Application of lysine methyltransferase DOT1L inhibitor in preparing medicine for treating liver ischemia-reperfusion injury

Abstract

The present invention relates to the use of a lysine methyltransferase DOT1L inhibitor in the preparation of a medicament for treating liver ischemia-reperfusion injury, belonging to the technical field of biomedicine. The lysine methyltransferase DOT1L inhibitor is SGC0946. The present invention discloses for the first time the new use of a lysine methyltransferase DOT1L inhibitor, especially SGC0946, in the preparation of a medicament for treating liver ischemia-reperfusion injury. The present invention confirms that the expression level of DOT1L in mice with liver ischemia-reperfusion injury is significantly increased, which in turn leads to a significant up-regulation of the H3K79 methylation level, a significant increase in the levels of alanine aminotransferase and aspartate aminotransferase, and the ferroptosis marker genes Ptgs2 and Chac1 are significantly up-regulated, indicating that DOT1L can be used as a target for treating liver ischemia-reperfusion injury, and a lysine methyltransferase DOT1L inhibitor can be used to prepare a medicament for treating liver ischemia-reperfusion injury.

Description

Technical Field

[0001] The present invention relates to the application of a lysine methyltransferase DOT1L inhibitor in the preparation of a medicament for treating liver ischemia-reperfusion injury, and belongs to the field of biomedical technology. Background Art

[0002] Currently, the most commonly used and effective treatment methods for patients with early-stage liver cancer and end-stage liver disease are liver transplantation and liver resection surgery. However, in liver transplantation or liver resection surgery, liver ischemia-reperfusion injury (IRI) almost inevitably occurs, and even develops into acute liver failure, multiple organ dysfunction, etc., seriously affecting the prognosis of patients. Due to the complex pathogenesis and insidious onset of liver IRI, there is currently a lack of an effective early intervention treatment method. Therefore, exploring the pathogenesis of liver IRI and developing new drugs for treating IRI are current research hotspots.

[0003] Ferroptosis is a new type of cell death mode first proposed and defined by the laboratory of Brent R. Stockwell in 2012. It is a programmed cell death induced by the excessive accumulation of iron ion-dependent lipid peroxidation, and its classical feature is the excessive accumulation of lipid peroxides. Previous studies have reported that ferroptosis plays an important role in the occurrence and development of liver ischemia-reperfusion injury diseases, and the classical ferroptosis inhibitor Liproxstatin-1 can significantly improve liver IRI. Lysine methyltransferase (DOT1 like histone lysine methyl-transferase, DOT1L) is the only histone H3 lysine 79 (H3K79) methyltransferase, which plays an important role in transcriptional regulation, embryonic development, cell cycle regulation, maintaining normal physiological functions, etc. A large number of previous studies have focused on the close relationship between the dysregulation of H3K79 methylation mediated by DOT1L and the occurrence of neoplastic diseases, proving that DOT1L can be used as a prognostic marker and potential therapeutic target for neoplastic diseases, and a series of DOT1L methyltransferase inhibitors have been developed. However, no study has explored the role of DOT1L inhibitors in ferroptosis-induced liver ischemia-reperfusion injury.

[0004] Therefore, exploring the role of DOT1L inhibitors in the occurrence and development of liver ischemia-reperfusion injury and finding a safe, effective, and rapid drug for treating liver ischemia-reperfusion injury clinically have important strategic significance. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides the use of a lysine methyltransferase DOT1L inhibitor in the preparation of a medicament for treating liver ischemia-reperfusion injury.

[0006] The technical solution of the present invention is as follows:

[0007] The use of a lysine methyltransferase DOT1L inhibitor in the preparation of a medicament for treating liver ischemia-reperfusion injury.

[0008] Preferably according to the present invention, the lysine methyltransferase DOT1L inhibitor treats liver ischemia-reperfusion injury by inhibiting the expression of lysine methyltransferase DOT1L, reducing the level of H3K79 methylation, and reducing the content of serum glutamic-pyruvic transaminase and glutamic-oxaloacetic transaminase.

[0009] Preferably according to the present invention, the lysine methyltransferase DOT1L inhibitor is SGC0946, and its structural formula is shown as follows:

[0010] 。

[0011] Preferably according to the present invention, the liver ischemia-reperfusion injury is the liver injury caused by ischemia-reperfusion after liver transplantation or liver resection.

[0012] Preferably according to the present invention, in the medicament for treating liver ischemia-reperfusion injury, the lysine methyltransferase DOT1L inhibitor is the only active ingredient.

[0013] Preferably according to the present invention, the medicament for treating liver ischemia-reperfusion injury comprises a lysine methyltransferase DOT1L inhibitor and a pharmaceutically acceptable carrier.

[0014] Preferably according to the present invention, the dosage form of the medicament for treating liver ischemia-reperfusion injury is a granule, a tablet, a capsule, a pill or an oral liquid preparation.

[0015] Beneficial effects:

[0016] 1. The present invention discloses for the first time a new use of a lysine methyltransferase DOT1L inhibitor in the preparation of a medicament for treating liver ischemia-reperfusion injury. The present invention confirms that the expression level of DOT1L in mice with liver ischemia-reperfusion injury is significantly increased, which in turn leads to a significant up-regulation of the H3K79 methylation level, a significant increase in the levels of glutamic-pyruvic transaminase and glutamic-oxaloacetic transaminase, and the ferroptosis marker genes Ptgs2 and Chac1 are significantly up-regulated, indicating that DOT1L can be used as a target for treating liver ischemia-reperfusion injury, and a lysine methyltransferase DOT1L inhibitor can be used to prepare a medicament for treating liver ischemia-reperfusion injury.

[0017] 2. The present invention for the first time confirms the new use of the lysine methyltransferase DOT1L inhibitor SGC0946 in the preparation of drugs for treating liver ischemia-reperfusion injury. Based on the mouse liver ischemia-reperfusion injury model, by supplementing the lysine methyltransferase DOT1L inhibitor SGC0946 through intraperitoneal injection, it is effectively confirmed that the lysine methyltransferase DOT1L inhibitor SGC0946 can reduce the H3K79 methylation level by inhibiting DOT1L activity, reduce the contents of serum alanine aminotransferase and aspartate aminotransferase, and rescue liver function injury. And it can improve the injury caused by ischemia-reperfusion and the occurrence of ferroptosis at the histopathological level. Therefore, the lysine methyltransferase DOT1L inhibitor SGC0946 can be used in the preparation of drugs for treating liver ischemia-reperfusion injury, which has important significance in the future clinical treatment of liver ischemia-reperfusion injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Detection of DOT1L, histone H3 and H3K79 methylation levels in liver tissues of mice in each group.

[0019] Figure 2 Statistical results of the detection of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the sera of mice in each group.

[0020] Figure 3 Classical ferroptosis genes in liver tissues of mice in each group Ptgs2 and Chac1 Relative expression detection.

[0021] Figure 4 HE staining results of liver tissues of mice in each group.

[0022] Figure 5 Staining results of the classical ferroptosis markers MDA and 4-HNE in liver tissues of mice in each group. DETAILED DESCRIPTION OF THE INVENTION

[0023] The technical solutions of the present invention will be further described below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto. The reagents and materials involved in the embodiments are all ordinary commercially available products without special instructions.

[0024] The C57BL6J mice used in the embodiments were all purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0025] The DOT1L inhibitor SGC0946 and the ferroptosis inhibitor Liproxstatin-1 used in the embodiments were both purchased from Selleck.

[0026] The Glutamic Pyruvic Transaminase (GPT / ALT) Activity Detection Kit and Glutamic Oxaloacetic Transaminase (GOT / AST) Activity Detection Kit used in the examples were both purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0027] The Malondialdehyde (MDA) antibody used in the examples was purchased from Abcam, and the 4-Hydroxynonenal (4HNE) antibody was purchased from Invitrogen.

[0028] The collection and detection of mouse serum and liver tissue specimens used in the examples were approved by the Animal Ethics Committee of Shandong University.

[0029] Example 1: Construction of a Mouse Liver Ischemia-Reperfusion Injury Model

[0030] 1. Animal preparation: Place 7-week-old C57BL6J mice in the SPF-level animal house of Shandong University and keep them in a stable state with normal diet for one week for subsequent experiments.

[0031] 2. Construction of the liver IRI model: Anesthetize the mice by intraperitoneal injection of 200 μL of 0.5% sodium pentobarbital. After successful anesthesia, place the mice flat on the operating table and fix their limbs with tape. Disinfect the surgical area with iodophor. Make a 1-cm midline abdominal incision to open the abdominal cavity, and carefully separate the hepatic pedicle of the left middle lobe of the liver; clamp the portal vein and hepatic artery of the middle lobe and left lobe with a non-invasive vascular clamp to cause 70% liver ischemia; place the mice on a 37°C constant temperature heating pad for warming, and apply a wet compress to the abdominal cavity with normal saline gauze to avoid excessive fluid loss. After 60 minutes of continuous ischemia, quickly remove the vascular clamp and suture the abdominal cavity layer by layer. The sham operation group performed the same operations, but did not clamp the portal vein and hepatic artery of the middle lobe and left lobe of the liver. After the mice recovered, place them in the breeding cage and observe the drinking and eating status of the mice.

[0032] 3. Model grouping and drug treatment: Mix DMSO: PBS buffer = 5:95 (volume ratio) evenly to obtain a solvent; add the ferroptosis inhibitor Liproxstatin-1 (Lipro-1) to the solvent and mix evenly to obtain a Lipro-1 solution. Mix DMSO: PEG300: Tween80: PBS buffer = 5:30:10:55 (volume ratio) evenly, and add the DOT1L inhibitor SGC0946 to the solvent and mix evenly to obtain an SGC0946 solution.

[0033] The mice were divided into four groups. The first group was the sham operation group, which were intraperitoneally injected with 200 μL of PBS buffer 1 hour before the operation; the second group was the IRI group, which were intraperitoneally injected with 200 μL of PBS buffer 1 hour before the operation; the third group was the IRI+SGC0946 group, which were intraperitoneally injected with 200 μL of SGC0946 solution at a dose of 5 mg / kg one day before the operation; the fourth group was the IRI+Lipro-1 group, which were intraperitoneally injected with Lipro-1 solution at a dose of 10 mg / kg 1 hour before the operation.

[0034] 4. Sample collection: After 24 hours of liver ischemia-reperfusion, the four groups of mice were euthanized and blood was collected from the eyeballs. The blood was centrifuged at 4°C and 1000 g for 10 minutes, and the supernatant was collected as serum and frozen at -80°C. The liver was dissected, part of the liver tissue was frozen at -80°C, and part of the liver tissue was fixed with 4% paraformaldehyde at room temperature, and paraffin sections were prepared according to existing methods.

[0035] Example 2: Expression of DOT1L in Ischemia-Reperfusion Injured Liver Tissue of Mice

[0036] 1. Preparation of liver tissue protein samples: Weigh 0.03 g of liver tissue blocks from the sham group, IRI group, IRI+SGC0946 group and IRI+Lipro-1 group of mice obtained in Example 1, cut them into pieces, grind them, wash the blood impurities with PBS buffer, centrifuge and discard the PBS buffer; add 300 μL of protein lysis buffer according to the weight of the tissue blocks, let them stand on ice for 1 h, vortex and mix every 10 min during the period to prevent the tissue from completely settling to the bottom; centrifuge at 4°C, 12000 rpm, 10 min, and the supernatant is the desired protein; centrifuge again at 4°C, 12000 rpm, 10 min, and the supernatant is the desired protein; transfer the supernatant to a new EP tube for standby use.

[0037] 2. Protein concentration determination: prepare BCA working solution, which includes solution A and solution B, solution A: solution B = 50:1 (volume ratio), total volume = 200 × total number of wells (standard wells + sample wells), mix thoroughly to obtain BCA working solution;

[0038] Sample wells: First, add 1 μL of the protein sample obtained in step 1 + 19 μL of PBS buffer, then add 200 μL of BCA working solution to each well, control the sample addition time to 5-10 min, and incubate at 37°C for 30 min. Measure the absorbance at 562 nm with an ELISA reader, draw a standard curve, and substitute it into the equation to calculate the protein concentration of the sample.

[0039] 3. Western blot assay: SDS loading buffer was added to the protein samples of liver tissues from the 4 groups of mice obtained in Example 1, and heat denaturation was carried out in a metal bath at 100 °C. Electrophoresis gels were prepared according to the conventional method, and electrophoresis, membrane transfer, blocking, incubation with primary antibody, incubation with secondary antibody, and gel imaging were performed to observe the expression levels of DOT1L, H3K79me, histone H3, and heat shock protein HSP90 in the liver tissues of the 4 groups of mice described in Example 1. The results are as Figure 1 shown.

[0040] It can be Figure 1 seen that compared with the mice in the sham operation Sham group, the expression level of DOT1L in the liver tissues of the IRI group mice was significantly up-regulated, and at the same time, the level of H3K79 methylation was significantly up-regulated. Compared with the IRI group mice, the DOT1L methyltransferase activity in the IRI + SGC0946 group mice was significantly down-regulated, and at the same time, the level of H3K79 methylation was significantly inhibited. This proves that liver ischemia-reperfusion can induce an increase in the expression level of DOT1L, thereby increasing the level of H3K79 methylation, while SGC0946 can inhibit the activity of DOT1L methyltransferase through pharmacological effects, thereby reducing the level of H3K79 methylation.

[0041] Example 3. Detection of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum

[0042] The sera of the mice in the sham operation Sham group, IRI group, IRI + SGC0946 group, and IRI + Lipro-1 group obtained in Example 1 were added to 96-well plates, and then the sera of each group were detected using an alanine aminotransferase (GPT / ALT) activity detection kit and an aspartate aminotransferase (GOT / AST) activity detection kit. The specific detection method was referred to the kit instructions. Standard curves were drawn based on the absorbance detection results at 505 nm, and the levels of ALT and AST in the sera were calculated. The results are as Figure 2 shown. Among them, the experimental data were analyzed using GraphPad Prism 9 software, and t-tests were used for differential analysis of different groups. P <0.05 indicates that the difference is statistically significant.

[0043] It can be Figure 2 seen that compared with the mice in the sham operation Sham group, the levels of ALT and AST in the sera of the IRI group mice were significantly increased, indicating that the mouse liver ischemia-reperfusion injury model in Example 1 was successfully constructed. Compared with the IRI group, the levels of ALT and AST in the sera of the IRI + SGC0946 group and IRI + Lipro-1 group mice were significantly decreased, and there was no significant difference between the two groups, that is, SGC0946 can effectively rescue the liver function injury caused by liver ischemia-reperfusion.

[0044] Example 4. Evaluation of classical markers of ferroptosis in liver tissue

[0045] Liver tissue RT-qPCR: Take the liver tissues of the four groups of mice obtained in Example 1. Add 1 ml of TRNzol Universal reagent to every 30 mg, add 3 small magnetic beads, and grind with a homogenizer; let it stand at room temperature for 10 minutes to completely separate the nucleic acid-protein complex; extract mRNA according to the method of TRNzol Universal total RNA extraction reagent (Tiangen DP424), and use the two-step reverse transcription method (Vazyme R223); dilute the obtained reverse transcription product cDNA by 10 times, and configure the system according to 2×ChamQ SYBR Color qPCR Master Mix 5 μL + Reverse Primer 10 μM 0.2 μL + Forward Primer 10 μM 0.2 μL + cDNA 4.6 μl + DEPC 10 mL (Vazyme Q711). After centrifugation, mix well, pay attention to avoiding light during this period, and then place it in a real-time fluorescence quantitative QPCR instrument to record the internal reference gene Gapdh and the target gene ( Ptgs2 and Chac1 ) CT values and perform statistical analysis. The primer sequences used in specific fluorescence quantitative QPCR are shown in Table 1 below (SEQ ID NO.1-6), and the fluorescence quantitative QPCR results are as Figure 3 shown

[0046] Table 1. Primers used in fluorescence quantitative QPCR

[0047]

[0048] It can be seen from Figure 3 that compared with the mice in the sham operation Sham group, the ferroptosis marker genes Ptgs2 and Chac1 in the liver tissues of the IRI group mice were significantly up-regulated, which clarified the occurrence of ferroptosis in the mouse liver ischemia-reperfusion injury model in Example 1. Compared with the IRI group, the Ptgs2 and Chac1 mRNA levels in the liver tissues of the IRI+SGC0946 group and the IRI+Lipro-1 group mice were significantly decreased, and there was no significant difference between the two groups, indicating that SGC0946 can effectively rescue the liver ischemia-reperfusion injury induced by ferroptosis, and the effect is similar to that of the classical ferroptosis inhibitor Lipro-1

[0049] Example 5. Liver tissue staining

[0050] 1. Liver HE staining: The paraffin sections of the liver tissues of the mice in the sham operation Sham group, IRI group, IRI + SGC0946 group, and IRI + Lipro-1 group obtained in Example 1 were routinely dewaxed to water, stained with hematoxylin staining solution for 3 min, rinsed with running water, and the staining degree was observed under a microscope; differentiated with 1% hydrochloric acid alcohol for 1 s, washed with water, soaked in tap water for 3 min to return to blue, and the color was observed under a microscope; dripped with eosin staining solution, stained for 3 min, observed under a microscope after washing with distilled water, rapidly dehydrated with a series of ethanol starting from 75%, cleared with xylene, and sealed with neutral gum and then observed and photographed under the microscope. The results are as Figure 4 shown.

[0051] 2. Liver immunohistochemical staining: The liver tissues of the above four groups of mice were routinely dewaxed to water, and then placed in a citrate solution at 95 °C for antigen retrieval and naturally cooled to room temperature; permeabilized with 2% Tween-20 at 37 °C, and endogenous peroxidase was removed by adding 3% hydrogen peroxide-methanol after washing with PBS buffer; the classic ferroptosis lipid peroxidation markers MDA primary antibody (1:200) and 4HNE primary antibody (1:300) were diluted with 5% BSA blocking solution respectively and incubated overnight in a wet box at 4 °C; the primary antibody was recovered after rewarming the next day, and the biotin-labeled secondary antibody and SABC were incubated successively; the positive chromogenic time was recorded by DAB staining, and the staining was terminated with PBS buffer; then hematoxylin-eosin staining was performed, dehydrated with alcohol from low concentration to high concentration gradient, cleared with xylene, and then sealed with neutral gum, and bright-field pictures were taken with an Oplympus microscope. The results are as Figure 5 shown.

[0052] From the Figure 4 HE staining, it can be seen that compared with the mice in the sham operation Sham group, the liver tissue sections of the mice in the IRI group showed karyopyknosis, fragmentation or dissolution of the liver nuclei, enhanced cytoplasmic eosinophilia, and blurred or disappeared cell boundaries. Compared with the IRI group, there was no obvious damage to the liver tissue sections of the mice in the IRI + SGC0946 group and the IRI + Lipro-1 group. This shows that SGC0946 can significantly resist the damage caused by liver ischemia-reperfusion.

[0053] From Figure 5It can be seen that in the MDA staining of lipid peroxidation markers, compared with the mice in the Sham group of sham operation, a large number of damaged areas in the liver tissue sections of the mice in the IRI group were stained brownish yellow or brown, and the intensity was positively correlated with the degree of damage. Compared with the IRI group, there were no obvious positive areas in the liver tissue sections of the mice in the IRI+SGC0946 group and the IRI+Lipro-1 group. In the staining of another lipid peroxidation marker 4HNE, a large number of positive areas in the liver tissue of the mice in the IRI group were also shown, while there were no obvious positive areas in the liver tissue sections of the mice in the IRI+SGC0946 group and the IRI+Lipro-1 group, indicating that SGC0946 can significantly improve the oxidative stress injury caused by ferroptosis.

[0054] In summary, the present invention first discloses that the expression level of DOT1L in mice with liver ischemia-reperfusion injury is significantly increased, which in turn leads to a significant up-regulation of the H3K79 methylation level, a significant increase in the levels of alanine aminotransferase and aspartate aminotransferase, and the ferroptosis marker genes Ptgs2 and Chac1 are significantly up-regulated, indicating that DOT1L can be used as a target for treating liver ischemia-reperfusion injury, and lysine methyltransferase DOT1L inhibitors can be used to prepare drugs for treating liver ischemia-reperfusion injury.

[0055] And it is first confirmed that the lysine methyltransferase DOT1L inhibitor SGC0946 has a new use in the preparation of drugs for treating liver ischemia-reperfusion injury. Based on the mouse model of liver ischemia-reperfusion injury, the lysine methyltransferase DOT1L inhibitor SGC0946 was supplemented by intraperitoneal injection, effectively confirming that the lysine methyltransferase DOT1L inhibitor SGC0946 can inhibit the up-regulation of the H3K79 methylation level by inhibiting the activity of the DOT1L enzyme, reduce the content of serum alanine aminotransferase and aspartate aminotransferase, and rescue liver function injury. And it can improve the injury caused by ischemia-reperfusion and the occurrence of ferroptosis at the histopathological level. Therefore, the lysine methyltransferase DOT1L inhibitor SGC0946 can be used to prepare drugs for treating liver ischemia-reperfusion injury, which has important significance in the future clinical treatment of liver ischemia-reperfusion injury.

[0056] The above embodiments are only the preferred specific implementation manners of the present invention and are not used to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a lysine methyltransferase DOT1L inhibitor in the preparation of a medicament for treating liver ischemia-reperfusion injury, characterized in that, The lysine methyltransferase DOT1L inhibitor is SGC0946, and its structural formula is shown as follows: 。 2. Use of the lysine methyltransferase DOT1L inhibitor as claimed in claim 1 in the preparation of a medicament for treating liver ischemia-reperfusion injury, characterized in that, The lysine methyltransferase DOT1L inhibitor treats liver ischemia-reperfusion injury by inhibiting the expression of lysine methyltransferase DOT1L, reducing the level of H3K79 methylation, and decreasing the contents of serum glutamic-pyruvic transaminase and glutamic-oxaloacetic transaminase.

3. Use of the lysine methyltransferase DOT1L inhibitor as claimed in claim 1 in the preparation of a medicament for treating liver ischemia-reperfusion injury, characterized in that, The liver ischemia-reperfusion injury is the liver injury caused by post-ischemic reperfusion resulting from liver transplantation or liver resection.

4. Use of the lysine methyltransferase DOT1L inhibitor according to claim 1 in the preparation of a medicament for treating liver ischemia-reperfusion injury, characterized in that, In the drug for treating liver ischemia-reperfusion injury, the lysine methyltransferase DOT1L inhibitor is the only active ingredient.

5. Use of the lysine methyltransferase DOT1L inhibitor as described in claim 1 in the preparation of a medicament for treating liver ischemia-reperfusion injury, characterized in that, The drug for treating liver ischemia-reperfusion injury comprises a lysine methyltransferase DOT1L inhibitor and a pharmaceutically acceptable carrier.

6. Use of the lysine methyltransferase DOT1L inhibitor as claimed in claim 1 in the preparation of a medicament for treating liver ischemia-reperfusion injury, characterized in that, The dosage form of the drug for treating liver ischemia-reperfusion injury is a granule, a tablet, a capsule, a pill or an oral liquid preparation.

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