Application of miR375-3p protein in preparation of medicine for treating AD

Abstract

The application belongs to the technical field of biotechnology and provides application of miR375-3p protein in preparation of AD treatment drugs, wherein the nucleotide sequence of the miR375-3p is shown in SEQ ID NO. 1: 3'-AGUGCGCUCGGCUUGCUUGUUU-5'. 25‑35 induced SH-SY5Y cell apoptosis, reduced Aβ deposition, inhibited mTOR phosphorylation, promoted microglial cell activation in early AD, repaired cognitive function and memory defects and reduced neuroinflammation. The application proves that miR375-3p plays an active role in AD, which provides a feasible scheme and a valuable reference case for preparation of AD treatment drugs.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to the application of a miR375-3p protein in the preparation of drugs for treating Alzheimer's disease (AD). Background Technology

[0002] Alzheimer's disease (AD) is the most common neurological disease affecting the elderly. The specific mechanisms by which AD causes neurological damage and cognitive impairment remain unclear, and clinical drug development has been less than ideal. The reasons for failure can be summarized as poor targeting, strong side effects, and inability to penetrate the blood-brain barrier. Current research indicates that normal autophagy is crucial for the clearance of Aβ in the brain. In AD mice, the mTOR signaling pathway is enhanced, and the mTORC1 complex can inhibit the formation of the downstream ULK1 complex, preventing autophagy. When autophagy is downregulated, intracellular and extracellular Aβ load increases, leading to a worsening of AD. Researchers have found that autophagy can activate microglia in AD models, thereby accelerating the processing of Aβ deposits. Furthermore, inducing normal autophagy can prevent the aging of microglia in the brains of AD model mice and restore cognitive abilities. These results demonstrate the important roles of microglia and autophagy in AD.

[0003] Non-coding single-stranded RNA molecules, microRNAs (miRNAs), are widely distributed in organisms, ranging in length from 18 to 25 nt, and play an important role in neurodegenerative diseases. miRNAs exert their functions by binding to their target mRNAs, either inhibiting mRNA translation or promoting mRNA degradation. As one of the in vivo treatment methods for Alzheimer's disease (AD), miRNAs have the advantage of being able to cross the blood-brain barrier and exert their effects via an endogenous pathway. Furthermore, miRNAs in vivo are highly stable and resistant to many harsh conditions, such as high temperatures, acidic or alkaline environments, and repeated freeze-thaw cycles.

[0004] This invention identified miR375-3p through screening. miR375-3p plays a role in AD, autophagy regulation, and microglia activation. We evaluated the effect of miR375-3p on Aβ. 25-35 The therapeutic effects of induced SH-SY5Y cells and APP / PS1 TG mice were investigated, and the mechanism of action under both in vivo and in vitro environments was elucidated. In APP / PS1 TG mice, we introduced rapamycin, and its combination with miR375-3p-agomir significantly improved cognitive impairment and memory deficits, and reduced Aβ deposition in the model mice. Summary of the Invention

[0005] The purpose of this invention is to provide an application of miR375-3p protein in the preparation of drugs for treating Alzheimer's disease (AD), thereby addressing the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The application of a miR375-3p protein in the preparation of a drug for treating Alzheimer's disease (AD), wherein the nucleotide sequence of miR375-3p is shown in SEQ ID NO.1: 3'-AGUGCGCUCGGCUUGCUUGUUU-5'.

[0007] Furthermore, the treatment refers to one or more of the following: inhibiting Aβ 25-35 It induces apoptosis in SH-SY5Y cells, reduces Aβ deposition, inhibits mTOR phosphorylation, promotes microglial activation in early AD, repairs cognitive function and memory deficits, and reduces neuroinflammation.

[0008] Furthermore, the inhibition of Aβ 25-35 Specifically, the induced apoptosis in SH-SY5Y cells is due to miR375-3p inhibiting Aβ. 25-35 Induced apoptosis in SH-SY5Y cells or antagonism of Aβ by promoting autophagy when rapamycin is used in combination with miR375-3p-mimic. 25-35 Induced apoptosis in SH-SY5Y cells.

[0009] Furthermore, the reduction of Aβ deposition specifically refers to the combined use of rapamycin and miR375-3p-mimic-agomir to reduce Aβ deposition.

[0010] Furthermore, the inhibition of mTOR phosphorylation specifically refers to miR375-3p promoting autophagy in the AD environment by inhibiting mTOR phosphorylation.

[0011] Furthermore, the promotion of microglia activation in early AD specifically involves rapamycin and miR375-3p-mimic promoting microglia activation in early AD.

[0012] Furthermore, the repair of cognitive function and memory deficits specifically involves the combined use of rapamycin and miR375-3p-mimic-agomir to repair cognitive function and memory deficits.

[0013] Furthermore, the reduction of neuroinflammation specifically involves the combined use of rapamycin and miR375-3p-mimic-agomirr to inhibit neuropathological markers at the transcriptional and translational levels.

[0014] Compared with the prior art, the beneficial effects of the present invention are: The application of the miR375-3p protein in the preparation of drugs for treating Alzheimer's disease (AD) demonstrates that miR375-3p plays a positive role in AD, autophagy regulation, and microglial activation. The combined use of rapamycin and miR375-3p-agomir can better improve cognitive dysfunction and memory deficits in model mice and reduce Aβ deposition. This invention provides a feasible approach and valuable reference case for the preparation of drugs for treating AD. Attached Figure Description

[0015] Figure 1A This is a diagram showing the miRNA prediction results obtained in this invention.

[0016] Figure 1B This is a heatmap summarizing the miRNAs predicted by this invention.

[0017] Figure 1C This is the KEGG graph obtained from the predictive analysis of this invention.

[0018] Figure 1D This is the GO diagram obtained from the predictive analysis of this invention.

[0019] Figure 1E The target protein that miR375-3p binds to, as predicted and analyzed in this invention.

[0020] Figure 1F To demonstrate the effect of miR375-3p on bioinformatics prediction target proteins in SHSY5Y cells.

[0021] Figure 1G To demonstrate the effect of miR375-3p on bioinformatics prediction target proteins in wild-type mice.

[0022] Figure 2A For Aβ 25-35 Western blot results showing the changes in various parameters of SHSY5Y cells at different time points after induction.

[0023] Figure 2B miR375-3p for Aβ 25-35 Western blot results showing the effects of various indicators on SHSY5Y cells induced.

[0024] Figure 2C miR375-3p for Aβ 25-35 Image showing the results of TUNEL assay on induced SHSY5Y cells.

[0025] Figure 2D miR375-3p for Aβ 25-35 Figure showing the changes in oxidative stress levels induced in SHSY5Y cells.

[0026] Figure 2EmiR375-3p for Aβ 25-35 Figure showing the results of induced changes in mitochondrial membrane potential levels in SHSY5Y cells.

[0027] Figure 2F miR375-3p for Aβ 25-35 Image showing the results of confocal staining of different key proteins in induced SHSY5Y cells.

[0028] Figure 3A miR375-3p and the autophagy activator rapamycin for Aβ 25-35 Image showing the results of TUNEL assay on induced SHSY5Y cells.

[0029] Figure 3B miR375-3p and the autophagy activator rapamycin for Aβ 25-35 Figure showing the changes in oxidative stress levels induced in SHSY5Y cells.

[0030] Figure 3C miR375-3p and autophagy activator for Aβ 25-35 Western blot results showing changes in various parameters of induced SHSY5Y cells.

[0031] Figure 4A The schedule for administering drugs to mice.

[0032] Figure 4B HE staining results of major organs in mice after drug injection.

[0033] Figure 4C In vivo imaging results after mice were injected with miR375-3p-mimic-agomir-CY3.

[0034] Figure 4D The results of the seven-day incubation period for escaping the water maze in each group of mice were summarized.

[0035] Figure 4E The results of the escape latency period on the seventh day in the water maze for each group of mice are shown in the figure.

[0036] Figure 4F Mapping the route for finding hidden platforms in the water maze for each group of mice on day 7.

[0037] Figure 4G To obtain the statistical results of the seven-day latency period for escaping the water maze in each group of mice.

[0038] Figure 4H The results of the spontaneous alternation experiment in the Y-maze for each group of mice are shown in the figure.

[0039] Figure 4I The results of the exploration time of the new arm in the Y-maze for each group of mice are shown in the figure.

[0040] Figure 4J The results of the exploration of the novel arm of the Y-maze for each group of mice are shown in the figure.

[0041] Figure 5A The results of Aβ immunohistochemical staining of mouse brain tissue.

[0042] Figure 5B The results of Nissl body staining in mouse brain tissue.

[0043] Figure 5C A bar chart showing the number of Nissl bodies in the brain tissue of mice in each group.

[0044] Figure 5D The image shows the results of TUNEL staining of brain tissue from mice in each group.

[0045] Figure 6A The image shows the results of BDNF immunofluorescence staining in the hippocampus of mice in each group.

[0046] Figure 6B The image shows the results of Syn immunofluorescence staining of the hippocampus in mice from each group.

[0047] Figure 6C Western blot results for different indicators of hippocampal tissue in mice from each group.

[0048] Figure 6D The image shows the immunofluorescence staining results of Beclin1 in the hippocampus of mice in each group.

[0049] Figure 6E The image shows the results of LC3B immunofluorescence staining in the hippocampus of mice in each group.

[0050] Figure 6F The image shows the results of PS1 immunofluorescence staining in the hippocampus of mice in each group.

[0051] Figure 6G Real-time PCR results of AD-related indicators in each group of mice.

[0052] Figure 6H Real-time PCR results for autophagy-related indicators in each group of mice.

[0053] Figure 7A For Aβ 25-35 Western blot results showing changes in phosphorylation activity of mTOR pathway-related proteins in SHSY5Y cells at different time points.

[0054] Figure 7B miR375-3p and rapamycin for Aβ 25-35 Western blot results of induction of mTOR pathway and microglial cell marker protein in SHSY5Y cells.

[0055] Figure 7C Western blot results of mTOR pathway and microglial cell marker protein activity in hippocampal tissues of mice in each group.

[0056] Figure 7D To obtain the sequence alignment results of miR375-3p with CD68, one of the bioinformatics predicted target proteins.

[0057] Figure 7E To demonstrate the effect of miR375-3p on the bioinformatics prediction target protein CD68 in SHSY5Y cells.

[0058] Figure 7F To demonstrate the effect of miR375-3p on the bioinformatics prediction target protein CD68 in wild-type mouse brain tissue.

[0059] Figure 7G The image shows the results of CD68 immunofluorescence staining in the hippocampus of mice in each group.

[0060] Figure 7H The image shows the results of CD80 immunofluorescence staining in the hippocampus of mice in each group.

[0061] Figure 7I The image shows the results of Iba1 immunofluorescence staining in the hippocampus of mice in each group.

[0062] Figure 7J Real-time PCR results for microglial cell-related indicators in each group of mice. Detailed Implementation

[0063] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0064] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0065] One embodiment of the present invention provides the application of miR375-3p protein in the preparation of drugs for treating Alzheimer's disease (AD), wherein the nucleotide sequence of miR375-3p is shown in SEQ ID NO.1: 3'-AGUGCGCUCGGCUUGCUUGUUU-5'.

[0066] In this embodiment of the invention, the first step is the enrichment and analysis of miR375-3p-related proteins: A gene set was used to screen miRs. This set included 20 AD-related pathways, 17 autophagy-related pathways, and multiple pathways related to microglia activation, including 7 JNK-related pathways, 9 MAPK-related pathways, 9 NF-κB-related pathways, and the PI3K pathway. All pathways were derived from GSEA. These gene sets were then used to predict interacting miRs using three mRNA-miRNA interaction databases: ENCORI, miRTarBase, and RNA22. During the prediction process, R packages such as ggsci, dplyr, and data.table were used to assist in the analysis. The predicted miRs were counted, and each group of screened miRs was sorted by frequency of occurrence. The top 100 miRs by frequency of occurrence were selected for further analysis.

[0067] Genes interacting with miR-375 were predicted using the TargetScan database. The predicted results were sorted from highest to lowest Aggregate PCT, and the top 580 genes were selected for further validation. Using DAVID, the official gene symbol was chosen as the identifier, and *Hommosapiens* was selected as the species. Pathway enrichment analyses were then performed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Genes were filtered according to P-value (P≤0.5) and then sorted by name, with the top 18 presented in ascending order.

[0068] The result showed 49 miRNAs present in the intersection of all gene sets. Figure 1A Based on a dataset of differentially expressed miRNAs in the cerebrospinal fluid of AD patients published by researchers, the predicted miRNAs were summarized, and only miR375-3p was found to be within the intersection of the two (…). Figure 1B This indicates that miR375-3p is associated with AD and autophagy, and is significantly downregulated in AD patient samples. Simultaneously, the target proteins predicted by miR375-3p were summarized and enriched, and KEGG analysis showed (…). Figure 1C The target proteins of miR375-3p are mainly enriched in pathways including the AMPK signaling pathway, AD, mTOR signaling pathway, PI3K-AKT signaling pathway, and TGF-β signaling pathway. GO biological process analysis results show that ( Figure 1DThe target proteins of miR375-3p are mainly involved in biological processes such as transcriptional regulation, cell proliferation, intracellular signal transduction, nervous system development, and protein phosphorylation. Therefore, we believe that miR375-3p plays a role in AD, autophagy, and microglia activation-related pathways, and its specific functions and molecular mechanisms can be determined through further experiments.

[0069] Among the predicted miR375-3p target proteins, we identified several proteins closely related to AD and autophagy, namely: PSEN1, Syn, BDNF, and ATG14. Figure 1E After transfecting SH-SY5Y cells with miR375-3p-mimic, compared with the NC group, the expression levels of PS1, Syn, BDNF, and ATG14 proteins in the miR375-3p group were significantly decreased. Figure 1F After miR375-3p was injected into wild-type mice, analysis of tissue samples from the hippocampus revealed that, compared to the control group, the expression levels of PS1, Syn, BDNF, and ATG14 were all decreased in the miR375-3p injection group. Figure 1G The results from cell and tissue samples were consistent with the predicted results, indicating that the addition of miR375-3p-mimic can inhibit the expression of miR375-3p target protein, which is consistent with the predicted results.

[0070] II. Cell Culture: SH-SY5Y neuroblastoma cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum (penicillin 100 IU / mL, streptomycin 100 μg / mL). After the SH-SY5Y cells were transferred to new culture dishes, the culture medium was changed every 48 hours. When the cells grew to more than 90%, they were passaged.

[0071] III. Total Protein Extraction from Cell and Brain Tissue Samples: Cell lysis buffer was added to each group of cell samples, cells were scraped and incubated on ice for 20 min. Mouse brain tissue was collected from each group, lyophilized in liquid nitrogen, and the parietal and frontal lobes were excised. Cell lysis buffer was added, and the tissues were incubated on ice for 20 min. Cell and tissue samples were then sonicated to obtain total protein extract. The concentration of total protein extract was determined using a BCA protein assay kit. Simultaneously, 4× loading buffer was added in proportion, and the mixture was treated in a boiling water bath for 8 min. The cell lysis buffer contained 50 mM Tris-HCl (pH 6.8), 8 M urea, 0.1 mM MDT, 2% (w / v) SDS, 1% (v / v) protease inhibitor, 1% (v / v) PMSF, and 1% (v / v) phosphatase inhibitor.

[0072] The following are the experiments conducted in this application, specifically including: 1. Western Blot: Select an appropriate gel concentration based on the molecular weight of the target protein. After SDS-PAGE, transfer the protein to a PVDF membrane: block at 100 mA constant current for 120 min, 4°C, with 5% skim milk and slow shaking at room temperature for 1 h, incubate with primary antibody overnight at 4°C, wash the membrane, and then incubate with HRP-labeled IgG antibody with slow shaking at room temperature for 1 h. Finally, perform ECL staining, and analyze the results using ImageJ software.

[0073] 2. Immunofluorescence staining: a. Cell slide fluorescence staining: Fix with 4% paraformaldehyde at room temperature for 30 min, incubate with Triton × 100 at room temperature for 5 min to permeate the cell membrane, incubate with 5% goat serum at room temperature for 45 min, then incubate with primary antibody (1:200) overnight at 4°C with gentle shaking. Incubate with IgG antibody with FITC or PE tag (1:300) at room temperature in the dark for 2 h, add anti-fluorescence attenuation mounting medium, and observe using a laser confocal microscope.

[0074] b. Fluorescent staining of tissue sections: Fresh tissue was fixed in 4% paraformaldehyde, embedded, and paraffin sections were prepared. The sections were then immersed in xylene at room temperature for 10 min, followed by another 10 min in fresh xylene. The xylene was recovered, and solutions of anhydrous ethanol, 90% ethanol, 80% ethanol, and 70% ethanol were prepared. First, the sections were immersed in fresh anhydrous ethanol for 5 min, then in 90%, 80%, and 70% ethanol solutions for 5 min each. 50 μL–100 μL of proteinase K was added to each section, and the sections were incubated at 37 °C for 20 min. After incubation with 5% goat serum at room temperature for 45 min, the sections were incubated with primary antibody (1:200) and slowly shaken overnight at 4 °C. Incubation with FITC or PE-tagged IgG antibody (1:300) was performed at room temperature in the dark for 2 h. Antifluorescence attenuation mounting medium was added, and the sections were observed using a laser confocal microscope.

[0075] 3. JC-1 staining: Using the mitochondrial membrane potential detection kit (JC-1), the JC-1 probe aggregates in the normal mitochondrial matrix to produce red fluorescence. When the membrane potential decreases, JC-1 cannot be properly retained in the mitochondrial matrix, thus producing monomers and emitting green fluorescence. The specific steps are as follows: prepare the JC-1 staining working solution according to the instructions, incubate the cell slides at 37°C for 30 min, and observe under a confocal microscope.

[0076] 4. ROS detection and Tunel assay: The DCFH-DA probe (diluted to 10 μM in serum-free medium, ROS) and Tunel assay solution were used to incubate the cells at 37°C in the dark for 30 min (ROS) and 60 min (Tunel), respectively, and the cells were detected by excitation light at 488 nm.

[0077] 5. Total RNA extraction and reverse transcription: Total RNA was extracted from cells and mouse blood, and injected into each group of cells (3×10⁻⁶). 6 Add 1 mL of TransZol Up to the sample from the sample (number of mice) and 200 μL of mouse blood, let stand for 5 min, add 200 μL of chloroform, shake vigorously for 30 s, incubate at room temperature for 3 min, centrifuge at 10000 g for 15 min at 4 ℃, collect the intermediate layer, add an equal volume of anhydrous ethanol, mix well, add to the centrifuge column, centrifuge at 12000 g for 30 s, discard the eluent, wash twice with washing buffer, and add 50 μL of N-Nase-free water to elute RNA. After obtaining total RNA, use it as a template to synthesize cDNA using a reverse transcription kit. In this experiment, Anchored Oligo(dT)18 was used as the primer. After adding each component, incubate at 42 ℃ for 15 min, heat at 85 ℃ for 5 s, and determine the concentration of the product for qPCR.

[0078] 6. Real-time PCR: Real-time PCR experiments were performed using the BlasTaq™ 2X qPCR MasterMix. Data collection and analysis were conducted using an Applied Biosystems 7500 real-time quantitative PCR system. Each sample was tested in triplicate, using 2... -ΔΔCT The Actin cDNA was normalized using a method.

[0079] 7. AD Model Mouse Grouping and Drug Administration: The AD model mice used were SPF-grade APP / PS1 TG mice, purchased from Shanghai Southern Model Biotechnology Co., Ltd.; the wild-type mice used were SPF-grade, purchased from Beijing Spaford Biotechnology Co., Ltd. All experimental animal models were bred according to the requirements of the Animal Welfare and Ethics Committee of the College of Life Sciences, Jilin University, and experiments were conducted in accordance with the Regulations on the Management of Experimental Animals of the People's Republic of China. Sufficient measures were taken to reduce pain and discomfort in the mice during the experiments. All mouse strains were C57BL / 6J. Both APP / PS1 TG mice and wild-type mice were male. They were grouped and drug administered at 28 weeks of age. There were 20 APP / PS1 transgenic mice and 20 wild-type mice. The APP / PS1 transgenic mice were divided into 4 groups of 5 mice each; the wild-type mice were divided into 4 groups. The drug administration period was the same as that for the APP / PS1 transgenic mice. The miR375-3p-agomir (genepharma) and NC-agomir (genepharma) solutions used in this experiment were prepared with sterile DEPC water, with each injection being 0.5 nmol via tail vein injection. The rapamycin (MCE) concentration used was 1.5 mg / 1 kg, and the rapamycin solution and 0.8% saline were administered via intraperitoneal injection.

[0080] 8. Behavioral testing: a. Morris water maze: This experiment was conducted after the injection cycle was completed. Edible whitening agent was added to the water tank. The water maze experimental system was divided into 4 zones: A1, A2, A3, and A4. In this experiment, the hidden platform was placed in zone A3. The water level in the tank should be 1-2 cm higher than the hidden platform, and the water temperature should be heated to 28-30 ℃ using a heating device. The experiment lasted for 10 days, with the first 3 days being the training phase and the last 7 days being the experimental phase. Four experiments were conducted each day. Mice were released from A1, A2, A4, and the center of the tank, and given 60 seconds to find the hidden platform. If they did not find it within the specified time, they were guided to the hidden platform. The experiment ended after 10 seconds of adaptation. The escape latency and path of the mice in finding the hidden platform were recorded.

[0081] bY maze spontaneous alternation experiment: Before the experiment, each mouse was first introduced into the Y maze system for 5 minutes to adapt. After 1 hour, the mouse was put into the middle of the Y maze system and the alternation order of exploring the three arms within 10 minutes was recorded. The proportion of mice entering three different arms consecutively was calculated. The experimental environment must be completely cleaned after each experiment.

[0082] cY Maze Novel Arm Exploration Experiment: The novel arm was completely blocked with a partition, while the starting arm and other arms were left open. Mice were placed in the system to explore freely for 5 minutes. After 1 hour, the partition of the novel arm was removed, and the mice were placed in the starting arm. The time to enter the novel arm and the exploration distance within 3 minutes were recorded. The experimental environment must be completely cleaned after each experiment.

[0083] 9. In vivo fluorescence imaging: One day before the fluorescence experiment, miR375-3p-cy3 and 0.8% physiological saline were injected respectively. Before the experiment, mice were anesthetized with tribromoethanol, and the hair on the back and head of the mice was removed with a hair removal device. The mice were then placed in a small animal in vivo imaging system (FOBI) to observe the distribution of miR375-3p-cy3.

[0084] 10. Tissue embedding and sectioning: Fresh tissue was fixed in 4% paraformaldehyde. After 24 hours, it was removed and subjected to a gradient dehydration process using a dehydrator: 75% ethanol solution, 4 hours - 85% ethanol solution, 2 hours - 90% ethanol solution, 2 hours - 95% ethanol solution, 1 hour - anhydrous ethanol, 30 minutes - anhydrous ethanol, 30 minutes - benzene, 10 minutes - xylene, 10 minutes - xylene, 10 minutes - paraffin solution, 1 hour - paraffin solution, 1 hour - paraffin solution, 1 hour. The paraffin-impregnated tissue was then embedded in an embedding machine. Melted paraffin was placed in the embedding frame. Before the paraffin solidified, the tissue was removed from the dehydration box and placed in the embedding frame. The tissue was cooled at -20°C. After the paraffin solidified, the tissue was removed and trimmed. The tissue was then sectioned using a paraffin microtome to a thickness of 4 μm.

[0085] 11. Immunohistochemical staining: Tissue sections were dewaxed (see the procedure for fluorescent staining of tissue sections). Antigen retrieval was performed using antigen retrieval buffer (pH 6.0). The sections were washed three times with PBS for 5 min each time, incubated with 3% BSA at room temperature for 30 min, washed once with PBS, incubated overnight with primary antibody at 4°C, washed three times with PBS for 5 min each time, covered with secondary antibody of the corresponding species, incubated at room temperature in the dark for 50 min, washed three times with PBS for 5 min each time, and then developed with DAB chromogenic agent. After washing once with distilled water, hematoxylin was used for counterstaining, followed by differentiation with 1% hydrochloric acid alcohol (approximately 1 s). The sections were washed once with distilled water, and then blued with ammonia. After washing with distilled water, the sections were observed under a microscope, and images were acquired using CaseViewer software. Antibodies used for immunohistochemical analysis included: anti-Aβ, anti-CD80, anti-Beclin-1, and anti-IBA-1.

[0086] 12. HE staining: Dewax tissue sections (see Tissue Section Fluorescence Staining for steps), stain with Harris hematoxylin for 6 min, wash sections twice with distilled water, differentiate with 1% hydrochloric acid alcohol for a few seconds, wash twice with distilled water, return to blue with 0.6% ammonia solution, wash three times with distilled water, stain sections with eosin solution for 3 min, wash twice with distilled water, and then sequentially immerse sections in 95% ethanol solution for 5 min, followed by 95% ethanol solution, 5 min, anhydrous ethanol, 5 min, anhydrous ethanol, xylene, 5 min, and 5 min. After dehydration and permeability, place sections at room temperature to air dry, mount with neutral resin, observe under a microscope, and acquire images using CaseViewer software.

[0087] 13. Nissl body staining: Dewax tissue sections (see Fluorescent staining of tissue sections for steps), stain with Nissl stain for 5 min, wash twice with distilled water, differentiate with 1% glacial acetic acid, wash twice with distilled water to terminate the reaction, dry the sections, permeate with xylene for 5 min, mount with neutral resin, observe under a microscope, and acquire images using CaseViewer software.

[0088] 14. For subsequent Real-time PCR, Western Blot, and various staining experiments, at least three independent samples were used for testing. Data were summarized using Excel and analyzed and plotted using GraphPad 8.0. For comparisons between two or more groups, p-values ​​were calculated using SPSS 22.0. Data are expressed as mean ± SEM. A p-value < 0.05 was considered statistically significant.

[0089] In a preferred embodiment of the present invention, miR375-3p inhibits Aβ. 25-35 Induced apoptosis in SH-SY5Y cells: To clarify Aβ 25-35 The precise timing of SH-SY5Y cell induction was determined by Western blot analysis of Aβ levels at different time points (18 h, 24 h, and 48 h). 25-35 Changes in PS1, apoptosis representative proteins, and autophagy markers in SH-SY5Y cells after treatment, with the most significant changes observed at 24 h. Figure 2A ).

[0090] Western blot results showed that miR375-3p overexpression could slightly induce total PARP protein cleavage and significantly block Aβ. 25-35 Induced PARP cleavage and Caspase-3 activation. miR375-3p can restore Aβ. 25-35The miR375-3p induces PS1 hydrolysis, inhibits the expression of Syn and the key autophagy protein Beclin1, and blocks the production of APP-CTFα, but has little effect on ATG14 expression. This indicates that miR375-3p is crucial for Aβ... 25-35 It has a significant impact on induced apoptosis, AD risk genes, and key autophagy proteins. Figure 2B The results from TUNEL, ROS, and JC-1 also confirmed this, showing that after transfection with miR375-3p-mimic, Aβ... 25-35 The number of fragmented DNA in induced SH-SY5Y cells was significantly downregulated. Figure 2C ), intracellular reactive oxygen species levels decreased ( Figure 2D mitochondrial membrane potential levels also recovered significantly. Figure 2E This indicates that the obstruction of ATP synthesis and mitochondrial damage have been alleviated. Single-channel immunofluorescence assay results show ( Figure 2F The addition of miR375-3p-mimic can promote Aβ 25-35 Induced degradation (phagocytosis) of Aβ in SH-SY5Y cells and enhanced LC3B levels, but no significant effect on Beclin1.

[0091] In a preferred embodiment of the present invention, rapamycin is used in combination with miR375-3p-mimic to antagonize Aβ by promoting autophagy. 25-35 Induced apoptosis in SH-SY5Y cells: TUNEL and ROS experiments further clarified the role of rapamycin. TUNEL results showed that after the addition of rapamycin, Aβ... 25-35 The number of fragmented DNA was significantly reduced in induced SH-SY5Y cells. After transfection with miR375-3p-mimic, the group using the combination of miR375-3p-mimic and rapamycin showed stronger anti-apoptotic activity compared to AD model cells transfected only with miR375-3p-mimic. Figure 3A Similar situations have also occurred in the detection of reactive oxygen species (ROS) levels. Figure 3B Western blot results showed ( Figure 3C The addition of rapamycin enhanced the expression of Mfn2 and Grp75 proteins, and also had a slight effect on the recovery of Beclin1. Most significantly, it enhanced the effect of LC3B. miR375-3p can protect Aβ. 25-35 It induced BDNF degradation, but the addition of rapamycin did not further enhance the effect of miR375-3p, and its effect on PS1 was not significant.

[0092] Evaluation of biosafety: Rapamycin and miR375-3p-mimic-agomir were transduced into wild-type mice via intraperitoneal injection and tail vein injection, respectively, with the injection cycle consistent with that of APP / PS1 Tg mice. Figure 4A After the cycle is completed, hematoxylin / eosin staining is performed. Figure 4B The results showed no significant damage to the heart, liver, spleen, lungs, and kidneys of mice, indicating that rapamycin and miR375-3p-mimic-agomir are safe to use in vivo.

[0093] In a preferred embodiment of the present invention, the repair of cognitive function and memory deficits specifically involves the combined use of rapamycin and miR375-3p-mimic-agomir to repair cognitive function and memory deficits.

[0094] In this embodiment of the invention, before completing the injection cycle, we determined the miR375-3p-mimic- through in vivo imaging experiments. Distribution of agomir in APP / PS1 TG mice Figure 4C The results showed that after tail vein injection of miR375-3p-mimic- agomir-CY3 was mainly distributed in the limbs, brain, and ear, indicating that miR375-3p-mimic-agomir-CY3 can accumulate in the main lesion areas of APP / PS1 TG mice. The injection cycle was completed ( Figure 4A First, the effects of rapamycin / miR375-3p-mimic-agomir alone and in combination on spatial memory and learning ability in AD model mice were evaluated using the Morris water maze and Y-maze experiments. The water maze results showed that the AD+miR375-3p group ( Figure 4D The escape latency (middle blue line) showed a significantly shorter trend compared to the AD group and the AD+rapamycin group. Furthermore, based on the escape latency results on day 7 of the experimental phase, compared to the AD group and the AD+rapamycin group (…), the escape latency was significantly shorter. Figure 4E Compared to the red and yellow bars, the AD+miR375-3p group and the AD+rapamycin+miR375-3p group ( Figure 4E The escape latency used by the blue and green bars was shorter, approaching that of the wildtype group. Furthermore, in choosing routes to find hidden platforms, the AD+miR375-3p group and the AD+rapamycin+miR375-3p group selected shorter routes compared to the AD and AD+rapamycin groups, demonstrating a stronger search strategy. Figure 4FAfter analyzing the average escape latency during the 7-day experimental phase, the results were consistent with the above findings. The average escape latency in the AD+miR375-3p group and the AD+rapamycin+miR375-3p group was significantly shorter than that in the AD group during the experimental phase. Figure 4G The blue and green bars in Figure 4G show the results, while the AD+rapamycin group showed no significant memory repair (yellow bars in Figure 4G).

[0095] The results of the Y-maze spontaneous alternation experiment showed that, compared with the AD group, the other four groups of mice exhibited a more rational exploration order during the spontaneous alternation exploration process, demonstrating a recovery in spatial cognitive ability. Figure 4H ), and the results of the new heteroarm exploration experiment showed that, compared with the AD group, the AD+miR375-3p group and the AD+rapamycin+miR375-3p group ( Figure 4I and 4J The blue and green pillars in the middle section represent longer exploration time and distance in the new heterogeneous arm, while the AD+rapamycin group ( Figure 4I and 4J The yellow bars (in the middle section) showed no significant difference compared to the AD group. These results indicate that intervention with miR375-3p-mimic-agomir can significantly alleviate cognitive and memory deficits in APP / PS1 Tg mice, and it also has the same effect when used in combination with rapamycin.

[0096] In a preferred embodiment of the present invention, the reduction of Aβ deposition specifically involves: rapamycin and miR375-3p- mimic-agomir reduces Aβ deposition and repairs nerve damage when used in combination.

[0097] In this embodiment of the invention, the immunohistochemical staining results of the mouse brain region showed ( Figure 5A The AD group had the most Aβ-positive plaques, which were multi-spherical, dense, and highly visible. In contrast, the AD+rapamycin group had the most plaques. The AD+miR375-3p group and the AD+miR375-3p group had fewer Aβ-positive plaques, and the brown plaques were relatively scattered. The AD+rapamycin+miR375-3p group had the fewest Aβ-positive plaques, while the AD+rapamycin group had no significant difference in Aβ-positive plaques compared to the AD group. This indicates that the combined use of rapamycin and miR375-3p-mimic-agomir can reduce Aβ deposition in the brains of APP / PS1 Tg mice, and the effect is the best compared to other experimental groups.

[0098] We stained Nissl bodies in the brain regions of each group of mice. Figure 5B and 5C Compared to the AD group, AD+ The number of Nissl bodies was significantly increased in the rapamycin+miR375-3p group and the AD+miR375-3p group, indicating that the nerve damage was repaired, while there was no significant difference between the AD+rapamycin group and the AD group.

[0099] Brain TUNEL results showed that ( Figure 5D Compared to the AD group, the combined use of rapamycin and miR375-3p-mimic-agomir could reverse apoptosis in the brains of APP / PS1 TG mice and repair neuronal damage. The effect of miR375-3p-mimic-agomir alone was similar, but not as good as when used in combination with rapamycin. The use of rapamycin alone had a positive effect on apoptosis in the brain, but had no significant effect on the clearance of Aβ plaques and the recovery of Nissl bodies.

[0100] In a preferred embodiment of the present invention, the reduction of neuroinflammation specifically comprises: rapamycin and miR375-3p- mimic-agomirr inhibits neuropathological markers at both the transcriptional and translational levels through a combined approach.

[0101] In this embodiment of the invention, the expression of brain-derived neurotrophic factor (BDNF) and synaptophysin (Syn) are important indicators for assessing AD brain pathology. Immunofluorescence staining experiments showed that the combined use of rapamycin and miR375-3p-mimic-agomir significantly increased the expression of BDNF in the brains of APP / PS1 TG mice, and the morphology of positive expression was more similar to that of wild-type mice. The groups treated with miR375-3p-mimic-agomir alone had similar effects. Figure 6A Syn's results are consistent with this. Figure 6B We performed Western blot analysis on the parietal and frontotemporal lobes of the mouse brain. The results showed that mice receiving miR375-3p alone or in combination with rapamycin exhibited very similar outcomes, blocking AD-related PS1 overexpression, APP degradation, CTFα and β production, decreased BNDF and SYN protein levels, restoring the expression levels of autophagy markers Beclin1 and LC3B, and restoring Grp75 and Mfn2 protein expression. Figure 6C The immunofluorescence results for Beclin1, LC3B, and PS1 were similar to those for Western blot. Figure 6D -F).

[0102] Quantitative real-time PCR results showed that rapamycin, in combination with miR375-3p-mimic-agomir and miR375-3p-mimic-agomir alone, inhibited PS1 at the transcriptional level, promoted the expression of BDNF and Syn, and the expression level of APP was close to that of wild-type mice. Rapamycin alone could inhibit the expression of PS1 and APP, but other indicators were not significantly different from those in the AD group. Figure 6G In the regulation of autophagy, compared to the AD group, rapamycin and miR375- The combined use of 3p-mimic-agomir and the use of miR375-3p-mimic-agomir alone promoted the expression of LC3B, Beclin1, Mfn2, and pink-1 at the transcriptional level, manifested as promotion of macroautophagy and mitophagy. Rapamycin alone promoted LC3B expression but had no significant effect on Mfn2, Pink-1, or Beclin1. Figure 6H In summary, both rapamycin and miR375-3p-mimic-agomir can affect neuropathological markers in APP / PS1 TG mice at the transcriptional and translational levels, and their combined effects can inhibit neuropathological features in APP / PS1 TG mice, promote synapsis, and repair autophagy disorders.

[0103] In a preferred embodiment of the present invention, the inhibition of mTOR phosphorylation specifically means that miR375-3p promotes autophagy in the AD environment by inhibiting mTOR phosphorylation.

[0104] In this embodiment of the invention, to further clarify the role of miR375-3p, we investigated the role of the mTOR pathway in this process. Western blot results showed that, via Aβ... 25-35 After treatment, the phosphorylation levels of mTOR (S2448) and AKT in SH-SY5Y cells at different time points were significantly increased, and the phosphorylation level of p70S6 (T389) increased with the extension of induction time. Figure 7A Aβ 25-35 After 24 h of treatment, rapamycin induces phosphorylation of mTOR and p70S6, dephosphorylation of ULK1, and inhibits ATG14 expression. Rapamycin's functions include blocking p70S6 phosphorylation and inhibiting mTORC1 activity by binding to FKBP12; therefore, when Aβ is treated with rapamycin... 25-35 Following induced SH-SY5Y cell induction, mTOR and p70S6 were dephosphorylated, while ULK1 (S638) dephosphorylation was slightly affected. Figure 7BThe combined use of miR375-3p-mimic and rapamycin, although it did not inhibit p-mTOR and pAKT, blocked p70S6 phosphorylation, ULK1 dephosphorylation, and restored ATG14 protein expression. Figure 7B (lane6), but in Aβ 25-35 Under the conditions of treatment, the combined application of the two can block the phosphorylation of mTOR. Figure 7B In lane 8, the autophagy level of cells in the experimental group with the addition of rapamycin was significantly increased, indicating that rapamycin and miR375-3p-mimic act in a parallel relationship, rather than upstream and downstream. Meanwhile, in Aβ... 25-35 In induced SH-SY5Y cells, after rapamycin and miR375-3p-mimic acted together, mTOR and p70S6 were dephosphorylated, while the phosphorylation level of downstream ULK-1 remained unchanged, and ATG14 expression increased. Figure 7B The lanes 3 and 8 indicate that miR375-3p can inhibit mTOR activation and activate autophagy in a simulated AD environment. In APP / PS1 TG mice, compared with the AD group, the AD+rapamycin+miR375-3p group showed decreased p-mTOR, significantly increased pULK-1, and no significant difference in ATG14 expression levels. Figure 7C This indicates that the combined action of rapamycin and miR375-3p-mimic-agomir can inhibit the activation of mTOR in the brain of APP / PS1 TG mice, thereby affecting the occurrence of downstream autophagy.

[0105] In a preferred embodiment of the present invention, the promotion of microglia activation in early AD specifically involves: rapamycin and miR375-3p-mimic promoting microglia activation in early AD.

[0106] In this embodiment of the invention, microglia often perform neuroprotective functions in early-stage AD, promoting the clearance of Aβ and Tau proteins. According to bioinformatics predictions, CD68 is also one of the target genes of miR375-3p. Figure 7D In SH-SY5Y cells and wild-type mice, overexpression of miR375-3p inhibited CD68 protein expression. Figure 7E and 7F The results of immunofluorescence staining of cells were also consistent with this. Figure 7GTo further clarify the role of microglia, we first determined whether astrocytes also played a role. Western blot results showed that miR375-3p-mimic had little effect on GFAP, indicating that astrocytes also had little influence in this process. However, miR375-3p-mimic could inhibit the expression of CD80 and IBA-1 in SH-SY5Y cells, and this phenomenon was reversed upon the addition of rapamycin. Figure 7B (lane2 and lane6), in Aβ 25-35 Under induced conditions, both rapamycin and miR375-3p-mimic can promote the expression of CD80 and IBA-1 in SH-SY5Y cells, and their combined use has an additive effect. Figure 7B In lanes 3 / 4 and 7 / 8 of the APP / PS1 TG mice, Western blot results showed that the expression levels of CD80, IBA-1, and CD68 proteins in the AD group were lower than those in the WT group. Figure 7C After intervention with rapamycin or miR375-3p-mimic-agomir, the expression levels of the three proteins were significantly increased, and the combined use of the two produced an additive effect, indicating that the addition of rapamycin and miR375-3p-mimic-agomir can simultaneously induce M1 and M2 polarization in microglia; quantitative real-time PCR results showed that rapamycin and miR375-3p-mimic-agomir promoted the expression of CD68, CD80 and IBA-1 at the transcriptional level, but the pro-inflammatory factor IL-6 was significantly downregulated in the AD+rapamycin+miR375-3p group. Figure 7J This indicates that although the combined use of rapamycin and AD+rapamycin+miR375-3p significantly activated the M1 type, the corresponding inflammatory response was not aggravated.

[0107] In summary, in Aβ 25-35 In induced SH-SY5Y cells, the addition of rapamycin and miR375-3p-mimic can induce a stress state similar to microglia activation in SH-SY5Y cells; in vivo experiments, rapamycin and miR375-3p-mimic can promote the activation of microglia in the brain of early APP / PS1 TG mice and perform neuroprotective functions.

[0108] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. The application of a miR-375-3p protein in the preparation of drugs for treating Alzheimer's disease, characterized in that, The nucleotide sequence of miR-375-3p is shown in SEQ ID NO.1: 3'-AGUGCGCUCGGCUUGCUUGUUU-5'.

2. The application of the miR-375-3p protein according to claim 1 in the preparation of drugs for treating Alzheimer's disease, characterized in that, The treatment refers to one or more of the following: inhibiting Aβ 25-35 It induces SH-SY5Y cell apoptosis, reduces Aβ deposition, inhibits mTOR phosphorylation, promotes microglia activation in early Alzheimer's disease, repairs cognitive function and memory deficits, and reduces neuroinflammation.

3. The application of the miR-375-3p protein according to claim 2 in the preparation of drugs for treating Alzheimer's disease, characterized in that, The inhibition of Aβ 25-35 The induced apoptosis in SH-SY5Y cells is specifically as follows: miR-375-3p inhibits Aβ 25-35 Induced apoptosis in SH-SY5Y cells or antagonism of Aβ by promoting autophagy when rapamycin is used in combination with miR-375-3p-mimic. 25-35 Induced apoptosis in SH-SY5Y cells.

4. The use of the miR-375-3p protein according to claim 2 in the preparation of drugs for treating Alzheimer's disease, characterized in that, The reduction of Aβ deposition specifically refers to: Rapamycin, when used in combination with miR-375-3p-mimic-agomir, reduces Aβ deposition.

5. The use of the miR-375-3p protein according to claim 2 in the preparation of drugs for treating Alzheimer's disease, characterized in that, The inhibition of mTOR phosphorylation specifically refers to: miR-375-3p promotes autophagy in the Alzheimer's environment by inhibiting mTOR phosphorylation.

6. The use of the miR-375-3p protein according to claim 2 in the preparation of drugs for treating Alzheimer's disease, characterized in that, The specific steps to promote microglial cell activation in early Alzheimer's disease are as follows: Rapamycin and miR-375-3p-mimic promote microglia activation in early Alzheimer's disease.

7. The use of the miR-375-3p protein according to claim 2 in the preparation of drugs for treating Alzheimer's disease, characterized in that, The repair of cognitive function and memory deficits specifically includes: Rapamycin, when used in combination with miR-375-3p-mimic-agomir, repairs cognitive and memory deficits.

8. The use of the miR-375-3p protein according to claim 2 in the preparation of drugs for treating Alzheimer's disease, characterized in that, The reduction of neuroinflammation specifically refers to: Rapamycin, when used in combination with miR-375-3p-mimic-agomirr, inhibits neuropathological markers at both the transcriptional and translational levels.

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