Application of mexatinib as copper death inhibitor in preparation of medicine for preventing and treating copper death related diseases

Abstract

The invention discloses an application of mexatinib as a copper death inhibitor in preparation of a medicine for preventing and treating copper death related diseases, and relates to the technical field of medicines. Cell and mouse experiments prove that the Merestinib remarkably recovers the levels of Fe-S cluster protein (LIAS / DLAT) and esterification protein (FDX1) related to copper death and reduces the level of heat shock protein HSP70, so that the sensitivity of various cells to copper death is reduced, and the recovery effect on acute liver injury is enhanced. Meanwhile, the Merestinib can also obviously reduce the levels of glutamic oxalacetic transaminase and glutamic-pyruvic transaminase in serum and the levels of inflammatory factors IL-1beta, IL-6 and TNF-alpha, effectively promotes the repair of liver tissues and plays an anti-inflammatory role. The invention provides theoretical support for treating copper death related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of merestinib as a copper death inhibitor in the preparation of drugs for preventing and treating copper death-related diseases. Background Art

[0002] Programmed cell death (PCD) is essential for maintaining body homeostasis, growth and development, and clearing infected, damaged or senescent cells. Common pathways of programmed cell death include apoptosis, necroptosis, autophagy, ferroptosis, and pyroptosis. In 2022, Tsvetkov et al. revealed a new type of programmed cell death triggered by reduced copper that is different from apoptosis, necrosis, pyroptosis, and ferroptosis, and named it copper death. This process is caused by Cu(I) directly binding to the acylated components in the tricarboxylic acid (TCA) cycle, leading to lipid protein aggregation and instability of Fe-S cluster proteins, triggering protein toxic stress, and ultimately leading to cell death. Studies have shown that copper death is closely related to the occurrence and development of many diseases, such as tumors, neurodegenerative diseases, cardiovascular diseases, ischemia-reperfusion injury, and acute damage to tissues and organs. Therefore, in-depth exploration of the relevant mechanisms in the copper death process is of great significance for finding new therapeutic targets and developing new drugs and treatments.

[0003] Copper is an essential element of living organisms. As a cofactor of many key metabolic enzymes, it participates in various biological processes of the body, including mitochondrial energy production, tyrosine and neurotransmitter metabolism, redox homeostasis and extracellular matrix remodeling. [5] . However, when copper ions accumulate in the body beyond a certain level, it can lead to the occurrence of diseases. For example, in the Wilson disease model, excessive copper accumulation in the liver and brain can lead to severe liver and nervous system diseases. In addition, high levels of copper have also been detected in some neurodegenerative disease models (Alzheimer's disease (AD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS), etc.). Studies in recent decades have confirmed that copper can promote the death of cancer cells through apoptosis and / or free radical accumulation.

[0004] The latest research shows that excessive copper increases the level of lipidated TCA enzymes (especially pyruvate dehydrogenase complexes), leading to lipidated protein aggregation, reducing the level of Fe-S cluster proteins and inducing the expression of HSP70, which can serve as a potential target for killing cancer cells with active mitochondrial metabolism. In the body, the liver is the main organ for storing copper, which plays an important role in the body's metabolism and internal environment homeostasis. However, when the body is damaged by certain stimuli (such as viral infection, excessive use of acetaminophen, ingestion of toxins, autoimmune diseases / metabolic diseases, etc.), it will cause metabolic disorders of copper ions, which will further aggravate the damage to liver cells and may lead to the occurrence of chronic liver diseases such as cirrhosis and liver fibrosis. Therefore, reducing the level of copper death of cells under pathological conditions is of great significance for alleviating liver damage and inhibiting the occurrence and development of chronic liver diseases, but so far there are no reports on copper death inhibitors. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to provide a copper death inhibitor and use it in the preparation of copper death-related disease drugs, thereby providing a basis for new drug development and new clinical therapies.

[0006] The present invention solves the above technical problems through the following technical means:

[0007] The present invention proposes the use of merestinib as a copper death inhibitor in the preparation of products for preventing and treating copper death-related diseases.

[0008] Preferably, the copper death-related diseases specifically refer to: neurodegenerative diseases, cardiovascular diseases, ischemia-reperfusion injuries, and acute and chronic injuries of tissues and organs, including but not limited to the above diseases, and acute liver injury is further preferred.

[0009] Preferably, the product includes one or more of food, medicine, and health care products.

[0010] The present invention also proposes any of the following uses of Merestinib:

[0011] (1) Application in the preparation of drugs for increasing the levels of Fe-S cluster proteins (LIAS / DLAT) and / or esterified proteins (FDX1);

[0012] (2) Application in the preparation of drugs for inhibiting the production of heat shock protein HSP70;

[0013] (3) Use in the preparation of drugs for reducing serum aspartate aminotransferase and / or alanine aminotransferase levels;

[0014] (4) Use in the preparation of drugs for reducing the levels of inflammatory factors IL-1β, IL-6 and / or TNF-α;

[0015] (5) Application in the preparation of drugs for inhibiting cell copper death;

[0016] (6) Application in the preparation of drugs for reducing the sensitivity of cells to copper death;

[0017] (7) Use in the preparation of drugs for alleviating acute liver injury;

[0018] (8) Application in the preparation of drugs for inhibiting liver tissue damage in mice and improving the repair effect of acute liver damage.

[0019] Preferably, in (5), the cell copper death is ES-Cu-induced cell copper death; the cells include B16-F10 and MDA-MB-231 cells.

[0020] Preferably, in (1) to (8), the drug further comprises a pharmaceutically acceptable salt, specifically an organic salt or an inorganic salt.

[0021] Preferably, in (1) to (8), the drug further comprises a pharmaceutically acceptable excipient.

[0022] Preferably, the 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.

[0023] Preferably, in (1) to (8), the drug is prepared into a pharmaceutically acceptable dosage form.

[0024] Preferably, the dosage form is tablet, pill, paste, oral solution or granule.

[0025] The beneficial effects of the present invention are:

[0026] 1. The present invention proposes a new use of an inhibitor, Merestinib (an effective orally bioavailable c-Met inhibitor with anti-tumor activity), in the preparation of drugs for the prevention and treatment of copper death-related diseases. Merestinib significantly restored the levels of Fe-S cluster proteins (LIAS / DLAT) and esterified proteins (FDX1) in the copper death process, reduced the level of heat shock protein HSP70, and simultaneously reduced the sensitivity of multiple cells to copper death and enhanced the recovery effect of acute liver injury.

[0027] 2. At the same time, Merestinib can also significantly reduce the levels of serum aspartate aminotransferase and alanine aminotransferase, and the levels of inflammatory factors (IL-1β, IL-6, TNF-α), promote the repair of liver tissue and play an anti-inflammatory role. In summary, the application of the inhibitor Merestinib in the preparation of drugs for copper death-related diseases can provide theoretical support for the development of new drugs and new clinical therapies. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The figures are analysis diagrams of Merestinib inhibiting ES-Cu-induced copper death in various cells in Example 1 of the present invention, wherein A is the effect of Merestinib on ES-Cu-induced B16-F10 cells; B is the effect of Merestinib on ES-Cu-induced MDA-MB-231 cells; C is a comparison diagram of each group treating B16-F10 cells; and D is a comparison diagram of each group treating MDA-MB-231 cells;

[0029] Figure 2 The analysis diagram of Merestinib's specific inhibition of copper death in Example 2 of the present invention, wherein A is the analysis of ES-Cu-induced cell copper death; B is the analysis of RSL3-induced ferroptosis; C is H 2 O 2 Analysis of induced cell necrosis.

[0030] Figure 3 This is a graph showing the mRNA levels of Fe-S cluster protein FDX1 (A) and esterified proteins LIAS (B) and DLAT (C) in the process of copper death in MDA-MB-231 cells restored by Merestinib in Example 3 of the present invention.

[0031] Figure 4 This is a graph showing the mRNA levels of Fe-S cluster protein FDX1 (A) and esterified proteins LIAS (B) and DLAT (C) during the process of Merestinib restoring Con A-induced copper death in Example 4 of the present invention.

[0032] Figure 5 This is a graph showing the levels of proteins (FDX1 / HSP70) related to the process of Merestinib restoring Con A-induced copper death in Example 5 of the present invention.

[0033] Figure 6 This is a diagram showing that Merestinib restores Con A-induced liver tissue damage in Example 6 of the present invention.

[0034] Figure 7 This is a graph showing that Merestinib reduces Con A-induced ALT (A) and AST (B) levels in Example 7 of the present invention.

[0035] Figure 8 This is a graph showing that Merestinib reduces the levels of Con A-induced inflammatory factors IL-1β (A), IL-6 (B) and TNF-α (C) in Example 8 of the present invention. DETAILED DESCRIPTION

[0036] 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.

[0037] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0038] If no specific technology or conditions are specified in the examples, they can be carried out according to the technology or conditions described in the literature in the field or according to the product instructions. Unless otherwise specified, the quantitative tests in the following examples are repeated more than three times, and the results are averaged.

[0039] Reagents and drug preparation methods used in the examples:

[0040] Merestinib: Catalog number T3455, purchased from Shanghai Taoshu Biotechnology;

[0041] Elesclomol: Catalog No. T6170, purchased from Shanghai Taoshu Biological;

[0042] Copper chloride dihydrate: Product No. A603090-0250, purchased from Shanghai Sangon Biotechnology Co., Ltd.;

[0043] Dimethyl sulfoxide (DMSO): Catalog No. A610163-0250, purchased from Sangon Biotechnology (Shanghai) Co., Ltd.;

[0044] CCK-8: Product No. c0005, purchased from Shanghai Taoshu Biological Technology Co., Ltd.

[0045] Propidium iodide: Product No. P4170, purchased from MilliporeSigma;

[0046] All-in-one RT SuperMix: Product No. R223, purchased from Vazyme;

[0047] qPCR SYBR Green Master Mix: Catalog No. 11201ES08, purchased from Yisheng Biotechnology;

[0048] RSL3: Product No. T3646, purchased from Shanghai Taoshu Biological Technology Co., Ltd.

[0049] Hydrogen peroxide solution: Product No. 7722-84-1, purchased from MacLean Reagent;

[0050] Concanavalin A: Product No. 11028-71-0, purchased from Sigma;

[0051] RNA extraction reagent (Trizol): Catalog number B511311-0100, purchased from Sangon Biotech (Shanghai) Co., Ltd.;

[0052] Mouse aspartate aminotransferase (ALT) kit: Catalog number C009-2-1, purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.;

[0053] Mouse alanine aminotransferase (AST) kit: Product No. C010-2-1, purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.;

[0054] Mouse IL-1β ELISA kit: Catalog number DY401, purchased from R&D Systems Inc;

[0055] Mouse IL-6 ELISA kit: Catalog number DY406-05, purchased from R&D Systems Inc;

[0056] Mouse TNF-α Duoset ELISA kit: Catalog number DY410-05, purchased from R&D Systems Inc;

[0057] Adrenodoxin Rabbit mAb: Product No. R23394, purchased from Chengdu Zhengneng Biotechnology Co., Ltd.;

[0058] beta Actin Rabbit pAb: Catalog No. 380624, purchased from Chengdu Zhengneng Biotechnology Co., Ltd.;

[0059] HSP70 Mouse mAb: Catalog No. A1507, purchased from ABclonal;

[0060] Preparation of copper death inducer ES-Cu working solution: First, Elesclomol and cupric chloride dihydrate were dissolved in DMSO to make 100mM stock solution, the Elesclomol stock solution was stored at -20℃, and the cupric chloride dihydrate stock solution was stored at 4℃. Then, 1μL of each stock solution was mixed in 8μL DMSO, and then added to 990μL serum-free and anti-drug-free DMEM culture medium. After mixing, 100μM ES-Cu working solution was prepared.

[0061] Instrument models and companies used in the examples:

[0062] Zeiss inverted fluorescence microscope: model Axio Vert A1, Carl Zeiss, Germany;

[0063] Zeiss fully automated upright fluorescence microscope: model Axio Imager Z2, Carl Zeiss, Germany;

[0064] Full-function microplate reader (ELISA reader): Model: Synergy H4, Berton Instruments, Inc., USA;

[0065] Fully automated fluorescence quantitative PCR system: LightCycler 480Ⅱ, 96 / 384-well plate, Roche, USA;

[0066] Fully automatic chemiluminescence / fluorescence image analysis system: model Tanon-5200Multi, Shanghai Tianneng Technology Co., Ltd.

[0067] Each experimental treatment group was subjected to three biological replicates, and the data of each group were expressed as the mean ± standard deviation (mean ± SD) of three independent experiments. The experimental results were visualized and statistically analyzed using GraphPad Prism 9.0 software. The differences between the two groups were analyzed by unpaired two-tailed t-test. Compared with the control group, the significance levels were expressed as *p<0.05, **p<0.01, and ***p<0.001.

[0068] Example 1: Merestinib inhibits ES-Cu-induced copper death in various cells

[0069] 1. Merestinib inhibits ES-Cu-induced copper death of B16-F10 and MDA-MB-231 cells.

[0070] 1. Cell plating: B16-F10 and MDA-MB-231 cells growing in the logarithmic phase were digested, centrifuged, resuspended and counted, and then the cells were evenly seeded in 96-well plates (10 4 cell / well), cells were cultured overnight;

[0071] 2. On the next morning, the well-growing cells were divided into groups: A. control group, no treatment; B. groups treated with different concentrations of Merestinib;

[0072] 3. Merestinib was added to treatment group B at final concentrations of 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10, and 20 μM per well and incubated for 2 h;

[0073] 4. After incubation, add ES-Cu working solution to the A control group and B treatment group at a final concentration of 1 μM per well to induce cell death for 8-10 hours;

[0074] 5. Add 10 μL CCK-8 / well according to the cell status, incubate in the incubator for 1 hour, and then use a microplate reader to detect the absorbance at a wavelength of 450 nm.

[0075] 6. Calculate cell viability according to the formula. Use GraphPad Prism 9 to calculate IC 50 value.

[0076] According to the results Figure 1 A and Figure 1 B concluded that the half-inhibitory concentrations of Merestinib on ES-Cu-induced copper death of B16-F10 and MDA-MB-231 cells were 1.507μM and 1.657μM, respectively, and the inhibitory effect was concentration-dependent.

[0077] 2. Morphology and fluorescence staining verified that Merestinib inhibited ES-Cu-induced copper death of B16-F10 and MDA-MB-231 cells.

[0078] 1. Cell plating: B16-F10 and MDA-MB-231 cells growing in the logarithmic phase were digested, centrifuged, resuspended and counted, and then the cells were evenly seeded in 24-well plates (10 5 cell / well), cells were cultured overnight;

[0079] 2. On the next morning, the well-growing cells were divided into groups: A. Control group (Mock), no treatment; B. ES-Cu treatment group; C. Merestinib + ES-Cu treatment group;

[0080] 3. Add Merestinib to the C treatment group at a final concentration of 10 μM per well and incubate for 2 h;

[0081] 4. After incubation, add ES-Cu working solution to the B treatment group and the C treatment group at a final concentration of 1 μM per well to induce cell death for 8-10 hours;

[0082] 5. Add 10 μg / mL propidium iodide according to the cell status and stain at room temperature in the dark for 30 minutes, then use a Zeiss inverted fluorescence microscope to photograph.

[0083] 6. After the shooting is completed, count the cells in each group.

[0084] According to the results Figure 1 C and Figure 1 D concluded that Merestinib can significantly inhibit ES-Cu-induced copper death of B16-F10 and MDA-MB-231 cells. In summary, Merestinib inhibits ES-Cu-induced copper death of various cells and can inhibit cell morphological changes.

[0085] Example 2: Merestinib specifically inhibits cell copper death.

[0086] 1. Cell plating: Digest, centrifuge, resuspend and count the MDA-MB-231 cells growing in the logarithmic phase, and then evenly inoculate the cells in a 96-well plate (10 4 cell / well), cells were cultured overnight;

[0087] 2. On the second day, the well-growing cells were divided into groups: A. control group (Mock), no treatment; B. inducer-treated group; C. Merestinib + inducer-treated group;

[0088] 3. Add Merestinib to the C treatment group at a final concentration of 10 μM per well and incubate for 2 h;

[0089] 4. After incubation, add ES-Cu (1 μM), RSL3 (1 μM), H 2 O 2 (10 μM) was added to the B-treated group and the C-treated group to induce cell death;

[0090] 5. Add 10 μL CCK-8 / well according to the cell status, incubate in the incubator for 1 hour, and then use an enzyme reader to detect the absorbance value of each well at a wavelength of 450 nm.

[0091] 6. Calculate cell viability according to the formula and use GraphPad Prism 9 to calculate IC 50 value.

[0092] According to the results Figure 2 A. Figure 2 B and Figure 2 C concluded that Merestinib could not inhibit RSL3-induced ferroptosis and H 2 O 2Merestinib induced cell necrosis, but could inhibit ES-Cu-induced cell copper death, indicating the specificity of merestinib in inhibiting cell death type.

[0093] Example 3: Merestinib restored the mRNA levels of FDX1, LIAS and DLAT in MDA-MB-231 cells.

[0094] 1. Cell plating: Digest, centrifuge, resuspend and count the MDA-MB-231 cells in the logarithmic phase, and then evenly inoculate the cells in a 12-well plate (3×10 5 cell / well), cells were cultured overnight;

[0095] 2. On the next morning, the well-growing cells were divided into groups: A. control group, no treatment; B. ES-Cu treatment group; C. Merestinib + ES-Cu treatment group;

[0096] 3. Add Merestinib to the C treatment group at a final concentration of 10 μM per well and incubate for 2 h;

[0097] 4. After incubation, add ES-Cu working solution to the B treatment group and the C treatment group at a final concentration of 1 μM per well to induce cell death for 6-8 hours;

[0098] 5. Lyse cells using Trizol and extract total RNA.

[0099] 6. RNA reverse transcription to cDNA: (1) cDNA template 1 μg, 4 μL 4×gDNA wiper Mix, add ddH 2 0 to 20 μL, mix gently with a pipette, and incubate at 42°C for 2 min. (2) Pipette 16 μL of the product from step (1) and add 4 μL of reverse transcriptase RT Super Mix, mix gently with a pipette, and incubate at 37°C for 15 min, and 85°C for 5 s.

[0100] 7. qPCR detection: (1) cDNA 0.5 μg, Primer F 0.5 μg, Primer R 0.5 μg, qPCR SYBRGreen Master Mix 10 μL, add ddHO 2 0 to 20 μL, mix gently with a pipette and then test on the instrument. (2) Program: Stage 1 pre-denaturation: 95℃ 3 min, Stage 2 cycle reaction: 95℃ 10 s, 60℃ 10 s, 72℃ 10 s, Stage 3 melting curve: 95℃ 15 s, 60℃ 60 s, 95℃ continuous.

[0101] 8. Perform data analysis after the test is completed.

[0102] According to the results Figure 3 A. Figure 3 B and Figure 3 C concluded that Merestinib can affect the mRNA levels of Fe-S cluster protein (FDX1) and esterification protein (LIAS / DLAT) related to copper death in cells, suggesting that Merestinib's inhibition of copper death may be related to its effect on the transcription levels of genes such as FDX1.

[0103] Example 4: Merestinib reduces Con A-induced FDX1, LIAS, and DLAT mRNA levels.

[0104] 1. Eighteen 8-week-old C57BL / 6 wild-type mice were selected and divided into three groups, with 6 mice in each group. The specific grouping and drug treatment methods are as follows:

[0105] (1) Control group: 0h: intraperitoneal injection of PBS with the same volume as the drug in the treatment group; 0.5h: tail vein injection of PBS with the same volume as the drug in the treatment group; 1h: intraperitoneal injection of PBS with the same volume as the drug in the treatment group;

[0106] (2) Con A group: 0h: intraperitoneal injection of PBS with the same volume as the drug in the treatment group; 0.5h: tail vein injection of Con A (15 mg / kg); 1h: intraperitoneal injection of PBS with the same volume as the drug in the treatment group;

[0107] (3) Merestinib+Con A group: 0h: intraperitoneal injection of half dose of MTB (12 mg / kg), 0.5h: tail vein injection of Con A (15 mg / kg), 1h: intraperitoneal injection of the other half dose of MTB (12 mg / kg);

[0108] 2. 24 hours after ConA injection, the eyeballs were removed to collect blood and the mice were killed. The mouse livers were quickly isolated. After all the mice were sampled, part of the liver was cut and added with appropriate amount of Trizol to grind the tissue and extract RNA.

[0109] 3. Then, the mRNA levels of FDX1, LIAS and DLAT in the liver were detected according to the reverse transcription and qPCR methods in Example 3.

[0110] According to the results Figure 4 A. Figure 4 B and Figure 4 C concluded that Merestinib can affect the mRNA levels of Fe-S cluster protein (FDX1) and esterified protein (LIAS / DLAT) associated with copper death in liver tissue.

[0111] Example 5: Merestinib reduces the levels of proteins related to Con A-induced copper death.

[0112] 1. Eighteen 8-week-old C57BL / 6 wild-type mice were selected and divided into three groups, with 6 mice in each group. The specific grouping and drug treatment methods are as follows:

[0113] A. Control group: 0h: intraperitoneal injection of PBS with the same volume as the drug in the treatment group; 0.5h: tail vein injection of PBS with the same volume as the drug in the treatment group; 1h: intraperitoneal injection of PBS with the same volume as the drug in the treatment group;

[0114] B. Con A group: 0h: intraperitoneal injection of PBS with the same volume as the drug in the treatment group, 0.5h: tail vein injection of Con A (15mg / kg), 1h: intraperitoneal injection of PBS with the same volume as the drug in the treatment group;

[0115] C. Merestinib+Con A group: 0h: intraperitoneal injection of half dose of MTB (12 mg / kg), 0.5h: tail vein injection of Con A (15 mg / kg), 1h: intraperitoneal injection of the other half dose of MTB (12 mg / kg);

[0116] 2. 24 hours after ConA injection, the eyeballs were removed to collect blood and the mice were killed. The mouse livers were quickly freed. After all the mice were sampled, part of the liver was cut and added with appropriate amount of NP40 for tissue grinding and crushing, and then the supernatant was obtained after centrifugation.

[0117] 3. Quantify the supernatant protein. After completion, add an appropriate amount of SDS loading buffer (1.5×) and boil in a metal bath at 101°C for 10 minutes.

[0118] 4. Then load the sample for western blot detection.

[0119] According to the results Figure 5 It was concluded that Merestinib can also affect the expression of Fe-S cluster protein (FDX1) and heat shock protein (HSP70) in liver tissue, further illustrating the effectiveness of Merestinib in inhibiting copper death.

[0120] Example 6: Merestinib restores Con A-induced liver damage.

[0121] 1. Eighteen 8-week-old C57BL / 6 wild-type mice were selected and divided into three groups, with 6 mice in each group. The specific grouping and drug treatment methods are as follows:

[0122] A. Control group: 0h: intraperitoneal injection of PBS with the same volume as the drug in the treatment group; 0.5h: tail vein injection of PBS with the same volume as the drug in the treatment group; 1h: intraperitoneal injection of PBS with the same volume as the drug in the treatment group;

[0123] B. Con A group: 0h: intraperitoneal injection of PBS with the same volume as the drug in the treatment group, 0.5h: tail vein injection of Con A (15mg / kg), 1h: intraperitoneal injection of PBS with the same volume as the drug in the treatment group;

[0124] C. Merestinib+Con A group: 0h: intraperitoneal injection of half dose of MTB (12 mg / kg), 0.5h: tail vein injection of Con A (15 mg / kg), 1h: intraperitoneal injection of the other half dose of MTB (12 mg / kg);

[0125] 2. 24 hours after ConA injection, the eyeballs were removed to collect blood and the mice were killed. All mouse livers were quickly freed and then the tissues were fixed, dehydrated, embedded, sliced, and HE stained. The livers of mice in different groups were stained and the pathological changes of liver tissue were observed under a microscope.

[0126] According to the results Figure 6 The results showed that Merestinib can effectively alleviate the acute damage to the liver caused by Con A, for example, it can maintain the basic morphology of organ tissues (hepatic sinusoids, hepatic plates and central veins, etc.), and reduce inflammatory infiltration of liver tissues.

[0127] Example 7: Merestinib reduces Con A-induced ALT / AST levels.

[0128] 1. Eighteen 8-week-old C57BL / 6 wild-type mice were selected and divided into three groups, with 6 mice in each group. The specific grouping and drug treatment methods are as follows:

[0129] A. Control group: 0h: intraperitoneal injection of PBS with the same volume as the drug in the treatment group; 0.5h: tail vein injection of PBS with the same volume as the drug in the treatment group; 1h: intraperitoneal injection of PBS with the same volume as the drug in the treatment group;

[0130] B. Con A group: 0h: intraperitoneal injection of PBS with the same volume as the drug in the treatment group, 0.5h: tail vein injection of Con A (15mg / kg), 1h: intraperitoneal injection of PBS with the same volume as the drug in the treatment group;

[0131] C. Merestinib+Con A group: 0h: intraperitoneal injection of half dose of MTB (12 mg / kg), 0.5h: tail vein injection of Con A (15 mg / kg), 1h: intraperitoneal injection of the other half dose of MTB (12 mg / kg);

[0132] 2. 24 hours after ConA injection, the eyeballs were removed to collect blood and the mice were killed. The collected whole blood was allowed to stand for 2 hours and then centrifuged at 4°C, 1500 rpm for 10 minutes to collect the serum.

[0133] 3. Detection of ALT / AST levels in serum should be performed according to the instructions of the ALT / AST detection kit and using an ELISA reader.

[0134] According to the results Figure 7 Conclusion: Merestinib can significantly reduce the damage caused by Con A to the liver.

[0135] Example 8: Merestinib reduces the levels of Con A-induced inflammatory factors IL-1β, IL-6 and TNF-α.

[0136] 1. Eighteen 8-week-old C57BL / 6 wild-type mice were selected and divided into three groups, with 6 mice in each group. The specific grouping and drug treatment methods are as follows:

[0137] A. Control group: 0h: intraperitoneal injection of PBS with the same volume as the drug in the treatment group; 0.5h: tail vein injection of PBS with the same volume as the drug in the treatment group; 1h: intraperitoneal injection of PBS with the same volume as the drug in the treatment group;

[0138] B. Con A group: 0h: intraperitoneal injection of PBS with the same volume as the drug in the treatment group, 0.5h: tail vein injection of Con A (15mg / kg), 1h: intraperitoneal injection of PBS with the same volume as the drug in the treatment group;

[0139] C. Merestinib+Con A group: 0h: intraperitoneal injection of half dose of MTB (12 mg / kg), 0.5h: tail vein injection of Con A (15 mg / kg), 1h: intraperitoneal injection of the other half dose of MTB (12 mg / kg);

[0140] 2. 24 hours after ConA injection, the eyeballs were removed to collect blood and the mice were killed. The collected whole blood was allowed to stand for 2 hours and then centrifuged at 4°C, 1500 rpm for 10 minutes to collect the serum.

[0141] 3. Serum levels of inflammatory factors IL-1β, IL-6, and TNF-α were tested according to the instructions of the IL-1β, IL-6, and TNF-α detection kits and using an ELISA reader.

[0142] According to the results Figure 8 The results showed that Merestinib can significantly reduce the levels of inflammatory factors IL-1β, IL-6 and TNF-α induced by Con A in the liver.

[0143] 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. The use of mesartinib as a copper death inhibitor in the preparation of products for preventing and treating copper death-related diseases.

2. The use according to claim 1, characterized in that: The copper death-related diseases specifically refer to: neurodegenerative diseases, cardiovascular diseases, ischemia-reperfusion injury, and acute and chronic injuries of tissues and organs.

3. The use according to claim 1, characterized in that: The product includes one or more of food, medicine, and health care products.

4. Any of the following uses of mesartinib: (1) Application in the preparation of drugs for increasing the levels of Fe-S cluster proteins (LIAS / DLAT) and / or esterified proteins (FDX1); (2) Application in the preparation of drugs for inhibiting the production of heat shock protein HSP70; (3) Use in the preparation of drugs for reducing serum aspartate aminotransferase and / or alanine aminotransferase levels; (4) Use in the preparation of drugs for reducing the levels of inflammatory factors IL-1β, IL-6 and / or TNF-α; (5) Application in the preparation of drugs for inhibiting cell copper death; (6) Application in the preparation of drugs for reducing the sensitivity of cells to copper death; (7) Use in the preparation of drugs for alleviating acute liver injury; (8) Application in the preparation of drugs for inhibiting liver tissue damage in mice and improving the repair effect of acute liver damage.

5. The use according to claim 4, characterized in that: In (5), the cell copper death is the cell copper death induced by ES-Cu.

6. The use according to claim 4, characterized in that: In (1) to (8), the drug also includes pharmaceutically acceptable salts, specifically organic salts or inorganic salts.

7. The use according to claim 4, characterized in that: In (1) to (8), the drug further comprises a pharmaceutically acceptable excipient.

8. The use according to claim 7, characterized in that: The 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 4, characterized in that: In (1) to (8), 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, oral liquid or granule.

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