Application of CX3CR1 as target to development / screening of drugs for relieving depression or construction of depression model animals

Abstract

The invention provides application of CX3CR1 as a target spot in developing / screening drugs for relieving depression or constructing depression model animals. Compared with the prior art, the invention has the following advantages: based on the discovery that the occurrence of depression can be caused by high expression or overexpression of CX3CR1, the invention provides the application of CX3CR1 as a target spot in developing / screening drugs for relieving depression, B6 / JGpt-Cx3cr1em1Cflox / Gpt and B6 / JGpt-Cx3cr1em1Cin (iCre) / Gpt mice are hybridized, and the CX3CR1 can be used as a target spot in developing / screening drugs for relieving depression. Meanwhile, temoxifen is used for inducing the temoxifen to specifically knock out CX3CR1 in microglial cells in the brain, so that the occurrence of the depression can be resisted, which indicates that the CX3CR1 possibly becomes a potential treatment target of the depression, and the temoxifen has important significance on clinical treatment of the depression.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of CX3CR1 as a target in the development / screening of drugs for alleviating depression or in the construction of depression model animals. Background Art

[0002] Depression is a common mental disorder, known as the "cold in psychiatry". Its lifetime prevalence is as high as 10% to 20%, and there are 350 million patients worldwide. Depression is characterized by high incidence, high recurrence, and high disability. It is the leading cause of disability around the world and a major factor in the global burden of disease. The biological etiology and pathogenesis of depression are still unclear, but it is generally believed that its onset is the result of the combined effects of genetic and environmental factors. The heritability of major depression is about 40%-50%. The pathogenesis of depression involves a variety of highly complex and interrelated pathways, mainly including monoamines, HPA axis, neurotrophic factors and neurogenesis, excitatory and inhibitory neurotransmission, mitochondrial dysfunction, epigenetics, inflammation, opioid system, myelination and gut-brain axis. The genetic etiology and pathological mechanism of depression are unclear, and there is a lack of universal drug targets, which brings great difficulties to clinical treatment. Intergenerational inheritance is one of the best models for studying the mutual regulation process of genetic and environmental factors in diseases. Previous studies have found that the offspring of male depressed mice exhibit depressive-like behaviors, proving that depression is inherited from generation to generation, and that small RNA in sperm directly mediates the occurrence of intergenerational inheritance of depression (Sci Adv, 2021); further studies have found that sperm small RNA can cause abnormalities in the early embryonic methylation reprogramming process and transmit this "abnormal information" to adulthood, leading to the occurrence of depressive traits (Nat Genet, Under Review); in this series of studies, through omics data screening, it was found that 20 genes represented by CX3CR1 are closely related to the occurrence of depressive behavior in offspring, and can be used as a candidate target gene library for the treatment of depression; currently, no research has systematically clarified the relationship between CX3CR1 and the occurrence of depression. Summary of the invention

[0003] In view of the above-mentioned technical limitations, the applicant conducted a comprehensive investigation into the relationship between the CX3CR1 gene and the occurrence of depression, and found that CX3CR1 is a key gene for the occurrence of depression and may become a potential therapeutic target for depression. Therefore, the present invention was proposed, that is, the application of CX3CR1 as a target in the development / screening of drugs for alleviating depression or in the construction of depression model animals; it overcomes the deficiencies and defects mentioned in the background technology and is of great significance to basic research and clinical depression treatment.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] The inventive point of the present invention is to provide the application of CX3CR1 as a target in the development / screening of drugs for alleviating depression.

[0006] Optionally, in the above application, the CX3CR1 is the CX3CR1 gene and / or the CX3CR1 protein.

[0007] Optionally, in the above application, the drug targets CX3CR1.

[0008] Optionally, in the above application, the drug can reduce or eliminate the relative expression level of CX3CR1 mRNA, and reduce or eliminate the protein expression of CX3CR1 in cells.

[0009] The second inventive aspect of the present invention is to provide the use of an agent for knocking down CX3CR1 in the preparation of a drug for treating / alleviating depression.

[0010] Optionally, in the above application, the drug for treating / alleviating depression further includes a pharmaceutically acceptable carrier and pharmaceutical excipients.

[0011] The third inventive point of the present invention is to provide the use of CX3CR1 as a target in constructing depression model animals.

[0012] Optionally, in the above application, the CX3CR1 is the CX3CR1 gene and / or the CX3CR1 protein.

[0013] Optionally, in the above application, the depression model animal is a commonly used depression model animal.

[0014] Optionally, in the above application, the animal is a mouse.

[0015] Compared with the prior art, the present invention has the following advantages: Based on the following findings: 1) CX3CR1 is highly expressed in different depression models; 2) Adeno-associated virus AAV-DIO-CX3CR1-GFP specifically overexpresses CX3CR1 in the hippocampus and prefrontal cortex of CX3CR1-Cre mice, which can lead to the occurrence of depression; 3) Overexpression of CX3CR1 overactivates microglia, produces proinflammatory cytokines, over-prunes neuronal synapses, and leads to a significant reduction in the number of synapses and dendritic spines, thereby leading to the occurrence of depression. The present invention provides the use of CX3CR1 as a target in the development / screening of drugs for relieving depression, and hybridizes B6 / JGpt-Cx3cr1em1Cflox / Gpt and B6 / JGpt-Cx3cr1em1Cin(iCre) / Gpt mice, and uses tamoxifen to induce specific knockout of CX3CR1 in microglia in the brain to resist the occurrence of depression, indicating that CX3CR1 may become a potential therapeutic target for depression, which is of great significance for the clinical treatment of depression. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of the modeling and behavioral testing of the CUMS model of mouse depression in one embodiment of the present invention.

[0017] Figure 2 The present invention shows the behavioral phenotype detection results of depression CUMS model mice (n=15) in one embodiment of the present invention. The behavioral phenotype detection experiments include tail suspension test TST, forced swimming test FST and novel environment feeding suppression test NSFT.

[0018] Figure 3 This is the QT-PCR detection result of CX3CR1 mRNA level in the prefrontal cortex PFC and hippocampus HPC of depression CUMS model mice in one embodiment of the present invention.

[0019] Figure 4 This is the WT detection result of CX3CR1 protein level in the prefrontal cortex PFC and hippocampus HPC of depression CUMS model mice in one embodiment of the present invention.

[0020] Figure 5 This is an immunofluorescence image of activated microglia using CD68 / iba1 marker in a mouse depression CUMS model according to an embodiment of the present invention, and a statistical graph of the intensity of CD68 / iba1 marker.

[0021] Figure 6 This is an immunofluorescence image of syn / psd95 labeled synapses in a mouse depression CUMS model according to an embodiment of the present invention.

[0022] Figure 7 This is a Golgi staining image of dendritic spines in the CUMS model of mouse depression according to an embodiment of the present invention.

[0023] Figure 8 This is the statistical result of the number of dendritic spines in the mouse depression CUMS model according to an embodiment of the present invention.

[0024] Fig. 9 This is a schematic diagram of the modeling of the LPS model and the LH model of mouse depression in one embodiment of the present invention, as well as the behavioral phenotype detection results of the depressed mice in the two models (n=15).

[0025] Fig.10 In one embodiment of the present invention, the WT detection results and relative quantitative analysis results of CX3CR1 protein levels in hippocampal HPCs of LPS model and LH model mice of depression are shown.

[0026] Fig.11 The results of QT-PCR detection of CX3CR1 mRNA levels in hippocampal HPCs of mice with LPS model and LH model of depression in one embodiment of the present invention.

[0027] Fig.12 In one embodiment of the present invention, in order to verify that overexpression of CX3CR1 can indeed increase susceptibility to depression, AAV vectors were used to inject the prefrontal cortex PFC and hippocampus HPC of CX3CR1-cre mice. One week later, the co-localized immunofluorescence images of microglia labeled with AAV vector GFP and iba1 were presented.

[0028] Fig.13 This is the behavioral phenotype test result of CX3CR1-cre mice four weeks after AAV vector injection in one embodiment of the present invention (n=14).

[0029] Fig.14 In one embodiment of the present invention, the WT detection result of CX3CR1 protein level and the QT-PCR detection result of CX3CR1 mRNA level in hippocampal HPCs after AAV vector injection into CX3CR1-cre mice are shown.

[0030] Fig.15 This is the QT-PCR detection result of the mRNA expression level of the inflammatory factors IL-6, IL-1β and TNF-α in the brain of CX3CR1-cre mice after AAV vector injection in one embodiment of the present invention.

[0031] Fig.16 In one embodiment of the present invention, after AAV vector was injected into CX3CR1-cre mice, immunofluorescence images of microglia activated by CD68 / iba1 marker and intensity statistics of CD68 / iba1 marker were presented.

[0032] Fig.17 This is an immunofluorescence image of syn / psd95 labeled synapses after AAV vector injection into CX3CR1-cre mice in one embodiment of the present invention.

[0033] Fig.18 In one embodiment of the present invention, the modeling of microglia-specific knockout CX3CR1 mouse model and the behavioral and molecular detection processes after four weeks are described. The CX3CR1-specific knockout mice are obtained by hybridizing CX3CR1-iCreERT2+ / - and CX3CR1flox / flox mice and then inducing with tamoxifen.

[0034] Fig.19 The present invention shows the behavioral phenotype detection results of the microglia-specific CX3CR1 knockout mouse model in one embodiment (n=14), wherein the behavioral phenotype detection experiments include tail suspension test TST, forced swim test FST, novel environment feeding suppression test NSFT, and open field test OFT. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the present invention more clear, the present invention is further described in detail below. However, it should be understood that the description herein is only used to explain the present invention and is not used to limit the scope of the present invention.

[0036] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention, and the terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The reagents and instruments used herein are all commercially available, and the characterization means involved can refer to the relevant descriptions in the prior art, which will not be repeated herein.

[0037] In order to further understand the present invention, the present invention is further described in detail below in conjunction with the best embodiment.

[0038] Example 1

[0039] Application of CX3CR1 as a target in the development / screening of drugs for the relief of depression.

[0040] CX3CR1 is CX3CR1 gene and / or CX3CR1 protein.

[0041] The drug targets CX3CR1.

[0042] The drug can reduce or eliminate the relative expression level of CX3CR1 mRNA and the protein expression of CX3CR1 in cells.

[0043] The present invention also provides use of a reagent for knocking down CX3CR1 in preparing a drug for treating / alleviating depression.

[0044] The drug used to treat / alleviate depression also includes pharmaceutically acceptable carriers and pharmaceutical excipients.

[0045] The present invention also provides the use of CX3CR1 as a target in constructing depression model animals.

[0046] CX3CR1 is CX3CR1 gene and / or CX3CR1 protein.

[0047] The present invention finds that CX3CR1 is highly expressed in depression model animals.

[0048] The animals are mice.

[0049] Example 2

[0050] This product studies and explains the genetic laws and pathogenesis of depression from a new intergenerational genetic perspective, and uses this as a basis to discover new targets for depression.

[0051] 1. Modeling:

[0052] 1. CUMS modeling:

[0053] Eight-week-old C57 BL / 6J male mice were subjected to chronic unpredictable stress modeling for 6 weeks, with 1-3 stimulations per day, including noise, strobe light, cage tilt, restraint, wet cage, and day and night reversal. Behavioral testing was performed after the modeling was completed.

[0054] The detailed modeling process of CUMS (García-Gutiérrez MS, Pérez-Ortiz JM, Gutiérrez-Adán A, Manzanares J. Depression-resistant endophenotype in mice overexpressing cannabinoid CB(2) receptors. Br J Pharmacol. 2010.) is shown in Table 1.

[0055] Table 1

[0056]

[0057] 2. LPS modeling:

[0058] Eight-week-old C57 BL / 6J male mice were intraperitoneally injected with 0.5 mg / kg LPS and saline for seven consecutive days, and behavioral tests were performed after the model was established (Cui Y, Yang Y, Ni Z, Dong Y, Cai G, Foncelle A, Ma S, Sang K, Tang S, Li Y, Shen Y, Berry H, Wu S, Hu H. Astroglial Kir4.1 in the lateralhabenula drives neuronal bursts in depression. Nature. 2018.).

[0059] 3. LH modeling:

[0060] The mice were stimulated with 0.15 mA current on their paws every day, randomly at 1-3 s / time, with 1-15 s interval, for a total of 360 stimulations, with a total time of 52 minutes. Behavioral tests were performed after two days of continuous stimulation (Chourbaji S, Zacher C, Sanchis-Segura C, Dormann C, Vollmayr B, Gass P. Learned helplessness: validity and reliability of depressive-like states in mice. Brain Res Brain Res Protoc. 2005.).

[0061] 4. Preparation of CX3CR1 knockout mice by hybridization:

[0062] CX3CR1-iCreERT2+ / - and CX3CR1flox / flox mice were hybridized to generate CX3CR1- / - whole-body knockout mice. When the mice were 30 days old, 150 mg / kg / day of tamoxifen was subcutaneously injected for three consecutive days to obtain CX3CR1-specific knockout mice of brain microglia.

[0063] Tamoxifen is a modeling drug. Tamoxifen (a kind of estrogen receptor molecule drug) regulates recombinase (CreER and FlpER) and is a more precise induction system that can achieve gene-specific knockout. Without tamoxifen, the CX3CR1 knockout mice produced by hybridization of B6 / JGpt-Cx3cr1em1Cflox / Gpt and B6 / JGpt-Cx3cr1em1Cin(iCre) / Gpt are systemic knockouts without tissue specificity. Intraperitoneal injection of tamoxifen for three days can specifically knock out CX3CR1 in brain microglia, and other tissues can restore the expression of CX3CR1, ultimately achieving specific knockout of CX3CR1 in the brain region.

[0064] 5. Behavioral testing methods:

[0065] 1) Open field experiment: The experimental device consists of an open field reaction box and an automatic data acquisition and processing system. The mouse open field reaction box is 25-30cm high, 72cm long at the bottom, the inner wall is painted black, and the bottom is evenly divided into 64 small squares. A digital camera is set up 2m above, and its field of view can cover the entire open field. The experiment is carried out in a quiet environment. Place the animal in the center of the bottom of the box, and take pictures and time it for 10 minutes. Clean the inner wall and bottom of the box to prevent the remaining information of the animal from the last time (such as the animal's urine, feces, and odor) from affecting the results of the next test. Replace the animal and continue the experiment.

[0066] 2) Tail suspension test: Use a small clip to clamp the mouse's tail 1 cm away, suspend it for 5 minutes, and detect its struggling and immobility time.

[0067] 3) Forced swimming: The mice were taught to swim in 25°C water for 15 min in advance. During the test, the mice were placed in 25°C warm water and recorded with a behavioral instrument for 5 min, recording their swimming, floating, and struggling time.

[0068] 4) Inhibition of eating in a new environment: The mice were fasted for 24 hours. During the test, the mice were placed from the corner into the open field, allowed to adapt to the environment for 1 minute, then taken out and a sugar pill / mouse chow was placed in the center of the open field. The instrument recorded the time taken by the mice from the time they were placed in the open field to the time they ate within five minutes.

[0069] 2. Verification of the association between CX3CR1 and depression caused by external stimuli:

[0070] 1. Using the CUMS mouse model, three different stimulations were given every day for 6 weeks ( Figure 1 ), behavioral phenotype detection revealed that CUMS model mice showed obvious behavioral despair ( Figure 2 ), further testing revealed that CX3CR1 mRNA levels ( Figure 3 ) and protein levels ( Figure 4 ) was significantly increased, and microglial activation increased ( Figure 5 ), decreased number of synapses and dendritic spines ( Figure 6-Figure 8 ), increased levels of inflammatory factors ( Fig. 9 ).

[0071] 1) Real-time quantitative QT-PCR analysis method:

[0072] Total RNA was purified using TRIzol (Invitrogen, 15596018) according to the manufacturer’s instructions and then III RT SuperMix for qPCR (+gDNAwiper) (Novizen, R323-01) was used to reverse transcribe mRNA. 1 μg of total RNA was used to remove genomic DNA using 4×gDNA wiper under the following conditions: 42°C for 2 minutes; then the mixture from the previous step was III qRT SuperMix was reverse transcribed into cDNA under the following conditions: 50°C for 15 minutes and 85°C for 5 minutes. Next, the RT products were amplified using ChamQ Universal SYBR qPCR Master Mix (Novizen, Q711-02) and CX3CR1-specific primers, IL-6, IL-1β, TNFα, and GAPDH. The reaction was incubated at 95°C for 5 minutes, followed by 40 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute. After the reaction was completed, the CT value was determined, and the relative level of mRNA was normalized to GAPDH and calculated by the 2-ΔΔCT method.

[0073] CX3CR1-specific primers:

[0074] Forward (SEQ ID No. 1): 5′-CTTCCCATCTGCTCAGGACC-3′;

[0075] Reverse (SEQ ID No. 2): 5′-ACCAGACCGAACGTGAAGAC-3′;

[0076] IL-6:

[0077] Forward (SEQ ID No. 3): 5′-TGTGCAATGGCAATTCTGAT-3′;

[0078] Reverse (SEQ ID No. 4): 5'-GGTACTCCAGAAGACCAGAGGA-3';

[0079] IL-1β:

[0080] Forward (SEQ ID No. 5): 5′-CCTGCAGCTGGAGAGTGTGGAT-3′;

[0081] Reverse (SEQ ID No. 6): 5′-TGTGCTCTGCTTGTGAGGTGCT-3′;

[0082] TNFα:

[0083] Forward (SEQ ID No. 7): 5′-AGCCCACGTCGTAGCAAACCAC-3′;

[0084] Reverse (SEQ ID No. 8): 5'-AGGTACAACCCATCGGCTGGCA-3';

[0085] GAPDH:

[0086] Forward (SEQ ID No. 9): 5′-TTGGCAGCACCAGTGGATGCAGGGA-3′;

[0087] Reverse (SEQ ID No. 10): 5'-AACTTTGGCATTGTGGAAGGGCTCA-3'.

[0088] Western blotting: Cells were rinsed with PBS (pH 7.4) and then lysed on ice for 30 min in RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with a cocktail of protease and phosphatase inhibitors (Thermo Scientific, Rockford, IL). Tissue samples were frozen solid in liquid nitrogen, ground into powder, and lysed on ice for 30 min in RIPA lysis buffer containing a cocktail of protease and phosphatase inhibitors. When necessary, samples were sonicated in an ice bath. Cell lysates and tissue homogenates were centrifuged at 4°C for 10 min (12,000 × g). Supernatants were collected and protein concentrations were determined using the Pierce BCA protein assay kit (Thermo Scientific, 23225, CA, USA). Subsequently, protein lysates (20 μg) were applied to acrylamide gels, proteins were separated by electrophoresis under denaturing conditions, and transferred to PVDF membranes. Total proteins were stained with Ponceau (Solarbio, P0012, Beijing, China) to ensure uniform loading. The membrane was blocked for 1 hour at room temperature with 5% skim dry milk (TBS) containing 0.1% Tween-20 (for phosphorylated proteins, the membrane was blocked for 1 hour with 5% bovine serum albumin in TBS containing 0.1% Tween-20) and incubated with the primary antibody overnight at 4°C. Washed 3 times with 1x TBST, 20 min each time. Incubated with the secondary antibody at room temperature for 1 hour, and then washed 3 times with 1x TBST, 10 minutes each time. The membrane was incubated with ECL substrate (Thermo Fisher, 46640, CA, USA) according to the manufacturer's instructions and detected with auto Article 643. The antibodies used for analyzing protein bands with ImageJ are as follows: anti-CX3CR1 (invitrogen, 1H14L7, 1:2000), anti-GAPDH (proteintech, 10494-1-AP, 1:10000).

[0089] 2) Immunofluorescence staining:

[0090] Mice were deeply anesthetized with sodium pentobarbital (50 mg / kg, intraperitoneal injection), perfused with 1× PBS and then cryopreserved with 4% PFA. The brain was fixed in 4% PFA overnight, dehydrated with 15% and 30% sucrose in 1× PBS at 4°C, and then frozen and sectioned. For immunofluorescence analysis, the sections were washed with 1× PBS and then blocked with 10% normal horse serum and 0.25% Triton X-100 in PBS (1 hour). Then, the sections were incubated overnight with primary antibodies diluted in blocking solution for CD68 (Bio-mad, MCA1957, 1:500) and Iba1 (Wako, 019-19714, 1:1000) as well as psd95 (Millipore, MAB1596, 1:500) and Synaptic I (Millipore, AB1543, 1:1000). On the second day, the sections were washed 6 times with 1x PBS for 5 min each time, and incubated in a blocking solution containing a secondary antibody for 1 hour. The sections were then washed 6 times with 1x PBS for 5 min each time, and mounted in an antifade containing DAPI (Biyuntian, P0131-25ml).

[0091] 3) Golgi staining:

[0092] FD Rapid GolgiStainTM Kit (FD rapid Golgi staining method) (PK401) was used. Mice were deeply anesthetized with sodium pentobarbital (50 mg / kg, intraperitoneal injection), and 1x PBS was perfused through the myocardium. The mouse brain was removed from the skull, and the surface blood was quickly rinsed in PBS solution. The brain was immersed in A / B solution prepared 24 hours in advance, and the solution was changed after 24 hours. After two weeks, the brain was immersed in C solution, and the solution was changed after 24 hours. The slices were stained after 7 days.

[0093] 2. In the mouse LPS model and LH model, the behavioral phenotype was detected and it was found that the model mice also showed obvious behavioral despair ( Fig.10 ); after further testing, it was found that the CX3CR1 protein level ( Fig.11 ) and mRNA levels ( Fig.12 ) increased significantly.

[0094] 1) Real-time quantitative QT-PCR analysis method:

[0095] Total RNA was purified using TRIzol (Invitrogen, 15596018) according to the manufacturer’s instructions and then III RT SuperMix for qPCR (+gDNAwiper) (Novizen, R323-01) was used to reverse transcribe mRNA. Briefly, 1 μg of total RNA was used to remove genomic DNA using 4×gDNA wiper under the following conditions: 42°C for 2 minutes; III qRT SuperMix was reverse transcribed into cDNA under the following conditions: 50°C for 15 minutes and 85°C for 5 minutes. Next, the RT products were amplified using ChamQ Universal SYBR qPCR Master Mix (Novizen, Q711-02) and CX3CR1-specific primers, IL-6, IL-1β, TNFα, and GAPDH. The reaction was incubated at 95°C for 5 minutes, followed by 40 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute. After the reaction was completed, the CT value was determined, and the relative level of mRNA was normalized to GAPDH and calculated by the 2-ΔΔCT method.

[0096] CX3CR1-specific primers:

[0097] Forward (SEQ ID No. 1): 5′-CTTCCCATCTGCTCAGGACC-3′;

[0098] Reverse (SEQ ID No. 2): 5′-ACCAGACCGAACGTGAAGAC-3′;

[0099] IL-6:

[0100] Forward (SEQ ID No. 3): 5′-TGTGCAATGGCAATTCTGAT-3′;

[0101] Reverse (SEQ ID No. 4): 5'-GGTACTCCAGAAGACCAGAGGA-3';

[0102] IL-1β:

[0103] Forward (SEQ ID No. 5): 5′-CCTGCAGCTGGAGAGTGTGGAT-3′;

[0104] Reverse (SEQ ID No. 6): 5′-TGTGCTCTGCTTGTGAGGTGCT-3′;

[0105] TNFα:

[0106] Forward (SEQ ID No. 7): 5′-AGCCCACGTCGTAGCAAACCAC-3′;

[0107] Reverse (SEQ ID No. 8): 5'-AGGTACAACCCATCGGCTGGCA-3';

[0108] GAPDH:

[0109] Forward (SEQ ID No. 9): 5′-TTGGCAGCACCAGTGGATGCAGGGA-3′;

[0110] Reverse (SEQ ID No. 10): 5'-AACTTTGGCATTGTGGAAGGGCTCA-3'.

[0111] 3. Experimental verification that CX3CR1 overexpression directly leads to depression:

[0112] AAV-DIO-CX3CR1-GFP virus (purchased from Shumi Technology, trade name rAAV-CX3CR1-EGFP-WPRE-hGHpolyA, trade number PT-0971) was microinjected into the prefrontal cortex and hippocampus of CX3CR1-Cre mice. One week later, it was observed that the virus began to express CX3CR1 specifically in microglia ( Fig.13 ), behavioral tests four weeks later revealed that overexpression of CX3CR1 caused mice to exhibit obvious depressive-like behaviors ( Fig.14 ), CX3CR1 protein level and mRNA level increased significantly ( Fig.15 ), and the level of inflammatory factors increased abnormally ( Fig.16 ), we also observed an increase in abnormal activation of microglia and a significant decrease in the number of synapses ( Figure 17-Figure 18 ).

[0113] 1) Real-time quantitative QT-PCR analysis method:

[0114] Total RNA was purified using TRIzol (Invitrogen, 15596018) according to the manufacturer’s instructions and then III RT SuperMix for qPCR (+gDNAwiper) (Novizen, R323-01) was used to reverse transcribe mRNA. Briefly, 1 μg of total RNA was used to remove genomic DNA using 4×gDNA wiper under the following conditions: 42°C for 2 minutes; III qRT SuperMix was reverse transcribed into cDNA under the following conditions: 50°C for 15 minutes and 85°C for 5 minutes. Next, the RT products were amplified using ChamQ Universal SYBR qPCR Master Mix (Novizen, Q711-02) and CX3CR1-specific primers, IL-6, IL-1β, TNFα, and GAPDH. The reaction was incubated at 95°C for 5 minutes, followed by 40 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute. After the reaction was completed, the CT value was determined, and the relative level of mRNA was normalized to GAPDH and calculated by the 2-ΔΔCT method.

[0115] CX3CR1-specific primers:

[0116] Forward (SEQ ID No. 1): 5′-CTTCCCATCTGCTCAGGACC-3′;

[0117] Reverse (SEQ ID No. 2): 5′-ACCAGACCGAACGTGAAGAC-3′;

[0118] IL-6:

[0119] Forward (SEQ ID No. 3): 5′-TGTGCAATGGCAATTCTGAT-3′;

[0120] Reverse (SEQ ID No. 4): 5'-GGTACTCCAGAAGACCAGAGGA-3';

[0121] IL-1β:

[0122] Forward (SEQ ID No. 5): 5′-CCTGCAGCTGGAGAGTGTGGAT-3′;

[0123] Reverse (SEQ ID No. 6): 5′-TGTGCTCTGCTTGTGAGGTGCT-3′;

[0124] TNFα:

[0125] Forward (SEQ ID No. 7): 5′-AGCCCACGTCGTAGCAAACCAC-3′;

[0126] Reverse (SEQ ID No. 8): 5'-AGGTACAACCCATCGGCTGGCA-3';

[0127] GAPDH:

[0128] Forward (SEQ ID No. 9): 5′-TTGGCAGCACCAGTGGATGCAGGGA-3′;

[0129] Reverse (SEQ ID No. 10): 5'-AACTTTGGCATTGTGGAAGGGCTCA-3'.

[0130] 2) Western blotting:

[0131] The cells were rinsed with PBS (pH 7.4) and then lysed on ice for 30 minutes in RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with a mixture of protease and phosphatase inhibitors (Thermo Scientific, Rockford, IL). Tissue samples were frozen solid in liquid nitrogen, ground into powder, and lysed on ice for 30 minutes in RIPA lysis buffer containing a mixture of protease and phosphatase inhibitors. When necessary, samples were sonicated in an ice bath. Cell lysates and tissue homogenates were centrifuged at 4°C for 10 min (12,000×g). The supernatant was collected and the protein concentration was determined using the Pierce BCA protein assay kit (ThermoScientific, 23225, CA, USA). Subsequently, the protein lysate (20 μg) was added to an acrylamide gel, the proteins were separated by electrophoresis under denaturing conditions, and transferred to a PVDF membrane. Total protein was stained with Ponceau (Solarbio, P0012, Beijing, China) to ensure uniform loading. The membrane was blocked for 1 hour at room temperature with 5% skim dry milk (TBS) containing 0.1% Tween-20 (for phosphorylated proteins, the membrane was blocked for 1 hour with 5% bovine serum albumin in TBS containing 0.1% Tween-20) and incubated with the primary antibody overnight at 4°C. Washed 3 times with 1x TBST, 20 min each time. Incubated with the secondary antibody at room temperature for 1 hour, and then washed 3 times with 1x TBST, 10 min each time. The membrane was incubated with ECL substrate (ThermoFisher, 46640, CA, USA) according to the manufacturer's instructions and detected with auto Article 643. The antibodies used for analyzing protein bands with ImageJ are as follows: anti-CX3CR1 (invitrogen, 1H14L7, 1:2000), anti-GAPDH (proteintech, 10494-1-AP, 1:10000).

[0132] 3) Immunofluorescence staining:

[0133] Mice were deeply anesthetized with sodium pentobarbital (50 mg / kg, intraperitoneal injection), perfused with 1× PBS and then cryopreserved with 4% PFA. The brain was fixed in 4% PFA overnight, dehydrated with 15% and 30% sucrose in 1× PBS at 4°C, and then frozen and sectioned. For immunofluorescence analysis, the sections were washed with 1× PBS and then blocked with 10% normal horse serum and 0.25% Triton X-100 in PBS (1 hour). Then, the sections were incubated overnight with primary antibodies diluted in blocking solution for CD68 (Bio-mad, MCA1957, 1:500) and Iba1 (Wako, 019-19714, 1:1000) as well as psd95 (Millipore, MAB1596, 1:500) and Synaptic I (Millipore, AB1543, 1:1000). On the second day, the sections were washed 6 times with 1x PBS for 5 min each time, and incubated in a blocking solution containing a secondary antibody for 1 hour. The sections were then washed 6 times with 1x PBS for 5 min each time, and mounted in an antifade containing DAPI (Biyuntian, P0131-25ml).

[0134] IV. Experimental verification of CX3CR1 knockout for the treatment / relief of depression:

[0135] B6 / JGpt-Cx3cr1em1Cflox / Gpt (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., the product name is C57BL / 6JGpt-Cx3cr1 em1Cflox / Gpt, product number T005904) and B6 / JGpt-Cx3cr1em1Cin(iCre) / Gpt (purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., product name C57BL / 6JGpt-Cx3cr1 em1Cin (iCre) / Gpt, product number T006768) mice were crossed, and when the offspring mice were 30 days old, 150 mg / kg tamoxifen was injected subcutaneously for three days to induce CX3CR1 in microglia cells in the brain, and depression modeling was started (CUMS modeling, by giving knockout mice depression modeling stimulation to confirm whether the gene knockout can prevent or resist the occurrence of depression). Four weeks later, behavioral tests ( Fig.19 ). Behavioral statistical results verified that microglia-specific knockout of CX3CR1 could resist the occurrence of depression.

[0136] 1. Behavioral testing methods:

[0137] 1) Open field experiment: The experimental device consists of an open field reaction box and an automatic data acquisition and processing system. The mouse open field reaction box is 25-30cm high, 72cm long at the bottom, the inner wall is painted black, and the bottom is evenly divided into 64 small squares. A digital camera is set up 2m above, and its field of view can cover the entire open field. The experiment is carried out in a quiet environment. Place the animal in the center of the bottom of the box, and take pictures and time it for 10 minutes. Clean the inner wall and bottom of the box to prevent the remaining information of the animal from the last time (such as the animal's urine, feces, and odor) from affecting the results of the next test. Replace the animal and continue the experiment.

[0138] 2) Tail suspension test: Use a small clip to clamp the mouse's tail 1 cm away, suspend it for 5 minutes, and detect its struggling and immobility time.

[0139] 3) Forced swimming: The mice were taught to swim in 25°C water for 15 min in advance. During the test, the mice were placed in 25°C warm water and recorded with a behavioral instrument for 5 min, recording their swimming, floating, and struggling time.

[0140] 4) Inhibition of eating in a new environment: The mice were fasted for 24 hours. During the test, the mice were placed from the corner into the open field, allowed to adapt to the environment for 1 minute, then taken out and a sugar pill / mouse chow was placed in the center of the open field. The instrument recorded the time taken by the mice from the time they were placed in the open field to the time they ate within five minutes.

[0141] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of CX3CR1 as a target in the development / screening of drugs for alleviating depression.

2. The use according to claim 1, characterized in that: The CX3CR1 is the CX3CR1 gene and / or the CX3CR1 protein.

3. The use according to claim 2, characterized in that: The drug targets CX3CR1.

4. The use according to any one of claims 1 to 3, characterized in that: The drug can reduce or eliminate the relative expression level of CX3CR1 mRNA, and reduce or eliminate the protein expression of CX3CR1 in cells.

5. Use of reagents for knocking down CX3CR1 in the preparation of drugs for treating / alleviating depression.

6. The use according to claim 5, characterized in that: The drug for treating / alleviating depression also includes a pharmaceutically acceptable carrier and pharmaceutical excipients.

7. Application of CX3CR1 as a target in constructing depression model animals.

8. The use according to claim 7, characterized in that: The CX3CR1 is the CX3CR1 gene and / or the CX3CR1 protein.

9. The use according to claim 8, characterized in that: The depression model animal is a commonly used depression model animal.

10. The use according to any one of claims 7 to 9, characterized in that: The animal is a mouse.

Images