Products for reducing KLHL22 gene expression or inhibiting KLHL22 protein action and use thereof in preparing drugs for preventing or treating metabolic-related fatty liver disease

Abstract

The present invention belongs to the field of medical molecular biology technology, and specifically relates to products that reduce the expression of the KLHL22 gene or inhibit the action of the KLHL22 protein and their use in the preparation of drugs for preventing or treating metabolic-related fatty liver disease. The present invention knocks out the KLHL22 gene systemically or specifically in the liver, and finds that the lipid deposition in the liver of mice with KLHL22 gene deletion is reduced, and the mice can resist insulin resistance induced by a high-fat diet. The present invention also finds at the cellular level that interfering with the expression of KLHL22 can reduce lipid deposition in hepatocytes induced by oleic acid. The present invention finds that inhibiting the KLHL22 gene or protein can be used as a therapeutic target for clinical metabolic-related fatty liver disease, providing new ideas for the research and development of new anti-metabolism-related fatty liver disease drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical molecular biology, and specifically relates to a product for reducing the expression of KLHL22 gene or inhibiting the action of KLHL22 protein and its application in preparing a drug for preventing or treating metabolism-related fatty liver disease. Background Art

[0002] Metabolic Dysfunction Associated Steatotic Liver Disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), is a group of pathological diseases mainly caused by ectopic lipid deposition in hepatocytes. The global prevalence of MASLD is about 30%. As of 2019, it is estimated that about 1.66 billion people suffer from MASLD worldwide, making it the most common liver disease in the world.

[0003] The liver is an important organ for lipid metabolism. As a central regulator of lipid balance, the liver is responsible for coordinating the synthesis of nascent fatty acids, their export and subsequent redistribution to other tissues, and their utilization as energy substrates. These processes are subject to complex interactions and regulation between hormones, nuclear receptors, and transcription factors, so that hepatic lipid homeostasis is strictly controlled. Disruption of one or more of these pathways may lead to fat deposition in the liver and the subsequent development of MASLD. Hepatic lipid accumulation is due to an imbalance between lipid acquisition and lipid metabolism, which is mainly regulated by four major pathways: circulating lipid uptake, de novo fatty acid synthesis (DNL), fatty acid oxidation (FAO), and lipid efflux in very low-density lipoprotein (VLDL). However, the molecular mechanisms of pathological lipid accumulation in the liver have not been fully elucidated. Once MASLD develops into end-stage liver disease, the treatment effect is limited and liver transplantation may be required. Early detection and intervention are essential.

[0004] The cornerstone of MASLD management is lifestyle intervention, including reducing energy intake and increasing physical exercise, which can reduce metabolic overload, improve adipose tissue dysfunction and the resulting metabolic inflammatory stress, and enable the liver repair mechanism to function. On March 14, 2024, the U.S. Food and Drug Administration (FDA) approved the thyroid hormone beta receptor agonist (THR-β) Resmetirom (MGL-3196) for the first time for the treatment of MASH patients with fibrosis. This is also the only drug currently approved for the treatment of MASLD, so exploring the pathogenesis of MASLD and seeking new potential targets is crucial for the treatment of MASLD.

[0005] Protein ubiquitination is a type of post-translational modification of proteins, which refers to the addition of ubiquitin molecules to substrate proteins. The most critical protein for ubiquitination is E3 ubiquitin ligase. Protein ubiquitination can participate in the regulation of DNA damage repair, cellular endocytosis, protein transport and degradation, etc. It plays an extremely important role in the occurrence and development of diseases, and the development of drugs targeting ubiquitination modification targets is also gaining attention. Kelch-like protein 22 (KLHL22) is a specific protein adapter for E3 ubiquitin ligases of the Cullin3RING domain family. It consists of 634 amino acids and has three domains, namely the N-terminal BTB, the middle BACK, and the C-terminal Kelch repeat sequence. KLHL22 binds to Cullin3RING E3 ubiquitin ligase through the BTB domain at the N-terminus and to substrate proteins through the Kelch repeat sequence domain at the C-terminus. Cullin3-RING binds to RBX1 to connect to E2 ubiquitin conjugating enzymes, and finally ubiquitinates the substrate proteins through enzymatic cascade amplification. The ability of KLHL22 to bind to specific substrate proteins makes it an extremely attractive intervention target. Summary of the invention

[0006] In view of the above prior art, the present invention has found that whole body KLHL22 gene knockout mice (KLHL22 - / - ) and liver-specific KLHL22 gene knockout mice (AAV-shKLHL22) can resist high-fat diet-induced metabolic-related fatty liver disease liver lipid deposition. Cell experiments showed that interfering with the expression of KLHL22 in hepatocytes can reduce oleic acid-induced hepatocyte lipid deposition. The present invention also found that compared with the control group of mice, KLHL22 - / - The glucose tolerance of AAV-shKLHL22 mice was significantly improved, and the insulin sensitivity of AAV-shKLHL22 mice was significantly increased.

[0007] Based on the above research results, the primary purpose of the present invention is to provide the following applications of KLHL22 gene or its protein as a target:

[0008] 1) Use of a product for reducing the expression of the KLHL22 gene in the preparation of a drug for preventing or treating metabolism-related fatty liver disease, wherein the CDS sequence of the KLHL22 gene is shown in SEQ ID NO.1; the product for reducing the expression of the KLHL22 gene comprises at least one of an agent for knocking out, inactivating by mutation, and inhibiting the KLHL22 gene.

[0009] Further,

[0010] The reagent for knocking out the KLHL22 gene includes: a vector for knocking out the KLHL22 gene; the reagent for mutating and inactivating the KLHL22 gene includes: a vector for mutating and inactivating the KLHL22 gene; the reagent for inhibiting the KLHL22 gene includes: siRNA for inhibiting the expression of the KLHL22 gene.

[0011] Furthermore,

[0012] The drugs for preventing or treating metabolism-related fatty liver disease include: drugs that enhance insulin sensitivity.

[0013] 2) Use of products that inhibit the action of KLHL22 protein in the preparation of drugs for preventing or treating metabolic-related fatty liver disease;

[0014] The amino acid sequence of KLHL22 protein is shown in SEQ ID NO.2; the product for inhibiting the action of KLHL22 protein comprises: at least one of an inhibitor for inhibiting the activity of KLHL22 protein and an inhibitor for blocking the interaction between KLHL22 and other proteins.

[0015] Further,

[0016] The drugs for preventing or treating metabolism-related fatty liver disease include: drugs that enhance insulin sensitivity.

[0017] The secondary purpose of the present invention is to provide the following products with KLHL22 gene or its protein as the target:

[0018] 1) A product that reduces the expression of the KLHL22 gene, which is a drug for preventing or treating metabolic-related fatty liver disease, including at least one of agents for knocking out, inactivating by mutation, and inhibiting the KLHL22 gene, and the CDS sequence of the KLHL22 gene is shown in SEQ ID NO.1.

[0019] Further,

[0020] The drugs for preventing or treating metabolism-related fatty liver disease include: drugs that enhance insulin sensitivity.

[0021] 2) Products that inhibit the action of KLHL22 protein are drugs for preventing or treating metabolic-related fatty liver disease, including: at least one of an inhibitor that inhibits the activity of KLHL22 protein and an inhibitor that blocks the interaction between KLHL22 and other proteins. The amino acid sequence of KLHL22 protein is shown in SEQ ID NO.2.

[0022] Further,

[0023] The drugs for preventing or treating metabolism-related fatty liver disease include: drugs that enhance insulin sensitivity.

[0024] In the present invention, the nucleotide sequence of the KLHL22 gene is referred to GenBank: NC_000082.7. The deletion, inhibition or inactivation of the KLHL22 gene or its protein can be achieved by conventional means in the art, such as a vector for knocking out the KLHL22 gene, a vector for inactivating the KLHL22 gene mutation and / or an inhibitor for inhibiting the activity of the KLHL22 protein, an inhibitor for blocking the interaction of KLHL22 with other proteins, and the like.

[0025] The present invention knocks out the KLHL22 gene systemically or specifically in the liver, and finds that the lipid deposition in the liver of KLHL22 gene-deficient mice is reduced, and the mice can resist insulin resistance induced by a high-fat diet. The present invention also finds at the cellular level that interfering with the expression of KLHL22 can reduce lipid deposition in hepatocytes induced by oleic acid. The present invention finds that inhibiting the KLHL22 protein can be used as a therapeutic target for clinical metabolic-related fatty liver disease, providing new ideas for the development of new anti-metabolism-related fatty liver disease drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Example 1 High-fat diet fed KLHL22 for 16 weeks + / + 、KLHL22 - / - , gross morphology, HE staining and Oil Red O staining results of the liver of AAV-shRNA and AAV-shKLHL22 mice, n=6-8, scale bar=50μm;

[0027] Figure 1 A is KLHL22 + / + and KLHL22 - / - Gross morphology, HE staining, and Oil Red O staining results of mouse liver; Figure 1 B shows the gross morphology, HE staining, and Oil Red O staining results of the liver of AAV-shRNA and AAV-shKLHL22 mice.

[0028] Figure 2 : Example 2 High-fat diet fed 16 weeks KLHL22 + / + 、KLHL22 - / - , AAV-shRNA and AAV-shKLHL22 mice resist high-fat diet-induced insulin resistance, n=6-8, , , ;

[0029] Figure 2 A is the GTT results of KLHL22+ / + and KLHL22- / - mice. Figure 2 B is KLHL22 + / + and KLHL22 - / -ITT results in mice; Figure 2 C is the GTT results of AAV-shRNA and AAV-shKLHL22 mice. Figure 2 D is the ITT results of AAV-shRNA and AAV-shKLHL22 mice.

[0030] Figure 3 : Detection results of siRNA interference in primary hepatocytes of 6-week-old C57 mice 24 hours after Example 3; n=6, , scale bar = 50 μm;

[0031] Figure 3 A is the result of Oil Red O staining of primary hepatocytes from 6-week-old C57 mice after siRNA interference for 24 hours and then treated with oleic acid for 12 hours. Figure 3 B is the absorbance OD value measured at a wavelength of 510 nm with isopropanol. DETAILED DESCRIPTION

[0032] In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making innovative work are within the scope of protection of the present invention.

[0033] Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels.

[0034] Example 1. Systemic and liver-specific KLHL22 knockout mice are resistant to high-fat diet-induced metabolic-related fatty liver disease.

[0035] Experimental methods

[0036] 1. Take photos of the gross morphology of mouse liver

[0037] Male KLHL22 - / - (whole-body KLHL22 knockout mice) and KLHL22 + / + (control mice) as well as AAV-shKLHL22 (liver-specific KLHL22 gene knockout mice) and AAV-shRNA (control mice) mice were fed a high-fat diet at 6 weeks of age. When the mice were fed a high-fat diet for 16 weeks, the livers were dissected and photographed.

[0038] Male KLHL22 - / - (whole-body KLHL22 knockout mice) and KLHL22 + / +(Control mice) Mouse acquisition process: CRISPR / Cas9 technology was used to cut out the fourth exon of KLHL22. According to Mendel's law of inheritance, heterozygous male mice and heterozygous female mice were mated to obtain knockout mice (KLHL22 - / - ) and littermate control mice (KLHL22 + / + ) was commissioned to be completed by Saiye Biotechnology Co., Ltd.

[0039] The process of obtaining AAV-shKLHL22 (liver-specific KLHL22 gene knockout mice) and AAV-shRNA (control mice) mice: First, three siRNA sequences targeting KLHL22 were designed, and the interference efficiency of the three siRNAs on primary liver cells KLHL22 was tested respectively, siRNA1: TGTCAACAATACCTATCGA; siRNA2: GCCAAATCCTGCACTTCAT; siRNA3: GCATCGTGTGATTATTTCA, as shown in SEQ ID NO.3-5 respectively. The siRNA sequence was designed and synthesized by Ruibo Biotechnology Co., Ltd., and the siRNA2 with the best interference effect was selected. Based on this sequence, AAV8-shKLHL22 adeno-associated virus V.GAAV8-shKLHL22 was constructed (commissioned by Shanghai Heyuan Biotechnology Co., Ltd.). 6-week-old C57 mice began to be fed with a high-fat diet (HFD). After 6 weeks of HFD feeding, 5×10 11 VG AAV8-shKLHL22 adeno-associated virus (AAV8 is a hepatotropic virus serotype that mainly targets the liver) or control empty adeno-associated virus were used, and samples were collected after continuing HFD feeding for 10 weeks.

[0040] 2. Hematoxylin-Eosin (HE) staining

[0041] Cut a bean-sized fresh liver tissue sample into a 5 mL centrifuge tube, add 5 mL of 4% paraformaldehyde fixative, and fix at room temperature for 2 hours or at 4°C overnight. Paraffin embedding: Remove the tissue from the fixative, put it into a dehydration box, put it into a dehydrator, and dehydrate it in a manner of increasing alcohol concentration and decreasing dehydration time. The specific steps are to dehydrate in 75% alcohol for 4 hours, then dehydrate in 85% alcohol for 2 hours, dehydrate in 90% alcohol for 2 hours, dehydrate in 95% alcohol for 1 hour, dehydrate twice in anhydrous ethanol, dehydrate for 30 minutes each time, rinse in anhydrous ethanol: xylene = 1:1 solution for 5-10 minutes, soak in xylene solution twice, each time for 5-10 minutes, and then dip in wax 3 times, each time for 1 hour. Embed the wax-soaked tissue, put it in a -20℃ refrigerator to cool and solidify, slice the solidified paraffin tissue with a paraffin slicer, stick the tissue slices on a glass slide, put it in a 60℃ oven, take it out after the wax is baked, and store it at room temperature. HE staining: The paraffin sections are soaked in xylene twice, each for 20 minutes, then soaked in anhydrous ethanol twice, each for 5 minutes, then transferred to 75% alcohol solution for 5 minutes, rinsed with running tap water for 2 minutes, stained with hematoxylin solution for 5 minutes, then washed with running tap water for 2 minutes, then treated with 1% hydrochloric acid alcohol differentiation solution, then washed with running tap water, treated with dilute lithium carbonate blueing solution, and rinsed with running tap water for 2 minutes. The slices were dehydrated in 85% alcohol for 5 minutes, and then in 95% alcohol for 5 minutes. The slices were stained with eosin for 5 minutes. The slices after HE staining should be dehydrated and transparent. The specific operation was to first soak them in anhydrous ethanol for 3 times, 5 minutes each time, and then soak them in xylene for 2 times, 5 minutes each time. The HE slices after transparent treatment were sealed with neutral gum.

[0042] 3. Tissue Oil Red O Staining

[0043] Cut a bean-sized fresh liver tissue sample into a 5 mL centrifuge tube, add 5 mL 4% paraformaldehyde fixative, and fix at room temperature for 2 hours or at 4°C overnight. Rinse the fixed liver sample 3 times with 1×PBS, 15 minutes each time, soak the rinsed liver tissue sample in 30% sucrose solution, incubate overnight at 4°C, 110 rpm shaker, remove the dehydrated liver tissue, put it in an embedding box, use clean filter paper to absorb the residual sucrose solution on the liver surface, and place it in OCT (Optimal cutting temperature The embedding agent of OCT compound was added dropwise into the embedding box. Note that no bubbles should be generated during this process. The embedding box was placed in a -80℃ refrigerator for freezing. The frozen embedding box was taken out of the -80℃ refrigerator and placed in a pre-cooled slicer. The slice thickness was about 6-8μm. The frozen slices were placed at room temperature and dried for 10 minutes. Then, the slices were fixed with fixative for 15 minutes, washed with running tap water for 2 minutes, and placed at room temperature until dry. The slices were placed in a dyeing tank filled with oil red dye and soaked for 10 minutes. Pay attention to avoid light. Then, the slices were taken out, soaked in 60% isopropanol for 3 seconds, washed with tap water for 10 seconds, and then soaked in 60% isopropanol for 5 seconds. After differentiation, they were washed with tap water for 10 seconds, re-stained with hematoxylin stain for 5 minutes, washed with tap water for 10 seconds, differentiated with hydrochloric acid for 8 seconds, and blued with bluing solution for 3 minutes, then washed with tap water for 10 seconds. The staining effect was checked under a microscope and sealed with glycerol gelatin.

[0044] The above experimental results can be found in Figure 1 .

[0045] 2. Experimental Results

[0046] like Figure 1 As shown in A, under high-fat diet feeding conditions, compared with the control group mice (KLHL22 + / + ) compared with whole-body KLHL22 knockout mice (KLHL22 - / - The liver tissue of the mice with KLHL22 gene knockout was smaller in size, darker in color, and lighter in fat. The results of HE and Oil Red O staining showed that the fatty liver model of mice was successfully induced under high-fat feeding conditions, and compared with the control group, the liver lipid deposition of the mice with systemic KLHL22 gene knockout was significantly improved. - / - The liver tissue of AAV-shKLHL22 mice was smaller, darker, and lighter in fat than that of the control group under high-fat diet. Both HE and Oil Red O staining showed less lipid deposition in the liver ( Figure 1 B). The above results indicate that systemic and liver-specific KLHL22 knockout mice are resistant to high-fat diet-induced metabolic-related fatty liver disease.

[0047] Example 2: Systemic and liver-specific KLHL22 knockout mice resist high-fat diet-induced insulin resistance

[0048] 1. Experimental Methods

[0049] 1. Glucose tolerance test (GTT) in mice

[0050] GTT was performed on mice fed a high-fat diet for 16 weeks.

[0051] (1) Pre-stimulation: All mouse experiments were pre-stimulated 3-5 days before the experiment to allow the mice to adapt to the experimental operation and reduce the errors caused by mouse excitement when grabbing the mice, drawing blood from the tail, and measuring blood sugar.

[0052] (2) Fasting: Fasting began in the afternoon of the day before the experiment. Clean bedding and feed racks were replaced. During the period when food was removed, mice were ensured to have adequate drinking water. The experiment began after fasting for about 16 hours.

[0053] (3) Weighing mice and measuring fasting blood glucose: Weigh each mouse with a weight scale and record the weight. Use scissors to cut a small hole at the end of the mouse's tail. Stroke the tail from the base to the distal end several times. Wipe off the first drop of blood that flows out with a sterile cotton ball. Squeeze a drop of fresh blood from the mouse's tail and drop it on the blood glucose test strip. Record the blood glucose value.

[0054] (4) After measuring fasting blood glucose, place the experimental mice in individual cages, lay down bedding, and allow them to adapt for 10 minutes.

[0055] (5) Intraperitoneal injection of glucose: Prepare 20% glucose injection solution with 0.9% saline, that is, dissolve 2g glucose powder in 10mL saline, filter with a 0.22μm filter, and prepare for use immediately. Calculate the amount of glucose solution required in advance according to the weight of the mouse (1g / kg body weight), and the injection volume of glucose solution is [5×mouse weight (g)]μL.

[0056] (6) Start timing after the first mouse is injected and complete the injection of all mice within 10 minutes.

[0057] (7) The blood glucose levels of mice were measured at 15 min, 30 min, 60 min, 90 min and 120 min after glucose injection.

[0058] (8) After the experiment is completed, put the mice back into the cage and provide them with sufficient sterilized water and feed.

[0059] 2. Insulin tolerance test (ITT) in mice

[0060] (1) Pre-stimulation: The steps are the same as step (1) of the GTT test above.

[0061] (2) Fasting: Fasting began at 8:00 a.m. on the day of the experiment. Clean bedding and feed racks were replaced and sufficient drinking water was ensured during the food removal period. The experiment began after fasting for about 6 hours.

[0062] (3) Place the experimental mice in individual cages, lay down the bedding, and allow them to acclimate for 10 minutes.

[0063] (4) Weighing mice and measuring fasting blood glucose: The steps are the same as step (3) of the GTT test above.

[0064] (5) Intraperitoneal injection of insulin: The insulin injection dose for high-fat fed mice is 1.0 IU / kg body weight, that is, 10 μL of Humulin is added to 10 mL of normal saline. After complete mixing, filter with a 0.22 μm filter and use it immediately. The insulin injection volume is [10×mouse weight (g)] μL.

[0065] (6) The time and steps for blood glucose testing are the same as steps (6) to (7) of GTT above.

[0066] The above experimental results can be found in Figure 2 .

[0067] 2. Experimental Results

[0068] The results of GTT and ITT experiments showed that under high-fat diet conditions, KLHL22 - / - Glucose tolerance of mice and AAV-shKLHL22 mice ( Figure 2 A and 2C) were significantly better than those in the control group, and the insulin sensitivity of AAV-shKLHL22 mice was significantly improved compared with that in the control group ( Figure 2 D). The above results indicate that systemic and liver-specific KLHL22 knockout mice are resistant to high-fat diet-induced insulin resistance.

[0069] Example 3: siRNA interference with the expression of KLHL22 in primary mouse hepatocytes can resist oleic acid-induced lipid deposition in hepatocytes

[0070] 1. Experimental Methods

[0071] 1. Extraction and culture of primary mouse liver cells

[0072] (1) Anesthetize the mice by intraperitoneal injection of an appropriate volume of 10% chloral hydrate (the dosage of chloral hydrate is: 4 μL / g mouse body weight). The mice will enter the anesthesia state in about 3 minutes.

[0073] (2) Fix the mouse in a supine position on a foam surgical board. Spray 75% alcohol to disinfect the chest and abdomen. Make a small cut in the middle of the abdomen. Cut the abdominal skin along the midline of the abdomen to expose the area from the sternum to the reproductive cavity. Carefully move the stomach, kidneys, intestines and other organs to the left side of the mouse to fully expose the inferior vena cava and portal vein of the mouse.

[0074] (3) Insert the intravenous needle parallel to the inferior vena cava close to the liver, fix the intravenous needle with a medical vascular clamp, cut the portal vein, and immediately inject 1× HBSS buffer. Note that the speed should be fast at the beginning, otherwise it is easy to cause blood vessel blockage in the organ. Then, slowly inject HBSS. After the blood in the liver is flushed clean and the liver turns khaki, stop injecting HBSS and start injecting digestive fluid.

[0075] (4) Connect the syringe containing tissue digestion solution to the end of the intravenous needle hose. Be careful not to generate bubbles. Slowly push in about 1 mL of digestion solution. Clamp the liver end of the portal vein with forceps and slowly push in about 0.5 mL of digestion solution. The liver will swell. Wait for half a minute, release the forceps, and continue to slowly push in about 0.5 mL of digestion solution. Repeat this three times. Be careful not to push the liquid too fast, otherwise it will easily cause liver cell damage.

[0076] (5) Use the back of the forceps to gently press down on the surface of the liver. If you see that the liver capsule cannot rebound and is in a collapsed state, it means that the liver tissue has been digested successfully.

[0077] (6) Cut off the entire liver and place it in a cell culture dish containing 1× HBSS buffer. Move it to the cell operation table, wash off the blood clots and other fascia tissues attached to the surface of the liver, and transfer it to a cell culture dish containing pre-cooled separation solution.

[0078] (7) Use forceps to tear open the liver capsule, lift the liver pedicle, and gently shake the liver tissue to allow the hepatocytes to be free from the liver capsule. Be careful not to rupture the gallbladder. Use a Pasteur pipette to transfer the separation solution containing a large number of hepatocytes to a 50 mL centrifuge tube. Use a 100 μm Falcon cell sieve to filter out impurities such as the liver capsule to obtain a cell suspension.

[0079] (8) Centrifuge at 50 g for 3 min at 4°C.

[0080] (9) Discard the supernatant and resuspend the cells in pre-cooled cell culture medium.

[0081] (10) Centrifuge at 50 g for 3 min at 4°C.

[0082] (11) Discard the supernatant, resuspend the cells in 37°C cell culture medium, and count them using a cell counter.

[0083] (12) Take an appropriate volume of cells (the density of primary liver cells is 5×105 Cells / mL) were plated into 6-well or 12-well plates, and the cells were shaken by the "cross method" and placed in a cell culture incubator. After the primary liver cells adhered to the wall (about 2 hours), the cells were washed three times with PBS and replaced with 37°C complete cell culture medium to continue culturing overnight for subsequent experimental processing.

[0084] 2. siRNA interference with KLHL22 gene expression in primary hepatocytes

[0085] Taking a 12-well plate as an example, before transfection, add 800 μL of serum-free and anti-dual antibody-free MEMα complete medium to each well; transfect primary hepatocytes: 1) dilute siRNA with 100 μL Opti-MEM, gently blow twice with the tip, and be gentle; 2) dilute 2.5 μL LipofectamineTM 3000 with 100 μL Opti-MEM, gently blow twice with the tip, and let stand at room temperature for 5 minutes; 3) add Lipo3000 transfection reagent to the siRNA dilution, gently blow twice with the tip to mix, and let stand at room temperature for 20 minutes; finally, add 200 μL of transfection complex to the 12-well plate, and the siRNA concentration is 200 nM; gently shake the 12-well plate using the cross method; put the 12-well plate in a cell culture incubator, and replace with new complete medium 6 hours after transfection for subsequent experiments. siRNA and control siRNA were purchased from Ruibo Biotechnology Co., Ltd. The sequences of siRNA1, 2, and 3 were: siRNA1:TGTCAACAATACCTATCGA; siRNA2: GCCAAATCCTGCACTTCAT; siRNA3: GCATCGTGTGATTATTTCA. Among them, siRNA2 had the best effect.

[0086] 3. Inducing lipid deposition in primary hepatocytes

[0087] (1) Solution preparation:

[0088] 1) 0.1M NaOH solution: Dissolve 0.2g NaOH in 50mL deionized water. After it is fully dissolved, incubate in a 70℃ water bath for 10min to dissolve oleic acid (OA).

[0089] 2) Preparation of 0.1M OA sodium salt: Dissolve 32μL of oleic acid stock solution in 968μL of preheated 0.1M NaOH solution. After dissolution, filter with a 0.22μm filter head and store at -20℃.

[0090] 3) Preparation of 10% FFA-free BSA solution: Dissolve 1 g FFA-free BSA in 10 mL deionized water, filter with a 0.22 μm filter, and heat in a 55°C water bath before use to prepare for binding with oleic acid.

[0091] 4) Preparation of 4 mM OA / BSA stock solution: Add 40 μL of 0.1 M OA sodium salt to 960 μL of 10% free fatty acid-free BSA solution, mix well and store at -20°C.

[0092] 5) Preparation of 0.4 mM OA / BSA working solution: Add 1 mL of 4 mM OA / BSA stock solution to 9 mL of complete culture medium and mix well. Prepare and use immediately.

[0093] (2) Add an appropriate volume of 0.4 mM OA / BSA working solution to the primary liver cell culture dish transfected with siRNA and culture for 12 hours.

[0094] 4. Oil Red O Staining of Cells

[0095] (1) Solution preparation: 1) Preparation of Oil Red O stock solution: dissolve 0.25 g of Oil Red O powder in 50 mL of isopropanol, place in a 60°C water bath, invert and mix every 10 minutes until the Oil Red O powder is completely dissolved, protect from light, and store at 4°C; 2) Preparation of Oil Red O working solution: prepare according to the ratio of Oil Red O stock solution to deionized water = 3:2 (volume ratio), filter with a 0.22 μm filter head, and use immediately after preparation.

[0096] (2) Rinse the cells twice with pre-cooled 1× PBS, discard the remaining PBS liquid as much as possible, and fix the cells with 4% paraformaldehyde at room temperature in the dark for 1 hour, or at 4°C in the dark overnight.

[0097] (3) Use negative pressure to remove the paraformaldehyde fixative solution, add an appropriate volume of Oil Red O working solution (enough to cover the cell surface) to each well, incubate at room temperature in the dark for 30 minutes, then use 1× PBS several times to wash away excess Oil Red O dye. Finally, add an appropriate volume of 1× PBS to protect the cells and prevent lipid droplets from rupturing.

[0098] (4) Observe under a microscope and take photos.

[0099] (5) Add isopropanol into the cell wells and measure the OD value at a wavelength of 510 nm using an ELISA reader.

[0100] The above experimental results can be found in Figure 3 .

[0101] 2. Experimental Results

[0102] like Figure 3 As shown, compared with the control group (Scramble), the lipid deposition in the hepatocytes of the group interfering with the KLHL22 gene expression in primary mouse hepatocytes was significantly reduced, and the OD value was significantly reduced. The above results show that siRNA interference with the expression of KLHL22 in primary mouse hepatocytes can resist oleic acid-induced lipid deposition in hepatocytes.

Claims

1. Use of a product for reducing the expression of the KLHL22 gene in the preparation of a drug for preventing or treating high-fat-induced metabolic-related fatty liver disease, wherein the CDS sequence of the KLHL22 gene is as shown in SEQ ID NO.1; the product for reducing the expression of the KLHL22 gene is: SiRNA for inhibiting the expression of the KLHL22 gene; the specific sequence of the SiRNA is: at least one of SiRNA1:TGTCAACAATACCTATCGA; SiRNA2:GCCAAATCCTGCACTTCAT; SiRNA3: GCATCGTGTGATTATTTCA.

2. The use according to claim 1, characterized in that: The drugs for preventing or treating high-fat-induced metabolic-related fatty liver disease include: drugs that enhance insulin sensitivity.

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