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

Abstract

The application provides application of FBXO22 in diagnosis and treatment of non-alcoholic fatty liver disease, discloses that FBXO22 is highly expressed in non-alcoholic fatty liver disease, and verifies that reducing the expression level of FEXO22 can delay the progress of non-alcoholic fatty liver disease, reduce liver inflammation and slow down liver lipid accumulation. FBXO22 can be used for diagnosis and treatment of non-alcoholic fatty liver disease, screening of candidate drugs for non-alcoholic fatty liver disease, and also can be used for efficacy evaluation, research and revelation of the pathogenesis of non-alcoholic fatty liver disease. The application has important significance for clinical research of non-alcoholic fatty liver disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of FBXO22 in the diagnosis and treatment of non-alcoholic fatty liver disease. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) has become one of the leading causes of liver disease worldwide, and the primary cause of chronic liver disease and hepatocellular carcinoma has gradually shifted from chronic viral hepatitis to NAFLD. NAFLD encompasses a range of metabolic disorders, including non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) or their associated cirrhosis. With the increasing prevalence of type 2 diabetes and obesity, the incidence of NAFLD is rising annually, while the incidence of NASH has increased exponentially in recent years. In non-alcoholic steatohepatitis, the accumulation of fat in hepatocytes leads to hepatocyte degeneration / necrosis, and the resulting inflammation and liver fibrosis can progress to cirrhosis and liver cancer. Therefore, NASH is considered a serious disease worldwide, and currently, there is a lack of effective treatments. Summary of the Invention

[0003] To overcome the shortcomings of existing technologies, the present invention aims to provide a method for diagnosing and treating non-alcoholic fatty liver disease (NAFLD). This invention discloses that FBXO22 is highly expressed in NAFLD and verifies that reducing FBXO22 expression levels can slow the progression of NAFLD, alleviate liver inflammation, and reduce lipid accumulation in the liver. FBXO22 can be used to diagnose and treat NAFLD, screen candidate drugs for NAFLD, and also for pharmacodynamic evaluation, research, and elucidation of the pathogenesis of NAFLD. This invention has significant implications for clinical research on NAFLD.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] The first aspect of this invention provides the use of FBXO22 in the preparation of a medicament for treating non-alcoholic fatty liver disease.

[0006] In this invention, "treatment" means achieving the desired pharmacological and / or physiological effect, covering any treatment of a pathological condition or disease in mammals, including humans. Treatment can mean treatment and / or prevention; prevention is understood when a disease or its symptoms are completely or partially prevented, and treatment is understood when a disease is partially or completely cured. Treatment covers any treatment of diseases in mammals, particularly 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 it; (d) inhibiting the disease, i.e., stopping its development (e.g., slowing the rate of disease progression); and (e) alleviating the disease, i.e., causing disease remission.

[0007] Furthermore, the drug includes an inhibitor of FBXO22.

[0008] In this invention, "inhibitor" can be understood as any substance or compound capable of specifically silencing, reducing, blocking, and / or inhibiting gene expression, or by preventing transcription of the FBXO22 gene, thereby avoiding the formation of any transcripts of the gene, or by promoting the degradation of any transcripts of the FBXO22 gene, or by specifically reducing, blocking, and / or inhibiting the expression of the protein encoding FBXO22, and inhibiting the activity of the FBXO22 protein. Transcription products refer to RNA derived from gene transcription. Furthermore, the inhibitor can reduce the expression level or functional activity of FBXO22.

[0009] A protein or nucleic acid expression level can be considered reduced when the expression level 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%, or at least 100% (i.e., not present) relative to a reference value. A “reference value” can refer to the expression level of a protein or nucleic acid prior to administration of the inhibitor. A reference value can be an absolute value, a relative value, a value with an upper and / or lower limit, a series of values, an average, a median, a mean, or a value expressed by a reference control or reference value. A reference value can be based on a value obtained from a single sample, such as a value obtained from a study sample but at a previous time point. A reference value can be based on a large sample, such as a value obtained in a sample population, or based on a sample pool including or excluding the sample to be tested.

[0010] A protein's functional activity can be considered reduced when it is 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%, or at least 100% (i.e., non-existent) relative to a reference value. The reference value may refer to the protein's functional activity prior to the administration of the inhibitor.

[0011] Furthermore, the inhibitors include nucleic acid inhibitors, protein inhibitors, and compounds.

[0012] In this invention, "nucleic acid" refers to a biopolymer of nucleotides linked together by phosphodiester bonds (polynucleotides, polynucleic acids). During chemical synthesis, the nucleotides of nucleic acids may additionally or alternatively be linked via thiophosphate or dithiophosphate bonds. Nucleic acids are classified into two categories: ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), depending on the type of sugar in the nucleotide (ribose or deoxyribose). Nucleic acids include any natural or non-natural nucleic acid. Natural nucleotides refer to nucleotides that can be purified from natural sources. Non-natural nucleotides are those produced using recombinant expression systems, and optionally, purified, chemically synthesized. Nucleic acids may include nucleoside analogs, such as analogs with chemically modified bases or sugars, or backbone modifications. Unless otherwise stated, nucleic acid sequences are shown in a 5'-3' orientation.

[0013] Furthermore, the nucleic acid inhibitors include shRNA, siRNA, sgRNA, and antisense nucleic acids.

[0014] Furthermore, the nucleic acid inhibitor is selected from siRNA.

[0015] Furthermore, the sequence of the siRNA is shown in SEQ ID NO:3-6.

[0016] In this invention, the non-alcoholic fatty liver disease includes non-alcoholic simple fatty liver and non-alcoholic steatohepatitis or related cirrhosis. Non-alcoholic fatty liver disease is acute or chronic liver damage that typically develops 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.

[0017] A second aspect of the present invention provides a pharmaceutical composition for treating non-alcoholic fatty liver disease, said pharmaceutical composition comprising an inhibitor of FBXO22.

[0018] Furthermore, the inhibitor can reduce the expression level or functional activity of FBXO22.

[0019] Furthermore, the inhibitors include, but are not limited to, reagents used in gene editing, reagents used in RNA interference, protein inhibitors, and compounds. Regardless of the type of inhibitor, any inhibitor capable of reducing the expression level or functional activity of FBXO22 falls within the scope of protection of this invention.

[0020] Furthermore, the reagents used in the gene editing include, but are not limited to, those used in 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.

[0021] Furthermore, the reagents used in the gene editing are selected from those used in Cre-LoxP.

[0022] Furthermore, the reagents used in Cre-LoxP include the Cre enzyme used in Cre-LoxP.

[0023] RNA interference (RNAi) is a highly conserved evolutionary phenomenon characterized by the efficient and specific degradation of homologous mRNA induced by double-stranded RNA. Reagents used in RNAi include, but are not limited to, siRNA, shRNA, and miRNA. Furthermore, the reagents used in RNAi are selected from siRNA.

[0024] In this invention, protein inhibitors refer to substances that bind to certain groups on the active site of a protein molecule, thereby reducing or even eliminating the protein's activity and function. Protein inhibitors include, but are not limited to, antibodies, leucine, anti-pain agents, chymotrypsin inhibitors, elastase inhibitors, pepsin inhibitors, and phosphatidylcholine.

[0025] Furthermore, the non-alcoholic fatty liver disease includes non-alcoholic simple fatty liver and non-alcoholic steatohepatitis or related cirrhosis.

[0026] Furthermore, the non-alcoholic fatty liver disease is selected from non-alcoholic steatohepatitis.

[0027] Furthermore, the pharmaceutical composition of the present invention can be administered alone or in combination with other drugs. Other drugs that can be administered with the pharmaceutical composition of the present invention are not limited, as long as they do not impair the efficacy of the pharmaceutical composition of the present invention.

[0028] Furthermore, the pharmaceutical compositions of the present invention can be prepared into various dosage forms as needed. These include, but are not limited to, tablets, solutions, granules, patches, ointments, capsules, aerosols, or suppositories for use via the skin, mucous membranes, nose, cheeks, sublingual application, or oral administration.

[0029] Furthermore, the route of administration of the pharmaceutical composition of the present invention is not limited, as long as it can achieve the desired therapeutic or preventive effect, including but not limited to intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesical, intramuscular, intratracheal, subcutaneous, through the skin, through the pleura, locally, inhaled, through mucous membranes, skin, gastrointestinal tract, intra-articular, intracardiac, rectal, vaginal, intraskull, intraurethral, ​​intrahepatic, and intratumoral administration. In some cases, it can be administered systemically. In some cases, it is administered locally.

[0030] Furthermore, the dosage of the pharmaceutical composition of the present invention is not limited, as long as the desired therapeutic effect is achieved, and can be appropriately determined based on 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.

[0031] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier and / or excipients.

[0032] A third aspect of the present invention provides a method for screening candidate drugs for treating non-alcoholic fatty liver disease (NAFLD), the method comprising: treating a culture system expressing or containing the FBXO22 gene or its encoded protein with a screening substance; detecting the expression or activity of the FBXO22 gene or its encoded protein in the system; wherein, when the screening substance reduces the expression level or activity of the FBXO22 gene or its encoded protein, the screening substance is a candidate drug for treating NAFLD.

[0033] The fourth aspect of this invention provides the use of FBXO22 in screening candidate drugs for the treatment of non-alcoholic fatty liver disease.

[0034] Furthermore, the method for screening candidate drugs is as follows: A culture system expressing or containing the FBXO22 gene or its encoded protein is treated with the substance to be screened. The expression or activity of the FBXO22 gene or its encoded protein in the system is detected. Wherein, when the substance to be screened reduces the expression level or activity of the FBXO22 gene or its encoded protein, the substance to be screened is a candidate drug for treating non-alcoholic fatty liver disease.

[0035] The fifth aspect of the present invention provides a product for diagnosing non-alcoholic fatty liver disease, the product comprising a reagent capable of detecting FBXO22 expression levels.

[0036] In this invention, "diagnosis" refers both to the process of attempting to identify and / or characterize a possible disease in a subject, i.e., a diagnostic procedure, and to the opinion derived from that process, i.e., a diagnostic opinion. Therefore, diagnosis can also be viewed as an attempt to classify an individual's condition into separate and distinct categories, thereby enabling medical decisions regarding treatment and prognosis.

[0037] Furthermore, FBXO22 includes wild-type, mutant, or fragments thereof.

[0038] Furthermore, FBXO22 includes full-length, unprocessed FBXO22, as well as any form of FBXO22 derived from cells and processed.

[0039] Furthermore, FBXO22 includes naturally occurring variants of FBXO22 (e.g., splice variants or allelic variants).

[0040] Furthermore, FBXO22 includes, for example, the FBXO22 gene, human FBXO22, and FBXO22 from any other vertebrate source, including mammals such as primates and rodents (e.g., mice and rats).

[0041] Furthermore, the product also includes chips, reagent kits, or nucleic acid membrane strips.

[0042] Furthermore, the chip includes gene chips and protein chips.

[0043] Furthermore, the gene chip includes a solid support and oligonucleotide probes immobilized on the solid support.

[0044] Furthermore, the oligonucleotide probe includes an oligonucleotide probe targeting the FBXO22 gene for detecting the transcriptional level of the FBXO22 gene.

[0045] Furthermore, the protein chip includes a solid support and a specific binder for the FBXO22 protein immobilized on the solid support.

[0046] Furthermore, the protein chip can be used to detect the expression levels of multiple proteins, including human FBXO22 protein (e.g., multiple proteins associated with non-alcoholic fatty liver disease). By simultaneously detecting multiple biomarkers of non-alcoholic fatty liver disease, the accuracy of non-alcoholic fatty liver disease diagnosis can be greatly improved.

[0047] Furthermore, the kit includes a gene detection kit and a protein immunoassay kit; the gene detection kit includes reagents for detecting the transcriptional level of the FBXO22 gene; the protein immunoassay kit includes a specific binding agent for the FBXO22 protein.

[0048] Furthermore, the kit includes reagents for detecting the expression level of the FBXO22 gene or protein by RT-PCR, qRT-PCR, microarray detection, DNA blotting, in situ hybridization, immunoblotting, and mass spectrometry.

[0049] Furthermore, the kit includes instruments or reagents for processing samples.

[0050] Furthermore, the sample includes cells, tissues, blood, urine, saliva, or mucus.

[0051] Furthermore, the sample is selected from tissues.

[0052] The sixth aspect of this invention provides the use of reagents for detecting FBXO22 and its expression products in the preparation of products for diagnosing non-alcoholic fatty liver disease.

[0053] Furthermore, the reagents include oligonucleotide probes that specifically recognize FBXO22, primers that specifically amplify FBXO22, binding agents that specifically bind to proteins encoded by FBXO22, or chips that specifically analyze FBXO22.

[0054] In this invention, the oligonucleotide probe that specifically recognizes the FBXO22 gene can be DNA, RNA, a DNA-RNA chimera, PNA, or other derivatives. The length of the probe is not limited; any length is acceptable as long as specific hybridization and binding to the target nucleotide sequence are achieved. The probe length can be as short as 25, 20, 15, 13, or 10 base pairs. Similarly, the probe length can be as long as 60, 80, 100, 150, 300 base pairs or longer, even the entire gene. Since different probe lengths have different effects on hybridization efficiency and signal specificity, the probe length is typically at least 14 base pairs and generally does not exceed 30 base pairs. The optimal length for complementarity with the target nucleotide sequence is 15-25 base pairs. The probe's own complementary sequence is preferably less than 4 base pairs to avoid affecting hybridization efficiency.

[0055] In this invention, primers refer to single-stranded polynucleotides capable of hybridizing with nucleic acids and allowing the polymerization of complementary nucleic acids (typically by providing a free 3'-OH group). The primers enable specific amplification of the target sequence. Specific amplification means that the primer set amplifies the target sequence at a statistically significant level compared to non-target sequences.

[0056] In this invention, a binder refers to a naturally occurring or non-naturally occurring molecule that specifically binds to a target. Examples of specific binders include, but are not limited to, proteins, peptides, nucleic acids, carbohydrates, and lipids.

[0057] Furthermore, the reagent is selected from primers that specifically amplify FBXO22.

[0058] Furthermore, the primer sequences for the specific amplification of FBXO22 are shown in SEQ ID NO:1-2.

[0059] The 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.

[0060] The 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.

[0061] Furthermore, FBXO22 and OPA1 are negatively correlated in their regulation. 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.

[0062] Furthermore, the FBXO22 regulator includes FBXO22 inhibitors and FBXO22 promoters.

[0063] Furthermore, FBXO22 expression levels can be reduced using FBXO22 inhibitors or by knockout. Regardless of the method used, any method that reduces FBXO22 expression levels falls within the scope of this invention. "Knockout" refers to altering the target gene sequence, making its expression undetectable or insignificant. Knockout also includes conditional knockout, such as exposing animals to substrates that promote target gene alteration, introducing enzymes that promote recombination at the target gene site (e.g., Cre in the Cre-LoxP system), or other methods used to guide target gene alteration after birth.

[0064] Furthermore, the FBXO22 inhibitors include nucleic acid inhibitors, protein inhibitors, and compounds.

[0065] Furthermore, the nucleic acid inhibitors include shRNA, siRNA, sgRNA, and antisense nucleic acids.

[0066] Furthermore, the nucleic acid inhibitor is selected from siRNA.

[0067] Furthermore, the sequence of the siRNA is shown in SEQ ID NO:3-6.

[0068] Furthermore, the expression level of FBXO22 can be increased by using an FBXO22 promoter, or by knock-up / knock-in. Regardless of the method used, as long as the expression level of FBXO22 can be increased, it falls within the protection scope of this invention.

[0069] In this invention, knock-in refers to the targeted insertion of a transgene into the host cell genome, resulting in transgene expression and / or altered expression of the endogenous target gene (e.g., increased (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 an endogenous copy of the target gene to enhance expression. Knock-in transgenes may comprise heterozygous knock-in or homozygous knock-in of the transgene. Knock-in also encompasses conditional knock-in, wherein transgene expression and / or altered expression of the endogenous target gene can occur, for example, by exposing an 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.

[0070] Furthermore, the FBXO22 promoter includes nucleic acid molecules, carbohydrates, liposomes, small molecule chemicals, antibody drugs, peptides, proteins, or interfering lentiviruses. In this invention, "liposomes" refers to small vesicles composed of various types of lipids, phospholipids, and / or surfactants, which can be used for drug delivery to mammals. Antibodies cover complete monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two complete antibodies, and antibody fragments, as long as they exhibit the desired antigen-binding activity, all fall within the scope of protection of this invention.

[0071] Furthermore, the FBXO22 promoter is selected from nucleic acid molecules and interfering lentiviruses.

[0072] The ninth aspect of this invention provides a method for constructing a non-alcoholic fatty liver disease model, wherein the model exhibits increased expression of FBXO22.

[0073] Furthermore, the increased expression of FBXO22 can be achieved by knocking in and / or knocking up FBXO22, or by applying an FBXO22 promoter.

[0074] Furthermore, the knock-in and / or knock-up can be achieved through 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.

[0075] Furthermore, the FBXO22 promoter can increase the expression level of the FBXO22 gene.

[0076] Furthermore, the FBXO22 promoter includes nucleic acid molecules, carbohydrates, liposomes, small molecule chemicals, antibody drugs, peptides, proteins, or interfering lentiviruses.

[0077] Furthermore, the FBXO22 promoter is selected from nucleic acid molecules and interfering lentiviruses.

[0078] Furthermore, the models include non-human animal models and cell models.

[0079] Furthermore, the non-human animal model refers to a non-human animal that has or displays characteristics of disease or symptom.

[0080] Furthermore, the non-human animal in question is a mammal.

[0081] Furthermore, the mammals include mice, rats, rabbits, dogs, pigs, monkeys, and sheep.

[0082] Furthermore, the mammal is selected from mice.

[0083] 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 this invention.

[0084] Furthermore, the cells used in the cell model are selected from mammalian cells.

[0085] Furthermore, the mammalian cells can be normal cells or cancer cells.

[0086] Furthermore, the mammalian cells can be undifferentiated stem cells or tissue cells.

[0087] Furthermore, the mammalian cells are selected from L-O2 cells, LX-2 cells, OSG-7701 cells, WRL68 cells, CHO cells, 293T cells, and 293F cells.

[0088] The advantages and beneficial effects of this invention are as follows:

[0089] This invention provides the application of FBXO22 in the diagnosis and treatment of non-alcoholic fatty liver disease (NAFLD). It discloses the high expression of FBXO22 in NAFLD and verifies that reducing FBXO22 expression levels can slow the progression of NAFLD, alleviate liver inflammation, and reduce lipid accumulation in the liver. FBXO22 can be used for the diagnosis and treatment of NAFLD, as well as for screening candidate drugs for NAFLD. It can also be used for pharmacodynamic evaluation, research, and to elucidate the pathogenesis of NAFLD. This invention has significant implications for clinical research on NAFLD. Attached Figure Description

[0090] Figure 1These are graphs analyzing the expression levels of FBXO22 in NASH. Graph A shows the expression levels of FBXO22 in normal and NASH tissues from the GEO database; Graph B shows the expression levels of FBXO22 in the livers of wild-type mice fed with normal diet and FFC at different time points; Graph C shows the diagnostic efficacy of FBXO22 for NASH; Graph D shows the H&E staining of liver sections from wild-type mice fed with normal diet and FFC at different time points; Graph E shows the Sirius red staining of liver sections from wild-type mice fed with normal diet and FFC at different time points.

[0091] Figure 2 These are diagrams analyzing mouse liver development and function. Figure A shows a schematic diagram of the construction of a liver-specific FBXO22 knockout mouse model; Figure B shows agarose gel electrophoresis of mouse genotypes; Figure C shows the mRNA expression level in mouse liver; Figure D shows the FBXO22 protein expression level in mice; Figure E shows the appearance of mice of the three genotypes; Figure F shows the weight statistics of mice of the three genotypes; Figure G shows the serum ALT statistics of mice of the three genotypes; Figure H shows the liver weight statistics of mice of the three genotypes; Figure I shows the appearance of the livers of mice of the three genotypes; Figure J shows the H&E staining of liver sections from mice of the three genotypes; and Figure K shows the expression statistics of FBXO22, GATA4, HNF1α, HNF4α, Cyp2e, and ATF1 in mice of two genotypes.

[0092] Figure 3 This is a graph analyzing the effects of FBXO22 on NASH. Figure A shows the liver appearance of mice with different genotypes at different induction time points; Figure B shows the body weight statistics of mice with different genotypes at different induction time points; Figure C shows the liver weight statistics of mice with different genotypes at different induction time points; Figure D shows the blood glucose statistics of mice with different genotypes at different induction time points; Figure E shows the plasma ALT statistics of mice with different genotypes at different induction time points; Figure F shows the plasma AST statistics of mice with different genotypes at different induction time points; and Figure G shows the H&E staining of liver sections from mice with different genotypes at different induction time points.

[0093] Figure 4 This is a graph analyzing the impact of FBXO22 deficiency on NASH progression. Figure A shows Sirius red staining and quantitative analysis of liver sections from mice with different genotypes at different induction time points; Figure B shows Oil Red staining of liver sections from mice with different genotypes at different induction time points; and Figure C shows Ki67 staining and statistical analysis of liver sections from mice with different genotypes at different induction time points.

[0094] Figure 5The graph shows the effects of FBXO22 on liver inflammation in mice. Figure A shows the statistical graph of IL-6, TNF-α and STAT3 expression levels in the livers of mice with different genotypes at different induction time points detected by q-PCR; Figure B shows the p-STAT3 staining and statistical graph of liver sections of mice with different genotypes at different induction time points; Figure C shows the p-p65 staining and statistical graph of liver sections of mice with different genotypes at different induction time points.

[0095] Figure 6 The effects of FBXO22 on lipid accumulation in cells are shown in Figure A, which is a mass spectrometry result of lipids in the livers of mice of different genotypes induced by normal feeding and FFC feeding for 20 weeks; Figure B is a statistical graph showing the effect of altered FBXO22 expression on lipid accumulation in normal mouse hepatocytes.

[0096] Figure 7 Figure 1 shows the mechanism by which FBXO22 promotes lipid accumulation by degrading OPA1 through ubiquitination. Figure A is the secondary mass spectrometry of OPA1. Figure B verifies that FBXO22 plays a negative regulatory role in OPA1 expression in two liver cancer cell lines. Figure C shows the results of treatment with the protease inhibitor MG132, which blocks the regulatory effect of FBXO22 on OPA1. Figure D shows the results of treatment with the protein synthesis inhibitor CHX, which accelerates the degradation of OPA1. Figure E shows the results of overexpression of FBXO22 promoting OPA1 ubiquitination. Figure F is a schematic diagram showing that simultaneous knockdown of FBXO22 and OPA1 in normal mouse hepatocytes can reverse the lipid reduction caused by knockdown of OPA1 alone. Detailed Implementation

[0097] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0098] Example 1: Study on the expression level of FBXO22 in NASH samples

[0099] 1. Experimental Methods

[0100] (1) Search for and download the GSE89632 dataset in the GEO database. This dataset contains 19 NASH patient samples and 24 normal healthy control samples.

[0101] (2) NASH was induced in wild-type mice C75 using FFC diet, and the correlation between FBXO22 expression and NASH score was analyzed.

[0102] (3) NASH was induced in wild-type C57 mice using an FFC diet. After inducing wild-type mice for 20, 26, and 28 weeks, mouse livers were harvested, proteins were extracted, and Western blot analysis was performed. Liver tissue sections from mice fed a normal diet and FFC mice were stained with H&E and Sirius red.

[0103] 2. Experimental Results

[0104] (1) After analyzing the expression level of FBXO22 in the samples, the results showed that, compared with the healthy control samples, the NASH samples showed significantly higher expression of FBXO22. Figure 1 A) suggests that FBXO22 may play an important role in NASH.

[0105] (2) Western blot analysis revealed that compared with mice fed a normal diet, the expression of FBXO22 in the liver of FFC-fed mice was significantly increased. Figure 1 B). After inducing NASH in 30 wild-type mice, the expression of FBXO22 in the liver and the NASH score were analyzed. The results showed that FBXO22 has significant diagnostic efficacy for NASH. Figure 1 C).

[0106] (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 revealed varying degrees of fibrosis in the livers of mice fed FFC. Figure 1 E).

[0107] The above experimental results show that the FFC-induced NASH model has been successfully established.

[0108] Note: * (p < 0.05), ** (p < 0.01), *** (p < 0.001), **** (p < 0.0001)

[0109] Example 2: Establishment of a liver-specific knockout FBXO22 mouse strain and analysis of liver development and function.

[0110] 1. Experimental Methods

[0111] (1) The Cre / LoxP system was introduced to construct a conditional knockout FXBO22 mouse strain ( Fbxo22 fl / fl The resulting mice are then crossed with liver-specific recombinase Alb-Cre transgenic mice to obtain liver-specific knockout FBXO22 mice. Fbxo22 fl / fl Alb-cre )strain( Figure 2A).

[0112] (2) Extract rat tail DNA and identify the genotype by PCR. The PCR primers are shown in Table 1, the PCR amplification reaction system is shown in Table 2, and the PCR program for the FBXO22 gene is shown in Table 3.

[0113] Table 1 PCR amplification primers

[0114]

[0115] Table 2 PCR amplification reaction system for mouse genotype identification

[0116]

[0117] Table 3 PCR program for the FBXO22 gene

[0118]

[0119] (3) Verification at the mRNA level using qRT-PCR experiments Fbxo22 - / - The knockout efficiency of FBXO22 in mouse liver was determined, and the knockout effect of FBXO22 was verified at the protein level by Western blotting experiments.

[0120] (4) After collecting adult mice of the three genotypes that were fed normal feed, the overall appearance of the mice of the three genotypes was observed. Next, the mice of the three genotypes were weighed, and then the ALT in the serum of the mice of the three genotypes was detected. After collecting the livers of the mice, the size, shape and color of the livers of the mice of the three genotypes were observed and the livers were weighed.

[0121] (5) After paraffin sectioning the liver, H&E staining was performed.

[0122] (6) Genotype 1 was analyzed at the mRNA level using qRT-PCR. Fbxo22 + / + and Fbxo22 - / - The expression of FBXO22, GATA4, HNF1α, HNF4α, Cyp2e, and ATF1 in mice was detected.

[0123] 2. Experimental Results

[0124] (1) Identification was performed based on DNA extracted from the rat tail. Figure 2 B) can divide mice into the following three genotypes: wild-type ( Fbxo22 + / + Alb-Cre abbreviation Fbxo22 + / + ,+ / +), heterozygote ( Fbxo22 + / fl Alb-Cre abbreviation Fbxo22 + / - + / -), homozygous ( Fbxo22 fl / fl Alb-Cre abbreviation Fbxo22 - / - Mice of different genotypes born in the same litter were selected as a group of model mice for subsequent modeling and related experiments.

[0125] (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 expression levels among the three genotypes in the spleen and kidneys.

[0126] (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 mice of the three genotypes. Figure 2 E). Meanwhile, the body weights of the three genotype mice ( Figure 2 F) There was no significant difference in liver weight. Figure 2 H). After detecting ALT in the serum of mice of three different genotypes, no significant difference in ALT levels was observed among the different genotypes. Figure 2 G). After collecting mouse livers, it was observed that there were no significant abnormalities or differences in the size, shape, or color of the livers from the three genotypes of mice. Figure 3 I).

[0127] (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).

[0128] (5) Using qRT-PCR at the mRNA level, the genotype was analyzed. Fbxo22 + / + and Fbxo22 - / - The expression of liver development-related genes in mice was detected. It was found that after FBXO22 knockout, there was no significant difference in the expression of GATA4, HNF1α, HNF4α, Cyp2e, and ATF1, which are related to liver development. Figure 2 K).

[0129] The above results indicate that liver-specific knockout of FBXO22 does not affect liver development and function in mice.

[0130] Note: * (p < 0.05), ** (p < 0.01), *** (p < 0.001), **** (p < 0.0001)

[0131] Example 3: Liver-specific knockout of FBXO22 slows down FFC-induced NASH progression in mice

[0132] 1. Experimental Methods

[0133] (1) Mice were fed FFC diet at 8 weeks of age. The mouse models were collected at 16, 20, 24 and 28 weeks of induction feeding. The feed and bedding in the mouse cage were emptied in advance, and the model mice were starved for more than 12 hours. After isoflurane anesthesia, blood was collected from the orbital area of ​​the mice, serum was separated, and the mouse liver was completely removed.

[0134] (2) Statistical analysis was performed on the final weight, blood glucose and liver weight of mice with different genotypes at the modeling endpoint.

[0135] (3) The levels of ALT and AST in mouse serum were measured.

[0136] (4) After sectioning the livers of mice of different genotypes at the above induction time points, H&E staining was performed.

[0137] (5) Sirius red dye can bind to the basic groups in collagen molecules, thereby staining collagen fibers red. Sirius red staining was performed on liver sections of mice of different genotypes at the above induction time points.

[0138] (6) Oil red staining was performed on frozen sections of mouse liver at different induction time points, the staining was observed and the data were statistically analyzed.

[0139] (7) Immunohistochemistry was performed on liver sections of mice after 16, 20, 24 and 28 weeks of induction to detect ki67 expression.

[0140] 2. Experimental Results

[0141] (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, but by 28 weeks, Fbxo22 + / + The mice had passed through the fatty liver and hepatitis stages and entered the liver fibrosis stage; therefore, after 28 weeks of induction, Fbxo22 - / - Mouse liver Fbxo22 + / + The mice actually increased in size ( Figure 3 A).

[0142] (2) After statistical analysis of the final body weight, blood glucose, and liver weight of mice with different genotypes, it can be seen that at 16, 20, and 24 weeks of induction, Fbxo22+ / + Fasting blood glucose levels in mice were lower than those in homozygotes. Fbxo22 - / - The mice showed increased levels of final body weight and final liver weight. Fbxo22 - / - The level increased in mice; however, at 28 weeks of induction, Fbxo22 + / + As mice entered the liver fibrosis stage, their final body weight, liver weight, and fasting blood glucose were all lower than at week 24. Figure 3 BD).

[0143] (3) After measuring ALT and AST levels in mouse serum, at 16, 20, 24, and 28 weeks of induction, Fbxo22 + / + The levels of ALT and AST in mouse serum were both lower than those in mice. Fbxo22 - / - The level increased in mice, suggesting Fbxo22 + / + Mouse liver injury Fbxo22 - / - The mice were more severely affected ( Figure 3 EF).

[0144] (4) H&E staining results ( Figure 3 G) showed that after 16 weeks of induction, Fbxo22 + / + Fat vacuoles began to appear in the livers of mice at 20 weeks of induction. Fbxo22 + / + Localized ballooning degeneration began to appear in the livers of mice, indicating hepatocellular damage. By week 24, extensive hepatocellular ballooning degeneration had developed, and by week 28, the area of ​​ballooning degeneration had further increased. Fbxo22 - / - Hepatocyte ballooning degeneration only began to appear in mice at 24 weeks of age, while the area of ​​hepatocyte damage was significantly smaller at 28 weeks of age. Fbxo22 + / + Mice.

[0145] (5) Stain the liver sections of mice with different genotypes at each of the above induction time points with Sirius red ( Figure 4 A) It can be observed that at 16 weeks of induction... Fbxo22 + / + mice and Fbxo22 - / - No obvious fibrosis was observed in the livers of mice, but at 20 weeks of induction, Fbxo22 + / + Mice began to show localized fibrosis in the portal area, and at 24 weeks of induction, Fbxo22 + / + The degree of fibrosis in mice worsened at this time point. Fbxo22 - / -Localized fibrosis began to appear in the livers of mice; at 28 weeks of induction, Fbxo22 + / + The liver fibrosis in mice worsened further, and significant fibrosis was also observed in the liver parenchyma. Fbxo22 - / - The degree of fibrosis in the mouse liver also increased compared to previous time points, but was still relatively high. Fbxo22 + / + The condition improved in the mice.

[0146] (6) It can be observed at 16, 20 and 24 weeks, Fbxo22 + / + Fat deposition expression in mouse liver is stronger than Fbxo22 - / - Mice, at 28 weeks, Fbxo22 + / + When mice entered the fibrotic stage of NASH, fat deposition was somewhat alleviated, while Fbxo22 - / - The disease progressed slowly in mice, and localized fat deposition remained severe. Figure 4 B).

[0147] 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.

[0148] (7) Immunohistochemical staining results showed that at 16, 20, 24, and 28 weeks, Fbxo22 + / + Ki67 positive expression was stronger in mouse liver than Fbxo22 - / - mice ( Figure 4 C)

[0149] The above results show that Fbxo22 - / - The overall NASH course in mice was relatively long. Fbxo22 + / + The slow progression in mice suggests that liver-specific knockout of FBXO22 can slow the course of NASH in mice.

[0150] Note: * (p < 0.05), ** (p < 0.01), *** (p < 0.001), **** (p < 0.0001)

[0151] Example 4: Liver-specific knockout of FBXO22 reduces liver inflammation in mice

[0152] 1. Experimental Methods

[0153] (1) Q-PCR was used to detect the effects of induction at 16, 20, 24, and 28 weeks. Fbxo22 - / -The levels of IL-6, TNF-α, and STAT3 in mouse liver.

[0154] (2) Immunohistochemistry was performed on liver sections of mice after induction at 16, 20, 24 and 28 weeks to detect p-STAT3 and p-p65.

[0155] 2. Experimental Results

[0156] (1) Q-PCR results ( Figure 5 A) shows that at 16, 20, and 24 weeks of induction, Fbxo22 - / - The levels of IL-6, TNF-α, and STAT3 in mouse liver were relatively low. Fbxo22 + / + The levels in mice were significantly reduced.

[0157] (2) Immunohistochemical staining results showed that at 16, 20, and 24 weeks, Fbxo22 + / + In mouse livers, the positive expression of inflammatory markers p-STAT3 and p-p65 was stronger than that of p-STAT3 and p-p65. Fbxo22 - / - Mice, and at 28 weeks, Fbxo22 + / + The mice have passed the acute inflammatory phase. Fbxo22 - / - The positive expression of p-STAT3 and p-p65 in mice gradually increased ( Figure 5 BC).

[0158] The above results suggest that knocking out FBXO22 can significantly slow down the activation of inflammatory signaling pathways and the expression of pro-inflammatory factors such as cytokines in the mouse liver, thereby slowing down the progression of NASH in mice.

[0159] Example 5: FBXO22 deficiency slows down hepatic lipid accumulation.

[0160] 1. Experimental Methods

[0161] 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 hepatocytes was changed by overexpression and knockout and then stained with Oil Red. The staining was observed and the data were statistically analyzed. The siRNA sequence used for knockout is shown in Table 4.

[0162] Table 4 siRNA sequences

[0163]

[0164] 2. Experimental Results

[0165] (1) Mass spectrometry results showed that the lipid content in the liver of mice with specific FBXO22 knockout was significantly lower than that in wild-type mice. Figure 6 A).

[0166] (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).

[0167] Example 6: FBXO22 promotes lipid accumulation by ubiquitinizing and degrading OPA1.

[0168] 1. Experimental Methods

[0169] (1) Use FBXO22 combined with mass spectrometry to find proteins related to lipid metabolism.

[0170] (2) Overexpression and knockout of FBXO22 were used to detect whether FBXO22 affected the expression of the proteins found in (1) by Western Blot. The siRNAs used for knockout are shown in Table 4.

[0171] (3) Through MG132 and CHX experiments, it was verified that the regulation of this protein by FBXO22 depends on the proteasome pathway.

[0172] (4) The immunoprecipitation experiment verified that FBXO22 can promote the ubiquitination modification of the protein.

[0173] (5) Simultaneously interfere with FBXO22 and this protein in cells, and verify whether the regulation of lipids by FBXO22 depends on this protein by Oil Red staining.

[0174] 2. Experimental Results

[0175] (1) FBXO22 binding mass spectrometry results ( Figure 7 A) shows that OPA1 combines with FBXO22.

[0176] (2) Results ( Figure 7 B) shows that overexpression of FBXO22 reduces OPA1 expression, while knockout of FBXO22 increases OPA1 expression.

[0177] (3) Results ( Figure 7 C) shows that blocking the proteasome pathway with MG132 significantly weakens the regulatory effect of FBXO22 on OPA1.

[0178] (4) Results ( Figure 7 D) shows that FBXO22 can accelerate the degradation of OPA1 after cells are treated with CHX.

[0179] (5) Results ( Figure 7 E) shows that FBXO22 promotes OPA1 ubiquitination modification.

[0180] (6) Results ( Figure 7 F) shows that simultaneously knocking down FBXO22 and OPA1 can reverse the reduction in lipid accumulation caused by knocking down FBXO22 alone.

[0181] The above results suggest that knocking out FBXO22 can slow down lipid accumulation in the liver, and that FBXO22 can promote lipid accumulation by ubiquitinizing and degrading OPA1.

[0182] Note: * (p < 0.05), ** (p < 0.01), *** (p < 0.001), **** (p < 0.0001)

[0183] This experiment used GraphPad Prism 9.5 and SPSS 20.0 to perform corresponding statistical comparisons on the experimental data and generate statistical graphs after the comparisons. All statistical data are represented by mean ± standard deviation. Comparisons between groups were evaluated using independent samples t-tests, while differences between two or more groups were evaluated using one-way ANOVA. The significance level was set at α = 0.05, and a p-value < 0.05 was considered statistically significant.

[0184] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. The use of FBXO22 inhibitors in the preparation of drugs for treating non-alcoholic fatty liver disease, wherein the inhibitor is a nucleic acid inhibitor selected from siRNA, and the sequence of the siRNA is shown in SEQ ID NO:3-6.

2. The application according to claim 1, characterized in that, The non-alcoholic fatty liver disease includes non-alcoholic simple fatty liver and non-alcoholic steatohepatitis or its associated cirrhosis.

3. The application according to claim 2, characterized in that, The non-alcoholic fatty liver disease mentioned is selected from non-alcoholic steatohepatitis.

4. A method for screening candidate drugs for the treatment of non-alcoholic fatty liver disease, characterized in that, The method includes: treating a culture system expressing or containing the FBXO22 gene or its encoded protein with a screening substance; detecting the expression or activity of the FBXO22 gene or its encoded protein in the system; wherein, when the screening substance reduces the expression level or activity of the FBXO22 gene or its encoded protein, the screening substance is a candidate drug for treating non-alcoholic fatty liver disease.

5. The method according to claim 4, characterized in that, The non-alcoholic fatty liver disease includes non-alcoholic simple fatty liver and non-alcoholic steatohepatitis or its associated cirrhosis.

6. The method according to claim 5, characterized in that, The non-alcoholic fatty liver disease mentioned is selected from non-alcoholic steatohepatitis.

7. Application of FBXO22 in screening candidate drugs for the treatment of non-alcoholic fatty liver disease.

8. The application according to claim 7, characterized in that, The method for screening candidate drugs is as follows: a culture system expressing or containing the FBXO22 gene or its encoded protein is treated with the substance to be screened; the expression or activity of the FBXO22 gene or its encoded protein in the system is detected; wherein, when the substance to be screened reduces the expression level or activity of the FBXO22 gene or its encoded protein, the substance to be screened is a candidate drug for the treatment of non-alcoholic fatty liver disease.

9. The application according to claim 8, characterized in that, The non-alcoholic fatty liver disease includes non-alcoholic simple fatty liver and non-alcoholic steatohepatitis or its associated cirrhosis.

10. The application according to claim 9, characterized in that, The non-alcoholic fatty liver disease mentioned is selected from non-alcoholic steatohepatitis.

11. Application of reagents for detecting FBXO22 and its expression products in the preparation of products for diagnosing non-alcoholic fatty liver disease.

12. The application according to claim 11, characterized in that, The reagents include oligonucleotide probes that specifically recognize FBXO22, primers that specifically amplify FBXO22, binding agents that specifically bind to proteins encoded by FBXO22, or chips that specifically analyze FBXO22.

13. The application according to claim 12, characterized in that, The reagents were selected from primers that specifically amplify FBXO22.

14. The application according to claim 13, characterized in that, The primer sequences for specifically amplifying FBXO22 are shown in SEQ ID NO:1-2.

15. The application according to claim 11, characterized in that, The non-alcoholic fatty liver disease includes non-alcoholic simple fatty liver and non-alcoholic steatohepatitis or its associated cirrhosis.

16. The application according to claim 15, characterized in that, The non-alcoholic fatty liver disease mentioned is selected from non-alcoholic steatohepatitis.

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