Application of Mps1-IN-1 as a ferroptosis inhibitor and in the preparation of drugs for preventing and treating ferroptosis-related diseases

Abstract

The present invention discloses the use of Mps1-IN-1 as a ferroptosis inhibitor and in the preparation of drugs for preventing and treating ferroptosis-related diseases, and belongs to the field of biomedicine technology. Specifically, any of the following applications of Mps1-IN-1 is proposed: 1. Application as a ferroptosis inhibitor; 2. Application in the preparation of drugs for preventing and / or treating ferroptosis-related diseases; 3. Application in constructing ferroptosis tumor cell models, etc. Beneficial effect: The inhibitor exhibits significant protective ability against cell fate under a variety of ferroptosis-inducing mechanisms. Its unique mechanism of action is not limited to simply inhibiting the ferroptosis process, but also lies in its ability to efficiently scavenge harmful oxygen free radicals and effectively curb the generation of lipid peroxides. This discovery has opened up a new way for the prevention and treatment of ferroptosis-related diseases. Its research and development and application not only have far-reaching scientific significance, but will also have a positive and far-reaching impact on human health.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine technology, mainly focusing on the innovative application of Mps1-IN-1 as a ferroptosis inhibitor, and deeply exploring its potential and value in the preparation, prevention and treatment of drugs related to ferroptosis. Background Art

[0002] Ferroptosis, a unique cell death mechanism, is characterized by a surge in toxic lipid peroxides (ROS) levels triggered by abnormal intracellular iron accumulation. This process differs significantly from traditional apoptosis pathways. Ferroptosis is fundamentally rooted in the severe depletion of glutathione (GSH). This reduction in this key antioxidant molecule directly impairs the function and activity of the GPX4 enzyme, a core component of the antioxidant defense system. This in turn blocks the effective metabolism of lipid peroxides through the glutathione-dependent pathway catalyzed by GPX4, leading to their continued accumulation.

[0003] What is particularly critical is that the continuously accumulated Fe2+ exacerbates the oxidative damage of unsaturated fatty acids on the cell membrane by activating the Fenton reaction. This chain reaction ultimately promotes the occurrence and development of ferroptosis. This complex biological process not only profoundly affects the normal physiological functions of the body, but is also widely associated with the pathological mechanisms of many serious diseases, including but not limited to neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's syndrome, as well as tumors, stroke, cerebral hemorrhage, traumatic brain injury, local ischemia-reperfusion injury and renal failure, demonstrating its importance in the medical field. Given the close connection between ferroptosis and the above-mentioned diseases, exploring and developing new therapeutic methods that can effectively inhibit cell ferroptosis has become a frontier hotspot in medical research and drug development. This strategy is not only expected to provide new ideas and approaches for the prevention and treatment of ferroptosis-related diseases, but may also bring revolutionary breakthroughs in improving patient prognosis and improving quality of life.

[0004] Acute liver injury, a global health challenge, is commonly caused by drug-induced hepatitis and post-traumatic hemorrhagic shock, which leads to a sharp decrease in liver blood perfusion and secondary damage to liver tissue. In the acute phase of the disease, patients often face the severe situation of significant liver dysfunction or even liver failure. In extreme cases, it is more likely to cause extensive necrosis of liver tissue, which directly threatens life safety. Current research has profoundly revealed the close relationship between acute liver injury and ferroptosis, a unique mode of cell death. Ferroptosis, as a pathological process that depends on iron metabolism imbalance, the collapse of the amino acid antioxidant defense system, and the abnormal accumulation of lipid peroxides, has left a distinct mark at all stages of liver disease from initiation to progression. This discovery not only deepens our understanding of the pathological mechanism of acute liver injury, but also provides valuable clues for exploring new treatment strategies.

[0005] Although there is now solid evidence that small molecule compounds such as ferrostatin-1, iron chelators (such as DFO), lipoxstatin-1, vitamin K, and free radical-trapping antioxidants are effective inhibitors of ferroptosis, selectively blocking the ferroptosis process through diverse molecular mechanisms, thereby significantly improving the clinical prognosis of various ferroptosis-related diseases, these inhibitors still face many challenges and limitations in terms of synthetic complexity, difficulty in large-scale production, unclear clinical indications, and clinical safety.

[0006] Mps1-IN-1 is a compound that belongs to a class of compounds that inhibit Mps1 (monopolar serine / threonine kinase 1). Its structural formula is Mps1 is an important cell cycle regulatory protein kinase associated with tumor development and abnormal cell division. To date, there have been no reports on the use of Mps1-IN-1 in inhibiting ferroptosis to treat ferroptosis-related diseases, particularly acute liver injury. Summary of the Invention

[0007] The technical problem to be solved by the present invention is how to solve the problems of complex preparation, poor effect and insufficient safety of existing ferroptosis inhibitors.

[0008] The present invention solves the above technical problems through the following technical means:

[0009] The present invention proposes any of the following applications of Mps1-IN-1:

[0010] (1) Application as a ferroptosis inhibitor;

[0011] (2) Application in the preparation of drugs for preventing and / or treating ferroptosis-related diseases;

[0012] (3) Application in constructing ferroptosis tumor cell models;

[0013] (4) Application in the preparation of drugs for inhibiting the increase of cellular reactive oxygen species (ROS) levels;

[0014] (5) Application in the preparation of drugs for reducing the levels of inflammatory factors IL-6, IL-1β, TNF-α, PTGS2, and INF-γ;

[0015] (6) Use in the preparation of drugs for lowering serum AST and ALT levels;

[0016] (7) Application in the preparation of drugs for maintaining stable glutathione (GSH) levels;

[0017] The structural formula of Mps1-IN-1 is

[0018] Preferably, the ferroptosis-related diseases include one or more of tumors, acute liver injury, stroke, neurodegenerative diseases, traumatic brain injury, mammalian degenerative diseases, spinal cord injury, congestive heart failure and blood system diseases, local ischemia-reperfusion injury, and renal failure-related diseases.

[0019] Preferably, the tumor cells are one or more selected from human fibrosarcoma cell lines, human breast cancer cell lines, mouse breast cancer cell lines, and mouse fibrosarcoma cell lines.

[0020] Preferably, in constructing the ferroptosis tumor cell model, the ferroptosis inducer used is selected from any one of Erastin, RSL3, and FIN56.

[0021] Preferably, the concentration of Mps1-IN-1 is 1-10 μM.

[0022] Preferably, the ferroptosis inhibitor includes a pharmaceutically acceptable salt, specifically an organic salt or an inorganic salt.

[0023] Preferably, in (2), (4), (5), (6), and (7), the drug includes pharmaceutically acceptable excipients.

[0024] Preferably, the pharmaceutically acceptable excipient is selected from one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier and a lubricant.

[0025] Preferably, in (2), (4), (5), (6), and (7), the drug is prepared into a pharmaceutically acceptable dosage form.

[0026] Preferably, the dosage form is tablet, pill, paste, capsule, oral solution or granule.

[0027] The beneficial effects of the present invention are:

[0028] 1. This invention pioneers a new application area for Mps1-IN-1 as a ferroptosis inhibitor, particularly its remarkable therapeutic effect in acute liver injury, a typical ferroptosis-related disease. Through empirical research, we not only confirmed the effectiveness of Mps1-IN-1 in preventing, treating, and delaying the onset and progression of acute liver injury, but also proposed a practical medication regimen. This discovery not only opens new avenues for the treatment of ferroptosis-related diseases, but also provides valuable insights and direction for the future exploration, development, and preparation of more efficient, advanced, and safer ferroptosis-inhibiting drugs.

[0029] 2. This invention explores and establishes the potential of Mps1-IN-1 as a novel and highly effective ferroptosis inhibitor. To achieve this innovative goal, the present invention introduces Mps1-IN-1, a unique compound that stands out for its remarkable potency as an ATP-competitive Mps1 kinase inhibitor.

[0030] 3. The research results of the present invention are remarkable, revealing that Mps1-IN-1 can significantly inhibit ferroptosis in various tumor cell models at an extremely low concentration (1 μM). Its inhibitory efficacy is not only comparable to currently known ferroptosis inhibitors, but even performs better in some cases.

[0031] 4. The present invention further demonstrates the remarkable efficacy of Mps1-IN-1 as a small molecule drug, demonstrating effective resistance to ferroptosis triggered by a variety of ferroptosis inducers (such as RSL3, Erastin, FIN56, etc., which are small molecule compounds that can induce ferroptosis in cells), thereby opening up a broader and more efficient treatment path for diseases driven by ferroptosis mechanisms. In the specific implementation strategy of the present invention, these ferroptosis inducers serve as important experimental tools to help evaluate the inhibitory efficacy and potential application value of Mps1-IN-1.

[0032] 5. By optimizing synthesis pathways, improving drug purity and stability, and deeply exploring its mechanism of action and biocompatibility, we hope to overcome the limitations of existing inhibitors, provide patients with more precise and effective treatment options, and promote the innovation and development of treatment strategies for ferroptosis-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a graph showing the effect of different concentrations of Mps1-IN-1 on RSL3-induced ferroptosis in Example 1 of the present invention; Figure 1 A is the IC50 value of Mps1-IN-1 in inhibiting RSL3-induced ferroptosis. Figure 1 B is a graph showing the changes in cell survival rate at different concentrations of Mps1-IN-1;

[0034] Figure 2 This is a diagram showing the effect of Mps1-IN-1 on ferroptosis induced by different inducers in vitro in Example 2 of the present invention; Figure 2 A is a graph showing changes in cell survival rate when Mps1-IN-1 inhibits ferroptosis induced by different ferroptosis inducers (RSL3, erastin, and FIN56); Figure 2 B is the graph showing changes in LDH activity;

[0035] Figure 3 This is a graph showing changes in cell survival rates of Mps1-IN-1 in Example 3 of the present invention in inhibiting RSL3-induced ferroptosis in different cell types: MCA-205 (A), HT1080 (B), 4T1 (C), and MDA-MB-231 (D);

[0036] Figure 4 This is a graph showing changes in LDH activity in cells of different MCA-205 (A), HT1080 (B), 4T1 (C), and MDA-MB-231 (D) cells that were induced by RSL3 by Mps1-IN-1 in Example 3 of the present invention;

[0037] Figure 5 This is a graph showing the effect of Mps1-IN-1 on RSL3-induced ferroptosis tested by PI staining in Example 4 of the present invention; Figure 5 A is the result of PI staining of ferroptosis. Figure 5 B is the statistical graph of the results;

[0038] Figure 6 This is a flow cytometry diagram of the effect of Mps1-IN-1 on lipid peroxides in cells in Example 5 of the present invention; Figure 6 A is the flow cytometry graph of Mps1-IN-1 inhibiting RSL3-induced intracellular lipid peroxides. Figure 6 B is the statistical graph of the results;

[0039] Figure 7 This is a flow cytometry diagram of the effect of Mps1-IN-1 on ROS in cells in Example 6 of the present invention; Figure 7 A is the flow cytometry diagram of intracellular ROS. Figure 7 B is the statistical graph of the results;

[0040] Figure 8 The qPCR test results of Mps1-IN-1 in Example 7 of the present invention for reducing the levels of IL-6 (A), TNF-α (B), PTGS2 (C), and INF-γ (D) in the liver after ConA induction are shown;

[0041] Figure 9 Figure 8 shows the results of ELISA test of Mps1-IN-1 reducing the levels of IL-6 (A), IL-1β (B), and TNF-α (C) in serum after ConA induction in Example 8 of the present invention;

[0042] Figure 10 Graph showing the results of Mps1-IN-1 reducing serum AST levels (A) and ALT levels (B) after ConA induction in Example 9 of the present invention;

[0043] Figure 11 This is a graph showing the effect of Mps1-IN-1 on the GSH level in the liver of mice with acute liver injury model induced by concanavalin A (ConA) in Example 10 of the present invention. DETAILED DESCRIPTION

[0044] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0045] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0046] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0047] Table 1. Experimental reagents

[0048]

[0049]

[0050] Example 1: Effects of different concentrations of Mps1-IN-1 on RSL3-induced ferroptosis

[0051] HT 1080 cells with good growth were digested with trypsin, centrifuged at 1500 rpm for 3 min, and the supernatant was discarded. 3 Cells were prepared into a certain concentration of cell suspension, 100 μL of cell suspension was added to each well, and the cells were inoculated into 96-well plates. The groups and corresponding treatments were as follows:

[0052] (1) Normal control group (Control): HT1080 cells were cultured normally without special treatment.

[0053] (2) RSL3 treatment group: RSL3 was dissolved in DMSO and prepared into a working solution according to the required concentration. 1 μM RSL3 was added to each well and treated for 24 h.

[0054] (3) Mps1-IN-1 treatment groups with different concentrations + RSL3 groups: Each well was treated with 1 μM, 3 μM, and 10 μM Mps1-IN-1 for 2 h. 1 μM RSL3 was added to the culture medium after treatment with Mps1-IN-1 at different final concentrations for 2 h.

[0055] Discard the culture medium from the 96-well plate and wash twice with 1×PBS. Prepare CCK-8 working solution at a ratio of 1:10 (CCK-8:DMEM medium), adding 100 μL to each well. Place the 96-well plate in a 37°C incubator in the dark for 1 hour. Measure the OD value at a wavelength of 450 nm using a microplate reader. Calculate cell viability according to the formula:

[0056] Cell viability = [(AS-Ab) / (AC-Ab)] x 100% As: experimental wells (culture medium containing cells, CCK-8, and test substance); Ac: control wells (culture medium containing cells, CCK-8, and no test substance); Ab: blank wells (culture medium without cells or test substance, CCK-8).

[0057] See the results Figure 1 From the analysis of cell viability in the RSL3-treated group, 1 μM RSL3 successfully induced cell ferroptosis. From the cell viability assay in the Mps1-IN-1-treated group + RSL3 group, it was found that Mps1-IN-1 pretreatment could inhibit cell ferroptosis. This indicates that Mps1-IN-1 can inhibit RSL3-induced ferroptosis.

[0058] Example 2: Effects of Mps1-IN-1 on ferroptosis induced by different inducers in vitro

[0059] 1. Mps1-IN-1 inhibits cell death after treatment with different ferroptosis inducers.

[0060] HT 1080 cells with good growth were digested with trypsin, centrifuged at 1500 rpm for 3 min, and the supernatant was discarded. 3 Cells were prepared into a certain concentration of cell suspension, 100 μL of cell suspension was added to each well, and the cells were inoculated into 96-well plates. The groups and corresponding treatments were as follows:

[0061] (1) Blank control group.

[0062] (2) RSL3-treated group: 1 μM RSL3 was added to the culture medium and treated for 24 h.

[0063] (3) RSL3+Mps1-IN-1 treatment group: 1 μM Mps1-IN-1 was added to the culture medium for 2 h, and then 1 μM RSL3 was added for 24 h.

[0064] (4) Erastin-treated group: 10 μM erastin was added to the culture medium for 24 h.

[0065] (5) Erastin+Mps1-IN-1 treatment group: 1 μM Mps1-IN-1 was added to the culture medium and treated for 2 h. After 2 h of treatment, 10 μM erastin was added and treated for 24 h.

[0066] (6) FIN56-treated group: 50 μM FIN56 was added to the culture medium and treated for 24 h.

[0067] (7) FIN56+Mps1-IN-1 treatment group: 1 μM Mps1-IN-1 was added to the culture medium and treated for 2 h. After 2 h of treatment, 50 μM FIN56 was added and treated for 24 h.

[0068] The culture medium in the 96-well plate was discarded, and the plate was washed twice with 1x PBS. CCK-8 working solution was prepared at a ratio of 1:10 for CCK-8:DMEM medium, and 100 μL was added to each well. The 96-well plate was incubated in a 37°C incubator in the dark for 1 hour. The OD value was measured at a wavelength of 450 nm using a microplate reader. Cell viability was calculated according to the formula.

[0069] like Figure 2 As shown in Figure A, cell viability decreased significantly after treatment with different ferroptosis inducers alone. However, after pretreatment with Mps1-IN-1, cell viability significantly recovered. This result indicates that pretreatment with Mps1-IN-1 can effectively prevent cell death triggered by multiple ferroptosis inducers.

[0070] 2. Mps1-IN-1 inhibits the release of lactate dehydrogenase after induction by different ferroptosis inducers

[0071] HT1080 cells with good growth conditions were digested and resuspended. Centrifuged at 1500 rpm for 3 minutes and the supernatant was discarded.

[0072] According to 5 x 10 per well 3 A cell suspension of a certain concentration was prepared, 100 μL of cell suspension was added to each well, and the cells were seeded into 96-well plates. The groups and treatments were as follows:

[0073] (1) Blank control group.

[0074] (2) RSL3-treated group: 1 μM RSL3 was added to the culture medium and treated for 24 h.

[0075] (3) RSL3+Mps1-IN-1 treatment group: 1 μM Mps1-IN-1 was added to the culture medium and treated for 2 h. After 2 h of treatment, 1 μM RSL3 was added and treated for 24 h.

[0076] (4) Erastin-treated group: 10 μM erastin was added to the culture medium for 24 h.

[0077] (5) Erastin+Mps1-IN-1 treatment group: 1 μM Mps1-IN-1 was added to the culture medium and treated for 2 h. After 2 h of treatment, 10 μM erastin was added and treated for 24 h.

[0078] (6) FIN56-treated group: 50 μM FIN56 was added to the culture medium and treated for 24 h.

[0079] (7) FIN56+Mps1-IN-1 treatment group: 1 μM Mps1-IN-1 was added to the culture medium and treated for 2 h. After 2 h of treatment, 50 μM FIN56 was added and treated for 24 h.

[0080] (8) LDH maximum release pore: Add LDH release reagent 1 h before collecting cell culture supernatant.

[0081] Determination of lactate dehydrogenase (LDH) activity:

[0082] (1) After each group of cells were treated, the cell culture supernatant was collected and the procedure was performed according to the kit instructions;

[0083] (2) Measure the OD value using a microplate reader at a wavelength of 490 nm;

[0084] (3) Calculate LDH activity according to the formula;

[0085] like Figure 2 As shown in Figure B, in the experimental groups treated with different inducers alone, LDH (lactate dehydrogenase) release was significantly increased. However, after pretreatment with Mps1-IN-1, LDH release in each experimental group was significantly reduced. This observation suggests that pretreatment with Mps1-IN-1 can effectively inhibit the LDH release process triggered by different inducers.

[0086] Example 3: Effects of Mps1-IN-1 on RSL3-induced ferroptosis in different cells

[0087] HT1080 cells with good growth conditions were digested and resuspended. Centrifuged at 1500 rpm for 3 minutes and the supernatant was discarded.

[0088] According to 5 x 10 per well 3Prepare a cell suspension of a certain concentration, add 100 μL of cell suspension to each well, inoculate into 96-well plates, and group as follows:

[0089] (1) Blank control group.

[0090] (2) RSL3-treated group: 1 μM RSL3 was added to the culture medium and treated for 24 h.

[0091] (3) RSL3+Mps1-IN-1 treatment group: 1 μM Mps1-IN-1 was added to the culture medium and treated for 2 h. After 2 h of treatment, 1 μM RSL3 was added and treated for 24 h.

[0092] Calculate the cell survival rate according to the formula.

[0093] like Figure 3 The images show that treatment with the ferroptosis inducer RSL3 reduced cell viability in various cell lines. However, pre-treatment with Mps1-IN-1 significantly increased the viability of various cell lines, including MCA-205 (A), HT1080 (B), 4T1 (C), and MDA-MB-231 (D). This result indicates that pre-treatment with Mps1-IN-1 significantly inhibits RSL3-induced ferroptosis in these various cell lines.

[0094] The cell LDH release rate (%) was calculated according to the formula: enzyme activity units measured in cell culture medium / (enzyme activity units measured in cell lysate + enzyme activity units measured in cell culture medium) × 100%

[0095] like Figure 4 The results show that treatment with the ferroptosis inducer RSL3 significantly increased cellular LDH (lactate dehydrogenase) levels in various cell lines. However, when these cell lines were pretreated with Mps1-IN-1 before RSL3 treatment, LDH levels in cell lines such as MCA-205 (A), HT1080 (B), 4T1 (C), and MDA-MB-231 (D) all showed a significant decrease. This result fully demonstrates the effectiveness of Mps1-IN-1 pretreatment in inhibiting RSL3-induced LDH release in different cell lines.

[0096] Example 4: PI staining to examine the effect of Mps1-IN-1 on RSL3-induced ferroptosis

[0097] 1. Experimental Grouping and Treatment

[0098] Take HT1080 cells with good growth conditions, digest them and resuspend them. Centrifuge at 1500 rpm for 3 minutes and discard the supernatant. 5Cells were seeded in 24-well plates and grouped as follows:

[0099] (1) Blank control group.

[0100] (2) RSL3-treated group: 3 μM RSL3 was added to the culture medium and treated for 6 h.

[0101] (3) RSL3+Mps1-IN-1 treatment group: 1 μM Mps1-IN-1 was added to the culture medium and treated for 2 h. After 2 h of treatment, 1 μM RSL3 was added and treated for 6 h.

[0102] Then the Nikon upright fluorescence photography results were counted.

[0103] like Figure 5 The researchers demonstrated that, in a cell culture system, treatment with RSL3 alone led to a significant increase in cell death. However, pretreatment with Mps1-IN-1 before RSL3 treatment effectively inhibited RSL3-induced cell death. This result directly demonstrates that Mps1-IN-1 can inhibit RSL3-induced cell death.

[0104] Example 5: Flow cytometry detection of the effect of Mps1-IN-1 on lipid peroxides in cells

[0105] Take HT1080 cells with good growth conditions, digest them and resuspend them. Centrifuge at 1500rpm for 3min and discard the supernatant. 5 Cells were seeded in 6-well plates and grouped as follows:

[0106] (1) Normal control group.

[0107] (2) RSL3-treated group: 3 μM RSL3 was added to the culture medium and treated for 4 h.

[0108] (3) RSL3+Mps1-IN-1 treatment group: 1 μM Mps1-IN-1 was added to the culture medium and treated for 2 h. After 2 h of treatment, 1 μM RSL3 was added and treated for 4 h.

[0109] After each group of cells was treated, the cell culture supernatant was removed, 1 mL of serum-free DMEM and 5 μM BODIPY581 / 591C11 were added to each well, and the 6-well plate was placed in a 37°C incubator in the dark for 0.5 h. EDTA-free trypsin was added to digest the cells. Digestion was stopped after the cells became round. The cells were transferred to a centrifuge tube and centrifuged at 4°C for 5 min at 3000 rpm. After collecting the cells, they were resuspended in 1×PBS pre-cooled at 4°C and centrifuged again to collect the cells (this washing process was repeated 2-3 times).

[0110] like Figure 6As shown in Figure 3, when cells were treated with RSL3 alone, lipid peroxidation levels in the ferroptosis pathway increased significantly. However, when Mps1-IN-1 was added before treatment, significant inhibition of lipid peroxidation levels was observed. This finding supports the role of Mps1-IN-1 in suppressing the increased lipid peroxidation during ferroptosis.

[0111] Example 6: Flow cytometry detection of the effect of Mps1-IN-1 on ROS in cells

[0112] Take HT1080 cells with good growth conditions, digest them and resuspend them. Centrifuge at 1500rpm for 3min and discard the supernatant. 5 Cells were seeded in 6-well plates and grouped as follows:

[0113] (1) Normal control group.

[0114] (2) RSL3-treated group: 3 μM RSL3 was added to the culture medium and treated for 4 h.

[0115] (3) RSL3+Mps1-IN-1 treatment group: 1 μM Mps1-IN-1 was added to the culture medium and treated for 2 h. After 2 h of treatment, 1 μM RSL3 was added and treated for 4 h.

[0116] After each group of cells was treated, the cell culture supernatant was removed, 1 mL of serum-free DMEM and 5 μM H2DCFDA were added to each well, and the 6-well plate was placed in a 37°C incubator in the dark for 0.5 h. EDTA-free trypsin was added to digest the cells. Digestion was stopped after the cells became round. The cells were transferred to a centrifuge tube and centrifuged at 4°C for 5 min, 3000 rpm. After collecting the cells, they were resuspended in 1×PBS pre-cooled at 4°C and centrifuged again to collect the cells (this washing process was repeated 2-3 times).

[0117] according to Figure 7 The results shown in Figure 3 show that treatment of cells with RSL3 alone resulted in a significant increase in ferroptosis-related reactive oxygen species (ROS) levels. However, when Mps1-IN-1 was added before RSL3 treatment, a significant suppression of ROS levels was observed. This finding further confirms the effectiveness of Mps1-IN-1 in suppressing ROS elevation during ferroptosis.

[0118] Example 7. qPCR detection of the effect of Mps1-IN-1 on the levels of IL-6, TNF-α, PTGS2(C), and INF-γ in the liver after ConA induction

[0119] 1. Animal Experiment Grouping and Treatment

[0120] Eight-week-old C57BL / 6 wild-type mice were selected and divided into three groups, with 6 mice in each group, as follows:

[0121] (1) Group 1: Negative control group. First, a certain volume of 1×PBS was injected intraperitoneally, and half an hour later, a certain volume of 1×PBS was injected into the tail vein, and half an hour later, a certain volume of 1×PBS was injected intraperitoneally again.

[0122] (2) Group 2: Mps1-IN-1 treatment group. Mps1-IN-1 (10 mg / kg) was first injected intraperitoneally, followed by ConA (15 mg / kg) injected into the tail vein half an hour later, and then Mps1-IN-1 (10 mg / kg) was injected intraperitoneally again half an hour later.

[0123] (3) The third group: ConA-induced group: First, a certain volume of 1×PBS was injected intraperitoneally, and ConA (15 mg / kg) was injected into the tail vein half an hour later. Half an hour later, a certain volume of 1×PBS was injected intraperitoneally again.

[0124] 2. Detection of IL-6, TNF-α, PTGS2(C), and INF-γ Levels in Mouse Liver

[0125] (1) Remove the liver from the mouse tail lobe, add Trizol to crush and grind it, and extract RNA.

[0126] (2) RNA is reverse transcribed into cDNA.

[0127]

[0128] (3) qPCR detection of IL-6, TNF-α, PTGS2(C), and INF-γ expression

[0129] like Figure 8 As shown in the results, after ConA induction, the levels of inflammatory factors IL-6, TNF-α, PTGS2 (also known as COX-2), and INF-γ in tissues increased significantly. However, intraperitoneal injection of Mps1-IN-1 (at a dose of 10 mg / kg) can effectively reduce the increase in these inflammatory factors induced by ConA. This result indicates that Mps1-IN-1 has the effect of inhibiting the increase in the levels of key cytokines in the ConA-induced inflammatory response.

[0130] Example 8: ELISA detection of IL-6, IL-1β, and TNF-α levels in mouse serum

[0131] 1. 24 hours after ConA injection, use the thumb, index finger, and middle finger of your left hand to grab the mouse's neck scalp, and use the little finger and ring finger to fix the tail. Gently press the skin around the eye to be removed to make the eyeball congested and protruding. Use ophthalmic scissors to cut the mouse's whiskers to prevent blood from accumulating in the whiskers and causing hemolysis. Use ophthalmic curved forceps to clamp the eyeball and remove it quickly, allowing the blood to flow from the eye socket into a 0.5mL EP tube. After the blood sample has been allowed to stand at room temperature for 2 hours, centrifuge it at 4°C, 1500rpm, for 10 minutes.

[0132] 2. ELISA detection method

[0133] (1) Dilute the capture antibody coating antibody working solution to the corresponding concentration, add 50 μl of antibody dissolution solution to the corresponding wells, and let it stand at room temperature overnight.

[0134] (2) Wash three times with PBST and remove the remaining liquid.

[0135] (3) Add 150 μL of blocking solution (PBS containing 10% FBS or PBS containing 1% BSA) to each well and incubate at room temperature for 1 h.

[0136] (4) Wash three times with PBST and remove the remaining liquid

[0137] (5) Add 50 μL of mouse serum and standard to each well and incubate at room temperature for 2 h.

[0138] (6) Wash three times with PBST and remove the remaining liquid.

[0139] (7) Add 50 μL of detection antibody to each well (detection antibody is generally diluted with working solution at a dilution of 1:200) and incubate at room temperature for 2 h.

[0140] (8) Wash three times with PBST and remove the remaining liquid.

[0141] (9) Add 50 μL HRP (diluted with working solution, generally 1:100) to each well and incubate at room temperature for 20 min.

[0142] (10) Wash three times with PBST and remove the remaining liquid.

[0143] (11) Add 100 μL of substrate to each well and incubate at room temperature for 5–20 min (terminate according to the degree of color development).

[0144] (12) Add 50 μL of 1 M sulfuric acid stop solution to each well, taking care to prevent the formation of large bubbles. Read immediately at 450 nm.

[0145] according to Figure 9The results showed that after ConA induction, the levels of inflammatory markers IL-6 (A), IL-1β (B), and TNF-α (C) in tissues were significantly upregulated. However, by intraperitoneal injection of Mps1-IN-1 (at a dose of 10 mg / kg), the increase in the levels of these inflammatory cytokines (IL-6, IL-1β, TNF-α) induced by ConA was successfully inhibited. This finding synonymously illustrates the effectiveness of Mps1-IN-1 in alleviating ConA-induced inflammatory responses and reducing the levels of key inflammatory factors.

[0146] Example 9: Detection of Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT) in Mouse Serum

[0147] 24 hours after ConA injection, grasp the mouse's scalp by the neck with the thumb, index finger, and middle finger of the left hand, and secure the tail with the pinky and ring fingers. Gently press the skin around the eye to be removed, causing the eyeball to become congested and protrude. Use ophthalmic scissors to trim the mouse's whiskers to prevent blood from accumulating and causing hemolysis. Use ophthalmic curved forceps to grasp the eyeball and quickly remove it, allowing the blood to flow from the eye socket into a 0.5 mL EP tube. After the blood sample has been allowed to stand at room temperature for 2 hours, centrifuge it at 1500 rpm at 4°C for 10 minutes and use the AST / ALT kit to measure AST / ALT levels.

[0148] like Figure 10 The results showed that after ConA induction, serum AST and ALT levels increased significantly, a sign typically associated with liver damage. However, intraperitoneal injection of Mps1-IN-1 (at a dose of 10 mg / kg) effectively reduced the ConA-induced increases in AST and ALT levels. This finding suggests a positive role for Mps1-IN-1 in alleviating ConA-induced liver damage and restoring liver function.

[0149] Example 10. Effect of Mps1-IN-1 on Glutathione (GSH) Levels in the Liver of Mice with ConA-Induced Acute Liver Injury

[0150] First, select 8-10-week-old C57BL / 6J male mice weighing approximately 22-26g. A certain amount of ConA is weighed and dissolved in physiological saline to prepare a modeling agent at a specific concentration. The drug is then injected into the tail vein at a dose of 10-20 mg / kg. Samples are collected 2-8 hours later for pathological analysis. If inflammatory cell infiltration around the central vein and portal area of ​​the liver in the model group mice is observed, as well as significant hepatocyte necrosis, the model is successfully established.

[0151] Detection of GSH levels in mouse liver tissue

[0152] (1) The right lobe of the mouse liver was removed and the tissue was accurately weighed. 9 times the volume of PBS was added to prepare a tissue homogenate at a ratio of weight (g) / volume (mL) = 1 / 9. The mixture was centrifuged at 2500 rpm for 5 min and the supernatant was collected for testing.

[0153] (2) Take a portion of the homogenate and determine the protein concentration using the BCA method.

[0154] (3) Follow the instructions of the GSH kit and measure the absorbance at a wavelength of 405 nm.

[0155] (4) GSH content was determined according to the following formula.

[0156] GSH content in tissue = (OD of test well - OD of blank well) 202 / (OD of standard well - OD of blank well) protein concentration.

[0157] like Figure 11 Results showed that after ConA induction, GSH levels in tissues and cells decreased significantly. However, intraperitoneal injection of Mps1-IN-1 (at a dose of 10 mg / kg) effectively restored the ConA-induced decrease in GSH levels. This suggests that Mps1-IN-1 has a beneficial effect in alleviating ConA-induced oxidative stress and maintaining stable GSH levels.

[0158] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Use of Mps1-IN-1 in the preparation of a medicament for preventing and / or treating acute liver injury, characterized in that: described The structural formula of Mps1-IN-1 is .

2. The use according to claim 1, characterized in that The acute liver injury is acute liver injury induced by concanavalin A.

3. The use according to claim 1, characterized in that Mps1-IN-1 prevents and treats acute liver injury by reducing serum AST and ALT levels.

4. The use according to claim 1, characterized in that Mps1-IN-1 protects against acute liver injury by maintaining stable glutathione (GSH) levels.

5. The use according to claim 1, characterized in that The concentration of Mps1-IN-1 is 1-10 μM.

6. The use according to claim 1, characterized in that The medicine includes pharmaceutically acceptable pharmaceutical excipients.

7. The use according to claim 6, characterized in that The pharmaceutical excipient is selected from one or more of a diluent, a binder, a wetting agent, a disintegrant, an absorption accelerator, and a surfactant.

8. The use according to claim 1, characterized in that The drug is prepared into a pharmaceutically acceptable dosage form.

9. The use according to claim 8, characterized in that The dosage form is tablet, pill, paste, capsule, oral liquid or granule.

10. The use according to claim 9, characterized in that The dosage form is a tablet.

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