Application of PFKFB3 in preparation of medicine for diagnosing, preventing or treating fatty liver

Abstract

The invention discloses application of PFKFB3 in preparation of a medicine for diagnosing, preventing or treating fatty liver. According to the invention, PFKFB3 plays an important role in diagnosis and treatment of fatty liver, especially non-alcoholic fatty liver disease and non-alcoholic steatohepatitis. Researches prove that the deficiency of PFKFB3 can lead to abnormal accumulation of liver lipid, aggravated inflammation level and aggravated fibrosis degree. The PFKFB3 can be used as a drug target for screening, diagnosing and treating the non-alcoholic fatty liver disease, and an accelerant of the PFKFB3 can be used for preparing drugs for preventing and treating the non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the use of PFKFB3 in the preparation of drugs for diagnosing, preventing or treating fatty liver. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a metabolic stress-induced liver injury disease closely related to insulin resistance (IR) and genetic susceptibility. The typical feature of NAFLD is the excessive accumulation of liver lipids. Clinically, when more than 5% of hepatocytes show fatty lesions or the proton fat fraction in magnetic resonance analysis > 5.6, it can be defined as NAFLD. NAFLD is a progressive disease, and its disease process includes: liver steatosis, non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma (HCC) (Figure 1-1). Since 2000, the incidence of hepatocellular carcinoma in NAFLD patients has increased significantly worldwide. In the United States, NASH has become the second leading cause of liver transplantation for end-stage liver disease. Currently, there are more than 240 million NAFLD patients in China, accounting for more than one-fifth of the global NAFLD patients, and it has been regarded as a major public health problem threatening people's health. So far, there is no specific drug for NAFLD. Existing drugs only perform relevant hypoglycemic or lipid-lowering treatments for NAFLD, and new drugs with the effect of preventing and / or treating fatty liver are still to be discovered.

[0003] The 6-phosphofructo-2-kinase / fructose 2,6-bisphosphatases (PFK-2 / FBPase-2) family is an important enzyme in the process of glycolysis. On the one hand, it can exert PFK-2 activity and catalyze fructose 6-phosphate to produce fructose 2,6-bisphosphate. On the other hand, it can also exert FBPase-2 activity and hydrolyze fructose 2,6-bisphosphate to fructose 6-phosphate. It has 4 isozymes (PFKFB1-4), which are encoded by 4 different genes and located on different chromosomes, among which PFKFB3 is located on chromosome 10. The research on PFKFB3 in tumors is relatively extensive and has been reported in most tumors, such as breast cancer, cervical cancer, pancreatic cancer, colorectal cancer, lung cancer, kidney cancer, glioma, oral cancer, bladder cancer, etc. The research on PFKFB3 related to diabetes shows that overexpression of PFKFB3 in adipocytes can significantly improve hepatic insulin resistance. PFKFB3 can prevent nutrient overload-induced adipose tissue and systemic insulin resistance, and suggests that selective activation of PFKFB3 is feasible for the treatment and / or prevention of insulin resistance and type 2 diabetes. Summary of the Invention

[0004] The object of the present invention is to provide an application of PFKFB3 in the preparation of drugs for diagnosing, preventing or treating fatty liver, so as to provide a more effective method for the diagnosis, prevention and treatment of non-alcoholic fatty liver disease.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] In the first aspect, the present invention provides an application of PFKFB3 in the preparation of drugs for diagnosing, preventing or treating fatty liver.

[0007] In the second aspect, the present invention provides a composition for diagnosing fatty liver, and the composition contains a probe for detecting the expression level of PFKFB3 in a biological sample obtained from a patient suspected of having fatty liver.

[0008] Furthermore, detecting the expression level of PFKFB3 in a biological sample obtained from a patient suspected of having fatty liver includes detecting the PFKFB3 protein level or detecting the PFKFB3 mRNA level, and the probe includes an antibody against the PFKFB3 protein or a nucleic acid sequence that binds to the PFKFB3 mRNA.

[0009] In the third aspect, the present invention provides a composition for preventing or treating fatty liver, and the composition contains an agent that can up-regulate the PFKFB3 protein level.

[0010] Furthermore, the agent capable of upregulating the PFKFB3 protein level comprises an agent capable of upregulating the expression level of the PFKFB3 protein or PFKFB3 mRNA.

[0011] Preferably, the composition comprises the PFKFB3 protein or a vector containing the PFKFB3 protein.

[0012] In a fourth aspect, the present invention provides a method for screening a drug for preventing or treating fatty liver, the method comprising contacting a cell capable of expressing the PFKFB3 protein with a candidate material and detecting the effect of the candidate material on the level of the expressed PFKFB3 protein.

[0013] Furthermore, when the level of the expressed PFKFB3 protein is upregulated, the candidate material is determined to be a drug for preventing or treating fatty liver.

[0014] Even further, in the above-mentioned use, composition or method, the fatty liver is non-alcoholic fatty liver disease.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention discloses the function and application of the PFKFB3 gene in fatty liver diseases. According to the present invention, PFKFB3 plays an important role in the diagnosis and treatment of fatty liver, especially non-alcoholic fatty liver disease and non-alcoholic steatohepatitis. Research has confirmed that the deletion of PFKFB3 can lead to abnormal accumulation of liver lipids, increased inflammation levels, and aggravated fibrosis. PFKFB3 can be used as a drug target for screening the diagnosis and treatment of non-alcoholic fatty liver disease, and its promoter can be used to prepare drugs for preventing and treating non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 . indicates low expression of PFKFB3 in NAFLD;

[0017] Figure 2 . indicates low expression of PFKFB3 in the NAFLD mouse model;

[0018] Figure 3 . indicates low expression of PFKFB3 in the cell model of NAFLD;

[0019] Figure 4 . indicates the expression verification after downregulation and upregulation of PFKFB3;

[0020] Figure 5 . indicates that downregulation of PFKFB3 promotes lipid accumulation, and upregulation of PFKFB3 inhibits lipid accumulation;

[0021] Figure 6 . indicates that downregulation of PFKFB3 inhibits the expression of fatty acid oxidation factors and promotes an increase in the level of inflammatory factors;

[0022] Figure 7 . It indicates that the deletion of PFKFB3 inhibits the level of fatty acid oxidation;

[0023] Figure 8 . It indicates that the deletion of PFKFB3 promotes the pathogenesis of NAFLD;

[0024] Figure 9 . It indicates that the deletion of PFKFB3 promotes liver inflammation and fibrosis;

[0025] Figure 10 . It indicates that the deletion of PFKFB3 inhibits the expression of fatty acid oxidation factors;

[0026] Figure 11 . It indicates the endogenous verification of the binding of PFKFB3 to OGDH;

[0027] Figure 12 . It indicates the exogenous verification of the binding of PFKFB3 to OGDH;

[0028] Figure 13 . It indicates that the deletion of PFKFB3 inhibits the enzyme activity of OGDH and does not change the gene and protein expression levels of OGDH;

[0029] Figure 14 . It indicates that PFKFB3 may be used as a diagnostic marker for NAFLD. Detailed implementation manners

[0030] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0031] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "upper", "lower", "inner", "outer", "front end", "rear end", "both ends", "one end", "the other end", etc. is based on the orientation or positional relationship shown in the accompanying drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance.

[0032] The present invention studies the relationship between PFKFB3 expression and the progression of non-alcoholic fatty liver disease. Based on the research results, it provides a new use of PFKFB3 as a target gene for the treatment of non-alcoholic fatty liver disease, and further applies the PFKFB3 gene to the diagnosis and treatment of non-alcoholic fatty liver disease.

[0033] The present invention first selects clinical samples and detects the correlation between the expression level of PFKFB3 and NAFLD in animal models and cell models, and finds that the expression level of PFKFB3 in the NAFLD group is significantly lower than that in the control group. We further down-regulate the expression of PFKFB3 by gene interference and find that after the down-regulation of PFKFB3, cell lipid accumulation is severe, the inflammatory level is increased, and fatty acid oxidation is reduced; while after the over-expression of PFKFB3, cell lipid accumulation is reduced and fatty acid oxidation is increased. A mouse with liver-specific knockout of PFKFB3 is constructed, and an animal model is constructed by high-fat diet (HFD). It is found that after the knockout of PFKFB3, the liver lipid accumulation of the mouse is severe, the inflammation is aggravated, the fibrosis is aggravated, and fatty acid oxidation is reduced. We identify a PFKFB3-binding protein by mass spectrometry and find that PFKFB3 can bind to oxoglutarate dehydrogenase (OGDH) through co-immunoprecipitation verification. OGDH is an important gene in the process of fatty acid oxidation. Both at the cellular level and the animal level, it is confirmed that when lipid overload occurs, the reduction of PFKFB3 level can inhibit OGDH enzyme activity. PFKFB3 can affect fatty acid oxidation by binding to OGDH.

[0034] The research of the present inventors has confirmed that there is a significant negative correlation between PFKFB3 and the pathogenesis of NAFLD, and PFKFB3 has the effects of inhibiting liver lipid accumulation, alleviating inflammation, and protecting liver function.

[0035] For the above functions, the present invention provides a composition for diagnosing non-alcoholic fatty liver disease, and the composition contains a probe for measuring the level of PFKFB3 protein from a sample suspected of having fatty liver.

[0036] The present invention also provides a composition for preventing and treating non-alcoholic fatty liver disease, and the composition contains the protein of PFKFB3 or a vector containing the same.

[0037] The present invention also provides a method for screening for preventing and treating non-alcoholic fatty liver disease, and the method includes: contacting a cell containing PFKFB3 protein with a candidate material in vitro; and the change in the expression level of the gene or protein caused by the candidate material. When the level of PFKFB3 protein is up-regulated, the candidate material is determined to be a drug for preventing or treating fatty liver.

[0038] The present invention discovers the correlation between PFKFB3 and the pathogenesis of NAFLD, as well as the effect of PFKFB3 in inhibiting fatty liver. Based on the above functions, it can be used to prepare a kit or drug for diagnosing, preventing, and / or treating non-alcoholic fatty liver disease.

[0039] The present invention will be further illustrated with specific embodiments below.

[0040] Example 1: Verification of the correlation between PFKFB3 and NAFLD

[0041] Collect a part of liver tissue from patients with non-alcoholic fatty liver disease and a non-fatty liver (normal) control group. Extract proteins with RIPA lysis buffer (containing protease inhibitors and PMSF). Embed a part of the liver in paraffin to make paraffin blocks, then cut into sections, and perform HE staining and immunohistochemical analysis using a hematoxylin and eosin staining kit. After adjusting the proteins extracted from each sample to the same concentration, perform SDS-PAGE gel electrophoresis separation (electrophoresis conditions: 80V, constant voltage for 20 minutes, 120V, constant voltage for 60 minutes) and membrane transfer (membrane transfer conditions: 250mA, constant current for 80 minutes). Transfer the proteins to a PVDF membrane, block the PVDF membrane (blocking conditions: incubate with TBST solution containing 5% skim milk powder at room temperature for 2 hours), incubate the PVDF membrane with the primary antibody PFKFB3 antibody (Abcam, catalog number: ab181861, antibody dilution ratio 1:1000) at 4°C for 16 hours. Place the PVDF membrane on a horizontal shaker at room temperature and wash it with TBST buffer (rotation speed 80rpm) 3 times, 10 minutes each time. After washing, incubate with the secondary antibody (Abcam, catalog number: ab205718, antibody dilution ratio 1:10000) for 2 hours at room temperature, then repeat the above washing process. Finally, perform chemiluminescent color development with a chromogenic solution. After gray-scale analysis of the developed pictures, perform statistical analysis and find that the expression level of PFKFB3 in the NAFLD group is significantly lower than that in the normal control group. The results are shown in Figure 1 As Figure 1 Shown in A - B, the expression level of PFKFB3 in the NAFLD group is significantly lower than that in the control group, indicating that PFKFB3 is significantly down-regulated in NAFLD. Figure 1 C shows the immunohistochemical results, which also show the low expression of PFKFB3 in the NAFLD group. At the same time, analyze the relationship between the expression level of PFKFB3 protein and clinical physiological and biochemical indicators. As shown in Figure 1 D - F, it is found that the expression level of PFKFB3 is significantly negatively correlated with liver function indicators such as ALT and AST, and triglyceride.

[0042] After feeding 8-week-old male C57BL / 6 mice with a normal diet (ND) and a high-fat diet (HFD) for 16 weeks respectively to construct a NAFLD animal model, and treating Huh7 cells with 1 mmol / L free fatty acid (FFA, containing oleic acid: palmitic acid = 2:1) to construct a cytological model of NAFLD. RNA of each group of samples was extracted by an RNA extraction kit, and the change of PFKFB3 mRNA was detected by a reverse transcription kit and a fluorescence quantitative PCR kit (using the β-actin gene as an internal reference). The change of PFKFB3 protein was found by extracting proteins for Western blotting, and the change of PFKFB3 content was detected by HE staining and immunohistochemical staining of the liver. The results showed that the expression of PFKFB3 decreased significantly in the NAFLD group.

[0043]

[0044]

[0045] As Figure 2 shown, PFKFB3 was lowly expressed in the NAFLD mouse model. Figure 2 A, B, C, G were diet-induced NAFLD models, among which Figure 2 A showed that the mRNA of PFKFB3 in the HFD group was significantly lower than that in the control group. Figure 2 B - C showed that the protein of PFKFB3 in the HFD group was significantly lower than that in the control group. Figure 2 G further showed by immunohistochemical results that PFKFB3 was lowly expressed in the HFD group. Figure 2 D, E, F, H were obese mouse models, among which Figure 2 D showed that the mRNA of PFKFB3 in the ob / ob group was significantly lower than that in the control group. Figure 2 B - C showed that the protein of PFKFB3 in the ob / ob group was significantly lower than that in the control group. Figure 2 G further showed by immunohistochemical results that PFKFB3 was lowly expressed in the ob / ob group.

[0046] Figure 3 showed that PFKFB3 was lowly expressed in the NAFLD cell model. As Figure 3 seen in A, the mRNA of PFKFB3 decreased with the increase of FFA concentration. At the same time, as Figure 3 seen in B - C, the protein of PFKFB3 decreased with the increase of FFA concentration.

[0047] Example 2: Verify the effect of the change in PFKFB3 expression level on NAFLD

[0048] We first down-regulated and up-regulated the PFKFB3 gene respectively and verified them, and then observed the effects of changes in PFKFB3 expression levels on NAFLD. First, PFKFB3 was interfered by transfecting small interfering RNA of PFKFB3, and the small interfering sequence was: GTTGTGGCCTCCAATATCA. Transfection was carried out according to the instructions of Lipofectamine 2000; by constructing a lentiviral vector for overexpression of PFKFB3 and transfecting it according to the operating instructions of the lentiviral transfection kit, PFKFB3 was overexpressed. The gene level and protein level of PFKFB3 were verified by fluorescence quantitative PCR and Western blot respectively to determine whether they were effectively down-regulated and up-regulated. Figure 4 A-C indicate that the small interfering sequence of PFKFB3 can down-regulate the expression of PFKFB3. Figure 4 D-F indicate that the lentiviral vector for overexpression of PFKFB3 can up-regulate the expression of PFKFB3. On the basis of down-regulating or up-regulating PFKFB3, the effect diagram of PFKFB3 on lipid accumulation was observed under lipid overload in Huh7 cells by stimulating with FFA respectively. Figure 4 It shows that PFKFB3 affects lipid accumulation and inflammation by regulating fatty acid oxidation (*p<0.05, **p<0.01, NC-BSA vs NC-FFA; #p<0.05, ##p<0.01 NC-FFA vs si-PFKFB3-FFA or NC-FFA vs OE-PFKFB3-FFA, NC-BSA represents adding a fatty acid-free BSA control solution after the cells are transfected with the negative control sequence, si-PFKFB3-BSA represents adding a fatty acid-free BSA control solution after the cells are transfected with the small interfering sequence of PFKFB3, NC-FFA represents adding FFA after the cells are transfected with the negative control sequence, si-PFKFB3-FFA represents adding FFA after the cells are transfected with the small interfering sequence of PFKFB3, vec-BSA represents adding a fatty acid-free BSA control solution after transfection with the lentiviral empty vector, OE-PFKFB3-BSA represents adding a fatty acid-free BSA control solution after transfection with the lentiviral vector for overexpression of PFKFB3, vec-FFA represents adding FFA after transfection with the lentiviral empty vector, OE-PFKFB3-FFA represents adding FFA after transfection with the lentiviral vector for overexpression of PFKFB3). The detection results of intracellular TG content showed that when treated with FFA, the intracellular TG content increased significantly with the down-regulation of PFKFB3 ( Figure 5 Results of TG content determination under different treatment factors when PFKFB3 was down-regulated), and decreased significantly with the up-regulation of PFKFB3 ( Figure 5C Measurement results of TG content under different treatment factors when PFKFB3 is upregulated); Oil Red O staining results also showed that when treated with FFA, downregulating PFKFB3 could promote lipid accumulation( Figure 5 B Oil Red O staining results under different treatment factors when PFKFB3 is downregulated), upregulation of PFKFB3 could inhibit lipid accumulation( Figure 5 D Oil Red O staining results under different treatment factors when PFKFB3 is upregulated). Further, related markers of lipid metabolism were detected by qPCR. The results showed that after FFA treatment, downregulation of PFKFB3 significantly decreased the levels of PGC1α, ACOX1, and CPT1α( Figure 6 A Changes in markers of fatty acid oxidation process under different treatment factors when PFKFB3 is downregulated detected by qPCR), and the levels of SREBP1c, FASN, SCD1, FATP1, APOA4, and MTTP did not change significantly( Figure 6 D Changes in markers of lipid generation process under different treatment factors when PFKFB3 is downregulated detected by qPCR, Figure 6 F Changes in markers of fatty acid intake process under different treatment factors when PFKFB3 is downregulated detected by qPCR, Figure 6 H Changes in markers of lipid transport process under different treatment factors when PFKFB3 is downregulated detected by qPCR); upregulation of PFKFB3 significantly increased the levels of PGC1α, ACOX1, and CPT1α( Figure 6 B Changes in markers of fatty acid oxidation process under different treatment factors when PFKFB3 is upregulated detected by qPCR), and the levels of SREBP1c, FASN, SCD1, CD36, FATP1, APOA4, and MTTP did not change significantly( Figure 6 E Changes in markers of lipid generation process under different treatment factors when PFKFB3 is downregulated detected by qPCR, Figure 6 J Changes in markers of fatty acid intake process under different treatment factors when PFKFB3 is upregulated detected by qPCR, Figure 6 I Changes in markers of lipid transport process under different treatment factors when PFKFB3 is upregulated detected by qPCR). We also detected the changes in inflammation-related molecular markers. The results showed that after FFA treatment, downregulation of PFKFB3 significantly increased the levels of the inflammatory molecular markers IL1β, TNFα, and IL6( Figure 6C qPCR was used to detect the changes in inflammatory factors when PFKFB3 was downregulated). Since fatty acid β-oxidation mainly occurs in mitochondria and is significantly related to mitochondrial function and mitochondrial respiration, we detected the changes in mitochondrial respiration through Seahorse experiments and found that when treated with FFA, downregulation of PFKFB3 could significantly inhibit the maximal respiratory capacity of mitochondria, and the basal respiratory capacity, proton leak and ATP production also decreased, but not significantly ([ Figure 7 A OCR measurement under different treatment factors when PFKFB3 was downregulated was detected by Seahorse experiment, Figure 7 B Statistical analysis at different stages of the Seahorse experiment), indicating that PFKFB3 can indirectly affect fatty acid oxidation by influencing mitochondrial respiration. We further detected the direct effect of PFKFB3 on fatty acid oxidation through palmitic acid oxidation stress test, and the results showed that when treated with FFA, downregulation of PFKFB3 could significantly inhibit fatty acid oxidation ([ Figure 7 C OCR measurement under different treatment factors when PFKFB3 was downregulated was detected by palmitic acid oxidation stress test, Figure 7 D Effect of PFKFB3 downregulation on fatty acid oxidation).

[0049] Example 3 Verification of the effect of PFKFB3 knockout on NAFLD

[0050] We fed Pfkfb3 liver-specific knockout (Liver specific knockout, LKO) mice and wild-type (Wild type, WT) mice with HFD to observe the changes in lipid metabolism. Wild-type and liver-specific Pfkfb3 knockout mice were fed with HFD for 16 weeks. *, #P < 0.05, **, ##P < 0.01. * represents comparison of this group with the wild-type mice group on normal diet; # represents comparison of this group with the wild-type mice group on high-fat diet. Figure 8 It was shown that the body weight of LKO mice fed with HFD was higher than that of WT mice, and there was a significant difference starting from the 13th week of HFD feeding ( Figure 8 A Body weight curve graph), and the liver weight of mice fed with HFD in the LKO group was significantly higher than that in the WT group ( Figure 8 B Liver weight after 16 weeks), and the measurement result of liver TG content of mice fed with HFD showed that the LKO group was significantly higher than the WT group ( Figure 8 C Liver triglyceride level). The results of H&E staining showed that the fatty lesions in the liver of LKO mice fed with HFD were more obvious than those in the WT group, and the results of Oil Red O staining showed that the lipid deposition in the liver of LKO mice fed with HFD was more severe than that in the WT group ( Figure 8 D H&E and Oil Red O staining). The detection results of blood biochemical indexes of mice showed that the levels of serum ALT, AST, and TG in the LKO group fed with HFD were significantly higher than those in the WT group ([Figure 8 E-G serum TG, ALT, and AST levels). qPCR detection results showed that the levels of inflammatory factors Il1β, Il6, and Tnfα in the livers of LKO mice fed with HFD were significantly lower than those in the WT group ( Figure 9 A Pro-inflammatory factor mRNA levels), and the levels of fibrosis molecular markers α-SMA, TGFβ, and Col1a1 were significantly reduced ( Figure 9 C Fibrosis marker mRNA levels). F4 / 80 staining and Sirius red staining results showed that the degree of liver fibrosis in the LKO group of mice fed with HFD was more severe than that in the WT group ( Figure 9 B F4 / 80 staining, Figure 9 D Sirius red staining). Figure 10 It was shown that the deletion of PFKFB3 inhibited the level of fatty acid oxidation. qPCR detection results found that the levels of fatty acid oxidation molecular markers Pgc1α, Acox1, and Cpt1α in the livers of LKO mice fed with HFD were significantly lower than those in the WT group, and other lipid metabolism-related molecular markers Srebp1c, Fasn, Scd1, Cd36, Fatp1, Apoa4, and Mttp did not show significant changes.

[0051] Example 4: Verify that the binding of PFKFB3 to OGDH causes a decrease in OGDH enzyme activity

[0052] As Figure 11 shown, the enzyme activity of OGDH decreased after the binding of PFKFB3 to OGDH and the decrease of PFKFB3. We were able to detect the expression of OGDH in the immunoprecipitation products of the BSA treatment group and the FFA treatment group after overexpression of PFKFB3 in Huh7 cells ( Figure 11 A Immunoprecipitation to verify the binding of PFKFB3 to OGDH), and the expression of PFKFB3 was detected in the immunoprecipitation products of OGDH ( Figure 11 B Immunoprecipitation to verify that OGDH can bind to PFKFB3). When overexpressing PFKFB3 in Huh7 cells, it was found that the expression of OGDH protein did not change ( Figure 11 C Overexpression of PFKFB3 does not affect the change of OGDH protein). When overexpressing OGDH and PFKFB3 in 293T cells respectively, it was detected later that PFKFB3 and OGDH could bind to each other ( Figure 12 A Exogenous verification that PFKFB3 can bind to OGDH, Figure 12 B Exogenous OGDH can bind to PFKFB3). In gene knockout mice, it was found that neither the mRNA nor the protein of OGDH changed significantly after the knockout of PFKFB3 ( Figure 13 A At the animal level, the knockout of PFKFB3 does not affect the change of OGDH mRNA,Figure 13 B Protein alteration of OGDH is not affected by PFKFB3 knockout at the animal level). Further research found that after FFA treatment, the enzyme activity of OGDH decreased after the downregulation of the PFKFB3 gene in Huh7 cells ( Figure 13 C At the cellular level, after the downregulation of PFKFB3, FFA treatment can reduce the enzyme activity of OGDH); after HFD treatment, the enzyme activity of OGDH decreased in the liver of PFKFB3 knockout mice ( Figure 13 D At the animal level, after PFKFB3 knockout, HFD feeding can reduce the enzyme activity of OGDH).

[0053] Example 5: PFKFB3 can be used as a diagnostic indicator for patients with fatty liver.

[0054] Refer to the method of Example 1 to collect the livers of patients with non-alcoholic fatty liver and the livers of normal control groups without fatty liver, detect the expression level of PFKFB3 by immunoblotting, and construct a receiver operating characteristic curve (ROC) for fatty liver based on the expression level of PFKFB3, Figure 14 The area under the ROC curve (AUC) value was shown to be 0.955, indicating that PFKFB3 has a very high diagnostic value for non-alcoholic fatty liver. Based on the above results, PFKFB3 can be used as a target for screening drugs for the prevention or treatment of fatty liver. If the candidate material can increase the expression of PFKFB3, it may be used for the prevention or treatment of fatty liver. If the candidate material cannot increase the expression of PFKFB3, the candidate material cannot be used for the prevention or treatment of fatty liver.

[0055] Details not described in this invention are all well-known techniques to those skilled in the art.

[0056] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified and equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. Application of PFKFB3 in the preparation of drugs for diagnosis, prevention or treatment of fatty liver.

2. A composition for diagnosing fatty liver, characterized in that: The composition comprises a probe for detecting the expression level of PFKFB3 in a biological sample obtained from a patient suspected of having fatty liver.

3. The composition for diagnosing fatty liver according to claim 2, characterized in that: Detecting the expression level of PFKFB3 in a biological sample obtained from a patient suspected of having fatty liver includes detecting the level of PFKFB3 protein or detecting the level of PFKFB3 mRNA, and the probe includes an antibody against PFKFB3 protein or a nucleic acid sequence that binds to PFKFB3 mRNA.

4. A composition for preventing or treating fatty liver, characterized in that: The composition comprises an agent capable of upregulating PFKFB3 protein levels.

5. The composition for preventing or treating fatty liver according to claim 4, characterized in that: The agent capable of upregulating the level of PFKFB3 protein includes an agent capable of upregulating the expression level of PFKFB3 protein or PFKFB3 mRNA.

6. The composition for preventing or treating fatty liver according to claim 4, characterized in that: The composition comprises PFKFB3 protein or a vector comprising PFKFB3 protein.

7. A method for screening drugs for preventing or treating fatty liver, characterized in that: The method comprises contacting a cell capable of expressing PFKFB3 protein with a candidate material, and detecting the effect of the candidate material on the level of the expressed PFKFB3 protein.

8. The method for screening a drug for preventing or treating fatty liver according to claim 7, characterized in that: When the level of the expressed PFKFB3 protein is upregulated, the candidate material is determined to be a drug for preventing or treating fatty liver.

9. The use, composition or method according to any one of claims 1 to 8, characterized in that The fatty liver is non-alcoholic fatty liver disease.

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