Application of receptor tyrosine kinase-like orphan receptor-1 as target spot in preparation of medicine for treating depression

Abstract

The invention discloses application of receptor tyrosine kinase-like orphan receptor-1 as a target spot in preparation of a medicine for treating depression, and belongs to the technical field of biological medicine. The invention provides ROR1 as a new target for developing and treating depression. According to the invention, gene regulation and control viruses are respectively injected into island leaf cortex brain regions of mice, specific overexpression or knock-down of ROR1 in IC brain regions of the mice is carried out, and the depression behavior of the mice is evaluated by using behavioral technical means. Results show that specific overexpression of the IC brain region ROR1 causes depression behaviors of mice; on the contrary, the mouse depression phenotype caused by chronic unpredictable stress can be effectively improved by specifically knocking down the ROR1 in the IC brain region. The invention provides a new target for revealing new pathogenesis and treatment means of depression and developing a novel antidepressant drug, has potential clinical transformation application prospects, and can effectively improve the defects of short curative effect, large side effect and the like of the traditional antidepressant drug.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of receptor tyrosine kinase-like orphan receptor-1 as a target in the preparation of drugs for treating depression. Background Art

[0002] Depression is a common mental illness characterized by persistent symptoms, primarily low mood, decreased interest, slowed thinking, irritability, and intellectual disability, often accompanied by insomnia. Patients with severe depression may engage in suicidal behavior, severely impacting their work and personal lives and imposing a heavy financial burden. Currently, many medications used to treat depression are tricyclic antidepressants. However, all of these drugs must be taken for at least several weeks to manifest their antidepressant effects. Besides their slow onset, traditional antidepressants also suffer from short duration of action and significant side effects, making them less effective. Fewer than 50% of all patients with depression achieve complete remission with optimized treatment.

[0003] Currently, genes associated with the pathogenesis or treatment of depression include leptin and brain-derived neurotrophic factor. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a receptor tyrosine kinase. This protein was first isolated from the human neuroblastoma cell line SH-SY5Y in 1992. Studies have reported that ROR1 plays an important role in mouse embryonic development, regulating skeletal development, cell migration, and cell polarity, and is also involved in the cell signaling pathways of various cancer tumor cells. There are no reports on the development of drugs targeting ROR1 to treat depression. Therefore, there is an urgent need to provide a rapid, safe, and effective treatment for depression that targets ROR1, thereby effectively improving the shortcomings of traditional antidepressants, such as short efficacy and severe side effects. Summary of the Invention

[0004] The purpose of the present invention is to provide the use of receptor tyrosine kinase-like orphan receptor-1 as a target in the preparation of drugs for treating depression, so as to solve the problems existing in the above-mentioned prior art. The present invention finds that knocking down receptor tyrosine kinase-like orphan receptor-1 in the insular cortex (IC) can improve the depressive behavioral phenotype of mice, providing a new target for revealing new pathogenesis and treatment methods of depression and the development of new antidepressant drugs, and has potential clinical translation application prospects.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an application of receptor tyrosine kinase-like orphan receptor-1 as a target in the preparation of a drug for treating depression.

[0007] Preferably, the depression is depression caused by external stimuli.

[0008] The present invention also provides a use of a receptor tyrosine kinase-like orphan receptor-1 inhibitor in the preparation of a drug for treating depression.

[0009] Preferably, the depression is depression caused by external stimuli.

[0010] Preferably, the inhibitor is an agent that reduces the level and / or activity of receptor tyrosine kinase-like orphan receptor-1.

[0011] Preferably, the inhibitor is an agent that specifically knocks down receptor tyrosine kinase-like orphan receptor-1.

[0012] Preferably, the dosage form of the inhibitor is any one of injection, infusion, powder, tablet, capsule or ointment.

[0013] Preferably, the inhibitor is in the form of an injection.

[0014] The present invention discloses the following technical effects:

[0015] The present invention provides receptor tyrosine kinase-like orphan receptor-1 as a new target for the development of treatment for depression. The present invention specifically overexpresses or knocks down ROR1 in the IC brain region of mice by injecting gene regulatory viruses into the insular cortex of mice, and uses two behavioral techniques, sugar water preference and forced swimming, to evaluate the depressive behavior of mice. The results showed that the specific overexpression of ROR1 in the IC brain region led to a decrease in the ratio of mice drinking sugar water in the sucrose preference experiment and an increase in the immobility time in the forced swimming experiment, which manifested as depression behavior in mice; on the contrary, the specific knockdown of ROR1 in the IC brain region can effectively improve the depressive phenotype caused by chronic unpredictable stress. The present invention provides a new target for revealing new pathogenesis and treatment methods of depression and the development of new antidepressant drugs. It has potential prospects for clinical translation and application, and can effectively improve the shortcomings of traditional antidepressants such as short efficacy and large side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1Figure 1 shows the experimental statistical results of CUS-induced increased ROR1 protein expression in the insular cortex; A is the WB experimental result; B is the ROR1 expression statistical graph; data are expressed as mean ± SEM; compared with the control group, * indicates P < 0.05;

[0018] Figure 2 Figure 2 shows the statistical results of a depressive behavior experiment in mice specifically overexpressing ROR1 in the insular cortex; A represents sucrose preference; B represents forced swimming immobility time; data are expressed as mean ± SEM; * indicates P < 0.05 compared with the control group;

[0019] Figure 3 Statistical results of the depressive behavior experiment in CUS-treated and non-compression-treated insular cortex-specific ROR1 knockdown mice; A represents sucrose preference; B represents forced swimming immobility time; data are expressed as mean ± SEM; compared with the control group, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. DETAILED DESCRIPTION

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0022] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0023] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0024] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0025] The present invention uses transgenic mice CaMKIIα-Cre and ROR1 flox / flox Using viral microinjection, mice were injected with adeno-associated viruses (AAVs) HBAAV2 / 9-CAG-DIO-m-Ror1-3×flag-Null (manufactured by Shanghai Hanheng Biotechnology Co., Ltd.) and HBAAV2 / 9-CMV-CaMKII-Cre-GFP (purchased from Shanghai Hanheng Biotechnology Co., Ltd.), respectively, into the insular cortex. This resulted in specific overexpression or knockdown of ROR1 in the insular cortex (IC) of mice. Depressive behavior was assessed using behavioral tests such as sucrose preference and forced swimming. A control group of mice was injected with the corresponding empty vector virus (purchased from Shanghai Hanheng Biotechnology Co., Ltd.).

[0026] Virus preparation process:

[0027] 1. Preparation of adeno-associated virus vector.

[0028] 1. Select the corresponding vector according to your needs and design PCR primers for the target fragment.

[0029] 2. Select appropriate restriction endonucleases, digest the vector and recover the linearized vector using agarose gel.

[0030] 3. Perform PCR on the target fragment and recover the target fragment through agarose gel.

[0031] 4. Connect the linearized vector and the target fragment by homologous recombination or T4 ligation.

[0032] 5. Transform competent DH5a or stbl3, plate the bacterial solution and culture for 12-16 hours.

[0033] 6. Pick monoclonal colonies.

[0034] 7. Sequence the positive clones.

[0035] 8. Extract plasmids from samples that have been sequenced correctly.

[0036] 2. Molecular cloning experimental steps

[0037] 1. Determine the vector and target gene information.

[0038] 2. Vector enzyme digestion: add the vector enzyme digestion system, gently blow evenly, and place it in a 37°C water bath to react for 1-2 hours. After the reaction is completed, perform electrophoresis and recover the target fragment.

[0039] 3. Obtain the target fragment and amplify the target fragment by PCR.

[0040] 4. The target fragment was ligated to the vector. The ligation reaction solution was reacted at 50°C for 30 minutes, placed on ice for 5 minutes, and then transformed.

[0041] 5. For transformation, remove the DH5α competent cells from the -80°C freezer and immediately thaw on ice. After thawing, aliquot the competent cells and add the ligation product. Place on ice for 20-30 minutes. Heat shock the cells at 42°C for 90 seconds. Immediately place the cells on ice and incubate for 2-3 minutes. Incubate at 37°C with shaking for 45-60 minutes. Spread the bacterial solution onto a solid plate of the appropriate resistance, then incubate the plate upside down at 37°C in a 37°C incubator for 12-16 hours.

[0042] 6. PCR identification of bacterial liquid.

[0043] 7. Sequencing: Select two positive clones from the screening for sequencing and compare the sequencing results.

[0044] 3. Plasmid extraction.

[0045] After successful sequencing, the bacterial solution is amplified and the plasmid is extracted and purified according to requirements.

[0046] IV. Adeno-associated virus packaging and quality testing

[0047] Hanbio uses a three-plasmid adeno-associated virus system for virus packaging.

[0048] 5. Adeno-associated virus packaging, concentration and purification

[0049] AAV-293 cells were passaged into 100 mm dishes for transfection. After transfection, the cells were placed in an incubator at 37°C, 5% CO2, and 95% relative humidity. Transfection was performed when the cells reached approximately 80-90% confluence.

[0050] 6. Adeno-associated virus purification

[0051] Add 0.1 μL of Benonase per 1 mL of crude viral extract and incubate in a 37°C water bath for 1 hour to remove the cellular genome and residual plasmid DNA from the viral fluid. Centrifuge at 600 × g at 4°C for 10 minutes and collect the supernatant. Add 4 mL of the viral sample obtained from the column purification to an ultrafiltration tube and centrifuge at 1400 × g for 30 minutes to obtain approximately 1 mL of viral fluid. Collect the resulting purified virus and store at -80°C.

[0052] 7. Adeno-associated virus quality testing

[0053] Adeno-associated virus was tested for sterility, mycoplasma, and titer.

[0054] Example 1

[0055] 1. Method for establishing a chronic unpredictable stress (CUS) depression model mouse model

[0056] The mice were given two different stress stimuli at different times of the day, and the experiment lasted for 21 days.

[0057] Stress stimuli included: 2 hours of restraint; 24 hours of continuous light; 24 hours of wet box; 15 minutes of tail clamp; 10 minutes of intermittent foot shock and 30 minutes of high platform.

[0058] 2. CaMKIIα-Cre and ROR1 flox / flox Identification and breeding of mice

[0059] The mice strain required for the experiment were introduced and bred from Jackson Laboratory: ROR1 flox / flox (StockNo: 018353), CaMKIIα-Cre (StockNo: 005359).

[0060] The present invention uses adult male or female mice weighing 25±5g, housed in an environment with alternating 12h light and 12h dark cycles, with adequate water and food. The animal room temperature is maintained at 24±1°C and the humidity is maintained at 50-60%. The experimental design of the present invention adopts the principle of reasonable and optimal number to minimize animal suffering during the experimental operation. All experimental animals used are randomly divided into groups. The experimental mice required for the present invention are bred in the SPF animal room of this hospital and meet the standards of the hospital animal ethics committee.

[0061] 3. Immunoblotting to detect ROR1 expression in the insular cortex

[0062] C57BL / 6 mice were given CUS for 21 days, and the insular cortex brain tissue was removed. The target tissue was lysed with lysis buffer (NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, phenylmethylsulfonyl fluoride, and PhosSTOP Phosphatase Inhibitor Cocktail), followed by low-temperature high-speed centrifugation, removal of the precipitate, and BCA protein concentration determination.

[0063] The treated protein samples were subjected to SDS-PAGE gel electrophoresis, and the target proteins were separated according to their molecular size and transferred to PVDF membranes. The membranes were blocked with 5% skim milk powder at room temperature for 1 hour, incubated with ROR1 primary antibody on a shaker at 4°C overnight, washed with TBST buffer, incubated with goat anti-rabbit (goat anti-rabbit IRDye680LT) fluorescent secondary antibody at room temperature for 1 hour, washed with TBST buffer, and imaged and quantitatively analyzed using the Odyssey dual infrared fluorescence imaging system.

[0064] 4. Depressive Behavior Measurement

[0065] 4.1 Modeling Grouping

[0066] CaMKIIα-Cre mice were injected with the adeno-associated virus HBAAV2 / 9-CAG-DIO-m-Ror1-3×flag-Null into the insular cortex, creating mice that specifically overexpress ROR1. CaMKIIα-Cre mice were injected with an empty vector virus into the insular cortex, serving as the control group.

[0067] ROR1 flox / flox The adeno-associated virus HBAAV2 / 9-CMV-CaMKII-Cre-GFP was injected into the insular cortex of mice to create ROR1-specific knockout mice. flox / flox Mice injected with empty vector virus into the insular cortex served as the control group.

[0068] The ROR1 knockout mice and their control group were randomly divided into a CUS group and a non-stress group. The CUS group was given stress stimulation according to the above method, and the non-stress group maintained normal culture.

[0069] 4.2 Detection methods

[0070] Sucrose preference: The sucrose preference test is an experimental method to test the anhedonia in depressed mice.

[0071] One week before testing, place two bottles of water in the mouse cage, as far apart as possible. Four hours before testing, remove the mice's drinking water. During testing, place drinking water on one side and 1% (w / v) sucrose solution on the other. The percentage of sucrose solution consumed by the mice within 2 hours after lights out was measured.

[0072] Forced swimming: Before testing, mice were individually caged for 2-3 hours. The mice were placed in a transparent Plexiglas cylinder (25 cm high, 10 cm diameter) filled with 24°C water to a height of 15 cm. The test lasted 6 minutes, and the duration of immobility within the last 4 minutes was recorded.

[0073] The length of time mice remain immobile reflects their degree of depression; the longer the immobility time, the higher the degree of depression, and vice versa.

[0074] 5. Depressive behavior test results

[0075] like Figure 1 As shown in the figure, compared with the control C57BL / 6 mice, the expression of ROR1 protein in the insular cortex of the CUS group mice was significantly increased.

[0076] like Figure 2As shown, compared with the control group, the sucrose preference of mice with insular cortex-specific overexpression of ROR1 was significantly reduced and the forced swimming immobility time was significantly increased, indicating that overexpression of ROR1 led to depressive behavior in mice.

[0077] like Figure 3 As shown, in CUS-treated mice, the sucrose preference of mice with insular cortex-specific knockdown of ROR1 was significantly increased and the forced swimming immobility time was significantly reduced compared with the control group, indicating that knockdown of ROR1 effectively improved the depressive behavioral phenotype of mice caused by CUS.

[0078] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of a receptor tyrosine kinase-like orphan receptor-1 as a target in the preparation of a drug for treating depression.

2. The use according to claim 1, characterized in that The depression is depression caused by external stimulation.

3. Use of a receptor tyrosine kinase-like orphan receptor-1 inhibitor in the preparation of a drug for treating depression.

4. The use according to claim 3, characterized in that The depression is depression caused by external stimulation.

5. The use according to claim 3, characterized in that The inhibitor is an agent that reduces the level and / or activity of receptor tyrosine kinase-like orphan receptor-1.

6. The use according to claim 5, characterized in that The reagent is a reagent for specifically knocking down receptor tyrosine kinase-like orphan receptor-1.

7. The use according to claim 3, characterized in that The dosage form of the inhibitor is any one of injection, infusion, powder, tablet, capsule or ointment.

8. The use according to claim 7, characterized in that The dosage form of the inhibitor is injection.

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