Use of ubiquitin ligase TRIM56

Abstract

The present application discloses the use of ubiquitin ligase TRIM56 in the preparation of a product for preventing, alleviating and / or treating non-alcoholic fatty liver disease and related diseases, reducing blood lipid or reducing body fat accumulation, and the product is a product for overexpressing TRIM56 protein in cells or tissues or a product for increasing the activity of TRIM56 protein in cells or tissues. Overexpression of the TRIM56 gene can significantly inhibit liver lipid deposition, inhibit the occurrence and development of non-alcoholic fatty liver disease, and has the effect of reducing blood lipid or reducing body fat accumulation, thus finding a new target for the preparation of drugs for preventing, alleviating and / or treating non-alcoholic fatty liver disease and related diseases, reducing blood lipid or reducing body fat accumulation.

Description

Technical Field

[0001] This application belongs to the field of bioengineering and relates to the application of the ubiquitin ligase TRIM56 in the preparation of products for preventing, alleviating and / or treating non-alcoholic fatty liver disease and related diseases, reducing blood lipid or reducing body fat accumulation. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is an increasingly serious health problem with a global incidence rate as high as 25%. Although with the development of human social economy and the improvement of medical level, the incidence rates of various chronic liver diseases basically remain stable or even decline, the incidence rate of NAFLD still shows a rapid upward trend and has become the primary type of chronic liver disease worldwide. NAFLD refers to the liver fat metabolism disorder of patients caused by factors other than alcohol and other clear liver-damaging factors, and is a clinicopathological syndrome mainly characterized by diffuse macrovesicular steatosis of hepatocytes. Its disease scope is wide, including the simplest steatosis, or further developing into non-alcoholic steatohepatitis (NASH), post-inflammatory reparative liver fibrosis, and even progressing to liver cirrhosis and even liver cancer (HCC) in the end. Epidemiological studies have found that the incidence rate of NAFLD in the general population reaches 25%-30%, among which, 10%-20% of patients can develop into NASH mainly characterized by inflammation and metabolic dysfunction. And among NASH patients, 26%-37% of patients will progress to liver fibrosis within the next 6 years, and 9% will progress to liver cirrhosis. Even, through long-term follow-up, it is found that: 0%-2.8% of NASH patients' conditions can further deteriorate and develop into liver cancer. At the same time, the latest epidemiology also shows that the influence of NASH as a risk factor for primary hepatocellular carcinoma (HCC) is showing an obvious upward trend. Therefore, considering that NAFLD has the characteristics of high incidence rate, many complications, and easy deterioration into a series of end-stage liver diseases with high mortality, NAFLD has become a major public health problem concerning the physical health and social stability of humans all over the world, and the exploration of its effective prevention methods and treatment means is urgent.

[0003] TRIM56 (tripartite motif containing 56) is one of the members of the TRIM family, containing a RING domain, a B-box domain and a coiled-coil domain. As a member of the TRIM family that mostly functions as an E3 ubiquitin enzyme, the conserved RING domain of TRIM56 endows it with E3 ubiquitin ligase activity. In previously reported studies, TRIM56 mainly plays a role in antiviral innate immunity through its E3 ubiquitin ligase activity, and there is no report on the relationship between TRIM56 and the treatment of non-alcoholic fatty liver. Summary of the Invention

[0004] To make up for the deficiencies in the clinical prevention and treatment of non-alcoholic fatty liver disease, the present application provides the use of the TRIM56 gene in the preparation of products for preventing, alleviating, and / or treating non-alcoholic fatty liver disease and related diseases, reducing blood lipids, or reducing body fat accumulation.

[0005] The object of the present application is achieved by the following technical solutions:

[0006] In the in vitro simulation model of primary liver fatty liver induced by PA+OA combined stimulation and the in vivo model of mice induced by HFD high-fat diet in the present application, it is found that the lipid droplets in primary hepatocytes with overexpression of TRIM56 are small and few, and the intracellular TG content is also significantly reduced. The liver weight and the TG content in the liver of mice with overexpression of the TRIM56 gene are both significantly reduced. The pathological HE and oil red staining results also show that the lipid degeneration in the liver is alleviated and the lipid accumulation is significantly reduced. The experimental results of TRIM56 in vivo and in vitro jointly show that overexpression of the TRIM56 gene can significantly inhibit liver lipid deposition and inhibit the occurrence and development of non-alcoholic fatty liver disease.

[0007] On this basis, the present application provides the use of the TRIM56 gene in the preparation of products for preventing, alleviating, and / or treating non-alcoholic fatty liver disease and related diseases, reducing blood lipids, or reducing body fat accumulation. The products include products for overexpressing the TRIM56 protein in cells or tissues or products for increasing the activity of the TRIM56 protein in cells.

[0008] In some embodiments provided by the present application, the product for overexpressing the TRIM56 protein in cells or tissues can enter the cells, and (i) can promote the overexpression of the TRIM56 gene to express the TRIM56 protein or (ii) the product can express or overexpress the TRIM56 protein in the cells.

[0009] In some embodiments provided by the present application, the products that can express or overexpress the TRIM56 protein in cells include one or more of polynucleotide sequences, plasmids, and viruses, and can be transcribed and translated into products containing the TRIM56 protein in the body of the subject.

[0010] In some embodiments provided by the present application, the products that can express or overexpress the TRIM56 protein in cells are suitable for autonomous replication in cells.

[0011] In some embodiments provided by the present application, the products that can promote the overexpression of the TRIM56 gene to express the TRIM56 protein include one or more of proteins, proteolysis-targeting chimeras, polynucleotides, and small molecule compounds.

[0012] In some embodiments provided by the present application, the products for enhancing the activity of intracellular TRIM56 protein include one or more of agonist antibodies of TRIM56, proteins, polypeptides, enzymes, and small molecule compounds that promote the activity of TRIM56 protein.

[0013] In some embodiments provided by the present application, the products can enter cells through one or more of the following methods: direct naked DNA injection, direct injection of liposome-encapsulated DNA, bombardment with a gene gun coated with gold and DNA, method of using replication-defective bacteria carrying plasmid DNA, method of using replication-defective adenovirus carrying the target DNA, injection of PEG-modified protein drugs, intravenous injection of liposome-encapsulated protein, subcutaneous injection of protein microsphere preparation.

[0014] In some embodiments provided by the present application, the products further include bacteria that express or overexpress TRIM56 protein.

[0015] In some embodiments provided by the present application, the products are used to inhibit the increase in blood lipid, body fat accumulation, or liver lipid deposition caused by a high-fat diet.

[0016] In some embodiments provided by the present application, non-alcoholic fatty liver disease is one or more of non-alcoholic simple fatty liver, non-alcoholic steatohepatitis, liver fibrosis, and cirrhosis.

[0017] In some embodiments provided by the present application, non-alcoholic fatty liver disease and related diseases further include one or more of insulin resistance, metabolic syndrome, obesity, diabetes, hyperglycemia, hyperlipidemia, and liver cancer.

[0018] The present application has the following advantages and effects compared with the prior art:

[0019] (1) The present application discovers a new use of the TRIM56 gene. Overexpression or increased activity of the TRIM56 protein can play a role in protecting the liver and inhibiting non-alcoholic fatty liver disease.

[0020] (2) Based on the role of the TRIM56 gene in inhibiting non-alcoholic fatty liver disease, it provides a new target for developing drugs for preventing, alleviating, and / or treating non-alcoholic fatty liver disease.

[0021] (3) Based on the role of TRIM56 in inhibiting non-alcoholic fatty liver disease, it can be used to prepare drugs for preventing, alleviating, and / or treating non-alcoholic fatty liver disease. Description of the Drawings

[0022] The drawings are used to provide an understanding of the technical solutions of the present application, and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solutions of the present application, and do not constitute a limitation to the technical solutions of the present application.

[0023] Figure 1 It is the Western Blot identification result diagram after primary mouse hepatocytes were infected with AdFlag and AdTrim56 adenoviruses;

[0024] Figure 2 It is the Nile red staining result diagram after primary mouse hepatocytes were infected with AdFlag and AdTrim56 adenoviruses and stimulated with PA and OA;

[0025] Figure 3 It is the result diagram of detecting the TG content in cells after primary mouse hepatocytes were infected with AdFlag and AdTrim56 adenoviruses and stimulated with PA and OA (*: p < 0.05);

[0026] Figure 4 It is the Western Blot identification result diagram of the liver of Trim56 - HepOE mice;

[0027] Figure 5 It is the statistical chart of liver weights of GFP mice and Trim56 - HepOE mice after being fed with NC and HFD for 16 weeks (*: p < 0.05);

[0028] Figure 6 It is the result diagram of the TG content in the liver of GFP mice and Trim56 - HepOE mice after being fed with HFD for 16 weeks (*: p < 0.05);

[0029] Figure 7 It is the representative pictures of HE staining and oil red staining of liver tissue sections of GFP mice and Trim56 - HepOE mice after being fed with HFD for 16 weeks.

[0030] Figure 8 It is the map of the entry vector used to construct AdTrim56.

[0031] Figure 9 It is the map of the adenovirus vector (AdFlag). Specific implementation manners

[0032] To make the objectives, technical solutions and advantages of this application clearer and more understandable, the embodiments of this application will be described in detail below. It should be noted that, without conflict, the embodiments and features in the embodiments of this application can be combined arbitrarily with each other.

[0033] The reagents involved in the experiments in this application are purchased in domestic and foreign markets or prepared according to the formulas in the instructions; the experimental methods not specially described are all conventional methods known in the art.

[0034] Experimental animals and feeding:

[0035] Male C57BL / 6J mice aged 6 - 8 weeks were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd. (Nanjing, China) or Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All mice were housed in individually ventilated cages (IVC) with the temperature controlled at 22 - 26°C and a 12 / 12-hour light / dark cycle. The mice had free access to food and water.

[0036] Animal feed formula:

[0037] Normal chow (NC) or high-fat diet (HFD, 60 kcal% fat, 20 kcal% protein, 20 kcal% carbohydrate, D12492 high-fat diet, Research Diets, New Brunswick, USA).

[0038] Experimental cells and culture:

[0039] Primary hepatocytes were isolated from male C57BL / 6J mice at 8 - 10 weeks of age under normal feeding conditions. The human embryonic kidney cell line (HEK) 293 cell line was purchased from the National Collection of Authenticated Cell Cultures in Shanghai, China. The above cells were cultured in DMEM (15140 - 122; Thermo Fisher Scientific; Waltham, Massachusetts, USA) high-glucose medium (containing 10% FBS and 1% penicillin-streptomycin) in a 37°C constant-temperature cell incubator with 5% CO2. The culture time of the experimental cells did not exceed three months, and mycoplasma detection was performed every three months. For cell cryopreservation, fetal bovine serum (FBS) containing 10% DMSO was used as the cell cryopreservation solution.

[0040] Example 1: Construction of a cell model of primary mouse hepatocytes infected with AdTrim56 adenovirus

[0041] 1. Isolation and culture of primary mouse hepatocytes

[0042] (1) Anesthetize the mice with 1% sodium pentobarbital, disinfect the area below the neck with 75% alcohol, and expose the liver by dissection;

[0043] (2) Perfuse the mouse liver with perfusion buffer (containing 137 mM NaCl, 5.4 mM KCl, 0.5 mM NaH2PO4, 0.4 mM Na2HPO4, 4.2 mM NaHCO3, 0.5 mM ethylene glycol-bis-(2-aminoethyl ether) tetraacetic acid (EGTA), 5 mM glucose, pH = 7.4) at a rate of 5 mL / min, and then continue to perfuse with liver digestion solution containing collagenase (17701 - 034; Thermo Fisher Scientific; Waltham, Massachusetts, USA) (2.5 mL / min) until the liver swells and enlarges;

[0044] (3) Cut the liver and transfer it to a cell culture dish pre-coated with rat-tail collagen. Tear open the liver capsule; filter it through a 70-μm cell strainer (BD Falcon) to remove undigested debris and collect the filtrate.

[0045] (4) Wash the filtrate obtained in step (3) twice with ice-cold DMEM medium. Centrifuge the washed cell pellet at 200 g for 3 min to obtain primary hepatocytes.

[0046] (5) Count the viable cells using trypan blue. Then, place the isolated primary hepatocytes in a cell culture dish pre-coated with rat-tail collagen according to their number. Culture them in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics in a humidified incubator at 5% CO2 and 37 °C for more than 2 h. After the viable cells adhere to the wall, wash them 2-3 times with PBS to remove dead cells. Add an appropriate amount of DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics and continue culturing until the cells are fully spread for the next experiment.

[0047] 2. Infection of primary hepatocytes with adenovirus overexpressing TRIM56

[0048] (1) Construction of recombinant adenovirus:

[0049] Construct an expression vector AdTrim56 for TRIM56 (CCDS 19762.1). Use the LR recombination method of the adenovirus expression system Getaway system to construct an adenovirus vector AdTrim56 expressing TRIM56; an adenovirus vector expressing an empty vector (AdFlag) is used as a control. The Gateway entry vector used for constructing AdTrim56 was purchased from Invitrogen. The map of the entry vector is as Figure 8 shown, and the map of the adenovirus vector (AdFlag) is as Figure 9 shown. The information of the target gene is shown in Table 1:

[0050] Table 1 Information of the target gene TRIM56

[0051]

[0052] (1.1) Clone the target gene into the entry vector:

[0053] (1.1.1) Design and synthesize primers according to the gene transcript of the target gene and experimental needs, as shown in Table 2:

[0054] Table 2

[0055]

[0056] (1.1.2) Obtaining the target gene

[0057] Table 3

[0058]

[0059] Experimental protocol:

[0060] (1.1.3) Construction of entry plasmid

[0061] ① Preparation of linearized vector: Double digestion of the entry vector with restriction enzymes: BamHI restriction enzyme + SpeI restriction enzyme.

[0062] ② Preparation of target gene fragment: PCR amplification of the target gene fragment with primers containing homologous arms.

[0063] ③ Recombination reaction of target gene fragment and entry vector: One Step Cloning Kit (purchased from Nanjing Novoprotein Scientific Inc.), 5 μL reaction system, recombination at 37 °C for 0.5 h.

[0064] ④ Transformation of competent cells, spreading on plates, overnight culture in a 37 °C incubator.

[0065] ⑤ Colony PCR identification, positive clones sent for sequencing, sequencing primers used are CMV-F, pEGFP-N-3'.

[0066] ⑥ Samples with correct sequencing were subjected to plasmid miniprep using the Tiangen Plasmid Mini Kit to obtain the entry plasmid.

[0067] (1.2) Construction of adenovirus plasmid by LR recombination reaction (Gateway cloning):

[0068] ① Add the following components to a 0.5 mL centrifuge tube and mix at room temperature:

[0069] Table 4

[0070]

[0071] ② Add 0.5 μL of LR Clonase II enzyme mixture to each of the above samples and mix well.

[0072] ③ Incubate the reaction at 25 °C for 1 h. Note: Extending the incubation time to 18 h usually produces more colonies.

[0073] ④ Add 0.25 μL of proteinase K solution to each reaction and incubate at 37 °C for 10 min.

[0074] ⑤ Transform the products of the LR recombination reaction into E. coli competent cells and select for expressing clones to obtain AdTrim56.

[0075] (2) Amplification of AdTrim56:

[0076] Inoculate HEK293 cells before transfection. When the cells reach 50%-70% confluence, change the medium, add fresh culture medium containing AdTrim56, and after culturing for 90 minutes, add fresh culture medium again. When approximately 50% of the cells detach from the culture plate, collect the cell suspension. Freeze-thaw repeatedly to prepare the crude virus extract, and process the crude virus extract by CsCl density gradient ultracentrifugation to obtain the purified virus solution.

[0077] (3) Determination of AdTrim56 titer:

[0078] Inoculate HEK293 cells in a 96-well plate. After 24 hours, add the virus solution diluted in a serial dilution. Add the diluted virus solution to columns 1-10, with 8 replicate wells for each concentration, and add virus-free complete culture medium to columns 11-12. After culturing for 10 days, observe the cytopathic effect (CPE) under a microscope and calculate the positive rate for each concentration. The virus titer is calculated using the Spearman-Karber Method: titer (pfu / mL) = 10 (x+0.8) , where x = the sum of the positive rates of each concentration. Prerequisites: There is no CPE and growth inhibition in the negative control; there is CPE in the lowest dilution concentration group; there is no CPE in the highest dilution concentration group.

[0079] (4) Identification of the effect of AdTrim56:

[0080] Use AdTrim56 and AdFlag at a concentration of 2×10 8 pfu / virus to infect primary hepatocytes cultured in a 6-well plate (about 80% confluence). After 24 hours of infection, collect the cells, add protein lysis buffer for lysis, and collect the supernatant after 50 minutes. Take 50 μg of the supernatant, separate it by 10% SDS-PAGE electrophoresis, and then perform Western Blot analysis using a TRIM56-specific antibody. Determine whether AdTrim56 can play the expected role based on the expression of the TRIM56 protein.

[0081] 3. Detection of TRIM56 protein expression by Western Blot

[0082] (1) Protein extraction from cells

[0083] Add lysis buffer to the cells. After lysis is complete, centrifuge and take the supernatant, and use a BCA protein quantification kit (BCA Protein Assay Kit) to quantitatively collect the protein samples.

[0084] (2) Loading and electrophoresis

[0085] Prepare an SDS-PAGE electrophoresis gel, add it to the electrophoresis tank, load the protein sample into the sample wells of the SDS-PAGE electrophoresis gel, and start electrophoresis after loading is completed.

[0086] (3) Transfer the membrane

[0087] ① Prepare the transfer buffer and pre-cool it at 4°C.

[0088] ② Immerse the polyvinylidene fluoride membrane (PVDF membrane) in methanol for 15 s and then place it in the transfer buffer for later use.

[0089] ③ Take out the SDS-PAGE electrophoresis gel, wash it with the transfer buffer, then lay the washed SDS-PAGE electrophoresis gel flat on the filter paper at the negative electrode, cover it with the PVDF membrane, clamp on the clamping plate, put it into the transfer tank, and fill it with the transfer buffer to submerge the SDS-PAGE electrophoresis gel.

[0090] ④ Connect the power supply to the transfer tank, set the voltage to 250 V and the current to 0.2 A, and transfer for 1.5 h.

[0091] ⑤ After the transfer is completed, take out the PVDF membrane covered with the protein membrane.

[0092] (4) Blocking

[0093] Place the PVDF membrane covered with the protein membrane into the pre-prepared TBST buffer to wash away the transfer buffer on the protein membrane, then put the protein membrane into the blocking solution, and slowly shake it on a shaker for blocking at room temperature for 1 - 4 h.

[0094] (5) Incubation with primary antibody

[0095] ① Wash the protein membrane with TBST buffer 3 times, 5 min each time.

[0096] ② Seal the protein membrane into a hybridization bag with a sealer and add the primary antibody.

[0097] ③ Place the hybridization bag into a shaker at 4°C overnight.

[0098] (6) Incubation with secondary antibody

[0099] ① Take out the protein membrane, wash it with TBST buffer 3 times, 5 min each time, and recover the primary antibody.

[0100] ② Place the protein membrane into the corresponding secondary antibody dilution solution containing the secondary antibody and incubate it in the dark for 1 h.

[0101] (7) Protein detection

[0102] After incubation with the secondary antibody, wash the protein membrane with TBST buffer 3 times, 5 min each time. Use the Bio-Rad Chemi DocXRS+ gel imaging system to detect the target band.

[0103] As Figure 1 shown in the Western Blot identification result graph, the expression of TRIM56 in primary hepatocytes of mice infected with AdTrim56 was significantly increased, indicating that the model of overexpressing primary hepatocytes of AdTrim56 was successfully established.

[0104] Example 2: Effect of TRIM56 overexpression on lipid deposition in primary hepatocytes

[0105] (1) Experimental cell grouping: AdFlag-infected primary hepatocyte control group, AdTrim56-infected primary hepatocyte control group, AdFlag-infected cell experimental group, AdTrim56-infected primary hepatocyte experimental group.

[0106] (2) Establishment and detection of fatty liver cell model: As soon as the primary hepatocytes adhered, the corresponding AdFlag or AdTrim56 was added for infection, and then palmitate (PA) and oleic acid (OA) (PA 0.5 mM + OA 1 mM) were added to the two experimental groups for stimulation, and the same amount of BSA was added to the control group. After 18 h, the cell samples of each group were collected and stained with Nile red.

[0107] The result of Nile red staining was as Figure 2 shown. There was no obvious staining in the control group cells, while after the addition of PA + OA for stimulation in the experimental group, the staining area of the experimental group cells was significantly increased compared with the control group, and the staining area of the AdTrim56-infected primary hepatocyte experimental group cells was significantly reduced compared with the AdFlag-infected primary hepatocyte experimental group. This result indicates that TRIM56 overexpression can inhibit lipid deposition in primary hepatocytes induced by PA + OA stimulation.

[0108] Example 3: Effect of TRIM56 overexpression on the content of serum triglyceride (TG) in primary hepatocytes

[0109] (1) Experimental cell grouping: AdFlag-infected primary hepatocyte control group, AdTrim56-infected primary hepatocyte control group, AdFlag-infected primary hepatocyte experimental group, AdTrim56-infected primary hepatocyte experimental group.

[0110] (2) Establishment and detection of fatty liver cell model: As soon as the primary hepatocytes adhered, the corresponding AdFlag or AdTrim56 was added for infection, and then palmitate (PA) and oleic acid (OA) (PA 0.5 mM + OA 1 mM) were added to the two experimental groups for stimulation, and the same amount of BSA was added to the control group. After 18 h, the cell samples of each group were collected. After cell counting, TG was detected using a TG detection kit (290-63701 Wako; Tokyo, Japan), and statistical plotting was performed.

[0111] The TG detection results are as follows Figure 3 As shown, compared with the control group cells, when PA + OA was added to the experimental group for stimulation, the TG content in the experimental group cells increased significantly. However, the TG content in the primary hepatocytes of the experimental group infected with AdTrim56 was significantly lower than that of the experimental group of primary hepatocytes infected with AdFlag. This result indicates that overexpression of TRIM56 can inhibit the increase of TG in primary hepatocytes induced by PA + OA stimulation.

[0112] Example 4: Construction of a Trim56-HepOE mouse model with overexpression of liver TRIM56

[0113] The Sleeping Beauty transposon system was used to construct Trim56-HepOE mice to achieve overexpression of TRIM56 in the mouse liver. Briefly, male C57BL / 6J mice at 8 weeks of age were treated by hydrodynamic tail vein injection with a PBS DNA solution containing 2 μg of the SB100 transposase-encoding plasmid (pCMV-SB100, Addgene #34879) and 50 μg of the pT3-alb-3×Flag-mTRIM56 (see SEQ ID: 3) plasmid for the Trim56-HepOE group. GFP control mice were constructed: male C57BL / 6J mice at 8 weeks of age were treated by hydrodynamic tail vein injection with a PBS DNA solution containing 2 μg of the SB100 transposase-encoding plasmid and 50 μg of the pT3-alb-GFP-control plasmid (see SEQ ID: 4). After construction, Western Blot was used to identify the expression of Flag-TRIM56 in the livers of the GFP control group and Trim56-HepOE mice. Note: In the above plasmid nomenclature, pT3 represents the vector backbone, alb is the promoter (liver-specific promoter), flag is the protein tag (3* represents 3 flags in tandem), and GFP represents green fluorescent protein.

[0114] The results are as follows Figure 4 As shown, the expression level of TRIM56 in the livers of the Trim56-HepOE group mice increased significantly, indicating that the Trim56-HepOE mouse model was successfully established.

[0115] Example 5: Animal experiment

[0116] Thirty-two male C57BL / 6J mice were randomly divided into 4 groups (8 mice in each group, namely GFPNC group, Trim56-HepOENC group, GFP HFD group, and Trim56-HepOEHFD group); mice in the GFPNC group and Trim56-HepOENC group were fed with NC diet for 16 weeks, and mice in the GFP HFD group and Trim56-HepOEHFD group were fed with HFD diet for 16 weeks.

[0117] After 16 weeks of feeding, the mice were quickly sacrificed by cervical dislocation. The mice were fixed in the supine position, and the hair on the chest and abdomen of the mice was moistened with distilled water. The skin in the middle of the abdomen of the mice was clamped with forceps, and the skin was cut open from the middle of the abdomen towards the head to below the xiphoid process, and then cut open towards the tail end to expose the subcutaneous fascia, muscles, etc. layer by layer. The abdominal cavity was opened, and all internal organs were fully exposed. The livers of the mice in each group were quickly found and removed and weighed, and then hematoxylin-eosin (HE) staining and oil red O staining were performed respectively, and the TG content in the livers of the mice in the GFP HFD group and Trim56-HepOEHFD group was measured.

[0118] Steps for measuring TG content: The TG detection kit (290-63701 Wako; Tokyo, Japan) was used to detect TG according to the instructions and the results were statistically analyzed and graphed. The main steps of hematoxylin-eosin (H&E) staining were as follows: dewax the paraffin specimen sections to water → stain the cell nuclei with hematoxylin → stain the cytoplasm with eosin → dehydrate and mount the slides → microscopic examination and image acquisition and analysis. The main steps of oil red O (Oilred O) staining were as follows: after the sections were dried, they were slightly washed with 50% ethanol → treated with oil red O ethanol staining solution → differentiated with 50% ethanol, and the differentiation was terminated with tap water → counterstained the nuclei with hematoxylin, blued with tap water, and mounted with glycerin gelatin.

[0119] Figure 5 It is a statistical chart of the liver weights of GFP mice and Trim56-HepOE mice after 16 weeks of feeding with NC and HFD diets. The results showed that there was no significant difference in the liver weights of GFP and Trim56-HepOE mice fed with NC diet. The liver weight of Trim56-HepOE mice fed with HFD diet was significantly lower than that of GFP mice, indicating that in the mouse model with 16-week HFD induction, overexpression of TRIM56 can reduce the liver weight of mice.

[0120] Figure 6 It is a result chart of the TG content in the livers of GFP mice and Trim56-HepOE mice after 16 weeks of feeding with HFD diet. The results showed that compared with GFP mice, the TG content in the livers of Trim56-HepOE mice was significantly reduced, indicating that in the mouse model with 16-week HFD induction, overexpression of TRIM56 can reduce the increase in TG in the liver caused by HFD diet in mice. Figure 7These are representative pictures of HE staining and Oil Red O staining of liver tissue sections from GFP mice and Trim56-HepOE mice after 16 weeks of feeding with a high-fat diet (HFD). HE staining showed that, under HFD feeding conditions, different from the hepatocytes of GFP mice that underwent fatty degeneration and produced a large number of vacuolizations, the morphological changes of hepatocytes in Trim56-HepOE mice were significantly milder. Oil Red O staining showed that large red patches appeared in the liver of GFP mice, indicating a large amount of lipid deposition, while the lipid deposition in the liver of Trim56-HepOE mice was significantly reduced. Overexpression of TRIM56 could inhibit liver fat deposition and inhibit liver fatty degeneration.

[0121] The above results showed that overexpression of TRIM56 could significantly inhibit lipid accumulation induced by PA+OA stimulation in primary hepatocytes in vitro and HFD induction in vivo, and inhibit the occurrence and development of fatty liver. Overexpression of the TRIM56 gene had a significant inhibitory effect on fatty liver disease.

[0122] In summary, this application first used primary hepatocytes as the research object and up-regulated the expression of the TRIM56 gene in primary hepatocytes by AdTrim56. A fatty liver cell model induced by palmitic acid (PA) and oleic acid (OA) was used to study the function of the TRIM56 gene in lipid deposition and fatty degeneration. The results showed that under the same PA+OA stimulation, compared with primary hepatocytes with normal expression of the TRIM56 gene, the lipid droplets in primary hepatocytes with overexpression of the TRIM56 gene were fewer and smaller, and the intracellular TG content was significantly reduced. The results indicated that the TRIM56 gene could significantly inhibit intracellular lipid deposition in hepatocytes and inhibit the occurrence of fatty liver. At the same time, this application also constructed Trim56-HepOE mice with overexpression of TRIM56, and used HFD induction to establish a disease model of non-alcoholic fatty liver disease in mice. It was found that compared with its control group, the liver weight and liver TG content of Trim56-HepOE mice with overexpression of TRIM56 were significantly down-regulated, and the pathological staining results also showed that the fatty liver lesions of mice with overexpression of TRIM56 were alleviated and the lipid accumulation was significantly reduced after high-fat diet induction. For the above functions of TRIM56, TRIM56 can be used to prepare drugs for preventing, alleviating and / or treating non-alcoholic fatty liver disease.

[0123] The above embodiments are preferred embodiments of this application, but the embodiments of this application are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of this application shall be equivalent replacement methods and are all included in the protection scope of this application.

Claims

1. Use of a product for overexpressing TRIM56 protein in cells or tissues in the preparation of a product for preventing, alleviating, and / or treating non-alcoholic fatty liver disease, characterized in that: The product for overexpressing the TRIM56 protein in cells or tissues is one or more of plasmids and adenoviruses.

2. The application according to claim 1, wherein: The product is suitable for autonomous replication in cells.

3. The application according to claim 1, characterized in that: The product allows entry into cells by one or more of the following methods: direct naked DNA injection, liposome-encapsulated DNA direct injection, gold-coated DNA gene gun bombardment, plasmid DNA carried by replication-defective bacteria, and replication-defective adenovirus carrying the target DNA.

4. The application according to claim 1, characterized in that: The product is used to inhibit the increase in blood lipids, body fat accumulation, or liver lipid deposition caused by a high-fat diet.

5. The application according to claim 1, wherein: The non-alcoholic fatty liver disease is one or more of non-alcoholic simple fatty liver, non-alcoholic steatohepatitis, liver fibrosis, and cirrhosis.

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