Application of reagent for inhibiting FOXO3a gene in preparation of medicine for treating homocysteine-induced metabolism-related fatty liver disease

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of a reagent for inhibiting an FOXO3a gene in preparation of a medicine for treating homocysteine induced metabolism-related fatty liver disease, and the base sequence of the FOXO3a gene is shown as SEQ ID NO.1. According to the application, Western blot is used for analyzing high methionine diet induced Cbs < + / -> mouse liver tissues and Hcy treatment group liver cells, it is clear that the expression of FOXO3a in the Hcy induced metabolism-related fatty liver diseases is remarkably up-regulated, and it is clear that FOXO3a can influence the occurrence of the metabolism-related fatty liver diseases through mediating mitochondrial damage.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the inhibition of... FOXO3a Application of gene-based reagents in the preparation of drugs for treating homocysteine-induced metabolic-related fatty liver disease. Background Technology

[0002] Metabolic dysfunction-associated fatty liver disease (MAFLD) is a chronic metabolic stress-induced liver disease caused by overnutrition, insulin resistance, and related metabolic dysfunction in genetically susceptible individuals. Currently, MAFLD is the leading chronic liver disease affecting more than one-third of the global adult population, and its prevalence is expected to rise further due to increased metabolic burden, high-calorie, low-nutrient diets, and reduced physical activity, making it an increasingly serious public health problem in my country and globally. The health risks posed by MAFLD should not be underestimated. Besides causing liver-specific complications such as cirrhosis and hepatocellular carcinoma, it is also closely related to systemic diseases such as obesity, metabolic syndrome, chronic kidney disease, and type 2 diabetes.

[0003] Homocysteine ​​(Hcy) is a sulfur-containing amino acid, a product of methionine demethylation. Various factors can lead to the accumulation of Hcy in plasma. The liver is a crucial organ for Hcy metabolism; when hepatocytes are damaged, Hcy levels rise synchronously. Abnormally elevated Hcy levels further enhance oxidative stress, causing hepatic lipid peroxidation, inducing hepatocyte damage and apoptosis, creating a vicious cycle that exacerbates metastatic liver injury and fibrosis (MAFLD). However, the mechanism by which Hcy levels regulate hepatocyte mitochondrial function and participate in the development of MAFLD remains unclear.

[0004] Currently, treatment options for homocysteine-induced metabolic fatty liver disease remain limited, and there is a lack of targeted therapies with clearly defined mechanisms of action in clinical practice. Therefore, elucidating the key molecular mechanisms of Hcy-induced liver injury, identifying potential targets, and developing corresponding treatment strategies are of great significance for the clinical prevention and treatment of this disease. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for suppressing... FOXO3a Application of gene-based reagents in the preparation of drugs for treating homocysteine-induced metabolic-related fatty liver disease.

[0006] To achieve the above objectives, the present invention adopts the following technical solution.

[0007] inhibition FOXO3a The application of gene-based reagents in the preparation of drugs for treating homocysteine-induced metabolic-related fatty liver disease;FOXO3a The base sequence is shown in SEQ ID NO.1.

[0008] Preferably, the reagent includes an inhibitor. FOXO3a Gene interference vectors.

[0009] Preferably, the interference carrier is the FOXO3a A gene siRNA interference vector; the siRNA is selected from one of the following primer pairs: The sense chain sequence is shown in SEQ ID NO.6, and the antisense chain sequence is shown in SEQ ID NO.7; or The sense chain sequence is shown in SEQ ID NO.8, and the antisense chain sequence is shown in SEQ ID NO.9; or The sense chain sequence is shown in SEQ ID NO.10, and the antisense chain sequence is shown in SEQ ID NO.11.

[0010] Preferably, the inhibition FOXO3a Gene reagents also include transfection reagents.

[0011] Preferably, the transfection aid is INVI DNA RNA Transfection Reagent.

[0012] Preferably, the drug inhibits FOXO3a The reagent for the gene is the only active ingredient.

[0013] Preferably, the drug further includes a pharmaceutically acceptable carrier.

[0014] Preferably, the pharmaceutically acceptable carrier is any one or more of chitosan, cholesterol, liposomes, and nanoparticles.

[0015] Preferably, the drug regulates mitochondrial damage in hepatocytes in the early stages of homocysteine-induced metabolic-related fatty liver disease.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention provides inhibition FOXO3a The application of gene-based reagents in the preparation of drugs for treating homocysteine-induced metabolic-related fatty liver disease; FOXO3a The gene's base sequence is shown in SEQ ID NO.1. This invention is the first to propose an inhibitory mechanism. FOXO3a The application of gene therapy in the treatment of homocysteine-induced metabolic-related fatty liver disease provides new targets and strategies for the drug treatment of this type of disease. By inhibiting... FOXO3aThe expression of [specific ingredient] can significantly alleviate Hcy-induced mitochondrial damage in hepatocytes, reduce oxidative stress levels, and improve lipid metabolism disorders, thereby exerting a therapeutic effect. This invention has demonstrated through in vitro and in vivo experiments that [specific ingredient] can [specifically] alleviate mitochondrial damage in hepatocytes, reduce oxidative stress levels, and improve lipid metabolism disorders, thereby exerting a therapeutic effect. FOXO3a Hcy expression was significantly upregulated in metabolically related fatty liver disease, and its expression level was closely related to mitochondrial dysfunction and the degree of lipid deposition. FOXO3a It can significantly improve mitochondrial membrane potential, reduce ROS production, alleviate mitochondrial morphological abnormalities, and reduce the accumulation of cholesterol and triglycerides in hepatocytes, thus clarifying the mechanism. FOXO3a The key role of this in the pathological process. The treatment strategy provided by this invention is characterized by its high specificity and clear mechanism, laying the foundation for the development of targeted therapies for homocysteine-induced metabolic-related fatty liver disease, and has significant clinical translational potential. Attached Figure Description

[0017] Figure 1 This invention utilizes HE and Oil Red O staining to observe the effects of a high-methionine diet on induced methionine absorption. Cbs + / - Liver tissue damage in mice; scale bar = 50 μm.

[0018] Figure 2 The figures show the lipid deposition level, total cholesterol (TC), and triglyceride (TG) levels in hepatocytes after Hcy intervention in this invention. In this figure, A represents the lipid deposition in hepatocytes observed by Oil Red O staining, with a scale bar of 20 μm; B represents the total cholesterol (TC) content in hepatocytes; and C represents the triglyceride (TG) content in hepatocytes.

[0019] Figure 3 In this invention FOXO3a Expression levels in Hcy-induced metabolic-associated fatty liver disease, where A represents... Cbs + / - A) Expression level of FOXO3a protein in mouse liver tissue; B) Statistical analysis of relative expression of FOXO3a protein; C) Expression level of FOXO3a protein in hepatocytes; D) Statistical analysis of relative expression of FOXO3a protein.

[0020] Figure 4 This invention describes the interference efficiency of qRT-PCR in detecting siRNA interference fragments transfected into hepatocytes.

[0021] Figure 5 Knockdown in this invention FOXO3aEffects of Hcy on mitochondrial damage in hepatocytes: A represents ROS levels in hepatocytes (scale bar = 50 μm); B represents changes in mitochondrial membrane potential in hepatocytes; C represents the percentage of JC-1 monomeric positive cells; D represents changes in mitochondrial morphology in hepatocytes (scale bar = 2 μm); and E represents the relative expression level of mtDNA in hepatocytes.

[0022] Figure 6 Knockdown in this invention FOXO3a Effects of Hcy on lipid deposition in hepatocytes: A shows lipid deposition in hepatocytes observed by Oil Red O staining (scale bar = 20 μm); B shows the total cholesterol (TC) content in hepatocytes; and C shows the triglyceride (TG) content in hepatocytes. Detailed Implementation

[0023] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0024] This invention provides FOXO3a It is used in the preparation of products for diagnosing homocysteine-induced metabolic-related fatty liver disease. Among them, FOXO3a

[0025] FOXO3a The base sequence of the positive-strand primer is shown in SEQ ID NO.2, and is as follows: 5′-CGGACAAACGGCTCACTCT-3′.

[0026] FOXO3a The base sequence of the antisense primer is shown in SEQ ID NO.3, and is as follows: 5′-GGACCCGCATGAATCGACTAT-3.

[0027] The base sequence of the positive strand of the GADPH internal reference primer is shown in SEQ ID NO.4, and is as follows: 5′-ACCCAGAAGACTGTGGAGG-3′.

[0028] The base sequence of the antisense strand of the GADPH internal reference primer is shown in SEQ ID NO.5, and is as follows: 5′-TTCTAGACGGCAGGTCAGGT-3′.

[0029] Example 1, Experimental Method I. Grouping and Culture of Hepatocytes 1. Experimental Grouping The study investigated the effects of homocysteine ​​(Hcy) intervention on mitochondrial damage in hepatocytes: control group and Hcy intervention group.

[0030] The control group was treated as follows: after the cells reached the logarithmic growth phase and the density reached 70% (V / V), they were randomly assigned to other groups and received no intervention. The Hcy intervention group was treated as follows: after the cells reached the logarithmic growth phase and the density reached 70% (V / V), they were randomly assigned to other groups and treated with 100 μmol / L Hcy working solution for 24 h.

[0031] Research interference FOXO3a Effects on hepatocyte mitochondrial damage: normal control group (Control group), interference control group (si-NC group), transfection FOXO3a Interference fragment group 1 (si- FOXO3a -1 group), transfection [[ID=3�]]FOXO3a Interference fragment group 2 (si- FOXO3a -2 groups) and transfection FOXO3a Interference fragment group 3 (si- FOXO3a-3 groups). Cells in the logarithmic growth phase were digested, counted, resuspended in DMEM medium (7% serum concentration), and seeded in equal volumes into 6-well plates. When the cell density reached 70% (V / V), transfection was performed. The normal control group (Control) was treated with DMEM high-glucose medium; the interference control group (si-NC) was treated with si-NC transfection reagent mixture. FOXO3a Interference group 1 (si- FOXO3a The treatment method for -1) is: add si- FOXO3a -1 transfection reagent mixture; FOXO3a Interference group 2 (si- FOXO3a -2) The treatment method is: add si- FOXO3a -2 transfection reagent mixture; FOXO3a Interference group 3 (si- FOXO3a -3) The treatment method is: add si- FOXO3a -3 transfection reagent mixture; return to incubator and continue incubation for 24 hours.

[0032] in accordance with FOXO3a Sequence design and construction of three interfering nucleic acids (siRNA) FOXO3a Knockdown model (Shanghai Gemma Gene Synthesis), denoted as si- FOXO3a The gene is then transferred into liver cells, thereby achieving the knockdown of the target gene.

[0033] si- FOXO3a The base sequence of the -1 positive strand primer is shown in SEQ ID NO.6, and is as follows: 5′-GCACCAUGAAUCUGAAUGATT-3′.

[0034] si- FOXO3a- The base sequence of the antisense primer is shown in SEQ ID NO.7, and is as follows: 5′-UCAUUCAGAUUCAUGGUGCTT-3′.

[0035] si- FOXO3a The base sequence of the -2 positive strand primer is shown in SEQ ID NO.8, and is as follows: 5′-GGAGCUUGGAAUGUGACAUTT-3′.

[0036] si- FOXO3a The base sequence of the -2 antisense primer is shown in SEQ ID NO.9, and is as follows: 5′-AUGUCACAUUCCAAGCUCCTT-3′.

[0037] si- FOXO3aThe base sequence of the -3 positive strand primer is shown in SEQ ID NO.10, and is as follows: 5′-GGAGUUUGGUCAAUCAGAATT-3′.

[0038] si- FOXO3a The base sequence of the -3 antisense primer is shown in SEQ ID NO.11, and is as follows: 5′-UUCUGAUUGACCAAACUCCTT-3′.

[0039] 2. Cell Culture (1) The cell culture medium is a complete culture medium, specifically: DMEM high glucose medium containing 10% horse serum and 1% penicillin-streptomycin by volume.

[0040] (2) Normoaerobic culture: in a cell culture incubator at 37°C and 5% CO2.

[0041] (3) Collect the cells after 24 hours for subsequent experiments.

[0042] 3. Cell cryopreservation Remove the cell culture flask from the incubator, select hepatocytes in the logarithmic growth phase, aspirate and discard the waste liquid from the culture flask on a clean bench, gently rinse twice, add 1 mL of trypsin to digest the cells, discard the trypsin after complete digestion, add DMEM high-glucose medium, disperse and mix the adherent cells, transfer to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add cryopreservation solution, mix the cells by pipetting, and then aliquot into 1.8 mL cryovials, labeling them with cell name, cryopreservation time and operator. Cryopreserve in a gradient order of 4℃ for 30 min, -20℃ for 2 h, and -80℃ overnight, and then transfer to a liquid nitrogen tank for storage.

[0043] The method for preparing the cryopreservation solution is as follows: the volume ratio of DMSO to fetal bovine serum is 1:9.

[0044] Overnight stays refer to stays of 12 hours or more.

[0045] 4. Interference with nucleic acid (siRNA) transfection Hepatocytes in the logarithmic growth phase were digested, counted, resuspended in DMEM medium containing 7% premium horse serum, and seeded in equal volumes into 6-well plates. Transfection was performed when the cell density reached 70% (V / V). The required volume of nucleic acid for transfection was calculated according to the transfection reagent instructions to prepare the Si- FOXO3a Working solution, transfection aid INVI DNA RNA Transfection Reagent and si- FOXO3aThe working fluid was added to the high-glucose DMEM medium at a ratio of 1:1 and allowed to stand at room temperature for 15 min. The si- FOXO3a transfection reagent mixture was added to the cell culture flask and then returned to the incubator for further culture. After 24 hours, hepatocyte RNA was extracted and the transfection efficiency was verified using PCR.

[0046] Among them, the name of the transfection reagent is INVI DNA RNA Transfection Reagent, which was purchased from Thermo Fisher Scientific, USA.

[0047] II. Feeding and grouping of experimental animals 1. Feeding of experimental animals SPF grade Cbs + / - Twelve male 6-week-old mice with a body weight of 23 g were purchased from Chengdu Medicine Kang Biotechnology Co., Ltd. [SYXK (Chuan) 2020-034] and raised in the SPF-grade modeling experiment barrier environment of the Experimental Animal Center of Ningxia Medical University [SYXK (Ning) 2025-0001]. The lighting was alternated with a 12-h day / 12-h night cycle, the environmental temperature inside the barrier was 23 °C, and the relative humidity was 60%. The mice were raised in the purified air-conditioning system's microenvironment of independent ventilated cages (IVC), and the bedding and normal drinking water were changed daily. During the animal experiment process, humane care was given in strict accordance with the 3R principle, and strict compliance with international experimental animal ethics requirements and experimental animal ethics norms was ensured, meeting the principles of animal protection, animal welfare, and ethics.

[0048] 2. Grouping of experimental animals 6 weeks old Cbs + / - The mice (n = 12) were randomly divided into a normal diet control group (ND group, n = 6) and a high-methionine diet group (HMD group, n = 6), and the experiment was conducted after 12 weeks of feeding.

[0049] III. Real-time fluorescence quantitative polymerase chain reaction, abbreviated as qRT-PCR 1. Extraction of total RNA from cells [[ID=得克萨斯州]] (1) Preparation: According to the instructions of the TIANGEN RNA preparation kit, the items required for RNA extraction were placed in a UV-irradiated laminar flow hood.

[0050] (2) Sample lysis: The pre-prepared hepatocytes transfected with si- FOXO3a interfering nucleic acid were placed in an EP tube containing 1 mL of RZ lysis buffer, and the cells were gently pipetted and mixed, and then left at room temperature for 5 min to allow sufficient lysis.

[0051] It should be noted that there seems to be an incorrect "得克萨斯州" in the translation of line 29 which is likely a misinput. The correct translation for that part should be: (1) Preparation: According to the instructions of the TIANGEN RNA preparation kit, the items required for RNA extraction were placed in a UV-irradiated laminar flow hood.(3) Place it in a pre-cooled centrifuge and centrifuge at 12000 rpm / min for 5 min. After taking it out, move it to the workbench and transfer the supernatant to a new enzyme-free EP tube that has been prepared in advance.

[0052] (4) Add 200 μL of chloroform to the sample, vortex vigorously for 30 seconds, let stand in the workbench for 3 min, centrifuge at 4℃ for 10 min and the sample can be seen to be divided into three layers: organic layer, intermediate layer and aqueous phase. RNA mainly aggregates in the aqueous phase. Take 450 μL of colorless aqueous phase layer and transfer it to a new EP tube.

[0053] (5) Add 225 μL of anhydrous ethanol to the EP tube and mix by inverting the tube. Transfer the mixed liquid to the labeled adsorption column and centrifuge at 12000 rpm / min for 30 seconds at 4℃.

[0054] (6) Discard the liquid in the collection tube and add 500 μL of protein removal elution buffer RD to the adsorption column. Centrifuge at 12000 rpm for 30 seconds at 4°C.

[0055] (7) Discard the liquid in the collection tube and add 500 μL of washing solution RW to the adsorption column. Place it in the workbench for 2 min and then centrifuge at 12000 rpm / min at 4℃ for 30 sec. Repeat this step twice.

[0056] (8) After discarding the waste liquid, centrifuge at 12000 rpm / min at 4℃ for 2 min. After centrifugation, remove the remaining liquid.

[0057] (9) In a clean bench, let the adsorption column stand for 10 minutes to dry completely. Finally, transfer it to a labeled RNase-free EP tube, add 40 μL of RNase-free ddH2O, let it stand at room temperature for 2 minutes, centrifuge at 12000 rpm / min at 4℃ for 30 seconds to collect RNA, detect the RNA concentration using a spectrophotometer, and store it in a -80℃ freezer for long-term preservation.

[0058] 2. Reverse transcription (1) After preparing the RNA, first put the RNA that has been extracted and stored at -80℃ on ice, thaw it, and then remeasure the RNA concentration. Prepare the reverse transcription system for each sample, as shown in Table 1.

[0059] Table 1 Reverse Transcription System (2) Shake the prepared reverse transcription system to mix well, remove air bubbles, and perform reverse transcription according to the conditions in Table 2: Table 2 Reverse transcription conditions (3) After the RNA is reverse transcribed into cDNA, the sample is collected and stored in a -20℃ refrigerator.

[0060] 3. PCR reaction (1) Prepare the reagents required for the PCR reaction according to the table below. Place the eight-pack on the pre-cooled constant temperature module to prepare the reaction solution. See Table 3 for details.

[0061] Table 3 Reaction System (2) Add the reaction solution to each of the eight-tube strips, gently shake to mix until there are no air bubbles, and then perform PCR amplification. The procedure is shown in Table 4.

[0062] Table 4 Amplification Procedure (8) Using 2 -ΔΔCt The method calculates the relative expression of the target gene.

[0063] IV. Observation of liver tissue damage by HE staining of liver tissue sections (1) Take the frozen liver slices out of the -80℃ freezer and let them stand at room temperature for 30 minutes.

[0064] (2) 4% (mass-volume ratio, which means that each 100 ml solution contains 4 g of paraformaldehyde) Paraformaldehyde fixation for 3 min.

[0065] (3) Stain with hematoxylin for 2 min, differentiate with differentiation solution for 1 s, and rinse with tap water for 30 s.

[0066] (4) Blueing solution for 1 minute, then rinse with tap water for 30 seconds.

[0067] (5) Stain with eosin for 1 min, then dehydrate in a gradient manner and clear.

[0068] (6) Mount the slide with neutral resin and take pictures of the field of view under a microscope.

[0069] V. Observation of the distribution and accumulation of lipid droplets in hepatocytes by Oil Red O staining of liver tissue sections. (1) Take the frozen liver slices out of the -80℃ freezer and let them stand at room temperature for 30 minutes.

[0070] (2) Rinse with distilled water for 2 min, then rinse with 60% isopropanol for 1 min.

[0071] (3) Stain with Oil Red O working solution for 5 min, rinse with 60% isopropanol and then immediately wash with water.

[0072] (4) Stain the nuclei with hematoxylin for 2 minutes, then rinse with running water to return to blue.

[0073] (5) Mount the slide with water-based mounting medium and take pictures of the field of view under a microscope.

[0074] VI. Cell Oil Red O Staining (1) Remove the cell culture medium and wash twice with PBS.

[0075] (2) Add ORO Fixative fixative and fix for 30 min.

[0076] (3) Remove the fixative and wash twice with distilled water.

[0077] (4) Wash with 60% isopropanol for 5 min, discard the isopropanol, add ORO Stain and soak for 20 min.

[0078] (5) Discard the staining solution and wash three times with distilled water.

[0079] (6) Add ORO Buffer and soak for 1 min, then discard the staining solution.

[0080] (7) Mount the slide with water-based mounting medium and take pictures of the field of view under a microscope.

[0081] VII. Detection of total cholesterol (TC) and triglyceride (TG) levels (1) Collect cells according to the group, centrifuge at 1000r for 10min, discard the supernatant and keep the cell pellet.

[0082] (2) Wash twice with PBS, centrifuge at 1000r for 10min, discard the supernatant and keep the cell pellet.

[0083] (3) Add 200 μL of PBS for homogenization, and sonicate under ice-water bath. The prepared homogenate is directly measured without centrifugation. The PBS contains 1.06 mM KH2PO4, 154.0 mM NaCl, and 5.60 mM Na2HPO4, with a pH of 7.4. It was purchased from Wuhan Saiweier.

[0084] (4) Take 2.5 μL of sample and 250 μL of TC / TG working solution into a 96-well plate, shake the plate to mix, and incubate at 37°C for 10 min.

[0085] (5) Measure the absorbance of each sample at 500 nm and calculate the TC and TG contents according to the protein concentration of the homogenate. The TC / TG detection kit was purchased from Nanjing Jiancheng.

[0086] 8. Western blot detection of FOXO3a protein expression level (1) Liver tissues from mice in the ND and HMD groups were ground according to their respective groups, and cells from the Control and Hcy groups were collected according to their respective groups. Cell lysis buffer was added to each group for protein extraction. The concentrations of each component in the cell lysis buffer were 1 mL of Lysis Buffer, 10 μL of phosphatase inhibitor, 1 μL of protease inhibitor and 10 μL of 100 mM PMSF, which were purchased from Jiangsu Kaiji Biotechnology.

[0087] (2) The protein concentrations obtained by the BCA method were uniformly diluted with Loading Buffer before loading.

[0088] (3) SDS-PAGE electrophoresis, membrane transfer, and blocking.

[0089] (4) Incubate the primary antibody at 4°C overnight, and the secondary antibody at room temperature for 2 hours. Overnight means ≥12 hours.

[0090] (5) The strips were washed and developed by electrochemical method, and the gray values ​​were statistically analyzed using ImageJ software.

[0091] IX. JC-1 Mitochondrial Membrane Potential Flow Cytometry Detection (1) Collect cells according to the group, add 0.5 mL of JC-1 staining working solution, and invert several times.

[0092] (2) Incubate at 37℃ for 20 min, centrifuge at 4℃ for 500g for 5 min, precipitate the cells, and discard the supernatant.

[0093] (3) Wash twice with JC-1 staining buffer.

[0094] (4) Add 1 mL of JC-1 staining buffer to resuspend the cells, centrifuge at 500g for 5 min at 4℃, precipitate the cells, and discard the supernatant.

[0095] (5) Resuspend the cells in an appropriate amount of JC-1 staining buffer. The JC-1 detection kit was purchased from Beijing Lanbolide.

[0096] (6) The mitochondrial membrane potential of JC-1 was detected by flow cytometry.

[0097] 10. ROS (Reactive Oxygen Spectrometer) Probe-Based Laser Confocal Detection of Intracellular Oxidative Stress Levels (1) According to the grouping, hepatocytes were spread on laser confocal dishes at a cell density of 70% (referring to the percentage of cells that the culture dish can hold).

[0098] (2) Discard the culture medium, add 1 mL of H2DCFDA working solution, and incubate at 37°C in the dark for 30 min.

[0099] (3) Add PBS and wash for 5 min, repeat twice.

[0100] (4) Add an appropriate amount of PBS to cover the area, and take a picture of the field of view using a laser confocal microscope.

[0101] XI. Observation of mitochondrial morphological changes by staining with Mito-tracker fluorescent probes (1) According to the grouping, hepatocytes were spread on laser confocal dishes at a cell density of 70% (referring to the percentage of cells that the culture dish can hold).

[0102] (2) Discard the culture medium and add culture medium containing Mito-tracker fluorescent probe working solution at 37℃. Incubate at 37℃ in the dark for 30 min.

[0103] (3) Add PBS at 37℃ and wash for 5 min. Repeat 3 times.

[0104] (4) Add anti-fluorescence quenching agent, and take a picture of the field of view with a laser confocal microscope.

[0105] XII. Statistical Methods Statistical analysis and graphing were performed using Prism 8.3.0 software. Normally distributed continuous data are expressed as mean ± standard deviation (s). Independent samples t-tests were used to compare data between two groups; one-way ANOVA was used to compare data among multiple groups. A p-value < 0.05 was considered statistically significant.

[0106] Example 2, Experimental Results 1. High-methionine diet induction Cbs + / - Mouse liver tissue injury Liver sections from the ND and HMD groups were stained with HE and Oil Red O to observe liver tissue damage and the distribution and accumulation of lipid droplets within hepatocytes. The results are as follows: Figure 1 As shown.

[0107] Figure 1 The results showed that, compared with the ND group, the HMD group exhibited disordered liver tissue structure, enlarged hepatocytes, disordered hepatic cords, loose and lightly stained cytoplasm, and numerous vacuolar degenerations within hepatocytes, accompanied by fatty degeneration. Oil Red O staining of the liver showed more fat droplets and an increased area of ​​Oil Red O positive staining. These results indicate that a high-methionine diet can induce... Cbs + / - Liver tissue damage in mice.

[0108] 2. Hcy regulates intracellular lipid deposition levels The number of positive lipid droplets in hepatocytes of the Control and Hcy-treated groups was observed using Oil Red O staining. Intracellular TC and TG levels were detected using total cholesterol (TC) and triglycerides (TG) kits. Results are as follows: Figure 2 As shown.

[0109] Figure 2 The results showed that, compared with the control group, the number of Oil Red O lipid droplets in hepatocytes was significantly increased in the Hcy treatment group; and the levels of total cholesterol (TC) and triglycerides (TG) in the cells were significantly increased, confirming that Hcy can induce MAFLD-related liver injury by promoting hepatic lipid synthesis and accumulation.

[0110] 3. FOXO3a Expression level in Hcy-induced MAFLD Western blot analysis was used to detect the protein expression levels of FOXO3a in the ND and HMD groups, and the Control and Hcy groups. The results are as follows: Figure 3 As shown.

[0111] Figure 3 The results showed that compared with the ND group, the expression level of FOXO3a protein was increased in the HMD group; compared with the Control group, the expression level of FOXO3a protein was upregulated in the Hcy group, suggesting that Hcy may participate in the progression of MAFLD by regulating FOXO3a.

[0112] 4. Construction FOXO3a Knockdown Model in accordance with FOXO3a Three interfering nucleic acids (siRNAs) were designed, and a normal control group (Control) and an interference control group (si-NC group) were set up. Transfection was performed... FOXO3a Interference fragment group 1 (si- FOXO3a -1 group), transfection FOXO3a Interference fragment group 2 (si- FOXO3a -2 groups) and transfection FOXO3a Interference fragment group 3 (si- FOXO3a -3 groups), the interference efficiency was verified using qRT-PCR, and the results are as follows: Figure 4 As shown.

[0113] Figure 4 The results showed that there was no difference in expression between the si-NC group and the control group; however, compared with the si-NC group, si- FOXO3a -1、si- FOXO3a -2、si- FOXO3a -3 groups FOXO3a mRNA expression was significantly reduced, suggesting FOXO3A The siRNA interference fragment was successfully constructed. Taking all factors into consideration, the siRNA fragment with the most significant interference effect was selected.FOXO3a -2 ( P Interference fragments with a value of <0.001 were used for subsequent experiments.

[0114] 5. FOXO3a Involved in Hcy-induced mitochondrial damage Si- FOXO3a Transfected into hepatocytes, and tested. FOXO3a Whether Hcy-induced MAFLD occurs by causing mitochondrial damage was investigated. Intracellular oxidative stress was detected using ROS-based laser confocal microscopy; mitochondrial membrane potential was detected using the JC-1 probe combined with flow cytometry; mitochondrial morphological changes were observed using Mito-tracker fluorescent probe staining; and mtDNA expression changes in each group were detected using qRT-PCR. Results are as follows: Figure 5 As shown.

[0115] Figure 5 The results showed that, compared with the control group, Hcy treatment significantly increased intracellular ROS levels; while knockdown... FOXO3a Subsequently, Hcy-induced excessive ROS production was significantly inhibited. JC-1 mitochondrial membrane potential flow cytometry analysis showed that, compared with the Control group, the Hcy group had a significant increase in JC-1 monomers (unhealthy mitochondria exist in the cytoplasm as monomers); compared with the Hcy+si-NC group, the Hcy+si- FOXO3a The number of JC-1 monomers was significantly reduced in the control group. Mito-tracker fluorescent labeling showed that, compared with the control group, mitochondria were broken and fragmented after Hcy treatment, while si- FOXO3a It can significantly reduce Hcy-induced mitochondrial morphological abnormalities. qRT-PCR results showed that compared with the Control group, the Hcy group had significantly increased mtDNA expression levels; compared with the si-NC+Hcy group, the Hcy+si- FOXO3a The mtDNA expression level in the group was reduced.

[0116] The above results indicate that FOXO3a It plays an important role in promoting Hcy-induced mitochondrial damage, and may mediate Hcy-induced MAFLD by regulating oxidative stress, mitochondrial membrane potential and structural integrity.

[0117] 9. Interference FOXO3a Post-intracellular lipid deposition levels The number of positive lipid droplets in hepatocytes of the Control and Hcy-treated groups was observed using Oil Red O staining. Intracellular TC and TG levels were detected using total cholesterol (TC) and triglycerides (TG) kits. Results are as follows: Figure 6 As shown.

[0118] Figure 6The results showed that compared with the Hcy+si-NC group, Hcy+si- FOXO3a Intracellular lipid deposition was significantly reduced in the Hcy+si-NC group. Compared with the Hcy+si-NC group, lipid deposition in Hcy+si-NC was significantly reduced. FOXO3a The levels of intracellular TC and TG in the group were significantly reduced, confirming that... FOXO3a The absence of Hcy can effectively reverse Hcy-induced intracellular lipid deposition.

[0119] This invention focuses on FOXO3a This key biomolecule, according to research, plays a role in homocysteine-induced metabolic-related fatty liver disease. FOXO3a The expression levels of [the substance] can change specifically and significantly. Advanced molecular biology techniques can be used to detect [the substance] in biological samples (such as blood, tissue fluid, etc.). FOXO3a The content of [the substance / method]. Based on this, the present invention provides [the following]. FOXO3a Its application in the preparation of drugs for the treatment of homocysteine-induced metabolic-related fatty liver disease lays the foundation for the development of targeted therapies for homocysteine-induced metabolic-related fatty liver disease and has significant clinical translational potential.

[0120] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0121] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. Inhibition FOXO3a The application of gene-based reagents in the preparation of drugs for treating homocysteine-induced metabolic-related fatty liver disease; characterized in that, The FOXO3a The base sequence of the gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The reagent includes inhibitors FOXO3a Gene interference vectors.

3. The application according to claim 2, characterized in that, The interference carrier is the FOXO3a A gene siRNA interference vector; the siRNA is selected from one of the following primer pairs: The sense chain sequence is shown in SEQ ID NO.6, and the antisense chain sequence is shown in SEQ ID NO.7; or The sense chain sequence is shown in SEQ ID NO.8, and the antisense chain sequence is shown in SEQ ID NO.9; or The sense chain sequence is shown in SEQ ID NO.10, and the antisense chain sequence is shown in SEQ ID NO.

11.

4. The application according to claim 1, characterized in that, The inhibition FOXO3a Gene reagents also include transfection reagents.

5. The application according to claim 4, characterized in that, The transfection aid is INVI DNA RNATransfection Reagent.

6. The application according to claim 1, characterized in that, The drug inhibits FOXO3a The reagent for the gene is the only active ingredient.

7. The application according to claim 6, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

8. The application according to claim 7, characterized in that, The pharmaceutically acceptable carrier is any one or more of chitosan, cholesterol, liposomes, and nanoparticles.

9. The application according to claim 1, characterized in that, The drug regulates mitochondrial damage in hepatocytes in the early stages of homocysteine-induced metabolic-related fatty liver disease.

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