Application of retinoic acid induced protein 6 (STRA6) inhibitor in prevention and / or treatment of metabolic dysfunction related fatty liver disease

Abstract

The invention discloses an application of a retinoic acid induced protein 6 (STRA6) inhibitor in prevention and / or treatment of metabolic dysfunction related fatty liver diseases, and particularly relates to an application of the retinoic acid induced protein 6 (STRA6) inhibitor in treatment of the metabolic dysfunction related fatty liver diseases. Experiments find that after STRA6 is knocked down on a mouse AML-12 hepatocyte line, palmitic acid (PA)-induced hepatocyte lipid accumulation can be remarkably improved, the content of triglyceride (TG) in hepatocytes is reduced, and finally occurrence and development of metabolic dysfunction related fatty liver diseases are relieved.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to the application of STRA6 inhibitors in the treatment of metabolic dysfunction-related fatty liver disease. Background Technology

[0002] Metabolic dysfunction-associated steatotic liver disease (MASLD) has become the most common chronic liver disease worldwide. This disease can progress from simple hepatic steatosis to metabolic dysfunction-associated steatohepatitis (MASLD). Approximately 20-30% of MASH patients will further develop cirrhosis, liver failure, and even hepatocellular carcinoma, leading to serious consequences. However, there are currently no specific drugs that can directly treat MASLD, and clinical treatment strategies are mainly limited to lifestyle interventions. Therefore, there is an urgent need to further explore the pathogenesis of MASLD and identify new therapeutic targets. Summary of the Invention

[0003] To address the shortcomings of existing methods, the purpose of this invention is to provide the application of STRA6 inhibitors in the treatment of metabolic dysfunction-related fatty liver disease.

[0004] The objective of this invention can be achieved through the following technical solutions:

[0005] The first aspect of this invention provides the use of a substance that inhibits the STRA6 target in the preparation of products related to the prevention and / or treatment of metabolic dysfunction-related fatty liver disease.

[0006] A second aspect of the present invention provides the use of substances that inhibit the STRA6 target in the preparation of products related to reducing lipid accumulation in hepatocytes.

[0007] The substance that inhibits the STRA6 target described in this invention can be a common inhibitor in the art, such as monoclonal antibodies, nucleic acid molecules, naturally purified substances, modified naturally purified substances, semi-synthetic substances, chemically synthesized substances, etc. that can inhibit STRA6 expression and / or activation.

[0008] In a specific embodiment of the present invention, the substance used to inhibit the STRA6 target is a small interfering RNA, such as siRNA-1, siRNA-2, or siRNA-3, wherein the sequence of siRNA-1 is shown in SEQ ID NO.1 and SEQ ID NO.2; the sequence of siRNA-2 is shown in SEQ ID NO.3 and SEQ ID NO.4; and the sequence of siRNA-3 is shown in SEQ ID NO.5 and SEQ ID NO.6.

[0009] The products described in this invention include pharmaceuticals, food, functional food, special medical food, health products, nutrient supplements, dietary supplements, or pharmaceutical compositions.

[0010] In some embodiments, the metabolic dysfunction-related fatty liver disease described in this invention is selected from one or more of fatty liver, hepatitis, liver injury, liver fibrosis, or cirrhosis.

[0011] The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a substance that inhibits the STRA6 target.

[0012] As a preferred technical solution of this application, the pharmaceutically acceptable carrier includes fillers, wetting agents, binders, disintegrants, lubricants, etc. Fillers include starch, lactose, mannitol, microcrystalline cellulose, etc.; binders include hydroxypropyl methylcellulose, povidone, microcrystalline cellulose, etc.; disintegrants include croscarmellose sodium, croscarmellose, surfactants, low-substituted hydroxypropyl cellulose, etc.; lubricants include magnesium stearate, talc, polyethylene glycol, sodium lauryl sulfate, micronized silica gel, talc, etc. Excipients also include colorants, sweeteners, etc.

[0013] As a preferred embodiment of this application, the pharmaceutical composition includes oral formulations, injectable formulations, transdermal formulations, or mucosal formulations.

[0014] Beneficial effects:

[0015] The experimental results of this invention show that administration of STRA6 small interfering RNA (siRNA) to AML-12 cells can significantly inhibit intracellular STRA6 expression at the mRNA level and significantly improve PA-induced lipid accumulation in hepatocytes and reduce intracellular TG content, indicating that the STRA6 inhibitor described in this invention can prevent and / or treat metabolic dysfunction-related fatty liver disease. Attached Figure Description

[0016] Figure 1 This refers to the interference efficiency of STRA6 small interfering RNA in AML-12 cells in this embodiment of the invention.

[0017] Figure 2This is a statistical analysis of Nile Red staining and relative fluorescence intensity of cells after STRA6 knockdown on AML-12 followed by PA administration in an embodiment of the present invention.

[0018] Figure 3 In this embodiment of the invention, TG detection of cells after knocking down STRA6 on AML-12 cells and then administering PA is performed. Detailed Implementation

[0019] To help those skilled in the art better understand the content of this invention, the embodiments of the invention are described in detail below. These embodiments are implemented based on the technical solution of this invention, providing detailed implementation methods and specific operating procedures. However, the content of this invention is not limited to the following examples. Reagents or instruments used without a specified manufacturer are considered to be conventional products that can be purchased on the market.

[0020] Unless otherwise specified, the amino acid sequence of retinoic acid-induced protein 6 (STRA6) used in the following examples is shown in SEQ ID NO. 7, and the NCBI number of the gene sequence encoding this protein is NC_000075.7 (https: / / www.ncbi.nlm.nih.gov / nuccore / NC_000075.7?report=fasta&from=57971076&to=58061287).

[0021] The cells used in this invention are mouse hepatocytes (AML-12). Experimental data were analyzed using GraphPad Prim 9.5 statistical software and are expressed as Mean ± SEM. One-way ANOVA was used to analyze significant differences between multiple experimental groups.

[0022] Example 1

[0023] AML-12 cells were cultured in Dulbecco's Modified EagleMedium: Nutrient Mixture F-12 medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics, at a growth rate of 2 × 10⁻⁶ cells / mL during the logarithmic growth phase. 4 Cells were seeded at a density of / wells in 24-well plates. The experiment consisted of a control group (NC) and a small interference group (siRNA-1, siRNA-2, siRNA-3). After cell adhesion, the small interference group was transfected with three different STRA6 small interference sequences using Lipofectamine 3000 transfection reagent.

[0024] siRNA-1:

[0025] f-GGCCAAUCUUCAAGCACUA (SEQ ID NO.1),

[0026] r- UAGUGCUUGAAGAUUGGCC (SEQ ID NO. 2);

[0027] siRNA-2:

[0028] f- GUCCUCAGGAUCCUAAGAU (SEQ ID NO. 3),

[0029] r-AUCUUAGGAUCCUGAGGAC (SEQ ID NO. 4);

[0030] siRNA-3:

[0031] f-GUCCUCUUCAGCAACCUAU (SEQ ID NO.5),

[0032] r-AUAGGUUGCUGAAGAGGAC (SEQ ID NO.6)

[0033] Simultaneously, the control group was transfected with the negative control sequence (NC: f-UUCUCCGAACGUGUCACGU, r-ACGUGACACGUUCGGAGAA). Cell RNA was collected 24 h later, and the efficiency of the STRA6 small interference was detected by RT-PCR. Results are as follows: Figure 1 As shown.

[0034] The results showed that all three different small interfering sequences of STRA6 could significantly knock down the expression of STRA6 in hepatocytes at the mRNA level. Among them, siRNA-1 had the best knockdown efficiency, and siRNA-1 was used as a representative for subsequent experiments.

[0035] Example 2

[0036] AML-12 cells grow at a rate of 2 × 10⁻⁶ during the logarithmic growth phase. 4Cells were seeded at a density of / wells in 24-well plates containing a climbing slide. The experiment was divided into a normal control group (Control), a small interference group (siStra6), a model drug treatment group (300 μM PA), and a small interference + model drug treatment group (siStra6 + 300 μM PA). After cell adhesion, the STRA6 small interference sequence siRNA-1 was transfected with Lipofectamine 3000. 24 h later, the model drug treatment group and the small interference + model drug treatment group were given 300 μM PA. 24 h later, Nile red staining was used to observe changes in lipid droplet content in hepatocytes in each group: staining was performed with Nile red dye, followed by mounting with DAPI-containing mounting medium, and observation and photography were conducted under an Olympus FV3000 microscope (Japan). Relative fluorescence intensity was statistically analyzed using ImageJ. Results are as follows: Figure 2 As shown.

[0037] The results showed that Nile red fluorescence intensity in hepatocytes increased significantly after PA administration, indicating significant lipid accumulation in hepatocytes. The Nile red fluorescence intensity in hepatocytes decreased significantly after small interference with STRA6, indicating that inhibiting STRA6 can significantly improve PA-induced lipid accumulation in hepatocytes.

[0038] Example 3

[0039] AML-12 cells grow at a rate of 5 × 10⁻⁶ during the logarithmic growth phase. 3 Cells were seeded at a density of / well in 96-well plates. The experiment included a normal control group (Control), a small interference group (siStra6), a model drug treatment group (300 μM PA), and a small interference + model drug treatment group (siStra6 + 300 μM PA). After cell adhesion, the STRA6 small interference sequence siRNA-1 was transfected with Lipofectamine 3000. 24 h later, the model drug treatment group and the small interference + model drug treatment group were given 300 μM PA. 24 h later, the culture medium was aspirated, and 2 μl of PBS was added, followed by three freeze-thaw cycles to lyse the cells. Then, 200 μl of TG working solution was added, and the cells were incubated at 37°C for 10 min. The absorbance was measured at 500 nm using a microplate reader, and the TG content of the cells was calculated based on the absorbance. Results are as follows: Figure 3 As shown.

[0040] The results showed that PA administration significantly increased intracellular TG levels in hepatocytes, leading to marked lipid accumulation. Small-scale STRA6 intervention significantly decreased intracellular TG levels in hepatocytes, indicating that STRA6 inhibition could significantly improve PA-induced lipid accumulation in hepatocytes. Therefore, this study provides a research basis for developing STRA6 inhibitors to treat metabolic dysfunction-related fatty liver disease.

[0041] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. Application of substances that inhibit the STRA6 target in the preparation of products for the prevention and / or treatment of metabolic dysfunction-related fatty liver disease.

2. The application according to claim 1, characterized in that, The substance that inhibits the STRA6 target is a monoclonal antibody, nucleic acid molecule, natural purified substance, modified natural purified substance, semi-synthetic substance or chemically synthesized substance that can inhibit STRA6 expression and / or activation.

3. The application according to claim 2, characterized in that, The substance that inhibits the STRA6 target is small interfering RNA.

4. The application according to claim 3, characterized in that, The small interfering RNAs are siRNA-1, siRNA-2, or siRNA-3, wherein the sequence of siRNA-1 is shown in SEQ ID NO.1 and SEQ ID NO.2; the sequence of siRNA-2 is shown in SEQ ID NO.3 and SEQ ID NO.4; and the sequence of siRNA-3 is shown in SEQ ID NO.5 and SEQ ID NO.

6.

5. The application according to claim 1, characterized in that, The metabolic dysfunction-related fatty liver disease mentioned above is selected from one or more of fatty liver, hepatitis, liver injury, liver fibrosis, or cirrhosis.

6. Application of substances that inhibit the STRA6 target in the preparation of products related to reducing lipid accumulation in hepatocytes.

7. The application according to claim 6, characterized in that, The substance that inhibits the STRA6 target is a monoclonal antibody, nucleic acid molecule, natural purified substance, modified natural purified substance, semi-synthetic substance or chemically synthesized substance that can inhibit STRA6 expression and / or activation.

8. The application according to claim 7, characterized in that, The substance that inhibits the STRA6 target is small interfering RNA.

9. The application according to claim 8, characterized in that, The small interfering RNAs are siRNA-1, siRNA-2, or siRNA-3, wherein the sequence of siRNA-1 is shown in SEQ ID NO.1 and SEQ ID NO.2; the sequence of siRNA-2 is shown in SEQ ID NO.3 and SEQ ID NO.4; and the sequence of siRNA-3 is shown in SEQ ID NO.5 and SEQ ID NO.

6.

10. The application according to any one of claims 1 to 9, characterized in that, The products mentioned are pharmaceuticals, food, functional food, special medical food, health products, nutrient supplements, dietary supplements, or pharmaceutical compositions.

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