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

Abstract

The invention discloses application of Mps1-IN-1 as a ferroptosis inhibitor and in preparation of drugs for preventing and treating ferroptosis-related diseases, and belongs to the technical field of biological medicines. The invention specifically provides any one of the following applications of Mps1-IN-1: 1, application as a ferroptosis inhibitor; 2, application in preparation of drugs for prevention and / or treatment of ferroptosis related diseases; 3, application in construction of a ferroptosis tumor cell model, and the like. The inhibitor has the beneficial effects that the inhibitor shows a remarkable protection capability on cell fate under various ferroptosis induction mechanisms, the unique action mechanism of the inhibitor is not limited to simple inhibition of a ferroptosis process, and the inhibitor can efficiently remove harmful oxygen free radicals and effectively inhibit the generation of lipid peroxides. The discovery opens up a new way for prevention and treatment of ferroptosis-related diseases, and the research and development and application have profound scientific significance and bring positive and profound influence on human health undertaking.

Description

Technical Field

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

[0002] Ferroptosis is a unique cell death mechanism, whose core feature is the surge in toxic lipid peroxide ROS levels triggered by abnormal accumulation of intracellular iron, a process that is significantly different from the traditional apoptosis pathway. The essence of ferroptosis is rooted in the severe depletion of glutathione (GSH) levels. The reduction of this key antioxidant molecule directly weakens the function and activity of the core of the antioxidant defense system, the GPX4 enzyme, and then blocks the channel for lipid peroxides to be effectively metabolized through the glutathione-dependent pathway catalyzed by GPX4, resulting in the continuous accumulation of lipid peroxides.

[0003] What is particularly critical is that the continuously accumulated Fe2+ activates the Fenton reaction, exacerbating the oxidative damage of unsaturated fatty acids on the cell membrane. 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 a variety of 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, etc., showing its importance in the medical field that cannot be ignored. In view of 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 is a global health challenge. Its common causes include the invasion of drug-induced hepatitis and post-traumatic hemorrhagic shock, which causes a sharp decrease in liver blood perfusion, resulting in 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 cell death mode. 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 in all stages of liver disease from the onset to the 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 solid evidence that small molecule compounds such as Ferrostatin-1, iron chelators (such as DFO), Liproxstatin-1, vitamin K, and free radical-trapping antioxidants, as effective inhibitors of ferroptosis, can selectively block the ferroptosis process of cells through diverse molecular mechanisms, thereby significantly improving the clinical prognosis of a variety of ferroptosis-related diseases, these inhibitors still face many challenges and limitations in terms of complexity of synthesis, 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) and has the structural formula Mps1 is an important cell cycle regulatory protein kinase that is associated with tumor development and abnormal cell division. So far, there has been no report on the application of Mps1-IN-1 in inhibiting ferroptosis to treat ferroptosis-related diseases, especially 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 ferroptosis inhibitor;

[0011] (2) Use 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 reducing 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 line, human breast cancer cell line, mouse breast cancer cell line, and mouse fibrosarcoma cell line.

[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 comprises a pharmaceutically acceptable salt, specifically an organic salt or an inorganic salt.

[0023] Preferably, in (2), (4), (5), (6) and (7), the drug comprises a pharmaceutically acceptable excipient.

[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 has innovatively revealed a new application field of Mps1-IN-1 as a ferroptosis inhibitor, especially its significant 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 occurrence and development of acute liver injury, but also proposed a set of practical medication regimens. This discovery not only opens up a new path for the treatment of ferroptosis-related diseases, but also provides valuable inspiration and direction for the future exploration, development and preparation of more efficient, advanced and safer ferroptosis inhibitory drugs.

[0029] 2. The present invention explores and establishes the potential application 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 efficacy 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 extremely low concentrations (1 μM). Its inhibitory efficacy is not only comparable to that of currently known ferroptosis inhibitors, but even performs better in some cases.

[0031] 4. The present invention further verifies the excellent efficacy of Mps1-IN-1 as a small molecule drug. It shows effective resistance to the ferroptosis process triggered by a variety of ferroptosis inducers (such as RSL3, Erastin, FIN56, etc., which are small molecule compounds that can induce cell ferroptosis), thereby opening up a broader and more efficient treatment path for the treatment of 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 the synthesis route, improving the purity and stability of the drug, 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; wherein Figure 1 A is the IC50 value of Mps1-IN-1 inhibiting RSL3-induced ferroptosis. Figure 1 B is the 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; wherein Figure 2 A is a graph showing the 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 the changes in cell survival rate of Mps1-IN-1 in Example 3 of the present invention in inhibiting RSL3-induced ferroptosis in different cells MCA-205 (A), HT1080 (B), 4T1 (C), and MDA-MB-231 (D);

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

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

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

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

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

[0041] Fig. 9 This is a graph showing the ELISA test results 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] Fig.10 The results of Mps1-IN-1 reducing the AST level (A) and ALT level (B) in serum after ConA induction in Example 9 of the present invention are shown;

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

[0044] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are 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 creative work are 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 art 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 a 96-well plate. The groups and corresponding treatments were as follows:

[0052] (1) Normal control group (Control): HT1080 cells were cultured normally without any 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) Different concentrations of Mps1-IN-1 treatment group + RSL3 group: 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 adding different final concentrations of Mps1-IN-1 for 2 h.

[0055] Discard the culture medium in the 96-well plate, wash twice with 1×PBS, prepare CCK-8 working solution according to the ratio of CCK-8:DMEM medium = 1:10, and add 100 μL to each well. Place the 96-well plate in a 37°C incubator in the dark for 1 hour, and measure the OD value at a wavelength of 450nm using an enzyme marker. Calculate the cell survival rate according to the formula:

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

[0057] Results Figure 1 From the analysis of the cell activity results of the RSL3 treatment group, 1μM concentration of RSL3 successfully induced cell ferroptosis. From the cell activity detection of the Mps1-IN-1 treatment group + RSL3 group, it can be seen that Mps1-IN-1 pretreatment can inhibit cell ferroptosis. This shows 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 a 96-well plate. 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 treatment 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] Then discard the culture medium in the 96-well plate, wash twice with 1×PBS, prepare CCK-8 working solution according to the ratio of CCK-8:DMEM medium = 1:10, and add 100μL to each well. Place the 96-well plate in a 37℃ incubator in the dark for 1h, and measure the OD value at a wavelength of 450nm using an enzyme marker. Calculate the cell survival rate according to the formula.

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

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

[0071] Take HT1080 cells with good growth conditions, digest and resuspend them, centrifuge at 1500 rpm for 3 minutes, and discard the supernatant.

[0072] According to 5 x 10 per well 3 A certain concentration of cell suspension was prepared, 100 μL of cell suspension was added to each well, and the cells were inoculated 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 treatment 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 well: 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 operated according to the instructions of the kit;

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

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

[0085] like Figure 2 As shown in B, in the experimental groups treated with different inducers alone, the release of LDH (lactate dehydrogenase) increased significantly. However, after pretreatment with Mps1-IN-1, the release of LDH in each experimental group decreased significantly. 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] Take HT1080 cells with good growth conditions, digest and resuspend them, centrifuge at 1500 rpm for 3 minutes, and discard the supernatant.

[0088] According to 5 x 10 per well 3A certain concentration of cell suspension was prepared, 100 μL of cell suspension was added to each well, and the cells were inoculated into 96-well plates. The groups were 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] The cell survival rate was calculated according to the formula.

[0093] like Figure 3 The results show that the cell activity decreased after treatment with the ferroptosis inducer RSL3 in different cell lines. However, under the condition of pre-treatment with Mps1-IN-1, the activity of various cell lines including MCA-205 (A), HT1080 (B), 4T1 (C) and MDA-MB-231 (D) showed a significant recovery. This result shows that pre-treatment with Mps1-IN-1 has a significant effect on inhibiting the ferroptosis process induced by RSL3 in these different cell lines.

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

[0095] like Figure 4 The results show that in various cell lines, treatment with the ferroptosis inducer RSL3 led to a significant increase in cellular LDH (lactate dehydrogenase) levels. However, when these cell lines were pretreated with Mps1-IN-1 before RSL3 treatment, the LDH levels of MCA-205 (A), HT1080 (B), 4T1 (C), and MDA-MB-231 (D) cell lines 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 1500rpm for 3min 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 results showed that in the cell culture system, RSL3 treatment alone led to a significant increase in the number of cell deaths. However, when Mps1-IN-1 was added for pretreatment before RSL3 treatment, the cell death caused by RSL3 was effectively inhibited. This result directly proves that Mps1-IN-1 can inhibit the cell death process caused by RSL3.

[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 were treated, the cell culture supernatant was removed, 1 mL of serum-free DMEM and 5 μM BODIPY581 / 591C11 were added to each well, the 6-well plate was placed in a 37°C incubator in the dark and cultured for 0.5 h, EDTA-free trypsin was added to digest the cells, and 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, and the cells were collected. After resuspending in 1×PBS pre-cooled at 4°C, the cells were centrifuged again to collect the cells (this washing process was repeated 2-3 times).

[0110] like Figure 6As shown in the figure, when cells were treated with RSL3 alone, the level of lipid peroxidation in the ferroptosis pathway increased significantly. However, if Mps1-IN-1 was added before treatment, a significant inhibition of lipid peroxidation levels was observed. This finding supports the role of Mps1-IN-1 in inhibiting 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 were treated, the cell culture supernatant was removed, 1 mL of serum-free DMEM and 5 μM H2DCFDA were added to each well, the 6-well plate was placed in a 37°C incubator in the dark and cultured for 0.5 h, EDTA-free trypsin was added to digest the cells, and 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, and the cells were collected. After resuspending in 4°C pre-cooled 1ⅹPBS, the cells were centrifuged again to collect the cells (this washing process was repeated 2-3 times).

[0117] according to Figure 7 As shown in the results, cells treated 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. The groups were divided as follows:

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

[0122] (2) Group 2: Mps1-IN-1 treatment group. Mps1-IN-1 (10 mg / kg) was first injected intraperitoneally, and ConA (15 mg / kg) was 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) Group 3: 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 caudal lobe of mice, 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, 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 caused by ConA induction. This result synonymously 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 to detect 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 fix the tail with the little finger and ring finger. Gently press the eye skin to be removed to make the eyeball congested and protruding. Use ophthalmic scissors to cut the mouse's whiskers to prevent blood from flowing from the whiskers and causing hemolysis. Use ophthalmic curved forceps to clamp the eyeball and remove it quickly, and let the blood flow from the eye socket into a 0.5mL EP tube. After the blood sample is allowed to stand at room temperature for 2 hours, centrifuge it at 4℃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 stand at room temperature overnight.

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

[0139] (7) Add 50 μL of detection antibody to each well (the 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 any 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 any remaining liquid.

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

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

[0145] according to Fig. 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, use the thumb, index finger and middle finger of the left hand to grab the neck scalp of the mouse, and fix the tail with the little finger and ring finger. Gently press the eye skin to be removed to make the eyeball congested and protruding. Use ophthalmic scissors to cut the mouse's whiskers to prevent blood from flowing from the whiskers and causing hemolysis. Use ophthalmic curved forceps to clamp the eyeball and remove it quickly, and let the blood flow from the eye socket into a 0.5mL EP tube. After the blood sample is allowed to stand at room temperature for 2 hours, it is centrifuged at 4℃1500rpm for 10 minutes and used for AST / ALT kit to detect AST / ALT levels.

[0148] like Fig.10 The results shown in the figure show that after ConA induction, the levels of AST and ALT in serum were significantly increased, which is usually associated with liver damage. However, treatment with intraperitoneal injection of Mps1-IN-1 (at a dose of 10 mg / kg) can effectively reduce the increase in AST and ALT levels caused by ConA induction. This finding synonymously reveals the positive role of 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 Model

[0150] First, select 8-10 weeks old C57BL / 6J male mice weighing about 22-26g, weigh a certain amount of ConA, dissolve it in physiological saline, prepare a certain concentration of modeling agent, and inject it into the tail vein at a dose of 10-20mg / kg. After 2-8h, samples were taken for pathological analysis. If there was inflammatory cell infiltration around the central vein and portal area of ​​the liver of the model group mice and obvious hepatocyte necrosis, it means that the model was successfully constructed.

[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. At a ratio of weight (g) / volume (mL) = 1 / 9, 9 times PBS was added to prepare a tissue homogenate. 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) The 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 Fig.11 The results showed that after ConA induction, the GSH level in tissues or cells decreased significantly. However, intraperitoneal injection of Mps1-IN-1 (at a dose of 10 mg / kg) can effectively restore the decrease in GSH levels caused by ConA induction. This shows 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 the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. 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 embodiments of the present invention.

Claims

1. Mps1-IN-1 is used in any of the following applications: (1) Application as a ferroptosis inhibitor; (2) Use in the preparation of drugs for preventing and / or treating ferroptosis-related diseases; (3) Application in constructing ferroptosis tumor cell models; (4) Application in the preparation of drugs for inhibiting the increase of cellular reactive oxygen species (ROS) levels; (5) Application in the preparation of drugs for reducing the levels of inflammatory factors IL-6, IL-1β, TNF-α, PTGS2, and INF-γ; (6) Use in the preparation of drugs for reducing serum AST and ALT levels; (7) Application in the preparation of drugs for maintaining stable glutathione GSH levels; The structural formula of Mps1-IN-1 is 2. The use according to claim 1, characterized in that: 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.

3. The use according to claim 1, characterized in that: 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.

4. The use according to claim 1, characterized in that: In constructing the ferroptosis tumor cell model, the ferroptosis inducer used is selected from any one of Erastin, RSL3, and FIN56.

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 ferroptosis inhibitor includes pharmaceutically acceptable salts, specifically organic salts or inorganic salts.

7. The use according to claim 1, characterized in that: In (2), (4), (5), (6) and (7), the drug includes pharmaceutically acceptable excipients.

8. The use according to claim 7, characterized in that: The pharmaceutically acceptable pharmaceutical 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.

9. The use according to claim 1, characterized in that: In (2), (4), (5), (6) and (7), the drug is prepared into a pharmaceutically acceptable dosage form.

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

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