CN115177726B - Use of GPR34 and its inhibitors in the preparation of therapeutic drugs for demyelination-related diseases - Google Patents

Abstract

The invention relates to GPR34 and application of an inhibitor thereof in preparation of a medicament for treating demyelinating related diseases. A series of in vivo experiments show that GPR34 can be used as a new target point for preparing a therapeutic drug for demyelinating related diseases, and a new scheme for screening and developing functions of the target GPR34 is effective for treating demyelinating related diseases such as experimental autoreactive encephalomyelitis, middle cerebral artery reperfusion, cyclohexanedihydrazone-induced demyelination, but has good application prospect in preparing a therapeutic drug for demyelinating related diseases due to GPR34 and inhibitors thereof, wherein the therapeutic drug is ineffective for treating anxiety central nerve inflammatory diseases induced by repeated electric shock on soles, chronic inflammation-mediated obesity induced by high fat and peripheral inflammation-mediated diseases such as lipopolysaccharide-induced acute inflammation-mediated sepsis. The research provides theoretical support for developing and designing the medicine taking GPR34 as a target spot, and can provide a new strategy for the treatment of demyelinating related diseases.

Description

Use of GPR34 and its inhibitors in the preparation of therapeutic drugs for demyelination-related diseases

Technical field

The invention belongs to the field of biotechnology, and specifically relates to the use of GPR34 and its inhibitors in the preparation of therapeutic drugs for demyelination-related diseases.

Background technique

During the process of central nervous system injury, myelination will be accompanied by demyelination, and demyelination has also been observed in a variety of neurological diseases, such as multiple sclerosis, ischemic stroke, Parkinson's disease, and Alzheimer's disease. Alzheimer's disease, spinal cord injury disease, traumatic brain injury, amyotrophic lateral sclerosis, frontotemporal dementia, schizophrenia, epilepsy (Adamo, 2014; Chen et al., 2020; and Lassmann, 2017; Shimizu et al., 2016; Werneburg et al., 2020). The connections between neurons are protected by peripheral myelin structures, and the integrity of myelin is critical to the maintenance of neuronal function. Past studies have shown that myelin fragments generated during demyelination can induce the activation of innate immune cells such as glial cells and macrophages, thereby promoting the occurrence of neuroinflammation and demyelination-related diseases (Clarner et al., 2012; Sun et al., 2010 ). In vitro experiments have shown that myelin fragments can induce the production of TNF-α, IL-6, IL-1b and other inflammatory factors after being phagocytosed by microglia, macrophages or endothelial cells (Sun et al., 2010; Wang et al., 2015; Williams et al., 1994; Zhou et al., 2019). In vivo experiments have found that the accumulation of myelin fragments caused by myelin fragment injection or brain injury can induce microglia activation and the expression of inflammatory factors (Clarner et al., 2012; Poliani et al., 2015; Sun et al., 2010). In fact, the efficiency of clearance of myelin fragments has important implications for multiple sclerosis, ischemic stroke, Parkinson's disease, Alzheimer's disease, spinal cord injury, traumatic brain injury, amyotrophic lateral sclerosis, frontotemporal Recovery from demyelination-related diseases such as dementia, schizophrenia, and epilepsy is also very important (Doyle and Buckwalter, 2020; Franklin and Ffrench-Constant, 2008; Neumann et al., 2009), but how do myelin fragments activate inflammatory responses and promote neural The pathological process of systemic diseases is not clear.

Microglia are a kind of tissue-resident macrophage unique to the central nervous system. They are the first line of defense for the central nervous system to eliminate infection or "danger" and play a role in regulating inflammation and homeostasis of the central nervous system. important role (Colonna and Butovsky, 2017; Heneka, 2019; Kierdorf et al., 2019). Like peripheral macrophages, overactivation of microglia can induce damage to neurons and oligodendrocytes by directly phagocytizing neurons or secreting cytokines, leading to myelin shedding, thereby exacerbating diseases such as multiple sclerosis. , ischemic stroke, Parkinson's disease, Alzheimer's disease, spinal cord injury, traumatic brain injury, amyotrophic lateral sclerosis, frontotemporal dementia, schizophrenia, epilepsy and other demyelination-related diseases progress. Myelin contains approximately 80% lipids, and some articles have reported that lipids can serve as danger-related signals to induce innate immune responses (Gong et al., 2019). So can microglia directly recognize lipids in myelin fragments, and what are their relevant receptors for lipid recognition?

G protein-coupled receptors (GPCRs) are a collective name for a class of seven-transmembrane membrane proteins. Many extracellular signal changes can be sensed by GPCRs, causing intracellular signal transduction to maintain body homeostasis (Gilman, 1987). GPCR dysfunction is closely related to many major human diseases. Therefore, many drugs are designed to target GPCR. According to statistics, drugs targeting GPCR account for more than 30% of all clinical drugs approved by the US FDA (O'Hayre et al., 2013). Therefore, searching for G protein-coupled receptors involved in regulating demyelination-related diseases can provide theoretical support for the later development and design of drugs to treat demyelination-related diseases and their effectiveness in the disease.

GPR34 is a G protein-coupled receptor and is downstream coupled to Gαi/o proteins. It is mainly expressed in microglia (Bedard et al., 2007; Butovsky et al., 2014; Engemaier et al., 2006), and studies have reported that its ligand is Lysophosphatidylserine LysoPS (Makide and Aoki, 2013; Sugo et al., 2006), and lysophosphatidylserine is produced by the hydrolysis of phosphatidylserine under the action of hydrolase. These works suggest that GPR34 of microglia may participate in the pathogenesis of demyelination-related diseases by recognizing lysophosphatidylserine derived from myelin fragments. Current basic research on GPR34 is mainly focused on its involvement in the inflammatory regulatory process. For example, after immunization with methylated BSA, compared with wild-type mice, the number of neutrophils and macrophages in the spleen of Gpr34-deficient mice was reduced. The number of cells was significantly reduced. In a delayed-type hypersensitivity test, Gpr34-deficient mice had significantly increased foot swelling compared with wild-type mice. Following Cryptococcus neoformans lung infection, Gpr34-deficient mice had a greater pathogen burden in extrapulmonary tissues compared with wild-type mice. However, there are no relevant reports on the role of GPR34 in demyelination-related diseases.

Experimental autoreactive encephalomyelitis (EAE) is a widely used animal model to study multiple sclerosis (Constantinescu et al., 2011; Xu et al., 2018), and middle cerebral artery reperfusion (MCAO) is a widely used For studying animal models of ischemic stroke (Joy et al., 2019; Liu et al., 2019), the cyclohexanoyl dihydrazone-induced demyelination model is an animal model widely used to study myelin loss and repair (Vega -Riquer et al., 2019; Wolf et al., 2018).

Contents of the invention

On the one hand, the object of the present invention is to provide the use of GPR34 and its inhibitors in the preparation of therapeutic drugs for demyelination-related diseases.

On the other hand, the present invention provides the use of substances that reduce the expression level of Gpr34 gene and/or functional inhibitors and/or antagonists of GPR34 protein in the preparation of drugs for treating demyelination-related diseases.

Further, the demyelination-related diseases include but are not limited to: multiple sclerosis, ischemic stroke, transverse myelitis, Hilder's disease, neuromyelitis optica, HTLV-I related myelopathy, Guillain-Barcelona Leigh syndrome, Barlow's disease, acute disseminated encephalomyelitis, central pontine myelinolysis, extrapontine myelinolysis, progressive multifocal leukoencephalopathy, posterior reversible encephalopathy syndrome, Canavan disease , Alexander disease, Lyme disease, heterogeneous leukodystrophy, adrenal neuropathy, glomerular leukodystrophy, Parkinson's disease, Alzheimer's disease, spinal cord injury disease, traumatic brain injury, amyotrophic lateral sclerosis , frontotemporal dementia, schizophrenia, epilepsy.

Further, the demyelination-related disease is selected from the group consisting of multiple sclerosis, ischemic stroke, transverse myelitis, Hilder's disease, neuromyelitis optica, HTLV-I related myelopathy, Guillain-Barré syndrome , Barlow's disease, acute disseminated encephalomyelitis, central pontine myelinolysis, extrapontine myelinolysis, progressive multifocal leukoencephalopathy, posterior reversible encephalopathy syndrome, Canavan disease, Alexander disease , Lyme disease, heterogeneous leukodystrophy, adrenoneuropathy, glomerular leukodystrophy, Parkinson's disease, Alzheimer's disease, spinal cord injury disease, traumatic brain injury, amyotrophic lateral sclerosis, frontotemporal in the group consisting of dementia, schizophrenia and epilepsy.

Further, the functional inhibitor and/or antagonist of the GPR34 protein is a GPR34 antibody or a small molecule inhibitor and/or antagonist of GPR34, preferably tyrosine, N-[((2E)-3-(4' -Chloro[1,1'-biphenyl]-4-yl)-1-oxo-2-propen-1-yl]-O-(phenylmethyl) or its structural analogues.

Further, the GPR34 antibodies include chimeric antibodies, monoclonal antibodies, polyclonal antibodies, humanized antibodies, bispecific antibodies and multispecific antibodies.

Furthermore, the substance that reduces the expression level of Gpr34 gene includes small interfering RNA that promotes the degradation of Gpr34 messenger RNA.

Further, the small interfering RNA is an RNAi fragment or siRNA fragment targeting the Gpr34 gene, or a CRISPR gRNA that promotes the degradation of Gpr34 messenger RNA.

On the other hand, the present invention provides a pharmaceutical composition for treating demyelination-related diseases. The pharmaceutical composition includes an active ingredient, which is a substance that reduces the expression level of Gpr34 gene and/or a functional inhibitor of GPR34 protein and /or antagonist.

In one embodiment, the pharmaceutical composition includes pharmaceutically acceptable excipients.

In one embodiment, the functional inhibitor and/or antagonist of the GPR34 protein is a GPR34 antibody or a small molecule inhibitor and/or antagonist of GPR34, preferably tyrosine, N-[((2E)-3 -(4'-Chloro[1,1'-biphenyl]-4-yl)-1-oxo-2-propen-1-yl]-O-(phenylmethyl) or its structural analogue.

In one embodiment, the substance that reduces the expression level of Gpr34 gene includes small interfering RNA that promotes degradation of Gpr34 messenger RNA.

In one embodiment, the small interfering RNA is an RNAi fragment or siRNA fragment targeting the Gpr34 gene.

In one embodiment, the demyelination-related diseases include, but are not limited to: multiple sclerosis, ischemic stroke, transverse myelitis, Hilder's disease, neuromyelitis optica, HTLV-I related myelopathy , Guillain-Barre syndrome, Barlow's disease, acute disseminated encephalomyelitis, central pontine myelinolysis, extrapontine myelinolysis, progressive multifocal leukoencephalopathy, posterior reversible encephalopathy syndrome, Canavan disease, Alexander disease, Lyme disease, heterogeneous leukodystrophy, adrenoneuropathy, glomerular leukodystrophy, Parkinson's disease, Alzheimer's disease, spinal cord injury, traumatic brain injury, amyotrophic side effects Thoracic sclerosis, frontotemporal dementia, schizophrenia, epilepsy.

In one embodiment, the demyelination-related disease is selected from the group consisting of multiple sclerosis, ischemic stroke, transverse myelitis, Hilder's disease, neuromyelitis optica, HTLV-I associated myelopathy, Guillain-Barcelona Leigh syndrome, Barlow's disease, acute disseminated encephalomyelitis, central pontine myelinolysis, extrapontine myelinolysis, progressive multifocal leukoencephalopathy, posterior reversible encephalopathy syndrome, Canavan disease , Alexander disease, Lyme disease, heterogeneous leukodystrophy, adrenal neuropathy, glomerular leukodystrophy, Parkinson's disease, Alzheimer's disease, spinal cord injury disease, traumatic brain injury, amyotrophic lateral sclerosis , frontotemporal dementia, schizophrenia, and epilepsy in the group.

In one embodiment, the pharmaceutical composition may be presented in unit dosage form containing a predetermined amount of active ingredient per unit dose. Preferred unit dosage compositions are those containing a daily dose or sub-dose, or an appropriate fraction thereof, of the active ingredient. Accordingly, such unit doses may be administered more than once a day. Preferred unit dosage compositions are those containing a daily dose or sub-dose (for administration more than once a day), or an appropriate fraction thereof, of an active ingredient, as herein recited.

In one embodiment, the pharmaceutical composition may be suitable for administration by any appropriate route, such as by oral, inhalational, parenteral (including subcutaneous, intramuscular, intravenous or intradermal) routes.

In one embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of tablets, capsules, pills, lozenges, powders, syrups, elixirs, suspensions, solutions, emulsions, and aerosols.

In one embodiment, the pharmaceutical composition is in a dosage form selected from the group consisting of tablets, capsules, pills, lozenges, powders, syrups, elixirs, suspensions, solutions, emulsions and aerosols.

In one embodiment, pharmaceutically acceptable excipients include the following types of excipients: diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents , wetting agent, solvent, co-solvent, suspending agent, emulsifier, sweetener, flavoring agent, odor masking agent, colorant, anti-caking agent, humectant, chelating agent, plasticizer, tackifier, Antioxidants, preservatives, stabilizers, surfactants, buffers. Those skilled in the art will appreciate that certain pharmaceutically acceptable excipients can serve more than one function and can serve alternative functions, depending on how many excipients are present in the formulation and which other excipients are present in the formulation middle.

In one embodiment, pharmaceutically acceptable excipients are selected from the group consisting of diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents , co-solvent, suspending agent, emulsifier, sweetener, flavoring agent, odor masking agent, colorant, anti-caking agent, humectant, chelating agent, plasticizer, tackifier, antioxidant, preservative, In the group consisting of stabilizers, surfactants and buffers.

In another aspect, the present invention provides a method of treating a demyelination-related disease or reducing the severity of a demyelination-related disease, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition as described above.

In one embodiment, the dosage of the pharmaceutical composition is, for example, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg , 14mg/kg, 15mg/kg, 16mg/kg, 17mg/kg, 18mg/kg, 19mg/kg, 20mg/kg, 21mg/kg, 22mg/kg, 23mg/kg, 24mg/kg, 25mg/kg, 26mg /kg, 27mg/kg, 28mg/kg, 29mg/kg, 30mg/kg or above.

In one embodiment, the dosage of the pharmaceutical composition is, for example, 5-30 mg/kg, 6-30 mg/kg, 7-30 mg/kg, 8-30 mg/kg, 9-30 mg/kg or above.

On the other hand, the present invention provides a method for treating demyelination-related diseases or reducing the severity of demyelination-related diseases, which includes knocking out the Gpr34 gene or inhibiting the expression of the Gpr34 gene in a subject in need thereof.

beneficial effects

By determining the role of GPR34 in experimental autoreactive encephalomyelitis, middle cerebral artery reperfusion and cyclohexanoyl dihydrazone-induced demyelination diseases, the present invention can provide treatment ideas and theoretical basis for demyelination-related diseases, and more It can provide molecular targets and preparation ideas for screening and preparing drugs for demyelination-related diseases.

Description of the drawings

In order to more clearly explain the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings needed to describe the embodiments or the prior art.

Figure 1 shows that in Example 1, myelin fragments derived from wild-type mice were used to stimulate microglia derived from wild-type mice and Gpr34-deficient mice, and then the cytokine interleukin 1b was detected by a real-time fluorescence quantitative PCR instrument. (Il1b) and interleukin 6 (Il6) expression results.

Figure 2 shows the detection of Gpr34 expression levels in microglia, astrocytes, neuronal cells, peritoneal macrophages, monocytes, and bone marrow-derived macrophages using a real-time fluorescence quantitative PCR instrument in Example 2. Result graph.

Figure 3 shows the results of exploring the changes in microglia-specific knockout of Gpr34 mice in the EAE model in Example 3, wherein Figure 3A shows that compared with control mice, microglia-specific knockout of Gpr34 The clinical scores of the mice were significantly reduced. Figure 3B shows that compared with control mice, lymphocyte infiltration in the spinal cord tissue of microglia-specific Gpr34 knockout mice was significantly reduced. Figure 3C shows that compared with control mice, microglia Demyelination was significantly reduced in the spinal cord tissue of mice with cell-specific knockout of Gpr34. Figure 3D shows the expression levels of cytokines Il1b and Il6 in the spinal cord tissue of mice with microglia-specific knockout of Gpr34 compared with control mice. Obvious reduction.

Figure 4 shows the results of exploring the changes in microglia-specific knockout of Gpr34 mice in the MCAO model in Example 3, wherein Figure 4A shows that compared with control mice, microglia-specific knockout of Gpr34 The clinical scores of the mice were significantly reduced. Figure 4B and Figure 4C show that the infarct area of the brain tissue of the microglia-specific Gpr34 knockout mice was significantly reduced compared with the control mice. Figure 4D shows that compared with the control mice, the infarct area of the brain tissue was significantly reduced. The expression levels of cytokines Il1b and Il6 were significantly reduced in the infarct tissue of glial cell-specific Gpr34 knockout mice.

Figure 5 shows the results of exploring the changes in microglia-specific Gpr34 knockout mice in the cyclohexanoyl dihydrazone-induced demyelination model in Example 3, wherein Figure 5A shows that compared with control mice, Microglia-specific knockout of Gpr34 mice had reduced demyelination in the cyclohexanoyl dihydrazone-induced model. Figure 5B shows that compared with control mice, microglia-specific knockout of Gpr34 mice had reduced demyelination in the cyclohexanoyl dihydrazone-induced model. Myelin-related synthesis genes myelin basic protein (Mbp), 2', 3'-cyclic nucleotide-3'-phosphodiesterase (Cnp), and myelin lipid protein in the corpus callosum tissue of the brain in the dihydrazone-induced model (Plp) expression levels were significantly reduced. Figure 5C shows that compared with control mice, microglia-specific knockout Gpr34 mice significantly reduced the expression levels of cytokines Il1b and Il6 in the brain corpus callosum tissue in the cyclohexanoyl dihydrazone-induced model. .

Figure 6 shows the results of exploring the changes in microglia-specific Gpr34 knockout mice in the anxiety model induced by repeated foot shock in Example 4, wherein Figure 6A shows that compared with control mice, microglia The total moving distance of the cell-specific knockout Gpr34 mice in the open field box did not change significantly. Figure 6B shows that compared with the control mice, the moving distance of the microglia-specific Gpr34 knockout mice in the center field did not change significantly. , Figure 6C shows that compared with control mice, microglia-specific knockout of Gpr34 mice has no significant change in the percentage of active time in the center field, Figure 6D shows that compared with control mice, microglia-specific knockout of Gpr34 There was no significant change in the number of times mice entered the central arena.

Figure 7 shows the results of exploring the changes of systemic knockout Gpr34 mice in high-fat-induced chronic inflammation-mediated obesity model in Example 5, wherein Figure 7A shows that compared with control mice, systemic knockout The food intake of Gpr34 mice did not change significantly. Figure 7B shows that the body weight of the systemic knockout Gpr34 mice did not change significantly compared with the control mice. Figure 7C shows that the systemic knockout Gpr34 mice compared with the control mice. There was no change in the fasting blood glucose test. Figure 7D shows that there was no significant change in the random blood glucose test in the systemic knockout Gpr34 mice compared with the control mice. Figure 7E shows that the insulin sensitivity in the systemic knockout Gpr34 mice was compared with the control mice. There was no significant change in the glucose tolerance test in the systemic knockout Gpr34 mice compared with control mice.

Figure 8 shows the results of the study in Example 5 of the changes in systemic Gpr34 knockout mice in the lipopolysaccharide-induced acute inflammation-mediated sepsis model.

Figure 9 shows the structural formula of the GPR34 inhibitor in Example 6, abbreviated as T602.

Figure 10 shows the results of exploring the therapeutic effect of GPR34 inhibitors in the EAE model in Example 6, wherein Figure 10A shows that compared with the control mice, the clinical scores of the mice in the inhibitor treatment group were significantly reduced, and Figure 10B shows that compared with the control mice Compared with mice in the inhibitor treatment group, lymphocyte infiltration in the spinal cord tissue was significantly reduced. Figure 10C shows that compared with the control mice, the demyelination in the spinal cord tissue of the mice in the inhibitor treatment group was significantly reduced. Figure 10D shows that compared with the control mice. Compared with mice, the expression levels of cytokines Il1b and Il6 in the spinal cord tissue of mice in the inhibitor treatment group were significantly reduced.

Figure 11 shows the results of exploring the therapeutic effect of GPR34 inhibitors in the MCAO model in Example 6. Figure 11A shows that compared with the control mice, the clinical scores of the mice in the inhibitor treatment group were significantly reduced. Figures 11B and 11C show that compared with the control mice, Compared with control mice, the infarct area of brain tissue of mice in the inhibitor treatment group was significantly reduced. Figure 11D shows that compared with control mice, the expression levels of cytokines Il1b and Il6 in the infarct tissue of mice in the inhibitor treatment group were significantly reduced.

Data statistical analysis

Unless otherwise noted in the illustrations, the data statistics used in this article are all statistically analyzed using GraphPad Prism (software version 6), and the analysis method uses two-tailed unpaired Student's t-test; *P<0.05, **P< 0.01, ***P<0.001, NS: no significant difference.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples can be purchased from conventional biochemical reagent stores unless otherwise specified.

MOG _35-55 peptide (Met-Glu-Val-Gly-Trp-Tyr-Arg-Ser-Pro-Phe-Ser-Arg-Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys): Made of Synthesized by Shanghai Qiangyao Biotechnology Co., Ltd.

GPR34 inhibitor: synthesized by Shanghai Taosu Biochemical Technology Co., Ltd., product number: T8848.

Pertussis toxin: List Biological, #181, 181238A1;

Inactivated Mycobacterium tuberculosis: Difico, 231141, 9347411.

Freund's incomplete adjuvant: Difico, 263910, 8008624.

MCAO thread bolt: Doccol.

High-fat food: Jiangsu Medison Biopharmaceutical Co., Ltd., MD1203.

Lipopolysaccharide: Invitrogen, LPS-EK Ultrapire.

Glucose: Sigma.

Insulin: Nova Nordisk.

Experimental animals: Wild-type C57BL/6J mice were purchased from Shanghai Slack Experimental Animal Co., Ltd., Gpr34 ^fl/fl mice and CX ₃ CR ₁ -Cre mice were purchased from Saiye Biotechnology Co., Ltd. Gpr34 ^fl/fl mice and CX ₃ CR ₁ -Cre mice were mated to produce CX ₃ CR ₁ -Cre.Gpr34 ^fl/fl mice. Because Gpr34 is highly expressed specifically in microglia and is hardly expressed in peripheral monocytes and macrophages, and CX ₃ CR ₁ is mainly expressed in monocytes and macrophages, it is considered that CX ₃ CR ₁ -Cre.Gpr34 ^{fl / The fl} mouse is a microglia-specific Gpr34-deficient mouse. The specific description is as follows: The construction process of the Gpr34 ^fl/fl mouse is to insert a 34-bp long DNA sequence (ATAACTTCGTATAGCATACATTATACGAAGTTAT) at both ends of exon 4 of the Gpr34 gene ( Contains two 13bp inverted repeat sequences and an 8bp core sequence, of which exon 4 of the Gpr34 gene is the expression region of the GPR34 protein). This 34bp sequence is the site recognized by the recombinase and is called the loxP site. Moreover, the loxp sites inserted into exon 4 of the Gpr34 gene have the same direction at both ends. Cre recombinase is a monomeric protein composed of 343 amino acids that can trigger DNA recombination at the loxP site. Any sequence of DNA that is located between two loxP sites with the same orientation will be deleted under the action of Cre recombinase. Because Cre recombinase is specifically expressed in monocytes and macrophages, and Gpr34 is specifically highly expressed in microglia and almost not expressed in peripheral monocytes and macrophages, it is believed that CX ₃ CR ₁ -Cre.Gpr34 ^{fl/ fl} mice are mice with microglia-specific deletion of Gpr34. Gpr34 ^fl/fl mice bred from CX ₃ CR ₁ -Cre.Gpr34 ^fl/fl mice are experimental control mice, and CX ₃ CR ₁ -Cre.Gpr34 ^fl/fl mice are microglia-specific deleted Gpr34 mice. mouse. Systemic Gpr34 knockout mice were obtained by technicians from the Animal Experiment Center of the School of Life Sciences of the University of Science and Technology of China through Crispr-Cas9 technology. Gpr34 ^+/+ mice bred from systemic Gpr34 knockout mice are experimental control mice, and Gpr34 ^−/− mice are Gpr34-deficient mice. All experimental animals in the experiments were kept in SPF facilities.

Example 1. Myelin debris stimulation upregulates the expression of cytokines Il1b and Il6 in microglia in a GPR34-dependent manner

1. Isolation of microglia: Take the brains of newborn mice that are 1-2 days old, remove the meninges and cut them into pieces. Digest them with 0.25% trypsin at 37°C for 10 minutes. Then add serum-containing medium to terminate the reaction. Use a 40-micron filter. Filter and transfer to a 15ml centrifuge tube, centrifuge at 1500rpm for 10 minutes, resuspend the pellet in serum-containing DMEM and transfer to a suspension culture bottle, replace the culture medium every 3 days. After 2 weeks, place the cells on a shaker and shake vigorously at 220 rpm and 37°C for 1 hour. Collect the supernatant and transfer to a 15 ml centrifuge tube. Centrifuge at 1500 rpm for 10 minutes. The precipitate is collected as microglia.

2. Preparation of myelin fragments: In order to avoid bacterial contamination, the following experimental procedures are carried out in a clean bench. Use sterile water to prepare a sucrose solution and process it with ToxinEraserTM Endotoxin Removal Kit (Company: GenScript, Cat. No.: L00308). The prepared sucrose solution was tested with an endotoxin detection kit (Company: GenScript, Cat. No.: L00350), and the result was lower than the minimum detection value. Kill the mice by cervical dislocation, then perfuse the heart with 10 ml of pre-cooled 1x PBS, peel out the brain tissue and spinal cord tissue, place them in a crushing tube, add 1 ml of 0.32M sucrose solution, crush the tissue, and then transfer it to a tube containing 4 ml of 0.32M sucrose solution. M sucrose solution in the ultracentrifuge tube, blow evenly, use a sterile Pasteur pipette to draw 4 ml of 0.85 M sucrose solution and slowly add it to the bottom of the ultracentrifuge tube, 75,000g, 30 minutes, 4°C. Use a Pasteur pipette to absorb the middle white layer, add it to an ultracentrifuge tube containing 8 ml of sterile water, keep in ice bath for 10 minutes, centrifuge at 75,000g for 30 minutes, and 4°C. Discard the supernatant, then add 8 ml of sterile water, incubate on ice for 10 minutes, centrifuge at 75,000g for 30 minutes, and 4°C. Discard the supernatant, add 5 ml of 0.32M sucrose solution, use a Pasteur pipette to draw 4 ml of 0.85M sucrose solution and slowly add it to the bottom of the ultracentrifuge tube, centrifuge at 75,000g for 15 minutes and 4°C. Aspirate the middle layer, add it to an ultracentrifuge tube containing 8 ml of 0.32M sucrose solution, and centrifuge at 75,000g for 10 minutes at 4°C. Discard the supernatant, add 300 μl of sterile water to resuspend, then freeze-dry and weigh.

3. Culture the isolated microglia in serum-free DMEM medium overnight. The next day, discard the medium and stimulate the microglia with myelin fragments at a concentration of 1 mg/ml. Collect the cells after 4 hours. Real-time fluorescence quantitative PCR was used to detect the expression of cytokines Il1b and Il6.

Figure 1 Experimental results show that when myelin fragments derived from wild-type mice are used to stimulate microglia derived from wild-type mice, the expression levels of cytokines Il1b and Il6 are increased. However, when microglia lack Gpr34, myelin fragments stimulate The up-regulated expression levels of cytokines Il1b and Il6 were significantly lower than those in wild-type mouse microglia. Among them, the expression of cytokine Il1b in Gpr34-deficient microglia accounted for 10% of the expression level in wild-type mouse microglia. The expression of cytokine Il1b in plasma cells accounts for about 37%, and the expression of cytokine Il6 accounts for about 31% of the expression of cytokine Il6 in microglia of wild-type mice, indicating that stimulation of myelin fragments upregulates the cytokines Il1b and Il6 in microglia. The expression of Il6 is dependent on GPR34.

Example 2. Gpr34 is highly expressed specifically in microglia

1. Isolation of astrocytes: Remove the meninges from 1-2-day-old mouse brains, cut into pieces, digest with 0.25% trypsin at 37°C for 10 minutes, then add DMEM (Gibco, C11965500BT) medium containing serum to terminate the reaction. , filter using a 40 micron filter mesh and transfer to a 15ml centrifuge tube, centrifuge at 1500rpm for 10 minutes, resuspend the pellet in DMEM containing serum and transfer to a suspension culture bottle, replace with fresh DMEM culture medium every 3 days. After 2 weeks, the cells were placed on a shaker and shaken vigorously at 220 rpm and 37°C for 1 hour. The supernatant was discarded, and then digested with 0.25% trypsin. The resulting cells were analyzed using flow cytometry antibody APC-ACSA1 (Company: Miltenyi Biotec, Catalog number: 130-098-803) and FITC-CD11b (Company: eBioscience, Catalog number: 11-0112-82, Clone number: M1/70) labeling, flow sorting CD11b ^- ACSA1 ⁺ cells into astrocytes .

2. Neuron isolation and culture: Coat the cell culture plate with polylysine (Company: Thermo Fisher Scientific, Catalog No.: A3890404) in advance, isolate the brains of newborn 1-2 day old mice, remove the meninges and separate the cerebral cortex. The cortex was digested with 0.25% trypsin for 10 minutes. Add serum-containing DMEM medium to terminate the reaction. Blow slightly and filter through a 40-micron filter mesh. Transfer the suspension to a 15ml centrifuge tube and centrifuge at 1500 rpm for 10 minutes. Use serum-containing medium. Resuspend the pellet in DMEM and distribute the cells in a 6-well plate at a volume of 5.0x10 ⁶ cells. After 24 hours, the culture medium was replaced with neuron matrix culture medium (Company: Thermo Fisher Scientific, Catalog No.: 21103049) containing B-27 (Company: ThermoFisher Scientific, Catalog No.: 17504044), and the neuronal matrix culture was replaced every 3 days. Basically, the cells obtained after 2 weeks are neuronal cells.

3. Isolation of peritoneal macrophages: Sacrifice the mice, and wash the peritoneal cavity with 20 ml of pre-cooled 1×PBS containing 1mM EDTA. The peritoneal lavage fluid was collected and then labeled with flow cytometry antibodies FITC-CD11b (Company: eBioscience, Catalog No.: 11-0112-82, Clone No.: M1/70) and APC-F4/80, (Company: eBioscience, Catalog No.: 17- 4801-82, clone number: BM8) CD11b ⁺ F4/80 ⁺ cells were sorted into peritoneal macrophages by flow cytometry.

4. Mononuclear cell isolation: Collect the mouse blood into a centrifuge tube containing anticoagulant. After lysing the red blood cells, label the flow cytometry antibody FITC-CD11b Company: eBioscience, Catalog No.: 11-0112-82, Clone No.: M1/ 70) and APC-Ly6C (Company: Biolegend, Catalog No.: 128016, Clone No.: HK1.4), CD11b ⁺ Ly6C ⁺ cells were sorted into monocytes by flow cytometry.

5. Isolation of bone marrow-derived macrophages: Isolate bone marrow from wild-type mice, lyse red blood cells, and mix monocytes and M-CSF (Company: Novus, Cat. No.: NBP1-99791, use concentration 10ng/ml) in a solution containing 10 After incubation in DMEM medium with % fetal bovine serum for 4 days, the differentiated cells were bone marrow-derived macrophages.

6. Collect microglia, astrocytes, neuronal cells, peritoneal macrophages, monocytes, and bone marrow-derived macrophages, and use a real-time fluorescence quantitative PCR instrument to detect the expression of Gpr34.

Figure 2 Experimental results show that the real-time fluorescence quantitative PCR instrument detects the expression level of Gpr34 in microglia, astrocytes, neuronal cells, peritoneal macrophages, monocytes, and bone marrow-derived macrophages. Gpr34 specificity Highly expressed in microglia.

Example 3. Microglia-specific Gpr34 knockout mice significantly reduce clinical symptoms in demyelination-related disease models

1. Microglia-specific knockout of Gpr34 mice significantly alleviated clinical symptoms in the EAE model

1. On day 0, prepare age- and gender-matched 8-week-old Gpr34 ^fl/fl and CX ₃ CR ₁ -Cre.Gpr34 ^fl/fl mice, where Gpr34 ^fl/fl is a control mouse derived from the same parent. . Dissolve pertussis toxin in physiological saline, dissolve inactivated Mycobacterium tuberculosis in Freund's incomplete adjuvant, prepare Freund's complete adjuvant containing 2.5 mg/ml inactivated Mycobacterium tuberculosis, and then dissolve MOG in the prepared In Freund's complete adjuvant, use a syringe to pipette repeatedly to form a milky liquid. 150 ng of pertussis toxin was injected intravenously, and then 150 μg of fully emulsified MOG peptide was injected subcutaneously on the back of both thighs.

2. On the second day, 150 ng of pertussis toxin was injected intravenously again, and the status of the mice was monitored daily for clinical scoring. 0 points, no clinical symptoms; 1 point, tail weakness; 2 points, tail paralysis and hind limb weakness; 3 points, hind limb paralysis; 4 points, front and rear limb paralysis; 5 points, imminent death.

Figure 3A results show that in the EAE model, the clinical scores of microglia-specific Gpr34 knockout mice were significantly reduced compared with wild-type mice. The clinical scores of microglia-specific Gpr34 knockout mice were is 0.9 points, and the clinical score of wild-type mice is 2.8 points.

3. On day 0, prepare age- and gender-matched 8-week-old Gpr34 ^fl/fl and CX ₃ CR ₁ -Cre.Gpr34 ^fl/fl mice, where Gpr34 ^fl/fl is a control mouse derived from the same parent. . Dissolve pertussis toxin in physiological saline, dissolve inactivated Mycobacterium tuberculosis in Freund's incomplete adjuvant, prepare Freund's complete adjuvant containing 2.5 mg/ml inactivated Mycobacterium tuberculosis, and then dissolve MOG in the prepared In Freund's complete adjuvant, use a syringe to pipette repeatedly to form a milky liquid. 150 ng of pertussis toxin was injected intravenously, and then 150 μg of fully emulsified MOG peptide was injected subcutaneously on the back of both thighs.

2. On the second day, 150 ng of pertussis toxin was injected intravenously again.

3. On the 13th day, sacrifice the mice, and then isolate the mouse spinal cord tissue, perform H&E staining, LFB staining, and detect the expression levels of cytokines Il1b and Il6 in the spinal cord tissue.

3.1H&E staining: Embed spinal cord tissue in paraffin, and cut 4-micron-thick spinal cord sections for pathological staining. Place the sections in an environmentally friendly tissue clearing agent for 20 minutes to remove paraffin from the tissue surface - absolute ethanol for 20 minutes - 90% ethanol for 5 minutes - 80% ethanol for 5 minutes - 70% ethanol for 5 minutes - rinse with distilled water for 5 minutes, and stain with hematoxylin For 5 minutes, use tap water to rinse the residual hematoxylin on the tissue surface, and then check whether it is rinsed thoroughly through a microscope. Treat with 80% ethanol for 2 minutes, stain with eosin for 2 minutes, 80% ethanol for 5 minutes, 95% ethanol for 5 minutes, and absolute ethanol. 10 minutes, environmentally friendly tissue clearing agent for 2 minutes to enhance the staining effect, seal the slide with neutral gum, dry it and observe the lymphocyte infiltration under a microscope.

3.2 LFB staining: Place the sections in environmentally friendly tissue clearing agent for 20 minutes to remove paraffin on the tissue surface - absolute ethanol for 20 minutes - 90% ethanol for 5 minutes - 80% ethanol for 5 minutes - 70% ethanol for 5 minutes - rinse with distilled water for 5 minutes minutes, incubate the sections in 0.1% LFB dye solution at 60°C overnight, rinse with tap water; place the sections in 70% ethanol for 5 minutes, immerse the sections in 0.05% lithium carbonate, and control the color separation time under the microscope until the color separation is complete (the gray matter part is visible) color becomes lighter), if the differentiation is not good, alternate these two steps several times until the microscopic examination is satisfactory, wash with water, counterstain with 0.1% eosin for 20 seconds, wash with environmentally friendly tissue clearing agent for 2 minutes to enhance the staining effect, and seal with neutral gum Take a film and observe the myelin sheath loss under the microscope.

3.3 Use real-time fluorescence quantitative PCR to detect the expression levels of cytokines Il1b and Il6 in spinal cord tissue.

Figure 3B Experimental results show that in the EAE model, lymphocyte infiltration in the spinal cord tissue of mice with microglia-specific knockout of Gpr34 was significantly reduced compared with wild-type mice, in which microglia-specific knockout of Gpr34 The number of infiltrating lymphocytes in mice accounts for about 30% of the number of infiltrating lymphocytes in wild-type mice (shown in the enlarged area of the box, the image scale is 100 μm), indicating that microglia-specific knockout of Gpr34 mice has a negative impact on the central nervous system of mice. Inflammation was significantly reduced compared with control mice.

The results of Figure 3C show that in the EAE model, compared with wild-type mice, the demyelination of the spinal cord tissue of mice with microglia-specific knockout of Gpr34 was significantly reduced, in which microglia-specific knockout of Gpr34 Myelin loss in mice accounts for about 20% of myelination loss in wild-type mice (box magnification area shown, image scale bar is 100 μm), indicating that microglia-specific Gpr34 knockout mice have obvious clinical symptoms. alleviate.

Figure 3D results show that in the EAE model, compared with wild-type mice, the expression of cytokines Il1b and Il6 in the spinal cord tissue of mice with microglia-specific knockout of Gpr34 was significantly reduced. In addition to the expression of cytokine Il1b in the spinal cord tissue of Gpr34 mice, which accounts for about 21% of the expression of cytokine Il1b in the spinal cord tissue of wild-type mice, the expression of cytokine Il6 in the spinal cord tissue accounts for about 21% of the expression of cytokine Il6 in the spinal cord tissue of wild-type mice. About 21%, indicating that the inflammation in the central nervous system of microglia-specific Gpr34 knockout mice is significantly reduced compared with control mice.

The above experimental results show that microglia-specific Gpr34 knockout mice significantly reduce clinical symptoms in the EAE model.

2. Microglia-specific knockout of Gpr34 mice significantly alleviated clinical symptoms in the MCAO model.

1. Prepare age- and gender-matched 8-week-old Gpr34 ^fl/fl and CX ₃ CR ₁ -Cre.Gpr34 ^fl/fl mice, where Gpr34 ^fl/fl is a control mouse derived from the same parent. After the mice were anesthetized with isoflurane, a small incision was made on the left side of the mouse's neck to separate the left external carotid artery and internal carotid artery. The distal end of the external carotid artery and superior thyroid artery were electrocoagulated. Insert a silicone suture plug (head diameter 0.23 mm) from the external carotid artery into the internal carotid artery. The insertion distance of the suture plug is about 10 mm to embolize the middle cerebral artery. During the operation, a heating pad was used to maintain the mouse's rectal temperature at approximately 37°C. After 60 minutes of embolization, the suture plug was pulled out to achieve reperfusion of the middle cerebral artery.

2. Clinical scores were performed 12 hours, 24 hours, and 48 hours after surgery: 0: No nerve damage; 1: The forelimb on the contralateral side of the ischemic brain cannot be fully extended during tail lifting; 2: Tail collision occurs when walking; 3: The body falls to the opposite side of the ischemic brain; 4: No spontaneous movement or roller-like movement.

3. 48 hours after surgery, sacrifice the mice and perfuse the heart with 1×PBS. Remove the brain from the olfactory bulb and cerebellum, cut the brain into 2 mm thick brain slices, and incubate with 0.5% 2,3,5-triphenyltetrazolium chloride (TTC for short, company: Sigma-Aldrich, product number: T8877) at 37°C Stain for 15 minutes in the dark, fix with 4% paraformaldehyde for 6 hours and then take pictures. Image J software is used to calculate the infarct ratio.

Figure 4A experimental results show that compared with wild-type mice, microglia-specific Gpr34 knockout mice have significantly lower clinical scores in the MCAO model, in which the clinical score of microglia-specific Gpr34 knockout mice is 1.3 points, and the clinical score of wild-type mice is 3.5 points.

The experimental results in Figure 4B and Figure 4C show that compared with wild-type mice, mice with microglia-specific knockout of Gpr34 have significantly reduced cerebral infarct area in the MCAO model, in which microglia-specific knockout of Gpr34 The average brain infarct area of mice is about 21%, and that of wild-type mice is about 45%.

4. Prepare age- and gender-matched 8-week-old Gpr34 ^fl/fl and CX ₃ CR ₁ -Cre.Gpr34 ^fl/fl mice, where Gpr34 ^fl/fl is a control mouse derived from the same parent. After the mice were anesthetized with isoflurane, a small incision was made on the left side of the mouse's neck to separate the left external carotid artery and internal carotid artery. The distal end of the external carotid artery and superior thyroid artery were electrocoagulated. Insert a silicone suture plug (head diameter 0.23 mm) from the external carotid artery into the internal carotid artery. The insertion distance of the suture plug is about 10 mm to embolize the middle cerebral artery. During the operation, a heating pad was used to maintain the mouse's rectal temperature at approximately 37°C. After 60 minutes of embolization, the suture plug was pulled out to achieve reperfusion of the middle cerebral artery.

4.1 48 hours after surgery, sacrifice the mice and perfuse them with 1×PBS through the heart. The brain was removed to remove the olfactory bulb and cerebellum, and the infarcted half-brain tissue was isolated, and the expression levels of cytokines Il1b and Il6 in the infarcted half-brain tissue were detected using a real-time fluorescence quantitative PCR instrument.

Figure 4D experimental results show that compared with wild-type mice, the expression levels of cytokines Il1b and Il6 in the infarcted half-brain tissue of microglia-specific Gpr34 mice in the MCAO model were significantly reduced, among which microglia The expression of cytokine Il1b in the infarcted half-brain tissue of mice with specific knockout of Gpr34 accounted for about 25% of the expression of cytokine Il1b in the infarcted half-brain tissue of wild-type mice, and the expression of cytokine Il6 in the infarcted half-brain tissue accounted for a smaller amount than that of wild-type mice. The expression level of cytokine Il6 in the infarcted half brain tissue of mice was about 27%.

The above experimental results show that microglia-specific Gpr34 knockout mice significantly reduce clinical symptoms in the MCAO model.

3. Microglia-specific knockout of Gpr34 mice significantly alleviated clinical symptoms in the cyclohexanoyl dihydrazone-induced demyelination model.

1. Prepare age- and gender-matched 8-week-old Gpr34 ^fl/fl and CX ₃ CR ₁ -Cre.Gpr34 ^fl/fl mice, where Gpr34 ^fl/fl is a control mouse derived from the same parent. Divided into four groups,

In the first group, 6 8-week-old Gpr34 ^fl/fl mice were fed normal food for 6 weeks.

In the second group, 6 8-week-old CX ₃ CR ₁ -Cre.Gpr34 ^fl/fl mice were fed normal food for 6 weeks.

The third group, 6 8-week-old Gpr34 ^fl/fl mice, were given diet containing 0.2% cyclohexanoyl dihydrazone (Company: Sigma-Aldrich, Product No.: C9012, the food was processed and synthesized by Beijing Keao Xieli Feed Co., Ltd.) Food feeding for 6 weeks.

The fourth group, 6 8-week-old CX ₃ CR ₁ -Cre.Gpr34 ^fl/fl mice, were fed with food containing 0.2% (i.e. 0.2g of cyclohexanoyl dihydrazone in 100g of food) cyclohexanoyl dihydrazone 6 weeks.

2. After feeding for 6 weeks, separate the brain tissue of the mice, embed the brain tissue in paraffin, cut out 6-micron-thick brain sections for LFB staining, and then count the myelin loss. The first group of data is represented by a solid circle, the second group of data is represented by an open circle, the third group of data is represented by an open square, and the fourth group of data is represented by a solid inverted triangle.

The experimental results in Figure 5A show that there is no significant change in the myelin condition of 8-week-old Gpr34 ^fl/fl and CX ₃ CR ₁ -Cre.Gpr34 ^fl/fl mice under physiological conditions. Six weeks after cyclohexanoyl dihydrazone induction, Gpr34 ^fl/fl mice had significantly more severe myelin loss than Gpr34 ^fl/fl mice under physiological conditions, indicating the rationality of the cyclohexanoyl dihydrazone-induced demyelination model. However, after 6 weeks of cyclohexanoyl dihydrazone induction, the demyelination of CX ₃ CR ₁ -Cre.Gpr34 ^fl/fl mice was significantly less than that of Gpr34 ^fl/fl mice, among which Gpr34 ^fl/fl was smaller. The myelin content of mice accounts for 50% of the myelin content of mice fed normal food, and the myelin content of CX ₃ CR ₁ -Cre.Gpr34 ^fl/fl mice accounts for 80% of the myelin content of mice fed normal food. It shows that microglia-specific Gpr34 knockout mice significantly reduce myelin demyelination in the cyclohexanoyl dihydrazone-induced demyelination model.

3. Prepare age- and gender-matched 8-week-old Gpr34 ^fl/fl and CX ₃ CR ₁ -Cre.Gpr34 ^fl/fl mice, where Gpr34 ^fl/fl is a control mouse derived from the same parent. Divided into four groups,

In the first group, 6 8-week-old Gpr34 ^fl/fl mice were fed normal food for 6 weeks.

In the second group, 6 8-week-old CX ₃ CR ₁ -Cre.Gpr34 ^fl/fl mice were fed normal food for 6 weeks.

The third group, 6 8-week-old Gpr34 ^fl/fl mice, were fed with food containing 0.2% cyclohexanoyl dihydrazone for 6 weeks.

The fourth group, 6 8-week-old CX ₃ CR ₁ -Cre.Gpr34 ^fl/fl mice, were fed with food containing 0.2% cyclohexanoyl dihydrazone for 6 weeks.

4. After 6 weeks of feeding, the corpus callosum tissue of the mouse brain was isolated, and the myelin-related synthesis genes myelin basic protein (Mbp) and 2', 3'-cyclic nucleotide-3'-phosphodiesterase (Cnp) were detected. ), myelin lipid protein (Plp), and the expression levels of cytokines Il1b and Il6. The first group of data is represented by a solid circle, the second group of data is represented by an open circle, the third group of data is represented by an open square, and the fourth group of data is represented by a solid inverted triangle.

Figure 5B experimental results show that under physiological conditions, the myelin-related synthesis genes myelin basic protein (Mbp), 2', 3 in 8-week-old Gpr34 ^{fl/fl and} CX ₃ CR ₁ -Cre. There were no significant changes in the expression of '-cyclic nucleotide-3'-phosphodiesterase (Cnp) and myelin lipid protein (Plp). Six weeks after ^{cyclohexanoyl} dihydrazone induction, the myelin-related synthesis genes myelin basic protein (Mbp) and 2', 3'-cyclic nucleotide-3'-phosphodiesterase ( The expression of Cnp) and myelin lipid protein (Plp) was significantly lower than that of Gpr34 ^fl/fl mice under physiological conditions, indicating the rationality of the cyclohexanoyl dihydrazone-induced demyelination model. However, after 6 weeks of cyclohexanoyl dihydrazone induction, the expression of the myelin-related synthesis gene myelin basic protein (Mbp) in CX ₃ CR ₁ -Cre.Gpr34 ^fl/fl mice accounted for the Mbp expression of mice fed normal food. 51%, but the Mbp expression in Gpr34 ^fl/fl mice only accounts for 13% of the Mbp expression in mice fed normal food. _The expression of the myelin-related synthesis gene 2', 3' _- cyclic nucleotide-3'-phosphodiesterase (Cnp) in CX 3 CR 1 -Cre.Gpr34 ^fl/fl mice accounted for Cnp in mice fed normal food. 60% of the expression amount, but the Cnp expression amount of Gpr34 ^fl/fl mice only accounts for 20% of the Cnp expression amount of mice fed normal food. The myelin-related synthesis genes of CX ₃ CR ₁ -Cre.Gpr34 ^fl/fl mice The expression of myelin lipid protein (Plp) accounts for 51% of the expression of Plp in mice fed normal food, but the expression of Plp in Gpr34 ^fl/fl mice only accounts for 21% of the expression of Plp in mice fed normal food, indicating that Microglia-specific Gpr34 knockout mice have less myelin loss and milder symptoms in the cyclohexanoyl dihydrazone-induced demyelination model.

Figure 5C experimental results show that the expression levels of cytokines Il1b and Il6 did not change significantly in 8-week-old Gpr34 ^fl/fl and CX ₃ CR ₁ -Cre.Gpr34 ^fl/fl mice under physiological conditions. After 6 weeks of cyclohexanoyl dihydrazone induction, the expression levels of cytokines Il1b and Il6 in Gpr34 ^fl/fl mice were significantly higher than those in Gpr34 ^fl/fl mice under physiological conditions, and the expression level of cytokine Il1b was increased 28 times. The expression of cytokine Il6 was increased by 9 times, indicating the rationality of the cyclohexanoyl dihydrazone-induced demyelination model. However, after 6 weeks of cyclohexanoyl dihydrazone induction, the expression levels of cytokines Il1b and Il6 in CX ₃ CR ₁ -Cre.Gpr34 ^fl/fl mice were up-regulated more than those in Gpr34 ^fl/fl mice. The expression level increased significantly, with the expression of cytokine Il1b increased by 12-fold and the expression of cytokine Il6 by 4-fold in CX ₃ CR ₁ -Cre.Gpr34 ^fl/fl mice. It was shown that microglia-specific knockout of Gpr34 mice had reduced inflammation in brain tissue in the cyclohexanoyl dihydrazone-induced demyelination model.

The above experimental results show that microglia-specific Gpr34 knockout mice significantly reduce clinical symptoms in the cyclohexanoyl dihydrazone-induced demyelination model.

To summarize Example 3, the above experimental results show that microglia-specific Gpr34 knockout mice are associated with demyelination such as experimental autoreactive encephalomyelitis, middle cerebral artery reperfusion, and cyclohexanoyl dihydrazone-induced demyelination. Clinical symptoms were significantly reduced in disease models.

Example 4. Microglia-derived GPR34 does not play a role in the anxiety model induced by repeated foot shock.

1. In the anxiety model induced by repeated foot shock, the behavioral characteristics of microglia-specific Gpr34 knockout mice did not change significantly compared with control mice.

1. Prepare age- and gender-matched 8-week-old Gpr34 ^fl/fl and CX ₃ CR ₁ -Cre.Gpr34 ^fl/fl mice, where Gpr34 ^fl/fl is a control mouse derived from the same parent. Before the experiment, the mice were placed in the laboratory for 1 hour to adapt to the environment, and then the mice were placed individually in a chamber with a grid floor connected to the shock generator. After being placed in the compartment for 2 minutes, the mice were randomly exposed to 0.6 mA footshocks 5 times for 3 seconds each within 120 seconds for 8 consecutive days. Before and after each experiment, clean the compartment with 75% alcohol to avoid any olfactory-induced experimental errors.

2. After 8 consecutive days of foot shock, the mice were moved from the feeding area to the experimental area room to adapt for 1 hour before conducting an open field experiment. Put the mouse from a corner of the open field box and let it move freely in the open field box. Record its movement trajectory for 5 minutes. Analyze the total moving distance of the mouse in the open field box and the movement distance in the central field. moving distance, the percentage of active time in the central venue, and the number of times entering the central venue. The results are shown in Figure 6. After each mouse experiment, wipe the open field box with 75% alcohol to eliminate the impact of odor on the mice.

Figure 6A Experimental results show that in the anxiety model induced by repeated foot shocks, compared with control mice, the total moving distance of microglia-specific Gpr34 knockout mice in the open field box during the experiment was not significant. Variety.

Figure 6B Experimental results show that in the anxiety model induced by repeated electric shock on the soles of the feet, compared with control mice, the moving distance of microglia-specific Gpr34 knockout mice in the central field did not change significantly during the experiment.

Figure 6C Experimental results show that in the anxiety model induced by repeated foot shocks, compared with control mice, microglia-specific Gpr34 knockout mice had no significant change in the percentage of active time in the central field during the experiment. .

Figure 6D Experimental results show that in the anxiety model induced by repeated foot shocks, compared with control mice, the number of microglia-specific Gpr34 knockout mice did not change significantly during the experiment.

The above experimental results indicate that microglia-derived GPR34 does not play a role in the anxiety model induced by repeated foot shock.

Example 5. Systemic knockout of Gpr34 mice has no obvious pathological changes in peripheral acute/chronic induced inflammation model.

1. In the chronic inflammation-mediated obesity model induced by high-fat food, compared with control mice, the obesity-related indicators of systemic Gpr34 knockout mice did not change significantly.

1. Prepare age- and gender-matched 6-week-old Gpr34 ^+/+ and Gpr34 ^-/- mice, where Gpr34 ^+/+ is a wild-type control mouse derived from the same parent, and then feed them high-fat food for 19 weeks. And record weight and diet. After 19 weeks, the mice were subjected to fasting blood glucose testing, random blood glucose testing, insulin sensitivity test, and glucose tolerance test.

2. Insulin sensitivity test: After the mice fasted for 4 hours, blood glucose was measured at 0 minutes, and then insulin 0.75U/kg was injected, and blood glucose was measured at 15 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes. Blood glucose was measured using tail vein bleeding and the OneTouchUltra Blood Glucose Test System Kit (purchased from Johnson & Johnson) was used for testing.

3. Glucose tolerance test: After the mice were fasted for 14 hours, blood glucose was measured at 0 minutes, and then glucose 1.5g/kg was injected, and blood glucose was measured at 15 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes.

Figure 7A Experimental results show that in the high-fat food-induced chronic inflammation-mediated obesity model, the food intake of systemic knockout Gpr34 mice did not change significantly compared with control mice.

Figure 7B Experimental results show that in the high-fat food-induced chronic inflammation-mediated obesity model, the body weight of systemic Gpr34 knockout mice did not change significantly compared with control mice.

Figure 7C experimental results show that in the high-fat food-induced chronic inflammation-mediated obesity model, there is no significant change in fasting blood glucose detection in systemic Gpr34 knockout mice compared with control mice.

Figure 7D Experimental results show that in the high-fat food-induced chronic inflammation-mediated obesity model, there is no significant change in random blood glucose detection in systemic Gpr34 knockout mice compared with control mice.

Figure 7E Experimental results show that in the high-fat food-induced chronic inflammation-mediated obesity model, there is no significant change in the insulin sensitivity test of systemic Gpr34 knockout mice compared with control mice.

Figure 7F Experimental results show that in the high-fat diet-induced chronic inflammation-mediated obesity model, there is no significant change in the glucose tolerance test of systemic Gpr34 knockout mice compared with control mice.

The above experimental results indicate that GPR34 is not involved in the obesity process mediated by chronic inflammation induced by high-fat food.

2. In the lipopolysaccharide-induced acute inflammation-mediated sepsis model, the survival rate of systemic Gpr34 knockout mice did not change significantly compared with control mice.

1. Prepare age- and gender-matched 8-week-old Gpr34 ^+/+ and Gpr34 ^-/- mice, where Gpr34 ^+/+ is a wild-type control mouse derived from the same parent, and then intraperitoneally inject 20 mg/kg lipopolysaccharide. The survival rate of mice was monitored every 12 hours.

Figure 8 Experimental results show that in the lipopolysaccharide-induced acute inflammation-mediated sepsis model, the survival rate of systemic knockout Gpr34 mice did not change significantly compared with control mice, indicating that GPR34 is not involved in lipopolysaccharide Induced acute inflammation mediated sepsis process.

To summarize Example 5, GPR34 is not involved in the chronic inflammation-mediated obesity process induced by high-fat food and the acute inflammation-mediated sepsis process induced by lipopolysaccharide.

Example 6. GPR34 inhibitors can effectively alleviate the pathogenesis of demyelination-related diseases

1. The structural formula of GPR34 inhibitor, referred to as T602, is shown in Figure 9 (Schoneberg et al., 2018).

T602 molecular formula: C ₃₁ H ₂₆ ClNO ₄ ;

T602 Chemical name: Tyrosine, N-[((2E)-3-(4'-chloro[1,1'-biphenyl]-4-yl)-1-oxo-2-propen-1-yl ]-O-(phenylmethyl).

2. GPR34 inhibitors can effectively alleviate the pathogenesis of EAE

1. On day 0, 8-week-old wild-type C56BL/6J mice of similar body weight were randomly divided into two groups, with 5 mice in each group. The specific grouping procedures are as follows:

Group 1: Intraperitoneal injection of 1xPBS containing 10% DMSO on days 0, 2, 4, 6, 8, 10, and 12, which is the control group.

Group 2: Intraperitoneal injection of GPR34 inhibitor (injection volume: 10 mg/kg, GPR34 inhibitor dissolved in 1xPBS containing 10% DMSO) on days 0, 2, 4, 6, 8, 10, and 12, that is, inhibitor treatment Group.

Then perform EAE induction, dissolve pertussis toxin in physiological saline, dissolve inactivated Mycobacterium tuberculosis in Freund's incomplete adjuvant, prepare Freund's complete adjuvant containing 2.5 mg/ml inactivated Mycobacterium tuberculosis, and then add MOG Dissolve in the prepared Freund's complete adjuvant and pipette repeatedly with a syringe to form a milky liquid. 150 ng of pertussis toxin was injected intravenously, and then 150 μg of fully emulsified MOG peptide was injected subcutaneously on the back of both thighs.

2. On the second day, 150 ng of pertussis toxin was injected intravenously again, and the status of the mice was monitored daily for clinical scoring. 0 points, no clinical symptoms; 1 point, tail weakness; 2 points, tail paralysis and hind limb weakness; 3 points, hind limb paralysis; 4 points, front and rear limb paralysis; 5 points, imminent death.

Figure 10A experimental results show that in the EAE model, compared with the control group of mice, the clinical symptom scores of the mice in the inhibitor treatment group were significantly reduced. The clinical score of the mice in the inhibitor treatment group was 1.2 points, and the clinical score of the mice in the control group was 1.2 points. The clinical score is 3 points, indicating that GPR34 inhibitors can effectively alleviate the pathogenesis of EAE.

3. On day 0, 8-week-old wild-type C56BL/6J mice of similar body weight were randomly divided into two groups, with 5 mice in each group. The specific grouping procedures are as follows:

Group 1: Intraperitoneal injection of 1xPBS containing 10% DMSO on days 0, 2, 4, 6, 8, 10, and 12, which is the control group.

Group 2: Intraperitoneal injection of GPR34 inhibitor (injection volume: 10 mg/kg, GPR34 inhibitor dissolved in 1xPBS containing 10% DMSO) on days 0, 2, 4, 6, 8, 10, and 12, that is, inhibitor treatment Group.

Then perform EAE induction, dissolve pertussis toxin in physiological saline, dissolve inactivated Mycobacterium tuberculosis in Freund's incomplete adjuvant, prepare Freund's complete adjuvant containing 2.5 mg/ml inactivated Mycobacterium tuberculosis, and then add MOG Dissolve in the prepared Freund's complete adjuvant and pipette repeatedly with a syringe to form a milky liquid. 150 ng of pertussis toxin was injected intravenously, and then 150 μg of fully emulsified MOG peptide was injected subcutaneously on the back of both thighs.

4. On the second day, 150 ng of pertussis toxin was injected intravenously again.

5. On the 13th day, sacrifice the mice, then isolate the mouse spinal cord tissue, perform H&E staining, LFB staining, and detect the expression levels of cytokines Il1b and Il6 in the spinal cord tissue.

5.1 H&E staining: Embed spinal cord tissue in paraffin, and cut 4-micron-thick spinal cord sections for pathological staining. Place the sections in an environmentally friendly tissue clearing agent for 20 minutes, remove paraffin from the tissue surface - absolute ethanol for 20 minutes - 90% ethanol for 5 minutes - 80% ethanol for 5 minutes - 70% ethanol for 5 minutes - rinse with distilled water for 5 minutes, and stain with hematoxylin for 5 minutes minutes, tap water microscopy returns to blue for 10 minutes, 80% ethanol treatment for 2 minutes, eosin staining for 2 minutes, 80% ethanol for 5 minutes, 95% ethanol for 5 minutes, absolute ethanol for 10 minutes, environmentally friendly tissue clearing agent for 2 minutes, To enhance the staining effect, seal the slide with neutral gum, dry it and observe the lymphocyte infiltration under a microscope.

5.2 LFB staining: Place the sections in environmentally friendly tissue clearing agent for 20 minutes to remove paraffin on the tissue surface - absolute ethanol for 20 minutes - 90% ethanol for 5 minutes - 80% ethanol for 5 minutes - 70% ethanol for 5 minutes - rinse with distilled water 5 minutes, incubate the sections in 0.1% LFB dye solution at 60°C overnight, rinse with tap water, put the sections in 70% ethanol for 5 minutes, immerse the sections in 0.05% lithium carbonate, and control the color separation time under the microscope until the color separation is complete (the gray matter part is visible) color becomes lighter), if the differentiation is not good, alternate these two steps several times until the microscopic examination is satisfactory, wash with water, counterstain with 0.1% eosin for 20 seconds, wash with water, use environmentally friendly tissue clearing agent for 2 minutes, seal with neutral gum, and observe under the microscope Myelin sheath loss.

5.3 Use real-time fluorescence quantitative PCR to detect the expression levels of cytokines Il1b and Il6 in spinal cord tissue.

Figure 10B experimental results show that in the EAE model, compared with mice in the control group, lymphocyte infiltration in the spinal cord tissue of mice in the inhibitor treatment group was significantly reduced, in which the number of infiltrating lymphocytes in the mice in the inhibitor treatment group accounted for 10% of the total number of infiltrating lymphocytes in the control group. About 20% of the number of infiltrating lymphocytes in mice (shown in the enlarged area of the box, the picture scale is 100 microns) shows that the inflammation in the central nervous system of the mice in the inhibitor treatment group is significantly reduced compared with the control mice.

Figure 10C experimental results show that in the EAE model, compared with the control group of mice, the demyelination of the spinal cord tissue of the mice in the inhibitor treatment group was significantly reduced, in which the demyelination of the mice in the inhibitor treatment group accounted for 10% of the control group. About 30% of the mice in the group lost myelin (shown in the enlarged area of the box, the picture scale is 100 microns), indicating that the clinical symptoms of the mice in the inhibitor treatment group were significantly reduced.

Figure 10D experimental results show that in the EAE model, compared with the control group of mice, the expression of cytokines Il1b and Il6 in the spinal cord tissue of the mice in the inhibitor treatment group was significantly reduced, among which the cells in the spinal cord tissue of the mice in the inhibitor treatment group were significantly reduced. The expression of factor Il1b accounted for about 25% of the expression of Il1b in the spinal cord tissue of mice in the control group. The expression of cytokine Il6 in the spinal cord tissue of mice in the inhibitor treatment group accounted for about 19% of the expression of Il6 in the spinal cord tissue of mice in the control group. It showed that the inflammation in the central nervous system of the mice in the inhibitor treatment group was significantly reduced compared with the control mice.

The above experimental results show that GPR34 inhibitors can effectively alleviate the pathogenesis of EAE.

3. GPR34 inhibitors can effectively alleviate the pathogenesis of MCAO

1. Take 8-week-old wild-type C56BL/6J mice of similar body weight and randomly group them into two groups, with 5 mice in each group. The specific grouping procedures are as follows:

Group 1: 1x PBS containing 10% DMSO was injected intraperitoneally 12 hours before MCAO induction, 0 hours, and 12 hours after MCAO induction, which is the control group.

Group 2: Intraperitoneal injection of GPR34 inhibitor (injection volume 10 mg/kg, GPR34 inhibitor dissolved in 1xPBS containing 10% DMSO) 12 hours before MCAO induction, and 12 hours after MCAO induction, that is, inhibitor treatment Group.

MCAO was induced. After anesthetizing the mice with isoflurane, a small incision was made on the left side of the mouse's neck to separate the left external carotid artery and internal carotid artery. The distal end of the external carotid artery and superior thyroid artery were electrocoagulated. Insert the silicone suture plug (head diameter 0.23mm) from the external carotid artery into the internal carotid artery. The insertion distance of the suture plug is about 10mm to embolize the middle cerebral artery. During the operation, a heating pad was used to maintain the mouse's rectal temperature at approximately 37°C. After 60 minutes of embolization, the suture plug was pulled out to achieve reperfusion of the middle cerebral artery.

2. Clinical scores were performed 12 hours, 24 hours, and 48 hours after surgery: 0: No nerve damage; 1: The forelimb on the contralateral side of the ischemic brain cannot be fully extended during tail lifting; 2: Tail collision occurs when walking; 3: The body falls to the opposite side of the ischemic brain; 4: No spontaneous movement or roller-like movement.

3. 48 hours after surgery, sacrifice the mouse, perfuse it with 1×PBS through the heart, remove the olfactory bulb and cerebellum from the brain, cut the brain into 2 mm thick slices, and stain in 0.5% TTC at 37°C for 15 minutes in the dark, 4 % paraformaldehyde was fixed for 6 hours and pictures were taken, and the infarct ratio was calculated using Image J software.

Figure 11A experimental results show that compared with mice in the control group, the clinical scores of the mice in the inhibitor treatment group were significantly reduced in the MCAO model. The clinical score of the mice in the inhibitor treatment group was 1.4 points, and the clinical score of the mice in the control group was 1.4. It's 3.8 points.

Figure 11B and Figure 11C Experimental results show that compared with mice in the control group, the area of cerebral infarction area in the inhibitor-treated mice in the MCAO model was significantly reduced, and the average area of cerebral infarction area in the inhibitor-treated mice was 17%. Around 45% of the brain infarction area of mice in the control group was on average.

4. Take 8-week-old wild-type C56BL/6J mice of similar weight and randomly group them into two groups, with 5 mice in each group. The specific grouping procedures are as follows:

Group 1: 1xPBS containing 10% DMSO was injected intraperitoneally 12 hours before MCAO induction, 0 hours, and 12 hours after MCAO induction, which is the control group.

Group 2: Intraperitoneal injection of GPR34 inhibitor (injection volume 10 mg/kg, GPR34 inhibitor dissolved in 1xPBS containing 10% DMSO) 12 hours before MCAO induction, and 12 hours after MCAO induction, that is, inhibitor treatment Group.

MCAO was induced. After anesthetizing the mice with isoflurane, a small incision was made on the left side of the mouse's neck to separate the left external carotid artery and internal carotid artery. The distal end of the external carotid artery and superior thyroid artery were electrocoagulated. Insert the silicone suture plug (head diameter 0.23mm) from the external carotid artery into the internal carotid artery. The insertion distance of the suture plug is about 10mm to embolize the middle cerebral artery. During the operation, a heating pad was used to maintain the mouse's rectal temperature at approximately 37°C. After 60 minutes of embolization, the suture plug was pulled out to achieve reperfusion of the middle cerebral artery.

5. 48 hours after surgery, the mice were sacrificed, and after cardiac perfusion with 1×PBS, the brains were removed to remove the olfactory bulb and cerebellum, and the infarcted half-brain tissue was isolated. The expression of cytokines Il1b and Il6 in the infarcted half-brain tissue was detected by a real-time fluorescence quantitative PCR instrument. level.

Figure 11D experimental results show that compared with mice in the control group, the expression levels of cytokines Il1b and Il6 in the infarcted half-brain tissue of the mice in the inhibitor-treated group were significantly reduced in the MCAO model. The mice in the inhibitor-treated group had the infarcted half of the brain. The expression of cytokine Il1b in the tissue accounted for about 32% of the expression of Il1b in the infarct half-brain tissue of mice in the control group, and the expression of cytokine Il6 in the infarct half-brain tissue of mice in the inhibitor treatment group accounted for about 32% of the expression of cytokine Il1b in the infarct half-brain tissue of mice in the control group. It accounts for about 25% of the expression of Il6.

The above experiments show that GPR34 inhibitors can effectively alleviate the pathogenesis of MCAO.

Summarizing Example 6, GPR34 inhibitors can effectively alleviate the pathogenesis of demyelination-related diseases such as EAE and MCAO.

The above experiments further confirmed that drugs targeting GPR34 can be developed and designed to provide new strategies for the treatment of demyelination-related diseases.

The above examples are for illustrating the disclosed embodiments of the present invention and are not to be construed as limitations of the present invention. In addition, various modifications listed herein, as well as changes in methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention.

Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to achieve the invention should be included in the scope of the present invention.

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Claims (5)

1. Use of a substance that reduces the expression level of the Gpr34 gene and/or a functional inhibitor and/or antagonist of the Gpr34 protein for the manufacture of a medicament for the treatment of a demyelinating-related disease selected from the group consisting of multiple sclerosis, ischemic stroke, alzheimer's disease, amyotrophic lateral sclerosis.

2. Use according to claim 1, wherein the functional inhibitor and/or antagonist of GPR34 protein is a GPR34 antibody or a small molecule inhibitor and/or antagonist of GPR 34.

3. Use according to claim 2, wherein the small molecule inhibitor and/or antagonist of GPR34 is T602, and wherein the structural formula of T602 is as follows:

4. the use of claim 1, wherein the agent that reduces the expression level of the Gpr34 gene comprises a small interfering RNA that promotes degradation of the Gpr34 messenger RNA.

5. The use of claim 4, wherein the small interfering RNA is an RNAi or siRNA fragment targeting the Gpr34 gene, or a CRISPR gRNA that promotes degradation of the Gpr34 messenger RNA.