Use of an inhibitor of ferroptosis for the treatment of hepatitis

Abstract

This invention belongs to the field of biomedical technology and provides an application of a ferroptosis inhibitor in the treatment of hepatitis. This invention reveals that a GRIM-19 gene defect specifically induces ferroptosis in hepatocytes, rather than other cell death mechanisms such as apoptosis, necrosis, or autophagy. This invention provides a ferroptosis inhibitor with Ferrostatin-1 as the active ingredient, solving the technical problem of insufficient intervention against the ferroptosis mechanism in existing hepatitis treatments. This inhibitor can significantly increase the expression level of GPX4 in liver tissue, effectively clear lipid peroxides, a core toxic product in the ferroptosis process, and block the ferroptosis process; in addition, it can significantly inhibit the activation of the NLRP3 inflammasome and reduce the expression of downstream inflammatory factors, thereby alleviating inflammatory cell infiltration and fibrotic lesions in the liver. This invention is applicable to the treatment of GRIM-19 deficiency-related chronic hepatitis, providing a new strategy for targeted therapy of liver diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the pharmaceutical use of ferroptosis inhibitors, specifically to the application of a ferroptosis inhibitor in the treatment of hepatitis. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a malignant tumor with a high incidence and mortality rate worldwide. Its development typically follows the classic pathological progression of "chronic hepatitis - liver fibrosis - cirrhosis - hepatocellular carcinoma." In this process, liver fibrosis, as a key reversible link, becomes a crucial intervention window to prevent the progression of chronic liver disease to end-stage liver disease. Various etiologies, such as viral hepatitis, alcoholic liver disease, and non-alcoholic steatohepatitis, can lead to chronic liver damage. Through the continuous activation of hepatic stellate cells, these cells transform into myofibroblasts and secrete large amounts of extracellular matrix, ultimately leading to liver fibrosis. Therefore, elucidating the molecular mechanisms of liver fibrosis and exploring effective early intervention targets are of great significance for the prevention and treatment of hepatocellular carcinoma.

[0003] GRIM-19 (NDUFA13), an essential subunit of mitochondrial respiratory chain complex I, is downregulated in various human cancers, including hepatocellular carcinoma, suggesting its function as a tumor suppressor gene. However, current research mainly focuses on the expression regulation of GRIM-19 in mature tumor tissues, while its role in the development of prodromal lesions, particularly chronic hepatitis and liver fibrosis, remains poorly understood.

[0004] Ferroprelation, a novel iron-dependent form of programmed cell death, has been found to be closely associated with the pathological process of liver fibrosis. This process is characterized by intracellular glutathione depletion and inhibition of glutathione peroxidase 4 (GPX4) activity, leading to the accumulation of lipid peroxides and causing cell membrane damage. Notably, activated hepatic stellate cells exhibit a unique sensitivity to ferroprelation, making ferroptosis a potential strategy for antifibrotic therapy. Ferrostatin-1, a synthetically produced potent ferroptosis inhibitor, inhibits the ferroptosis process by directly scavenging lipid free radicals through its antioxidant activity. Studies have shown that Ferrostatin-1 exhibits protective effects by inhibiting ferroptosis in various disease models, including lung injury and osteolysis.

[0005] Currently, it remains unclear whether and how GRIM-19 expression deficiency participates in the initiation and development of chronic hepatitis-liver fibrosis, and its key downstream pathological pathways also need to be elucidated. In particular, effective strategies for targeted intervention in GRIM-19 deficiency-related liver fibrosis are still unknown. Furthermore, the therapeutic role and application of the ferroptosis inhibitor Ferrostatin-1 in GRIM-19 deficiency-induced chronic hepatitis and liver fibrosis models require further investigation. Therefore, this invention reveals the mechanism by which GRIM-19 deficiency promotes the progression of hepatitis-liver fibrosis by inducing hepatocyte ferroptosis, and proposes the use of the ferroptosis inhibitor Ferrostatin-1 for the treatment of chronic hepatitis and liver fibrosis characterized by low GRIM-19 expression, providing a new theoretical basis and potential intervention strategy for the treatment of related liver diseases. Summary of the Invention

[0006] The purpose of this invention is to provide an application of a ferroptosis inhibitor in the treatment of hepatitis, which blocks the progression of hepatitis to liver fibrosis by inhibiting hepatocyte ferroptosis, thereby solving the problem of the lack of effective intervention strategies for GRIM-19 deficiency-related liver diseases in the prior art.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: The present invention provides an application of a ferroptosis inhibitor in the preparation of a drug for treating hepatitis, wherein the ferroptosis inhibitor reduces the expression level of inflammatory factors in liver tissue by increasing the protein expression level of glutathione peroxidase 4 in liver tissue.

[0008] Preferably, the inflammatory factor is at least one of NLRP3, ASC, Casapse-1, IL-1β, and IL-33.

[0009] Preferably, the hepatitis is hepatitis induced by a GRIM-19 gene defect.

[0010] Preferably, the active ingredient of the ferroptosis inhibitor includes Ferrostatin-1.

[0011] Preferably, the drug specifically catalyzes the conversion of lipid peroxides into lipid alcohols by selenoproteins and removes toxic products in the ferroptosis process to inhibit ferroptosis.

[0012] Preferably, the drug further includes a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient.

[0013] Preferably, the pharmaceutically acceptable excipient is selected from at least one of the following pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, osmotic pressure regulators, stabilizers, suspending agents, coating materials, anti-adhesives, binding agents, penetration enhancers, pH adjusters, buffers, surfactants, absorbents, diluents, filter aids, and controlled-release materials.

[0014] The beneficial effects of this invention are:

[0015] 1. Targeted regulation of key ferroptosis pathways: By upregulating GPX4 protein expression in liver tissue through the ferroptosis inhibitor (Ferrostatin-1), lipid peroxides can be effectively cleared and GRIM-19 deficiency-induced hepatocyte ferroptosis can be inhibited, thus effectively blocking the progression of hepatitis to liver fibrosis.

[0016] 2. Significantly inhibits inflammatory response: Intervention by ferroptosis inhibitors can reduce the expression of key factors in the NLRP3 inflammasome pathway (such as ASC, Caspase-1, IL-1β, and IL-33), alleviate liver inflammatory damage, and provide a precise treatment strategy for GRIM-19-related hepatitis.

[0017] 3. Improve liver pathological condition: By inhibiting ferroptosis and inflammatory response, it can reduce the infiltration of inflammatory cells and inflammatory factors in liver tissue, effectively inhibit the development of hepatitis, and prevent the pathological process of progression to liver fibrosis. Attached Figure Description

[0018] Figure 1 This invention describes the construction of the GRIM-19 gene knockout AML-12 cell line (A and B are plasmid construction diagrams; C is a Western blot analysis of GRIM19 protein expression in ANL-12 cells; D is a flow cytometry GFP positive detection diagram).

[0019] Figure 2 This invention describes the detection of apoptosis, necrosis, and white phagocytosis in AML-12 cells (A shows the flow cytometry detection of apoptosis and necrosis in AML-12 cells; B shows the immunoblotting analysis of LC3A / B protein expression, an autophagy marker, in AML-12 cells).

[0020] Figure 3 The AML-12-58 cells and GRIM-1 in this invention fl / - Transmission electron microscopy images of mouse gastric and liver tissues (A: Transmission electron microscopy analysis of mitochondrial phenotype in AML-12-58 cells; B: GRIM-1 cells). fl / - Transmission electron microscopy analysis of mitochondrial phenotype in mouse gastric and liver tissues.

[0021] Figure 4 The AML-12-58 cells and GRIM-19 cells used in this invention fl / -Detection of ferroptosis marker proteins in mouse gastric and liver tissues (A: Western blot analysis of expression levels of ferroptosis marker proteins (GPX4, FTH1, TFR) in AML-12-58 cells; B: Western blot analysis of GRIM-19...) fl / - Expression levels of ferroptosis marker proteins (GPX4, FTH1, TFR) in mouse gastric mucosa tissue.

[0022] Figure 5 The AML-12-58 cells and GRIM19 in this invention fl / - Detection of iron ions in mouse liver tissue (A represents Fe in AML-12 cells) 2+ Content detection; B is GRIM19 fl / - (Results of Prussian blue staining of mouse liver tissue)

[0023] Figure 6 This invention relates to AML-12-58 cells and GRIM19 cells after intervention with the ferroptosis inhibitor Ferrostatin-1. fl / - Detection of ferroptosis marker proteins in mouse liver tissue (A: Western blot analysis of expression levels of ferroptosis marker proteins (GPX4, FTH1, TFR) in AML-12-58 cells treated with the ferroptosis inhibitor Ferrostatin-1 (0, 5 μM, 10 μM); B: Western blot analysis of GRIM-19 cells treated with the ferroptosis inhibitor Ferrostatin-1). fl / - Expression levels of ferroptosis marker proteins (GPX4, FTH1, TFR) in mouse liver tissue.

[0024] Figure 7 This invention relates to AML-12-58 cells and GRIM19 cells after intervention with the ferroptosis inhibitor Ferrostatin-1. fl / - Detection of inflammatory factors in mouse liver tissue (A: Western blot analysis of NLRP3, ASC, Casapse-1, IL-1β, and IL-33 protein expression levels in AML-12-58 cells after Ferrostatin-1 intervention; B: Western blot analysis of GRIM-19f after intervention with the ferroptosis inhibitor Ferrostatin-1) l / - Expression levels of NLRP3, ASC, Casapse-1, IL-1β, and IL-33 proteins in mouse liver tissue. Detailed Implementation

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0028] Example 1: GRIM-19 gene defect-induced hepatitis

[0029] 1. Construction of Lentiviral Vector System

[0030] The CRISPR / CAS9 single-vector lentivirus system was used (see plasmid construction diagram). Figure 1 China A Figure 1 (B), purchased from Shanghai Jikai Gene Technology Co., Ltd.; the target gene information is shown in Table 1, and the sgRNA information of the target gene is shown in Table 2.

[0031] Table 1. Basic information about the GRIM-19 gene

[0032]

[0033] Table 2 GRIM-19 gene sgRNA information

[0034]

[0035] 2. Western blot analysis of GRIM-19 expression, the specific procedure is as follows:

[0036] (1) Lyse cells on ice for more than 30 minutes using RIPA lysis buffer (Beyotime);

[0037] (2) Collect the supernatant at 12000 rpm for 10 min;

[0038] (3) Add the corresponding volume of SDS-PAGE protein loading buffer, and incubate in a metal bath at 100 ℃ for 10 min.

[0039] (4) SDS-PAGE electrophoresis;

[0040] (5) Seal at room temperature for 30 min;

[0041] (6) Apply the first antibody and incubate overnight on a shaker;

[0042] (7) Wash the membrane with TBST three times, 10 min each time;

[0043] (8) Apply the secondary antibody and incubate on a shaker at room temperature for 1 hour;

[0044] (9) Wash the membrane with TBST three times, 10 min each time;

[0045] (10) Development.

[0046] The results showed that the GRIM-19 protein level in AML-12 cells was significantly reduced compared to the control group. Figure 1 The optimal knockout method was 11658 (C), and subsequent experiments were conducted using 58. This indicated that the normal hepatocyte cell line AML-12 (epithelial cell-like) GRIM-19 knockout model was successfully constructed.

[0047] 3. Cell sorting by flow cytometry

[0048] GFP-positive cells can be sorted using flow cytometry, diluted into single cells, and then subjected to monoclonal proliferation to obtain high-purity GFP-positive cells. Figure 1 (D).

[0049] 4. Analysis of apoptosis in AML-12-58 cells using an apoptosis kit.

[0050] (1) Collect 5×10 5 cell;

[0051] (2) Rinse twice with PBS (2000 rpm / 5 min);

[0052] (3) Binding Buffer: 7-AAD = 10:1, add 50 μL of dye solution, mix well, and react in the dark for 15 min;

[0053] (4) Binding Buffer: Annexin V-PE = 450:1, add 450 μL of dye solution, mix well, and react in the dark for 15 min;

[0054] (5) Flow cytometry detection within 1 hour

[0055] The results showed no significant difference in apoptosis analysis between AML-12-58 and the control AML-12-NC. Figure 2 (A) This demonstrates that GRIM-19 deficiency induces slower hepatocyte proliferation, not apoptosis.

[0056] 5. Autophagy analysis of AML-12-58 cells

[0057] The expression levels of autophagy marker proteins LC3A / B in AML-12-58 cells were detected using Western blotting. The results showed that the expression level of LC3A / B in AML-12-58 cells did not increase compared to the control group. Figure 2 (Middle B), confirming that the mechanism by which GRIM-19 deficiency induces slower proliferation and reduced activity of liver cells is not autophagy.

[0058] 6. AML-12-58 cells and GRIM-19 fl / - Electron microscopy analysis of liver tissue

[0059] (1) Cut fresh tissue into 1 mm pieces 3 Size, immediately add electron microscopy fixative, store at 4 ℃ (can be stored for 3-6 months), and send to the electron microscopy room for analysis at a later date;

[0060] (2) Cell digestion 1×10 6 1200 rpm / 10 min, to form a tight cell cluster at the bottom of the EP tube, aspirate the supernatant, and gently add fixative along the tube wall (do not break the sample cluster). Store at 4 ℃ (can be stored for 3-6 months) and then send to the electron microscopy room for further analysis.

[0061] Transmission electron microscopy analysis revealed significant changes in mitochondrial structure and number in AML-12-58 cells compared to AML-12-NC: reduced number, increased membrane density, and decreased cristae. Figure 3 (A), and these are consistent with the morphological characteristics of ferroptosis; compared with control mice, GRIM-19 fl / - Similar changes were observed in the mitochondrial structure of mouse liver tissue cells: increased mitochondrial membrane density, reduced cristae, and the appearance of disorganized lamellar cristae. Figure 3 (B)

[0062] The above in vitro and in vivo transmission electron microscopy analyses confirmed that GRIM-19 deficiency induced ferroptosis morphological changes in the mitochondria of liver epithelial cell lines and mouse hepatocytes.

[0063] 7. AML-12-58 cells and GRIM-19 fl / - Analysis of expression of ferroptosis markers in mouse liver tissue

[0064] Detection of AML-12-58 cells and GRIM-19 by Western blot fl / - Expression of ferroptosis-related marker proteins in mouse liver tissue.

[0065] The results showed that, compared with AML-12-NC cells, AML-12-58 cells had decreased GPX4 (glutathione peroxidase 4) and FTH1 (ferritin), and increased TFR (transferrin receptor). Figure 4 (A) This suggests that after GRIM-19 is knocked out of normal liver epithelial cells, Fe 2+ Overload, reduced lipid peroxide clearance capacity, and ferroptosis occur. Similarly, in mouse liver tissue, GRIM-19... fl / - Mouse liver tissue showed the same trend as in vitro compared to the control group in terms of ferroptosis marker proteins. Figure 4 (Middle B), which suggests that the mechanism by which GRIM-19 deficiency in mouse liver tissue cells induces hepatocyte damage is ferroptosis.

[0066] 8. Detection and analysis of ferrous ions in AML-12-58 cells

[0067] Intracellular excess Fe 2+ The Fenton reaction leads to the depletion of the antioxidant glutathione (GSH) and inactivation of GPX4, which in turn induces the accumulation of lipid peroxides, resulting in ferroptosis. Ferrous ion colorimetric assay kit (Elabscience) was used to detect Fe in AML-12-58 cells. 2+ Content. The specific steps are as follows:

[0068] (1) Collect 4-6×10 6 One cell;

[0069] (2) Add reagent one and homogenize;

[0070] (3) 1000 g / 10 min, collect the supernatant for later use;

[0071] (4) Add reagent two and incubate at 37 ℃ for 10 min;

[0072] (5) 12000 g / 10 min, collect the supernatant for later use;

[0073] (6) OD value was measured at 532 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0074] The results showed that AML-12-58 cells Fe 2+ increase( Figure 5 (A) suggests that GRIM-19 knockout induces Fe in hepatic epithelial cells. 2+ Increase, subsequent Fe 2+ It can promote ferroptosis via the Fenton reaction.

[0075] 9. GRIM-19 fl / - Detection and analysis of Prussian blue iron ions in mouse liver tissue

[0076] The expression of iron ions in mouse gastric mucosa was detected using a Prussian blue reagent kit (Solepro). The specific steps are as follows:

[0077] (1) Organizational stability;

[0078] (2) Conventional dehydration and embedding;

[0079] (3) Slice and bake;

[0080] (4) Stain with Perls staining working solution for 15 min;

[0081] (5) Wash with distilled water for 5 min;

[0082] (6) Stain the nuclear solid red solution for 10 min;

[0083] (7) Wash with tap water;

[0084] (8) Standard dehydration and transparency;

[0085] (9) Neutral resin sealing.

[0086] The results showed that GRIM-19 was significantly better than the control group. fl / - The iron content in mouse liver tissue was significantly increased ( Figure 5 (B) This suggests that iron overload in liver tissue further exacerbates ferroptosis.

[0087] Example 2: Effects of Ferrostatin-1 on Hepatitis

[0088] 1. Analysis of the expression of ferritin biomarkers

[0089] Ferrostatin-1 intervention in AML-12-58 cells and GRIM-19f l / - The expression of ferroptosis markers in mouse liver tissue was analyzed using Western blotting.

[0090] The results showed that treatment of AML-12-58 cells with the ferroptosis inhibitor Ferrostatin-1 increased the levels of ferroptosis marker proteins GPX4 and FTH1, and decreased TFR. Figure 6 (A) This suggests that Ferrostatin-1 effectively inhibits the pathological process of ferroptosis in GRIM-19 knockout hepatic epithelial cells AML-12. In vivo experiments showed Ferrostatin-1 injection into GRIM-19... fl / - In mice, the results showed that GPX4, FTH1, and TFR exhibited similar trends. Figure 6 (B) The above in vitro and in vivo experiments confirmed that the ferroptosis inhibitor Ferrostatin-1 effectively inhibited the pathological process of ferroptosis in liver tissue cells induced by GRIM-19 deficiency.

[0091] 2. Analysis of inflammatory factor expression

[0092] Analysis of inflammatory factor expression in AML-12-58 cells and GRIM-19fl / - mice after Ferrostatin-1 intervention; Western blotting was used to detect the expression of inflammatory factors in AML-12-58 cells and GRIM-19fl / - mice after intervention with the ferroptosis inhibitor Ferrostatin-1. fl / - The expression of inflammatory factors in mice after treatment.

[0093] The results showed that after in vitro intervention of AML-12-58 cells with the ferroptosis inhibitor Ferrostatin-1, the protein expression levels of inflammatory factors NLRP3, ASC, Casapse-1, IL-1β, and IL-33 were significantly reduced; in vivo experiments demonstrated that compared with the control group injected with physiological saline (0.9% Nacl), the Ferrostatin-1 group also showed a significant reduction in inflammatory factors such as LRP3 and ASC. Figure 7 The above results suggest that in vitro and in vivo experiments have confirmed that the ferroptosis inhibitor Ferrostatin-1 can reduce the expression of inflammatory factors in the liver tissue of GRIM-19 knockout hepatic epithelial cells and hepatocyte-specific GRIM-19 knockout mice, effectively alleviating the inflammatory response in liver tissue and inhibiting the pathological process of liver inflammation.

[0094] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. The application of a ferroptosis inhibitor in the preparation of a drug for treating hepatitis, characterized in that, The ferroptosis inhibitor reduces the expression level of inflammatory factors in liver tissue by increasing the protein expression level of glutathione peroxidase 4.

2. The application according to claim 1, characterized in that, The inflammatory factor is at least one of NLRP3, ASC, Casapse-1, IL-1β, and IL-33.

3. The application according to claim 1, characterized in that, The hepatitis mentioned is hepatitis induced by a defect in the GRIM-19 gene.

4. The application according to claim 1, characterized in that, The active ingredient of the ferroptosis inhibitor includes Ferrostatin-1.

5. The application according to claim 1, characterized in that, The drug specifically catalyzes the conversion of lipid peroxides into selenoproteins into lipid alcohols and removes toxic products in the ferroptosis process, thereby inhibiting ferroptosis.

6. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable carriers and / or pharmaceutically acceptable excipients.

7. The application according to claim 6, characterized in that, The pharmaceutically acceptable excipients are selected from at least one of the following pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, osmotic pressure regulators, stabilizers, suspending agents, coating materials, anti-adhesives, binding agents, penetration enhancers, pH adjusters, buffers, surfactants, absorbents, diluents, filter aids, and sustained-release materials.

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