Application of NR3A as protein target in screening or preparing medicine for preventing and / or treating depression-like behavior

Abstract

The invention belongs to the field of neurobiology and drug research and development, and discloses application of NR3A as a protein target in screening or preparing drugs for preventing and / or treating depression-like behaviors. According to the invention, the expression of NR3A in hippocampus is enhanced by stereotactic injection of adeno-associated virus containing Grin3a gene. A chronic constraint stress (CRS) is adopted to induce a depressive mouse, and a sucrose preference test (SPT), a forced swimming test (FST) and an open field test (OFT) are adopted for identification. The expression of proteins such as NR3A, calpain 2 (Calpain2), neuron nitric oxide synthase (nNOS), 3-nitrotyrosine (3-NT), an NLRP3 inflammation complex and the like is detected by WesternBlot; and performing DHE staining to detect reactive oxygen species (ROS). By enhancing hippocampus NR3A expression, depression-like behaviors induced by CRS can be improved, signal cascade activated by calcium is inhibited, ROS generation and 3-NT expression are reduced, and neuroinflammation driven by NLRP3 inflammasomes is inhibited. The NR3A participates in depression-like behaviors induced by CRS, and the mechanism of the NR3A is related to calcium homeostasis regulation, ROS generation and inflammatory response driven by NLRP3 inflammasomes.

Description

Technical Field

[0001] The invention belongs to the field of neurobiology and drug research and development, and discloses an application of NR3A as a protein target in screening or preparing drugs for preventing and / or treating depressive-like behaviors. Background Art

[0002] Depression is a widespread and complex chronic neurological disease that will persistently affect the mental, emotional and physical health of patients. It is characterized by low mood, lack of vitality, sadness, insomnia, etc. The pathophysiology of depression is related to oxidative stress, inflammation, etc. However, the molecular mechanism behind depression remains elusive. The onset of depression is closely related to the abnormality of N-methyl-D-aspartate (NMDA) receptors, which are a type of ionotropic receptor for the excitatory neurotransmitter glutamate (Glu). The NMDA receptor family includes seven subunits, NR1, NR2 (A / B / C / D), NR3A and NR3B. The classic NMDA receptor is a tetramer composed of two molecules of NR1 and NR2, which is activated by glutamate. Studies have shown that ketamine (an NMDA receptor antagonist) can exert a rapid antidepressant effect, suggesting that blocking NMDA receptors can exert an antidepressant effect. Studies have found that the selective NR2B antagonist Ro25-6981 can quickly improve chronic stress-induced depression model mice anhedonia and anxiety behavior, but antidepressants developed for NR2B have serious cardiac toxicity and extremely low oral bioavailability, far from meeting the requirements of clinical treatment. Therefore, finding a suitable target is crucial for the development of drugs to treat depression.

[0003] NMDA receptors containing NR3A include tetramers composed of NR1 / NR3A and NR1 / NR2(A / B / C / D) / NR3A. NMDA receptors composed of NR1 / NR2(A / B / C / D) / NR3A are also activated by glutamate or NMDA and require glycine (Gly) as a co-agonist. Unlike the classical NMDA receptor (NR1 / NR2(A / B / C / D) isomer), it has a smaller single-channel conductivity and a lower calcium ion (Ca) + ) permeability and magnesium ion blocking sensitivity. NMDA receptors containing NR3A are mainly expressed in the medial habenula, hippocampus and prefrontal cortex of mice after adulthood.

[0004] Chronic restraint stress (CRS) is an animal model that simulates depressive symptoms and has been widely reported. It is reported that CRS causes glutamate accumulation in the hippocampus. Glutamate, as a ligand, continuously activates NMDA receptors, ion channels open, and calcium ions flow in in large quantities. NMDA receptor-mediated calcium ion overload can cause a series of fatal downstream reactions. Nitric oxide synthase is an isoenzyme that is divided into neuronal, endothelial, and inducible types. Neuronal nitric oxide synthase (nNOS) is activated by calcium ions. nNOS catalyzes the production of nitric oxide (NO). Under physiological conditions, the catalytic production of NO is beneficial to the growth of endothelial cells, but under pathological conditions, its overactivation induces excessive production of NO. Excessive nitric oxide leads to its derivative product, peroxynitrite anion (ONOO - ) and other toxic products, thus damaging nerve cells. Excessive calcium ions in cells can cause abnormalities in the mitochondrial electron chain, leading to excessive production of reactive oxygen species (ROS). Reactive oxygen-mediated oxidative stress is a key factor in neurological diseases. ROS also induces the activation of the inflammasome NLRP3. NLRP3, ACS, and cleaved-caspase1 form an inflammatory complex that mediates the production of inflammatory factors such as IL-1β and IL-18.

[0005] Previous studies have found that NR3A in the hippocampus of CRS model mice was significantly downregulated, and then overexpression of NR3A in the hippocampus significantly alleviated the depressive-like behavior of mice, but the specific molecular mechanism is still unclear. Therefore, in this experiment, a CRS model and an NR3A overexpression + CRS model were constructed to detect the expression levels of related indicators in the hippocampus, and to further explore the role of NR3A in depression.

[0006] Through the above analysis, the problems and defects of the prior art are as follows:

[0007] It has been confirmed that the reduction of hippocampal NMDAR subunit NR3A is associated with depression. However, the underlying mechanism of NR3A involvement in depression is still unclear. Summary of the invention

[0008] In view of the problems existing in the prior art, the present invention provides an application of NR3A as a protein target in screening or preparing drugs for preventing and / or treating depressive-like behaviors.

[0009] The present invention is achieved by using NR3A as a protein target in screening or preparing drugs for preventing and / or treating depressive-like behaviors.

[0010] The present invention provides a method for treating depression based on the NR3A target, comprising the following steps:

[0011] The Grin3a gene is introduced into mammalian hippocampal tissue via an adeno-associated virus vector to specifically enhance the expression level of the NR3A target, thereby achieving the effect of alleviating depressive symptoms.

[0012] The present invention provides a high-throughput detection method for NR3A target screening, which uses CRISPR / Cas9 technology to knock out or overexpress the Grin3a gene in a cell line, and combines fluorescent labeling and two-photon microscopy imaging technology to screen out compounds or biological molecules that have a regulatory effect on the NR3A target.

[0013] The present invention provides a targeted drug delivery system for NR3A target, which includes a nanoparticle carrier coated with a targeting recognition peptide, and the carrier can specifically recognize and bind to the NR3A target on nerve cells to achieve directional release and precise treatment of the drug.

[0014] The present invention provides a diagnostic device for NR3A target-related diseases, the device comprising:

[0015] A biosensor module for detecting specific antibodies or ligands binding to the NR3A target in a body fluid sample;

[0016] The signal amplification module is used to amplify the signal detected by the biosensor to improve the accuracy of early diagnosis of NR3A-related diseases.

[0017] The present invention provides a neuroprotectant based on the NR3A target, characterized in that the neuroprotectant reduces neuronal damage by targeting the NR3A receptor, and is used for treating or preventing neuronal death associated with neurodegenerative diseases.

[0018] The present invention provides a drug screening method for the treatment of mental illness, which is characterized by establishing a cell model expressing the NR3A target to screen candidate drugs that have a regulatory effect on the NR3A target, thereby screening out potential antidepressant drugs or antianxiety drugs.

[0019] The present invention provides a gene therapy method based on the NR3A target, characterized in that the Grin3a gene is delivered to the patient's central nervous system via an adeno-associated virus vector to enhance the expression of the NR3A target, thereby improving mental or neurological diseases associated with NR3A deficiency or functional decline.

[0020] The present invention provides a personalized treatment plan design system related to the NR3A target, characterized in that the system provides personalized drug combinations and treatment strategies for the NR3A target according to the patient's gene test results and NR3A expression level, so as to improve the treatment effect.

[0021] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0022] First, the expression of NR3A in the hippocampus was enhanced by stereotaxic injection of adeno-associated virus containing the Grin3a gene. Chronic restraint stress (CRS) was used to induce depression in mice, and sucrose preference test (SPT), forced swimming test (FST) and open field test (OFT) were used for identification. Nissl staining was used for neuropathological examination. Western blot was used to detect the expression of NR3A, calpain 2 (Calpain2), neuronal nitric oxide synthase (nNOS), 3-nitrotyrosine (3-NT), and NLRP3 inflammatory complex protein. DHE staining was used to detect ROS.

[0023] Results: Overexpression of NR3A in the hippocampus improved CRS-induced depressive-like behavior, inhibited calcium-activated signaling cascades, reduced ROS generation and 3-nitrotyrosine expression, and suppressed NLRP3 inflammasome-driven neuroinflammation.

[0024] Conclusion: NR3A is involved in CRS-induced depressive-like behavior, and its mechanism is related to the regulation of calcium homeostasis, ROS generation, and inflammatory response driven by NLRP3 inflammasome, suggesting that NR3A alleviates the occurrence of depression by regulating calcium homeostasis.

[0025] Second, the technical solution of the present invention solves the technical problems that people have been eager to solve but have never succeeded in solving: the beneficial effects of the present invention are: based on a large number of clues and own experiments, it was found that CRS-induced depressive-like behavior was related to the reduction of NR3A expression in the hippocampus. By constructing a CRS model and a NR3A overexpression + CRS model, it was found that enhancing the expression of hippocampal NR3A can improve the depressive-like behavior of CRS-induced mice. The present invention establishes a potential mechanism for NR3A to improve depressive-like behavior by regulating calcium homeostasis, oxidative stress and neuroinflammation in nerve cells. Regulation of NR3A is expected to become a potential therapeutic approach for the treatment of depression, providing a new therapeutic target.

[0026] Third, the present invention mainly solves the following key problems in the treatment methods for depression and neurodegenerative diseases in the prior art:

[0027] 1) Insufficient target specificity: Traditional drug treatments often lack specificity and cannot effectively regulate specific targets closely related to depression and neurodegenerative diseases, such as NR2B, which leads to limited treatment effects and large side effects.

[0028] 2) Targeted drug delivery is difficult: Existing drug delivery systems are unable to achieve specific directional delivery of drugs in the nervous system, resulting in insufficient drug concentration in the target tissue, thus affecting efficacy.

[0029] 3) Difficulty in early detection of diseases: Traditional disease diagnosis methods lack sensitive detection of specific molecular targets, making it difficult to accurately diagnose and intervene in diseases in the early stages.

[0030] 4) Complex gene expression regulation: Existing gene therapy methods often face problems such as unstable gene expression regulation and low efficiency, making them difficult to be widely used in clinical practice.

[0031] Significant technological advancements:

[0032] Through the technical solution of the present invention, the above technical problems have been effectively solved, and the following significant technical progress has been achieved:

[0033] 1) Target specific enhancement: The present invention utilizes the NR3A target and designs a gene therapy method that specifically enhances NR3A expression, thereby directly regulating the pathological mechanism related to depression at the molecular level, significantly improving the targetedness and effect of the treatment.

[0034] 2) Efficient targeted drug delivery system: The targeted drug delivery system developed by the present invention, through the combination of nanoparticles and targeted recognition peptides, can achieve the directional release of drugs in specific parts of the nervous system, significantly improve the therapeutic effect of the drug, and reduce the side effects on non-target tissues.

[0035] 3) Sensitive early diagnostic device: The diagnostic device based on the NR3A target designed by the present invention combines biosensors and signal amplification technology to achieve high-sensitivity detection of disease-related targets, which helps in the early diagnosis and intervention of the disease and significantly improves the accuracy of diagnosis.

[0036] 4) Advanced gene therapy methods: The present invention utilizes adeno-associated virus vectors and CRISPR / Cas9 technology to provide an efficient and stable gene expression regulation method that can specifically restore or enhance the expression of NR3A in the central nervous system, providing new ideas and technical support for gene therapy.

[0037] In summary, the present invention not only solves the key problems in the prior art through target-specific drug design, effective drug delivery system, sensitive diagnostic device and advanced gene therapy technology, but also achieves significant technological progress in the treatment and diagnosis of depression and neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1It is a flow chart of the experimental method for treating depression based on NR3A provided in an embodiment of the present invention.

[0039] Figure 2 It is a structural block diagram of a depression treatment experimental device based on NR3A provided in an embodiment of the present invention.

[0040] Figure 3 According to an embodiment of the present invention, the virus is injected into the bilateral hippocampus of mice, and after CRS intervention, the expression level of NR3A protein in the hippocampus and the behavioral differences between different groups of mice are detected.

[0041] Figure 4 The expression levels of Calpain2, CAMKIIα and Calbindin (mRNA) in the CRS group provided in the embodiment of the present invention are significantly increased compared with the control group. Except for CAMKIIα, the expression level of the NR3A overexpression + CRS group is decreased.

[0042] Figure 5 3. The levels of nNOS, NO, ROS and 3-NT in the CRS group provided in the embodiment of the present invention are significantly increased, and the expression levels in the NR3A overexpression+CRS group are significantly decreased.

[0043] Figure 6 The embodiment of the present invention provides a diagram showing that NLRP3, ASC, Cleaved-Caspase1, IL-1β, and IL18 in the hippocampus of CRS group mice are significantly increased, and overexpression of NR3A reduces their expression levels.

[0044] Figure 7 This is a figure provided by an embodiment of the present invention showing that the number of neurons in the CA3 region of the CRS group is reduced and the nuclei are darkly stained, the neurons in the CA1 region are not affected, and the BAX expression level is significantly increased; the neuronal damage and apoptosis in the NR3A overexpression+CRS group are significantly reduced. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0046] like Figure 1 As shown, an experimental method for treating depression based on NR3A provided in an embodiment of the present invention comprises the following steps:

[0047] Step 1: NR3A overexpression virus injection and CRS intervention

[0048] The mice provided in the embodiment of the present invention selected 36 male C57BL / 6N mice, which were randomly divided into a control group, a CRS group, and a NR3A overexpression + CRS group (n=12) after adapting to a normal environment for 1 week, and then injected with adeno-associated virus (rAAV) containing the Grin3a gene into the bilateral hippocampal region of the NR3A overexpression + CRS group mice by stereotactic injection technology. After 7 days of viral expression, CRS intervention was performed on mice in the CRS group and the NR3A overexpression + CRS group for 2 hours a day for 14 days. During this period, the expression of NR3A repaired its decreased level in the hippocampus, laying the foundation for subsequent behavioral and molecular biological detection.

[0049] Step 2: Behavioral assessment

[0050] Mice were characterized by the following behavioral tests:

[0051] Sucrose preference test (SPT): Assessing anhedonia in mice, one of the core symptoms of depression.

[0052] Forced swim test (FST): detects the despair behavior of mice, reflecting their degree of depression.

[0053] Open field test (OFT): Assess the locomotor activity and anxiety level of mice. Through these behavioral tests, it is confirmed whether the depression model is successfully established in mice.

[0054] Step 3: Pathology and molecular biology testing

[0055] Nissl staining: used for neuropathological analysis to observe pathological changes of neurons.

[0056] Western Blot assay: Detect the expression levels of Calpain2, nNOS, 3-NT, NLRP3 inflammatory complex and apoptosis-related proteins BAX and BCL-2, and evaluate the activity of related molecular pathways.

[0057] DHE staining: used to detect the level of reactive oxygen species (ROS) to evaluate the impact of oxidative stress on depression.

[0058] The program combines behavioral, pathological, and molecular biological testing to systematically evaluate the therapeutic effect of NR3A on depression.

[0059] The mice provided in the embodiments of the present invention are 7-8 weeks old and weigh about 20-23 g.

[0060] like Figure 2 As shown, an experimental device for treating depression provided by an embodiment of the present invention comprises:

[0061] The injection module is used to inject the adeno-associated virus containing the Grin3a gene into the bilateral hippocampal regions of mice in advance through stereotactic injection technology to construct the NR3A overexpression + CRS model;

[0062] The identification module is used to induce depression in mice using chronic restraint stress CRS, and to conduct behavioral identification of three groups of mice using the sucrose preference test SPT, forced swimming test FST, and open field test OFT;

[0063] The detection module is used for neuropathological examination using Nissl staining; western blot is used to detect the expression of NR3A, Calpain2, neuronal nitric oxide synthase (nNOS), 3-nitrotyrosine (3-NT), and NLRP3 inflammatory complex protein; DHE staining is used to detect ROS.

[0064] The NR3A-based depression treatment experimental method proposed in the present invention aims to restore the expression of NR3A in the hippocampus, study its effect on improving depressive symptoms, and explore the related molecular mechanisms. The specific working principle is as follows:

[0065] First, in step S101, an adeno-associated virus (rAAV) containing the Grin3a gene is precisely injected into the hippocampus of mice through stereotactic injection technology. The Grin3a gene encodes the NR3A subunit, which plays an important role in the NMDAR complex of neurons. In CRS-induced depressed mice, the expression of NR3A is usually reduced, and by injecting rAAV adenovirus containing the Grin3a gene in advance, the expression of NR3A in the hippocampus can be enhanced, thereby inhibiting the reduction of NR3A subunits in the hippocampus of mice induced by CRS, thereby improving the depressive-like behavior of mice.

[0066] Next, in step S102, a chronic restraint stress (CRS) model is used to induce depressive symptoms in mice. CRS is a commonly used animal model of depression that simulates the state of depression in humans through continuous stress exposure. After a period of stress treatment, the mice are behaviorally identified by sucrose preference test (SPT), forced swim test (FST) and open field test (OFT) to evaluate the depressive behavior of the mice. These behavioral tests are used to measure the loss of pleasure, despair behavior and spontaneous activity levels of mice, respectively, to confirm the establishment of the depression model.

[0067] In step S103, key molecular markers, including NR3A, Calpain2, neuronal nitric oxide synthase (nNOS), 3-nitrotyrosine (3-NT), and the expression levels of NLRP3 inflammatory complex and apoptosis-related proteins BAX and BCL-2, are detected by Western blot, DHE staining and other experiments. Calpain2 is related to calcium homeostasis in neurons, while ROS, 3-NT and NO are related to oxidative stress. NLRP3 is an important inflammatory complex that is closely related to the role of neuroinflammation in depression, while BAX and BCL-2 are related to cell damage and apoptosis. The expression levels of these molecular markers can reflect the molecular changes in depressed mice after the restoration of NR3A.

[0068] Finally, the neural tissue of mice was pathologically examined by Nissl staining. Nissl staining is a commonly used neural tissue staining method that can clearly show the structure of neurons, especially the morphology of cell bodies and nucleoli. Through this method, the morphological changes of neurons in the hippocampus region after restoring NR3A expression can be observed to evaluate whether it has the effect of improving depressive symptoms.

[0069] Through the above steps, the present invention provides a systematic experimental method to explore the mechanism of action of NR3A in the treatment of depression, aiming to provide new molecular targets and strategies for the treatment of depression. The present invention is specifically implemented as follows:

[0070] Materials and methods:

[0071] 1.1 All experimental animals were fed with standard laboratory chow and water, placed in standard cages and ordinary cages, and subjected to a 12-h light-dark cycle (lights on at 8:00 a.m.). The temperature was 22°C to 24°C, and the relative humidity was 30% to 70%. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals. The experimental procedures were approved by the Animal Care Committee of Chongqing Medical University.

[0072] 1.2 Thirty-six C57BL / 6 mice were selected for the experiment. After one week of adaptive feeding, they were randomly divided into a control group (n=12), a CRS group (n=12), and a NR3A overexpression + CRS group (n=12). The NR3A overexpression + CRS group was injected with rAAV-CMV-Grin3a-HA-WPREs (rAAV-Grin3a) virus. The virus material was purchased from Wuhan Shumi Technology Co., Ltd., China. The titer of rAAV-Grin3a was 5.8×10 12vg / ml. After anesthesia, each mouse was placed in a stereotaxic apparatus and the virus was injected at 0.3ul / min (1.5ul on each side) using a 5ul titer syringe. The injection coordinates of Bregma were: AP, -2.3mm; ML, ±1.8mm; DV, -2mm. After injection, the syringe was placed for 4 minutes and then slowly withdrawn. One week after the injection of the virus, the mice in the CRS group and the NR3A overexpression + CRS group were subjected to the above treatment.

[0073] 1.3 Mice were stressed in rodent plastic filters for 2 h every day from 9:00 to 11:00 AM for 14 days. Behavioral testing was performed within 24 hours after the last stress test to assess the depressive-like behavior of the mice.

[0074] 1.4 All tests were performed between 9:00 and 18:00. Mice were subjected to a battery of behavioral tests, namely the sucrose preference test (SPT), forced swimming test (FST), and open field test (OFT). Before the SPT, mice were placed in individual cages and allowed to be observed for 3 days. Throughout the process, one of the two bottles containing 1% sucrose water (w / v) was placed in each cage. Before the start of the test, mice were deprived of water for 24 hours, and then each mouse in a separate cage was exposed to two bottles for 24 hours, one filled with 1% sucrose water solution and the other with water, and the bottles were weighed before and after the experiment. SPT is expressed as the percentage of sucrose water consumption (sucrose consumption / total liquid consumption × 100%). FST was performed by placing the experimental mice in a transparent cylindrical water tank (20 cm in diameter and 35 cm in height), which was filled with water at a temperature of 23–25°C and a depth of 20 cm. Each mouse was tested for 6 minutes, and the test process was recorded by video. The immobility time of the last four minutes was measured. During the OFT test, each mouse was gently placed in a corner of an open field apparatus (50 cm × 50 cm × 40 cm) and allowed to move freely for 5 minutes. The total distance moved, total moving time, and time spent in the center area of ​​the mouse were automatically recorded and calculated using the ANY maze software (Stoelting, USA).

[0075] 1.5 Total RNA was extracted from tissues using TRIzol reagent according to the manufacturer's instructions. Total RNA was first reverse transcribed into cDNA using PrimerScriptTM RT kit with gDNAEraser (Takara, RR047A). Fluorescence quantitative PCR was performed using SYBRGreen PCR mix (Takara, RR820A). The microtubule gene actin was used as an internal reference for standardization.

[0076] 1.6 The other half of the brain used for RNA extraction was used for western blot analysis. The frozen brain samples were homogenized and proteins were extracted using Tris-EDTA lysis buffer and freshly added protease inhibitor cocktail. After protein quantification, SDS-PAGE electrophoresis was performed and the separated proteins were transferred to polyacrylamide membrane (PVDF). Anti-β-actin antibody or anti-β-tubulin antibody was used as an internal control to standardize the loading amount. After incubation at 4°C overnight and washing three times with TBST, the bands were then exposed to HRP-conjugated secondary antibodies for 60 minutes at room temperature. Subsequently, the immunoblots were visualized using an ultra-high sensitivity ECL kit. Protein quantification was performed using ImageJ software.

[0077] 1.7 Antigen retrieval was performed using citrate antigen retrieval solution by microwave heating for 8 minutes. Next, the sections were exposed to 0.1% TritonX-100 for 30 minutes and subsequently blocked with goat serum for 2 hours. Subsequently, the sections were incubated with the primary antibodies listed below at 4°C overnight: guinea pig anti-Iba-1, and after three rounds of PBS washing, the sections were treated with the corresponding secondary antibodies for 1 hour at room temperature in a dark environment. DAPI staining of cell nuclei was performed for 10 minutes. Finally, the sections were carefully examined and images were taken using a fluorescence microscope by a blinded observer. The micrographs taken were evaluated with the help of ImageJ software.

[0078] 1.8 Dihydroethidium (DHE) staining was performed to evaluate ROS production in brain tissue. In brief, 10 μm frozen brain sections were treated with the fluorescent dye dihydroethidium at a concentration of 3 μmol / L. Incubated in a humidified 37°C room in the dark for 30 minutes. After washing twice with PBS, the sections were placed in DAPI for 10 minutes and then examined under a fluorescence microscope. ImageJ software was used to quantify the fluorescence intensity.

[0079] 1.9 Mice were killed by cervical dislocation, and brain tissue was fixed with 4% paraformaldehyde. The fixed brain tissue was dehydrated with xylene, embedded in paraffin, and cut into 5μm thick slices. The slices were placed in xylene I for 20min, xylene II for 20min, anhydrous BI for 5min, anhydrous ethanol II for 5min, 75% alcohol for 5min, and finally rinsed with tap water. The treated samples were stained with Nissl stain for 2-5min. Rinse under running water until colorless, seal with neutral resin after drying, and observe the Nissl bodies of neurons in the CA1 and CA3 regions of the hippocampus with a microscope slide scanner and take pictures.

[0080] 1.10 All statistical analyses were performed using SPSS 25.0 software and Prism 8. Data are presented as mean ± SEM. Two-group comparisons were analyzed using a two-tailed unpaired Student's t-test. Three-group comparisons were performed using a one-way analysis of variance (ANOVA) followed by a LSD post hoc test. Differences were considered significant when p < 0.05.

[0081] Conclusion 1: Overexpression of NR3A in the hippocampus improves CRS-induced depressive behavior and decreased expression of NR3A in mice

[0082] To further confirm the relationship between NR3A deficiency and depression, rAAV-Grin3a adenovirus was injected into the hippocampus of mice before exposure to CRS, and one week after the virus injection, the mice were subjected to CRS intervention for 2 hours at the same time every day for 14 days. Compared with the control group, the expression level of NR3A in the hippocampus of mice in the CRS group was significantly reduced, and the expression level in the NR3A+CRS group was restored. Subsequently, behavioral tests were performed on the three groups of mice. The immobility time of mice in the NR3A+CRS group in the FST was restored, the sugar water preference of mice in the NR3A+CRS group in the SPT was increased compared with the CRS group, and the time of the NR3A+CRS group in the central area in the OFT was restored compared with the CRS group, but the total movement distance did not change.

[0083] The above results demonstrate that enhancing NR3A expression in the hippocampus can improve CRS-induced depressive-like behavior in mice.

[0084] Conclusion 2: Overexpression of NR3A in the hippocampus inhibits calcium-activated signaling cascades

[0085] In order to explore the activity of specific calcium-dependent signals, calcium-dependent related signals were detected. The expression levels of Calpain2 and Calbindin (PCR) in the CRS group were significantly higher than those in the control group, and recovered in the NR3A+CRS group. The expression level of CAMKIIα in the NR3A+CRS group was not significantly lower than that in the CRS group, but there was a downward trend.

[0086] Conclusion 3: NR3A overexpression significantly reduced the increase of nNOS and ROS induced by CRS

[0087] Then, in order to further clarify the role of NR3A in depression, the expression levels of nNOS, NO, ROS and 3-NT in the mouse hippocampus were detected. Compared with the control group, the levels of ROS, 3NT and NO in the CRS group were significantly increased, and their expression levels were significantly restored in the NR3A overexpression group, but the ROS level in the hippocampal CA3 region was not restored. Compared with the control group, the levels of nNOS and NO in the CRS group were significantly increased, and overexpression of NR3A reduced their expression levels. This result proves that Figure 5 .

[0088] Conclusion 4: NR3A overexpression alleviates CRS-induced hippocampal neuroinflammation

[0089] The NLRP3 inflammasome is an inflammatory molecule signaling complex that plays an important role in depression. In order to explore the relationship between NR3A and neuroinflammation, the levels of inflammatory factors in the hippocampus were detected. Figure 6 The data showed that compared with the control group, the levels of NLRP3, ASC, Cleaved-caspase1, IL-1β, and IL18 in the hippocampus of mice in the CRS group were significantly increased. Except for IL-1β, overexpression of NR3A significantly reduced the expression levels of the remaining proteins.

[0090] Conclusion 5: NR3A overexpression significantly alleviated CRS-induced neuronal pathological damage and apoptosis

[0091] Next, in order to more clearly elucidate the role of NR3A in CRS-induced depression, Nissl staining was used to detect neuronal damage in the CA1 and CA3 regions of the hippocampus. Figure 7 As shown in the figure, in the control group, neurons were clearly visible, with intact structures and neat arrangement. Compared with the control group, the number of neurons in the CA3 region of the CRS group decreased, the nuclei were darkly stained, and the neurons in the CA1 region were not affected; the neuronal damage in NR3A+CRS was significantly reduced. The expression of apoptosis-related protein BAX in the NR3A+CRS group was significantly lower than that in the CRS group.

[0092] Analysis: Abnormal expression of NR3A in the brain is involved in the occurrence and development of a variety of neurodegenerative diseases and psychiatric disorders, such as AD, bipolar disorder, etc. However, the mechanism of action of NR3A in depression is still unclear. In this study, it was demonstrated that enhancing the expression of NR3A in the hippocampus can alleviate CRS-induced depressive-like behavior, inhibit the production of ROS and the activation of the NLRP3 complex, suggesting that NR3A may be involved in the pathogenesis of depression by regulating intracellular calcium homeostasis and the ROS-NLRP3 signaling pathway.

[0093] Previous research evidence suggests that plasma glutamate levels are elevated during the pathogenesis of depression, and neuroimaging and a small number of genetic studies have shown an association between glutamate-related gene variants in the brain and depression in patients with depression. In addition, it has been shown that exposure of rats to various stressors or administration of corticosterone induces increases in glutamate content in several regions, including the hippocampus, amygdala, and prefrontal cortex. Disturbances in glutamate homeostasis or chronic exposure to excess glutamate can have significant effects on neuronal cells by triggering neurotoxic or excitotoxic cascades.

[0094] Single-cell sequencing previously revealed that the downstream pathways of NR3A knockout mice are enriched in pathways related to calcium homeostasis and calcium ion concentration regulation. Therefore, it is inferred that NR3A may alleviate CRS-induced hippocampal damage by regulating intracellular calcium homeostasis. The results also showed that overexpression of NR3A in the hippocampus can alleviate the signal cascade activated by calcium ions. + It is closely related to ROS. Calcium overload can cause mitochondrial damage and further release Ca2 + Triggering mitochondrial-dependent apoptosis. Mitochondrial damage releases large amounts of ROS. Interestingly, many of these systems can be regulated by calcium. ROS can affect intracellular Ca2 + homeostasis, while calcium can increase the production of ROS.

[0095] The increase of ROS leads to the activation of NLRP3 and induces neuroinflammation. In addition, neuroimmunology also shows that neuroinflammation can mediate the occurrence of depression. Studies have reported that the CRS-induced depression model activates the NLRP3 inflammasome. Our study found that CRS caused ROS production and NLRP3 activation, which is consistent with previous reports. Interestingly, overexpression of NR3A in the hippocampus reduced the production of ROS and inhibited the activation of NLRP3 inflammasome induced by CRS. These results indicate that NR3A plays a role in the occurrence of depression.

[0096] The results showed that after overexpression of NR3A in the hippocampus, calcium homeostasis in the hippocampus of CRS-induced depression model mice was improved, Calpain2 activation was inhibited, ROS generation was significantly reduced, and NLRP3 inflammasome activation and its mediated inflammatory response were weakened. These results indicate that NR3A alleviates CRS-induced hippocampal damage by regulating intracellular calcium homeostasis and neuroinflammation.

[0097] The following are two specific embodiments, based on the technical content of the above claims:

[0098] Example 1: Gene therapy method for depression based on NR3A target

[0099] This example aims to develop a gene therapy method using the NR3A target to alleviate depressive symptoms caused by chronic stress. Studies have found that the expression of the NR3A subunit is decreased in the hippocampal tissue of patients with depression. Therefore, by restoring the expression of NR3A, the symptoms of depression can be significantly improved.

[0100] 1) Implementation of gene therapy:

[0101] Eight-week-old male C57BL / 6N mice were used and randomly divided into a control group, a CRS group, and a NR3A overexpression + CRS group after seven days of adaptive feeding. rAAV-Grin3a virus was injected into the hippocampus of mice in the NR3A overexpression + CRS group.

[0102] 2) Establishment of animal model:

[0103] One week after viral expression, mice in the CRS group and NR3A overexpression + CRS group were subjected to chronic restraint stress (CRS) for 2 hours per day for 14 days to induce depressive symptoms.

[0104] 3) Behavioral testing:

[0105] After 14 days of CRS intervention, the three groups of mice were subjected to sucrose preference test (SPT), forced swimming test (FST) and open field test (OFT) to evaluate depressive-like behavior. The results showed that mice receiving NR3A gene therapy showed a higher preference for sugar water in SPT and a shorter immobility time in FST, indicating a significant improvement in depressive-like behavior.

[0106] 4) Molecular biology detection:

[0107] Western Blot analysis of NR3A protein expression levels and detection of downstream signaling molecules such as Calpain2, nNOS, 3-NT and NLRP3 inflammatory complex expression changes. The results showed that overexpression of NR3A inhibited neuroinflammation and oxidative stress associated with depression.

[0108] 5) Histopathological examination:

[0109] Nissl staining was used to analyze the mouse hippocampal tissue structure and neuronal morphology, and the results showed that overexpression of NR3A restored CRS-induced hippocampal neuronal damage.

[0110] This example successfully demonstrated that the gene therapy method of restoring NR3A expression can effectively improve the depressive symptoms caused by chronic stress, providing strong evidence for the application of NR3A target in the treatment of depression.

[0111] Example 2: High-throughput detection method for NR3A target screening

[0112] This example aims to screen candidate drugs or biomolecules that can regulate the expression of NR3A targets by establishing a cell model and combining CRISPR / Cas9 technology. This method can be applied to the development of antidepressant drugs.

[0113] 1) Establishment of cell model:

[0114] A human neuroblastoma cell line (SH-SY5Y) was selected, and the NR3A gene was knocked out in the cell line using CRISPR / Cas9 technology, or the NR3A gene was overexpressed by plasmid transfection, thereby establishing cell models with high and low expression of NR3A.

[0115] 2) Fluorescence labeling and imaging:

[0116] Using fluorescently labeled NR3A protein, the cell model was imaged in real time using a two-photon microscope to analyze changes in NR3A expression levels under different treatment conditions.

[0117] 3) Drug screening:

[0118] Various candidate compounds or biomolecules were added to cell models with low and high NR3A expression to observe their regulatory effects on NR3A expression. Compounds that can significantly restore NR3A expression were screened out using imaging technology.

[0119] 4) Verification and analysis:

[0120] The screened candidate compounds were further functionally validated, such as using Western Blot and RT-qPCR to analyze their effects on the NR3A downstream signaling pathway, and confirming their potential antidepressant effects through cell survival rate and neuronal function analysis.

[0121] This example successfully established a high-throughput screening method that can effectively screen candidate drugs that have a regulatory effect on the NR3A target, providing technical support for the development of new antidepressant drugs.

[0122] 1. Working principle of the biosensor module

[0123] The core component of the device is the biosensor module, which is used to detect specific antibodies or ligands that bind to the NR3A target in a body fluid sample. The biosensor introduces the body fluid sample into the detection area within the sensor and uses the NR3A target binding molecules fixed on the surface to capture the target antibodies or ligands in the sample. When these target molecules bind to the NR3A target, the sensor generates electrical or optical signals. These signals are a direct manifestation of the detection of NR3A target activity and reflect the pathological processes related to NR3A in the patient's body.

[0124] 2. Working principle of signal amplification module

[0125] The initial signal detected by the biosensor is weak, especially in the early stages of the disease, so a signal amplification module is needed to enhance the signal. This module uses electrical amplification technology to amplify the weak signal generated by the biosensor, making the detected signal clearer. The amplified signal is further analyzed by the data processing module, which can improve the accuracy of early diagnosis of NR3A-related diseases and ensure that even trace amounts of NR3A target binding can be identified and monitored.

[0126] 3. Mechanism of action of NR3A target in neuroprotection

[0127] NR3A receptors play an important role in regulating neuronal function, and their overactivation leads to neuronal damage. Based on this, the neuroprotectant targets NR3A receptors to reduce neuronal damage. The neuroprotectant can effectively block the excessive activity of NR3A receptors, reduce the calcium influx of neurons, and thus prevent cell damage and death caused by calcium ion overload. This mechanism is particularly suitable for preventing or treating neuronal death associated with neurodegenerative diseases.

[0128] 4. Potential application of NR3A target in neurodegenerative diseases

[0129] Neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease are often accompanied by a gradual loss of neurons. By targeting the NR3A receptor, neuroprotective agents can reduce neuronal damage caused by these diseases, thereby delaying disease progression. Regulation of the NR3A receptor can help reduce excitotoxicity, a key factor in the pathological process of neurodegeneration. This mechanism provides a new targeted therapy for the treatment of these diseases, especially in early intervention, which can show good neuroprotective effects.

[0130] 5. Drug screening method based on NR3A target

[0131] This screening method is used to screen candidate drugs that have a regulatory effect on the NR3A target by establishing a cell model expressing the NR3A target in the laboratory. In this model, the NR3A gene is introduced by transfection technology to make the cells express the target. Then, the candidate drug is applied to the cell model to monitor its regulatory effect on the NR3A target. By detecting cell signaling, ion channel activity, etc., it can be determined whether the candidate drug has potential antidepressant or antianxiety effects.

[0132] 6. Drug screening process for neuroprotection and psychiatric disease treatment potential

[0133] The screened drugs that have a regulatory effect on the NR3A target have potential neuroprotective and psychiatric therapeutic effects. This type of drug regulates the NR3A receptor, reduces neuronal damage, and improves the pathological process of mental illnesses such as depression and anxiety. Through the action of the NR3A target, the screened drugs can show excellent efficacy in preclinical and clinical studies, providing new ideas and drug options for the treatment of mental illness and neurodegenerative diseases.

[0134] The following are Figures 4 to 7 Specific description:

[0135] Figure 4 :Title: "Changes in the expression levels of Calpain2, CAMKIIα and Calbindin (mRNA) in the CRS group"

[0136] The embodiment of the present invention provides Figure 4 The results showed that the expression levels of Calpain2 and Calbindin in the CRS group were significantly higher than those in the control group, while the expression of CAMKIIα was also significantly increased. In addition to CAMKIIα, the expression levels of Calpain2 and Calbindin in the NR3A overexpression combined with CRS treatment group were significantly decreased, indicating that the overexpression of NR3A has a certain inhibitory effect on the increased protein expression caused by CRS.

[0137] Figure 5 :Title: "Changes in nNOS, NO, ROS, and 3-NT levels in the CRS group"

[0138] The embodiment of the present invention provides Figure 5 The results showed that the levels of nNOS, NO, ROS and 3-NT were significantly increased in the CRS group, indicating that oxidative stress and nitric oxide synthase activity were enhanced. In contrast, the levels of these indicators were significantly decreased in the NR3A overexpression combined with CRS treatment group, indicating that NR3A overexpression has a mitigating effect on oxidative stress induced by CRS.

[0139] Figure 6 :Title: "Changes in the expression of inflammatory markers NLRP3, ASC, Cleaved-Caspase1, IL-1β and IL18 in the hippocampus of mice in the CRS group"

[0140] The embodiment of the present invention provides Figure 6 The results showed that the expression levels of NLRP3, ASC, Cleaved-Caspase1, IL-1β and IL18 in the hippocampus of mice in the CRS group were significantly increased, reflecting the enhanced inflammatory response caused by CRS. In the NR3A overexpression combined with CRS treatment group, the expression levels of these inflammatory markers were significantly decreased, indicating that NR3A overexpression has an inhibitory effect on CRS-induced inflammation.

[0141] Figure 7 :Title: "Changes in the number of neurons and nuclear staining in the CA3 region of the CRS group"

[0142] The embodiment of the present invention provides Figure 7 The results showed that the number of neurons in the CA3 region of the hippocampus of mice in the CRS group was significantly reduced, and the nuclear staining of neurons was deepened, indicating that CRS caused damage to neurons in the CA3 region. However, neurons in the CA1 region were not affected. In contrast, in the NR3A overexpression combined with CRS treatment group, neuronal damage in the CA3 region was significantly reduced, indicating that NR3A overexpression has a protective effect on neuronal damage caused by CRS.

[0143] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. An application of NR3A as a protein target in screening or preparing drugs for preventing and / or treating depressive-like behaviors.

2. A method for treating depression based on the NR3A target used in claim 1, characterized in that: The Grin3a gene is introduced into mammalian hippocampal tissue via an adeno-associated virus vector to specifically restore and enhance the expression level of the NR3A target, thereby achieving the effect of alleviating depressive symptoms.

3. A high-throughput detection method for NR3A target screening based on the application of claim 1, characterized in that: The NR3A gene was knocked out or overexpressed in cell lines using CRISPR / Cas9 technology, and combined with fluorescent labeling and two-photon microscopy imaging technology, compounds or biological molecules that have regulatory effects on the NR3A target were screened.

4. A diagnostic device for NR3A target-related diseases based on the application of claim 1, characterized in that: The device comprises: A biosensor module for detecting specific antibodies or ligands binding to the NR3A target in a body fluid sample; The signal amplification module is used to amplify the signal detected by the biosensor to improve the accuracy of early diagnosis of NR3A-related diseases.

5. A neuroprotective agent based on the NR3A target for use according to claim 1, characterized in that: The neuroprotectant targets the NR3A receptor, thereby reducing neuronal damage, and is used to treat or prevent neuronal death associated with neurodegenerative diseases.

6. A method for screening drugs for treating mental illness based on the application of claim 1, characterized in that: By establishing a cell model expressing the NR3A target, it is used to screen candidate drugs that have a regulatory effect on the NR3A target, thereby screening potential antidepressant or antianxiety drugs.

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