Use of flavin-containing monooxygenase 2 in preparation of non-alcoholic fatty liver disease treatment drugs

Abstract

This invention relates to the field of biomedicine, and discloses the application of flavin-containing monooxygenase 2 in the preparation of drugs for the treatment of non-alcoholic fatty liver disease. This invention is the first to discover that flavin-containing monooxygenase 2 has a preventive or therapeutic effect on non-alcoholic fatty liver disease.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of a flavin-containing monooxygenase 2 in the preparation of a drug for the treatment of non-alcoholic fatty liver disease. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) is a clinical syndrome characterized by excessive lipid (mainly triglycerides) deposition in liver parenchymal cells, excluding alcohol consumption and other clearly defined causes. It is a progressive liver disease, progressing through four stages from mild to severe: non-alcoholic steatosis, non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma. According to relevant surveys, the global prevalence of NAFLD is 25%, and in my country, approximately 30% of the population is affected. In particular, with social progress and rising living standards, the high intake of high-fat foods and unbalanced diets have led to a year-on-year increase in the incidence of NAFLD and a rapid increase in the number of younger patients.

[0003] Nonalcoholic fatty liver disease (NAFLD) is not a single disease; its pathogenesis is complex. The classic "two-hit" theory posits that the accumulation of large amounts of fat in liver parenchymal cells due to various factors (especially elevated insulin levels) constitutes the first hit, while lipid peroxidation and oxidative stress constitute the second hit. In recent years, with in-depth research, the "multiple-hit" theory has gradually gained acceptance. This theory suggests that the pathogenesis of NAFLD is not limited to the aforementioned two hits but also includes other factors such as inflammatory cytokines, host-microbe interactions, genetic factors, and dietary factors. Currently, there are no truly effective drugs for treating NAFLD clinically; treatment primarily focuses on eliminating triggering factors and adjusting diet. Therefore, developing highly effective drugs with low side effects for the treatment of NAFLD is essential.

[0004] Flavin-containing monooxygenases (FMOs) belong to the flavin protease family and are a group of microsomal enzymes dependent on flavin adenine dinucleotide (FAD), reduced nicotinamide adenine dinucleotide phosphate (NADPH), and molecular oxygen. They are generally found in eukaryotes and have attracted widespread attention in heterologous biomass metabolism, pharmacokinetics, and biocatalytic synthesis due to their regioselectivity, chiral catalysis, and good stability. FMOs are the second most important drug-metabolizing enzyme after cytochrome P450 (CYP) enzymes, playing a crucial role in the detoxification and elimination of drugs and chemicals. They catalyze the oxidation of compounds and drugs containing nitrogen, sulfur, phosphorus, selenium, and other nucleophilic heteroatoms. They exist in many subtypes in mammals and humans, each with its own substrate specificity and tissue limitations. Although all isoforms of human FMOs share approximately 60% sequence similarity, their structural and functional similarity is very low. Studies have reported that the absence or dysfunction of FMO3 can lead to trimethylamine metabolism disorders, which is the cause of "fishy smell syndrome," also known as trimethylamineuria.

[0005] Furthermore, FMO2, a member of the flavin-containing monooxygenase family, is a metabolic enzyme with NADP+ and FAD as cofactors. After its discovery in 1992, its function remained largely unexplored for a long period. It wasn't until 2015 that studies found FMO2 to be associated with the lifespan and motility of nematodes. To date, research on FMO2 has primarily focused on its genetic polymorphisms, with limited functional studies. Compared to CYPs, FMOs have long been neglected. However, with increasing research into the characteristics of FMO family members and the consequences of rare FMO mutations and common polymorphic variations, the pharmacological and toxicological significance of these enzymes is becoming increasingly apparent. As an important enzyme in the liver, its research value is immense. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides the application of flavin-containing monooxygenase 2 in the preparation of a therapeutic drug for non-alcoholic fatty liver disease. This invention is the first to discover that flavin-containing monooxygenase 2 has a preventive or therapeutic effect on non-alcoholic fatty liver disease.

[0007] The specific technical solution of this invention is as follows:

[0008] In a first aspect, the present invention provides the application of flavin monooxygenase 2 in the preparation of a therapeutic drug for non-alcoholic fatty liver disease.

[0009] This invention discovers the application of flavin monooxygenase 2 in treating fatty liver by inhibiting de novo lipid synthesis mediated by the SREBP1 / ACC / FASN pathway in the liver.

[0010] Preferably, the drug is a drug that reduces hepatic lipid synthesis and storage or a drug that promotes hepatic lipid metabolism.

[0011] Preferably, the nucleotide sequence of the flavin monooxidase 2 is shown in SEQ ID NO.1 (mouse) or SEQ ID NO.3 (human), and the amino acid sequence is shown in SEQ ID NO.2 (mouse) or SEQ ID NO.4 (human).

[0012] Secondly, the present invention provides a treatment for non-alcoholic fatty liver disease containing flavin monooxygenase 2.

[0013] Preferably, the drug is a drug that reduces hepatic lipid synthesis and storage or a drug that promotes hepatic lipid metabolism.

[0014] Preferably, the nucleotide sequence of the flavin monooxidase 2 is shown in SEQ ID NO.1 (mouse) or SEQ ID NO.3 (human), and the amino acid sequence is shown in SEQ ID NO.2 (mouse) or SEQ ID NO.4 (human).

[0015] Preferably, the drug is an oral or injectable formulation.

[0016] Preferably, the dosage form of the oral preparation is a capsule, tablet, solution, or powder; the dosage form of the injectable preparation is an injection or powder.

[0017] Preferably, the drug also includes a pharmaceutically acceptable carrier, excipient, or solvent.

[0018] Compared with the prior art, the beneficial effects of the present invention are: the present invention discovers that flavin monooxygenase 2 has the effect of preventing or treating non-alcoholic fatty liver disease. Attached Figure Description

[0019] Figure 1 This figure shows the in vivo experimental results of C57 mice after 12 weeks of feeding with either a normal control diet or a high-fat diet. A represents wild-type mice versus FMO2 mice. - / - Statistical results of mouse liver weight, *P<0.05 WT Versus FMO2 - / - B represents wild-type mice and FMO2. - / - Statistical results of triglyceride content in mouse liver, *P<0.05 WT Versus FMO2 - / -- C represents wild-type mice and FMO2. - / - Comparison of gross liver findings in mice; D represents wild-type mice and FMO2 mice. - / -Comparison of HE staining results after paraffin sectioning of mouse liver. WT represents the wild-type mouse group, FMO2. - / - The FMO2 knockout transgenic mouse group. NCD is the normal control diet group, and HFD is the high-fat diet group.

[0020] Figure 2 This image shows the results of in vitro experiments using C57 mouse primary hepatocytes. A represents wild-type primary hepatocytes and FMO2 cells. - / - Statistical results of triglyceride content in primary hepatocytes, *P<0.05, **P<0.01 WT Versus FMO2 - / - B represents wild-type primary hepatocytes and FMO2. - / - Results of Oil Red staining of primary hepatocytes. BSA represents the normal control group, and OA represents the in vitro fatty liver model.

[0021] Figure 3 Figure A shows the results of in vitro experiments using primary hepatocytes from C57 mice. A represents the statistical results of triglyceride levels in the wild-type primary hepatocyte group, the primary hepatocyte group transfected with the FMO2 negative control virus, and the primary hepatocyte group transfected with the FMO2 overexpression virus. **P<0.01 WT Versus FMO2 - / - B represents the Oil Red staining results of wild-type primary hepatocytes, primary hepatocytes transfected with FMO2 negative control virus, and primary hepatocytes transfected with FMO2 overexpressing virus. WT represents wild-type mouse primary hepatocytes, FMO2-Vector represents primary hepatocytes transfected with FMO2 negative control virus, and FMO2-Over represents primary hepatocytes transfected with FMO2 overexpressing virus. BSA represents the normal control group, and OA represents the in vitro fatty liver model group.

[0022] Figure 4 Figure A shows the results of an in vivo mechanism study in C57 mice. A represents the results of a high-fat diet for 12 weeks in wild-type mice and FMO2 mice. - / - Expression of cholesterol synthesis-related genes in mouse liver; B represents wild-type mice and FMO2 mice after 12 weeks of high-fat diet feeding. - / - Expression of fatty acid uptake-related genes in mouse liver; C represents wild-type mice and FMO2 mice fed a high-fat diet for 12 weeks. - / - Expression of genes related to fatty acid and triglyceride synthesis in mouse liver; D represents wild-type mice and FMO2 mice after 12 weeks of high-fat diet feeding. - / - Expression of genes related to fatty acid and triglyceride oxidation metabolism in mouse liver; E represents wild-type mice and FMO2 mice after 12 weeks of high-fat diet feeding. - / - Western blot results of key proteins in the lipid synthesis pathway in mouse liver. WT represents the wild-type mouse group, FMO2. - / -The transgenic mouse group with FMO2 knockout.

[0023] Figure 5 Figure A shows the results of an in vitro mechanism study of mouse primary hepatocytes. A represents the results of a lipid synthesis experiment, recording the effects of wild-type primary hepatocytes and FMO2. - / - Lipid de novo synthesis capacity of primary hepatocytes; B represents wild-type primary hepatocytes and FMO2. - / - Western blot results of key proteins in the lipid synthesis pathway in primary hepatocytes; C represents the Western blot results of key proteins in the lipid synthesis pathway in primary hepatocytes transfected with FMO2 negative control virus and primary hepatocytes transfected with FMO2 overexpression virus. Detailed Implementation

[0024] The present invention will be further described below with reference to embodiments.

[0025] General Implementation Examples

[0026] A drug for the treatment of non-alcoholic fatty liver disease, containing flavin monooxygenase 2.

[0027] Preferably, the drug is a drug that reduces hepatic lipid synthesis and storage or a drug that promotes hepatic lipid metabolism.

[0028] Preferably, the nucleotide sequence of the flavin monooxidase 2 is shown in SEQ ID NO.1 (mouse) or SEQ ID NO.3 (human), and the amino acid sequence is shown in SEQ ID NO.2 (mouse) or SEQ ID NO.4 (human).

[0029] Preferably, the drug is an oral or injectable formulation. Further, the oral formulation is in the form of capsules, tablets, solutions, or powders; the injectable formulation is an injection or powder.

[0030] Preferably, the drug also includes a pharmaceutically acceptable carrier, excipient, or solvent.

[0031] Example 1

[0032] Preparation of virus overexpressing flavin monooxygenase 2 (FMO2):

[0033] The FMO2 overexpression lentivirus used in the embodiments of this invention

[0034] (Ubi-FMO2-3FLAG-CBh-gcGFP-IRES-puromycin), negative control lentivirus;

[0035] (Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin), all purchased from Shanghai Jikai Gene Technology Co., Ltd.;

[0036] Mouse FMO2 nucleotide sequence (SEQ ID NO. 1).

[0037] Example 2

[0038] I. Establishment of a mouse model of non-alcoholic fatty liver disease

[0039] In this invention, 8-week-old healthy C57BL / 6J mice were divided into four groups: wild-type mice with normal control diet, wild-type mice with high-fat diet, FMO2 knockout mice with normal control diet, and FMO2 knockout mice with high-fat diet. Five mice were fed in each group for 12 weeks.

[0040] II. Tissue sampling, fixation, sectioning, and staining

[0041] (1) Sample collection: After anesthetizing the mice with 1% sodium pentobarbital, the abdominal cavity was quickly opened, blood was taken from the heart, the liver was quickly separated, weighed, and an appropriate amount of tissue was cut and placed in the pre-prepared tissue lysis buffer, triglyceride lysis buffer and 4% paraformaldehyde fixative.

[0042] (2) Fixation and sectioning: After fixation with 4% paraformaldehyde, the liver was divided into two parts for paraffin embedding and OCT embedding, respectively. The paraffin-embedded tissue was serially sectioned (3 μm thick), and after being retrieved, it was baked in a 60°C oven for 30 min and placed in a clean slide box for later use. During OCT embedding, the tissue blocks were slowly immersed in liquid nitrogen to prevent them from freezing and cracking. The 6 μm thick frozen sections were mounted on cation exchange slides, slightly dried, and then frozen at -80°C for later use.

[0043] (3) HE staining: liver paraffin sections were fixed with 4% paraformaldehyde for 10 min, washed with PBS for 5 min / 3 times → hematoxylin staining for 40 s → 1% hydrochloric acid (1 mL concentrated hydrochloric acid added to 99 mL anhydrous ethanol) alcohol differentiation, immersed 3 times → washed with water for 5 min / 3 times → eosin staining for 1 min → washed with water for 5 min / 3 times → dehydration: gradient alcohol (75%, 2 s; 85%, 2 s; 90%, 2 min; 95%, 2 min; 100%, 5 min / 2 times) → xylene, 5 min / 2 times → resin mounting.

[0044] III. Triglyceride Detection in Liver Tissue

[0045] Take approximately 50 mg of liver tissue, add 1 ml of triglyceride lysis buffer, homogenize 30 times using a glass homogenizer, and lyse at room temperature for 10 min. Take 100 μL of the homogenate for BCA protein measurement, and inactivate lipase by incubating the remaining homogenate in a 70°C water bath for 10 min. Centrifuge at 2000 rpm for 5 min at room temperature, and use the supernatant for triglyceride quantification. Finally, convert the TG / protein ratio to µmol / g.

[0046] IV. RNA extraction, reverse transcription, and real-time quantitative PCR

[0047] Add 1 ml of Trizol (Invitrogen) to 20 mg of tissue, homogenize the tissue, add 200 μL of chloroform, vortex vigorously to mix, and centrifuge at 12000 rpm for 15 minutes. Transfer the supernatant to an equal volume of isopropanol, invert to mix, and centrifuge at 12000 rpm to obtain a precipitate. Wash the precipitate twice with 70% ethanol, and add 50-100 μL of DEPC water. After determining the RNA concentration, take 1500 ng of total RNA for reverse transcription. Reverse transcription was performed using the Takara PrimeScript™ RT MasterMix Reverse Transcription Kit. Quantitative Real-Time PCR was performed using the Takara TB Green® Premix Ex Taq™ II Kit. The instrument used was an ABI 7500 quantitative PCR instrument. The primer sequences used in the experiment are as follows:

[0048] ACTIN-F 5'-ACACTGTGCCCATCTACGAG-3'

[0049] ACTIN-R 5'-CAGCACTGTGTTGGCATAGAG-3'

[0050] HMGCR-F 5'-ATCATGTGCTGCTTCGGCTGCAT-3'

[0051] HMGCR-R 5'-AAATTGGACGACCCTCACGGCT-3'

[0052] CYP7A1-F 5'-TCAAAGAGCGCTGTCTGGGTCA-3'

[0053] CYP7A1-R 5'-TTTCCCGGGCTTTATGTGCGGT-3'

[0054] ABCG1-F 5'-TGAACCCGTTTCTTTGGCACCG-3'

[0055] ABCG1-R 5'-AGTCCCGCATGATGCTGAGGAA-3'

[0056] FATP1-F 5'-TGCACAGCAGGTACTACCGCAT-3'

[0057] FATP1-R 5′–TGCGCAGTACCACCGTCAAC-3′

[0058] FABP1-F 5'-TGGTCCGCAATGAGTTCACCCT-3'

[0059] FABP1-R 5’-CCAGCTTGACGACTGCCTTGACTT-3’

[0060] SREBP1c-F 5'-GGAGCCATGGATTGCACATT-3'

[0061] SREBP1c-R 5′-GCTTCCAGAGAGGAGGCCAG-3′

[0062] FASN-F 5′-TCCAAGACTGACTCGGCTACTGAC-3′

[0063] FASN-R 5′-GCAGCCAGGTTCGGAATGCTATC-3′

[0064] PDK4-F 5'-TTCACACCTTCACCACATGC-3'

[0065] PDK4-R 5'-AAAGGGCGGTTTTCTTGATG-3'

[0066] CPT1-F 5'-ACCACTGGCCGCATGT-3'

[0067] CPT1-R 5'-CTCCATGGCGTAGTAGTTGCT-3'

[0068] ACOX1-F 5'-CGGAAGATACATAAAGGAGACC-3'

[0069] ACOX1-R 5′-AAGTAGGACACCATACCACCC-3′

[0070] MCAD-F 5′-GCTAGTGGAGCACCAAGGAG-3′

[0071] MCAD-R 5'-CCAGGCTGCTCTCTGGTAAC-3'

[0072] UCP2-F 5'-GCTGTGGTGGTCGGAGATA-3'

[0073] UCP2-R 5'-ACTGGCCCAAGGCAGAGTT-3'

[0074] V. Detection of SREBP1 / ACC / FASN expression in liver tissue by Western blotting

[0075] Approximately 50 mg of liver tissue was collected and 1 ml of Ripa protein lysis buffer (containing protease inhibitors) was added. The tissue was thoroughly ground and centrifuged at 12000 g / 4℃ / 20 min. The supernatant was collected, and the protein concentration was determined using the BCA method. Protein concentration was quantified to 2 mg / ml using Ripa tissue protein lysis buffer and 5X SDS protein loading buffer, and the mixture was boiled in a metal bath at 98℃ for 5 min. 20 μg of the protein solution was electrophoresed on an 8% polyacrylamide gel and transferred to a PVDF membrane. The primary antibodies were FMO2 (NOVUS, NBP1-85952), SREBP1 (Abcam, ab28481), ACC (CST, 3676), and FASN (Abcam, ab99359). The secondary antibody was goat anti-rabbit antibody (CST, 7074). After thorough washing with PBST, the membrane was exposed in a dark room according to the ECL ultrasensitive luminescent solution instructions.

[0076] VI. Isolation of primary liver cells and establishment of in vitro models

[0077] (1) Isolation of primary liver cells

[0078] (1.1) Autoclave the necessary instruments in advance and prepare DHANKS and 0.03% collagenase IV. Soak the catheters in 75% alcohol; disinfect the laminar flow hood by UV irradiation for 30 minutes; preheat the culture medium, DHANKS and collagenase IV at 37ºC.

[0079] (1.2) Sodium pentobarbital (60 mg / kg) was administered intraperitoneally to anesthetize mice.

[0080] (1.3) Fill the sterilized tubing with preheated DHANKS.

[0081] (1.4) Prepare the skin and disinfect with iodine; open the abdominal cavity in the laminar flow hood, fully expose the portal vein, separate it by 1cm and thread a suture, and fix the portal vein cannula.

[0082] (1.5) DHANKS was perfused through the portal vein at a flow rate of 2 mL / min. After 20 mL of perfusion, the liver turned white. Then, preheated collagenase IV was used at a flow rate of 2 mL / min. After 20 mL of perfusion, the liver was removed.

[0083] (1.6) After washing the liver with preheated DHANKS, place it in a mixture of 10 mL collagenase IV and 10 mL DHANKS, and gently tear open the capsule with curved forceps to suspend the hepatocytes in the mixture.

[0084] (1.7) Digest the hepatocyte suspension by shaking in a 37ºC water bath for 20 minutes, and then filter it through a 200-mesh filter.

[0085] (1.8) Centrifuge the obtained hepatocyte suspension at 800 rpm for 5 min; discard the supernatant, then resuspend the hepatocytes in serum-free DMEM medium, centrifuge at 800 rpm for 5 min, and discard the supernatant.

[0086] (1.9) The obtained hepatocyte pellet was resuspended in high-glucose DMEM medium containing 10% FBS and its viability was observed by trypan blue staining. The proportion of viable cells was calculated, and the suspended cells were seeded into 6-well plates with 10^6 cells per well.

[0087] (1.10) Cultured at 37ºC for 1 hour, then changed to high-glucose DMEM with 10% FBS. After another 3 hours, the medium was changed to high-glucose DMEM with 2% FBS. Cell morphology was observed after 12 hours.

[0088] (2) Cell counting method: Drop the diluted cell suspension onto the counting plate, allowing the suspension to freely fill the gap below the coverslip, without leaving air bubbles. Then observe and count the number of cells in the four large squares under a light microscope. When pressing the lines, only count the cells in the top and right lines. Then calculate the cell concentration using the following formula: (Total number of cells in the large squares / 4) × 10000 × dilution factor = number of cells / mL

[0089] (3) Cell modeling: After 24 hours, the isolated primary liver cells were added with 500 uM oleic acid, cultured for another 24 hours, stained with Oil Red O, and the samples were collected to extract RNA, protein and triglycerides.

[0090] (4) FMO2 overexpression virus transfection

[0091] Twenty-four hours after the isolated primary liver cells were transfected, negative control lentivirus (FMO2-Vector) and FMO2 overexpression lentivirus (FMO2-Over) were added with an MOI of 20. The culture medium was changed 12 hours after transfection, and 500 uM oleic acid was added 48 hours later to induce the model.

[0092] VII. Cell Oil Red O Staining

[0093] (1) Preparation of working solution: Add saturated Oil Red O stock solution to double distilled water at a ratio of 3:2 (Oil Red O: double distilled water), mix well, let stand at room temperature for 5 minutes, filter and use.

[0094] (2) After the culture medium is removed from the cells, rinse them 3 times with PBS → fix them with 4% paraformaldehyde at room temperature for 10 min → wash with PBS for 5 min → stain with Oil Red O working solution for 15 min → wash with PBS for 5 min / 3 times → take pictures under an inverted optical microscope within 1 hour.

[0095] VIII. Triglyceride Detection in Primary Hepatocytes

[0096] Cells were washed three times with PBS. 200 μL of lipid lysis buffer was added to each well of a six-well plate. Cells were scraped off with a scraper and homogenized by sonication. Lysis was performed at room temperature for 10 min. 50 μL of the homogenate was used for protein measurement using the BCA method. The remaining homogenate was incubated at 70°C for 10 min to inactivate lipase. Cells were centrifuged at 2000 rpm for 5 min at room temperature. The supernatant was used for triglyceride quantification, and the result was converted to TG / protein in µmol / g.

[0097] IX. Detection of SREBP1 / ACC / FASN expression in primary hepatocytes by Western blotting

[0098] Cells were washed three times with PBS. 100 μL of Ripa protein lysis buffer (containing protease inhibitors) was added to each well of a six-well plate. After scraping with a scraper, the cells were homogenized by sonication and centrifuged at 12000 g / 4℃ / 20 min. The supernatant was collected, and protein concentration was determined using the BCA method. Protein was quantified to 1 mg / mL using Ripa tissue protein lysis buffer and 5X SDS protein loading buffer, and then boiled in a metal bath at 98℃ for 5 min. 20 μg of the protein solution was electrophoresed on an 8% polyacrylamide gel and transferred to a PVDF membrane. Primary antibodies were FMO2 (NOVUS, NBP1-85952), SREBP1 (Abcam, ab28481), ACC (CST, 3676), and FASN (Abcam, ab99359). The secondary antibody was goat anti-rabbit antibody (CST, 7074). After thorough washing with PBST, the cells were exposed in a dark room according to the ECL ultrasensitive luminescent solution instructions.

[0099] 10. De novo lipid synthesis experiments

[0100] Primary hepatocytes were isolated and seeded at a density of 10^6 cells per well in 6-well plates. After adhesion, they were cultured overnight in serum-free high-glucose DMEM. The next day, the medium was changed to high-glucose medium containing 0.2% BSA-free, and two hours later, the medium was changed to DMEM medium containing 0.5 mMOA. 0.5 μCi U-14C-labeled glucose was added to each well. After culturing at 37°C for four hours, lipids were extracted using the Folch method: cells were washed twice with pre-chilled PBS, and cells were scraped from each well with 200 μL of PBS containing 0.5% Triton. 150 μL of the scraped cells were added to 500 μL of chloroform:methanol solution, and the mixture was vigorously shaken for 30 seconds. After standing on ice for one hour, 125 μL of double-distilled water was added, and the mixture was vigorously shaken. After centrifugation at 4°C / 1000 RPM for 15 minutes, the lower organic phase was collected. The carbon-14 transition into lipids was measured using a Tri-Carb 2900 TR liquid scintillation counter. All readings were normalized to protein concentration.

[0101] XI. Data Analysis

[0102] 1. Deletion of the FMO2 gene exacerbates high-fat diet-induced NAFLD.

[0103] To investigate the role of the FMO2 gene in non-alcoholic liver disease, we administered FMO2 knockout mice (FMO2 knockout mice) to 8-week-old FMO2 knockout mice. - / - ) and its littermate wild-type control mice (FMO2) + / + Mice were fed a normal control diet (NCD) and a high-fat diet (HFD) for 12 weeks, after which they were sacrificed, and lipid accumulation in the liver was observed. In this study, FMO2 knockout mice were constructed using homologous recombination. Liver Western blot results showed that FMO2 was completely knocked out. Figure 4 E). After 12 weeks of HFD feeding, mice showed significant liver swelling and whitening, with a marked increase in liver weight, indicating excessive lipid accumulation in the liver and successful induction of fatty liver. Figure 1 A and Figure 1 C). Compared with wild-type mice, FMO2 after HFD feeding - / - The mouse livers were larger and whiter in gross appearance, and their weight was significantly increased. Figure 1 A and Figure 1 C). To further assess the severity of fatty liver, we measured the triglyceride content in liver tissue and stained paraffin sections of liver tissue with H&E. The results indicated that after 12 weeks of HFD feeding, the triglyceride content in the liver tissue of mice was significantly increased, and H&E staining revealed a large accumulation of lipid droplets within the liver lobules. Figure 1 B and Figure 1 D). Compared with wild-type mice, FMO2 after HFD feeding - / -The triglyceride content in mouse liver tissue was significantly increased, and H&E staining showed more severe hepatic steatosis. Figure 1 B and Figure 1 D). This indicates that the deletion of the FMO2 gene exacerbates NAFLD induced by a high-fat diet.

[0104] 2. Deletion of the FMO2 gene exacerbates oleic acid-induced hepatocellular steatosis.

[0105] To investigate the effect of FMO2 gene deletion on hepatocyte steatosis in vitro, we collected samples from wild-type mice and FMO2 mice. - / - Primary hepatocytes were isolated from mouse livers. Western blot results showed that the isolated FMO2 cells... - / - Primary hepatocytes lacking FMO2 gene expression ( Figure 5 B). Compared with the BSA control group, after 24 hours of induction with 500 µM oleic acid, the triglyceride content in hepatocytes was significantly increased, and Oil Red O staining revealed a large accumulation of lipid droplets in hepatocytes. Figure 2 A and Figure 2 B). Compared with control group hepatocytes, FMO2 - / - The triglyceride content in hepatocytes was significantly increased, and more lipid droplets accumulated in the cells. This difference was even more pronounced after oleic acid induction. Figure 2 A and Figure 2 B) indicates that the deletion of the FMO2 gene exacerbates oleic acid-induced hepatocellular steatosis.

[0106] 3. Overexpression of the FMO2 gene improves oleic acid-induced hepatocellular steatosis.

[0107] We observed in both an in vivo high-fat feeding model in mice and an in vitro oleic acid-induced hepatocyte model that FMO2 gene deletion exacerbates fatty liver. To verify this conclusion, we isolated primary hepatocytes from wild-type mice and overexpressed the FMO2 gene by infecting them with Ubi-FMO2-3FLAG-CBh-gcGFP-IRES-puromycin lentivirus. Simultaneously, control hepatocytes were infected with a negative control lentivirus, Ubi-MCS-3FLAG-CBhg-cGFP-IRES-puromycin. Western blot results showed that infection with the FMO2-overexpressing lentivirus effectively increased the protein level of FMO2 in hepatocytes. Figure 5 C). Based on the statistical analysis of triglyceride levels in hepatocytes and Oil Red O staining, infection with the FMO2 negative control virus did not affect hepatocyte steatosis. However, overexpression of FMO2 significantly improved hepatocyte steatosis, especially after oleic acid induction. Figure 3 (A and 3B). This demonstrates that overexpression of the FMO2 gene can improve oleic acid-induced hepatocellular steatosis.

[0108] 4. FMO2 improves NAFLD by inhibiting SREBP1 / ACC / FASN pathway-mediated de novo lipid synthesis.

[0109] Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive fat deposition in hepatocytes, inflammation, and abnormal liver function. Its pathogenesis involves numerous factors, including genetics, metabolism, environment, and lifestyle. Currently, the exact pathogenesis of NAFLD is not fully understood. As the metabolic center of animals, lipid metabolism in the liver is an extremely complex process, including lipid uptake, de novo lipid synthesis, and lipid oxidation. Abnormalities in any of these processes can lead to abnormal lipid deposition in the liver.

[0110] To investigate the specific mechanism by which FMO2 gene deletion exacerbates NAFLD and to identify which step in hepatic lipid metabolism is regulated by FMO2, we examined the expression of a series of lipid metabolism-related genes in the liver of mice fed a high-fat diet. PCR results showed that, compared with wild-type mice, FMO2 expression was significantly reduced. - / - There were no significant differences in cholesterol synthesis, lipid uptake, and lipid oxidation in the mouse liver, but the expression of genes related to de novo lipid synthesis was increased. Figure 4 A- Figure 4 D). To verify whether FMO2 affects hepatic de novo lipid synthesis and thus regulates NAFLD, we conducted a de novo lipid synthesis experiment using carbon-14 labeled glucose as a substrate. The results showed that oleic acid-induced FMO2... - / - The de novo synthesis capacity of hepatocytes was significantly increased compared to the control group. Figure 5 A). The de novo lipid synthesis (DNL) pathway in the liver plays a crucial role in the development of fatty liver disease, with sterol regulatory element-binding protein-1 (SREBP1) serving as its central hub. SREBP1 is an important transcription factor regulating fatty acid synthesis in the liver. During synthesis, it exists in an inactive form (p-SREBP1) on the endoplasmic reticulum. Upon specific signal regulation, it is transported to the Golgi apparatus, where it is further cleaved to release the transcriptionally active fragment (n-SREBP1), which enters the nucleus and promotes the transcription of a series of lipid synthesis-related genes (ACC / FASN / SCD). Liver Western blot results showed that FMO2 was significantly reduced after HFD feeding. - / - The levels of N-SREBP1 and its downstream proteins ACC and FASN in the liver of mice were significantly higher than those in wild-type mice, suggesting enhanced activation of the DNL pathway in the liver. Figure 4 E). We found the same results in primary hepatocytes in vitro, with Western blotting showing that FMO2 was induced by oleic acid. - / -The levels of N-SREBP1 and its downstream proteins ACC and FASN in hepatocytes were significantly higher than those in control hepatocytes. Conversely, the levels of N-SREBP1 and its downstream proteins ACC and FASN in hepatocytes of the oleic acid-induced FMO2 overexpression group were significantly lower than those in the control hepatocytes. This indicates that FMO2 mainly improves NAFLD by inhibiting the SREBP1 / ACC / FASN pathway and reducing de novo lipid synthesis in the liver.

[0111] Figure 1 This figure shows the in vivo experimental results of C57 mice after 12 weeks of feeding with either a normal control diet or a high-fat diet. A represents wild-type mice versus FMO2 mice. - / - Statistical results of mouse liver weight, *P<0.05 WT Versus FMO2 - / - B represents wild-type mice and FMO2. - / - Statistical results of triglyceride content in mouse liver, *P<0.05 WT Versus FMO2 - / -- C represents wild-type mice and FMO2. - / - Comparison of gross liver findings in mice; D represents wild-type mice and FMO2 mice. - / - Comparison of HE staining results after paraffin sectioning of mouse liver. WT represents the wild-type mouse group, FMO2. - / - The FMO2 knockout transgenic mouse group. NCD is the normal control diet group, and HFD is the high-fat diet group.

[0112] Figure 2 This image shows the results of in vitro experiments using C57 mouse primary hepatocytes. A represents wild-type primary hepatocytes and FMO2 cells. - / - Statistical results of triglyceride content in primary hepatocytes, *P<0.05, **P<0.01 WT Versus FMO2 - / - B represents wild-type primary hepatocytes and FMO2. - / - Results of Oil Red staining of primary hepatocytes. BSA represents the normal control group, and OA represents the in vitro fatty liver model.

[0113] Figure 3 Figure A shows the results of in vitro experiments using primary hepatocytes from C57 mice. A represents the statistical results of triglyceride levels in the wild-type primary hepatocyte group, the primary hepatocyte group transfected with the FMO2 negative control virus, and the primary hepatocyte group transfected with the FMO2 overexpression virus. **P<0.01 WT Versus FMO2 - / -B represents the Oil Red staining results of wild-type primary hepatocytes, primary hepatocytes transfected with FMO2 negative control virus, and primary hepatocytes transfected with FMO2 overexpressing virus. WT represents wild-type mouse primary hepatocytes, FMO2-Vector represents primary hepatocytes transfected with FMO2 negative control virus, and FMO2-Over represents primary hepatocytes transfected with FMO2 overexpressing virus. BSA represents the normal control group, and OA represents the in vitro fatty liver model group.

[0114] Figure 4 Figure A shows the results of an in vivo mechanism study in C57 mice. A represents the results of a high-fat diet for 12 weeks in wild-type mice and FMO2 mice. - / - Expression of cholesterol synthesis-related genes in mouse liver; B represents wild-type mice and FMO2 mice after 12 weeks of high-fat diet feeding. - / - Expression of fatty acid uptake-related genes in mouse liver; C represents wild-type mice and FMO2 mice fed a high-fat diet for 12 weeks. - / - Expression of genes related to fatty acid and triglyceride synthesis in mouse liver; D represents wild-type mice and FMO2 mice after 12 weeks of high-fat diet feeding. - / - Expression of genes related to fatty acid and triglyceride oxidation metabolism in mouse liver; E represents wild-type mice and FMO2 mice after 12 weeks of high-fat diet feeding. - / - Western blot results of key proteins in the lipid synthesis pathway in mouse liver. WT represents the wild-type mouse group, FMO2. - / - The transgenic mouse group with FMO2 knockout.

[0115] Figure 5 Figure A shows the results of an in vitro mechanism study of mouse primary hepatocytes. A represents the results of a lipid synthesis experiment, recording the effects of wild-type primary hepatocytes and FMO2. - / - Lipid de novo synthesis capacity of primary hepatocytes; B represents wild-type primary hepatocytes and FMO2. - / - Western blot results of key proteins in the lipid synthesis pathway in primary hepatocytes; C represents the Western blot results of key proteins in the lipid synthesis pathway in primary hepatocytes transfected with FMO2 negative control virus and primary hepatocytes transfected with FMO2 overexpression virus.

[0116] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0117] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention. sequence list <110> Zhejiang University <120> Application of flavin monooxygenase 2 in the preparation of drugs for the treatment of non-alcoholic fatty liver disease <160> 4 <170> SIPOSequenceListing 1.0 <210> 2 <211> 1608 <212> DNA / RNA <213> Mice-FMO2 (flavin monooxygenase 2) <400> 2 atggcaaaga aggttgtggt gattggagca ggggtcagtg gcctaatctc tctgaagtgc 60 tgcgtggacg agggtctgga gcccacctgc ttcgaaagga ctgaagatat agggggactg 120 tggagattca aagaaaacgt ggaagatggc cgcgctagca tctaccgctc tgtcattacc 180 aacactagca aagaaatgtc ctgtttcagt gacttcccga tgccggagga cttccccaac 240 ttcctgcaca actctaaact cctggaatat tttaggatct ttgccaagaa atttgatctc 300 ctaaaatata ttcagtttca gaccactgtc attagtgtga aaaaacgccc ggacttcgca 360 tcttctgggc aatgggaagt ttatactcag agcaacggaa aggagcaacg cactgtcttt 420 gacgctgtta tggtttgcag cggccatcac atccagcctc acctgccact caagtcattc 480 ccaggtattg agaggttccg aggccagtat ttccatagcc gcgaatacaa gcacccagtg 540 gggtttgagg ggaacgcat cctggtggtt ggaataggaa actcagctgc agatattgcc 600 tctgaactga gtaagacggc cgcgcaggtg ttgtcagca ccagacatgg atcctgggtc 660 atgagtcgaa tctctgaga tggctacct tgggacatgg tgttccacac tacttagc 720 tccatgctcc gaatgtcct gccccgcacg gtcgtcaagt ggatgatgga acaacagatg 780 aatcgatggt tcaccatga aaattatggt ctggtgccctc agaacaata ccttatgaaa 840 gaacctgtgc taaacgacga cctccccagc cgcctgctgt acggagccat caagtaaaa 900 acacggggtga aggagctcac ggagacagct gtggtcttcg aggatggcac ggtggaggaa 960 gacgtggata tcattgtct tgccacgggg tatacgttct ctttcttt cctggaagac 1020 tcacttgtta aagtggagga taatagggtt tccctgtaca aagccatgtt tcccctcac 1080 ctggagaagc cacccttgc gtgcataggt ctcatccagc ctctaggctc catctccca 1140 accgtagagc ttcaggcgcg ctgggcgaca agagttttca aaggcttgtg tagcttgcct 1200 tcggagacga ctatgatggc agacattgtt gaaagaatg aaaaagagt taacctgttt 1260 gggaaaagcc agagtcagat actgcagacc aactacgttg actacctgga tgaactcgcc 1320 ttggagatag gtgcaaagcc agatttcgtc tctctctttt tcaaagaccc taaactggct 1380 gtgaaactct acttcggacc ctgtaattcc taccagtacc gcctggttgg acctgggcag 1440 tgggaaggag ccaggaacgc catcctcacc cagaagcaga ggatcctgaa acccttaaag 1500 acccggactc tccagtcctc tgatagtgcc ccagtgtctt tcctgctcaa gatcctgggg 1560 ctgctcgctg ttgttctggc cttctttttc caacttcagg ggttttaa 1608 <210> 1 <211> 535 <212> PRT <213> Mouse flavin - containing monooxygenase 2 (Mice - FMO2) <400> 1 Met Ala Lys Lys Val Val Val Ile Gly Ala Gly Val Ser Gly Leu Ile 1 5 10 15 Ser Leu Lys Cys Cys Val Asp Glu Gly Leu Glu Pro Thr Cys Phe Glu 20 25 30 Arg Thr Glu Asp Ile Gly Gly Leu Trp Arg Phe Lys Glu Asn Val Glu 35 40 45 Asp Gly Arg Ala Ser Ile Tyr Arg Ser Val Ile Thr Asn Thr Ser Lys 50 55 60 Glu Met Ser Cys Phe Ser Asp Phe Pro Met Pro Glu Asp Phe Pro Asn 65 70 75 80 Phe Leu His Asn Ser Lys Leu Leu Glu Tyr Phe Arg Ile Phe Ala Lys 85 90 95 Lys Phe Asp Leu Leu Lys Tyr Ile Gln Phe Gln Thr Thr Val Ile Ser 100 105 110 Val Lys Lys Arg Pro Asp Phe Ala Ser Ser Gly Gln Trp Glu Val Tyr 115 120 125 Thr Gln Ser Asn Gly Lys Glu Gln Arg Thr Val Phe Asp Ala Val Met 130 135 140 Val Cys Ser Gly His His Ile Gln Pro His Leu Pro Leu Lys Ser Phe 145 150 155 160 Pro Gly Ile Glu Arg Phe Arg Gly Gln Tyr Phe His Ser Arg Glu Tyr 165 170 175 Lys His Pro Val Gly Phe Glu Gly Lys Arg Ile Leu Val Val Gly Ile 180 185 190 Gly Asn Ser Ala Ala Asp Ile Ala Ser Glu Leu Ser Lys Thr Ala Ala 195 200 205 Gln Val Phe Val Ser Thr Arg His Gly Ser Trp Val Met Ser Arg Ile 210 215 220 Ser Glu Asp Gly Tyr Pro Trp Asp Met Val Phe His Thr Arg Phe Ser 225 230 235 240 Ser Met Leu Arg Asn Val Leu Pro Arg Thr Val Val Lys Trp Met Met 245 250 255 Glu Gln Gln Met Asn Arg Trp Phe Asn His Glu Asn Tyr Gly Leu Val 260 265 270 Pro Gln Asn Lys Tyr Leu Met Lys Glu Pro Val Leu Asn Asp Asp Leu 275 280 285 Pro Ser Arg Leu Leu Tyr Gly Ala Ile Lys Val Lys Thr Arg Val Lys 290 295 300 Glu Leu Thr Glu Thr Ala Val Val Phe Glu Asp Gly Thr Val Glu Glu 305 310 315 320 Asp Val Asp Ile Ile Val Phe Ala Thr Gly Tyr Thr Phe Ser Phe Ser 325 330 335 Phe Leu Glu Asp Ser Leu Val Lys Val Glu Asp Asn Arg Val Ser Leu 340 345 350 Tyr Lys Ala Met Phe Pro Pro His Leu Glu Lys Pro Thr Leu Ala Cys 355 360 365 Ile Gly Leu Ile Gln Pro Leu Gly Ser Ile Phe Pro Thr Val Glu Leu 370 375 380 Gln Ala Arg Trp Ala Thr Arg Val Phe Lys Gly Leu Cys Ser Leu Pro 385 390 395 400 Ser Glu Thr Thr Met Met Ala Asp Ile Val Glu Arg Asn Glu Lys Arg 405 410 415 Val Asn Leu Phe Gly Lys Ser Gln Ser Gln Ile Leu Gln Thr Asn Tyr 420 425 430 Val Asp Tyr Leu Asp Glu Leu Ala Leu Glu Ile Gly Ala Lys Pro Asp 435 440 445 Phe Val Ser Leu Phe Phe Lys Asp Pro Lys Leu Ala Val Lys Leu Tyr 450 455 460 Phe Gly Pro Cys Asn Ser Tyr Gln Tyr Arg Leu Val Gly Pro Gly Gln 465 470 475 480 Trp Glu Gly Ala Arg Asn Ala Ile Leu Thr Gln Lys Gln Arg Ile Leu 485 490 495 Lys Pro Leu Lys Thr Arg Thr Leu Gln Ser Ser Asp Ser Ala Pro Val 500 505 510 Ser Phe Leu Leu Lys Ile Leu Gly Leu Leu Ala Val Val Leu Ala Phe 515 520 525 Phe Phe Gln Leu Gln Gly Phe 530 535 <210> 3 <211> 1608 <212> DNA / RNA <213> Human Flavin-containing Monooxygenase 2 (Human-FMO2) <400> 3 atggcaaaga aggtagctgt gattggagct ggggtcagtg gcctaatttc tctgaagtgc 60 tgtgtggatg agggacttga gcccacttgc tttgagagaa ctgaagatat tggaggagtg 120 tggaggttca aagagaatgt ggaagatggc cgagcaagta tctatcaatc tgtcgttacc 180 aacaccagca aagaaatgtc ctgtttcagt gactttccaa tgcctgaaga ttttccaaac 240 ttcctgcata attctaaact tctggaatat ttcaggattt ttgctaaaaa atttgatctg 300 ctaaaatata ttcagttcca gacaactgtc cttagtgtga gaaaatgtcc agatttctca 360 tcctctggcc aatggaaggt tgtcactcag agcaacggca aggagcagag tgctgtcttt 420 gacgcagtta tggtttgcag tggccaccac attctacctc atatcccact gaagtcattt 480 ccaggtatgg agaggttcaa aggccaatat ttccatagcc gccaatacaa gcatccagat 540 ggatttgagg gaaaacgcat cctggtgatt ggaatgggaa actcaggctc agatattgct 600 gttgagctga gtaagaatgc tgctcaggtt tttatcagca ccaggcatgg cacctgggtc atgagccgta tctctgaaga tggctatcct tgggactcag tgttccacac ccggtttcgt 720 tctatgctcc gcaatgtact gccacgaaca gctgtaaaat ggatgataga acaacagatg aatcggtggt tcaaccatga aaattatggc cttgagcctc aaaacaata cattatgaag gaacctgtac taaatgatga tgtcccaagt cgtctactct gtggagccat caaggtgaaa tctacagtga aagagctcac agaaacttct gccatctttg aggatggac aggatggag 960 aacattgatg tcatcatttt tgcaacagga tatagtttct cttttccctt ccttgaagat tcactcgtta father's fatherggtc tcactgtata father's father ccccgctcac ctggacaagt caacccctcgc gtgcattggt ctcatccagc ccctaggttc cattttccca actgctgaac ttcaagctcg ttgggtgaca agagttttca aaggcttgtg tagcctgccc tcagagaga ctatgatgat ggacattatc aaaaggaatg aaaaaagat tgacctgttt ggagaaagcc agagccagac gttgcagacc aattatgttg actacttgga cgagctcgcc ttagagatag gtgcgaagcc agatttctgc tctctcttgt tcaaagatcc taaactggct gtgagactct atttcggacc ctgcaactcc tatcagtatc gcctggttgg gcctgggcaa 1440 tgggaaggag ccagaaatgc catcttcacc cagaaacaaa gaatactgaa gccactcaag 1500 actcgggccc tgaaggattc atctaatttc tcagtttctt ttctgttgaa aatcctgggc 1560 cttcttgctg ttgttgtggc ctttttttgc caacttcaat ggtcctag 1608 <210> 4 <211> 535 <212> PRT <213> Human-FMO2 <400> 4 Met Ala Lys Lys Val Ala Val Ile Gly Ala Gly Val Ser Gly Leu Ile 1 5 10 15 Ser Leu Lys Cys Cys Val Asp Glu Gly Leu Glu Pro Thr Cys Phe Glu 20 25 30 Arg Thr Glu Asp Ile Gly Gly Val Trp Arg Phe Lys Glu Asn Val Glu 35 40 45 Asp Gly Arg Ala Ser Ile Tyr Gln Ser Val Val Thr Asn Thr Ser Lys 50 55 60 Glu Met Ser Cys Phe Ser Asp Phe Pro Met Pro Glu Asp Phe Pro Asn 65 70 75 80 [[ID=3 85 90 95 Lys Phe Asp Leu Leu Lys Tyr Ile Gln Phe Gln Thr Thr Val Leu Ser 100 105 110 Val Arg Lys Cys Pro Asp Phe Ser Ser Ser Gly Gln Trp Lys Val Val 115 120 125 Thr Gln Ser Asn Gly Lys Glu Gln Ser Ala Val Phe Asp Ala Val Met 130 135 140 Val Cys Ser Gly His His Ile Leu Pro His Ile Pro Leu Lys Ser Phe 145 150 155 160 Pro Gly Met Glu Arg Phe Lys Gly Gln Tyr Phe His Ser Arg Gln Tyr 165 170 175 Lys His Pro Asp Gly Phe Glu Gly Lys Arg Ile Leu Val Ile Gly Met 180 185 190 Gly Asn Ser Gly Ser Asp Ile Ala Val Glu Leu Ser Lys Asn Ala Ala 195 200 205 Gln Val Phe Ile Ser Thr Arg His Gly Thr Trp Val Met Ser Arg Ile 210 215 220 Ser Glu Asp Gly Tyr Pro Trp Asp Ser Val Phe His Thr Arg Phe Arg 225 230 235 240 Ser Met Leu Arg Asn Val Leu Pro Arg Thr Ala Val Lys Trp Met Ile 245 250 255 Glu Gln Gln Met Asn Arg Trp Phe Asn His Glu Asn Tyr Gly Leu Glu 260 265 270 Pro Gln Asn Lys Tyr Ile Met Lys Glu Pro Val Leu Asn Asp Asp Val 275 280 285 Pro Ser Arg Leu Leu Cys Gly Ala Ile Lys Val Lys Ser Thr Val Lys 290 295 300 Glu Leu Thr Glu Thr Ser Ala Ile Phe Glu Asp Gly Thr Val Glu Glu 305 310 315 320 Asn Ile Asp Val Ile Ile Phe Ala Thr Gly Tyr Ser Phe Ser Phe Pro 325 330 335 Phe Leu Glu Asp Ser Leu Val Lys Val Glu Asn Asn Met Val Ser Leu 340 345 350 Tyr Lys Tyr Ile Phe Pro Ala His Leu Asp Lys Ser Thr Leu Ala Cys 355 360 365 Ile Gly Leu Ile Gln Pro Leu Gly Ser Ile Phe Pro Thr Ala Glu Leu 370 375 380 Gln Ala Arg Trp Val Thr Arg Val Phe Lys Gly Leu Cys Ser Leu Pro 385 390 395 400 Ser Glu Arg Thr Met Met Met Asp Ile Ile Lys Arg Asn Glu Lys Arg 405 410 415 Ile Asp Leu Phe Gly Glu Ser Gln Ser Gln Thr Leu Gln Thr Asn Tyr 420 425 430 Val Asp Tyr Leu Asp Glu Leu Ala Leu Glu Ile Gly Ala Lys Pro Asp 435 440 445 Phe Cys Ser Leu Leu Phe Lys Asp Pro Lys Leu Ala Val Arg Leu Tyr 450 455 460 Phe Gly Pro Cys Asn Ser Tyr Gln Tyr Arg Leu Val Gly Pro Gly Gln 465 470 475 480 Trp Glu Gly Ala Arg Asn Ala Ile Phe Thr Gln Lys Gln Arg Ile Leu 485 490 495 Lys Pro Leu Lys Thr Arg Ala Leu Lys Asp Ser Ser Asn Phe Ser Val 500 505 510 Ser Phe Leu Leu Lys Ile Leu Gly Leu Leu Ala Val Val Val Ala Phe 515 520 525 Phe Cys Gln Leu Gln Trp Ser 530 535

Claims

1. The application of flavin-containing monooxygenase 2 in the preparation of drugs for the treatment of non-alcoholic fatty liver disease, characterized in that, The nucleotide sequence of the flavin monooxidase 2 is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.

4.

2. The application as described in claim 1, characterized in that, The drug is either a drug that reduces hepatic lipid synthesis and storage or a drug that promotes hepatic lipid metabolism.

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