Use of RPN11 inhibitor capzimin in the preparation of a medicament for treating nonalcoholic steatohepatitis

Abstract

The application discloses application of an RPN11 inhibitor Capzimin in preparation of a medicine for treating non-alcoholic steatohepatitis. The application proves for the first time that the RPN11 inhibitor Capzimin can significantly improve NASH related symptoms, including reducing liver triglyceride deposition, relieving liver damage, improving liver inflammation and fibrosis, through in-vivo experiments in a high-fat high-cholesterol diet fed adult NASH mouse model, and therefore, the application provides a new medicine for effectively treating non-alcoholic steatohepatitis, and the RPN11 inhibitor Capzimin proposed in the application shows potential clinical application prospect and application value as a medicine for treating non-alcoholic steatohepatitis.

Description

Technical Field

[0001] This invention relates to the application of the RPN11 inhibitor Capzimin in the preparation of a drug for treating non-alcoholic steatohepatitis, and belongs to the field of biomedical technology. Background Technology

[0002] Nonalcoholic steatohepatitis (NASH) has become a major chronic liver and metabolic disease worldwide. The main clinical features of NASH include hepatic triglyceride deposition, chronic inflammation, and persistent liver damage. Furthermore, with the development of fibrosis, NASH can progress to cirrhosis, becoming one of the leading causes of end-stage liver diseases such as liver cancer and liver failure. However, to date, there is still a lack of effective and specific treatments in clinical practice, making NASH a disease that seriously threatens the physical and mental health of the Chinese population.

[0003] RPN11 belongs to the deubiquitinase family and exerts its effects by removing ubiquitination modifications of target proteins and increasing their stability. Previous studies have developed Capzimin, a specific inhibitor of RPN11, which has been found to inhibit tumor cell growth. However, whether Capzimin can be used as a treatment for NASH has not been reported domestically or internationally. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the RPN11 inhibitor Capzimin in the preparation of a medicament for treating non-alcoholic steatohepatitis (NASH). In adult NASH mice fed a high-fat, high-cholesterol diet, intraperitoneal injection of Capzimin significantly alleviated NASH-related symptoms, including reduced liver weight, decreased liver lipid content, and reduced liver inflammation and fibrosis.

[0005] To achieve the above objectives, this invention provides the use of the RPN11 inhibitor Capzimin in the preparation of a medicament for treating non-alcoholic steatohepatitis (NASH). The CAS number of the RPN11 inhibitor Capzimin is 2084868-04-0, and its chemical structural formula is shown below:

[0006]

[0007] Preferably, the drug comprises a medically acceptable carrier and an effective amount of an active ingredient, wherein the active ingredient is Capzimin or a pharmaceutically acceptable salt form thereof.

[0008] Preferably, the dosage form of the drug includes injections, tablets, powders, suspensions, capsules, pills, or syrups.

[0009] Preferably, the drug improves non-alcoholic steatohepatitis by reducing liver weight, reducing triglyceride levels in the liver and plasma, reducing plasma alanine aminotransferase and aspartate aminotransferase levels, and improving liver inflammation and collagen fiber deposition in the liver.

[0010] This invention also provides the use of the RPN11 inhibitor Capzimin in the preparation of a drug that reduces triglyceride levels in the liver and plasma. The CAS number of the RPN11 inhibitor Capzimin is 2084868-04-0, and its chemical structure is shown below:

[0011]

[0012] This invention also provides the use of the RPN11 inhibitor Capzimin in the preparation of a medicament for treating liver injury. The CAS number of the RPN11 inhibitor Capzimin is 2084868-04-0, and its chemical structure is shown below:

[0013]

[0014] This invention also provides the use of the RPN11 inhibitor Capzimin in the preparation of a drug to improve liver inflammation and hepatic collagen fiber deposition. The CAS number of the RPN11 inhibitor Capzimin is 2084868-04-0, and its chemical structure is shown below:

[0015]

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] In an adult NASH mouse model fed a high-fat, high-cholesterol diet, the present invention significantly improved NASH-related symptoms, including reduced liver weight, reduced liver and plasma triglyceride levels, and reduced liver inflammation and fibrosis, by using the RPN11 inhibitor Capzimin. Therefore, the present invention provides a novel therapeutic agent for the clinical treatment of non-alcoholic steatohepatitis (NASH)—the RPN11 inhibitor Capzimin. Attached Figure Description

[0018] Figure 1 The mRNA expression of RPN11 in the liver tissue of NASH mice was increased (A), and the protein expression was increased (B).

[0019] Figure 2 Capzimin significantly reduced liver weight (A), liver weight to body weight ratio (B), liver triglyceride content (C), and plasma triglyceride content (D) in NASH mice.

[0020] Figure 3 Capzimin significantly reduced plasma alanine aminotransferase (ALT) levels (A) and plasma aspartate aminotransferase (AST) levels (B) in NASH mice.

[0021] Figure 4 Capzimin significantly improved hepatic ballooning degeneration and lipid deposition in NASH mice;

[0022] Figure 5 Capzimin significantly reduced inflammatory cell infiltration in the liver of NASH mice (A, B). A: F4 / 80 immunohistochemical staining, indicating inflammatory cell infiltration and activation. B: Statistical analysis of F4 / 80 staining in the two groups of mice;

[0023] Figure 6 Capzimin significantly reduced collagen fiber deposition in the liver of NASH mice; A: Sirius red staining, indicating collagen fiber deposition; B: Statistical analysis of Sirius red staining in the livers of the two groups of mice;

[0024] Figure 7 Capzimin significantly reduced the mRNA expression of inflammation-related genes and fibrosis-related genes in the liver of NASH mice. Detailed Implementation

[0025] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0027] The experimental materials and methods involved in the following embodiments of the present invention are as follows:

[0028] 1.8-week-old male C57BL / 6 mice were purchased from Shanghai Silec Laboratory Animal Co., Ltd. Mouse housing and experiments were conducted at the Department of Laboratory Animal Science, Shanghai Jiao Tong University School of Medicine. The normal diet was purchased from Shanghai Silec Laboratory Animal Co., Ltd., catalog number M01-F, containing 10 kcal% fat, 70 kcal% carbohydrates, and 20 kcal% protein. The high-fat, high-cholesterol diet was purchased from Research Diet Pharmaceuticals, USA, catalog number D09100310, containing 40 kcal% fat, 2% cholesterol, and 20 kcal% fructose. All mice were euthanized upon reaching the experimental endpoint. Whole blood was anticoagulated, centrifuged, and the supernatant was collected to obtain plasma, which was stored at -80 degrees Celsius. In subsequent experiments, plasma triglyceride levels were measured using a kit. Fresh liver tissue was collected, flash-frozen in liquid nitrogen, and stored at -80°C. In subsequent experiments, the tissue was lysed at high temperature (95°C) in NP40 lysis buffer, and the supernatant was obtained after centrifugation. Triglyceride content was detected using a kit. Part of the frozen liver tissue was used to extract RNA for real-time quantitative PCR and Western blotting to detect the mRNA and protein expression abundance of the target genes. Fresh liver tissue was also collected, fixed in 4% paraformaldehyde for 48 hours, and then prepared into paraffin sections for eosin hematoxylin staining; frozen sections were also prepared for Oil Red O staining.

[0029] 2. Triglyceride content detection: The detection was performed according to the instructions of the triglyceride quantitative detection kit provided by Abcam, UK, catalog number ab65336.

[0030] 3. Detection of plasma alanine aminotransferase and aspartate aminotransferase: The tests were performed according to the instructions of the quantitative detection kits provided by Shanghai Kehua Co., Ltd., China.

[0031] 4. F4 / 80 Immunofluorescence Staining: F4 / 80 is a marker protein of inflammatory cells. Immunofluorescence staining of paraffin-embedded liver sections allows observation of the activation and infiltration of inflammatory cells in the liver. The F4 / 80 antibody was purchased from Abcam (UK), catalog number ab6640.

[0032] 5. Sirius Red Staining: Sirius red is an acidic dye that reacts with alkaline collagen to produce color, allowing observation of collagen fiber deposition in the liver. Sirius red dye was purchased from Shanghai Ruiyu Biotechnology Co., Ltd., catalog number Bry-0013.

[0033] 6. Detection of liver gene expression: The expression of the following genes was detected by real-time quantitative PCR: RPN11, tumor necrosis factor (TNF), interleukin 1beta (IL1b), interleukin 6 (IL6), interleukin 12beta (IL12b), inducible nitric oxide synthase 2 (Nos2), chemokine (C-Cmotif) ligand 2 (CCL2), chemokine (CC motif) ligand 5 (CCL5), and adhesion G protein-coupled receptor E1 (Adgre1) are hallmark genes of liver inflammation. Type I collagen fibers (Collagen, type I, alpha 1, Col1a1; Colagen, type I, alpha 2, Col1a2), smooth muscle cell-specific alpha-actin (Actin alpha 2, smooth muscle, Acta2), transforming growth factor beta 1 (TGFb1), and matrix metalloproteinase 13 (MMP13) are hallmark genes of liver fibrosis. The 36B4 gene was used as an internal control in real-time quantitative PCR experiments. Primer sequences are shown below.

[0034] Primers for real-time quantitative PCR experiments:

[0035] Mouse RPN11:

[0036] Forward: 5'-GGAGGTGGTGATAGCCGGTAT-3' (SEQ ID NO: 1);

[0037] Reverse:5'-TGGGTAATCCATAGAGCCCAG-3' (SEQ ID NO: 2);

[0038] Mouse 36B4:

[0039] Forward: 5'-AGATTCGGGATATGCTGTTGGC-3' (SEQ ID NO: 3);

[0040] Reverse:5'-TCGGGTCCTAGACCAGTGTTC-3' (SEQ ID NO: 4);

[0041] Mouse TNF:

[0042] Forward: 5'-CAGGCGGTGCCTATGTCTC-3' (SEQ ID NO: 5);

[0043] Reverse: 5'-CGATCACCCCGAAGTTCAGTAG-3' (SEQ ID NO: 6);

[0044] Mouse IL1b:

[0045] Forward: 5'-GAAATGCCACCTTTTGACAGTG-3' (SEQ ID NO: 7); Reverse: 5'-TGGATGCTCTCATCAGGACAG-3' (SEQ ID NO: 8);

[0046] Mouse IL6:

[0047] Forward: 5'-TAGTCCTTCCTACCCCAATTTCC-3' (SEQ ID NO: 9); Reverse: 5'-TTGGTCCTTAGCCACTCCTTC-3' (SEQ ID NO: 10);

[0048] Mouse IL12b:

[0049] Forward: 5'-TGGTTTGCCATCGTTTTGCTG-3' (SEQ ID NO: 11);

[0050] Reverse:5'-ACAGGTGAGGTTCACTGTTTCT-3' (SEQ ID NO: 12); Mouse Nos2:

[0051] Forward: 5'-CGGCAAACATGACTTCAGGC-3' (SEQ ID NO: 13);

[0052] Reverse:5'-TAGGTCGATGCACAACTGGG-3' (SEQ ID NO: 14);

[0053] Mouse CCL2:

[0054] Forward: 5’-TTAAAAACCTGGATCGGAACCAA-3’ (SEQ ID NO: 15); Reverse: 5’-GCATTAGCTTCAGATTTACGGGT-3’ (SEQ ID NO: 16); Mouse CCL5:

[0055] Forward: 5’-GCTGCTTTGCCTACCTCTCC-3’ (SEQ ID NO: 17);

[0056] Reverse: 5’-TCGAGTGACAAACACGACTGC-3’ (SEQ ID NO: 18); Mouse Adgre1:

[0057] Forward: 5’-CCCCAGTGTCCTTACAGAGTG-3’ (SEQ ID NO: 19); Reverse: 5’-GTGCCCAGAGTGGATGTCT-3’ (SEQ ID NO: 20);

[0058] Mouse Col1a1:

[0059] Forward: 5’-GCTCCTCTTAGGGGCCACT-3’ (SEQ ID NO: 21);

[0060] Reverse: 5’-CCACGTCTCACCATTGGGG-3’ (SEQ ID NO: 22);

[0061] Mouse Acta2:

[0062] Forward: 5’-GTCCCAGACATCAGGGAGTAA-3’ (SEQ ID NO: 23); Reverse: 5’-TCGGATACTTCAGCGTCAGGA-3’ (SEQ ID NO: 24);

[0063] Mouse TGFb1:

[0064] Forward: 5’-CTCCCGTGGCTTCTAGTGC-3’ (SEQ ID NO: 25);

[0065] Reverse: 5’-GCCTTAGTTTGGACAGGATCTG-3’ (SEQ ID NO: 26);

[0066] Mouse MMP13:

[0067] Forward: 5'-GGAGCCCTGATGTTTCCCAT-3' (SEQ ID NO: 27);

[0068] Reverse:5'-GTCTTCATCGCCTGGACCATA-3' (SEQ ID NO: 28);

[0069] 7. Detection of RPN11 protein expression: RPN11 protein expression was detected by Western blotting. The RPN11 antibody was purchased from Proteintech, catalog number 12059-1-AP; β-Actin protein was used as an internal control, and the antibody was purchased from Santa Cruz, catalog number sc-47778.

[0070] 8. The RPN11 inhibitor Capzimin is a commercially available reagent with CAS number 2084868-04-0. Its chemical structure is shown below:

[0071]

[0072] 9. Statistical Analysis: In this study, all numerical variables are expressed as mean plus standard error. A two-tailed Student's t-test was used to compare variables between the two groups. A p-value < 0.05 was considered statistically significant. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0073] Example 1

[0074] Increased RPN11 expression in liver tissue of NASH mice:

[0075] Feeding C57BL / 6 mice with a high-fat, high-cholesterol diet (HFHC diet) is a common method for establishing a NASH animal model. Eight-week-old male C57BL / 6 mice were randomly divided into two groups of six each, fed a normal diet or an HFHC diet, respectively. After 24 weeks, the mice were euthanized, and liver tissue from both groups was collected. Gene expression was detected using quantitative real-time PCR and Western blotting. The results showed that compared with the normal diet group, the liver RPN11 messenger RNA (mRNA) and protein expression were significantly increased in the HFHC diet-fed mice. Figure 1 (A and 1B). This result indicates that RPN11 expression is increased in the liver tissue of the NASH mouse model.

[0076] Example 2

[0077] RPN11 inhibitor Capzimin improves NASH:

[0078] The results of Example 1 suggest that inhibiting RPN11 may be a novel approach to improving NASH. In this example, a NASH model was induced by feeding 8-week-old male C57BL / 6 mice a HFHC diet for 24 weeks. Subsequently, the mice were randomly divided into two groups of six each, receiving either the RPN11 inhibitor Capzimin (2.5 mg / kg) or a control solvent (corn oil) via intraperitoneal injection once daily for two weeks. After two weeks, the mice were euthanized, and plasma and liver tissue were collected for analysis. The results showed that compared with the control group, the Capzimin-treated group had significantly lower liver weight, liver weight-to-body weight ratio, liver triglyceride content, and plasma triglyceride levels. Figure 2 A-2D). Both plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly decreased. Figure 3 A and 3B) indicate that Capzimin can alleviate liver damage. Eosin hematoxylin staining shows a reduction in hepatocyte ballooning degeneration (A and B). Figure 4 Oil Red O staining showed reduced lipid deposition in the liver. Figure 4 F4 / 80 staining showed a decrease in inflammatory cell infiltration in the liver. Figure 5 A and 5B), Sirius red staining indicates improved liver fibrosis. Figure 6 A and 6B). Gene expression analysis showed that the mRNA expression of genes related to liver inflammation and fibrosis was significantly reduced (A and 6B). Figure 7 The above results confirm that Capzimin can effectively improve NASH-related symptoms.

[0079] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. The use of the RPN11 inhibitor Capzimin in the preparation of a drug for treating non-alcoholic steatohepatitis, characterized in that, The CAS number for the RPN11 inhibitor Capzimin is 2084868-04-0, and its chemical structure is shown below: 。 2. The application as described in claim 1, characterized in that, The drug comprises a medically acceptable carrier and an effective amount of an active ingredient, wherein the active ingredient is Capzimin or a pharmaceutically acceptable salt form thereof.

3. The application as described in claim 1 or 2, characterized in that, The dosage forms of the drug include injections, tablets, powders, suspensions, capsules, pills, or syrups.

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