Application of lysine methyltransferase DOT1L inhibitor in preparation of medicine for treating hepatic ischemia-reperfusion injury

Abstract

The invention relates to application of a lysine methyltransferase DOT1L inhibitor in preparation of a medicine for treating hepatic ischemia reperfusion injury, and belongs to the technical field of biological medicine. The lysine methyltransferase DOT1L inhibitor is SGC0946, and the lysine methyltransferase DOT1L inhibitor is The invention discloses a lysine methyltransferase DOT1L inhibitor for the first time, and particularly discloses new application of SGC0946 in preparation of a medicine for treating liver ischemia reperfusion injury. The invention proves that the expression level of the DOT1L in a mouse suffering from the hepatic ischemia reperfusion injury is obviously improved, so that the methylation level of H3K79 is obviously increased, the levels of glutamic-pyruvic transaminase and glutamic oxalacetic transaminase are obviously increased, and ferroptosis marker genes Ptgs2 and Cac1 are obviously increased, which indicates that the DOT1L can be used as a target spot for treating the hepatic ischemia reperfusion injury and can be used for treating the hepatic ischemia reperfusion injury. The lysine methyltransferase DOT1L inhibitor can be used for preparing the medicine for treating the hepatic 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] Liver cancer is the third leading cause of cancer death worldwide. China is a major country with liver cancer and the age of onset shows a trend of getting younger. In addition, the total number of patients with other types of liver diseases in China also exceeds 300 million, and the number of new cases of liver failure each year exceeds 500,000. The serious burden of liver diseases has become a social public health problem that China urgently needs to pay attention to and solve. Currently, the most commonly used and effective treatment methods for early-stage liver cancer and end-stage liver disease patients 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 Brent R. Stockwell laboratory 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, and 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, and have proved 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 the treatment of 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 lysine methyltransferase DOT1L inhibitors in the preparation of drugs for the treatment of liver ischemia-reperfusion injury.

[0006] The technical solution of the present invention is as follows: Use of lysine methyltransferase DOT1L inhibitors in the preparation of drugs for the treatment of liver ischemia-reperfusion injury.

[0007] 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.

[0008] Preferably according to the present invention, the lysine methyltransferase DOT1L inhibitor is SGC0946, and its structural formula is shown as follows: 。

[0009] 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.

[0010] Preferably according to the present invention, in the drug for the treatment of liver ischemia-reperfusion injury, the lysine methyltransferase DOT1L inhibitor is the only active ingredient.

[0011] Preferably according to the present invention, the drug for the treatment of liver ischemia-reperfusion injury comprises a lysine methyltransferase DOT1L inhibitor and a pharmaceutically acceptable carrier.

[0012] Preferably according to the present invention, the dosage form of the drug for the treatment of liver ischemia-reperfusion injury is granules, tablets, capsules, pills or oral liquid preparations.

[0013] Beneficial Effects: 1. The present invention discloses for the first time the new use of lysine methyltransferase DOT1L inhibitors in the preparation of drugs for the treatment of 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 Chac1Significantly upregulated, indicating that DOT1L can be used as a therapeutic target for liver ischemia-reperfusion injury, and lysine methyltransferase DOT1L inhibitors can be used to prepare drugs for treating liver ischemia-reperfusion injury.

[0014] 2. The present invention first confirmed the new use of the lysine methyltransferase DOT1L inhibitor SGC0946 in the preparation of drugs for treating liver ischemia-reperfusion injury. Based on a mouse model of liver ischemia-reperfusion injury, by intraperitoneal injection, the lysine methyltransferase DOT1L inhibitor SGC0946 was supplemented, effectively demonstrating that the lysine methyltransferase DOT1L inhibitor SGC0946 can reduce the level of H3K79 methylation by inhibiting DOT1L activity, decrease 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 ferroptosis at the histological 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Detection of DOT1L, histone H3 and H3K79 methylation levels in the liver tissues of mice in each group.

[0016] Figure 2 Statistical results of the detection of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the sera of mice in each group.

[0017] Figure 3 Is the classical ferroptosis gene in the liver tissues of mice in each group Ptgs2 AND Chac1 Relative expression detection.

[0018] Figure 4 HE staining results of the liver tissues of mice in each group.

[0019] Figure 5 Staining results of the classical ferroptosis markers MDA and 4-HNE in the liver tissues of mice in each group. DETAILED DESCRIPTION OF THE INVENTION

[0020] The technical solutions of the present invention will be further described below in conjunction with the embodiments. However, 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.

[0021] The C57BL6J mice used in the examples were all purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0022] Both the DOT1L inhibitor SGC0946 and the ferroptosis inhibitor Liproxstatin-1 used in the examples were purchased from Selleck.

[0023] The kits for detecting the activity of alanine aminotransferase (GPT / ALT) and aspartate aminotransferase (GOT / AST) used in the examples were both purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0024] The Malondialdehyde (MDA) antibody used in the examples was purchased from Abcam, and the 4-Hydroxynonenal (4HNE) antibody was purchased from Invitrogen.

[0025] 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.

[0026] Example 1: Construction of a mouse liver ischemia-reperfusion injury model 1. Animal preparation: Place 7-week-old C57BL6J mice in the SPF-level animal house of Shandong University and keep them in a normal diet and stable state for one week for subsequent experiments.

[0027] 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 wet saline gauze to the abdominal cavity 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. 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.

[0028] The mice were divided into four groups. The first group was the Sham group (sham operation), and 200 μL of PBS buffer was intraperitoneally injected 1 hour before the operation. The second group was the IRI group, and 200 μL of PBS buffer was intraperitoneally injected 1 hour before the operation. The third group was the IRI+SGC0946 group, and 200 μL of SGC0946 solution was intraperitoneally injected 1 day before the operation at a dose of 5 mg / kg. The fourth group was the IRI+Lipro-1 group, and 10 mg / kg of Lipro-1 solution was intraperitoneally injected into the mice 1 hour before the operation.

[0029] 4. Sample collection: After 24 hours of liver ischemia-reperfusion in the above four groups of mice, the mice were euthanized, blood was collected from the eye socket, centrifuged at 4°C and 1000 g for 10 minutes, and the supernatant was collected as serum and stored at -80°C; the liver was dissected, part of the liver tissue was stored at -80°C, and part of the liver tissue was fixed with 4% paraformaldehyde at room temperature, and paraffin sections were prepared according to the existing method.

[0030] Example 2. Expression of DOT1L in liver tissue of mice with ischemia-reperfusion injury 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 respectively, cut them into small pieces, grind them, wash the blood impurity components with PBS buffer, and centrifuge to discard the PBS buffer; according to the weight of the tissue block, add 300 μL of protein lysate, and let it stand on ice for 1 h to lyse. During this period, vortex and mix every 10 min to prevent the tissue from completely precipitating to the bottom; centrifuge at 4°C, 12000 rpm for 10 min, and the supernatant is the required protein; centrifuge again at 4°C, 12000 rpm for 10 min, and the supernatant is the required protein; transfer the supernatant to a new EP tube for standby.

[0031] 2. Protein concentration determination: Prepare the BCA working solution. The BCA working solution includes solution A and solution B. Solution A:solution B = 50:1 (volume ratio), and the total volume = 200×total number of wells (standard wells + sample wells). After thorough mixing, the BCA working solution is obtained; Sample wells: First, add 1 μL of the protein sample obtained in step 1 + 19 μL of PBS buffer, and then add 200 μL of the BCA working solution to each well. The sample addition time is controlled within 5 - 10 min, and incubate at 37°C for 30 min. Measure the absorbance at 562 nm with an enzyme-linked immunosorbent assay (ELISA) reader, draw a standard curve, and substitute it into the equation to calculate the protein concentration of the measured sample.

[0032] 3. Western blot assay: SDS loading buffer was added to the protein samples of the liver tissues of the 4 groups of mice obtained in Example 1, and heat denaturation was carried out in a metal bath at 100 °C. The electrophoresis gel was prepared according to the conventional method, and electrophoresis, membrane transfer, blocking, incubation with primary antibody, incubation with secondary antibody and gel imaging were carried out 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 shown as Figure 1 follows.

[0033] As Figure 1 can be seen, compared with the mice in the 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.

[0034] Example 3. Detection of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum The sera of the mice in the 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. The standard curve was drawn according to the absorbance results detected at 505 nm, and the levels of ALT and AST in the serum were calculated. The results are shown as Figure 2 follows. Among them, the experimental data were analyzed using GraphPad Prism 9 software, and the t-test was used for the differential analysis of different groups. P P < 0.05 indicates that the difference is statistically significant.

[0035] As Figure 2 can be seen, compared with the mice in the 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 obvious difference between the two groups, that is, SGC0946 can effectively rescue the liver function injury caused by liver ischemia-reperfusion.

[0036] Example 4. Evaluation of classical markers of ferroptosis in liver tissue 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 a two-step reverse transcription (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), mix well after centrifugation, pay attention to avoid 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 ), record the 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). The results of fluorescence quantitative QPCR are as Figure 3 shown.

[0037] Table 1. Primers used in fluorescence quantitative QPCR It can be Figure 3 seen 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 confirmed the occurrence of ferroptosis in the mouse liver ischemia-reperfusion injury model in Example 1. Compared with the IRI group, the liver tissues of the IRI + SGC0946 group and the IRI + Lipro-1 group mice Ptgs2 and Chac1 mRNA levels were significantly decreased, and there was no significant difference between the two groups, which indicated that SGC0946 could effectively rescue the liver ischemia-reperfusion injury induced by ferroptosis, and the effect was similar to that of the classical ferroptosis inhibitor Lipro-1.

[0038] Example 5. Liver tissue staining 1. Liver HE staining: Paraffin sections of liver tissues from 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 turn blue, and the color was observed under a microscope; dropped eosin staining solution, stained for 3 min, observed under a microscope after washing with distilled water, dehydrated rapidly 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.

[0039] 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 dropping 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.

[0040] From the Figure 4 HE staining, it can be seen that compared with the sham operation Sham group of mice, the liver tissue sections of the IRI group of mice showed karyopyknosis, fragmentation or dissolution of liver cell nuclei, enhanced cytoplasmic eosinophilia, and blurred or disappeared cell boundaries. Compared with the IRI group, there was no obvious damage in the liver tissue sections of the IRI + SGC0946 group and the IRI + Lipro-1 group of mice. This shows that SGC0946 can significantly resist the damage caused by liver ischemia-reperfusion.

[0041] 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 IRI group mice showed brownish yellow or brown staining, and the intensity was positively correlated with the degree of injury. Compared with the IRI group, there were no obvious positive areas in the liver tissue sections of the IRI+SGC0946 group and the IRI+Lipro-1 group mice. In the staining of another lipid peroxidation marker 4HNE, a large number of positive areas were also presented in the liver tissue of the IRI group mice, while there were no obvious positive areas in the liver tissue sections of the IRI+SGC0946 group and the IRI+Lipro-1 group mice, indicating that SGC0946 can significantly improve the oxidative stress injury caused by ferroptosis.

[0042] 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.

[0043] 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 liver ischemia-reperfusion injury model, 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 DOT1L enzyme activity, 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.

[0044] The above embodiments are only the preferred specific implementation schemes 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 within the protection scope of the present invention.

Claims

1. Application of lysine methyltransferase DOT1L inhibitors in the preparation of drugs for the treatment of liver ischemia-reperfusion injury.

2. Use of the lysine methyltransferase DOT1L inhibitor according to claim 1 in the preparation of a drug for treating liver ischemia-reperfusion injury, characterized in that: The lysine methyltransferase DOT1L inhibitor inhibits the expression of lysine methyltransferase DOT1L, reduces the H3K79 methylation level, and reduces the serum alanine aminotransferase and aspartate aminotransferase levels, thereby treating liver ischemia-reperfusion injury.

3. Use of the lysine methyltransferase DOT1L inhibitor according to claim 1 in the preparation of a drug for treating liver ischemia-reperfusion injury, characterized in that: The lysine methyltransferase DOT1L inhibitor is SGC0946, and its structural formula is shown below: 。 4. Use of the lysine methyltransferase DOT1L inhibitor according to claim 1 in the preparation of a drug for treating liver ischemia-reperfusion injury, characterized in that: The liver ischemia-reperfusion injury is liver damage caused by ischemia-reperfusion after liver transplantation or liver resection.

5. Use of the lysine methyltransferase DOT1L inhibitor according to claim 1 in the preparation of a drug 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.

6. Use of the lysine methyltransferase DOT1L inhibitor according to claim 1 in the preparation of a drug 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.

7. Use of the lysine methyltransferase DOT1L inhibitor according to claim 1 in the preparation of a drug for treating liver ischemia-reperfusion injury, characterized in that: The dosage form of the medicine for treating liver ischemia-reperfusion injury is granules, tablets, capsules, pills or oral liquid preparations.

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