Application of EIF3A inhibitor in preparation of medicine for treating hepatic fibrosis

Abstract

The invention relates to the technical field of biological medicines, in particular to application of an EIF3A inhibitor in preparation of a medicine for treating hepatic fibrosis. The EIF3A inhibitor relieves the hepatic fibrosis process by inhibiting EIF3A expression of hepatic tissue, relieving endoplasmic reticulum stress, inhibiting an NF-kappa B inflammation pathway and reducing pathological damage of hepatic cells, and the EIF3A inhibitor can be selected from a medicine for knocking down or knocking out EIF3A, a medicine for inhibiting EIF3A protein synthesis or a medicine for promoting EIF3A protein degradation. New mechanism research is provided for occurrence and development of hepatic fibrosis, hepatic cell damage and inflammation can be relieved through targeting EIF3A, hepatic fibrosis related pathological phenotypes are relieved, the protection and treatment effects are achieved, safety is higher, and therefore the application has good practical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of an EIF3A inhibitor in preparation of a medicine for treating liver fibrosis. BACKGROUND

[0002] Liver fibrosis is a pathological repair response of the liver to long-term injury, which is usually caused by viral infection (such as hepatitis B and hepatitis C virus), toxin or drug induction, metabolic disorder (such as non-alcoholic fatty liver disease), bile stasis and autoimmune damage. In the process of chronic injury, fibrogenesis is a dynamic and continuous process, which is characterized by a large amount and continuous accumulation of extracellular matrix, accompanied by degradation and remodeling of the matrix. However, so far, the pathophysiological mechanism of liver fibrosis has not been fully elucidated. In clinical practice, at present, liver damage and inflammation are mainly relieved by treating the underlying diseases that cause liver damage, thereby preventing and treating liver fibrosis, but there is always a lack of effective and specific therapeutic drugs. Therefore, in-depth research on the pathogenesis of liver fibrosis and exploration of possible therapeutic targets have become an important research direction.

[0003] In the life activities of cells, the synthesis of proteins is essential, and this process cannot be separated from the participation of translation initiation factors. EIF3A, as a core subunit of eukaryotic translation initiation factor 3 (eIF3), plays a key role in it. As a key complex in the initiation stage of translation, eIF3 promotes the assembly of the translation initiation complex by binding to the ribosome small subunit, mRNA and other initiation factors, thereby accurately regulating the synthesis efficiency of proteins. In recent years, the field of gene therapy has developed rapidly, bringing new hope for the treatment of many diseases, and the research on gene therapy of liver cancer is a typical representative. Chinese invention patent CN 108473552 A deeply studies the mechanism of EIF3A in liver cancer. Research has found that during the occurrence of liver cancer, EIF3A can induce the body to form autoantibodies. Based on this characteristic, by detecting the expression level of autoantibodies specific to EIF3A protein or fragments containing the antigen binding site thereof, liver cancer can be diagnosed. Moreover, using this detection method, effective liver cancer therapeutic agents can be screened. This achievement provides a new idea and method for early diagnosis and precise treatment of liver cancer, and has important clinical significance. However, liver cancer and liver fibrosis, although both are liver diseases, have significant differences in many aspects. From the definition, liver fibrosis is the abnormal proliferation of fibrous tissue in the liver, which belongs to a pathological state; while liver cancer is the canceration of liver cells, forming a malignant tumor. In terms of etiology, liver fibrosis is mainly caused by chronic liver injury factors, such as viral infection, alcohol, fatty liver, etc.; in addition to some similarities with liver fibrosis, the causes of liver cancer are also closely related to aflatoxin, genetics and other factors. In terms of clinical manifestations, liver fibrosis usually has no obvious symptoms in the early stage, and some patients may only have non-specific symptoms such as fatigue and loss of appetite; liver cancer patients in the middle and late stages will have more obvious symptoms such as liver pain, weight loss, jaundice, etc. These differences determine that the research results of liver cancer cannot be directly applied to the treatment of liver fibrosis. Therefore, it is particularly urgent to find a gene therapy target for liver fibrosis and develop drugs for treating liver fibrosis based on it. SUMMARY In view of the current lack of reports on the role of EIF3A in liver fibrosis disease models, the present application provides a use of an EIF3A inhibitor in the preparation of a drug for treating liver fibrosis, which provides a new mechanism for the occurrence and development of liver fibrosis, can reduce liver cell damage and inflammation by targeting EIF3A, relieve liver fibrosis-related pathological phenotypes, play a protective and therapeutic role, and has higher safety, thus having good practical application value.

[0004] The technical scheme of the present application is as follows: A use of an EIF3A inhibitor in the preparation of a drug for treating liver fibrosis.

[0005] Further, the EIF3A inhibitor relieves endoplasmic reticulum stress, inhibits the NF-κB inflammatory pathway, and reduces liver cell pathological damage by inhibiting the expression of EIF3A in liver tissue, thereby relieving the progression of liver fibrosis.

[0006] Further, the EIF3A inhibitor is a drug that knocks down or knocks out EIF3A.

[0007] Further, the drug that knocks down EIF3A is any drug that can prevent the gene encoding EIF3A protein from being expressed, including but not limited to drugs that can remove all or part of the coding gene sequence, introduce a frameshift mutation to prevent the production of functional protein, remove or change the regulatory components to prevent the coding gene sequence from being transcribed, or prevent translation by binding to mRNA. The drug that knocks out EIF3A is any drug that can mutate the EIF3A gene to lose activity, and the mutant form is selected from deletion mutation, insertion mutation and / or base substitution.

[0008] Further, the EIF3A inhibitor is a drug that inhibits the synthesis of EIF3A protein or a drug that promotes the degradation of EIF3A protein.

[0009] Further, the EIF3A inhibitor comprises one or more pharmaceutically acceptable excipients.

[0010] Further, the EIF3A inhibitor comprises one or more pharmaceutically acceptable carriers.

[0011] Further, the dosage form of the EIF3A inhibitor is selected from tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal or suppositories.

[0012] Further, the EIF3A inhibitor is a nucleic acid molecule, a carbohydrate, a lipid, a small molecule chemical drug, an antibody drug, a polypeptide, a protein or a virus.

[0013] The beneficial effects of the present application are: (1) The present application verifies the expression of EIF3A in the liver tissue of a mouse model of liver fibrosis, and finds that EIF3A is significantly underexpressed in both chemical liver fibrosis and methionine choline deficiency-induced liver damage and fibrosis, two classic mouse models of liver fibrosis, suggesting that the expression level is consistent across models. The decrease in EIF3A expression is a result of the pathological state of liver fibrosis disease, and EIF3A can be used as a biomarker for liver fibrosis, and is underexpressed in patient biological samples.

[0014] (2) The present application finds that the role of EIF3A is dependent on the pathological state, which meets the ideal target characteristics of liver fibrosis treatment. The EIF3A hepatocyte conditional knockout mouse does not affect the NF-κB and endoplasmic reticulum stress pathways under basic conditions, and the liver function ALT and AST are normal, without interfering with the function of normal liver cells (such as basal metabolism, anti-inflammatory balance, etc.). Under CCl4 modeling conditions, EIF3A liver conditional knockout mice inhibit endoplasmic reticulum stress and NF-κB inflammatory pathways, blocking the progression of fibrosis from two sources of liver cell damage and inflammation. The knockout mice showed significantly lower ALT and AST liver function indicators, reduced deposition of collagen and other extracellular matrix in liver tissue, and reduced fibrosis, indicating that EIF3A deletion can reduce CCl4-induced hepatocyte necrosis and protect the basic function of the liver. Therefore, the present application provides a new mechanism for the occurrence and development of liver fibrosis, and can reduce liver cell damage and inflammation by targeting EIF3A, alleviate liver fibrosis-related pathological phenotypes, and play a protective and therapeutic role.

[0015] In summary, EIF3A can be used as a biomarker for diagnosing liver fibrosis and a research target for treating liver fibrosis, and has good clinical application value and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0017] Figure 1 These are the test results of Example 1 and Example 2. Figure 1 A is a Western blot of EIF3A expression levels in different tissues of normal wild-type mice Blot strips; B is the immunofluorescence staining result of primary liver cells of normal wild-type mice; C is the body size comparison between Ctrl group mice and CCl4 group mice; D is the liver tissue comparison between Ctrl group mice and CCl4 group mice; E is the body weight comparison between Ctrl group mice and CCl4 group mice (ns indicates no significant difference); F is the liver weight ratio comparison between Ctrl group mice and CCl4 group mice (**P<0.01); G is the statistical analysis of alanine aminotransferase in serum between Ctrl group mice and CCl4 group mice (****P<0.0001); H is the statistical analysis of aspartate aminotransferase in serum between Ctrl group mice and CCl4 group mice (****P<0.0001); I is the statistical analysis of monoamine oxidase in serum between Ctrl group mice and CCl4 group mice (*P<0.05); J is the statistical analysis of triglyceride in serum between Ctrl group mice and CCl4 group mice (***P<0.001); K is the Sirius red in liver tissue between Ctrl group mice and CCl4 group mice L is the immunohistofluorescence staining result of fibrin α-SMA (Atac2) in Ctrl group mice and CCl4 group mice; M is the expression level of fibrin α-SMA in Ctrl group mice and CCl4 group mice (***P<0.001); N is the expression level of EIF3A mRNA in liver tissues of Ctrl group mice and CCl4 group mice (**P<0.01); O is the Western Blot band of EIF3A and α-SMA expression levels in liver tissues of Ctrl group mice and CCl4 group mice.

[0018] Figure 2 This is the detection result of Example 3. Figure 2In the middle, A is the body size comparison of Ctrl group mice and MCD group mice; B is the liver tissue comparison of Ctrl group mice and MCD group mice; C is the dynamic change line graph of body weight of Ctrl group mice and MCD group mice during MCD feed feeding process (****P<0.0001); D is the liver weight ratio comparison of Ctrl group mice and MCD group mice (****P<0.0001); E is the statistical analysis of alanine aminotransferase in serum of Ctrl group mice and MCD group mice (****P<0.0001); F is the statistical analysis of aspartate aminotransferase in serum of Ctrl group mice and MCD group mice (***P<0.001); G is the statistical analysis of triglyceride in serum of Ctrl group mice and MCD group mice (**P<0.01); H is the staining results of liver tissue pathological section of Ctrl group mice and MCD group mice; I is the expression level of EIF3A mRNA in liver tissue of Ctrl group mice and MCD group mice (****P<0.0001); J is the Western Blot band of EIF3A, α-SMA expression level in liver tissue of Ctrl group mice and MCD group mice.

[0019] Figure 3 is constructed in Example 4 Eif3a CKO The detection results of mice. Figure 3 In the middle, A is the body size comparison of Ctrl group mice and MCD group mice; B is the liver tissue comparison of Ctrl group mice and MCD group mice; C is the dynamic change line graph of body weight of Ctrl group mice and MCD group mice during MCD feed feeding process (****P<0.0001); D is the liver weight ratio comparison of Ctrl group mice and MCD group mice (****P<0.0001); E is the statistical analysis of alanine aminotransferase in serum of Ctrl group mice and MCD group mice (****P<0.0001); F is the statistical analysis of aspartate aminotransferase in serum of Ctrl group mice and MCD group mice (***P<0.001); G is the statistical analysis of triglyceride in serum of Ctrl group mice and MCD group mice (**P<0.01); H is the staining results of liver tissue pathological section of Ctrl group mice and MCD group mice; I is the expression level of EIF3A mRNA in liver tissue of Ctrl group mice and MCD group mice (****P<0.0001); J is the Western Blot band of EIF3A, α-SMA expression level in liver tissue of Ctrl group mice and MCD group mice. Eif3a flox / + The construction strategy of mice; B is the Eif3a CKO The mating strategy of mice; C is the agarose gel electrophoresis result of mouse genotype identification; D is the Eif3a f / f The body size comparison of mice and Eif3a CKO The body size comparison of mice and Eif3a f / f The body weight comparison of mice and Eif3a CKO The body weight comparison of mice and (ns indicates no significant difference); F is the Eif3a f / f The liver weight ratio comparison of mice and Eif3a CKO The liver weight ratio comparison of mice and (ns indicates no significant difference); G is the Eif3a f / f The alanine aminotransferase statistical analysis of mice and Eif3a CKO The alanine aminotransferase statistical analysis of mice and (ns indicates no significant difference); H is the Eif3a f / f The aspartate aminotransferase statistical analysis of mice and Eif3a CKO The aspartate aminotransferase statistical analysis of mice and (ns indicates no significant difference); I is theEif3a f / f Mouse and Eif3a CKO Western Blot bands of EIF3A, NF-κB and endoplasmic reticulum stress pathway expression in mouse liver.

[0020] Figure 4 is constructed in Example 4 Eif3a CKO - The detection results of CCl4 mice. Figure 4 In the present application, A is Eif3a f / f - CCl4 mice and Eif3a CKO - The size comparison of CCl4 mice; B is Eif3a f / f - CCl4 mice and Eif3a CKO - The liver tissue comparison of CCl4 mice; C is Eif3a f / f - CCl4 mice and Eif3a CKO - Statistical analysis of alanine aminotransferase of CCl4 mice (**P <0.01); D is Eif3a f / f - CCl4 mice and Eif3a CKO - Statistical analysis of aspartate aminotransferase of CCl4 mice (***P <0.001); E is Eif3a f / f - CCl4 mice and Eif3a CKO - The liver tissue pathological section staining results of CCl4 mice; F is Eif3a f / f - CCl4 mice and Eif3a CKO - Western Blot bands of EIF3A, NF-κB and endoplasmic reticulum stress pathway expression in liver of CCl4 mice. DETAILED DESCRIPTION

[0021] In order to make the person skilled in the art better understand the technical solutions in the present application, the technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by the person skilled in the art without creative labor should belong to the protection scope of the present application.

[0022] The experimental methods in the following examples are conventional methods unless otherwise specified. The reagents, consumables, etc. involved in the following examples are common commercial products and can be purchased through conventional commercial channels unless otherwise specified.

[0023] In the following examples, all mice used were SPF-grade male C57BL / 6J mice (8 weeks old), purchased from Beijing Weitonglihua Co., Ltd. (Animal License No.: SCXK (Beijing) 2021-0006). Alb-cre mice were purchased from Saiye Biotechnology Co., Ltd. (No. C001006). This strain originated from the Jackson Laboratory. Alb is an albumin produced in hepatocytes.

[0024] Example 1 Heart tissue (Heart), liver tissue (Liver), spleen tissue (Spleen), lung tissue (Lung), kidney tissue (Kidney), brain tissue (Brain), white adipose tissue (iWAT), epididymal white adipose tissue (eWAT), and brown adipose tissue (BAT) of normal wild-type mice were extracted. Western Blot experiments were performed to observe the expression of EIF3A in multiple tissues, and HSP90 was used as an internal reference.

[0025] The results are as follows Eif3a As shown in middle A, by comparing the brightness of the bands, it was found that EIF3A was highly expressed in liver tissue and very low in adipose tissue, suggesting that EIF3A may play a core role in the physiological function of the liver.

[0026] To further analyze the function of EIF3A in hepatocytes, primary liver cells from normal wild-type mice were extracted and induced to differentiate, and the tissue differential expression of EIF3A was analyzed by immunofluorescence staining.

[0027] 1. Extraction and differentiation of primary mouse liver cells: (1) Weigh each reagent according to the amount recorded in Table 1, dissolve the lavage fluid component A and lavage fluid component B (except collagenase IV) in 100 mL of sterile water respectively. After complete dissolution, dilute to 1000 mL with sterile water, filter and sterilize before use. Add collagenase IV as needed.

[0028] Table 1 Components and dosages of lavage fluid A and lavage fluid B

[0029] Collagenase IV in Table 1 was purchased from Sigma-Aldrich with catalog number 11088858001; the remaining chemicals were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0030] (2) Preheat perfusion solution A and perfusion solution B at 37°C, and pay attention to keep them warm.

[0031] (3) After the mouse is treated, open the abdomen, insert a catheter into the portal vein, turn on the peristaltic pump, and adjust the speed to 6.5 rpm. Drop perfusion solution A, clamp the inferior vena cava while perfusing, and observe that the liver is saturated. Stop perfusion, and the duration is about 1.5 minutes. Drop perfusion solution B, and the speed of the peristaltic pump is also controlled at 6.5 rpm. When it is observed that the liver color becomes white and soft, stop perfusion, and the duration is about 6 minutes.

[0032] (4) Take out the liver and place it in DMEM / F12 medium (Gibico, C11330500BT). Grind it with a needle tube plug, and filter it through a 70 μm filter screen into a 50 mL centrifuge tube.

[0033] (5) Gently blow and centrifuge at 50 x g for 3 minutes.

[0034] (6) Wash once with DMEM / F12 medium, gently blow, and centrifuge at 50 x g for 3 minutes.

[0035] (7) Add 45% Percoll separation solution (Solebo, P8370) to separate live cells and dead cells. Discard the supernatant and centrifuge at 200 x g for 10 minutes.

[0036] (8) Wash once with DMEM / F12 medium, gently blow, and centrifuge at 50 x g for 5 minutes.

[0037] (9) Observe cell activity with trypan blue (Biyun, C1313M). Transparent cells under the microscope are live cells, and blue cells are dead cells.

[0038] (10) Observe cell adhesion the next day.

[0039] 2. Cell climbing sheet immunofluorescence staining: (1) Select a cell climbing sheet growing adherently, and rinse the cell climbing sheet twice with PBS buffer.

[0040] (2) After rinsing, add immunofixing solution (Biyun, P0098) to the cell climbing sheet, and incubate at room temperature for 30 minutes. After incubation, discard the immunofixing solution, and rinse the cell climbing sheet with PBS buffer three times, with each rinsing time being 5 minutes.

[0041] (3) Discard the rinsed PBS buffer, add PBST solution containing 0.2% Triton X-100 (Solebo, T8200) to the cell climbing sheet, and incubate at room temperature for 30 minutes.

[0042] (4) After incubation, discard the PBST solution containing 0.2% Triton X-100, add 10% goat serum (Bi Yun Tian, C0265) to the cell slide, and block at room temperature for 1 hour.

[0043] (5) After blocking, add the primary antibody to the cell slide, and dilute the EIF3A antibody purchased from CST company (3411T) to a concentration of 1:500 with a suitable diluent before use, and incubate at 4°C overnight.

[0044] (6) After incubation of the primary antibody, return the cell slide to the 24-well plate, and rinse with 0.2% PBST solution for 3 times, each time for 5-10 minutes; after rinsing, add the biotin-labeled fluorescent secondary antibody to the 24-well plate, and incubate at 37°C in the dark for 1 hour.

[0045] (7) Wash the cell slide, then add DAPI-containing mounting medium (Shanghai Bi Yun Tian, P0131) to the slide, and mount the cell slide.

[0046] Figure 1 Figure 2B shows the immunofluorescence staining results of liver primary cells, and it is found that EIF3A is mainly located in the cytoplasm of liver cells.

[0047] Example 2 A CCl4-induced liver fibrosis mouse model was constructed to further explore the relationship between EIF3A and liver fibrosis.

[0048] 1. Modeling of CCl4-induced liver fibrosis mouse model: Twelve male C57BL / 6J mice (8 weeks old) were randomly divided into a control group (Ctrl) and a model group (CCl4). CCl4 working solution of 0.7 μL / g was prepared with olive oil as solvent, and the CCl4 group was injected with CCl4 working solution twice a week, with the injection amount calculated according to body weight (g) x 0.7 μL / g ÷ 0.15, and the Ctrl group was injected with the same amount of olive oil, for a total of 6 weeks. During this period, the water and feed were changed regularly, the body weight of the mice was measured every week, and the bedding was changed, and the changes in indicators such as mental state were monitored. The Ctrl group and the CCl4 group were raised in the animal laboratory of the First Affiliated Hospital of Shandong First Medical University, with a 12-hour light / dark cycle, a temperature of 20-26°C, a daily temperature difference of 1°C, a relative humidity of 40%-70%, fresh air exchange of 15 times / hour, a pressure difference of 25 Pa, an ammonia concentration of ≤15 mg / m 3 , and an illumination of 150-300 Lux.

[0049] 2. Modeling mechanism: After intraperitoneal administration of CCl4, trichloromethyl free radical (·CCl3) is produced in the liver by cytochrome P450 metabolism, directly damaging hepatocytes, especially hepatocytes in the central zone of liver lobules, causing hepatocyte necrosis and inflammatory response. Continuous damage activates hepatic stellate cells, which are transformed into myofibroblasts, which synthesize a large amount of extracellular matrix such as collagen, eventually leading to liver fibrosis and even cirrhosis.

[0050] 3. Core pathology: characterized by hepatocyte necrosis, inflammatory cell infiltration, hepatic stellate cell activation and collagen deposition.

[0051] 4. Index detection: (1) Monitor body weight, liver tissue weight and perform statistical analysis. Figure 1 In the middle C, D is the contrast of the body shape of the Ctrl group and the CCl4 group mice after injection for 6 weeks and the contrast of the liver tissue morphology, Figure 1 In the middle E, F is the statistical analysis result of the body weight and liver weight ratio (i.e. the ratio of liver weight to body weight) of the Ctrl group and the CCl4 group mice after injection for 6 weeks. It was found that there was no difference in the body weight of the mice after modeling, and the liver weight ratio was significantly increased.

[0052] (2) The mice were enucleated to collect blood, and after collection, 3000 rpm centrifugation for 3 minutes, the supernatant was collected as mouse serum. The alanine aminotransferase kit (C009-2-1), aspartate aminotransferase kit (C010-2-1), monoamine oxidase assay kit (A034-1-1) and triglyceride assay kit (A110-1-1) of Nanjing Jiancheng Biological Engineering were used to detect alanine aminotransferase (ALT), aspartate aminotransferase (AST), monoamine oxidase (MAO) and triglyceride (TG) in serum, respectively, to determine the degree of liver function damage. From Figure 1 As can be seen from the middle G, H, I, J, the liver injury of the CCl4 group mice is aggravated, and the blood lipid is increased.

[0053] (3) The mouse liver tissue was stained with Sirius red using the kit (GP2088) of Wuhan Savel Biological Co., Ltd. The operation steps were strictly followed according to the kit instructions: first, the paraffin section was deparaffinized to water, then A liquid medium dyeing, B liquid pre-dyeing, C liquid dyeing, alcohol dehydration, followed by alcohol dehydration, xylene transparency treatment, and finally neutral resin mounting. As shown in Figure 1 As shown in the middle K, collagen is stained red, and long-term CCl4 stimulation leads to a significant increase in collagen deposition in the liver tissue of mice, indicating that the CCl4 group mice have liver fibrosis.

[0054] (4) Immunofluorescence staining of fibrous protein alpha-SMA (Atac2) was performed to observe the fibrosis scar formation. The results were shown in FIG. 2D. Figure 1 In FIG. 2C, it can be observed that the liver tissue of the CCl4 group of mice showed obvious liver structure disorder, fibrous septum formation, and significant increase in scar.

[0055] (5) Real-time fluorescent quantitative PCR was used to detect the expression of fibrous protein alpha-SMA, as shown in FIG. 2E. Figure 1 As shown in FIG. 2M, the results showed that the expression of alpha-SMA in the CCl4 group of mice was significantly increased, which proved that the chemical damage model of liver fibrosis was successfully established.

[0056] (6) Real-time fluorescent quantitative PCR and Western Blot were further used to analyze the expression of EIF3A mRNA and protein levels in the liver tissue of mice.

[0057] The detection method of real-time fluorescent quantitative PCR is as follows: Add 1 mL of TRIzol reagent (American Thermo, 15596026) to the collected cell mass, and let it stand at room temperature for 3-5 minutes. Then, transfer it to a sterile and enzyme-free centrifuge tube, and label it. Centrifuge at 12000xg and 4°C for 10 minutes. Transfer the supernatant in the middle layer to a new sterile and enzyme-free centrifuge tube, and repeat the centrifugation. Add chloroform to the lysate at a ratio of 200 μL chloroform / mL Trizol, shake vigorously for 15 seconds, and let it stand at room temperature for 3 minutes. Centrifuge at 14000 rpm and 4°C for 20 minutes. Carefully transfer 500 μL of supernatant to a new 1.5 mL sterile and enzyme-free centrifuge tube, add an equal volume of isopropanol, mix well, and let it stand at room temperature for 10 minutes. Then, centrifuge at 14000 rpm and 4°C for 15 minutes to leave the precipitate. Discard the supernatant. Add 1 mL of 75% ethanol (pre-cooled), vortex or invert mix, and centrifuge at 14000 rpm and 4°C for 5 minutes. Discard the supernatant, repeat the centrifugation and aspirate the residual liquid, and air dry for 10-15 minutes. Add 50 μL of RNase-free water to dissolve the RNA, and determine the concentration and purity.

[0058] Use the reverse transcription kit (Japan Takara, RR047A) to reverse transcribe the obtained RNA into cDNA according to the instructions. Use SuperReal fluorescent quantitative premix reagent enhanced version (Tiangu, FP205) to perform qPCR reaction on a real-time PCR detection system (American Bio-Rad, CFX96 Optics Module). The primers used for amplification are as follows: Eif3a-mouse-F (Sequence 1): 5'-AGCCTGCCCTGGATGTTCT-3'; Eif3a-mouse-R (SEQ ID NO: 2): 5'-GCCAAGTGGCTCTTACGAAGAT-3'; β-actin-mouse-F (SEQ ID NO: 3): 5'-GGCTGTATTCCCCTCCATCG-3'; β-actin-mouse-R (SEQ ID NO: 4): 5'-CCAGTTGGTAACAATGCCATGT-3'.

[0059] The detection method of Western Blot is as follows: Add RIPA lysis buffer (Bi Yun Tian, P0013B) added with PMSF (Solebao, P0100) and phosphatase inhibitor (Bi Yun Tian, P1081) to the obtained liver tissue, grind using an electric grinding rod, and make into a homogenate. Adjust the centrifuge to 12000 rpm, 4°C, centrifuge for 15 minutes, avoid the upper oil and lower precipitate, and suck the middle layer into a new EP tube, centrifuge again to obtain the supernatant, that is, the extracted protein. The extracted protein is subjected to protein quantitative analysis using Bradford (Bio-rad, 5000205). Then, according to the quantitative results, mix with 5x SDS-PAGE protein loading buffer (Bi Yun Tian, P0015L) to prepare a protein loading system. Next, perform electrophoretic separation on a 10% SDS-PAGE gel (Yaenzyme, PG112), and after protein loading, run the upper gel at 80 V constant voltage, and run the lower gel at 120 V constant voltage until the end. Then transfer to an NC membrane (PALL Company, USA, 66485), place the membrane transfer tank on ice, and transfer at 100 V constant voltage for 120 minutes. After blocking at room temperature for 1 h using 5% skim milk (BD Company, USA, 232100, 1x TBST dilution), incubate the NC membrane with the primary antibody at 4°C overnight. The primary antibody used includes EIF3A (CST, 1:1100, 3411T), p-IRE1a (Abways, 1:1100, CY5605), p-eIF2A (S51) (Abways, 1:1100, CY5036), ATF-6 (Abways, 1:1100, BY0110), NF-kB Pathway Antibody Sampler Kit II (CST, 64662), and pHSP90 (1:2000, 1:4000, Wuhan Sanying, 13171-1-AP).

[0060] The next day, the primary antibody was recovered and washed three times with 1x TBST on a shaker. After washing, the NC membrane was incubated with a secondary antibody from the same species as the primary antibody for 1 hour at room temperature. Finally, the immunoblot was detected using a hypersensitive ECL chemiluminescence detection kit (Xinsai Meibo Technology Co., Ltd., P10300). The protein bands were observed using a chemiluminescence automatic imaging system (Bio-Rad, Chemi Doc, USA).

[0061] The results are shown in Table 1. Figure 1 As shown in Table 1, EIF3A expression was significantly reduced in the liver tissue of CCl4-induced liver fibrosis mice.

[0062] Example 3 To further clarify that EIF3A is involved in the progression of liver fibrosis disease, a MCD diet (methionine and choline deficiency) induced liver injury mouse model was constructed.

[0063] 1. MCD diet induced liver injury mouse model: Twelve male C57BL / 6J mice (8 weeks old) were randomly divided into a control group (Ctrl) and a MCD diet feeding group (MCD). The MCD group was gradually changed to MCD diet by transition feeding, with 30% MCD diet + 70% normal diet for the first 3 days, 50% MCD diet + 50% normal diet for the next 3 days, 70% MCD diet + 70% normal diet for the next 3 days, and 100% MCD diet from the 10th day. The MCD diet was purchased from Xiaoshuoyoutai (Beijing) Biotechnology Co., Ltd.; the Ctrl group was fed with normal diet. The feeding lasted for 4 weeks, during which the water and feed were changed regularly, the body weight of the mice was measured every week, and the bedding was changed, and the changes in mental state and other indicators were monitored. The Ctrl group and the model group were raised in the animal laboratory of the First Affiliated Hospital of Shandong First Medical University, with a 12-hour light / dark cycle, a temperature of 20-26°C, a daily temperature difference of 1°C, a relative humidity of 40%-70%, fresh air exchange of 15 times / hour, a pressure difference of 25 Pa, an ammonia concentration of ≤15 mg / m 3 , and an illumination of 150-300 Lux.

[0064] 2. Mechanism of modeling: methionine and choline are key nutrients for liver lipid metabolism. Choline is involved in the synthesis of very low density lipoprotein, promoting the output of triglycerides in the liver; when deficient, triglycerides in the liver cannot be effectively transported to the periphery, leading to lipid accumulation. Methionine is involved in glutathione synthesis and methyl donor metabolism; when deficient, the antioxidant capacity of the liver decreases, and oxidative stress caused by lipid accumulation intensifies, leading to liver cell damage and inflammation. MCD diet induces a cascade reaction from lipid accumulation → oxidative stress → inflammation → liver cell damage → fibrosis, simulating the pathological progression of liver injury.

[0065] 3. Core pathology: steatosis (accumulation of lipid in hepatocytes), lobular inflammation, hepatocellular ballooning, and often fibrosis.

[0066] 4. Index detection: (1) During the feeding process, the body weight of the Ctrl group and the MCD group mice was dynamically recorded. After 4 weeks of feeding, the mice and their liver tissue morphology were photographed and weighed, and the liver weight ratio of the mice was statistically analyzed. The experimental results are shown in A, B, C, and D in Figure 1 , the body weight of the MCD group mice was significantly reduced, and the liver weight ratio was also reduced.

[0067] (2) The mice were enucleated to collect blood, and after collection, centrifuged at 3000 rpm for 3 minutes, and the supernatant was collected as mouse serum. The alanine aminotransferase kit (C009-2-1), aspartate aminotransferase kit (C010-2-1), and triglyceride determination kit (A110-1-1) of Nanjing Jiancheng Biological Engineering were used to detect the alanine aminotransferase (ALT), aspartate aminotransferase (AST), and triglyceride (TG) in the serum, respectively. It was found that the alanine aminotransferase and aspartate aminotransferase of the MCD group mice were significantly increased (as shown in E and F in Figure 2 ), and the triglyceride level was reduced (as shown in G in Figure 2 ).

[0068] (3) Pathological analysis of liver tissue was performed by HE staining, Masson's trichrome staining, and Sirius red staining.

[0069] The HE staining method for paraffin sections is as follows: Place the paraffin sections in a 65°C oven for 2-3 hours. After removal, soak them in xylene I and then xylene II for 10 minutes each to remove the paraffin. Then, soak the xylene-soaked tissue sections in anhydrous ethanol I and then in anhydrous ethanol II for 5 minutes each to elute the xylene. The sections are then immersed in 95%, 85%, and 75% ethanol for 5 minutes each to fully hydrate the sections. After hydration, soak the sections in PBS three times for 5 minutes each. Add 100 μL of hematoxylin solution to each section and stain thoroughly for 7 minutes. Excess hematoxylin solution is then washed off with running water (3DW water). Sections are then incubated with 1% hydrochloric acid in alcohol for 1-3 seconds to remove excess stain from the nuclei and cytoplasm, enhancing staining clarity. After incubation, rinse the sections immediately with tap water to restore the blue color. Add eosin stain and stain thoroughly for 2 minutes, then gently rinse 1-2 times with running water (3DW water) to remove excess eosin. After staining, dehydrate the sections in a gradient process: soak in 75% ethanol, 85% ethanol, 95% ethanol, and anhydrous ethanol for 1-2 seconds each, then dehydrate in anhydrous ethanol for 1 minute. Soak in xylene twice for 1 minute each to make the tissue transparent for easier observation. Remove the sections from the xylene and allow them to dry slightly. Mount with neutral gum and cover with a glass slide before observing and photographing under a microscope.

[0070] Paraffin sections were stained with Masson trichrome staining using the kit (G1006) from Wuhan Sevier Biological Co., Ltd. The specific staining method is as follows: The paraffin sections were deparaffinated to water; the frozen sections stored at -20°C were allowed to recover to room temperature. Then the sections were immersed in Masson A and incubated in a 65°C oven for 30 minutes. After removal, the sections were rinsed in tap water for 30 seconds until the yellow color on the tissue faded. Meanwhile, Masson D and Masson F were preheated in a 65°C oven. Then, Masson B and Masson C were mixed in equal volume (freshly prepared, not stored), and the sections were immersed in the mixed solution for 1 minute, and then rinsed in running water. The sections were differentiated in 1% hydrochloric acid alcohol for about 1 minute until the cell nucleus was blackish gray and the background was nearly colorless or light gray. After rinsing in tap water, the sections were gently drained of excess water, and then immersed in Masson D for 6 minutes. If the red color was too light, the time could be appropriately extended, and for frozen sections (8 μm), the time was shortened to 3 minutes. Then the sections were rinsed in running water for about 20 seconds until the running water was colorless. The sections were gently drained of water (not dried), and then immersed in Masson E for differentiation until the collagen fibers were light red and the fibers were red. The time could be adjusted according to the depth of the color, and was generally 1-2 minutes. After gently draining the Masson E, the sections were directly immersed in Masson F without water washing. The time was 2-30 seconds for conventional tissues, 30 seconds for bone tissues, and 60 seconds for skin tissues. For frozen sections (8 μm), the time was appropriately shortened, and the specific time could be adjusted according to the degree of staining. Then the sections were successively rinsed in three cylinders of 1% acetic acid water solution for about 7 seconds each to remove excess aniline blue. The third cylinder was examined under a microscope to avoid over-differentiation of the blue color. Finally, the sections were successively dehydrated in three cylinders of anhydrous ethanol for about 3 seconds, 5 seconds and 5 seconds, respectively, and then transparentized in xylene for 5 minutes, and then mounted with neutral balsam.

[0071] The method of staining the paraffin sections with picrosirius was the same as that of Example 2.

[0072] The results of staining are shown in FIG. 2, and the results of HE staining, Masson trichrome staining and picrosirius staining are shown in FIGS. 3A, 3B and 3C, respectively. Figure 2 As shown in FIG. 2, it was found by HE staining that the liver tissue sections of the MCD mice had obvious vacuolation and lipid droplet infiltration. It was found by Masson trichrome staining that the lipid droplet infiltration in the liver tissue of the MCD mice was verified, and the fibrous septa were preliminarily formed. It was found by picrosirius staining that the liver tissue of the MCD mice had mild fibrous accumulation and scar formation.

[0073] The above results show that the liver tissue of the MCD mice has severe fatty degeneration, hepatocyte ballooning, and mild liver fibrosis, and is in the development stage of progression from non-alcoholic steatohepatitis to liver fibrosis.

[0074] (4) The expression of EIF3A mRNA and protein levels in the liver tissue of the MCD mice was further analyzed by real-time fluorescent quantitative PCR and Western Blot.

[0075] The specific experimental method is the same as Example 2, and the results are as follows Figure 2 As shown in I and J of the middle, EIF3A has shown a significant downward trend, indicating that EIF3A is a sensitive indicator of liver fibrosis. Therefore, the decrease in EIF3A expression is a concomitant result of the pathological state of liver fibrosis disease, and EIF3A can be used as a biomarker for liver fibrosis, and is lowly expressed in patient biological samples.

[0076] Example 4 In order to further explore the function of the EIF3A gene in the liver and its role in liver injury, more accurately observe and analyze the effect of EIF3A gene deletion on the physiological and pathological state of the liver, we constructed a liver conditional EIF3A knockout mouse model. Using Crisper Cas9 technology, we constructed Figure 2 flox / + Mice, such as Eif3a As shown in A of the middle, a gRNA plasmid is designed for the intron region before the second exon and after the fifth exon of the EIF3A gene to achieve site-specific cleavage of the target gene. At the same time, a donor plasmid is designed to insert a loxP sequence and an endonuclease sequence at the two intron cleavage sites of EIF3A for PCR-RELP experimental identification of positive mouse genotypes. Subsequently, sgRNAs are transcribed in vitro, and the donor plasmid is linearized by restriction endonuclease. The two sgRNAs, the donor plasmid, and the Cas9 plasmid are microinjected into mouse zygotes in vitro. By genotyping the born mice, F0 positive chimeric mice are obtained, and the positive mice are mated with wild-type mice to obtain F1 Figure 3 flox / + Mice.

[0077] The F1 Eif3a flox / + Mice are mated with each other to breed Eif3a flox / flox Mice (referred to as Eif3a f / f Mice), the Alb-cre mice are mated with Eif3a flox / + Mice to obtain liver parenchymal cell conditional knockout Eif3a of Eif3a flox / flox Alb cre Mice, referred to as Eif3a CKO Mice. For the daily breeding of mice, the Eif3a CKO Mice are mated with Eif3a f / f Mice for breeding (such as Eif3aMouse tail DNA was extracted and subjected to qPCR and agarose gel electrophoresis. The 234 bp band was for wild-type mice, and the 302 bp band was for knockout mice, confirming the successful knockout of EIF3A at the DNA level (as shown in Figure 2B). Figure 3 Mouse genotype identification primers (ordered from Sangon Biotech (Shanghai) Co., Ltd.) are as follows: EIF3A flox-F (sequence 5): 5′-GTGAAATGTTTGAAAGAGGCCAGG-3′; EIF3A flox-R (sequence 6): 5′-TAGCTGTGAGTTTGAAGCTGTGA-3′; Alb cre-F (sequence 7): 5'-GAAGCAGAAGCTTAGGAAGATGG-3'; Alb cre-R (sequence 8): 5′-TTGGCCCCTTACCATAACTG-3′.

[0078] Take 6-8 weeks old mice from the same litter and observe Figure 3 f / f Mice and Eif3a CKO The basic conditions of mice such as body size and liver weight were found to be Eif3a f / f Comparison with mice, Eif3a CKO There were no significant differences in body size, liver weight, or liver-to-body weight ratio among the mice (e.g. Eif3a Further serological analysis of mouse eye blood was performed to detect liver function indicators. The results are shown in Figure 4. Figure 3 As shown in G and H, it was found Figure 3 f / f Mice and Eif3a CKO There were no significant differences in the alanine aminotransferase levels and triglyceride content of the mice. The expression levels of EIF3A, NF-κB and endoplasmic reticulum stress pathway in the liver were detected by Western Blot. Eif3a As shown in Figure 1, under basal conditions, EIF3A knockout did not affect the expression levels of NF-κB (P65, p-P65, IKKα, IKKβ, IKBα) and ER stress (p-EIF2α, p-IRE1α) pathways. These results suggest that EIF3A deficiency does not interfere with normal hepatocyte functions, such as basal metabolism and anti-inflammatory balance.

[0079] Pick Figure 3 f / f Mice and Eif3a CKOFour mice were used to prepare a 0.7 μL / g CCl4 working solution using olive oil as solvent. The CCl4 working solution was injected into the two mice. The injection volume was calculated based on body weight (g) × 0.7 μL / g ÷ 0.15. The CCl4 working solution was injected twice a week for a total of 6 weeks. The injected mice were named Eif3a f / f -CCl4 mice and Eif3a CKO -CCl4 mice.

[0080] Comparing the body size and liver tissue morphology of the two groups of mice after injection, it was found that Eif3a f / f -CCl4 mice and Eif3a CKO -CCl4 mice showed no significant differences in body size and weight, but Eif3a CKO -CCl4 mice showed a reduced miliary sensation on the liver surface (e.g. Eif3a (as shown in A and B).

[0081] The liver function indicators of mouse serum were tested, such as Figure 4 As shown in C and D, Figure 4 CKO The levels of alanine aminotransferase and aspartate aminotransferase in CCl4-treated mice were significantly reduced, suggesting that liver tissue-specific EIF3A knockout can improve CCl4-induced liver damage.

[0082] right Eif3a f / f -CCl4 mice and Eif3a CKO The liver tissues of CCl4-bearing mice were stained with HE, Masson's trichrome and picrosirius red respectively. Eif3a As shown in E, HE staining results indicate that under long-term CCl4 stimulation, EIF3A deficiency can alleviate the more serious liver structural damage in mice, reduce the degree of tissue lesions, including lipid droplet infiltration and fiber formation; Sirius red staining observed Figure 4 CKO -CCl4 mice showed reduced red collagen fibril formation; Masson trichrome staining showed Eif3a CKO -CCl4 mice had reduced blue fiber bundle formation. The above pathological analysis found Eif3a CKO The collagen fibers in the liver tissue of -CCl4 mice were significantly improved compared with those in the Ctrl group, suggesting that liver tissue-specific EIF3A knockout can alleviate CCl4-induced liver damage and lipid accumulation.

[0083] Next, Western Blot analysisEif3a f / f -CCl4 mice and Eif3a CKO -CCl4 mice and Eif3a Figure 4 The expression levels of key molecules of NF-κB (P65, p-P65, IKKα, IKKβ, IKBα) and endoplasmic reticulum stress (p-EIF2α, p-IRE1α, ATF6) pathways of CCl4 mice were found, and it was found that EIF3A deletion could inhibit endoplasmic reticulum stress and NF-κB inflammatory pathway (as shown in FIG. 6F), thereby blocking the progression of fibrosis from two sources of hepatocyte injury and inflammation. This shows that the role of EIF3A in the process of liver fibrosis is not a simple linear relationship, and the influence of the change of the expression level on liver fibrosis may depend on the pathological state of the cell and the activation of the related signal pathway. One of the core driving factors of liver fibrosis is chronic inflammation, that is, persistent inflammation stimulates the activation of hepatic stellate cells and promotes collagen synthesis. NF-κB is a core pathway for regulating the release of pro-inflammatory factors (such as TNF-α, IL-6), and the reduction of its activity can reduce the secretion of inflammatory factors. After EIF3A knockout, the key molecules (P65, phosphorylated p-P65, IKKα) of the NF-κB signaling pathway are reduced. Excessive activation of endoplasmic reticulum stress can lead to liver cell dysfunction, apoptosis, and release of pro-fibrosis signals (such as TGF-β1), which is an important auxiliary mechanism of liver fibrosis. After EIF3A knockout, the key indicators (p-IRE1α, p-EIF2α) of endoplasmic reticulum stress are reduced, indicating that it can reduce the stress injury of liver cells.

[0084] From the above experimental results, it can be found that under normal physiological conditions, the NF-κB and endoplasmic reticulum stress pathways of the liver parenchymal cell conditional knockout EIF3A mouse are not affected, and the liver function ALT, AST is normal, and it will not interfere with the function of normal liver cells. However, under the pathological condition of liver fibrosis, such as CCl4 modeling, conditional knockout of EIF3A in liver parenchymal cells can play a protective role.

[0085] Therefore, using an EIF3A inhibitor has no adverse effects on the liver under normal conditions, but under the pathological condition of liver fibrosis, it can play a therapeutic role by inhibiting the related pathogenic pathways. The present application provides a new mechanism for the occurrence and development of liver fibrosis, and can reduce liver cell damage and inflammation by targeting EIF3A, relieve liver fibrosis-related pathological phenotypes, and play a protective and therapeutic role.

[0086] Example 5 On the basis of the above examples, the present example 5 provides a use of EIF3A as a target for preparing a liver fibrosis drug, and the specific liver fibrosis drug is an EIF3A inhibitor. By inhibiting the expression of EIF3A in liver tissue, endoplasmic reticulum stress is relieved, the NF-κB inflammatory pathway is inhibited, and liver cell pathological damage is reduced, thereby relieving the progression of liver fibrosis.

[0087] The EIF3A inhibitor can be a drug that knocks down or knocks out EIF3A, a drug that inhibits the synthesis of EIF3A protein, or a drug that promotes the degradation of EIF3A protein. The drug that knocks down EIF3A can be any drug that can prevent the expression of the gene encoding EIF3A protein, such as removing all or part of the coding gene sequence, introducing a frameshift mutation to prevent it from producing a functional protein, removing or changing the promoter or other regulatory components to prevent the transcription of the coding gene sequence, or preventing translation by binding to mRNA, etc.

[0088] The drug that knocks out EIF3A can be any drug that can cause the gene to have a deletion mutation, an insertion mutation, or a base substitution mutation, resulting in loss of activity.

[0089] In terms of substance type, the EIF3A inhibitor can be selected from nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, antibody drugs, polypeptides, proteins, viruses, etc.

[0090] Meanwhile, in order to meet the needs of pharmaceutical applications, the EIF3A inhibitor can also contain one or more pharmaceutically acceptable excipients and carriers, and can be made into tablets, capsules, aerosols, pills, powders, solutions, etc. Various dosage forms are suitable for different drug delivery needs.

[0091] The excipient can be selected from glucose, sucrose, sorbitol, mannose, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, and / or water, etc. The carrier can be selected from DNA plasmid vectors, lentivirus vectors, retrovirus vectors, poxvirus vectors, herpes simplex virus vectors, adenovirus vectors, adeno-associated virus vectors, DNA plasmid-bound liposomes, DNA plasmid-bound molecular conjugates, and / or DNA plasmid-bound polymers, etc.

[0092] Although the present application has been described in detail by referring to the preferred embodiments thereof, it is to be understood that the present application is not limited to them. It is apparent that various equivalents, modifications and / or improvements can be made by those skilled in the art without departing from the spirit and scope of the present application. Any modification or replacement made by those skilled in the art within the technical scope disclosed by the present application should be covered by the scope of the present application.

Claims

1. Use of an EIF3A inhibitor in the preparation of a drug for treating liver fibrosis.

2. The use according to claim 1, characterized in that EIF3A inhibitors are drugs that knock down or eliminate EIF3A.

3. The use according to claim 2, characterized in that The drug for knocking down EIF3A is any drug that can prevent the expression of the gene encoding the EIF3A protein.

4. The use according to claim 2, characterized in that The drug for knocking out EIF3A is any drug that can cause the EIF3A gene to mutate and thereby lose its activity, and the mutation form is selected from deletion mutation, insertion mutation and / or base substitution.

5. The use according to claim 1, characterized in that EIF3A inhibitors are drugs that inhibit the synthesis of EIF3A protein or promote the degradation of EIF3A protein.

6. The use according to any one of claims 1 to 5, characterized in that The EIF3A inhibitor includes one or more pharmaceutically acceptable excipients.

7. The use according to any one of claims 1 to 5, characterized in that The EIF3A inhibitor includes one or more pharmaceutically acceptable carriers.

8. The use according to any one of claims 1 to 5, characterized in that The dosage form of the EIF3A inhibitor is selected from tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal formulations or suppositories.

9. The use according to any one of claims 1 to 5, characterized in that EIF3A inhibitors are nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, peptides, proteins or viruses.

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