Application of E3 ubiquitin enzyme inhibitor in non-alcoholic fatty liver related drugs

Abstract

The invention relates to the technical field of biomedicine, in particular to application of an E3 ubiquitin enzyme inhibitor in non-alcoholic fatty liver related drugs. The TRIM23 has obvious influence on the non-alcoholic fatty liver disease, and hepatic cell lipid deposition induced by mixing of palmitic acid and oleic acid can be improved by inhibiting expression of the TRIM23 at the cellular level; on the contrary, promoting the expression level of the TRIM23 can aggravate hepatocyte lipid accumulation induced by mixing of palmitic acid and oleic acid. Therefore, the TRIM23 inhibitor can be used for preparing the medicine for preventing or treating the 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 liver cell fat, 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: The present application discloses a new function of TRIM23 gene, and the expression level of the gene in non-alcoholic fatty liver is increased. 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, a TRIM23 inhibitor can be applied to the preparation of a drug for preventing or treating non-alcoholic fatty liver. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 FIG. 1 is a detection result diagram of Western blotting in the present application, wherein β-Actin is an internal reference protein.

[0017] 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 present application, wherein *** indicates P<0.001.

[0018] Figure 3 FIG. 3 is a result diagram of oil red O staining in the present application.

[0019] Figure 4 FIG. 4 is a detection result diagram of triglyceride in the present application, wherein * indicates P<0.05.

[0020] Figure 5 Figure for detection result of Western blotting in Example 2 of the present application, wherein β-Actin is the internal reference protein.

[0021] Figure 6 Figure for quantitative analysis statistics of TRIM23 protein expression amount in detection result of Western blotting in Example 2 of the present application, wherein ** represents P<0.01.

[0022] Figure 7 Figure for oil red O staining result in Example 2 of the present application.

[0023] Figure 8 Figure for triglyceride detection result in Example 2 of the present application, wherein ** represents P<0.01.

[0024] Figure 9 Figure for detection result of Western blotting in Example 3 of the present application, wherein β-Actin is the internal reference protein.

[0025] Figure 10 Figure for quantitative analysis statistics of TRIM23 protein expression amount in detection result of Western blotting in Example 3 of the present application, wherein ** represents P<0.01.

[0026] Figure 11 Figure for hematoxylin-eosin staining and oil red O staining result in Example 3 of the present application.

[0027] Figure 12 Figure for triglyceride detection result in Example 3 of the present application, wherein ** represents P<0.01. DETAILED DESCRIPTION

[0028] The concept and technical effects of the present application will be described clearly and completely below to fully explain the purpose, scheme and effects of the present application.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] Example 1 Effect of TRIM23 knockdown on cell lipid accumulation 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 per 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 per 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.

[0033] 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.

[0034] 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 stimulation with compound fatty acids, oil red O staining was performed to detect 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.

[0035] After the control group and the experimental group were stimulated with complex fatty acids for 24 hours, the cells were washed twice with 1x PBS buffer, and then the intracellular triglyceride content was detected using a tissue cell glycerol triesterase method kit (purchased from Beijing Puli Lei Gene Technology Co., Ltd., item number E1013). The triglyceride content detection results are shown in Figure 4 As shown in the figure, the triglyceride content of the experimental group (knockdown group) was significantly lower than that of the control group, indicating that knocking down TRIM23 can alleviate the increase in triglyceride content induced by complex fatty acids.

[0036] Example 2 Effect of TRIM23 overexpression on cell lipid accumulation First, the TRIM23 target gene was amplified by PCR, and after agarose gel electrophoresis, the DNA recovery kit (purchased from Nanjing Nuowezan Biotechnology Co., Ltd.) was used for gel recovery; pcDNA3.1 was used as a vector, and the vector and the recovered TRIM23 fragment were double-digested, respectively. After agarose gel electrophoresis of the enzyme digestion products, the enzyme-digested vector backbone and TRIM23 fragment were recovered; the recovered target gene and linear vector were ligated, 16°C overnight, to obtain the ligation product; then the ligation product was transformed, single colonies were picked, colony PCR and agarose gel electrophoresis were performed to verify positive colonies, and the positive colonies were inoculated in LB liquid medium containing ampicillin, 37°C shaking for 12h, and the endotoxin-free plasmid extraction kit (purchased from Tian Gen Biochemical Technology (Beijing) Co., Ltd.) was used for plasmid extraction, and the TRIM23 overexpression plasmid was obtained.

[0037] AML12 cells were seeded and divided into control and overexpression groups. The TRIM23 overexpression plasmid was transfected into the overexpression group cells using LIPO3000 and P3000, and the control group was transfected with pcDNA3.1 empty plasmid. Western blotting was performed 48 hours after transfection to detect the TRIM23 protein level.

[0038] The results of Western blotting are shown in Figure 5 and Figure 6 The TRIM23 protein level of the overexpression group was significantly higher than that of the control group, indicating that the overexpression of TRIM23 in AML12 cells was successfully achieved.

[0039] Complex fatty acids were added to the control and overexpression groups for 24 hours, and then oil red O staining was performed to detect lipid accumulation. The results of oil red O staining are shown in Figure 7 As shown in the figure, there was a significant amount of lipid droplet accumulation in the control group, and the staining was obvious; while the lipid accumulation in the overexpression group was more severe, and the number and degree of lipid droplet staining increased significantly. This indicates that overexpression of TRIM23 can exacerbate the accumulation of lipids in hepatocytes induced by complex fatty acids.

[0040] After the control group and overexpression group cells were stimulated with complex fatty acids for 24 hours, the triglyceride content was also detected using a tissue cell glycerol triesterase kit.

[0041] The triglyceride content detection results are shown in Figure 8 The triglyceride content of the overexpression group was significantly higher than that of the control group, indicating that overexpression of TRIM23 can aggravate triglyceride accumulation induced by complex fatty acids.

[0042] Example 3: Effect of TRIM23 overexpression on lipid accumulation in mouse liver The animal experiment was performed according to the following steps: (1) TRIM23 overexpression plasmid acquisition The TRIM23 overexpression plasmid was purchased from Beijing Genki Biotechnology Co., Ltd. The plasmid uses pLive as a vector and inserts the TRIM23 target gene. Using plate streaking method, dip the glycerol bacteria of the purchased plasmid in solid medium containing kanamycin for four-zone streaking, and invert in a 37°C incubator overnight. The next day, single colony bacteria were picked and inoculated in LB liquid medium containing antibiotics, and shaken at 200 rpm for 12-16 hours. Then the plasmid was extracted using a kit to obtain the TRIM23 overexpression plasmid.

[0043] (2) Experimental animals and feeding The experimental animals were C57BL / 6 (C57) mice, 6-8 weeks old, male, purchased from Jisui Yekang Biotechnology Co., Ltd. All experimental mice were raised in the SPF animal room of the Transgenic Animal Center of the Nanchang University Translational Medicine Research Institute, with license number SYXK (Jiangxi) 2021-0001. The light was alternated every 12 hours, the temperature was 24±2°, the humidity was 40%-70%, and the mice were free to drink water and eat.

[0044] The mice were randomly divided into control and experimental groups, and both groups of mice were fed with high-fat diet for 12 weeks. The experimental group mice were injected with TRIM23 overexpression plasmid in the tail vein at the first and fifth weeks, and the control group mice were injected with pLive empty plasmid. After 12 weeks, the samples were collected and the mouse liver was taken.

[0045] 20 mg of liver tissue was weighed, the tissue protein was extracted, and Western blotting was performed to detect the TRIM23 expression level of the control and experimental groups. The results are shown in Figure 9 and Figure 10 Compared with the control group, the TRIM23 protein level of the experimental group increased significantly, indicating that high expression of TRIM23 in mouse liver tissue was successfully achieved.

[0046] The mouse liver tissue was sectioned, and then stained with hematoxylin-eosin (HE) and oil red O. The staining results are shown in Figure 11As shown, the liver tissue of the high-fat-fed mice was obviously steatotic and vacuolization was significant; overexpression of TRIM23 aggravated this phenomenon, indicating that promoting the expression of TRIM23 can aggravate the steatosis of the liver of the high-fat-fed mice.

[0047] The triglyceride content was detected by using a tissue cell glycerol triesterase method detection kit, and the results are shown in Table 1. Figure 12 As shown, the triglyceride content of the liver tissue of the experimental mice was significantly higher than that of the control group, indicating that overexpression of TRIM23 can aggravate the accumulation of triglyceride in the liver tissue of the high-fat-fed mice.

[0048] According to the experimental results of the above embodiments, inhibiting the expression level of TRIM23 can effectively alleviate the lipid accumulation of hepatocytes, and overexpression can aggravate the lipid accumulation in cells, suggesting that TRIM23 plays a promoting role in non-alcoholic fatty liver. Therefore, TRIM23 is taken as a target, and the expression is inhibited as a guide, and non-alcoholic fatty liver is relieved or treated by a TRIM23 inhibitor.

[0049] The above is only a preferred embodiment of the present application, and the present application is not limited to the above-described embodiments. As long as the same or equivalent means achieve the technical effects of the present application, they should belong to the protection scope of the present application. Within the protection scope of the present application, the technical solutions and / or embodiments can have various modifications and changes.

Claims

1. The use of TRIM23 inhibitors in the preparation of drugs for the prevention or treatment of non-alcoholic fatty liver disease, characterized in that, The TRIM23 inhibitor is an inhibitor that inhibits the mRNA expression level of TRIM23 and / or an inhibitor that inhibits the protein expression level of TRIM23.

2. The application according to claim 1, characterized in that, The inhibitors that suppress the mRNA expression level of TRIM23 include at least one of siRNA, shRNA, and miRNA.

3. The application according to claim 2, characterized in that, The inhibitor that suppresses the mRNA expression level of TRIM23 is siRNA, and the sequence of siRNA is shown in SEQ ID No.

1.

4. A drug for the prevention or treatment of non-alcoholic fatty liver disease, characterized in that, This includes TRIM23 inhibitors, which are inhibitors that inhibit the mRNA expression level of TRIM23 and / or inhibitors that inhibit the protein expression level of TRIM23.

5. The medicament for preventing or treating non-alcoholic fatty liver disease according to claim 4, characterized in that, The inhibitors that suppress the mRNA expression level of TRIM23 include at least one of siRNA, shRNA, and miRNA.

6. The medicament for preventing or treating non-alcoholic fatty liver disease according to claim 5, characterized in that, The inhibitor that suppresses the mRNA expression level of TRIM23 is siRNA, and the sequence of siRNA is shown in SEQ ID No.

1.

7. The medicament for preventing or treating non-alcoholic fatty liver disease according to claim 4, characterized in that, The inhibitors that suppress the protein expression level of TRIM23 include at least one of plasmids, peptides, enzymes, antibodies, viruses, and polynucleotides.

8. The medicament for preventing or treating non-alcoholic fatty liver disease according to claim 4, characterized in that, The drugs for preventing or treating fatty liver also include at least one of the following: a drug delivery carrier, a buffer solution, and a stabilizer.

Images