Application of FBXO22 in diagnosis and treatment of non-alcoholic fatty liver disease

Abstract

The invention provides application of FBXO22 in diagnosis and treatment of a non-alcoholic fatty liver disease, discloses high expression of FBXO22 in the non-alcoholic fatty liver disease, and verifies that the process of the non-alcoholic fatty liver disease can be delayed, liver inflammation can be relieved and liver lipid accumulation can be slowed down by reducing the expression level of the FBXO22. The FBXO22 can be used for diagnosing and treating the non-alcoholic fatty liver disease and screening candidate drugs for the non-alcoholic fatty liver disease, and the FBXO22 can also be used for evaluating the drug effect and researching and revealing the pathogenesis of the non-alcoholic fatty liver disease. The invention has important significance for clinical research of the non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically, relates to the application of FBXO22 in the diagnosis and treatment of non-alcoholic fatty liver disease. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) has become one of the leading causes of liver disease worldwide. The leading cause of chronic liver disease and hepatocellular carcinoma worldwide has gradually shifted from chronic viral hepatitis to NAFLD. NAFLD includes a series of diseases related to metabolic disorders, including non-alcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH) or its related cirrhosis. With the gradual increase in the number of patients with type 2 diabetes and obesity, the incidence of NAFLD has also increased year by year, and the incidence of NASH has doubled in recent years. The accumulation of fat in non-alcoholic steatohepatitis in liver cells can cause liver cell degeneration / necrosis, and the resulting inflammation and liver fibrosis may develop into cirrhosis and liver cancer. Therefore, NASH is regarded as a serious disease worldwide, and there is currently a lack of effective treatment. Summary of the invention

[0003] In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a method for diagnosing and treating non-alcoholic fatty liver disease. The present invention discloses that FBXO22 is highly expressed in non-alcoholic fatty liver disease, and verifies that reducing the expression level of FEXO22 can delay the progression of non-alcoholic fatty liver disease, reduce liver inflammation, and slow down the accumulation of lipids in the liver. FBXO22 can be used to diagnose and treat non-alcoholic fatty liver disease and screen candidate drugs for non-alcoholic fatty liver disease. FBXO22 can also be used to evaluate the efficacy, study and reveal the pathogenesis of non-alcoholic fatty liver disease. The present invention is of great significance for the clinical research of non-alcoholic fatty liver disease.

[0004] In order to achieve the above object, the present invention adopts the following technical solution: The first aspect of the present invention provides the use of FBXO22 in the preparation of a medicament for treating non-alcoholic fatty liver disease.

[0005] In the present invention, "treatment" refers to obtaining the desired pharmacological and / or physiological effects, covering any treatment of pathological conditions or diseases in mammals, including humans. Treatment may refer to treatment and / or prevention, and when the disease or its symptoms are completely or partially prevented, it can be understood as prevention, and when it is a partial or complete cure of the disease, it can be understood as treatment. Treatment covers any treatment of a disease in mammals, especially in humans, and includes: (a) increasing survival time; (b) reducing the risk of death due to the disease; (c) preventing the disease from occurring in subjects who may be susceptible to the disease but have not yet been diagnosed with the disease; (d) inhibiting the disease, that is, stopping its development (e.g., reducing the rate of disease progression); and (e) alleviating the disease, that is, causing the disease to regress.

[0006] Furthermore, the drug includes an inhibitor of FBXO22.

[0007] In the present invention, "inhibitor" can be understood as any substance or compound that can specifically silence, reduce, prevent and / or block the expression of a gene, or by preventing the transcription of the FBXO22 gene, thereby avoiding the formation of any transcription product of the gene, or by promoting the degradation of any transcription product of the FBXO22 gene, or by specifically reducing, preventing and / or blocking the expression of the encoded protein, and any compound that inhibits the activity of the FBXO22 protein. The transcription product refers to the RNA derived from the transcription of the gene. Further, the inhibitor can reduce the expression level or functional activity of FBXO22.

[0008] When the expression level of a protein or nucleic acid is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% (i.e., absent) relative to a reference value, the expression level of a protein or nucleic acid can be considered to be reduced. A "reference value" can refer to the expression level of a protein or nucleic acid before administration of an inhibitor. A reference value can be an absolute value, a relative value, a value with an upper and / or lower limit, a range of values, an average, a median, an average, or a value represented by a reference control or a reference value. A reference value can be based on a value obtained from a single sample, such as, for example, a value obtained from a research sample but obtained at a previous time point. A reference value can be based on a large number of samples, such as, a value obtained in a sample population, or based on a sample pool including or excluding a sample to be tested.

[0009] The functional activity of a protein may be considered reduced when it is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% (i.e., absent) relative to a reference value. The reference value may refer to the functional activity of the protein prior to administration of the inhibitor.

[0010] Furthermore, the inhibitors include nucleic acid inhibitors, protein inhibitors, and compounds.

[0011] In the present invention, "nucleic acid" means a biopolymer of nucleotides, which are interconnected via phosphodiester bonds (polynucleotides, polynucleic acids). When chemically synthesized, the nucleotides of the nucleic acid can be additionally or alternatively connected via phosphorothioate or phosphorodithioate bonds. Depending on the type of sugar in the nucleotide (ribose or deoxyribose), it is distinguished into two categories of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Nucleic acids include any natural or non-natural nucleic acids. Natural nucleotides mean nucleotides that can be purified from natural sources. Non-natural nucleotides are those produced using a recombinant expression system, and optionally, purified, chemically synthesized. Nucleic acids can include nucleoside analogs, such as analogs with chemically modified bases or sugars, backbone modifications. Unless otherwise indicated, nucleic acid sequences are represented in a 5'-3' direction.

[0012] Furthermore, the nucleic acid inhibitors include shRNA, siRNA, sgRNA, and antisense nucleic acids.

[0013] Furthermore, the nucleic acid inhibitor is selected from siRNA.

[0014] Furthermore, the sequence of the siRNA is shown in SEQ ID NO: 3-6.

[0015] In the present invention, the non-alcoholic fatty liver disease includes non-alcoholic simple fatty liver and non-alcoholic steatohepatitis or its related cirrhosis. Non-alcoholic fatty liver disease is acute or chronic damage to the liver, usually developed in patients with at least one of the following risk factors: obesity, dyslipidemia and glucose intolerance. Further, the non-alcoholic fatty liver disease is selected from non-alcoholic steatohepatitis.

[0016] A second aspect of the present invention provides a pharmaceutical composition for treating non-alcoholic fatty liver disease, wherein the pharmaceutical composition comprises an inhibitor of FBXO22.

[0017] Furthermore, the inhibitor can reduce the expression level or functional activity of FBXO22.

[0018] Furthermore, the inhibitors include but are not limited to reagents used for gene editing, reagents used for RNA interference, protein inhibitors, and compounds. Regardless of the type of inhibitor, as long as it can reduce the expression level or functional activity of FBXO22, it falls within the protection scope of the present invention.

[0019] Furthermore, the reagents used for the gene editing include but are not limited to zinc finger nuclease technology, transcription activator-like effector nuclease technology, CRISPR-Cas9, Cre-LoxP, FLP / FRT, R / RS, Gin / gix, Cin H / RS2, Par A / MRS or reagents used for phiC31.

[0020] Furthermore, the reagent used for the gene editing is selected from the reagent used for Cre-LoxP.

[0021] Furthermore, the reagents used in the Cre-LoxP include the Cre enzyme used in the Cre-LoxP.

[0022] RNA interference refers to the phenomenon of highly conserved, double-stranded RNA-induced, efficient and specific degradation of homologous mRNA in the evolutionary process. The reagents used for RNA interference include but are not limited to siRNA, shRNA and miRNA. Further, the reagents used for RNA interference are selected from siRNA.

[0023] In the present invention, protein inhibitors refer to substances that bind to some groups on the active center of protein molecules, reducing the activity of proteins or even eliminating them. Protein inhibitors include, but are not limited to, antibodies, leupeptins, antipain, chymostatin, elastase inhibitor, pepstatin, and phosphodiesterin.

[0024] Furthermore, the non-alcoholic fatty liver disease includes non-alcoholic simple fatty liver disease and non-alcoholic steatohepatitis or related cirrhosis.

[0025] Furthermore, the non-alcoholic fatty liver disease is selected from non-alcoholic steatohepatitis.

[0026] Furthermore, the pharmaceutical composition of the present invention can be administered as a medicine alone or together with other drugs. Other drugs that can be administered together with the pharmaceutical composition of the present invention are not limited as long as they do not impair the effects of the pharmaceutical composition of the present invention.

[0027] Furthermore, the pharmaceutical composition of the present invention can be prepared into various dosage forms as required, including but not limited to tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories for transdermal, mucosal, nasal, buccal, sublingual or oral administration.

[0028] Further, the administration route of the pharmaceutical composition of the present invention is not limited, as long as it can exert the desired therapeutic effect or preventive effect, including but not limited to intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesicular, intramuscular, intratracheal, subcutaneous, through the skin, through the pleura, local, inhalation, through the mucosa, skin, gastrointestinal, intraarticular, intraventricular, rectal, vaginal, intracranial, intraurethral, ​​intrahepatic, intratumoral. In some cases, it can be administered systemically. In some cases, it is administered locally.

[0029] Furthermore, the dosage of the pharmaceutical composition of the present invention is not limited as long as the desired therapeutic effect is obtained, and can be appropriately determined according to symptoms, gender, age, etc. The dosage of the therapeutic pharmaceutical composition of the present invention can be determined using, for example, the therapeutic effect on the disease as an indicator.

[0030] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier and / or excipient.

[0031] The third aspect of the present invention provides a method for screening candidate drugs for treating non-alcoholic fatty liver disease, the method comprising: treating a culture system expressing or containing the FBXO22 gene or a protein encoded by it with a substance to be screened. Detecting the expression or activity of the FBXO22 gene or a protein encoded by it in the system. Wherein, when the substance to be screened reduces the expression level or activity of the FBXO22 gene or a protein encoded by it, the substance to be screened is a candidate drug for treating non-alcoholic fatty liver disease.

[0032] A fourth aspect of the present invention provides the use of FBXO22 in screening candidate drugs for treating non-alcoholic fatty liver disease.

[0033] Furthermore, the method for screening candidate drugs is as follows: treating a culture system expressing or containing the FBXO22 gene or a protein encoded by it with a substance to be screened, and detecting the expression or activity of the FBXO22 gene or a protein encoded by it in the system. Wherein, when the substance to be screened reduces the expression level or activity of the FBXO22 gene or a protein encoded by it, the substance to be screened is a candidate drug for treating non-alcoholic fatty liver disease.

[0034] A fifth aspect of the present invention provides a product for diagnosing non-alcoholic fatty liver disease, the product comprising a reagent capable of detecting the expression level of FBXO22.

[0035] In the present invention, "diagnosis" refers to both the process of attempting to determine and / or identify a possible disease in a subject, i.e., the diagnostic procedure, and the opinion reached through this process, i.e., the diagnostic opinion. Therefore, diagnosis can also be viewed as an attempt to classify an individual's condition into separate and distinct categories in order to make medical decisions regarding treatment and prognosis.

[0036] Furthermore, FBXO22 includes wild type, mutant type or fragments thereof.

[0037] Further, FBXO22 includes full-length, unprocessed FBXO22, as well as any form of FBXO22 that results from processing in cells.

[0038] Further, FBXO22 includes naturally occurring variants of FBXO22 (eg, splice variants or allelic variants).

[0039] Further, FBXO22 includes, for example, the FBXO22 gene, human FBXO22, and FBXO22 from any other vertebrate source, including mammals, such as primates and rodents (eg, mice and rats).

[0040] Furthermore, the product also includes a chip, a test kit or a nucleic acid membrane strip.

[0041] Furthermore, the chip includes a gene chip and a protein chip.

[0042] Furthermore, the gene chip includes a solid phase carrier and oligonucleotide probes fixed on the solid phase carrier.

[0043] Furthermore, the oligonucleotide probe includes an oligonucleotide probe targeting the FBXO22 gene for detecting the transcription level of the FBXO22 gene.

[0044] Furthermore, the protein chip includes a solid phase carrier and a specific binding agent of the FBXO22 protein fixed on the solid phase carrier.

[0045] Furthermore, the protein chip can be used to detect the expression levels of multiple proteins including human FBXO22 protein (e.g., multiple proteins related to non-alcoholic fatty liver disease). By simultaneously detecting multiple markers of non-alcoholic fatty liver disease, the accuracy of non-alcoholic fatty liver disease diagnosis can be greatly improved.

[0046] Furthermore, the kit includes a gene detection kit and a protein immunoassay kit; the gene detection kit includes a reagent for detecting the transcription level of the FBXO22 gene; and the protein immunoassay kit includes a specific binding agent for the FBXO22 protein.

[0047] Furthermore, the kit includes reagents for detecting the expression level of FBXO22 gene or protein by RT-PCR, qRT-PCR, biochip detection, Southern blotting, in situ hybridization, immunoblotting, and mass spectrometry.

[0048] Furthermore, the kit includes instruments or reagents for processing samples.

[0049] Furthermore, the sample includes cells, tissues, blood, urine, saliva or mucus.

[0050] Further, the sample is selected from tissue.

[0051] A sixth aspect of the present invention provides the use of a reagent for detecting FBXO22 and its expression product in the preparation of a product for diagnosing non-alcoholic fatty liver disease.

[0052] Furthermore, the reagents include an oligonucleotide probe that specifically recognizes FBXO22, a primer that specifically amplifies FBXO22, a binding agent that specifically binds to a protein encoded by FBXO22, or a chip that specifically analyzes FBXO22.

[0053] In the present invention, the oligonucleotide probe that specifically recognizes the FBXO22 gene can be DNA, RNA, DNA-RNA chimera, PNA or other derivatives. There is no limit to the length of the probe, as long as it completes specific hybridization and specifically binds to the target nucleotide sequence, any length is acceptable. The length of the probe can be as short as 25, 20, 15, 13 or 10 bases. Similarly, the length of the probe can be as long as 60, 80, 100, 150, 300 base pairs or longer, or even the entire gene. Since different probe lengths have different effects on hybridization efficiency and signal specificity, the length of the probe is usually at least 14 base pairs, and the longest is generally no more than 30 base pairs. The length complementary to the target nucleotide sequence is optimally 15-25 base pairs. The probe self-complementary sequence is preferably less than 4 base pairs to avoid affecting the hybridization efficiency.

[0054] In the present invention, primer refers to a single-stranded polynucleotide that can hybridize with a nucleic acid and allow polymerization of complementary nucleic acid (generally by providing a free 3'-OH group). The primer can achieve specific amplification of a target sequence. Specific amplification means that the primer set amplifies the target sequence more than the non-target sequence at a statistically significant level.

[0055] In the present invention, a binding agent refers to a naturally occurring or non-naturally occurring molecule that specifically binds to a target. Examples of specific binding agents include, but are not limited to, proteins, peptides, nucleic acids, carbohydrates, and lipids.

[0056] Furthermore, the reagent is selected from primers that specifically amplify FBXO22.

[0057] Furthermore, the primer sequences for the specific amplification of FBXO22 are shown in SEQ ID NO: 1-2.

[0058] A seventh aspect of the present invention provides a method for studying the pathogenesis of non-alcoholic fatty liver disease, the method comprising studying the pathogenesis of non-alcoholic fatty liver disease by studying FBXO22.

[0059] An eighth aspect of the present invention provides a method for regulating OPA1, the method comprising administering an FBXO22 regulator to regulate the expression level of FBXO22.

[0060] Furthermore, the FBXO22 is negatively correlated with the OPA1. When the expression level of FBXO22 increases, the expression level of OPA1 decreases, and when the expression level of FBXO22 decreases, the expression level of OPA1 increases.

[0061] Furthermore, the FBXO22 regulator includes a FBXO22 inhibitor and a FBXO22 promoter.

[0062] Further, the expression level of FBXO22 can be reduced by FBXO22 inhibitors or by knockout. No matter which method is used, as long as the expression level of FBXO22 can be reduced, it falls within the protection scope of the present invention. "Knockout" refers to changing the sequence of the target gene so that the expression of the target gene is undetectable or insignificant. Knockout also includes conditional knockout, such as exposing the animal to a substrate that promotes target gene change, introducing an enzyme that promotes recombination at the target gene site (such as Cre in the Cre-LoxP system), or other methods for guiding target gene change after birth, which can cause changes in the target gene.

[0063] Furthermore, the FBXO22 inhibitors include nucleic acid inhibitors, protein inhibitors, and compounds.

[0064] Furthermore, the nucleic acid inhibitors include shRNA, siRNA, sgRNA, and antisense nucleic acids.

[0065] Furthermore, the nucleic acid inhibitor is selected from siRNA.

[0066] Furthermore, the sequence of the siRNA is shown in SEQ ID NO: 3-6.

[0067] Furthermore, the expression level of FBXO22 can be increased by using a FBXO22 promoter, or by knock-up / knock-in. Regardless of the method, as long as the expression level of FBXO22 can be increased, it falls within the protection scope of the present invention.

[0068] In the present invention, knock-in refers to the targeted insertion of a transgene into the host cell genome, resulting in expression of the transgene and / or altered expression of an endogenous target gene (e.g., elevated (including ectopic) or decreased expression), for example, by introducing an additional copy of the target gene or by operatively inserting a regulatory sequence that provides enhanced expression of the endogenous copy of the target gene. Knock-in transgenes may comprise heterozygous knock-in of a transgene or homozygous knock-in of a transgene. Knock-in also encompasses conditional knock-ins, in which expression of a transgene and / or altered expression of an endogenous target gene may occur, for example, by exposing the animal to a substance that promotes such expression, by introducing an enzyme that promotes recombination at the targeted insertion site, or by some other method for altering the targeted insertion site.

[0069] Further, the FBXO22 promoter includes nucleic acid molecules, carbohydrates, liposomes, small molecule chemical drugs, antibody drugs, polypeptides, proteins or interfering lentiviruses. In the present invention, "liposomes" refer to small vesicles composed of various types of lipids, phospholipids and / or surfactants that can be used to deliver drugs to mammals. Antibodies cover complete monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies) formed by at least two complete antibodies, and antibody fragments, as long as they exhibit the desired antigen binding activity, they all fall within the scope of protection of the present invention.

[0070] Furthermore, the FBXO22 promoter is selected from nucleic acid molecules and interfering lentiviruses.

[0071] A ninth aspect of the present invention provides a method for constructing a non-alcoholic fatty liver disease model, wherein the expression of FBXO22 in the model is increased.

[0072] Furthermore, the increased expression of FBXO22 can be achieved by knocking in and / or knocking up FBXO22, or by administering a FBXO22 promoter.

[0073] Furthermore, the knock-in and / or knock-up can be achieved by any of the following technologies: zinc finger nuclease technology, transcription activator-like effector nuclease technology, CRISPR-Cas9, Cre-LoxP, FLP / FRT, R / RS, Gin / gix, Cin H / RS2, Par A / MRS, phiC31.

[0074] Furthermore, the FBXO22 promoter can increase the expression level of the FBXO22 gene.

[0075] Furthermore, the FBXO22 promoter includes nucleic acid molecules, carbohydrates, liposomes, small molecule chemical drugs, antibody drugs, polypeptides, proteins or interfering lentiviruses.

[0076] Furthermore, the FBXO22 promoter is selected from nucleic acid molecules and interfering lentiviruses.

[0077] Furthermore, the model includes non-human animal models and cell models.

[0078] Furthermore, the non-human animal model refers to a non-human animal that has or displays characteristics of a disease or condition.

[0079] Furthermore, the non-human animal is a mammal.

[0080] Furthermore, the mammals include mice, rats, rabbits, dogs, pigs, monkeys, and sheep.

[0081] Furthermore, the mammal is selected from mice.

[0082] Furthermore, the cells used in the cell model include mammalian cells, insect cells, plant cells or yeast cells. All cells capable of gene editing fall within the scope of protection of the present invention.

[0083] Furthermore, the cells used in the cell model are selected from mammalian cells.

[0084] Furthermore, the mammalian cells may be normal cells or cancer cells.

[0085] Furthermore, the mammalian cells may be undifferentiated stem cells or tissue cells.

[0086] Furthermore, the mammalian cells are selected from L-02 cells, LX-2 cells, OSG-7701 cells, WRL68 cells, CHO cells, 293T cells, and 293F cells.

[0087] The present invention has the following advantages and beneficial effects: The present invention provides the use of FBXO22 in the diagnosis and treatment of non-alcoholic fatty liver disease. The present invention discloses that FBXO22 is highly expressed in non-alcoholic fatty liver disease, and verifies that reducing the expression level of FBXO22 can delay the progression of non-alcoholic fatty liver disease, reduce liver inflammation, and slow down the accumulation of lipids in the liver. FBXO22 can be used to diagnose and treat non-alcoholic fatty liver disease and screen candidate drugs for non-alcoholic fatty liver disease. FBXO22 can also be used to evaluate the efficacy, study and reveal the pathogenesis of non-alcoholic fatty liver disease. The present invention is of great significance for the clinical research of non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1These are analysis graphs of FBXO22 expression levels in NASH, wherein Figure A is an analysis graph of FBXO22 expression levels in normal tissues and NASH tissues in the GEO database; Figure B is a graph showing the expression levels of FBXO22 in the livers of wild-type mice at different time points after normal feed feeding and FFC feeding; Figure C is a graph showing the diagnostic efficacy of FBXO22 for NASH; Figure D is an H&E staining graph of liver sections of wild-type mice at different time points after normal feed feeding and FFC feeding; Figure E is a Sirius red staining graph of liver sections of wild-type mice at different time points after normal feed feeding and FFC feeding.

[0089] Figure 2 It is a diagram of mouse liver development and function analysis, among which, Figure A is a schematic diagram of the construction of a liver-specific knockout FBXO22 mouse model; Figure B is an agarose gel electrophoresis diagram for mouse genotype identification; Figure C is a diagram of mRNA expression levels in mouse liver; Figure D is a diagram of mouse FBXO22 protein expression levels; Figure E is an appearance diagram of mice of three genotypes; Figure F is a statistical diagram of body weight of mice of three genotypes; Figure G is a statistical diagram of serum ALT of mice of three genotypes; Figure H is a statistical diagram of liver weight of mice of three genotypes; Figure I is an appearance diagram of the liver of mice of three genotypes; Figure J is an H&E staining diagram of liver sections of mice of three genotypes; Figure K is a statistical diagram of FBXO22, GATA4, HNF1α, HNF4α, Cyp2e, and ATF1 expression in two genotypes of mice.

[0090] Figure 3 These are analysis graphs of the effects of FBXO22 on NASH, among which, Figure A is the appearance of the liver of mice with different genotypes at different induction time points; Figure B is the body weight statistics of mice with different genotypes at different induction time points; Figure C is the liver weight statistics of mice with different genotypes at different induction time points; Figure D is the blood glucose statistics of mice with different genotypes at different induction time points; Figure E is the plasma ALT statistics of mice with different genotypes at different induction time points; Figure F is the plasma AST statistics of mice with different genotypes at different induction time points; Figure G is the H&E staining of liver sections of mice with different genotypes at different induction time points.

[0091] Figure 4 These are analysis graphs of the effect of FBXO22 deficiency on the progression of NASH, where Figure A is the Sirius red staining and quantitative analysis graph of liver sections of mice with different genotypes at different induction time points; Figure B is the Oil red staining graph of liver sections of mice with different genotypes at different induction time points; Figure C is the Ki67 staining and statistical graph of liver sections of mice with different genotypes at different induction time points.

[0092] Figure 5Figure 1 is a graph showing the effect of FBXO22 on mouse liver inflammation, where Figure A is a statistical graph showing the expression of IL-6, TNF-α and STAT3 in the livers of mice with different genotypes at different induction time points detected by q-PCR; Figure B is a graph showing p-STAT3 staining and statistical graphs of liver sections of mice with different genotypes at different induction time points; Figure C is a graph showing p-p65 staining and statistical graphs of liver sections of mice with different genotypes at different induction time points.

[0093] Figure 6 Figure 3 shows the effect of FBXO22 on lipid accumulation in cells. Figure A shows the mass spectrometry detection results of lipids in the livers of mice of different genotypes after normal feeding and FFC feeding for 20 weeks. Figure B shows the effect of changing FBXO22 expression on lipid accumulation in normal mouse hepatocytes and the statistical graph.

[0094] Figure 7 Figure 1 shows the mechanism by which FBXO22 promotes lipid accumulation by degrading OPA1 through ubiquitination, where Figure A is the secondary mass spectrum of OPA1; Figure B verifies that FBXO22 negatively regulates OPA1 expression in two liver cancer cell lines; Figure C shows that the regulatory effect of FBXO22 on OPA1 was blocked after treatment with the protease inhibitor MG132; Figure D shows that FBXO22 can accelerate the degradation of OPA1 after treatment with the protein synthesis inhibitor CHX; Figure E shows that overexpression of FBXO22 can promote OPA1 ubiquitination; Figure F is a schematic diagram of the result that simultaneous knockdown of FBXO22 and OPA1 in normal mouse liver cells can reverse the lipid reduction caused by knockdown of OPA1 alone. DETAILED DESCRIPTION

[0095] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0096] Example 1 Study on the expression level of FBXO22 in NASH samples 1. Experimental methods (1) Search and download the GSE89632 dataset from the GEO database. This dataset contains 19 NASH patient samples and 24 normal healthy control samples.

[0097] (2) FFC feed was used to induce NASH in wild-type C75 mice, and the correlation between FBXO22 expression and NASH score was analyzed.

[0098] (3) FFC feed was used to induce NASH in wild-type C57 mice. After 20, 26, and 28 weeks of induction, the livers of wild-type mice were collected, proteins were extracted, and Western Blot analysis was performed. The liver tissue sections of mice fed a normal diet and FFC mice were sliced ​​and stained with H&E and Sirius red.

[0099] 2. Experimental results (1) After analyzing the expression level of FBXO22 in the samples, the results showed that compared with the healthy control samples, FBXO22 was significantly higher in NASH samples ( Figure 1 A), suggesting that FBXO22 may play an important role in NASH.

[0100] (2) Western Blot analysis showed that the expression of FBXO22 in the liver of mice fed with FFC was significantly increased compared with that of mice fed with normal diet ( Figure 1 B). After inducing NASH in 30 wild-type mice, the expression of FBXO22 in the mouse liver and the NASH score were analyzed. The results showed that FBXO22 has a significant diagnostic efficacy for NASH ( Figure 1 C).

[0101] (3) H&E staining showed obvious fat vacuoles and varying degrees of hepatocyte ballooning degeneration in the livers of mice fed FFC ( Figure 1 D). Sirius red staining showed different degrees of fibrosis in the liver of mice fed with FFC ( Figure 1 E).

[0102] The above experimental results show that the FFC-induced NASH model has been successfully established.

[0103] Note: *(p<0.05), **(p<0.01), ***(p<0.001), ****(p<0.0001) Example 2 Establishment of liver-specific knockout FBXO22 mouse strain and analysis of liver development and function 1. Experimental methods (1) Introducing the Cre / LoxP system, constructing a conditional knockout FXBO22 mouse strain ( Fbxo22 fl / fl ), and then the obtained mice were hybridized with liver-specific recombinase Alb-Cre transgenic mice to obtain liver-specific knockout FBXO22 mice ( Fbxo22 fl / fl Alb-cre )strain( Figure 2 A).

[0104] (2) Extract mouse tail DNA and identify the genotype by PCR. The PCR primers are shown in Table 1, the PCR reaction system is shown in Table 2, and the PCR program of the FBXO22 gene is shown in Table 3.

[0105] Table 1 PCR amplification primers

[0106] Table 2 PCR amplification reaction system for mouse genotype identification

[0107] Table 3 PCR procedure for FBXO22 gene

[0108] (3) Verification at the mRNA level by qRT-PCR experiment Fbxo22 - / - The knockout efficiency of FBXO22 in mouse liver was investigated, and the knockout effect of FBXO22 was verified at the protein level by Western Blot experiments.

[0109] (4) After collecting the three genotype adult mice fed with normal feed, the overall appearance of the three genotype mice was observed. Next, the three genotype mice were weighed, and then the ALT in the serum of the three genotype mice was detected. After collecting the mouse livers, the size, shape, and color of the livers of the three genotype mice were observed and the livers were weighed.

[0110] (5) Paraffin sections of the liver were sectioned and stained with H&E.

[0111] (6) Genotype analysis at the mRNA level by qRT-PCR Fbxo22 + / + and Fbxo22 - / - The expression of FBXO22, GATA4, HNF1α, HNF4α, Cyp2e, and ATF1 in mice was detected.

[0112] 2. Experimental results (1) After identification based on DNA extracted from rat tail ( Figure 2 B), mice can be divided into the following three genotypes: wild type ( Fbxo22 + / + Alb-Cre , referred to as Fbxo22 + / + , + / +), heterozygous ( Fbxo22 + / fl Alb-Cre , referred to as Fbxo22 + / - , + / -), homozygous ( Fbxo22 fl / fl Alb-Cre , referred to as Fbxo22 - / - , - / -). Mice with different genotypes born in the same litter were selected as a group of model mice for subsequent modeling and related experiments.

[0113] (2) qRT-PCR results ( Figure 2 C) and Western Blot results ( Figure 2 D) Shows that FBXO22 expression was knocked out in the liver, while there was no significant difference in the expression levels among the three genotypes of mice in the spleen and kidney.

[0114] (3) After collecting adult mice of the three genotypes fed with normal feed, it can be seen that there is no significant difference in the overall appearance of the three genotypes of mice ( Figure 2 E). The body weight of mice of three genotypes ( Figure 2 F) and liver weight showed no significant difference ( Figure 2 H). After testing the ALT in the serum of the three genotype mice, it can be seen that there is no significant difference in the ALT in the serum of the different genotype mice ( Figure 2 G). After collecting the mouse livers, it can be observed that there are no obvious abnormalities or differences in the size, shape, and color of the livers of the three genotypes of mice ( Figure 3 I).

[0115] (4) H&E staining results of liver paraffin sections showed that knocking out FBXO22 did not affect the liver structure of mice ( Figure 2 J).

[0116] (5) Genotype analysis at the mRNA level by qRT-PCR Fbxo22 + / + and Fbxo22 - / - The expression of liver development-related genes in mice was detected. It can be seen that after FBXO22 knockout, there was no significant difference in the expression of genes related to liver development such as GATA4, HNF1α, HNF4α, Cyp2e, and ATF1 ( Figure 2 K).

[0117] The above results indicate that liver-specific knockout of FBXO22 does not affect mouse liver development and function.

[0118] Note: *(p<0.05), **(p<0.01), ***(p<0.001), ****(p<0.0001) Example 3 Liver-specific knockout of FBXO22 slows down the progression of FFC-induced NASH in mice 1. Experimental methods (1) The mice were fed FFC feed at 8 weeks of age, and the mice were collected at 16, 20, 24, and 28 weeks of induction feeding. The feed and bedding in the cages were emptied in advance, and the model mice were starved for more than 12 hours. After isoflurane anesthesia, the mice were bled from their orbits, the serum was separated, and the mouse livers were completely removed.

[0119] (2) The body weight, blood glucose and liver weight of mice with different genotypes at the end of modeling were statistically analyzed.

[0120] (3) Determination of ALT and AST in mouse serum.

[0121] (4) The livers of mice of different genotypes at the above-mentioned induction time points were sectioned and stained with H&E.

[0122] (5) Sirius red dye can bind to the basic groups in collagen molecules, thereby dyeing the collagen fibers red. Sirius red staining was performed on liver sections of mice of different genotypes at the above-mentioned induction time points.

[0123] (6) The frozen sections of mouse liver at different induction time points were stained with Oil Red, and the staining conditions were observed and statistically analyzed.

[0124] (7) Immunohistochemistry was performed on mouse liver sections after 16, 20, 24, and 28 weeks of induction to detect Ki67 expression.

[0125] 2. Experimental results (1) It can be seen that at 16, 20, and 24 weeks of induction, Fbxo22 + / + Mouse liver Fbxo22 - / - The mice's livers were enlarged and lighter in color, and at 28 weeks, Fbxo22 + / + The mice had passed the fatty liver and hepatitis stage and entered the liver fibrosis stage. Therefore, after 28 weeks of induction, Fbxo22 - / - Mouse liver Fbxo22 + / + Mice, on the contrary, have increased ( Figure 3 A).

[0126] (2) After statistical analysis of the body weight, blood glucose and liver weight at the end point of modeling of mice with different genotypes, it can be seen that at 16 weeks, 20 weeks and 24 weeks of induction, Fbxo22 + / + Fasting blood glucose in mice Fbxo22 - / - The end-point body weight and end-point liver weight of mice were also higher. Fbxo22 - / - mice; at 28 weeks after induction, Fbxo22 + / + As mice entered the liver fibrosis stage, the end-point body weight, liver weight and fasting blood glucose were lower than those at 24 weeks ( Figure 3 BD).

[0127] (3) After measuring ALT and AST in mouse serum at 16, 20, 24, and 28 weeks of induction, Fbxo22 + / + The levels of ALT and AST in mouse serum were Fbxo22 - / - The mice showed an increase in Fbxo22 + / + Liver damage in mice Fbxo22 - / - Mice were more severely Figure 3 EF).

[0128] (4) H&E staining results ( Figure 3 G) shows that at 16 weeks after induction, Fbxo22 + / + Fat vacuoles began to appear in the liver of mice. At 20 weeks of induction, Fbxo22 + / + Local ballooning degeneration began to appear in the liver of mice, indicating liver cell damage. Large areas of liver cell ballooning degeneration appeared at 24 weeks, and the area of ​​ballooning degeneration increased further at 28 weeks. Fbxo22 - / - Ballooning of liver cells began to occur in mice at 24 weeks, and the area of ​​liver cell damage was significantly less than that in mice at 28 weeks. Fbxo22 + / + Mouse.

[0129] (5) Sirius red staining was performed on liver sections of mice of different genotypes at each induction time point. Figure 4 A), it can be observed that at 16 weeks of induction, Fbxo22 + / + Mice and Fbxo22 - / - No obvious fibrosis was found in the liver of mice. However, at 20 weeks of induction, Fbxo22 + / + The mice began to develop local fibrosis in the portal area. At 24 weeks of induction, Fbxo22 + / + The degree of fibrosis in mice increased. At this time point, Fbxo22 - / - Local fibrosis began to appear in the mouse liver; at 28 weeks of induction, Fbxo22 + / + The liver fibrosis of mice was further aggravated, and obvious fibrosis was also observed in the liver parenchyma. Fbxo22 - / - The degree of fibrosis in the mouse liver was also aggravated compared with the previous time point, but still Fbxo22 + / + Mice were relieved.

[0130] (6) It can be observed that at 16 weeks, 20 weeks, and 24 weeks, Fbxo22 + / + Fat deposition in mouse liver is more strongly expressed than Fbxo22 - / - Mice, at 28 weeks, Fbxo22 + / + Mice entered the NASH fibrosis stage, fat deposition was alleviated, and Fbxo22 - / - The course of the disease in mice was slower, but local fat deposition was still severe ( Figure 4 B).

[0131] These results suggest that FBXO22 can affect the lipid metabolism process in mouse liver, and knocking out FBXO22 can significantly slow down the rate of lipid deposition in mouse liver.

[0132] (7) Immunohistochemistry results showed that at 16, 20, 24, and 28 weeks, Fbxo22 + / + Ki67 positive expression in mouse liver is stronger than Fbxo22 - / - Mouse ( Figure 4 C) The above results show Fbxo22 - / - The overall course of NASH in mice was Fbxo22 + / + This suggests that liver-specific knockout of FBXO22 can slow the progression of NASH in mice.

[0133] Note: *(p<0.05), **(p<0.01), ***(p<0.001), ****(p<0.0001) Example 4 Liver-specific knockout of FBXO22 reduces liver inflammation in mice 1. Experimental methods (1) Q-PCR detection at 16, 20, 24, and 28 weeks of induction: Fbxo22 - / - Contents of IL-6, TNF-α, and STAT3 in mouse liver.

[0134] (2) Immunohistochemistry was performed on mouse liver sections at 16, 20, 24, and 28 weeks after induction to detect p-STAT3 and p-p65.

[0135] 2. Experimental results (1) Q-PCR results ( Figure 5A) shows that at 16, 20, and 24 weeks of induction, Fbxo22 - / - The contents of IL-6, TNF-α and STAT3 in mouse liver Fbxo22 + / + mice were significantly reduced.

[0136] (2) Immunohistochemistry results showed that at 16, 20, and 24 weeks, Fbxo22 + / + The positive expression of inflammatory markers p-STAT3 and p-p65 in mouse liver was stronger than Fbxo22 - / - mice, and at 28 weeks, Fbxo22 + / + The mice had passed the acute phase of inflammation. Fbxo22 - / - The positive expression of p-STAT3 and p-p65 in mice gradually increased ( Figure 5 BC).

[0137] The above results suggest that knocking out FBXO22 can significantly slow down the activation of inflammatory signaling pathways in the liver of mice and the expression of pro-inflammatory factors such as cytokines, thereby slowing down the development of NASH in mice.

[0138] Example 5 FBXO22 deficiency reduces lipid accumulation in the liver 1. Experimental methods The livers of mice treated for 20 weeks were sent to the company for lipid mass spectrometry analysis. The expression of FBXO22 in normal mouse liver cells was changed by overexpression and knockout, and oil red staining was performed. The staining was observed and data statistics were performed. The siRNA sequences used for knockout are shown in Table 4.

[0139] Table 4 siRNA sequences

[0140] 2. Experimental results (1) Mass spectrometry results showed that the lipid content in the liver of mice with specific FBXO22 knockout was significantly lower than that of wild-type mice ( Figure 6 A).

[0141] (2) Overexpression of FBXO22 in normal mouse hepatocytes significantly increased Oil red staining; knockdown of FBXO22 significantly reduced Oil red staining, indicating that FBXO22 can promote lipid accumulation ( Figure 6 B).

[0142] Example 6 FBXO22 promotes lipid accumulation by degrading OPA1 through ubiquitination 1. Experimental methods (1) Search for proteins related to lipid metabolism by combining FBXO22 with mass spectrometry.

[0143] (2) Overexpression and knockout of FBXO22. Western Blot was used to detect whether FBXO22 affects the expression of the proteins found in (1). The siRNA used for knockout is shown in Table 4.

[0144] (3) Through MG132 and CHX experiments, it was verified that the regulation of FBXO22 on this protein depends on the proteasome pathway.

[0145] (4) Immunoprecipitation experiments verified that FBXO22 could promote the ubiquitination modification of this protein.

[0146] (5) Interfering with FBXO22 and the protein simultaneously in cells and verifying whether FBXO22's regulation of lipids depends on the protein by Oil Red staining 2. Experimental results (1) FBXO22 combined with mass spectrometry results ( Figure 7 A) shows that OPA1 binds to FBXO22.

[0147] (2) Results Figure 7 B) Overexpression of FBXO22 reduced OPA1 expression, while knockout of FBXO22 increased OPA1 expression.

[0148] (3) Results Figure 7 C) shows that blocking the proteasome pathway with MG132 significantly weakened the regulatory effect of FBXO22 on OPA1.

[0149] (4) Results Figure 7 D) shows that FBXO22 can accelerate the degradation of OPA1 after cells are treated with CHX.

[0150] (5) Results Figure 7 E) FBXO22 promotes OPA1 ubiquitination.

[0151] (6) Results Figure 7 F) shows that simultaneous knockdown of FBXO22 and OPA1 can reverse the reduction in lipid accumulation caused by single knockdown of FBXO22.

[0152] The above results suggest that knocking out FBXO22 can slow down lipid accumulation in the liver, and FBXO22 can promote lipid accumulation by degrading OPA1 through ubiquitination.

[0153] Note: *(p<0.05), **(p<0.01), ***(p<0.001), ****(p<0.0001) This experiment used GraphPad Prism 9.5 and SPSS 20.0 to compare the experimental data and make statistical graphs. All statistical data were represented by mean ± standard deviation. The comparison between groups was evaluated by independent sample t test, and the difference between two groups or more was evaluated by one-way variance test. The hypothesis test level was defined as α = 0.05, and the difference was considered statistically significant when P < 0.05.

[0154] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Application of FBXO22 in the preparation of drugs for the treatment of non-alcoholic fatty liver disease; Preferably, the drug comprises an inhibitor of FBXO22; Preferably, the inhibitor can reduce the expression level or functional activity of FBXO22; Preferably, the inhibitor includes a nucleic acid inhibitor, a protein inhibitor, or a compound; Preferably, the nucleic acid inhibitor includes shRNA, siRNA, sgRNA, and antisense nucleic acid; Preferably, the nucleic acid inhibitor is selected from siRNA; Preferably, the sequence of the siRNA is as shown in SEQ ID NO:3-6; Preferably, the non-alcoholic fatty liver disease includes non-alcoholic simple fatty liver disease and non-alcoholic steatohepatitis or related cirrhosis; Preferably, the non-alcoholic fatty liver disease is selected from non-alcoholic steatohepatitis.

2. A pharmaceutical composition for treating non-alcoholic fatty liver disease, characterized in that: The pharmaceutical composition includes an inhibitor of FBXO22; Preferably, the inhibitor can reduce the expression level or functional activity of FBXO22; Preferably, the inhibitor includes reagents used for gene editing, reagents used for RNA interference, protein inhibitors, and compounds; Preferably, the reagents used for gene editing include reagents used for zinc finger nuclease technology, transcription activator-like effector nuclease technology, CRISPR-Cas9, Cre-LoxP, FLP / FRT, R / RS, Gin / gix, Cin H / RS2, Par A / MRS or phiC31; Preferably, the reagent used for gene editing is selected from the reagent used for Cre-LoxP; Preferably, the reagent used in the Cre-LoxP comprises the Cre enzyme used in the Cre-LoxP; Preferably, the reagents used for RNA interference include siRNA, shRNA and miRNA; Preferably, the non-alcoholic fatty liver disease includes non-alcoholic simple fatty liver disease and non-alcoholic steatohepatitis or related cirrhosis; Preferably, the non-alcoholic fatty liver disease is selected from non-alcoholic steatohepatitis.

3. The drug combination according to claim 2, characterized in that: The pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

4. A method for screening candidate drugs for treating non-alcoholic fatty liver disease, characterized in that: The method comprises: treating a culture system expressing or containing the FBXO22 gene or a protein encoded by it with a substance to be screened; detecting the expression or activity of the FBXO22 gene or a protein encoded by it in the system; wherein, when the substance to be screened reduces the expression level or activity of the FBXO22 gene or a protein encoded by it, the substance to be screened is a candidate drug for treating non-alcoholic fatty liver disease; Preferably, the non-alcoholic fatty liver disease includes non-alcoholic simple fatty liver disease and non-alcoholic steatohepatitis or related cirrhosis; Preferably, the non-alcoholic fatty liver disease is selected from non-alcoholic steatohepatitis.

5. Application of FBXO22 in screening candidate drugs for the treatment of non-alcoholic fatty liver disease; Preferably, the method for screening candidate drugs is as follows: treating a culture system expressing or containing the FBXO22 gene or a protein encoded by it with a substance to be screened; detecting the expression or activity of the FBXO22 gene or a protein encoded by it in the system; wherein, When the substance to be screened reduces the expression level or activity of the FBXO22 gene or the protein encoded by it, the substance to be screened is a candidate drug for treating non-alcoholic fatty liver disease; Preferably, the non-alcoholic fatty liver disease includes non-alcoholic simple fatty liver disease and non-alcoholic steatohepatitis or related cirrhosis; Preferably, the non-alcoholic fatty liver disease is selected from non-alcoholic steatohepatitis.

6. A product for diagnosing non-alcoholic fatty liver disease, characterized in that: The product includes a reagent capable of detecting the expression level of FBXO22; Preferably, the product further comprises a chip, a kit or a nucleic acid membrane strip; Preferably, the chip includes a gene chip and a protein chip; Preferably, the gene chip comprises an oligonucleotide probe targeting FBXO22 for detecting the transcription level of FBXO22; Preferably, the protein chip comprises a specific binding agent for FBXO22 protein; Preferably, the kit includes reagents for detecting the expression level of FBXO22 gene or protein by RT-PCR, qRT-PCR, biochip detection, Southern blotting, in situ hybridization, immunoblotting, or mass spectrometry; Preferably, the kit includes an instrument or reagent for processing a sample; Preferably, the sample comprises cells, tissue, blood, urine, saliva or mucus; Preferably, the sample is selected from tissue; Preferably, the non-alcoholic fatty liver disease includes non-alcoholic simple fatty liver disease and non-alcoholic steatohepatitis or related cirrhosis; Preferably, the non-alcoholic fatty liver disease is selected from non-alcoholic steatohepatitis.

7. Application of reagents for detecting FBXO22 and its expression products in the preparation of products for diagnosing non-alcoholic fatty liver disease; Preferably, the reagents include an oligonucleotide probe that specifically recognizes FBXO22, a primer that specifically amplifies FBXO22, a binding agent that specifically binds to a protein encoded by FBXO22, or a chip that specifically analyzes FBXO22; Preferably, the reagent is selected from primers that specifically amplify FBXO22; Preferably, the primer sequence for specific amplification of FBXO22 is as shown in SEQ ID NO: 1-2; Preferably, the non-alcoholic fatty liver disease includes non-alcoholic simple fatty liver disease and non-alcoholic steatohepatitis or related cirrhosis; Preferably, the non-alcoholic fatty liver disease is selected from non-alcoholic steatohepatitis.

8. A method for studying the pathogenesis of non-alcoholic fatty liver disease, characterized in that: The method includes studying the pathogenesis of non-alcoholic fatty liver disease by studying FBXO22; Preferably, the non-alcoholic fatty liver disease includes non-alcoholic simple fatty liver disease and non-alcoholic steatohepatitis or related cirrhosis; Preferably, the non-alcoholic fatty liver disease is selected from non-alcoholic steatohepatitis.

9. A method for regulating OPA1, characterized in that: The method comprises administering an FBXO22 modulator to modulate the expression level of FBXO22; Preferably, the FBXO22 and the OPA1 Present Negative correlation regulation; Preferably, the FBXO22 regulator includes an FBXO22 inhibitor and an FBXO22 promoter; Preferably, the FBXO22 inhibitor includes nucleic acid inhibitors, protein inhibitors, and compounds; Preferably, the nucleic acid inhibitor includes shRNA, siRNA, sgRNA, and antisense nucleic acid; Preferably, the nucleic acid inhibitor is selected from siRNA; Preferably, the sequence of the siRNA is as shown in SEQ ID NO:3-6; Preferably, the FBXO22 promoter is capable of increasing the expression level of FBXO22; Preferably, the FBXO22 promoter includes nucleic acid molecules, carbohydrates, liposomes, small molecule chemical drugs, antibody drugs, polypeptides, proteins or interfering lentiviruses; Preferably, the FBXO22 promoter is selected from nucleic acid molecules and interfering lentiviruses.

10. A method for constructing a non-alcoholic fatty liver disease model, characterized in that: The model described has increased FBXO22 expression; Preferably, the increased expression of FBXO22 can be achieved by knocking in and / or knocking up FBXO22, or by administering a FBXO22 promoter; Preferably, the knock-in and / or knock-up can be achieved by any of the following technologies: zinc finger nuclease technology, transcription activator-like effector nuclease technology, CRISPR-Cas9, Cre-LoxP, FLP / FRT, R / RS, Gin / gix, Cin H / RS2, Par A / MRS, phiC31; Preferably, the FBXO22 promoter can increase the expression level of the FBXO22 gene; Preferably, the FBXO22 promoter includes nucleic acid molecules, carbohydrates, liposomes, small molecule chemical drugs, antibody drugs, polypeptides, proteins or interfering lentiviruses; Preferably, the FBXO22 promoter is selected from nucleic acid molecules and interfering lentiviruses; Preferably, the model includes a non-human animal model, a cell model; Preferably, the non-human animal model refers to a non-human animal that has or displays characteristics of a disease or condition; Preferably, the non-human animal is a mammal; Preferably, the mammals include mice, rats, rabbits, dogs, pigs, monkeys, and sheep; Preferably, the mammal is selected from mice; Preferably, the cells used in the cell model include mammalian cells, insect cells, plant cells or yeast cells; Preferably, the cells used in the cell model are selected from mammalian cells; Preferably, the mammalian cells are selected from L-02 cells, LX-2 cells, OSG-7701 cells, WRL68 cells, CHO cells, 293T cells, 293F cells; Preferably, the non-alcoholic fatty liver disease includes non-alcoholic simple fatty liver disease and non-alcoholic steatohepatitis or related cirrhosis; Preferably, the non-alcoholic fatty liver disease is selected from non-alcoholic steatohepatitis.

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