CN116370606A - Application of schistosome-derived peptide in preparation of medicament for treating depression - Google Patents
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Abstract
The invention provides application of schistosome source peptide in preparing a medicament for treating depression, and discovers the characteristic that schistosome source polypeptide SjDX5-271 induces the increase of the proportion of Treg cells, so that the invention can effectively improve the depression symptom of mice and is expected to solve the problem that the current depression cannot be treated in the future.
Description
Technical Field
The invention relates to the field of medical biology, mainly schistosome-derived polypeptides, which have the effect of inducing regulatory T cell increase, thereby improving host depression.
Background
The immune system includes innate immunity and adaptive immunity. Regulatory T cells (tregs) as a subset of adaptive immune cells can act on target cells by activating a series of immunosuppressive signals, thereby avoiding excessive activation of the immune system and homeostasis of the body. There is growing evidence that the course of central nervous system disorders is also accompanied by the activation of the adaptive immune system and plays an important role in the progression of neuropsychiatric disorders. Depression is a common mental disorder, and its etiology and pathology are not yet studied. In addition to the disturbance of the monoaminergic nervous system and the plastic impairment of synapses, there is also increasing evidence for the regulating action of the immune system.
The central nervous system directs the operation of the whole body, playing an irreplaceable role throughout the life cycle. Therefore, the protection of the body is particularly critical. In addition to the mechanical protection of the skull, the body itself has evolved a blood-brain barrier, a blood-cerebrospinal fluid barrier to protect against external infectious agents. In addition, some immune cells that have been present in the neural center since development, such as those that reside in the perivascular and choroid plexus, and microglial cells inherent in the brain, are of great importance in developing physiological functions of the central nervous system. Recent studies have shown that the dural sinus has a very rich number and variety of immune cells residing therein and can directly receive antigen information derived from the brain parenchyma and respond to the immune. Some of these cell populations are thought to play an important role in the progression of neuropsychiatric diseases, such as: anxiety disorders and multiple sclerosis. Thus, similar to peripheral blood tregs, tregs in the dural sinuses may develop in response to the occurrence of depression-like behaviors and exert unique effects that are distinct from peripheral blood tregs.
The invention discovers that the polypeptide SjDX5-271 can achieve the effect of relieving depression symptoms by inducing the clonal expansion of dural Treg. There is no report of depression treatment by meningo Treg.
Disclosure of Invention
The invention aims to develop a small molecule drug for treating depression through the function of inducing the increase of the Treg proportion of the polypeptide SjDX5-271.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
application of schistosome-derived polypeptide SjDX5-271 in preparing medicament for treating depression, wherein the amino acid sequence of schistosome-derived polypeptide SjDX5-271 is shown as SEQ ID NO. 1:
YKNLGGQQQSGSSQGQFPSGQMQQQQRPQQ。
further, the polypeptide SjDX5-271 is derived from eggs of Schistosoma japonicum.
Furthermore, the polypeptide SjDX5-271 is obtained by cell disruption, separation and purification of schistosoma japonicum eggs, or artificial expression by a genetic engineering method, or a chemical synthesis method.
The aforementioned polypeptide can induce the increase of Treg cells.
As a preferred technical scheme of the application, the medicine can comprise a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is selected from one or more of a filling agent, a wetting agent, a binding agent, a disintegrating agent and a lubricant.
As a preferred technical scheme of the present application, the above pharmaceutical dosage forms include oral preparations, injection preparations, transdermal administration preparations or mucosal administration preparations.
As a preferred technical scheme of the application, the pharmaceutical dosage forms comprise drops, oral liquid, tablets, capsules, granules, electuaries, films, gels, powders, emulsions, self-emulsifying preparations, dripping pills, suppositories, aerosols, sprays, powder mists, patches, plasters, solutions, ointments or creams and the like.
Further, the polypeptide is administered by injection.
The third object of the invention is to provide an application of the schistosome-derived polypeptide SjDX5-271 in preparing a reagent for inducing Treg cell increase in dura mater.
Advantageous effects
The application of the schistosome-derived polypeptide in treating depression has the following beneficial effects:
(1) The schistosome derived polypeptide SjDX5-271 may be obtained through cell disruption, separation and purification of schistosoma japonicum ovum, chemical synthesis, artificial expression via genetic engineering process, etc.
(2) Use of polypeptide SjDX5-271 for inducing Treg cells.
(3) Application of polypeptide SjDX5-271 in preparing depression.
In conclusion, the schistosome-derived peptide prepared by the invention can induce Treg cell increase and relieve the depression symptom of mice.
Drawings
FIG. 1 example 1 schistosome derived polypeptide SjDX5-271 induces in vitro an increase in the proportion of mouse spleen mononuclear cell Treg cells, while SjDX5-271 has a concentration dependence on the increase in Treg induction. FIG. 1A is a flow chart representative of the proportion of SjDX5-271 stimulated mice spleen mononuclear cell tregs, and FIG. 1B is a quantification of the proportion of SjDX5-271 stimulated mice spleen mononuclear cell tregs.
FIG. 2 example 2 expression level of the schistosome polypeptide SjDX5-271 in the culture supernatant IL-10 of mouse mononuclear cells.
Fig. 3 example 3 fig. 3A shows the results of the social experiment (social interaction) behavioural experiment.
Fig. 3B shows the results of tail-hanging for detecting emotional despair behavior.
Fig. 3C results of sugar water preference experiments for detecting emotional despair behavior.
FIGS. 3D-G show the results of open field experiments (open filtered) for detecting the level of depression in mice.
FIG. 4 example 4 ratio detection of mouse dural Treg cells induced by intraperitoneal injection of the schistosome polypeptide SjDX5-271.
FIG. 5 example 5 shows that the use of immunofluorescent staining assay demonstrates that the intraperitoneal injection of the schistosome polypeptide SjDX5-271 can induce an increase in the proportion of mouse dural Treg cells. Fig. 5A is the expression of mouse dura FOXP3, and fig. 5B is the quantification of positive proportion of mouse dura FOXP 3.
FIG. 6 example 6 mice were induced to have increased dural Treg by injection of SjDX5-271 into the cisterna magna, and observations were made of non-invasive memory cognition and social behavior of the cisterna magna administration. Fig. 6A is a behavioural experiment Y maze, a three-box social experiment and a new object experiment. Fig. 6B is a test of the proportion of mouse dural Treg cells. FIG. 6C shows the immunofluorescence detection of FOXP3 expression in mouse dura.
FIG. 7 example 7 mouse dural Treg induced by injection of SjDX5-271 into the medullary pool, wherein FIG. 7A is a flow-through representation of the ratio of the induction of mouse dural Treg cells by dural injected schistosome polypeptide SjDX5-271. Fig. 7B is a quantized graph of the streaming result.
FIG. 8 example 8 shows that the mouse dural Treg cell fraction can be induced to increase by immunofluorescent staining of the dural injected schistosome polypeptide SjDX5-271. Fig. 8A is the expression of mouse dura FOXP3, and fig. 8B is the quantification of positive proportion of mouse dura FOXP 3.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the invention in any way.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The therapeutic effects of the application of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.
The schistosoma japonicum according to the invention, wherein the literature name is schistosoma japonicum, law Ding Xueming is Schistosoma japonicum Katsurada,1904.
EXAMPLE 1 obtaining of peptide molecules of insect origin
The invention separates the polypeptide mixture of schistosoma japonicum eggs by high performance liquid chromatography in the early stage, and carries out mass spectrum analysis, and selects one polypeptide peptide segment from the polypeptide peptide segments for research, which is named as SjDX5-271.
The specific sequence is as follows:
YKNLGGQQQSGSSQGQFPSGQMQQQQRPQQ; molecular formula C 137 H 215 N 47 O 49 S, average molecular weight 3336.5g/mol, theoretical isoelectric point pI 10.39.
The present invention provides the above amino acid sequences, and the peptide chains in this example and the following examples are obtained by chemical synthesis by the division of biological engineering (Shanghai) Co., ltd. With a purity of > 95% unless otherwise specified.
The expression of Treg cells after the stimulation of mouse mononuclear cells by SjDX5-271 is detected by using a flow cytometer.
The experimental method is as follows:
example 1 ova-derived polypeptide SjDX5-271 induces Treg cytosis in vitro
Spleen mononuclear cells of mice were isolated and cell count was 1 x 10 6 After inoculation, the cells were incubated with SjDX5-271 at concentrations of 0, 0.01, 0.1, 1, 10 and 100. Mu.g/ml for 72h, and CD4 was detected + CD25 + FOXP3 + Cell ratio, CD4 + CD25 + FOXP3 + The cells are Treg cells.
The results showed that the proportion of Treg cells increased significantly after stimulation with SjDX5-271 and was concentration dependent (fig. 1A-B).
Example 2 in vitro IL-10 cytokine increase induced by the ovum-derived polypeptide SjDX5-271
Spleen mononuclear cells of mice were isolated and cell count was 1 x 10 6 After inoculation, the cells were incubated with SjDX5-271 at a concentration of 10. Mu.g/ml for 72 hours, and the expression of IL-10 cytokines was examined.
The results showed that SjDX5-271 induced increased expression of IL-10 cytokine (FIG. 2).
Example 3 intraperitoneal injection of polypeptide SjDX5-271 for alleviating depression symptoms in mice
Male C57BL/6 mice are used as study subjects and randomly divided into a blank control group and a CSDS depression model induction group; the model group of mice is 2 groups, namely a physiological saline injection group (saline) and a SjDX5-271 injection group, and 15 mice in each group. Mice were given an intraperitoneal injection of each group of the corresponding drug at a dose of 11 μg/d beginning on day 4 of molding. And performing depression behavioural detection after modeling, detecting the proportion of Treg cells at the dura mater of the mouse, and detecting the expression of FOXP3 at the dura mater of the mouse through experimental immunofluorescence.
The results showed that intraperitoneal injection of SjDX5-271 was effective in alleviating the symptoms of depression in mice (FIGS. 3A-F).
In fig. 3A, the tested mice and strange age-matched ICR mice are placed in a detection box, and the social interaction time of the tested mice is recorded, so that the result shows that the absolute social interaction time of the mice can be remarkably recovered after the mice are treated by SjDX5-271.
Fig. 3B shows the results of tail-hanging for detecting emotional despair behavior. Wherein, the tested mice were suspended upside down in the experimental place, and the tail suspension immobility time of the mice within 6 minutes and the last 5 minutes was monitored by detection software. The results show that the immobility time of the tail suspension experiment can be remarkably reduced after SjDX5-271 treatment.
Fig. 3C results of sugar water preference experiments for detecting emotional despair behavior. The experimental mice continuously drink normal water and 1% sucrose water for 24 hours in a molding cage, and the drinking condition and the proportion of the two types of water are recorded respectively. The results show that the sugar water preference rate can be remarkably recovered after treatment by SjDX5-271.
Fig. 3D to G show results of open field experiments (open field), wherein fig. 3D is a shuttle index of the open field central area, fig. 3E is a movement time index of the open field central area, fig. 3F is a total movement distance index of the open field, and fig. 3G is an average movement speed index of the open field for detecting a depression level of a mouse. Depressed mice will exhibit lower central area locomotion times and shuttling times due to the darkened nature of the mice. The result shows that the movement time and the shuttle times of the central area of the open field can be obviously recovered after the SjDX5-271 is treated, and the total movement distance and the average movement speed of the experimental mice in the open field experiment are not influenced. EXAMPLE 4 intraperitoneal injection of polypeptide SjDX5-271 induces an increase in the proportion of mouse dural Treg
As in example 3, male C57BL/6 mice were randomly divided into a placebo group and a CSDS depression model-induced group; wherein, the CSDS model group mice are divided into 2 groups, namely a physiological saline control group (sample) and a SjDX5-271 injection group, and 15 mice in each group are respectively arranged. Mice were given intraperitoneal injections of each group of the corresponding drug starting on day 4 of molding to the end of molding on day 10 (11 μg, i.p., qd). The mice are anesthetized and obtained immediately after three days of behavioral detection, and fresh dura mater tissues are separated and subjected to enzymolysis to obtain single-cell suspension. Detection of CD4 on-press using flow-type antibody-labeled Treg cells + CD25 + FOXP3 + Is selected from the group consisting of Treg cells.
The results showed that intraperitoneal injection of SjDX5-271 was effective in inducing an increase in the number of mouse dural Tregs (FIG. 4).
Example 5 intraperitoneal injection of polypeptide SjDX5-271 induces expression of mouse dura Foxp 3.
As in example 3, male C57BL/6 mice were randomly divided into a placebo group and a CSDS depression model-induced group; wherein, the CSDS model group mice are divided into 2 groups, namely a physiological saline control group (sample) and a SjDX5-271 injection group, and 15 mice in each group are respectively arranged. Mice were given intraperitoneal injections of each group of the corresponding drug starting on day 4 of molding to the end of molding on day 10 (11 μg, i.p., qd). Immediately after three days of behavioral testing, mice were anesthetized for sampling, intact cranium and meninges were separated and tissue fixed in 4% paraformaldehyde. After 24h, the membranes were removed and intact under a microscope using surgical forceps. After removal, the slide was rinsed for 5min at room temperature with PBS and 2 changes of solution, and attached to a clean slide. After the surface PBS is removed, 3% hydrogen peroxide is added dropwise, the solution is treated at room temperature for 15min and then washed away, the PBS is rinsed three times again, after the surface PBS is removed, a blocking solution is added dropwise, and the solution is treated at room temperature for 1.5h. And diluting the corresponding antibody by using an antibody diluent, replacing the blocking solution after the blocking is finished, and placing the mixture in a refrigerator at 4 ℃ for incubation for 24 hours. And taking out the mixture after the incubation is finished, rinsing the mixture for 3 times at room temperature by using PBS, dripping the secondary antibody corresponding to the source, and continuing to incubate at room temperature for 1.5h. After the incubation, the PBS rinse was continued 3 times for 5min each. The dye (1:100) was prepared using a multiple dye reaction solution. The brain pieces were treated at 8. Mu.L/brain piece for 1min, and at the end, the reaction was stopped by immersing in PBS. After completion of the staining, 8. Mu.L of antibody eluent was added dropwise to the brain slice, followed by elution in a water bath at 37℃for 0.5h. After the completion of the second antibody staining, the second antibody was incubated and stained in PBS for 10min for 3 times, and then the second antibody was stained until the staining was completed, and nuclear dye hochest (1:5000) diluted with deionized water was added dropwise, and after the brain pieces were treated at room temperature for 10min, 10. Mu.L of each brain piece was subjected to sealing. And (5) shooting with a Zeiss confocal fluorescence microscope after airing.
The results showed that the intraperitoneally injected polypeptide SjDX5-271 can significantly increase the dural foxp3+ cell number (fig. 5A).
Example 6 injection of SjDX5-271 via the cisterna magna does not affect short term memory and social tendencies
The experimental mice were anesthetized and placed in a 37 ℃ constant temperature operating table and the head was fixed and stereotactic in an injector. Raise the head and fully expose the neck skin. The neck hair of the mice was cut off with surgical scissors and sterilized with iodophor. The neck skin was cut about 7 mm and muscle groups were exposed, and three layers of muscles were gently scraped sequentially using surgical forceps while avoiding touching the veins as much as possible until the adventitia of the cerebellum medullary pool was exposed to the field of view. The microinjection needle head and the capillary needle are fixed and 2ul (5 mug) of SjDX5-271 is sucked, the same volume of physiological saline is injected into the control group, and the direction of needle insertion is adjusted to be a horizontal plane. The horizontal angle is kept to puncture into the medullary canal of the cerebellum, the needle insertion is stopped immediately when the capillary is broken through, the injection speed of the antibody is adjusted to be 1 mug/min, and the needle is kept for 5min after the antibody injection is finished. And then the needle is withdrawn, the mice are sewed and observed at a constant temperature table, and the mice are put back into a feeder cage for recovery after awakening. Detecting Y maze after 48 hours, three-compartment social and new object identification behavioural detection
The results show that injection of SjDX5-271 into the cisterna magna did not affect the short-term memory and social function of the mice.
Fig. 6A is a behavioural experimental Y maze test. Wherein i is the movement time of the tested mice in the novel arm, ii is the total times of the tested mice entering the novel arm, and the result shows that the short-term memory of the mice is not damaged by injecting SjDX5-271 into the medullary canal of the cerebellum
Fig. 6B is a behavioral three-compartment social experimental test, where i is the relative social rate of the mice tested, and ii is the relative social novelty rate of the mice tested, showing that the injection of SjDX5-271 into the cisterna magna did not impair the social and social novelty of the mice.
Fig. 6C shows a behavioral new object recognition test, where i is a training phase of new object recognition in the test mice and ii is a test phase of new object recognition in the test mice, and the results show that the new object recognition ability of the mice is not impaired by injecting SjDX5-271 into the cisterna magna.
EXAMPLE 7 Induction of increased numbers of mouse dural Tregs by SjDX5-271 injection through the cisterna magna
The experimental mice were injected with 5. Mu.g of SjDX5-271 via the medullary canal of the cerebellum, and the control group was given an equal volume of physiological saline as in example 6. Performing behavioral detection after 48 hours of injection, immediately and anaesthetizing the materials, separating fresh dural tissues, and obtaining single-cell suspension after enzymolysis. Detection of CD4 on-press using flow-type antibody-labeled Treg cells + CD25 + CD127 low Is selected from the group consisting of Treg cells.
The results showed that SjDX5-271 injected via the cisterna magna could effectively induce an increase in the mouse dural Treg number (FIG. 7A), and FIG. 7B is a quantification.
Example 8 Induction of increased numbers of mouse dural Tregs by SjDX5-271 injection through the cisterna magna
The experimental mice were injected with 5. Mu.g of SjDX5-271 via the medullary canal of the cerebellum, and the control group was given an equal volume of physiological saline as in example 6. Behavioural examination was performed 48 hours after injection and immediately and anaesthetizes the material, separating intact cranium from meninges and tissue fixation in 4% paraformaldehyde. Fixed tissue was stained for FOXP3 immunofluorescence on dura mater as in example 5.
The results show that the polypeptide SjDX5-271 injected through the cisterna magna can significantly increase the dura mater FOXP3 + Cell number (fig. 8A), fig. 8B is quantification.
The schistosome-derived peptide prepared by the invention can be used for treating depression and has important clinical value.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (3)
1. The application of the schistosome source polypeptide SjDX5-271 in preparing a medicament for treating depression is provided, wherein the amino acid sequence of the schistosome source polypeptide SjDX5-271 is shown as SEQ ID NO. 1.
2. The use according to claim 1, wherein the schistosome-derived polypeptide SjDX5-271 is administered by injection.
3. Application of schistosome-derived polypeptide SjDX5-271 in preparing reagent for inducing Treg cell increase in dura mater.
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| CN109789171A (en) * | 2016-08-10 | 2019-05-21 | 波比奥泰克股份公司 | For treating the composition of major depressive disorder |
| CN112707959A (en) * | 2020-11-11 | 2021-04-27 | 南京医科大学 | Polypeptide, preparation method and application |
| WO2022094719A1 (en) * | 2020-11-06 | 2022-05-12 | Pacific Myco Biosciences Ltd. | A method of treating depression by immune modulation |
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| CN109789171A (en) * | 2016-08-10 | 2019-05-21 | 波比奥泰克股份公司 | For treating the composition of major depressive disorder |
| WO2022094719A1 (en) * | 2020-11-06 | 2022-05-12 | Pacific Myco Biosciences Ltd. | A method of treating depression by immune modulation |
| CN112707959A (en) * | 2020-11-11 | 2021-04-27 | 南京医科大学 | Polypeptide, preparation method and application |
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