Application of RBCK1 inhibitor in preparation of medicine for preventing or treating metabolism-related fatty liver disease

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of an RBCK1 inhibitor in preparation of a medicine for preventing or treating metabolism-related fatty liver diseases. The inventor discovers the mutual relation between RBCK1 gene expression and MAFLD for the first time, and puts forward that the RBCK1 gene is applied to treatment of MAFLD for the first time. The medicine provided by the invention accurately targets the liver, reduces the adverse reaction of known medicines at present, performs intervention in the early stage of diseases, effectively relieves the symptoms of fatty liver, prevents further deterioration of the diseases, and plays an important role in preventing metabolic diseases. The long-term dependence of the patient on various drugs for treatment is avoided, and the treatment compliance of the patient can be improved. The comprehensive treatment scheme provides a new thought for the treatment of fatty liver and related metabolic diseases, can significantly improve the overall metabolic health of patients, and has important clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of RBCK1 inhibitors in the preparation of drugs for the prevention or treatment of metabolic-related fatty liver disease. Background Technology

[0002] Fatty liver disease is caused by excessive fat deposition in the liver and is commonly seen in obese individuals, people with diabetes, and those with metabolic syndrome. Metabolic-associated fatty liver disease (MAFLD) is the most common type of fatty liver disease. It often presents with no obvious symptoms in its early stages, but if left untreated, it can progress to liver fibrosis, cirrhosis, and even liver cancer. Its global prevalence is as high as 32.4%, while in China it reaches 29.2%. This disease is characterized by excessive fat accumulation in the liver and is a significant contributing factor to cirrhosis and liver cancer. The diagnostic criteria for MAFLD require a diagnosis of fatty liver along with one of three other conditions: obesity, type 2 diabetes, or metabolic dysfunction. Simultaneously, the clinical focus of MAFLD treatment is gradually shifting from emphasizing liver lesions and their related outcomes to the prevention and treatment of obesity, type 2 diabetes, and metabolic disorders.

[0003] Although various medications for fatty liver disease exist on the market, their efficacy is generally limited, especially in patients with intermediate to advanced stages, for whom there is no specific drug that can completely reverse or cure fatty liver. Currently, the FDA has only approved Rezdiffra (a thyroid hormone receptor agonist) for the treatment of adult patients with metabolic dysfunction-associated steatohepatitis (MASH) accompanied by moderate to advanced liver scarring (fibrosis). Its most common side effects include diarrhea, nausea, and elevated liver enzymes. However, in cases with comorbidities, medications already approved for treating comorbidities and potentially beneficial for MAFLD may be considered. For example, antidiabetic drugs such as PPARγ agonists, GLP-1 agonists, and SGLT2 inhibitors have been reported to improve fatty liver disease, but most of these drugs have certain side effects and limited therapeutic efficacy. Therefore, there is an urgent need to develop a novel, effective, and less side-effect-prone targeted therapy for fatty liver disease.

[0004] RING refers to the structure of BK protein 1 (RBCK1), an E3 ubiquitin ligase and a major component of the linear ubiquitin chain assembly complex (LUBAC). It couples linear polyubiquitin chains to substrates, promoting substrate ubiquitination and its degradation by the proteasome. RBCK1 plays a crucial role in nuclear factor κB (NF-kappa-B) activation and inflammation regulation. Furthermore, the LUBAC complex, including RBCK1, balances endothelial angiogenesis by specifically editing the linear ubiquitination of ALK1. Previous studies have also found increased expression of RBCK1 in renal cell carcinoma, breast cancer, and lung adenocarcinoma, with mechanisms related to the degradation of p53 and protein kinase C zeta (PKCζ).

[0005] The inventors first discovered the relationship between RBCK1 gene expression and MAFLD, and first proposed the application of RBCK1 gene in the treatment of MAFLD, thus forming this invention. Summary of the Invention

[0006] To address the shortcomings and deficiencies of existing technologies, this invention provides the application of RBCK1 inhibitors in the preparation of drugs for the prevention or treatment of metabolic-related fatty liver disease.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides the use of RBCK1 inhibitors in the preparation of medicaments for the prevention or treatment of metabolic-related fatty liver disease.

[0008] Alternatively, in the above applications, the drug is suitable for mammals.

[0009] Preferably, the drug is applicable to humans or mice, among other things.

[0010] Alternatively, in the above applications, the RBCK1 inhibitor is a drug that reduces RBCK1 gene expression or reduces the amount of RBCK1 protein.

[0011] Alternatively, in the above applications, the agent is selected from one or more of the following: nucleic acid molecules, small molecule compounds, traditional Chinese medicine, compound preparations, antibodies, peptides, proteins, gene editing vectors, lentiviruses, or adeno-associated viruses.

[0012] Alternatively, in the above applications, the agent is siRNA.

[0013] Alternatively, in the above applications, the nucleotide sequence of the siRNA is as shown in SEQ ID No. 1 (GCTGCACTTTCATCAACAA).

[0014] Alternatively, in the above applications, the drug may also comprise a pharmaceutically acceptable carrier.

[0015] Preferably, the carrier is any pharmaceutical agent or carrier medium capable of delivering an effective dose of the active substance of the present invention, without interfering with the biological activity of the active substance and without toxic side effects on the host or subject.

[0016] Alternatively, in the above applications, the metabolic-associated fatty liver disease is induced by a high-fat, high-cholesterol diet and is characterized by hepatomegaly and excessive lipid accumulation in the liver.

[0017] Alternatively, in the above applications, the metabolic-associated fatty liver disease includes one or more of metabolic-associated steatohepatitis, liver fibrosis, cirrhosis, and other chronic liver diseases.

[0018] In a second aspect, the present invention provides the use of RBCK1 as a therapeutic target for metabolic-associated fatty liver disease.

[0019] In a third aspect, the present invention provides the use of RBCK1 as a target in the development, screening, or preparation of medicaments for the prevention or treatment of metabolic-related fatty liver disease.

[0020] Compared with the prior art, the beneficial effects of the present invention are: (1) The inventors discovered for the first time the relationship between RBCK1 gene expression and MAFLD, and proposed for the first time to apply the RBCK1 gene to the treatment of MAFLD. This invention provides a novel target for the clinical treatment of MAFLD and offers new ideas and perspectives for the development of innovative drugs for MAFLD.

[0021] (2) The drug of this invention precisely targets the liver, reducing the adverse reactions of currently known drugs, and intervenes in the early stage of the disease, effectively alleviating fatty liver symptoms, preventing further deterioration of the disease, and playing an important role in the prevention of metabolic diseases. It avoids long-term dependence on multiple drug treatments and helps improve patient treatment compliance. This comprehensive treatment plan provides a new approach to the treatment of fatty liver and related metabolic diseases, can significantly improve the overall metabolic health of patients, and has important clinical application value. Attached Figure Description

[0022] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention, but do not constitute a limitation thereof. In the drawings: Figure 1 High-fat diet upregulates the mRNA expression level of RBCK1 in mouse liver tissue.

[0023] Figure 2RBCK1 overexpression can exacerbate hepatic lipid deposition. (A) The protein level of RBCK1 in the liver was significantly upregulated after AAV intervention; (B) Hepatic lipid droplets increased after RBCK1 overexpression.

[0024] Figure 3 Knockdown of RBCK1 in an in vitro model alleviated lipid deposition in AML12 cells. (A) Knockdown of RBCK1 with siRNA in AML12 cells significantly reduced RBCK1 protein levels; (B) Knockdown of RBCK1 with siRNA in AML12 cells significantly reduced PA-induced lipid droplet deposition.

[0025] Figure 4 Liver-specific knockout RBCK1 haploid mice (RBCK1 hep The protein level of RBCK1 in the liver of the infected animals was significantly decreased. (A) Compared with the negative control group (RBCK1) fl / fl Compared to RBCK1 hep (B) Liver protein imprinting bands of mice in group A; compared with the littermate negative control (RBCK1) fl / fl Compared to RBCK1 hep Statistical graph of liver protein imprinting in mice.

[0026] Figure 5 Liver-specific knockout of RBCK1 can significantly reverse high-lipid-induced hepatic lipid deposition. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the materials and reagents used are commercially available.

[0028] Example 1: High-fat diets were purchased from Research Diet (Catalog No.: D12492) in the United States. Mice were fed either a normal diet (NC) or a high-fat diet (HF) starting at 8 weeks of age, with 6 mice in each group, for 12 weeks. The mRNA expression level of RBCK1 in the liver tissues of mice in the NC and HF groups was detected using quantitative real-time PCR. The results were statistically analyzed using an unpaired t-test. This indicates a comparison with the NC group. p <0.05. The primer sequences used in the PCR detection are shown in Table 1.

[0029] Table 1 like Figure 1 As shown, the results indicate that the RBCK1 mRNA level in the HF group mice was significantly higher than that in the NC group mice.

[0030] Example 2: Male C57BL / 6J mice (purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were injected via tail vein with adeno-associated virus (pAAV-TBG-Rbck1(mouse)-FLAG-EGFP or pAAV-TBG-EGFP, 2 × 10⁻⁶ per mouse). 11 Vg, n=6 (synthesized by Quanyang Biotechnology), was used to specifically overexpress RBCK1 in the experimental group. RBCK1 expression levels were detected by Western blotting after 3 weeks (A). RBCK1 (purchased from Huaan Biotechnology, catalog number: ER1915-72) was corrected using Vinculin (purchased from Aibotek, catalog number: A2752) as an internal control. Liver sections from mice specifically overexpressing RBCK1 and control mice were embedded in OCT and frozen for Oil Red O staining (B) (magnification: 40x). Oil Red O staining: The Oil Red O staining kit (G1015) was purchased from Savill Biotechnology Co., Ltd. The testing method followed the kit instructions.

[0031] like Figure 2 As shown in Figure A, the results indicate that RBCK1 protein expression in the liver of mice (RBCK1 group) was significantly increased after AAV injection. Figure 2 As shown in Figure B, the results indicate that lipid droplet deposition is significantly increased in the liver of mice overexpressing RBCK1.

[0032] Example 3: RBCK1 knockdown was performed on normal AML12 mouse hepatocytes (purchased from Pronosei, catalog number CL-0602) using small interfering RNA (siRNA) (sequence: SEQ ID No. 1: GCTGCACTTTCATCAACAA) (synthesized by Sangon Biotech). AML12 cells were seeded into 6-well plates, and siRNA transfection was performed using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) at 60% cell density. The siRNA was mixed with Opti-MEM, and then Lipofectamine RNAiMAX was added. After incubation at room temperature for 20 minutes, this mixture was added to the cells. The mixture was evenly spread on the plates and cultured at 37°C for 48 hours. Protein was extracted after the intervention for Western blotting verification of protein levels. Subsequently, AML12 cells were stimulated with 0.2 mM palmitic acid (PA) (purchased from Xi'an Kunchuang Technology Development Co., Ltd.) for 24 hours to establish an in vitro model of fatty liver. RBCK1 (purchased from Huaan Biotechnology, catalog number: ER1915-72) in the liver tissues of normal and obese mice was detected using a validated siRNA sequence, with β-actin (purchased from Wuhan Sanying, catalog number: 20536-1-AP) as an internal control for correction. Lipid droplets were then fluorescently labeled with BODIPY (purchased from Beyotime, catalog number: C2053S) and imaged under confocal microscopy (magnification: 200x).

[0033] like Figure 3 As shown in Figure A, the results indicate that this sequence can significantly reduce RBCK1 protein expression. Figure 3 As shown in Figure B, the results indicate that RBCK1 knockdown significantly reduced lipid droplets in AML12 cells.

[0034] Example 4: C57BL / 6 male mice, ALB-cre mice, and RBCK1 conditional knockout mice were all purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. Liver-specific knockout RBCK1 mice were obtained by crossing RBCK1 conditional knockout mice with ALB-cre mice to obtain heterozygotes, resulting in haploid mice with liver-specific knockout RBCK1.

[0035] High-fat diet was purchased from Research Diet (product number: D12492) in the United States. Haploid mice with liver-specific RBCK1 knockout (RBCK1) were fed this diet. hep ) and its littermate negative control mice (RBCK1) fl / fl High-fat diet was introduced at 8 weeks of age, divided into HF + RBCK1. fl / fl and HF+RBCK1 hepGroups, n=6-7 animals / group, were euthanized and samples were collected after 12 weeks. The expression level of RBCK1 in the liver was detected by Western blotting. RBCK1 (purchased from Huaan Biotechnology, catalog number: ER1915-72) was corrected using Vinculin (purchased from Aibotek, catalog number: A2752) as an internal control.

[0036] The results were statistically analyzed using an unpaired t-test. Indicates to RBCK1 fl / f Compared to the group, p <0.001.

[0037] like Figure 4 A and Figure 4 As shown in B, the results indicate that RBCK1 hep The protein expression of RBCK1 in the liver of mice was significantly reduced.

[0038] Example 5: HF+RBCK1 in Example 4 above hep Group and HF+RBCK1 fl / fl Liver sections were embedded in OCT scans and then stained with Oil Red O. Oil Red O staining: The Oil Red O staining kit (G1015) was purchased from Sevier Biotechnology Co., Ltd. The test method was performed according to the kit instructions.

[0039] like Figure 5 As shown, the results indicate that lipid droplet deposition in the liver of liver-specific knockout mice is significantly reduced.

[0040] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several modifications or equivalent substitutions can be made to the technical solution without departing from the principle of the present invention, and these modifications or equivalent substitutions should also be considered within the scope of protection of the present invention.

Claims

1. Application of RBCK1 inhibitors in the preparation of drugs for the prevention or treatment of metabolic-related fatty liver disease.

2. The application according to claim 1, characterized in that: The RBCK1 inhibitor is a drug that reduces RBCK1 gene expression or reduces the amount of RBCK1 protein.

3. The application according to claim 2, characterized in that: The agent is selected from one or more of the following: nucleic acid molecules, small molecule compounds, antibodies, peptides, proteins, gene editing vectors, lentiviruses, or adeno-associated viruses.

4. The application according to claim 3, characterized in that: The drug is siRNA.

5. The application according to claim 4, characterized in that: The nucleotide sequence of the siRNA is shown in SEQ ID No.

1.

6. The application according to any one of claims 1 to 5, characterized in that: The drug also contains a pharmaceutically acceptable carrier.

7. The application according to claim 1, characterized in that: The metabolic-associated fatty liver disease is induced by a high-fat, high-cholesterol diet and is characterized by hepatomegaly and excessive lipid accumulation in the liver.

8. The application according to claim 1, characterized in that: The metabolic-associated fatty liver disease includes one or more of metabolic-associated steatohepatitis, liver fibrosis, cirrhosis, and other chronic liver diseases.

9. The use of RBCK1 as a therapeutic target for metabolic-associated fatty liver disease.

10. Use of RBCK1 as a target in the development, screening, or preparation of drugs for the prevention or treatment of metabolic-related fatty liver disease.

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