Use of e3 ubiquitin ligase inhibitors in non-alcoholic fatty liver related drugs

Abstract

The present application relates to the field of biological medical technology, and particularly relates to application of E3 ubiquitinase inhibitor in non-alcoholic fatty liver related drugs. TRIM23 has a significant influence on non-alcoholic fatty liver disease, and inhibition of expression of TRIM23 at a cell level can improve lipid deposition of hepatocytes induced by mixed palmitic acid and oleic acid; on the contrary, promotion of expression level of TRIM23 can aggravate lipid accumulation of hepatocytes induced by mixed palmitic acid and oleic acid. Therefore, the TRIM23 inhibitor can be used for preparing a drug for preventing or treating fatty liver.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to the application of E3 ubiquitinase inhibitors in non-alcoholic fatty liver related drugs. BACKGROUND

[0002] Non-alcoholic fatty liver disease (NAFLD) is a chronic progressive liver disease characterized by excessive deposition of hepatocyte lipids, which can develop into hepatitis, cirrhosis and liver cancer. Obesity is the most common and most clear risk factor for NAFLD, which drives the occurrence and development of the disease through mechanisms such as nutritional excess and insulin resistance.

[0003] However, the molecular pathogenesis of NAFLD has not been fully elucidated, and existing treatment methods have limited efficacy and obvious side effects. For example, lifestyle interventions have poor compliance; chemical drugs such as insulin sensitizers and lipid-lowering drugs can only improve some indicators, and may cause gastrointestinal reactions or liver toxicity. At present, there is still a lack of specific treatment targets and safe and effective drugs for the core pathological aspects of NAFLD, and new intervention strategies are urgently needed. SUMMARY

[0004] Therefore, the present application provides the application of E3 ubiquitinase inhibitors in non-alcoholic fatty liver related drugs, which at least solves one problem in the prior art.

[0005] In a first aspect, the present application provides the application of TRIM23 inhibitors in the preparation of drugs for preventing or treating fatty liver.

[0006] TRIM23 has a significant impact on non-alcoholic fatty liver disease, and inhibiting the expression of TRIM23 at the cellular level can improve the lipid deposition of hepatocytes induced by a mixture of palmitic acid and oleic acid; on the contrary, promoting the expression level of TRIM23 can aggravate the lipid accumulation of hepatocytes induced by a mixture of palmitic acid and oleic acid. Therefore, TRIM23 inhibitors can be used to prepare drugs for preventing or treating fatty liver.

[0007] In some optional embodiments, the drug for preventing or treating fatty liver is a drug for preventing or treating non-alcoholic fatty liver.

[0008] In a second aspect, the present application provides a drug for preventing or treating fatty liver, which comprises a TRIM23 inhibitor.

[0009] In some optional embodiments, the drug for preventing or treating fatty liver is a drug for preventing or treating non-alcoholic fatty liver.

[0010] In some alternative embodiments, the TRIM23 inhibitor is an inhibitor of mRNA expression level of TRIM23 and / or an inhibitor of protein expression level of TRIM23.

[0011] In some alternative embodiments, the inhibitor of mRNA expression level of TRIM23 comprises at least one of siRNA, shRNA, and miRNA.

[0012] In some alternative embodiments, the inhibitor of mRNA expression level of TRIM23 is siRNA, and the sequence of the siRNA is shown in SEQ ID No. 1. The SEQ ID No. 1 is GGAAGAAGGTTGTCAAACT.

[0013] In some alternative embodiments, the inhibitor of protein expression level of TRIM23 comprises at least one of plasmid, polypeptide, enzyme, antibody, virus, and polynucleotide.

[0014] In some alternative embodiments, the drug for preventing or treating fatty liver further comprises at least one of a carrier, a buffer solution, and a stabilizer.

[0015] With the above technical solutions, the embodiments of the present application have at least the following advantages:

[0016] A new function of TRIM23 gene is found, the expression level of the gene in non-alcoholic fatty liver is increased, and inhibition of the expression of TRIM23 can obviously improve lipid accumulation and reduce the content of triglyceride; overexpression of TRIM23 can aggravate lipid accumulation in liver and increase the content of triglyceride; based on the function of TRIM23 in non-alcoholic fatty liver, the TRIM23 inhibitor can be applied to the preparation of a drug for preventing or treating non-alcoholic fatty liver. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 FIG. 1 is a detection result diagram of Western blotting in the embodiment 1 of the present application, in which β-Actin is an internal reference protein.

[0018] Figure 2 FIG. 2 is a quantitative analysis statistical diagram of the expression amount of TRIM23 protein in the detection result of Western blotting in the embodiment 1 of the present application, in which *** indicates P<0.001.

[0019] Figure 3 FIG. 3 is a result diagram of oil red O staining in the embodiment 1 of the present application.

[0020] Figure 4 FIG. 4 is a result diagram of triglyceride detection in the embodiment 1 of the present application, in which * indicates P<0.05.

[0021] Figure 5 Figure for detection result of Western blotting in Example 2 of the present application, wherein β-Actin is the internal reference protein.

[0022] Figure 6 Figure for quantitative analysis of TRIM23 protein expression amount in detection result of Western blotting in Example 2 of the present application, wherein ** represents P<0.01.

[0023] Figure 7 Figure for oil red O staining result in Example 2 of the present application.

[0024] Figure 8 Figure for triglyceride detection result in Example 2 of the present application, wherein ** represents P<0.01.

[0025] Figure 9 Figure for detection result of Western blotting in Example 3 of the present application, wherein β-Actin is the internal reference protein.

[0026] Figure 10 Figure for quantitative analysis of TRIM23 protein expression amount in detection result of Western blotting in Example 3 of the present application, wherein ** represents P<0.01.

[0027] Figure 11 Figure for hematoxylin-eosin staining and oil red O staining result in Example 3 of the present application.

[0028] Figure 12 Figure for triglyceride detection result in Example 3 of the present application, wherein ** represents P<0.01. DETAILED DESCRIPTION

[0029] The concept and technical effects of the present application will be described clearly and completely below to fully set forth the purpose, scheme and effects of the present application.

[0030] Tri-motif protein 23 (TRIM23) has dual activities of E3 ubiquitin ligase and GTPase, and can regulate many different cell processes and pathways, especially plays an important role in antiviral defense, activation of innate immune response, cancer development, etc. The inventors found that TRIM23 has a significant impact on non-alcoholic fatty liver disease, suggesting that TRIM23 gene can be used as a new target for increasing the treatment strategy of non-alcoholic fatty liver disease.

[0031] The present application provides the use of TRIM23 inhibitor in the preparation of a drug for preventing or treating fatty liver, and the technical effects of the present application are verified by the following examples.

[0032] In the following examples, the mouse hepatocyte cell line (AML12) was purchased from Wuhan Zhishan Biotechnology Co., Ltd., with the item number STCC20037, and the growth medium was DMEM / F12 + 10% FBS + 1% penicillin-streptomycin + 40 ng / mL dexamethasone, which was cultured in a 5% CO2 37°C constant temperature cell special incubator; the cells were cryopreserved using serum-free cell freezing medium, and stored in liquid nitrogen. Among them, DMEM / F12 and FBS were purchased from Thermo Fisher Scientific, penicillin-streptomycin was purchased from Beijing Solabio Technology Co., Ltd., dexamethasone was purchased from Shanghai Biyun Tian Biotechnology Co., Ltd., and serum-free cell freezing medium was purchased from Suzhou Xinsaimai Biotechnology Co., Ltd.

[0033] Example 1 Effect of TRIM23 knockdown on cell lipid accumulation

[0034] The AML12 cells were cultured, and when the cells were in good growth state, the cells were plated (six-well plate) and divided into control group and knockdown group; when the cell density after plating reached 70%, si-TRIM23 was transfected into the experimental group using lipo3000 (5 μL was added to each well, so that the final concentration of si-TRIM23 was 50 nM), and si-NC was transfected into the control group using lipo3000 (5 μL was added to each well, so that the final concentration of si-NC was 50 nM); 48 hours after transfection, cell proteins were extracted for Western blotting experiment to detect the expression level of TRIM23. Among them, si-TRIM23 is a TRIM23 inhibitor, which was purchased from Guangzhou Ribo Biotechnology Co., Ltd. (hereinafter referred to as Ribo Biotechnology); si-TRIM23 is an siRNA, and the specific sequence is shown in SEQ ID No. 1.

[0035] The results of Western blotting detection are shown in Figure 1 and Figure 2 The TRIM23 protein level of the experimental group (knockdown group) was significantly lower than that of the control group, indicating that TRIM23 was successfully knocked down in AML12 cells.

[0036] Compound fatty acids, i.e. palmitic acid (PA) + oleic acid (OA) mixture, were added to the control group and the experimental group, respectively, with a final concentration of 0.5 mM; after 24 hours of compound fatty acid stimulation, oil red O staining was performed to detect the lipid accumulation. The results of oil red O staining are shown in Figure 3 As shown in , there was obvious lipid accumulation in the control group cells, and the lipid droplets were obviously colored and significantly reduced in number; while the lipid accumulation in the experimental group (knockdown group) was significantly improved, and the lipid droplets were colored and reduced in number. This indicates that knocking down TRIM23 can significantly improve the lipid accumulation in hepatocytes induced by compound fatty acids.

[0037] After 24 hours of stimulation with compound fatty acids in both the control and experimental groups, cells were washed twice with 1×PBS buffer. Intracellular triglyceride levels were then detected using a tissue cell triglyceride enzymatic assay kit (purchased from Beijing Pulilai Gene Technology Co., Ltd., catalog number E1013). The triglyceride content detection results are as follows: Figure 4 As shown, the triglyceride content in the experimental group (knockdown group) was significantly lower than that in the control group, indicating that knocking down TRIM23 can alleviate the increase in triglyceride content induced by complex fatty acids.

[0038] Example 2: Effect of TRIM23 overexpression on cellular lipid accumulation

[0039] First, the TRIM23 target gene was amplified by PCR. After agarose gel electrophoresis, the DNA was recovered using a DNA recovery kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.). Using pcDNA3.1 as a vector, both the vector and the recovered TRIM23 fragment were double-digested with enzymes. After agarose gel electrophoresis, the digested vector backbone and TRIM23 fragment were recovered. The recovered target gene and linear vector were ligated and incubated overnight at 16°C to obtain the ligation product. The ligation product was then transformed, and single colonies were picked for colony PCR and agarose gel electrophoresis to verify positive colonies. The positive colonies were then inoculated into LB liquid medium containing ampicillin and incubated at 37°C for 12 hours. The plasmid was extracted using an endotoxin-free plasmid extraction kit (purchased from Tiangen Biotech (Beijing) Co., Ltd.) to obtain the TRIM23 overexpression plasmid.

[0040] AML12 cells were seeded and divided into a control group and an overexpression group. The TRIM23 overexpression plasmid was transfected into the overexpression group cells using LIPO3000 and P3000, while the control group cells were transfected with the pcDNA3.1 empty vector plasmid. The TRIM23 protein level was detected by immunoblotting 48 hours after transfection.

[0041] Immunoblotting test results Figure 5 and Figure 6 As shown, the TRIM23 protein level in the overexpression group was significantly higher than that in the control group, indicating that TRIM23 overexpression in AML12 cells was successfully achieved.

[0042] Both the control and overexpression groups were stimulated with complex fatty acids for 24 hours, followed by Oil Red O staining to detect lipid accumulation. The Oil Red O staining results are shown below. Figure 7 As shown, the control group exhibited a significant accumulation of lipid droplets with marked staining; while the overexpression group showed a greater degree of lipid accumulation, with a significant increase in both the number and intensity of lipid droplet staining. This indicates that overexpression of TRIM23 can exacerbate lipid accumulation in hepatocytes induced by fatty acid synthesis.

[0043] After 24 hours of stimulation with complex fatty acids in the control group and overexpression group cells, the triglyceride content was also measured using a tissue cell triglyceride enzyme assay kit.

[0044] The results of the triglyceride content detection are as Figure 8 shown. The triglyceride content in the overexpression group was significantly higher than that in the control group, indicating that overexpression of TRIM23 can exacerbate triglyceride accumulation induced by complex fatty acids.

[0045] Example 3 Effect of TRIM23 overexpression on lipid accumulation in mouse liver

[0046] The animal experiment was carried out according to the following steps:

[0047] (1) Obtaining the TRIM23 overexpression plasmid

[0048] The TRIM23 overexpression plasmid was purchased from Beijing Tsingke Biotechnology Co., Ltd. This plasmid uses pLive as the vector and inserts the TRIM23 target gene. Using the plate streaking method, dip the glycerol bacteria of the purchased plasmid and perform four-zone streaking on a solid medium containing kanamycin. Invert it and incubate it in an incubator at 37°C overnight. The next day, pick a single colony and inoculate it into an LB liquid medium containing antibiotics. Shake the bacteria at 200 rpm for 12 - 16 h. Then use a kit to extract the plasmid, and the TRIM23 overexpression plasmid can be obtained.

[0049] (2) Experimental animals and feeding

[0050] The experimental animals were C57BL / 6 (C57) mice, 6 - 8 weeks old, male, and all were purchased from Jicuiyaokang Biotechnology Co., Ltd. All experimental mice were housed in the SPF-class animal room of the Translational Medicine Research Institute of Nanchang University, and the license number was SYXK (Gan) 2021 - 0001. The lighting was alternated every 12 hours, the temperature was 24 ± 2 °C, and the humidity was 40% - 70%. The mice drank water and ate freely.

[0051] The mice were randomly divided into a control group and an experimental group. Both groups of mice were fed a high-fat diet for 12 weeks. At the 1st week and the 5th week, the experimental group mice were injected with the TRIM23 overexpression plasmid via the tail vein, while the control group mice were injected with the pLive empty plasmid. After 12 weeks, the samples were collected and the livers of the mice were taken.

[0052] Weigh 20 mg of liver tissue, extract the tissue protein, and perform an immunoblotting experiment to detect the expression levels of TRIM23 in the liver tissues of the control group and the experimental group. The results are as Figure 9 and Figure 10 shown. Compared with the control group, the protein level of TRIM23 in the experimental group was significantly increased, proving that the high expression of TRIM23 in the mouse liver tissue was successfully achieved.

[0053] Mouse liver tissue sections were prepared and subsequently stained with hematoxylin-eosin (HE) and Oil Red O. The staining results are shown below. Figure 11 As shown, the liver tissue of mice fed a high-fat diet exhibited significant steatosis and vacuolation; overexpression of TRIM23 exacerbated this phenomenon, indicating that promoting TRIM23 expression can aggravate steatosis in the liver of mice induced by a high-fat diet.

[0054] The triglyceride content was detected using a tissue cell triglyceride enzymatic assay kit, and the results are as follows: Figure 12 As shown, the triglyceride content in the liver tissue of the experimental group mice was significantly higher than that in the control group, indicating that overexpression of TRIM23 can aggravate the accumulation of triglycerides in the liver tissue of mice induced by a high-fat diet.

[0055] The experimental results from the above embodiments show that inhibiting TRIM23 expression can effectively alleviate lipid accumulation in hepatocytes, while overexpression exacerbates intracellular lipid accumulation, suggesting that TRIM23 promotes non-alcoholic fatty liver disease. Therefore, TRIM23 can be used as a target to inhibit its expression, thereby alleviating or treating non-alcoholic fatty liver disease through TRIM23 inhibitors.

[0056] The above description is merely a preferred embodiment of the present invention. The present invention is not limited to the above-described embodiments. Any embodiment that achieves the technical effects of the present invention by the same or equivalent means should fall within the protection scope of the present invention. Within the protection scope of the present invention, various modifications and variations can be made to the technical solutions and / or implementation methods.

Claims

1. The application of TRIM23 inhibitors in the preparation of drugs for treating non-alcoholic fatty liver disease, characterized in that, The TRIM23 inhibitor is siRNA, and the sequence of the siRNA is shown in SEQ ID No.

1.

2. A drug for treating non-alcoholic fatty liver disease, characterized in that, The active ingredient of the drug for treating non-alcoholic fatty liver disease is a TRIM23 inhibitor, which is siRNA, and the sequence of the siRNA is shown in SEQ ID No.

1.

3. The medicament for treating non-alcoholic fatty liver disease according to claim 2, characterized in that, The medication for treating non-alcoholic fatty liver disease also includes a delivery carrier.

4. The medicament for treating non-alcoholic fatty liver disease according to claim 2, characterized in that, The medication for treating non-alcoholic fatty liver disease also includes at least one of buffer solutions and stabilizers.

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