Application of Nr4a3 as target spot in preparing and screening medicine for treating non-alcoholic fatty liver disease

Abstract

The invention provides an application of Nr4a3 as a target spot in preparing and screening a medicine for treating a non-alcoholic fatty liver disease. Experiments prove that in the NAFLD pathogenesis process, Nr4a3 expression is increased in the liver. Through knockout of Nr4a3 expression, the levels of alanine transaminase (ALT) and aspartate transaminase (AST) in NAFLD mouse blood can be reduced, the content of triglyceride in liver tissue and the content of collagen in the liver are reduced, and immune cell infiltration in the liver tissue is reduced. The invention provides a new way and exploration direction for the treatment of the non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a target for drug treatment, specifically, the use of Nr4a3 as a target in the preparation and screening of drugs for treating non-alcoholic fatty liver disease. Background Art

[0002] Nonalcoholic fatty liver disease (NAFLD) has emerged as a significant cause of chronic liver disease worldwide. NAFLD is defined as the presence of more than 5% steatosis in the liver in the absence of other causative factors for fatty liver disease, such as drug or alcohol abuse, autoimmune disease, or viral hepatitis. NAFLD is a benign condition; however, it may progress to more severe conditions, including nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. The overall prevalence of NAFLD in the Asian population is estimated to be approximately 29.6%, and its prevalence has steadily increased over time, from 25.3% between 1999 and 2005, to 28.5% between 2006 and 2011, and to 33.9% between 2012 and 2017. Rapid industrialization and socioeconomic growth experienced across Asia has led to the prevalence of a sedentary lifestyle and a high-calorie diet, which may have contributed to the increasing prevalence of NAFLD. However, there is a severe shortage of drugs for the treatment of NAFLD, and so far there is only one drug on the market.

[0003] Nuclear receptors are a family of transcriptional regulators that are abundant in mammals. They play an important regulatory role in metabolism, reproduction, and development. Ligand-bound nuclear receptors recruit coactivators to activate the expression of target genes. Nuclear receptor Nor1, also known as Nr4a3 (nuclear receptor subfamily 4group A member 3), has a gene located on the chromosome 9 chain of human chromosome. Its site information is as follows: Chromosome9, NC_000009.12 (99821885..99866891), its transcript (NM_006981.4) sequence is shown in SEQ ID No.1, and its protein amino acid sequence is shown in SEQ ID No.2. Nr4a3 is involved in the transduction of acute and chronic antigen receptor signals in T cells and B cells, and is related to the development of lymphocytes. The transcript sequence of mouse Nr4a3 is shown in SEQ ID No.3, and its protein amino acid sequence is shown in SEQ ID No.4.

[0004] Little is known about the role of Nr4a3 in the liver, but previous studies have shown that Nr4a3 plays an important regulatory role in cardiovascular disease. For example, Nr4a3 causes the formation of atherosclerosis in ApoE mice, mainly by increasing inflammatory cell infiltration. Other studies have shown that overexpression of Nr4a3 can aggravate AngII-induced myocardial fibrosis. The progression of NAFLD is accompanied by a series of pathological changes such as lipid metabolism disorders, immune infiltration, fibrosis and cell death, among which inflammation and fibrosis are important pathological processes that promote the occurrence and development of non-alcoholic fatty liver disease. Summary of the invention

[0005] In view of the above technical problems in the prior art, the present invention provides the use of Nr4a3 as a target in the preparation and screening of drugs for treating non-alcoholic fatty liver disease. The application is to solve the technical problem that the drugs in the prior art are not effective in treating non-alcoholic fatty liver disease.

[0006] The present invention provides the use of Nr4a3 as a target in the preparation and screening of drugs for treating non-alcoholic fatty liver disease.

[0007] Furthermore, the sequence of the Nr4a3 transcript is shown as SEQ ID No.1, and its amino acid sequence is shown as SEQ ID No.2.

[0008] Furthermore, the drug is a drug that inhibits the expression level of Nr4a3.

[0009] Furthermore, the drug is a drug that inhibits the activity of Nr4a3.

[0010] The present invention also provides the use of an agent for inhibiting or silencing Nr4a3 gene expression in the preparation of a drug for treating non-alcoholic fatty liver disease.

[0011] The present invention also provides the use of an inhibitor targeting Nr4a3 in the preparation of a drug for treating non-alcoholic fatty liver disease, wherein the inhibitor inhibits the expression level of Nr4a3 or inhibits the activity of Nr4a3.

[0012] The present invention provides a new idea and target for the use of Nr4a3 in the preparation of non-alcoholic fatty liver disease drugs. Experiments have confirmed that Nr4a3 expression increases in the liver during the onset of NAFLD. By knocking out the expression of Nr4a3, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the blood of NAFLD mice can be reduced, the triglyceride content in the liver tissue and the collagen content in the liver can be reduced, and the infiltration of immune cells in the liver tissue can be reduced.

[0013] Compared with the existing technology, the technical effect of the present invention is positive and obvious. The present invention finds that Nr4a3 can be a new target for treating non-alcoholic fatty liver disease, providing new ideas and targets, new pathways and exploration directions for preparing new non-alcoholic fatty liver disease drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The expression changes of Nr4a3 in liver tissue of NAFLD mice induced by CDAHFD diet and mice in the normal diet group.

[0015] Figure 2 To investigate the effects of Nr4a3 knockout on liver weight, hepatic triglycerides, and ALT and AST levels in peripheral serum of NAFLD mice induced by CDAHFD diet.

[0016] Figure 3 To investigate the effects of Nr4a3 knockout on liver fibrosis area, steatosis score, and inflammation score in CDAHFD diet-induced NAFLD mice.

[0017] Figure 4 To investigate the effect of Nr4a3 knockout on monocyte and neutrophil infiltration in the liver of NAFLD mice induced by CDAHFD diet. DETAILED DESCRIPTION

[0018] The present invention is further described below with examples, but should not be construed as limiting the present invention. The examples do not include a detailed description of conventional methods, which are well known to those skilled in the art and are described in many publications.

[0019] Unless otherwise specified, the percentages of the present invention are calculated by area; unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention, and the preferred implementation methods and materials described in the specific implementation methods are for demonstration purposes only.

[0020] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were sent to Sewell Biotech for testing. The brief experimental process is described below. Flow cytometry antibodies were purchased from biolegend, BD Biosciences and other companies, isoflurane was purchased from Runan Better China; RNA reverse transcription kit and qRT-PCR kit were purchased from Ruibo Biotech, triglyceride (TG) content detection kit was purchased from Prilai Biotech, and HE staining, Sirius red staining, and Oil Red O staining kits were purchased from Solebao Biotech.

[0021] Example 1: Nr4a3 expression in mouse liver tissue Wild-type mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.) were divided into two groups (8 mice / group): one group was a control group with a normal diet (NC group), and the other group was a non-alcoholic fatty liver disease group induced by feeding a high-fat methionine and trace choline-deficient diet (CDAHFD group) for 12 weeks. Liver tissues of the two groups were obtained and the changes in Nr4a3 expression in mouse liver tissues were detected by real-time fluorescence quantitative PCR. The results are shown in Figure 1 As shown, the expression of Nr4a3 in the liver tissue of mice in the CDAHFD group was significantly increased.

[0022] Example 2: Mouse liver weight detection Conditional knockout mice (Nr4a3 fl / f Nr4a3 knockout mice (Nr4a3 cKO ), and the control group mice were Nr4a3 fl / fl Two groups of mice (10 mice / group) were fed with a high-fat methionine and trace choline-deficient diet (CDAHFD group) for 12 weeks to induce non-alcoholic fatty liver disease.

[0023] The mice fed for 12 weeks were fixed on the surgical board, and the abdominal cavity of the mice was cut open with sterilized surgical scissors. The liver of the mice was cut out and placed on a micro electronic balance to weigh the liver weight. Compared with the control group of mice, the knockout of Nr4a3 significantly reduced the liver weight of the mice fed CDAHFD ( Figure 2 Middle A).

[0024] Example 3: Detection of triglyceride (TG) content in mouse liver tissue Two groups of mice (CDAHFD-Nr4a3 cKO group and CDAHFD-Nr4a3 fl / fl The liver tissue of the control group was weighed, 50 mg of liver tissue was weighed and placed in a 1.5 ml centrifuge tube, 20 µl of lysis buffer was added to every 1 mg of liver tissue, and the tissue was homogenized using a tissue homogenizer. After homogenization, the mixture was allowed to stand for 10 minutes, and an appropriate amount of supernatant was transferred to a new 1.5 ml centrifuge tube. The remaining lysis buffer was used for protein quantification using the BCA method.

[0025] According to the instructions of the kit (Cat: E1013, Pulilai Biotech), 4 ml of reagent R1 and 1 ml of reagent R2 were mixed in a ratio of 4:1 to form a detection working solution for immediate use. The 4 mM glycerol standard was diluted with sample buffer to a concentration gradient of 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 0 µmol / L. 10 µl of sample and 190 µl of detection working solution were added to a 96-well plate and reacted at 37°C for 15 minutes. After obtaining the OD value, the sample concentration was calculated after calibration with a blank tube. Finally, according to the aforementioned BCA protein quantification, the triglyceride content in the liver tissue was converted into micrograms per milligram of protein.

[0026] The results showed that knocking out Nr4a3 could reduce the content of triglycerides (TG) in mice fed with CDAHFD ( Figure 2 Middle B).

[0027] Example 4: Detection of ALT and AST in serum Mice (CDAHFD-Nr4a3 cKO group and CDAHFD-Nr4a3 fl / fl The mice (group) were weighed on an electronic balance and placed in an anesthesia tank. After the anesthesia was stable, the peripheral blood of the mice was obtained by removing the eyeballs and centrifuging after the blood coagulated. The centrifugal speed was 1000g and the centrifugation was performed for 2 minutes. After the centrifugation, the supernatant was obtained. If it was not tested immediately, it was stored in a deep low temperature refrigerator. If it was tested immediately, the serum was placed in a fully automatic animal blood biochemical instrument for testing.

[0028] Biochemical analysis of peripheral blood revealed that knockout of Nr4a3 could reduce serum ALT and AST levels in CDAHFD-fed mice ( Figure 2 Medium CD).

[0029] Example 5: Pathological staining under a microscope 5.1 Hematoxylin-eosin staining Obtain liver tissues from two groups of mice (CDAHFD-Nr4a3 cKO group and CDAHFD-Nr4a3 fl / fl The samples were placed in 4% paraformaldehyde and fixed at room temperature for 24 hours before routine paraffin embedding. The samples were placed on a paraffin slicer for sectioning, with a thickness of 4-5 μm, and then stained. The staining process is as follows: (1) Dewaxing: xylene (I) 15 min → xylene (II) 15 min → anhydrous ethanol 5 min*2 times → 95% ethanol 5 min*2 times → 80% ethanol 5 min*2 times → 75% ethanol 5 min*2 times → double distilled water washing several times; (2) Hematoxylin staining: After absorbing the water, stain with hematoxylin for 5 minutes and rinse with tap water; (3) Acidification: Differentiation in hydrochloric acid and alcohol for 60 seconds; (4) Eosin staining: Soak in tap water for 15 minutes, dry the water and place in eosin solution for 3 minutes; (5) Dehydration: After washing with distilled water to remove the floating color, dehydrate with 75% ethanol for 40 seconds, then 95% ethanol for 40 seconds, and then dehydrated ethanol for 40 seconds. (6) Transparent and fixation: xylene (I) 2 min → xylene (II) 2 min → xylene (II) 2 min → neutral resin sealing and fixation. After drying, take photos under an optical microscope.

[0030] 5.2 Sirius red staining Obtain mouse liver tissue (CDAHFD-Nr4a3 cKO group and CDAHFD-Nr4a3 fl / fl The samples were placed in 4% paraformaldehyde and fixed at room temperature for 24 hours before routine paraffin embedding. The samples were placed on a paraffin slicer for sectioning, with a thickness of 4-5 μm. The staining process was as follows: (1) Dewaxing: xylene (I) 15 min → xylene (II) 15 min → anhydrous ethanol 5 min*2 times → 95% ethanol 5 min*2 times → 80% ethanol 5 min*2 times → 75% ethanol 5 min*2 times → double distilled water washing several times; (2) Dye with Sirius red solution for 10-15 minutes, and then rinse off the solution with running water; (3) Prepare iron hematoxylin staining solution, add it dropwise for 5-10 minutes, and wash with running water for 10-20 seconds; (4) Dehydration: 75% ethanol for 40 s → 95% ethanol for 40 s → anhydrous ethanol for 40 s; (5) Transparent and fixation: xylene (I) 2 min → xylene (II) 2 min → xylene (II) 2 min → neutral resin sealing and fixation. After drying, take photos under an optical microscope.

[0031] 5.3 Oil Red O staining Obtain mouse liver tissue (CDAHFD-Nr4a3 cKO group and CDAHFD-Nr4a3 fl / fl The embedded blocks were then frozen in OCT embedding medium and placed on a cryostat for rapid refrigeration. The embedded blocks were then frozen and sectioned with a thickness of 10 μm. The staining process was as follows: (1) Place the frozen sections in 4% paraformaldehyde fixative and fix at room temperature for 20-30 minutes; (2) Then, wash the slices in 75% ethanol solution for 5 seconds; (3) Stain the sections with oil red dye for 5-10 minutes, wash off the excess dye with 60% isopropanol solution, and rinse with tap water for 30 seconds; (4) Stain with hematoxylin for 1 minute, differentiate with hydrochloric acid and alcohol for 1-5 seconds, and rinse with tap water for 30 seconds to return to blue; (5) Seal the slide with glycerol gelatin. After it dries, take a photo under an optical microscope.

[0032] The staining results are as follows Figure 3 The results showed that knocking out Nr4a3 could reduce the collagen content in the liver of CDAHFD-fed mice ( Figure 3 A, B), and improved liver steatosis index ( Figure 3 C) and inflammation index ( Figure 3 Middle D).

[0033] Example 6. Detection of mouse liver immune cells Mice (CDAHFD-Nr4a3 cKO group and CDAHFD-Nr4a3 fl / fl (Group) After anesthesia is stable, place the mice on the surgical board, use the ex vivo perfusion system, use type IV collagenase to enzymatically digest the liver, and the digestion fluid enters through the superior vena cava and exits from the portal vein. The overall time is controlled within 10 minutes to maintain cell activity. The digested liver is placed in a culture dish, and the liver is torn open with tweezers. The hepatocytes are gently shaken out, and the cells are filtered through a 100μm cell filter, washed with PBS and centrifuged. Repeat 3 times, and the precipitate is resuspended for the last time and filtered through a 40μm cell filter to make a single cell suspension. The content of monocytes and neutrophils in liver tissue is detected using dead cell dyes, CD45, CD11b, ly-6c, ly-6g and other flow cytometry antibodies.

[0034] Single cell suspensions of mouse liver were obtained through an ex vivo perfusion system, and immune cells were labeled with corresponding flow cytometry antibodies and then detected by flow cytometry ( Figure 4 Middle A), flow cytometry results showed that knocking out Nr4a3 could reduce the number of monocytes and neutrophils in the liver tissue of mice fed CDAHFD ( Figure 4 Middle B).

Claims

1. The use of Nr4a3 as a target in the preparation and screening of drugs for the treatment of non-alcoholic fatty liver disease.

2. The use of Nr4a3 as a target in the preparation and screening of drugs for treating non-alcoholic fatty liver disease according to claim 1, characterized in that: The sequence of the Nr4a3 transcript is shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No.

2.

3. The use of Nr4a3 as a target in the preparation and screening of drugs for treating non-alcoholic fatty liver disease according to claim 1, characterized in that: The drug is a drug that inhibits the expression level of Nr4a3.

4. The use of Nr4a3 as a target in the preparation and screening of drugs for treating non-alcoholic fatty liver disease according to claim 1, characterized in that: The drug is a drug that inhibits the activity of Nr4a3.

5. Use of an agent for inhibiting or silencing Nr4a3 gene expression in the preparation of a drug for treating non-alcoholic fatty liver disease.

6. Use of an inhibitor targeting Nr4a3 in the preparation of a drug for treating non-alcoholic fatty liver disease, wherein the inhibitor inhibits the expression level of Nr4a3 or inhibits the activity of Nr4a3.

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