Use of a natural small molecule compound in the preparation of a medicament for treating a disease associated with CSF1R loss of function

Abstract

The application of a natural small molecule compound in the preparation of a drug for treating a disease related to CSF1R function loss belongs to the technical field of medicine. The natural small molecule compound Diosmetin-7-O-beta-D-glucopyranoside (DDG) can effectively improve the disease phenotype of a CSF1R function loss model mouse by up-regulating the transcription and protein expression of CSF1R in microglia through enhancing the activity of a super-enhancer FIRE in the second intron of the CSF1R gene. The drug can be prepared into injection preparations, oral preparations, spray preparations, ointment preparations or patches, and provides a natural small molecule treatment scheme for diseases related to the weakening / loss of CSF1R content or function, and has a wide clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biotechnology, specifically relating to the application of a natural small molecule compound in the preparation of drugs for treating diseases related to CSF1R function deficiency. Background Technology

[0002] Colony-stimulating factor 1 receptor (CSF1R), also known as c-FMS, is a tyrosine kinase receptor. Colony-stimulating factor 1 (CSF-1) and interleukin 34 (IL-34) are ligands of CSF1R. CSF1R-mediated signaling is crucial for the survival, function, proliferation, and differentiation of myeloid cells, including microglia, osteoclasts, monocytes / macrophages, Langerhans cells in the skin, and Paneth cells in the intestine. CSF1R plays a crucial role in the development and maturation of oocytes, trophoblasts, and neural progenitor cells in the female reproductive tract. Mice lacking the CSF1R gene exhibit multiple defects in macrophage development, reproduction, and tissue remodeling (Konno, T., et al., Clinical and genetic characterization of adult-onset leukoencephalopathy with axonal spheroids and pigmented glia associated with CSF1R mutation. Eur J Neurol, 2017. 24(1): p.37-45). Clinically, heterozygous mutations in the CSF1R gene can lead to CSF1R-related disorder (CSF1R-RD), a neurodegenerative disease characterized by progressive cognitive impairment, for which effective treatments are still lacking. Currently, clinical treatment for CSF1R-RD is limited to symptomatic and supportive care. The effectiveness of dopamine-like drugs or antidepressants in treating Parkinson's-like symptoms and depression has not been clinically proven. As the disease progresses, patients experience significant changes in personality, psychological state, and motor function, impacting their quality of life. Therefore, finding effective treatments for CSF1R-RD is crucial for prolonging the lives of CSF1R-RD patients and improving their quality of life.

[0003] The second intron downstream of the first exon of CSF1R contains a conserved sequence called the fms-intron regulation element (FIRE), which was identified as a super enhancer in whole-genome analysis of mouse macrophage chromatin. It is an important regulatory element of the CSF1R gene in macrophages and controls transcript elongation during macrophage-specific transcription of CSF1R (Yue X, Favot P, Dunn TL, et al. Expression of mRNA encoding the macrophage colony-stimulating factor receptor (c-fms) is controlled by a constitutive promoter and tissue-specific transcriptionelongation[J]. Mol Cell Biol, 1993,13(6):3191-201). In recent years, several research teams have published genome-wide analyses analyzing the locations of promoter- and enhancer-related modified histones in monocytes and macrophages, and detecting binding sites for various transcription factors through chromatin immunoprecipitation (Tada, M., etal., Characteristic microglial features in patients with hereditary diffuseleukoencephalopathy with spheroids. Ann Neurol, 2016. 80(4): p. 554-65). This sequence contains binding sites for a large number of macrophage-expressed transcription factors, including Ets, PU.1, ATF, C / EBP, RUX, AP-1, IRF, STAT, KLF, REL, and FUS / TLS (Pixley, FJ and ER Stanley, CSF-1 regulation of the wandering macrophage: complexity in action. Trends Cell Biol, 2004. 14(11): p. 628-38). During the differentiation of immature precursor cells into macrophages, transcription factor recruitment and chromatin remodeling first occur at the proximal CSF1R promoter and then at FIRE, resulting in differentiated macrophages expressing higher levels of CSF1R.In mouse macrophages, the antisense promoter activity of FIRE is induced by stimuli such as LPS, phorbol ester, or CSF-1. These stimuli alter the transcription factor occupancy of FIRE cis-acting elements, including the binding sites of RUNX1, AP1, and Sp1, thereby inhibiting CSF1R transcription, demonstrating that FIRE acts as an antisense promoter in macrophages. Furthermore, changes in the orientation of FIRE significantly reduce its activity in macrophages, and mutations in the transcription start site within FIRE also downregulate CSF1R mRNA transcription, indicating that FIRE is a direction-specific transcriptional enhancer element (Mouchemore, KA and FJPixley, CSF-1 signaling in macrophages: pleiotrophy through phosphotyrosine-based signaling pathways. Critical Reviews in Clinical Laboratory Sciences, 2012. 49(2): p. 49-61).

[0004] A recent study showed that the absence of FIRE in mice selectively affects CSF1R expression and macrophage development in tissues such as the brain, skin, kidneys, heart, and peritoneum, demonstrating that FIRE is functionally important only in specific macrophage populations (Munro DAD, Bradford BM, Mariani SA, Hampton DW, Vink CS, Chandran S, Hume DA, Pridans C, Priller J. CNS macrophages differentially rely on an intronic CSF1R enhancer for their development. Development. 2020 Dec 15;147(23):dev194449). FIRE-deficient mice were healthy, fertile, and exhibited normal growth, neural development, or other developmental characteristics compared to CSF1R-deficient mice, suggesting that FIRE is not required for CSF1R expression in all types of myeloid cells.

[0005] Clinically, CSF1R mutations are mostly heterozygous mutations with loss of function. Therefore, it is essential to enhance FIRE activity through high-throughput drug screening to increase CSF1R allele expression and upregulate CSF1R protein levels in order to treat CSF1R-RD. Summary of the Invention

[0006] The purpose of this invention is to address the lack of effective therapeutic drugs for CSF1R loss-of-function related diseases (such as CSF1R-RD) in the prior art, especially the lack of drugs that can upregulate CSF1R expression and improve disease phenotype by regulating key regulatory elements of the CSF1R gene (such as the super enhancer FIRE in the second intron). This invention provides a natural small molecule compound DDG based on enhancing CSF1R expression, its screening method, pharmaceutical composition and application, for improving, alleviating or treating CSF1R loss-of-function related diseases.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0008] This invention provides a natural small molecule compound, Diosmetin-7-O-β-D-glucopyranoside (DDG for short), whose chemical structure is shown in formula (I):

[0009]

[0010] (I)

[0011] The natural small molecule compound DDG was obtained through high-throughput screening. DDG itself or its modifiers that enhance CSF1R expression activity are the core active ingredients. DDG can specifically enhance the activity of the super enhancer FIRE in the second intron of the CSF1R gene. DDG also has good biocompatibility and low toxicity risk.

[0012] This invention provides a method for screening natural small molecule compound DDG, comprising the following steps:

[0013] (1) Construction of pGL3-FIRE plasmid: The FIRE fragment of the second intron of the CSF1R gene was amplified from the human genome, inserted into the pGL3 vector, and obtained by double digestion with KpnI / XhoI, ligation with T4 ligase, transformation with DH5α and sequencing verification to obtain the recombinant plasmid pGL3-FIRE;

[0014] (2) Cell transfection: The recombinant plasmid pGL3-FIRE constructed in step (1) was transfected into HEK 293T host cells;

[0015] (3) Compound treatment: Add the compounds from the natural small molecule compound library to be screened into the transfected host cells to make the final concentration of the compounds 20 μM, and set up a blank control group without adding the compounds;

[0016] (4) Screening and verification: The activity was detected by dual luciferase reporter system, and compounds with FIRE activity upregulated by ≥1.5 times were screened. DDG was confirmed after re-screening and gradient verification.

[0017] In step (3), the library of natural small molecule compounds to be screened includes, but is not limited to, the TargetMol library.

[0018] In step (4), the secondary screening preferably selects the top 20 compounds in terms of activity from the initial screening, and after gradient verification, DDG is determined to be the best FIRE agonist.

[0019] The present invention provides a pharmaceutical composition for improving, alleviating or treating diseases related to CSF1R deficiency, wherein the active ingredient of the pharmaceutical composition is the natural small molecule compound DDG or a modifier thereof that enhances CSF1R expression activity.

[0020] The modified form that enhances CSF1R expression activity is a derivative of DDG that, after being modified by hydroxyl substitution, methylation, or esterification, can still enhance the activity of the CSF1R gene superenhancer FIRE.

[0021] The pharmaceutical composition further comprises pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients consist of 10% DMSO, 40% PEG400 and 50% physiological saline; the final concentration of DDG in the injectable pharmaceutical composition is 1.25 mg / mL.

[0022] The dosage form of the pharmaceutical composition is selected from injectable preparations, oral preparations, spray preparations, ointment preparations, or patches; when the dosage form is an injectable preparation, the administration method includes, but is not limited to, intraperitoneal injection, and the preferred dosage is 10 mg / kg body weight / day.

[0023] The natural small molecule compound DDG or its modified form that enhances CSF1R expression activity described in this invention can be used in the preparation of drugs to improve, alleviate or treat diseases related to CSF1R dysfunction.

[0024] In vitro and in vivo experiments have verified that DDG can significantly improve microglial homeostasis, reduce axonal damage, and improve motor and learning / memory function deficits; DDG can significantly upregulate the mRNA and protein expression levels of CSF1R in microglia.

[0025] The drug can improve the neuropathological phenotype and behavioral defects in CSF1R dysfunction model animals.

[0026] This invention provides a method for upregulating CSF1R expression, comprising treating cells or individuals expressing CSF1R with a natural small molecule compound that enhances the activity of the superenhancer FIRE within the second intron of the CSF1R gene; wherein the natural small molecule compound is DDG or a modifier thereof that enhances CSF1R expression activity.

[0027] Furthermore, when treating cells, the concentration of DDG or its modifier that enhances CSF1R expression activity is 40 μM, and the treatment time is 36 h; when treating individuals, the continuous administration time of DDG or its modifier that enhances CSF1R expression activity is at least 28 days.

[0028] The CSF1R loss-of-function related diseases include, but are not limited to, neurodegenerative diseases, myeloid cell developmental disorders, and tumor microenvironment-related diseases caused by reduced or weakened / absent CSF1R content; among them, neurodegenerative diseases are preferably those caused by heterozygous deletion of the CSF1R gene or CSF1R I792T mutation, and more preferably, adult-onset axonoglobulinosis and glioblastoma pigmentosum leukoencephalopathy (ALSP).

[0029] This invention provides a FIRE luciferase reporter gene plasmid, which is constructed by inserting the FIRE fragment from the second intron of the CSF1R gene into the pGL3 vector.

[0030] Multiple experimental results confirm that the present invention screens and obtains a natural small molecule compound DDG that can specifically regulate the activity of the super enhancer FIRE in the second intron of the CSF1R gene, and confirms that it can significantly upregulate the expression of CSF1R. This compound can improve the disease phenotype of mice with CSF1R loss of function and can be used to prepare therapeutic drugs for patients with CSF1R loss of function.

[0031] Compared with the prior art, the significant advantages of the present invention are:

[0032] This invention is the first to discover a natural small molecule compound that enhances the protein expression of the colony-stimulating factor 1 receptor (CSF1R) gene by increasing the activity of a super-enhancer within the second intron. A high-throughput screening strategy based on FIRE activity is precise and efficient; the screened compound DDG significantly upregulates the transcriptional and protein levels of CSF1R. This compound can be used to treat or alleviate diseases related to weakened CSF1R function. It can effectively improve, alleviate, or treat neuropathological phenotypes and motor and cognitive deficits in CSF1R dysfunction models, filling the treatment gap for CSF1R-RD and related diseases. Furthermore, the natural small molecule compound exhibits good biocompatibility and can be formulated into various dosage forms. This invention provides a method for developing a natural small molecule drug based on enhancing CSF1R expression and its application in the preparation of drugs for diseases related to weakened CSF1R levels or function, showing promising clinical application prospects. Attached Figure Description

[0033] Figure 1The images show the construction and activity verification results of the FIRE luciferase reporter gene plasmid. A shows a schematic diagram of the pGL3-FIRE recombinant plasmid construction; B shows the Sanger sequencing verification results of the pGL3-FIRE plasmid; and C shows the dual-luciferase reporter gene detection results.

[0034] Figure 2 High-throughput drug screening identified DDG as a FIRE agonist. A shows the FIRE agonist screening flowchart; B shows the FIRE activity heatmap of the initial screening of 1500 natural small molecule compounds; C shows the rescreening results of the top 20 compounds; D shows the FIRE activity verification results of 7 positive compounds; and E shows the dose-effect verification of DDG on FIRE activity.

[0035] Figure 3 DDG upregulated the transcriptional level of the human microglia cell line CSF1R. A shows the mRNA transcriptional level of CSF1R in HMC3 cells detected by RT-PCR; B shows the mRNA level of the microglia homeostasis marker P2RY12 detected by RT-PCR; and C shows the mRNA levels of ARG1 and TMEM119 detected by RT-PCR.

[0036] Figure 4 Upgrade CSF1R for DDG + / - Expression levels of CSF1R in microglia. A represents CSF1R as detected by Western blot. + / + and CSF1R + / - Protein expression of CSF1R in primary microglia; B represents quantitative statistics of mature CSF1R; C represents quantitative statistics of total CSF1R.

[0037] Figure 5 Upgrade CSF1R for DDG I792T / + Expression levels of CSF1R in microglia. A represents CSF1R as detected by Western blot. + / + and CSF1R I792T / + B shows the protein expression of CSF1R in primary microglia; C shows the quantitative statistics of total CSF1R; D shows the quantitative statistics of mature CSF1R; and D shows the mRNA level of CSF1R detected by RT-PCR.

[0038] Figure 6 Upgrade CSF1R for DDG I792T / +Expression levels of CSF1R in mouse brain tissue. In the figures, A shows a schematic diagram of in vivo drug administration in mice; B shows the Western blot results of CSF1R in the cerebral cortex; C shows the quantitative statistics of total CSF1R in the cortex; D shows the quantitative statistics of mature CSF1R in the cortex; E shows the Western blot results of CSF1R in the hippocampus; F shows the quantitative statistics of total CSF1R in the hippocampus; and G shows the quantitative statistics of mature CSF1R in the hippocampus.

[0039] Figure 7 To promote CSF1R for DDG I792T / + Microglial homeostasis and survival in mouse brain. A shows the mRNA levels of cortical microglial homeostasis markers detected by RT-PCR; B shows the number of cortical microglia as indicated by Iba1 immunofluorescence staining; C shows the three-dimensional reconstruction of microglial morphology stained with Iba1; D shows the number of microglia in a single field of view in the cortex; E shows the number of microglia in a single field of view in the hippocampus; F shows the number of intersections between microglial branches and concentric circles; G shows the Western blot results of cortical Cyclin D1 and c-myc; H shows the quantitative statistics of Cyclin D1; and I shows the quantitative statistics of c-myc.

[0040] Figure 8 Improve CSF1R for DDG I792T / + Neuropathological phenotypes in the mouse brain. A shows axonoid degeneration (indicated by arrows) as revealed by p-NFH immunofluorescence staining (left) and immunohistochemistry (right); B shows the total number of axonoid degenerations in the coronal section of the left hemisphere; C shows hippocampal astrocytes as revealed by GFAP immunofluorescence staining; D shows the number of astrocytes in a single field of view in the hippocampus.

[0041] Figure 9 Improve CSF1R for DDG I792T / + Motor deficits in mice. A shows a gait diagram; B shows average walking speed statistics; C shows the maximum rate of change in movement statistics; D shows a balance beam diagram; E shows the average number of slips on each foot; and F shows the time taken to cross the balance beam.

[0042] Figure 10 Improve CSF1R for DDG I792T / + Deficiencies in learning and memory in mice. Where A is a schematic diagram of the T-maze experiment; B is a statistical representation of the percentage of T-maze transitions. p<0.05, N=11-12); C is a schematic diagram of the Y maze experiment; D is the percentage of Y maze transformations (no significant difference between groups, ns, N=11-12); E is a schematic diagram of the new location recognition experiment (object position change); F is the percentage of time spent exploring new locations (no significant difference between groups, ns, N=11-12); G is a representative image of the nest building experiment (left: unprocessed, right: DDG processed); H is the nest building score statistics ( p<0.05, N=11-12). Detailed Implementation

[0043] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications and variations can be made to the technical solutions of the present invention without departing from the concept of the present invention, and all such modifications and variations should fall within the scope of protection of the present invention. Matters not described in detail below can be handled using conventional techniques in the art.

[0044] This embodiment uses experiments to illustrate the screening of small molecule natural compounds based on enhanced CSF1R expression and their therapeutic effects.

[0045] 1. Construction and identification of FIRE luciferase reporter gene plasmid

[0046] The FIRE fragment was amplified from the human genome using primers containing KpnI / XhoI restriction sites. The amplification system is shown in Table 1.

[0047] Table 1

[0048]

[0049] The PCR reaction procedure is shown in Table 2.

[0050] Table 2

[0051]

[0052] The amplified pGL3 vector and the insert fragment were subjected to double enzyme digestion (incubated at 37 °C for 5 min), and the reaction system is shown in Table 3.

[0053] Table 3

[0054]

[0055] The enzyme digestion products were separated by electrophoresis and purified by gel extraction. The linearized vector and insert fragment were mixed in a specific ratio, and T4 ligase was added. Ligation was performed at 37°C for 1 h to generate the recombinant plasmid pGL3-FIRE. The pGL3-FIRE plasmid was added to DH5α competent cells, incubated on ice for 30 min, then heat-shocked at 42°C for 40 s. After removal, the cells were placed on ice for 2 min, and then 600 μL of LB medium was added for a 30 min recovery period. The mixture was then plated on LB agar plates containing antibiotics and incubated at 37°C for 14 h. Ten single colonies were picked and cultured in LB liquid medium for 6 h. The bacterial culture was sent for sequencing to verify the successful plasmid construction. Subsequently, the positive bacterial culture was expanded (200 mL LB, cultured for 16–18 h). Plasmids were extracted in large quantities using a plasmid extraction kit (HLingene), and the concentration and purity (A260 / A280) were determined by UV spectrophotometry.

[0056] 2. Detection and analysis of luciferase reporter gene activity

[0057] Before the experiment, prepare 1×PLB lysis buffer (dilute 5×PLB with double-distilled water), LAR II (Luciferase Assay Substrate dissolved in Buffer II), and Stop Buffer (200 μL Stop & Glo Substrate mixed with 10 mL Stop & Glo Buffer). For detection, aspirate the culture medium from the 96-well plate, add 20 μL of 1×PLB lysis buffer to each well, and incubate for 15 min at room temperature on a shaker. Then add 100 μL of LAR II, incubate at 37°C for 5 min, and measure the F-Luc fluorescence value at 560 nm using a microplate reader. Immediately add 100 μL of Stop Buffer, incubate at 37°C for 5 min, and measure the R-Luc fluorescence value at 450 nm. Data analysis corrects for transfection efficiency using the F-Luc / R-Luc ratio.

[0058] 3. High-throughput screening of natural small molecule drugs

[0059] HEK 293T cells were used at a rate of 2 × 10⁻⁶ 6 Cells were seeded in six-well plates, and pGL3-Basic (control) or pGL3-FIRE plasmid (2 μg / well) was transfected into the cells using PEI transfection reagent. After 24 hours, the cells were digested and 1.5 × 10⁶ cells / well were added. 4Cells were seeded in 384-well plates. The next day, 0.1 μL of TargetMol library's 1500 natural small molecule compounds (final concentration 20 μM) were added to the cell culture medium. The control group received no drug. Cells were cultured for another 24 hours. Luciferase activity (F-Luc / R-Luc ratio) was measured using a dual-luciferase reporter system kit (Promega). Compounds with FIRE activity upregulated ≥1.5-fold were screened for further verification.

[0060] 4. Cell Culture and Processing

[0061] After disinfecting newborn mice with alcohol, their heads were quickly severed, and the brain parenchyma tissue was removed and placed in pre-cooled HBSS buffer. The meninges were removed under a stereomicroscope, and complete culture medium was added. The brain tissue was pipetted until no particles were present and then seeded into culture flasks and cultured at 37°C. After 24 hours of culture, the medium was replaced with complete medium containing GM-CSF, and fresh medium was added every two days thereafter. On day 10, floating microglia were collected, shaken at 220 r / min for 30 min, centrifuged (500 g, 5 min), resuspended, and counted. Cells were seeded into six-well plates as needed. HEK293T and HMC3 cell lines were cultured in complete medium containing 10% (v / v) FBS and 1% penicillin antibiotics. When the cell density reached 80%, the cells were digested with trypsin and passaged at a ratio of 1:3 to 1:5. DDG was prepared as a 10 mM stock solution with DMSO (stored at -80°C). When treating primary microglia or cell lines with the drug, the drug was diluted to 40 μM in serum-free medium and the treatment time was 36 h. The control group was treated with an equal amount of DMSO.

[0062] 5. In vivo administration to animals

[0063] Preparing CSF1R + / + and CSF1R I792T / + Twenty-four male mice of each genotype were used. The following solutions were added sequentially: 10% DMSO, 40% PEG400, and 50% physiological saline, to achieve a final drug concentration of 1.25 mg / mL, followed by sonication to aid dissolution. DDG was then administered intraperitoneally to the mice at a dose of 10 mg / kg body weight (CSF1R). + / + and CSF1R I792T / + 12 mice each), control group (CSF1R) + / + and CSF1R I792T / + Twelve mice were given an equal dose of physiological saline once a day for four weeks before behavioral experiments were conducted.

[0064] 6. Western blotting

[0065] Fresh or frozen mouse cerebral cortex and hippocampal tissues were collected, added to PBS buffer, and homogenized at 50 Hz for 2 min using a tissue homogenizer. Tissue homogenates or cells were collected by lysing with immunoprecipitation-free analysis lysis buffer (Boster) and a mixture of EDTA-free protease inhibitors (Roche). Total protein concentration was determined using a BCA protein assay kit (Boster). Equal-mass protein samples or supernatants were tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) millipore membrane. Proteins bound to primary antibody (1:1000 CSF1R or 1:1000 Cyclin D1 or 1:1000 c-Myc or 1:4000 Anti-β-actin or 1:10000 Anti-α-Tubulin) and secondary antibody (1:5000 Goat Anti-Rabbit) were visualized by ECL. Quantitative analysis of the immunoreaction bands was performed using ImageJ software.

[0066] 7. Immunofluorescence staining pathological analysis

[0067] Mice were anesthetized and fixed. The heart was exposed by opening the chest along the midline of the abdomen. PBS was infused through a cannula inserted into the apex of the heart. After cervical transection, the entire brain was dissected on ice. The left hemisphere was fixed with 4% PFA at 4°C for 24 hours, followed by dehydration with a 20% and 30% sucrose gradient. The dehydrated tissue was embedded in OCT and stored at -80°C. Coronal brain sections with thicknesses of 15 μm and 30 μm were cut using a cryostat. After rewarming, the sections were washed with PBS buffer, blocked and cleared with 5% BSA / 0.5% Triton X-100 blocking solution at room temperature for 1 hour, and then incubated overnight at 4°C with primary antibody (1:200 Iba1, 1:200 GFAP, or 1:200 p-NFH). The next day, the sections were incubated with fluorescent secondary antibody (containing DAPI) in the dark for 1–2 hours. After mounting, the sections were imaged using a confocal microscope and analyzed using ImageJ software.

[0068] 8. RT-PCR method

[0069] (1) RNA extraction: Transfer 60 μL of tissue homogenate to a 1.5 mL RNase-free EP tube, add 1 mL of Trizol reagent, and incubate on ice for 10 min to ensure complete lysis. Add 100 μL of chloroform, invert and mix for 15 s, incubate for 15 min, and then centrifuge at 12000 r / min for 10 min. After centrifugation, the solution separates into three layers. Slowly aspirate the upper aqueous phase (about 200 μL) into an RNase-free EP tube, add 200 μL of isopropanol, mix gently, and incubate for 5-10 min to allow the RNA to precipitate completely. Centrifuge at 12000 r / min for 10 min and discard the supernatant. Wash the precipitate with freshly prepared 75% ethanol, centrifuge at 7500 g for 10 min, and discard the supernatant. Air dry for 10 min until the precipitate is clear. Add 15 μL of enzyme-free sterile water and incubate at 56 ℃ for 5 min to fully dissolve the RNA. RNA concentration was determined using a micro-spectrophotometer, and the A260 / A280 ratio and A260 / A230 ratio were simultaneously detected to assess nucleic acid purity and residual organic solvents.

[0070] (2) cDNA synthesis: Using the All-in-one qRT SuperMix kit, the reverse transcription reaction system was prepared in RNase-free eight-tube strips according to Table 4, and reverse transcription was performed in the PCR instrument according to the program of 50℃ for 15 min and 85℃ for 5 sec.

[0071] Table 4

[0072]

[0073] (3) RT-PCR

[0074] Dilute the cDNA to 5 ng / μL with enzyme-free sterile water and prepare the reaction system in a 96-well plate according to Table 5.

[0075] Table 5

[0076]

[0077] Place the 96-well plate in a real-time PCR instrument and amplify according to the reaction procedure in Table 6.

[0078] Table 6

[0079]

[0080] Calculate the relative expression level of each gene (2-ΔΔCt method), with at least 3 biological replicates per group.

[0081] Example 1. Construction and activity verification of FIRE luciferase reporter gene plasmid

[0082] The study aimed to identify CSF1R agonists targeting the super-enhancer FIRE, upregulate or restore CSF1R expression, and observe whether this reversed the disease phenotype. In this embodiment, a dual-luciferase reporter gene system was used to verify FIRE activity, detecting changes in FIRE activity by measuring changes in the fluorescence intensity of the reporter gene system. First, the FIRE fragment was amplified from the human genome. Then, using homologous recombination, FIRE was inserted into the firefly luciferase reporter gene plasmid. Figure 1 In the A section, Sanger sequencing sequence alignment showed that the FIRE sequence in the FIRE luciferase reporter gene plasmid (pGL3-FIRE) matched the FIRE fragment in the human genome, proving the successful construction of the pGL3-FIRE plasmid. Figure 1 (B) Luciferase assay confirmed the activation of FIRE activity in the pGL3-FIRE plasmid. Dual-luciferase reporter gene assays revealed that the fluorescence intensity of pGL3-FIRE was increased approximately 3-fold compared to the empty firefly reporter gene plasmid (pGL3-Basic). Figure 1 (C) This demonstrates that FIRE activity was successfully activated in the pGL3-FIRE plasmid. Statistical analysis was performed using an unpaired t-test. p<0.05; N=3 / group. All experimental data are expressed as mean ± standard error.

[0083] Example 2. High-throughput drug screening revealed that DDG is an agonist of FIRE.

[0084] To screen for natural small molecule compounds that can specifically activate the FIRE activity of the super-enhancer within the second intron of the CSF1R gene, this embodiment employs a high-throughput screening strategy based on the pGL3-FIRE luciferase reporter gene system. The specific steps are as follows: The successfully constructed pGL3-FIRE recombinant plasmid was transfected into HEK 293T cells, allowing intracellular FIRE activity to be quantified by the expression level of firefly luciferase (ratio to Renilla luciferase). Subsequently, 1500 natural small molecule compounds (final concentration 20 μM) from the TargetMol library were added to the cells, with untreated cells serving as a control. Compounds that significantly enhance FIRE activity were screened using a dual-luciferase reporter gene detection system.

[0085] The screening process and results are as follows: Figure 2 As shown: In the initial screening stage, 38 positive compounds that could significantly activate FIRE activity were detected among 1500 compounds. Figure 2(AB in the original screening); to eliminate false positives, the top 20 compounds in terms of activity in the initial screening were selected for secondary screening, and finally 7 positive compounds with good stability were identified ( ). Figure 2 (C in the text); further, the FIRE activation activities of these 7 compounds were validated using a gradient method. By comparing the enhancement magnitude and stability of luciferase activity, DDG was ultimately determined to be the optimal FIRE agonist (C in the text). Figure 2 (DE in the text). Statistical analysis used unpaired t-tests, and experimental data are expressed as mean ± standard error. p<0.05 p<0.01 p<0.001 p<0.0001, the results confirm that the activation effect of DDG on FIRE is significant and reliable.

[0086] Example 3. DDG upregulates the transcriptional level of human microglia line CSF1R.

[0087] The aforementioned experimental results have confirmed that DDG can activate the activity of FIRE, the super enhancer of CSF1R. This embodiment further explores whether the FIRE agonist DDG can upregulate the transcriptional level of CSF1R by activating FIRE, and observes its effect on microglial homeostasis markers.

[0088] Human microglia cell line HMC3 (Human Microglia Clone 3) was cultured in vitro. After reaching a suitable cell density, the cells were treated with 40 μM DDG, while the control group received an equal volume of DMSO. After treatment, total RNA was extracted from each group of cells, and the mRNA transcription levels of CSF1R and microglia homeostasis-related markers (P2RY12 and ARG1) were detected by RT-PCR. The experiment was performed in triplicate (N=3). Statistical analysis was performed using an unpaired t-test. Data are expressed as mean ± standard error (SEM).

[0089] The results are as follows Figure 3 As shown: Compared with the control group, treatment with 40 μM DDG significantly upregulated the mRNA transcription level of CSF1R in HMC3 cells. p<0.05, Figure 3 (A in the text); At the same time, the mRNA level of the microglial homeostasis marker P2RY12 was also significantly increased (A in the text). p<0.05, Figure 3In B), the mRNA level of ARG1 showed an upregulation trend. Figure 3 (C in the text). The above results confirm that DDG can significantly upregulate the transcriptional level of CSF1R in human microglia by activating FIRE, and may participate in regulating the homeostasis of microglia, providing in vitro evidence for its further functional verification in in vivo experiments.

[0090] Example 4. DDG upregulates CSF1R + / - expression level of CSF1R in microglia

[0091] To verify the role of DDG in the CSF1R functional partial loss model, this embodiment uses CSF1R heterozygous loss (CSF1R) as an example. + / - This study investigated the effects of DDG on CSF1R protein expression levels under different treatment times, using primary microglia as the research subject. Simultaneously, wild-type (CSF1R) microglia were used as the control group. + / + Microglia were used as a control.

[0092] Experimental method: Separation of CSF1R + / + and CSF1R + / - Primary microglia of mice were cultured to a stable state in vitro and then treated with 40 μM DDG for 0, 12, 24, and 36 h, respectively. The control group was treated with an equal volume of solvent (DMSO). After treatment, total protein was extracted from each group of cells, and the protein expression level of CSF1R was detected by Western blot, focusing on the changes in the expression of mature CSF1R and total CSF1R. The experiment was performed in triplicate (N=3), and statistical analysis was performed using intergroup comparisons. Data are expressed as mean ± standard error (SEM).

[0093] The results are as follows Figure 4 As shown: Western blot analysis revealed that in CSF1R + / - In primary microglia, the expression level of CSF1R protein gradually increased with prolonged DDG treatment time. Figure 4 (A in the text); Quantitative analysis showed that, compared with the 0 h treatment group (untreated group), the protein expression level of the mature form of CSF1R was significantly upregulated after 36 h DDG treatment ( p<0.05, Figure 4 In B), and the total CSF1R protein expression level was also significantly increased ( p<0.05, Figure 4 (C in the text). And CSF1R + / + In microglia, DDG treatment had no significant effect on CSF1R expression. Figure 4 (A in the middle).

[0094] Experiments showed that DDG upregulated the protein expression level of CSF1R in primary microglia with CSF1R heterozygous deletion in a time-dependent manner. Compared with the untreated group, the expression level of CSF1R in microglia was significantly increased after 36 h of DDG treatment, providing direct evidence for the therapeutic potential of DDG in CSF1R partial loss-of-function models.

[0095] Example 5. DDG upregulates CSF1R I792T / + expression level of CSF1R in microglia

[0096] Clinically, the I794T heterozygote of the CSF1R gene (CSF1R I794T / + Point mutations are hotspot mutations that lead to adult-onset axonoglobulin-like changes and glioblastoma pigmentosum leukoencephalopathy (ALSP). To mimic the pathological characteristics of human diseases, this embodiment constructs a mouse model (CSF1R) corresponding to the human I794T mutation using homologous recombination technology. I792T / + The mouse CSF1R gene I792T mutation is highly homologous in functional domain to the human I794T mutation, making it an ideal model for studying diseases related to CSF1R function loss. Previous experiments have confirmed that DDG can upregulate CSF1R. + / - This example further investigates the effect of DDG on CSF1R expression in microglia. I792T / + The effect of CSF1R expression in primary microglia.

[0097] Separate CSF1R + / + (Wild type) and CSF1R I792T / + Primary microglia from (mutant) mice were cultured in vitro to the logarithmic growth phase and then treated with 40 μM DDG. The control group received an equal volume of DMSO. Treatment lasted 36 h. After treatment, CSF1R protein expression levels (including total protein and mature form) were detected by Western blot, and CSF1R mRNA transcription levels were detected by RT-PCR. The experiments were performed in quadruplicates (N=4). Statistical analysis was performed using the two-way ANOVA test. Data are expressed as mean ± standard error (SEM).

[0098] The results are as follows Figure 5 As shown: Western blot analysis revealed that, compared to CSF1R + / + Compared to microglia, untreated CSF1R I792T / + The expression levels of both total and mature CSF1R protein in microglia were significantly reduced. Figure 5 (A in the text); and after treatment with 40 μM DDG for 36 h, CSF1R I792T / +The total protein expression level of CSF1R in microglia was significantly upregulated. p<0.05, Figure 5 In B), the protein level of the mature form CSF1R was also significantly increased ( p<0.05, Figure 5 (C in the text). Meanwhile, RT-PCR results showed that CSF1R after DDG treatment... I792T / + The mRNA transcription level of CSF1R in microglia was significantly increased compared with the control group. p<0.01, Figure 5 (D in the middle).

[0099] Experiments show that, in CSF1R that simulates hotspot mutations in human ALSP... I792T / + In primary microglia, DDG can simultaneously and significantly upregulate both the transcriptional and protein expression levels (including the mature functional form) of CSF1R, further confirming that the upregulation effect of DDG on CSF1R expression is effective in pathological mutation models.

[0100] Example 6. DDG upregulates CSF1R I792T / + Expression level of CSF1R in mouse brain tissue

[0101] To verify the regulatory role of DDG on CSF1R expression in vivo, this embodiment uses CSF1R... I792T / + Using mutant mice as a model, the effect of DDG on CSF1R expression in brain tissue was investigated by intraperitoneal injection.

[0102] Experimental method: Five-month-old male CSF1R were selected. + / + (Wild type) and CSF1R I792T / + (Mice) mutant, 12 mice per group, were divided into a solvent control group and a DDG treatment group. Mice in the DDG treatment group were intraperitoneally injected with DDG (dissolved in 10% DMSO + 40% PEG400 + 50% physiological saline) at a dose of 10 mg / kg, while the control group was injected with an equal volume of the solvent. The administration was once daily for 28 consecutive days. Figure 6 (A) After drug administration, mice were anesthetized and cerebral cortex and hippocampal tissues were collected. The expression levels of total CSF1R protein and mature form were detected by Western blot. The experiment was set up with 6-7 biological replicates (N=6-7 / group). Statistical analysis was performed using the two-way ANOVA test. Data are expressed as mean ± standard error.

[0103] In the cerebral cortex, Western blot results showed ( Figure 6 (B) Compared with the wild-type control group, CSF1R I792T / +The expression levels of total CSF1R protein and mature form were significantly reduced in the mutant mouse control group (p<0.05); while after DDG treatment, the expression levels of CSF1R were significantly increased. I792T / + The expression level of total CSF1R protein in the mouse cerebral cortex was significantly upregulated. p<0.001, Figure 6 In C), the expression of mature CSF1R was also significantly increased (in C). p<0.01, Figure 6 (D in the text). Similar results were observed in the hippocampus region (…). Figure 6 (E in the text): DDG treatment significantly improved CSF1R I792T / + Total CSF1R protein in mouse hippocampus ( p<0.0001, Figure 6 (F in the middle) and mature form ( p<0.05, Figure 6 The expression level of CSF1R in wild-type mice was measured. No significant change in CSF1R expression was observed after DDG treatment.

[0104] Experiments show that in mutant mice with partial loss of CSF1R function, DDG can effectively penetrate the blood-brain barrier through intraperitoneal injection, significantly upregulating the expression level of CSF1R in key brain regions such as the cerebral cortex and hippocampus, especially the biologically active mature form of CSF1R. This provides direct in vivo experimental evidence for the therapeutic application of DDG in neurodegenerative diseases related to loss of CSF1R function.

[0105] Example 7. DDG promotes CSF1R I792T / + Microglial cell homeostasis and survival in mouse brain

[0106] In the central nervous system, CSF1R regulates microglial homeostasis, neurogenesis, and neuronal survival. In mice, CSF1R deficiency leads to a complete loss of macrophages, including microglia in the brain, and results in severe developmental abnormalities and shortened lifespan. This study investigates the upregulation of CSF1R by DDG. I792T / + Does CSF1R in mouse brain tissue affect microglial homeostasis and survival?

[0107] Select 5-month-old male CSF1R + / + (Wild type) and CSF1R I792T / + (Mutant) mice were divided into a control group and a treatment group (N=6-7 / group) according to the administration regimen of Example 6 (10 mg / kg DDG intraperitoneal injection, for 28 consecutive days). After administration:

[0108] Microglial homeostasis marker detection: Total RNA was extracted from the cerebral cortex, and the transcriptional levels of microglial homeostasis-related markers (Tmem119, P2ry12, Cx3cr1, Tgf-β) were detected by RT-PCR;

[0109] Analysis of microglia number and morphology: Coronal sections of brain tissue were prepared, and the number of microglia (counting in a single field of view in the cortex and hippocampus) and morphology (number of intersections of branches with concentric circles of different diameters, reflecting the complexity of branches) were observed by immunofluorescence staining (Iba1, a microglia-specific marker), and three-dimensional reconstruction was performed.

[0110] Cell proliferation capacity assay: Total protein was extracted from the cerebral cortex and the expression levels of cell cycle-related proteins (Cyclin D1, c-myc) were detected by Western blot.

[0111] Experimental data are expressed as mean ± standard error, and statistical analysis was performed using a two-way ANOVA test. RT-PCR detection showed that, compared with untreated CSF1R... I792T / + Compared with mice, the mRNA transcription levels of Tmem119 (p<0.05) and Cx3cr1 (p<0.05), core markers of microglial homeostasis in the cerebral cortex, were significantly upregulated in the DDG-treated group, while P2ry12 and Tgf-β showed an upregulated trend. Figure 7 The presence of A in the image suggests that DDG may promote microglial homeostasis. Immunofluorescence Iba1 staining showed (…). Figure 7 (B in the middle), unprocessed CSF1R I792T / + The number of microglia in the brain of mice (counted in a single visual field of the cortex and hippocampus) was significantly lower than that in wild-type mice; while after DDG treatment, CSF1R I792T / + Mouse cerebral cortex (p<0.01, Figure 7 D in the middle and hippocampus (p<0.05, Figure 7 The number of microglia in E) was significantly increased, approaching wild-type levels. Morphological analysis showed ( Figure 7 (C in the middle), unprocessed CSF1R I792T / + Mouse microglia showed sparse branching and low complexity; after DDG treatment, the number of intersections between microglia branches and concentric circles of different diameters significantly increased (p<0.0001). Figure 7 The F-values ​​indicate increased branching complexity and a morphology more closely resembling normal microglia. Western blot analysis showed ( Figure 7 (G in DDG), CSF1R after DDG processing I792T / +The expression level of cyclin D1 (a key molecule regulating cell proliferation) in the mouse cerebral cortex was significantly upregulated (p<0.05). Figure 7 H), while c-myc expression showed no significant change (ns, Figure 7 The I in the figure suggests that DDG may enhance the proliferative capacity of microglia by upregulating Cyclin D1.

[0112] Experiments showed that, compared to the untreated group, the transcriptional levels of microglia homeostasis markers Tmem119 and Cx3cr1 were significantly upregulated in the DDG-treated group. Immunofluorescence Iba1 staining was used to observe the number of microglia in mouse brain tissue sections, and the results showed that after DDG administration, CSF1R... I792T / + The number of microglia in the mouse brain (Iba1) + The number of cells increased significantly, and the number of microglia branches increased significantly; DDG can upregulate CSF1R. I792T / + Expression of CSF1R in the mouse brain significantly increased the transcriptional levels of microglial homeostasis markers, increased the number of microglia and improved their morphology (increased branching complexity), and enhanced cell proliferation by upregulating Cyclin D1. This indicates that DDG treatment enhances cell proliferation in the mouse brain. DDG can significantly promote CSF1R expression. I792T / + Microglial cell homeostasis and survival in mouse brain.

[0113] Example 8. DDG improves CSF1R I792T / + Neuropathological phenotypes in the mouse brain

[0114] Axonal bulboid degeneration is one of the main pathological features of CSF1R-RD. This example investigates whether DDG treatment can improve CSF1R. I792T / + Mouse axonal bulboid degeneration.

[0115] Select 5-month-old male CSF1R + / + (Wild type) and CSF1R I792T / + (Mutant) mice were divided into a control group and a treatment group (N=4 / group) according to the administration regimen of Example 6 (10 mg / kg DDG intraperitoneal injection, for 28 consecutive days). After administration, coronal sections of mouse brain tissue were prepared, and the pathological phenotype was detected by the following methods:

[0116] Axonoid degeneration detection: Immunofluorescence staining (p-NFH, phosphorylated neurofilament heavy chains, axonal injury markers) and immunohistochemical staining were used to observe the number of axonoid degenerations in the brain and to count the total number of axonoid degenerations in the coronal section of the entire left hemisphere.

[0117] Astrocyte activation detection: Immunofluorescence staining (GFAP, astrocyte-specific marker) was used to observe changes in the number of astrocytes in the hippocampus (counting in a single field of view).

[0118] Experimental data are expressed as mean ± standard error, and statistical analysis was performed using the two-way ANOVA test. Axonal bulbiform degeneration is the most characteristic pathological change in CSF1R-RD. Immunofluorescence p-NFH staining ( Figure 8 (Left image of A in the image) and immunohistochemical staining ( Figure 8 The right image of A in the figure shows the unprocessed CSF1R. I792T / + Numerous axonospheric degenerations were observed in the mouse brain (cortex, hippocampus, etc.), while CSF1R showed significant changes after 28 days of DDG treatment. I792T / + The number of axonoid degenerations was significantly reduced in the coronal section of the entire left hemisphere of the mouse brain (p<0.05). Figure 8 The presence of B in the image suggests that DDG can improve axonal damage caused by CSF1R dysfunction. Astrocyte activation is another pathological feature of CSF1R-RD. Immunofluorescence GFAP staining showed (…). Figure 8 In C), compared with wild-type mice, untreated CSF1R I792T / + The number of astrocytes in the hippocampus of mice was significantly increased (suggesting activation); however, after DDG treatment, CSF1R... I792T / + The number of hippocampal astrocytes in mice was not significantly different from that in the untreated group (ns, Figure 8 The D in the figure indicates that DDG has no significant effect on improving astrocyte activation.

[0119] Experiments show that DDG can specifically improve CSF1R. I792T / + The axonoglobulin-like changes in the mouse brain are a core pathological feature of CSF1R-RD, but have no significant effect on astrocyte activation. These results suggest that DDG can specifically alleviate axonal damage caused by CSF1R dysfunction by upregulating CSF1R expression and improving microglia function, providing key evidence for its treatment of the pathological basis of CSF1R-RD.

[0120] Example 9. DDG Improves CSF1R I792T / + Motor defects in mice

[0121] The initial clinical presentation of patients with CSF1R-related disorders (CSF1R-RD) is memory loss, cognitive impairment, and motor dysfunction. Previous research by the inventors indicated that 9-month-old CSF1R-related disorders... I792T / + Mice exhibited impaired learning, memory, and motor abilities. Based on these results, DDG was indicated for CSF1R. I792T / +The study showed significant improvement in the pathology of mice. This embodiment further evaluated the effect of DDG on CSF1R through behavioral experiments. I792T / + Does it improve cognition and motor function in mice?

[0122] Nine-month-old male CSF1R were selected. + / + (Wild type) and CSF1R I792T / + (Mutant) mice were divided into a control group and a treatment group (N=10-12 / group) according to the administration regimen of Example 6 (10 mg / kg DDG intraperitoneal injection for 28 consecutive days). After administration, motor function was assessed by gait test and beam-walking test.

[0123] (1) Gait Test: Gently place the mouse at the beginning of the gait analysis (CatWalk system) channel, ensuring the mouse's head faces the other end of the channel, and allow the mouse to walk freely without human intervention or interference. Use a high-speed camera to record the mouse's walking process in the channel, ensuring that the complete gait cycle is captured (including parameters such as stride length, stride frequency, and stride width of the forelimbs and hindlimbs). Record at least 3 valid walking data for each mouse, and record the mouse's average walking speed and maximum rate of change of movement.

[0124] (2) Beam-walking Test: A cylindrical balance beam with a diameter of 17 mm and a length of 1.5 m was fixed at a 25° angle to a 40 cm high support. A 1 m test section was marked on the balance beam, and a concealed box was set at the end as the target for animal behavior reinforcement. Mice underwent three adaptive training sessions, during which they were guided from the starting line to the concealed box. Then, three formal tests were conducted, and the time (s) required for the mice to walk across the 1 m balance beam and the number of times their hind limbs slipped were recorded.

[0125] Experimental data are expressed as mean ± standard error. Statistical analysis was performed using a two-way ANOVA test. p<0.05, p<0.01; N=10-12 / group.

[0126] A schematic diagram of the gait experiment is shown below. Figure 9 As shown in A. Unprocessed CSF1R I792T / + The average walking speed of mice was significantly lower than that of wild-type mice (p<0.01), while after DDG treatment, CSF1R... I792T / + The average walking speed of the mice was significantly higher than that of the untreated group (p<0.05). Figure 9(B in the text); Meanwhile, the maximum rate of change in movement (reflecting stability during movement) was abnormal in untreated mutant mice, and this indicator tended to normalize after DDG treatment (p<0.05, ...). Figure 9 The C in the figure suggests that DDG can improve motor coordination and stability in mice. A schematic diagram of the balance beam experiment is shown below. Figure 9 As shown in D in the figure. Unprocessed CSF1R I792T / + Mice took significantly longer to cross the balance beam than wild-type mice (p<0.05), while DDG treatment significantly shortened the time compared to the untreated group (p<0.05). Figure 9 The average number of slips between the left and right feet was not significantly different among the groups (F in the original text). Figure 9 The E in the text suggests that DDG primarily improves athletic performance by enhancing balance rather than improving limb control precision.

[0127] Experimental results showed that DDG could significantly improve CSF1R in 9-month-old infants. I792T / + The motor deficits in mice, specifically increased average walking speed, enhanced motor stability, and shortened balance beam crossing time, are consistent with the improvement in previous neuropathological phenotypes (such as reduced axonal bulbiform degeneration). These results further confirm that DDG, by upregulating CSF1R expression, not only alleviates intracranial pathological damage but also effectively restores motor dysfunction caused by CSF1R dysfunction, providing crucial behavioral evidence for its application in the treatment of motor symptoms in CSF1R-RD.

[0128] Example 10. DDG improves CSF1R I792T / + Deficiencies in learning and memory in mice

[0129] This embodiment assesses the learning, recognition, and memory abilities of mice through T-maze, Y-maze, and novel object recognition experiments, and further explores the effect of DDG on improving learning and memory-related functional deficits.

[0130] Nine-month-old male CSF1R were selected. + / + (Wild type) and CSF1R I792T / + (Mutant) mice were divided into control and treatment groups (N=11-12 / group) according to the administration regimen of Example 6 (10 mg / kg DDG intraperitoneal injection, for 28 consecutive days). After administration, learning, memory, and related behavioral functions were assessed using the T / Y maze test, novel location recognition (NLR) test, and nest building test.

[0131] (1) T / Y maze test: A 30 cm arm-length T / Y maze was placed in the center of the camera's field of view and the lighting was adjusted to be uniform. Before the experiment, the maze was sprayed with 75% alcohol to disinfect it and eliminate odor interference. Mice were placed in the center of the maze and their behavior was recorded for 5 minutes after 10 seconds of adaptation. The maze was cleaned and disinfected after each test. Spatial memory ability was assessed by analyzing the alternation triplet (%) using the CleverSys system.

[0132] (2) Novel location recognition (NLR) experiment: Two identical cylinders were fixed diagonally in an open field box (10 cm from the side wall), and disinfected with alcohol to eliminate odor interference. Mice were released between the two cylinders and allowed to explore freely for 10 min. The time and frequency of the mice's exploration of each object were tracked and recorded in real time using a behavior analysis system. After 3 h, one of the cylinders was moved to the adjacent side wall, and the mice were released again to explore for 10 min. The novel location recognition index (NLR Index, novel location recognition time / total recognition time × 100%) was calculated using the behavior analysis system to assess hippocampal-dependent spatial memory function.

[0133] (3) Nest Building Test: Fresh bedding was placed in a home cage. 3-gram square pieces of absorbent cotton were pre-cut as nesting material. The test mice were placed individually in the cage, with the absorbent cotton placed inside. The mice were allowed to move freely within the cage and build nests using the absorbent cotton. Nesting activity was recorded over 24 hours. Scoring was based on the degree of cotton damage and the integrity of the nest structure (1 point: over 90% of the absorbent cotton remained intact; 2 points: some material was slightly torn but did not form a nest, 50%–90% of the cotton remained intact; 3 points: the absorbent cotton was clearly torn but did not accumulate, over 50% of the nesting material was torn and scattered around the cage; 4 points: over 90% of the absorbent cotton was damaged, the material accumulated into an incomplete nest structure, lower than the height of the mouse; 5 points: over 90% of the absorbent cotton was damaged and accumulated into a complete nest taller than the mouse). This experiment reflects daily behavioral abilities and cognitive-related executive functions.

[0134] Experimental data are expressed as mean ± standard error, and statistical analysis was performed using a two-way ANOVA test.

[0135] A schematic diagram of the T-maze experiment is shown below. Figure 10 As shown in A in the diagram. In the T-maze experiment, compared to wild-type mice, CSF1R... I792T / + The percentage of transitions in the T-maze (reflecting the flexibility of spatial working memory) in mice showed a downward trend, while CSF1R was reduced after DDG treatment. I792T / +The conversion percentage in mice returned to normal levels (p<0.05). Figure 10 (B in the text) suggests that DDG can improve spatial working memory deficits. Y maze experiment ( Figure 10 The C) statistics showed that there was no significant difference in the percentage of conversion in different arms among the four groups of mice. Figure 10 D in the middle); new location recognition experiment ( Figure 10 In E), there was no statistically significant difference in the proportion of time spent exploring objects at new locations among the different groups of mice. Figure 10 The F in the figure indicates that DDG had no significant effect on the memory function assessed in these two experiments. A representative diagram of the nesting experiment is shown below. Figure 10 As shown in G. Unprocessed CSF1R I792T / + The nesting score (reflecting daily behavioral organization and executive function) of mice was significantly lower than that of wild-type mice (p<0.05), while after DDG treatment, CSF1R I792T / + The nesting score of mice was significantly higher than that of the untreated group (p<0.05). Figure 10 (H in the text), the above behavioral results indicate that CSF1R I792T / + Mice exhibited impaired motor function, as well as reduced learning, recognition, and memory abilities, while DDG treatment could improve CSF1R. I792T / + Deficiencies in motor function and learning / memory in mice.

[0136] Experimental results show that DDG can selectively improve CSF1R in 9-month-old infants. I792T / + The study revealed learning and memory-related functional deficits in mice, specifically in the recovery of spatial working memory conversion ability in the T-maze and improved executive function scores in the nesting test, while having no significant impact on memory function assessed in the Y-maze and novel location recognition tests. These results are consistent with previous findings of improved neuropathology and motor function, further confirming that DDG, by upregulating CSF1R expression, can alleviate cognitive-related behavioral disorders caused by CSF1R dysfunction, providing experimental evidence for its treatment of cognitive impairment in CSF1R-RD patients.

[0137] This invention addresses the treatment needs of diseases related to CSF1R function loss (especially CSF1R-RD) and provides a novel treatment approach based on natural small molecule compounds through an innovative research strategy. Its core innovations are as follows:

[0138] 1. Innovative Mechanism of Action: Most research on CSF1R-related diseases focuses on directly regulating CSF1R protein activity or downstream signaling pathways. This invention, however, is the first to discover and verify that natural small molecule compounds can upregulate CSF1R expression at the transcriptional level by enhancing the activity of the super-enhancer FIRE within the second intron of the CSF1R gene. This mechanism breaks through the traditional approach of targeting proteins, achieving precise regulation of CSF1R expression by modulating key cis-regulatory elements (FIREs) in the gene, providing a new paradigm for disease treatment at the gene expression regulation level.

[0139] 2. Innovation of the Screening Strategy: This invention innovatively constructs a FIRE luciferase reporter gene system, and establishes a high-throughput screening platform based on this system. It directionally screens agonists capable of activating FIRE activity from 1500 natural small molecule compounds, ultimately identifying DDG as the optimal candidate compound. This strategy is highly targeted, directly linked to the core regulatory element of CSF1R expression, significantly improving the efficiency and specificity of drug screening, and providing a referable technical path for drug development targeting gene regulatory elements.

[0140] 3. Innovative Application Value: CSF1R-RD is a progressive neurodegenerative disease for which there is currently no effective treatment. This invention systematically demonstrates through in vitro and in vivo experiments that DDG can be effective in the CSF1R heterozygous loss model (CSF1R... + / - ) and models simulating human hotspot mutations (CSF1R) I792T / + In this study, the transcriptional and protein levels of CSF1R were significantly upregulated, microglial homeostasis and survival were improved, core pathological damage such as axonoid degeneration was reduced, and motor function and learning and memory-related behavioral deficits in model animals were restored. This finding provides, for the first time, a potential therapeutic agent for CSF1R-RD with both a clear mechanism and efficacy.

[0141] 4. Advantages of the Compound Type: The DDG screened in this invention is a natural small molecule compound, which has potential advantages such as good biocompatibility and low toxicity risk compared to chemically synthesized drugs. Furthermore, it can be formulated into various dosage forms such as injections and oral preparations to meet different clinical needs, laying a solid foundation for subsequent drug development.

[0142] The present invention includes a sequence list, which includes SEQ ID NO:1 to SEQ ID NO:23, and the specific information of each sequence is shown in Table 7.

[0143] Table 7

[0144]

[0145] This invention innovatively develops a natural small molecule compound, DDG, that upregulates CSF1R expression by targeting the super enhancer FIRE of the CSF1R gene. From mechanism and method to application, it breaks through the limitations of existing technologies and provides a new treatment strategy and candidate drug for CSF1R loss-of-function related diseases (especially CSF1R-RD), which has important scientific value and clinical translation prospects.

[0146] The above embodiments are merely preferred embodiments of the present invention and should not be considered as limiting the scope of the present invention. All equivalent variations and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention. Those skilled in the art can modify and process the natural small molecule compounds that enhance CSF1R expression as described in this invention; as long as the modified drug can enhance CSF1R expression, it should fall within the scope of protection of this invention.

Claims

1. The application of a natural small molecule compound in the preparation of a drug for treating diseases related to CSF1R function loss, characterized in that, The natural small molecule compound is Diosmetin-7-O-β-D-glucopyranoside. The drug upregulates the transcription and / or protein expression of CSF1R by enhancing the activity of the super-enhancer FIRE in the second intron of the CSF1R gene.

2. The application according to claim 1, characterized in that, The diseases associated with the loss of CSF1R function are neurodegenerative diseases.

3. The application according to claim 2, characterized in that, The neurodegenerative diseases mentioned are adult-onset axonoglobulinosis and glioblastoma.

4. The application according to claim 3, characterized in that, The adult-onset axonoglobulinization and glioblastoma are caused by heterozygous deletion of the CSF1R gene or heterozygous mutation of the CSF1R gene I792T.

5. The application according to claim 1, characterized in that, The drug also contains pharmaceutically acceptable excipients.

6. The application according to claim 1, characterized in that, The dosage form of the drug is an injectable preparation, an oral preparation, a spray preparation, an ointment preparation, or a patch.

7. The application according to claim 1, characterized in that, The drug can improve the relevant neuropathological phenotypes and / or behavioral defects in CSF1R dysfunction model animals.

8. The application according to claim 7, characterized in that, The neuropathological phenotypes include axonoglobulin-like degeneration; the behavioral deficits include motor function deficits and / or learning and memory function deficits.

9. A method for upregulating CSF1R expression, characterized in that, This includes administering an effective amount of the natural small molecule compound Diosmetin-7-O-β-D-glucopyranoside to cells or individuals expressing CSF1R, wherein Diosmetin-7-O-β-D-glucopyranoside upregulates the CSF1R gene by enhancing the activity of the superenhancer FIRE within the second intron.

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