Furthermore, the gastrointestinal dosage forms include, but are not limited to, enteric-coated tablets, coated tablets, film-coated tablets, sugar-coated tablets, dispersible tablets, sucking tablets, chewable tablets, effervescent tablets, scored tablets, sustained-release and controlled-release formulations such as sustained-release tablets, sustained-release coated tablets, controlled-release tablets, orally disintegrating tablets, lozenges, oral patches, etc.

[0014] In some embodiments of the present invention, the parenteral dosage forms include at least one of injection dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, and cavity dosage forms.

[0015] Furthermore, the injection dosage forms include, but are not limited to, injection solutions, injection suspensions, intravenous drip injection solutions, sterile powders for injection, intravenous injection needles, aqueous injections, injection emulsions, powder injections, injections, sterile powder injections, freeze-dried powder injections, etc.

[0016] In some embodiments of the present invention, the subject of administration of the product is a mammal.

[0017] In some embodiments of the present invention, the mammal includes humans and mice.

[0018] An object of the second aspect of the present invention is to provide a pharmaceutical composition comprising neural stem cells and melatonin.

[0019] In some embodiments of the present invention, using mice as the subject, the usage ratio of the neural stem cells to melatonin is (1×10 5 ~1×10 8 ) cells / mouse : (10~50) mg / kg.

[0020] The beneficial effects of the present invention are as follows:

[0021] The present invention discovers that, compared with the blank group, the movement time of the C57 mouse depression model group in the central area of the open field is significantly reduced (P<0.05); the immobile time in the tail suspension test and the forced swimming test is significantly increased (P<0.05). Compared with the model group, the movement time of the stem cell treatment group in the central area of the open field is significantly increased (P<0.05); compared with the model group, the immobile time in the tail suspension test and the forced swimming test of the stem cell treatment group is significantly reduced (P<0.05). The pathological results show that the nerve cells in the CA1 region of the hippocampus of the model group mice are significantly necrotic, while the number of necrotic cells is significantly reduced after NSCs treatment. And the combination of NSCs and melatonin can improve the treatment effect. Description of the Drawings

[0022] The following further describes the present invention in conjunction with the drawings and embodiments, wherein:

[0023] Figure 1 It is the open field trajectory diagram of the depression model mice, where A: blank group; B: chronic depression group; C: acute 0.5 mg / kg depression group; D: acute 2.0 mg / kg depression group.

[0024] Figure 2 It is the open field test result of the depression model mice.

[0025] Figure 3 It is the tail suspension test result of the depression model mice.

[0026] Figure 4 It is the forced swimming test result of the depression model mice.

[0027] Figure 5 It is the schematic diagram of the treatment process.

[0028] Figure 6Open-field trajectory map of mice after NSCs treatment, where A: blank group; B: chronic depression NSCs treatment group; C: acute 0.5 mg / kg depression NSCs treatment group; D: acute 2.0 mg / kg depression NSCs treatment group.

[0029] Figure 7 Open-field test results of mice after NSCs and melatonin treatment.

[0030] Figure 8 Tail suspension test results of mice after NSCs and melatonin treatment.

[0031] Figure 9 Forced swimming test results of mice after NSCs and melatonin treatment.

[0032] Figure 10 Pathological staining results of the blank control group.

[0033] Figure 11 Pathological staining results of the acute (0.5 mg / kg LPS) NSCs treatment for depression group.

[0034] Figure 12 Pathological staining results of the acute (2 mg / kg LPS) NSCs treatment for depression group.

[0035] Figure 13 Pathological staining results of the chronic depression NSCs treatment group.

[0036] Figure 14 Pathological staining results of the acute (0.5 mg / kg LPS) depression group.

[0037] Figure 15 Pathological staining results of the chronic depression group.

[0038] Figure 16 Pathological staining results of the acute (2 mg / kg LPS) depression group.

[0039] Figure 17 Treatment comparison results of the chronic depression group, where A: chronic depression treatment group; B: results after one week of chronic depression NSCs treatment.

[0040] Figure 18 Statistical chart of the levels of hippocampal inflammatory factor IL-10 in mice of different groups.

[0041] Figure 19 Statistical chart of the levels of hippocampal inflammatory factor IL-1β in mice of different groups.

[0042] Figure 20 Statistical chart of the levels of hippocampal inflammatory factor TNF-α in mice of different groups.

[0043] Figure 21 Statistical chart of the level of oxidative factor GAH-Px in the hippocampus of mice in different groups.

[0044] Figure 22 Statistical chart of the level of oxidative factor T-SOD in the hippocampus of mice in different groups.

[0045] Figure 23 Statistical chart of the level of oxidative factor MDA in the hippocampus of mice in different groups.

[0046] Figure 24 Open field trajectory maps of different groups during melatonin treatment, where A: chronic depression model group; B: melatonin treatment group; C: NSCs + 10 mg / kg melatonin treatment group; D: NSCs + 50 mg / kg melatonin treatment group; E: blank control group.

[0047] Figure 25 Results of the open field experiment for the neural stem cell treatment group and the melatonin combined with neural stem cell treatment group.

[0048] Figure 26 Results of the tail suspension test for the neural stem cell treatment group and the melatonin combined with neural stem cell treatment group.

[0049] Figure 27 Results of the forced swimming test for the neural stem cell treatment group and the melatonin combined with neural stem cell treatment group. Detailed implementation manners

[0050] The concept and technical effects of the present invention will be clearly and completely described below in combination with embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0051] Example 1 Construction of a mouse model of depression

[0052] In this example, two mouse models, namely a chronic unpredictable mild stress depression model and an acute LPS drug-induced depression model, were constructed as follows:

[0053] 1. Experimental materials

[0054] 58 eight-week-old C57 mice were divided into seven groups, with 8 mice in each group. They were divided into: blank group; chronic depression group; chronic depression stem cell treatment group; acute (0.5 mg / kg LPS) depression group; acute (0.5 mg / kg LPS) depression stem cell treatment group; acute (2.0 mg / kg LPS) depression group; acute (2.0 mg / kg LPS) depression stem cell treatment group.

[0055] 2. Experimental methods

[0056] Chronic unpredictable mild stress depression model: cage tilting; fasting for 24 h; water deprivation for 24 h; tail clamping for 2 min; wet bedding for 24 h; overnight lighting; swimming in 4°C ice water.

[0057] Acute LPS drug-induced depression model: injecting 0.5 mg / kg LPS for two weeks; injecting 2.0 mg / kg LPS for one week.

[0058] 3. Detection criteria

[0059] 1) Open field test: Place C57 mice in an open 50 cm × 50 cm × 50 cm square space and observe their behavioral performances within a certain period of time, including their movement paths in the field, the staying times in the central and marginal areas of the field, and behaviors such as rearing and self-cleaning. A longer staying time of animals in the central area is usually interpreted as showing a lower anxiety level, while a longer staying time in the marginal area (referred to as "wall-hugging behavior") may indicate a higher anxiety level. Compare the staying times in the central area of the depression group and the treatment group with those of the blank control group respectively to see if there are significant differences.

[0060] 2) Tail suspension test: Fix the tail of C57 mice so that their heads hang downwards. The animals struggle in this environment, attempting to get out of this predicament. After failing to get out after efforts, they show intermittent immobility, indicating a state of "behavioral despair". The experimental animals struggle desperately to escape in this environment but are unable to do so, thus providing an inescapable oppressive environment. After a period of the experiment, record a series of parameters during the process of the animals in this environment showing the immobile state of despair. This typical "immobile state" shown by the animals reflects a state called "behavioral despair state". Compare the times of the behavioral despair state of the depression group and the treatment group with those of the blank control group respectively to see if there are significant differences.

[0061] 3) Forced swimming test: Put C57 mice into a container filled with water, and they must keep swimming to avoid drowning. Usually, observe the behaviors, swimming times, and immobile times of the mice in the water to study their behavioral and physiological changes under stress conditions. Compare the immobile times of the depression group and the treatment group with those of the blank control group respectively to see if there are significant differences.

[0062] 4. Modeling results

[0063] As shown in the open field test such as Figure 1 、 2 shown, the results of the tail suspension test and the forced swimming test are respectively as shown in Figure 3 、4 As shown, the summary data is shown in Table 1.

[0064] Table 1 Behavioral evaluation of depression indicators before treatment (compared with the blank group; x±s, n = 8)

[0065]

[0066] Note: Compared with the blank control group, *P<0.05; #P>0.05.

[0067] In this study, the therapeutic effect of NSCs on the C57 mouse depression model was evaluated through open field experiments, tail suspension experiments, and forced swimming experiments. The results showed that compared with the blank group, the movement time and total moving distance of the model group mice in the central area of the open field were significantly reduced (P<0.05), indicating a decrease in activity ability. In addition, the immobility time of the model group mice in the tail suspension experiment and the stationary time in the forced swimming experiment were both significantly increased (P<0.05), further indicating that they showed depressive-like behavioral characteristics, and the depression mouse model was successfully constructed.

[0068] Example 2 Effects of neural stem cells on the behavioral phenotypes of the C57 mouse depression model

[0069] In this example, three groups of mice with depression models constructed in Example 1 were taken, and one group was used as the neural stem cell treatment group: chronic depression treatment group; acute (0.5 mg / kg) depression treatment group; acute (2.0 mg / kg) depression treatment group. Neural stem cells (0.2 - 0.4 ml / mouse) were continuously injected into the tail vein for three days. After the mice rested for 5 - 7 days, behavioral tests were performed (the same as in Example 1), and compared with the blank group and the previous depression group to observe whether neural stem cells had a therapeutic effect on depression. In addition, melatonin was added to the chronic depression model for observation, and it was divided into melatonin treatment group (10 mg / kg chronic depression group), NSCs + 10 mg / kg melatonin treatment group (chronic depression group), and NSCs + 50 mg / kg melatonin treatment group (chronic depression group).

[0070] Cultivation method of neural stem cells: Neural stem cells were cultured and passaged normally. When the cells reached confluence (80% - 90% density) in a T75 culture dish (the number of cells was approximately 2×10 7 ), the cells were taken out and placed in a centrifuge tube, and dispersed with 1 mL of cold PBS to make an injection solution.

[0071] The treatment experimental process is as Figure 5 shown.

[0072] The treatment results are as Figures 6 - 9 shown.

[0073] Table 2 Behavioral evaluation of depression indicators after treatment (compared with the blank group; x±s, n = 8)

[0074]

[0075]

[0076] Note: Compared with the blank control group, *P < 0.05; #P > 0.05.

[0077] In the stem cell treatment group, these behavioral indices were significantly improved: the movement time and total moving distance in the center area of the open field increased (P < 0.05), and the immobile time in the tail suspension test and the immobile time in the forced swimming test decreased (P < 0.05). These results indicate that NSCs can effectively alleviate the depressive symptoms in the C57 mouse model of depression.

[0078] Example 3 Anatomopathological Results of the Hippocampus in Depressed Mice

[0079] Mice in the depression model group, the neural stem cell treatment model group, and the blank control group were dissected, and the brains were taken for pathological examination to observe the pathological changes in the hippocampus of the brain, so as to detect whether neural stem cells have a therapeutic effect.

[0080] Histopathological examination showed that in the model group (see Figure 14 , Figure 15 , Figure 16 ), a large number of nerve cell necroses (dark purple circular structures, with nuclear atrophy) appeared in the CA1 region of the hippocampus of mice, while only sporadic injuries were seen in the blank group (see Figure 10 ). After NSCs treatment, the number of necrotic cells in the CA1 region of the hippocampus in the acute LPS model groups (0.5 mg / kg and 2.0 mg / kg) decreased significantly (see Figure 11 , Figure 12 , Figure 13 ), indicating that NSCs can repair neuron damage caused by inflammation or oxidative stress.

[0081] It can be seen from Figure 17 that individual nerve stem cells in the CA1 region of the hippocampus in the chronic depression treatment group showed necrosis (+), showing a light purple ring. After one week of normal feeding after treatment in the depression treatment group, a large number of the injected nerve stem cells in the CA1 region of the hippocampus showed necrosis (+++), showing a dark purple ring, and the nuclei of the nerve stem cells shrank and disappeared. By comparison, it can be known that the treatment duration of nerve stem cells is about one week.

[0082] Example 4 Results of Changes in the Levels of Inflammatory Factors in the Hippocampus of Mice

[0083] The levels of inflammatory factors in mice of each group were detected, and the results are shown in Figures 18 - 23 , Tables 3 and 4.

[0084] There were significant differences in the levels of inflammatory factors among the three groups of depressed mice and the normal group, indicating that the mice were depressed. There were no significant differences in the levels of inflammatory factors among the three groups of treated depressed mice and the normal group, indicating that the depression in the mice had been cured.

[0085] Neural stem cells were injected via the tail vein and reached the brain through the blood circulation to supplement neural stem cells in the damaged hippocampus, replenishing and repairing the reduction of neural stem cells in the hippocampus caused by inflammation or oxidation. Neural stem cells have the ability to secrete a variety of neuroprotective factors, increase new GABAergic interneurons, and maintain hippocampal neurogenesis. From the data of inflammatory factor levels, it can be seen that neural stem cells can effectively inhibit inflammatory factors and suppress the hyperactivity of the hypothalamic-pituitary-adrenal axis-glutamate-N-methyl-D-aspartate receptor-nitric oxide pathway. Neural stem cells can stimulate the proliferation of endogenous neural stem cells and promote the differentiation of neural stem cells into neurons, and can generate oligodendrocytes to repair demyelination. Based on the close correlation between hippocampal neurogenesis and cognition and emotion, the increased hippocampal neurogenesis can re-promote the recovery of the mice's vitality.

[0086] In the hippocampus of the depression model group, the inflammatory factors IL-10, IL-1β, TNF-α and the oxidative factors GAH-Px and T-SOD were significantly increased (chronic depression group: IL-1β 0.35±0.01 pg / ml, TNF-α 0.37±0.01 pg / ml, GAH-Px 0.28±0.01 pg / ml, T-SOD 0.30±0.01 pg / ml; blank group: IL-1β 0.30±0.01 pg / ml, TNF-α 0.26±0.01 pg / ml, GAH-Px 0.32±0.01 pg / ml, T-SOD 0.35±0.01 pg / ml), and the anti-inflammatory factor IL-10 was decreased (chronic depression group: 0.33±0.01 pg / ml vs. blank group: 0.41±0.01 pg / ml; P<0.05). After NSCs treatment, the levels of inflammatory factors and antioxidant factors were close to normal (acute low-dose LPS treatment group: IL-1β 0.35±0.01 pg / ml, TNF-α 0.31±0.01 pg / ml; P>0.05), indicating that it plays a role by inhibiting neuroinflammation and antioxidation (Tables 3 and 4, Figures 18 - 23 )

[0087] The sources of the cytokine detection kits are as follows:

[0088] IL-10 (Interleukin-10), manufacturer: Abcam, catalog number: ab46034.

[0089] IL-1β (Interleukin-1β), manufacturer: R&D Systems, catalog number: MLB00C.

[0090] TNF-α (Tumor Necrosis Factor-α), Manufacturer: R&D Systems, Catalog Number: MTA00B.

[0091] GSH-Px, Manufacturer: Nanjing Jiancheng Bioengineering Institute, Catalog Number: A005-1-2.

[0092] T-SOD, Manufacturer: Sigma-Aldrich, Catalog Number: 19160.

[0093] MDA, Manufacturer: Sigma-Aldrich, Catalog Number: MAK085.

[0094] Table 3 Inflammatory Factor Levels in the Hippocampus of Depressed Mice (Compared with the Normal Group; x±s, n = 3 - 5)

[0095]

[0096] Note: Compared with the blank control group, *P < 0.05; #P > 0.05.

[0097] Table 4 Oxidation Factor Levels in the Hippocampus of Depressed Mice (Compared with the Normal Group; x±s, n = 3 - 5)

[0098]

[0099]

[0100] Note: Compared with the blank control group, *P < 0.05; #P > 0.05.

[0101] Example 5 Changes in the Therapeutic Effect of Neural Stem Cells Combined with Melatonin

[0102] According to Table 5, Figures 24 - 27 Based on the behavioral data analysis, in the tail suspension test and forced swimming test of the group treated with neural stem cells combined with melatonin, the immobility time was lower than that of the mice treated with neural stem cells or melatonin alone for depression, and the movement time in the central area of the open field test was also lower. It can be seen that the treatment with melatonin combined with neural stem cells is better than the treatment with neural stem cells or melatonin alone.

[0103] Table 5 Behavioral Data of the Neural Stem Cell Treatment Group and the Melatonin Combined with Neural Stem Cell Treatment Group (x±s, n = 5)

[0104]

[0105] Note: Compared with the chronic depression model group, *P < 0.05; compared with the blank control group, #P > 0.05.

[0106] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the scope of knowledge possessed by those of ordinary skill in the relevant art. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. The application of neural stem cells and melatonin in combination in the preparation of products for the treatment of depression.

2. The use according to claim 1, characterized in that: The neural stem cells are derived from umbilical cord blood stem cells and induced pluripotent stem cells.

3. The use according to claim 1, characterized in that: The products include pharmaceuticals.

4. The use according to claim 3, characterized in that: The drug includes pharmaceutically acceptable excipients.

5. The use according to claim 4, characterized in that: The pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

6. The use according to claim 3, characterized in that: The dosage form of the product includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration.

7. The use according to claim 6, characterized in that: The gastrointestinal dosage form includes at least one of powders, tablets, granules, capsules, sustained-release agents, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets.

8. The use according to claim 6, characterized in that: The non-intestinal administration dosage form includes at least one of an injection dosage form, a respiratory tract administration dosage form, a skin administration dosage form, a mucosal administration dosage form, and a cavity administration dosage form.

9. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises neural stem cells and melatonin.

10. The pharmaceutical composition according to claim 9, characterized in that: Taking mice as the subjects, the ratio of the neural stem cells to melatonin is (1×10 5 ~1×10 8 ) piece / piece: (10~50)mg / kg.

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