Use of a setdb1 activator in the manufacture of a medicament for treating organ injury and medicaments

Abstract

The application relates to an application of a SETDB1 activator in a medicine for treating organ injury and the medicine. The application of the SETDB1 activator in the medicine for treating organ injury is (R, R)-59. The application further provides a medicine for treating organ injury, and the medicine comprises the SETDB1 activator (R, R)-59. The application provides a new application of the SETDB1 activator (R, R)-59, and the SETDB1 activator (R, R)-59 can effectively relieve liver toxicity caused by hunger or other factors by regulating lipid toxicity and promoting lipid autophagy, the SETDB1 activator (R, R)-59 can inhibit inflammatory response and hepatocyte apoptosis caused by APAP overdose, promotes liver repair, and provides a new medicine for treating drug-induced liver injury.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of a SETDB1 activator in a medicine for treating organ injury and the medicine. BACKGROUND

[0002] The liver plays a crucial role in the human body, as a multifunctional metabolic organ, mainly responsible for detoxification, metabolism, synthesis and storage of various important substances. Liver health is essential for maintaining normal physiological functions of the human body. However, with the rapid changes in modern lifestyle, liver health is facing unprecedented threats.

[0003] Firstly, one kind of liver injury is under the influence of bad living habits such as hunger, malnutrition, metabolic disorders and drug abuse, the toxicity level of liver rises significantly, which poses a serious threat to liver health. Studies have shown that lipotoxicity is a major factor leading to liver injury, and this toxicity is mainly caused by the excessive accumulation of fatty acids and their metabolites in hepatocytes. In addition, lipophagy, as a key physiological process for removing excess lipids in cells, its dysfunction has also been found to be closely related to liver toxicity.

[0004] In current medical practice, the treatment methods for the above-mentioned liver toxicity mainly focus on symptomatic treatment, such as relieving liver inflammation or reducing liver burden through drugs. Although these methods can alleviate symptoms to some extent, they lack specificity and cannot fundamentally solve the problem of liver toxicity.

[0005] In addition, acetaminophen (APAP) is a commonly used antipyretic and analgesic drug in clinical practice, however, excessive intake can cause severe acute liver injury (ALI), which is one of the main causes of liver failure. The existing treatment method mainly relies on N-acetylcysteine (NAC), but its efficacy is limited by the use of time window (usually within 8 hours of excessive intake), and the treatment effect on the liver cell damage that has occurred is limited.

[0006] Therefore, it is urgent to develop a therapeutic drug that can target liver toxicity and treat acute liver injury, in order to achieve more effective treatment effect and improve liver health status. SUMMARY

[0007] Therefore, the application aims to provide a SETDB1 activator in the treatment of organ damage and a drug, to provide a new use of the SETDB1 activator, and the SETDB1 activator can effectively alleviate liver toxicity caused by starvation or other factors by regulating lipotoxicity and promoting lipid autophagy, to provide a new drug for liver detoxification, and the SETDB1 activator can treat acute liver injury caused by acetaminophen overdose, to effectively improve liver injury caused by APAP, and to provide a new drug for acute liver injury caused by acetaminophen overdose.

[0008] In order to achieve the above-mentioned purpose, the technical scheme adopted by the application is as follows:

[0009] The application of a SETDB1 activator in the treatment of organ damage, wherein the SETDB1 activator is (R, R)-59, and the structural formula of (R, R)-59 is shown in formula (1).

[0010]

[0011] It is found through experiments that (R, R)-59 can be used as a SETDB1 activator, can effectively reduce the level of lipotoxic substances by regulating the expression of genes, thereby reducing liver cell damage and inflammatory response, and (R, R)-59 can be used as a SETDB1 activator, effectively promotes liver repair and reduces liver cell apoptosis.

[0012] Preferably, the SETDB1 activator is used in the treatment of organ detoxification.

[0013] Preferably, when used as a liver detoxification drug, the amount of (R, R)-59 is 0.6-0.7 mg / kg.

[0014] (R, R)-59 can be used as a SETDB1 activator, can make liver cells more effectively process fatty acids by regulating the expression of genes, reduce the accumulation of fatty acids in liver cells, inhibit the accumulation of free fatty acids and triglycerides in liver cells, reduce the level of lipotoxic substances, thereby reducing liver cell damage and inflammatory response, and regulate lipotoxicity and promote lipid autophagy to effectively alleviate liver toxicity caused by starvation or other factors.

[0015] Preferably, the amount of (R, R)-59 is 0.655 mg / kg.

[0016] Preferably, the organ includes at least one of liver, kidney, heart, lung and intestine.

[0017] Preferably, the SETDB1 activator (R, R)-59 is used in the treatment of acute liver injury caused by acetaminophen overdose.

[0018] (R,R)-59 can be used as a SETDB1 activator, which can significantly up-regulate downstream anti-apoptotic protein Bcl-2 and down-regulate pro-apoptotic protein Cleaved-caspase3, thereby effectively inhibiting oxidative stress, inflammatory response and hepatocyte apoptosis caused by APAP overdose, and promoting liver repair.

[0019] Preferably, the amount of (R,R)-59 used in the treatment of acute liver injury caused by paracetamol overdose is 0.6-0.7 mg / kg.

[0020] Preferably, the (R,R)-59 is used in the treatment of fatty acid, which can activate SETDB1 to increase lipid autophagy, so that the cell can decompose lipids, thereby reducing the accumulation of fat in the cell.

[0021] Experimental studies have found that (R,R)-59 can up-regulate gene expression by methylation and up-regulate the lipid autophagy pathway to promote the decomposition of lipids in the cell. This dual mechanism makes (R,R)-59 show significant superiority in regulating lipid metabolism.

[0022] Preferably, the (R,R)-59 is used in the treatment of reducing serum alanine aminotransferase (ALT).

[0023] Experimental studies have found that (R,R)-59 can effectively reduce the level of serum alanine aminotransferase (ALT) and reduce hepatocyte apoptosis.

[0024] Preferably, the (R,R)-59 is used in the treatment of reducing serum aspartate aminotransferase (AST).

[0025] Experimental studies have found that (R,R)-59 can effectively reduce the level of serum aspartate aminotransferase (AST) and reduce hepatocyte apoptosis.

[0026] Preferably, the (R,R)-59 is used in the treatment of inhibiting the accumulation of free fatty acids and triglycerides in the cell. The (R,R)-59 can inhibit the accumulation of free fatty acids and triglycerides in the liver cell.

[0027] Preferably, the (R,R)-59 is used in the treatment of inducing the activation of the lipid autophagy pathway. The (R,R)-59 can induce the activation of the lipid autophagy pathway, enhance the formation and function of lipid autophagosomes, and promote the degradation of lipids in the liver cell.

[0028] Among them, (R, R)-59 can significantly improve the expression level of lipid autophagy-related genes in the liver, such as microtubule-associated protein light chain 3 (LC3) and lysosome-associated protein 1 (LAMP1). The up-regulation of these genes helps to promote the decomposition and removal of intracellular lipids, thereby effectively relieving the accumulation of fat in the liver.

[0029] The present application also provides a medicine for treating organ damage, which comprises a SETDB1 activator (R, R)-59.

[0030] Preferably, the medicine further comprises a pharmaceutically acceptable carrier.

[0031] Preferably, the carrier comprises a diluent and / or a pH adjuster.

[0032] Preferably, the medicine can be used to treat diseases related to liver lipid metabolism disorders, such as non-alcoholic fatty liver disease (NAFLD).

[0033] Preferably, the medicine can be used to prevent or treat fat accumulation in the liver, by reducing the synthesis of fatty acids and promoting the degradation of lipids, thereby protecting the liver from the damage of fat accumulation. The dosage of the medicine can be adjusted according to the patient's body weight, age, gender, health status and severity of the disease.

[0034] Preferably, the medicine can be administered orally or by injection, such as tablets, capsules, suspensions or injection solutions. In addition, the medicine can also be used in combination with other drugs to enhance the therapeutic effect or reduce side effects, such as combined use with lipid-lowering drugs or anti-inflammatory drugs.

[0035] The beneficial effects of the present application are as follows:

[0036] The application of the SETDB1 activator of the present application in the medicine for treating organ damage, (R,R)-59 can be used as a SETDB1 activator, by regulating the expression of genes, so that liver cells can more effectively process fatty acids, reduce the accumulation of fat in liver cells, and at the same time, inhibit the accumulation of free fatty acids and triglycerides in liver cells, reduce the level of lipotoxic substances, thereby reducing liver cell damage and inflammatory response, and regulating lipotoxicity and promoting autophagy of lipids to effectively alleviate liver toxicity caused by starvation or other factors. At the same time, (R,R)-59 can be used as a SETDB1 activator, which activates the downstream anti-apoptotic protein Bcl-2 to significantly up-regulate, and the pro-apoptotic protein Cleaved-caspase to down-regulate, thereby effectively inhibiting oxidative stress, inflammatory response and liver cell apoptosis caused by excessive APAP, and promoting liver repair. Therefore, (R,R)-59 is expected to become a new drug for treating lipotoxicity and related metabolic diseases, and is particularly suitable for patients with non-alcoholic fatty liver disease, oxidative stress, inflammatory response and liver cell apoptosis caused by excessive APAP, and acute liver injury caused by malnutrition, and has popularization and application value in the field of biological medicine technology. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 A graph of the effect of the SETDB1 activator (R,R)-59 on the expression level of SETDB1;

[0038] Figure 2 A comparison chart of morphological observation of the effect of (R,R)-59 on liver lipid accumulation;

[0039] Figure 3 A graph of the experimental results of pathological observation and detection of triglycerides in liver tissue of the effect of (R,R)-59 on liver lipid accumulation;

[0040] Figure 4 A graph of the experimental results of the effect of (R,R)-59 on promoting autophagy of liver lipids;

[0041] Figure 5 A graph of the results of serum detection of the effect of (R,R)-59 on liver function in an acute liver injury model;

[0042] Figure 6 A graph of the results of morphological observation of the effect of (R,R)-59 on acute liver injury in pathological detection;

[0043] Figure 7 A graph of the results of the effect of (R,R)-59 on the inflammatory level in liver tissue;

[0044] Figure 8 A graph of the results of apoptosis detection in liver tissue in an acute liver injury model affected by (R,R)-59;

[0045] Figure 9 Results of Western blot detection. DETAILED DESCRIPTION

[0046] Other advantages and benefits of the present application will become apparent to those skilled in the art upon consideration of the disclosure herein with the accompanying drawings and preferred embodiments. The present application can be embodied in other different specific embodiments and applications and modifications and alterations can be made therein without departing from the spirit of the present application. It should be understood that the preferred embodiments are only for illustrating the present application and are not intended to limit the scope of protection of the present application.

[0047] The present application intends to disclose the application of a SETDB1 activator in a drug for treating organ damage, to provide a new use of SETDB1, and the SETDB1 activator effectively alleviates liver toxicity caused by starvation or other factors by regulating lipotoxicity and promoting lipid autophagy, providing a new drug for liver detoxification, and the SETDB1 activator (R,R)-59 is used to treat acute liver damage caused by acetaminophen overdose, to effectively improve liver damage caused by APAP, and to provide a new drug for acute liver damage caused by acetaminophen overdose.

[0048] In the application, the SETDB1 activator is (R,R)-59, and the structural formula of (R,R)-59 is shown as formula (1).

[0049]

[0050] In some embodiments, the SETDB1 activator is used in a drug for organ detoxification.

[0051] In some embodiments, when used as a drug for liver detoxification, the amount of the SETDB1 activator (R,R)-59 is 0.6-0.7 mg / kg.

[0052] For example, when used as a drug for liver detoxification, the amount of the SETDB1 activator (R,R)-59 is 0.655 mg / kg.

[0053] In some embodiments, the organ includes at least one of a liver, a kidney, a heart, a lung, and an intestine. In particular, the organ is a liver.

[0054] In some embodiments, the SETDB1 activator is used in a drug for treating acute liver damage caused by acetaminophen overdose.

[0055] For example, when used as a drug for treating acute liver injury caused by an overdose of acetaminophen, the amount of (R,R)-59 used is in the range of 0.6-0.7 mg / kg.

[0056] In some embodiments, the SETDB1 activator (R,R)-59 is used in a drug for treating fatty acids by activating SETDB1 to increase lipid autophagy, so that the cell can decompose lipids and reduce the accumulation of fat in the cell.

[0057] In some embodiments, the SETDB1 activator (R,R)-59 is used in a drug for reducing serum alanine aminotransferase (ALT).

[0058] In some embodiments, the SETDB1 activator (R,R)-59 is used in a drug for reducing serum aspartate aminotransferase (AST).

[0059] In some embodiments, the SETDB1 activator (R,R)-59 is used in a drug for inhibiting the accumulation of free fatty acids and triglycerides in the cell, so that (R,R)-59 can inhibit the accumulation of free fatty acids and triglycerides in the liver cell, reduce the level of lipotoxic substances, and thus reduce liver cell damage and inflammatory response.

[0060] In some embodiments, the SETDB1 activator (R,R)-59 is used in a drug for inducing the activation of the lipid autophagy pathway. Thus, (R,R)-59 can induce the activation of the autophagy pathway, promote the degradation of lipids in the liver cell by enhancing the formation and function of lipid autophagosomes, help the liver to clear excess lipids, and thus maintain the metabolic homeostasis and functional health of the liver.

[0061] In some embodiments, a drug for treating organ damage is also provided, which comprises the SETDB1 activator (R,R)-59.

[0062] In some embodiments, the drug further comprises a pharmaceutically acceptable carrier.

[0063] In some embodiments, the carrier comprises a diluent and / or a pH adjuster, such as water for injection, sodium citrate, etc.

[0064] Specific experimental detection and analysis

[0065] I. Experiment of the effect of SETDB1 activator (R,R)-59 on the expression level of SETDB1

[0066] The experiment of this project is divided into three groups: normal control group, adjuvant (DMSO: corn oil = 1:9) injection group and (R, R)-59 injection group. (R, R)-59 is treated as an SETDB1 activator. The normal control group of mice is not treated, and the adjuvant (DMSO: corn oil = 1:9) injection group and (R, R)-59 injection group are all fasting model mice, specifically, non-alcoholic fatty liver model is established by fasting for 48 hours, and then adjuvant and (R, R)-59 are injected intraperitoneally, with an injection amount of 10 mg / Kg. After 24 hours, the mice are decapitated and dissected, and liver tissue samples are taken. Part of the liver tissue is fixed with 4% paraformaldehyde for 24 hours, and paraffin-embedded paraffin sections and frozen sections are prepared, respectively. Part is directly frozen in liquid nitrogen and stored in a -80°C refrigerator for use as samples for other index detection.

[0067] The specific operation steps are as follows: western blot

[0068] (I) Extraction of protein sample

[0069] (1) RIPA lysis buffer (Radio Immunoprecipitation Assay Lysis buffer) is used to lyse cell membranes (including nuclear membranes) and extract soluble proteins from tissues or cells. After mixing various inhibitors and lysis buffer uniformly, it is placed on ice for use. The lysis buffer is prepared and used immediately.

[0070] (2) Weigh 20 mg of liver tissue stored at -80°C into a 1.5 mL EP tube, and add 400 μL of the above lysis buffer.

[0071] (3) Put a magnetic bead in the EP tube, and perform physical crushing in the grinder at 60HZ for 3 minutes (to prevent protein sample degradation during processing, the whole operation should be performed on an ice brick taken from low temperature).

[0072] (4) Pre-cool the low-temperature centrifuge at 4°C, and place the protein sample after completing the grinding process into the low-temperature centrifuge, set the speed to 8000g, and centrifuge for 10 minutes. The supernatant is separated during centrifugation.

[0073] (5) After centrifugation, the supernatant is taken out and transferred to a new EP tube, and the sample is stored in a -80°C refrigerator.

[0074] (II) Determination of protein concentration

[0075] (1) The protein concentration determination method is BCA protein concentration detection kit. Before determining the concentration, a standard curve needs to be determined using a standard concentration of BSA solution.

[0076] (2) After the protein is diluted, mix it evenly with a vortex instrument, add 9 μL of PBS to each well of the perimeter microplate cuvette, and then add 1 μL of the protein standard, with three duplicate wells for each concentration gradient.

[0077] (3) Configuration of the color developing reagent in the kit, according to the instructions, the ratio of A liquid to B liquid is 50:1, and the color developing reagent is prepared. During the sampling process, some loss will be generated, so more reagent than the required volume is prepared to reserve the amount of process loss.

[0078] (4) Add 100 μL of the working solution to each well of the microplate, and place it in a 37°C incubator for 30 min. Use an enzyme marker to measure the absorbance at 570 nm. According to the OD value and the standard protein concentration, a standard curve is drawn (the statistical value R2 is greater than or equal to 0.99).

[0079] (Three) SDS-PAGE protein gel electrophoresis

[0080] (1) The collected protein sample is quantified using the BCA kit, and the method is based on the protein concentration standard curve. To reduce experimental error, three duplicate wells are set for each sample. The protein OD value is measured at 570 nm, and the protein concentration is calculated by entering the standard curve.

[0081] (2) Sample preparation: after calculation, the volume of the required protein sample is calculated based on the general loading mass of 20 ug, and the final volume of the sample is generally 10-20 μL. After adding the protein sample, the remaining volume is filled with SDS loading and 3d H2O to the same volume.

[0082] (3) Protein denaturation: centrifuge the prepared protein sample in the centrifuge to ensure that the liquid is at the bottom of the EP tube. Place the tube in a 95°C metal bath for 10 min to denature the protein. During the process, pay attention to prevent the heating from causing the EP tube cover to collapse and the liquid to splash out, affecting the experimental results. After heating, cool and centrifuge for standby.

[0083] (4) Configuration of the appropriate experimental concentration of SDS-PAGE gel: according to the molecular weight of different proteins, select different concentrations of gels, which are composed of two parts: concentrated gel and separation gel. According to the sample volume and the number of samples required, select the thickness of the gel plate and add different specifications of plastic combs to reserve the sample wells. A 10 μL volume of sample generally requires a 1.0 mm thick gel plate, while a 20 μL volume of sample requires a 1.5 mm thick gel plate.

[0084] (5) Sample loading: After the gel plate is solidified, place the two gel plates in the electrophoresis tank at the same time, and add SDS electrophoresis buffer in the electrophoresis tank. Slowly add the sample in the gel hole reserved during gel preparation. Avoid generating bubbles during the process to affect the sample loading volume and cause errors in the results. Add protein marker in one sample hole to facilitate the differentiation of the target protein band later.

[0085] (6) Electrophoresis: Set the constant voltage of the electrophoresis instrument. Use 70V voltage for electrophoresis of the upper concentrated gel. The voltage of the lower separation gel can be adjusted appropriately, but should not exceed 120V. High voltage will affect the neatness of the protein band.

[0086] (Four) Electrophoresis transfer

[0087] (1) Cut the PVDF membrane to the appropriate size according to the size of the gel plate separation gel part, and place it in methanol. Shake well on a shaker for about 20s to activate.

[0088] (2) Use wet transfer method for transfer. Add transfer electrophoresis buffer, transfer clamp, sponge, filter paper and PVDF membrane in the transfer container. Carefully close the transfer clamp after removing the air bubbles in the arranged transfer filter paper and PVDF membrane. Place the transfer clamp in the correct electrode order to prevent it from being placed in the transfer electrophoresis tank.

[0089] (3) Electrophoresis: After adding 1x electrophoresis buffer in the electrophoresis tank, set the constant current of the electrophoresis instrument for electrophoresis. The current size needs to be adjusted flexibly according to the size of the protein molecular weight (current size 200mA-300mA). The basic principle is to use slightly high current for large proteins and low current for small proteins. The transfer process will generate heat, which will affect the transfer effect, so the transfer equipment needs to be operated in a low temperature environment.

[0090] (4) Treatment and blocking of target band: After electrophoresis is completed, take out the protein on the PVDF membrane without obvious characteristics. If the target band needs to be differentiated, use Ponceau red dye to mark. First, take the PVDF membrane and activate it in methanol for 15-20s. Then place the membrane in Ponceau red dye. After the protein band is colored, cut the membrane according to the size of the experimental protein. Then wash the Ponceau red to white color on the PVDF membrane with TBST working solution. Block with 5% skim milk. The purpose is to combine protein in the blank part of the PVDF membrane in advance to avoid non-specific binding of the subsequent antibody to produce blurred band background. Place the incubation box on the shaker at a speed of 20-25rpm and block at room temperature for 1h.

[0091] (Five) Antibody incubation:

[0092] (1) Dilute the protein primary antibody with the primary antibody diluent according to the nature of the antibody. The general ratio range is 1:2000-1:10000. After preparation, place it on ice for standby.

[0093] (2) After the completion of the closed protein band is washed with TBST twice to remove the remaining milk, put into the antibody incubation box, add the prepared primary antibody solution, and incubate slowly in the shaker at 4°C. The incubation time is more than 8 hours, and generally overnight incubation.

[0094] (3) After the incubation of the primary antibody, it is recovered (stored in the 4°C refrigerator, can be reused), and the residual primary antibody is washed with TBST solution. The shaker is at 60-80 rpm, and the washing is 4X8 min. New TBST solution is needed for each time.

[0095] (4) After the incubation of the primary antibody, the secondary antibody is incubated according to the resistance of the primary antibody. The secondary antibody is diluted with 2.5% skimmed milk. The recovered blocking milk can be diluted by half, and different concentrations of secondary antibodies are prepared according to the type of protein. The general concentration range is 1:5000-1:15000. The secondary antibody is added to the incubation box, the shaker is gently shaken, and the room temperature is incubated for 2 hours.

[0096] (5) Recover the secondary antibody, and repeat the membrane washing step. TBST solution, wash 4 times, 8 min each time. Store in TBST solution at 4°C. The subsequent steps should be performed as soon as possible.

[0097] (Six) Chemiluminescence development

[0098] (1) Use the chemiluminescence development kit. Mix the luminescent liquids A and B in a ratio of 1:1, vortex well, and wait for use (since the luminescence principle is enzyme reaction, so prepare it immediately for use to ensure the luminescence effect).

[0099] (2) Place a smooth plastic film on the plane, use a pipette to take an appropriate amount of luminescent liquid and place it evenly on the plastic film. Take the PVDF film from the TBST solution, use enzyme-free paper to absorb as much water as possible from the side without protein, and then contact the protein side with the luminescent liquid. Gently lift and place the film a few times to make the luminescent liquid contact evenly. Incubate for 2 minutes, then move to the dark clamp and enter the dark developing room for fluorescence exposure.

[0100] (3) After entering the darkroom, adjust the exposure time according to the brightness of the band until the protein band shows the appropriate image. The results of the effect of the mouse liver tissue SETDB1 activator (R, R)-59 on the expression level of SETDB1 are shown in Figure 1 .

[0101] Figure 1 The protein expression results of SETDB1, H3K9me3 and ACTB (internal reference) in western blot experiment are shown in

[0102] The results of the effect of the mouse liver tissue SETDB1 activator (R, R)-59 on the expression level of SETDB1 are shown in Figure 1From the analysis, it can be seen that the expression level of SETDB1 in the adjuvant injection group is higher than that in the normal control group. In the (R,R)-59 injection group, the expression of SETDB1 is further significantly enhanced, which proves that (R,R)-59 can effectively activate the expression of SETDB1. H3K9me3 is a histone modification marker catalyzed by SETDB1, and in the (R,R)-59 injection group, the level of H3K9me3 is significantly increased, which is consistent with the increase of SETDB1 expression, thereby proving that the activity of SETDB1 is also significantly enhanced. Comprehensive analysis shows that (R,R)-59 as a SETDB1 activator, significantly improves the expression of SETDB1 and the level of H3K9me3 catalyzed by it. This shows that (R,R)-59 has a significant effect on the activation and regulation of epigenetic modification of SETDB1.

[0103] II. Morphological observation experiment of the effect of (R,R)-59 on liver lipid accumulation

[0104] The specific operation steps are as follows:

[0105] Adjuvant injection group and (R,R)-59 injection group. The adjuvant (DMSO: corn oil = 1:9) injection group and the (R,R)-59 injection group are both starved model mice, specifically non-alcoholic fatty liver model mice starved for 48 hours, and then injected with adjuvant and (R,R)-59 into the abdominal cavity, respectively, with an injection amount of 10 mg / Kg. After 24 hours, the mice were decapitated and dissected to observe the characteristics of the liver. The experiment is divided into an adjuvant intraperitoneal injection group and an (R,R)-59 intraperitoneal injection group, and the results are shown in Figure 2 .

[0106] Figure 2 A shows the liver of the adjuvant intraperitoneal injection group, and Figure 2 A observation, the liver volume is large, accompanied by color lightening, which is usually related to liver fatty degeneration, indicating that the lipid accumulation in the liver is more obvious.

[0107] Figure 2 B is the liver of the (R,R)-59 intraperitoneal injection group, which is smaller in volume, darker in color, and healthier in appearance, indicating that the lipid accumulation in the liver has been reduced, thereby proving that the SETDB1 activator (R,R)-59 plays an effective role in reducing lipid accumulation.

[0108] III. Pathological observation of the effect of (R,R)-59 on liver lipid accumulation and detection of triglycerides in liver tissue

[0109] 1. HE staining

[0110] (I) Sample preparation: paraffin section: the section thickness is 5 μm.

[0111] (II) Staining

[0112] (I) Routine dewaxing

[0113] Removing paraffin: Place the slides in a xylene solution for 2 immersions of 5 minutes each to remove paraffin.

[0114] (II) Gradient hydration: Reduce the concentration of the solvent at each change, from xylene to different concentrations of ethanol, until distilled water. The order is as follows:

[0115] 100% ethanol: 2 times, 2 minutes each.

[0116] 95% ethanol: 2 times, 2 minutes.

[0117] 85% ethanol: 2 times, 2 minutes.

[0118] 70% ethanol: 1 time, 2 minutes.

[0119] Distilled water: 1 time, 2 minutes.

[0120] (III) Staining

[0121] 1) Hematoxylin staining: Stain the slides with Hematoxylin dye.

[0122] Place the slides in the Hematoxylin staining solution, the staining time is usually 5-10 minutes depending on the type of tissue and requirements. After the staining is finished, rinse the slides with tap water to remove excess dye.

[0123] 2) Differentiation and bluing:

[0124] Stain the slides with Hematoxylin dye, the time is usually 10-20 seconds until the tissue presents an appropriate blue color.

[0125] Subsequently, place the slides in running water for 2-3 minutes for counterstaining.

[0126] Staining (Oil Red O)

[0127] 1) Dissolve Oil Red O powder in isopropyl alcohol at a concentration of 0.3%.

[0128] 2) Heat the solution in a water bath at 45°C for 15 minutes, the incompletely dissolved Oil Red O can be removed with filter paper. Immerse the slides in the Oil Red O staining solution, the staining time is usually 10-15 minutes. Gently shake the slides to ensure uniform distribution of the Oil Red O dye.

[0129] 3) Rinse: Rinse the slides with 70% isopropyl alcohol (or 70% ethanol) to gently remove excess dye, taking care not to rub the slides. And let the slides dry.

[0130] 4) Hydrating and Mounting: Transfer the slides to distilled water to rinse off excess isopropyl alcohol.

[0131] (4) Dehydrating and Mounting

[0132] Gradual Dehydration: Soak the slides in 70%, 85%, 95%, and 100% ethanol for 2 minutes each. Soak in xylene solution for 5 minutes.

[0133] (5) Mounting: Use an appropriate amount of neutral resin mounting medium to drop it onto the surface of the slide. Gently cover the coverslip and remove air bubbles.

[0134] (III) Final Inspection

[0135] Use a microscope to observe the staining effect, ensuring that the cell nuclei are blue-purple and the cytoplasm is pink. Also observe pathological changes.

[0136] 2. Bodipy Staining Experiment:

[0137] Bodipy staining is a small-molecule fluorescent probe used to detect lipids, commonly used for labeling lipids in tissue sections or cell cultures. Bodipy dyes can bind to specific parts of lipids within cells, especially interacting with fatty acids, triglycerides, and phospholipids, and emit fluorescence at specific wavelengths.

[0138] (I) Sample Preparation

[0139] Tissue Sections: Use a cryostat to prepare tissue sections, with a thickness of generally 5 μm.

[0140] (II) Slide Preparation and Fixation

[0141] (1) Fixation: Frozen sections can be fixed using a fixative, commonly 4% paraformaldehyde (PFA). Fixation time is 10-15 minutes (at room temperature). Avoid excessive fixation to prevent affecting lipid staining.

[0142] (2) Rinsing: Wash the slides with PBS (phosphate buffered saline) twice for 5 minutes each to remove fixative and residual impurities.

[0143] (III) Permeabilization

[0144] (1) Permeabilization Solution: Use 0.2% Triton X-100 as a permeabilization solution to properly permeabilize the cell membrane, helping BODIPY dye better enter the tissue. Time is 5-10 minutes (at room temperature).

[0145] (IV) BODIPY Staining

[0146] (1) BODIPY dye solution: Prepare a BODIPY staining solution, generally at a concentration of 2 ng / mL. Staining is performed by diluting the BODIPY staining solution 1:4000 in PBS solution, and staining in the dark. Place the sections in the BODIPY dye solution, and the staining time is generally 15 minutes.

[0147] (2) Washing: Remove excess dye: Wash the sections with PBS 2-3 times for 5 minutes each time to remove excess BODIPY dye.

[0148] (3) DAPI staining (optional): If you want to stain the nucleus, you can choose DAPI staining. DAPI can bind to DNA to show the nucleus. DAPI staining solution: 1 pg / mL DAPI solution, staining for 5-10 minutes.

[0149] (4) Washing: Wash with PBS 2-3 times.

[0150] (V) Mounting: Mounting medium: Use a mounting medium suitable for fluorescent staining, such as a mounting medium containing anti-fluorescence decay. Add an appropriate amount of mounting medium to the surface of the section, and gently cover the coverslip to avoid air bubbles.

[0151] (VI) Result observation: Microscopic observation: Use a fluorescence microscope to observe the section. The lipid part stained by BODIPY usually presents green fluorescence (the emission wavelength is usually 510-520 nm), and is observed under 488 nm excitation. If DAPI staining is performed, the nucleus presents blue fluorescence. The results are shown in Figure 3 .

[0152] Figure 3 A hematoxylin-eosin staining (H&E staining). From Figure 3 analysis in A, it can be seen that the adjuvant injection group shows a large number of lipid droplets accumulated in hepatocytes, and there are obvious vacuolar structures in the liver, indicating that the liver has excessive accumulation of lipids. In contrast, the number of lipid droplets in hepatocytes in the (R,R)-59 injection group is significantly reduced, showing fewer vacuolar structures, indicating that the lipid accumulation is lighter.

[0153] 3. TG detection experiment

[0154] Operation steps:

[0155] (I) Sample preparation

[0156] Experimental materials: mouse liver tissue: generally weighing 50-100 mg.

[0157] (II) Lipid extraction

[0158] (1) Tissue homogenate

[0159] Take about 50-100 mg of liver tissue and place it in a 1.5 mL centrifuge tube. Add 500 μL of ice-cold PBS or normal saline. Homogenize thoroughly using a homogenizer or ultrasonic disrupter until the tissue is completely uniform.

[0160] (2) Lipid extraction

[0161] Organic solvent extraction method: Add chloroform-methanol (2:1, v / v) mixed solution to the tissue homogenate solution at a ratio of 2:1 (e.g., 500 μL of homogenate solution added to 1 mL of chloroform-methanol solution). Mix well by vortexing for 10 minutes. Let it stand at room temperature until the lipids are completely dissolved. Add 0.2 times the volume of deionized water (e.g., 400 μL), mix well by vortexing, and let it stand for 10 minutes before phase separation. Centrifuge at low speed (1500 rpm for 10 minutes), and take the lower organic phase (chloroform phase) as the lipid extract.

[0162] (3) Triglyceride (TG) detection using a commercially available triglyceride detection kit (brand Solarbio). Enzymatic method is used, i.e., triglyceride content is determined by glycerol oxidase reaction. Follow the kit instructions: add enzyme reaction reagents (including glycerol ester hydrolysis enzyme and oxidase, etc.), and the reaction product generates colored substances. The reaction is carried out at 37°C constant temperature, and the time is 10-30 minutes.

[0163] (4) Colorimetric method: After the reaction is completed, measure the absorbance value at the specified wavelength (usually 490-550 nm) using a spectrophotometer or enzyme marker. Calculate the TG content in the sample according to the standard curve. The results are shown in Figure 3 .

[0164] Figure 3 B is oil red O staining, BODIPY staining, wherein oil red O staining is specifically used to detect neutral lipids in tissues. From Figure 3 the comparison of oil red O staining group in B, it can be seen that in the liver sections of the adjuvant injection group, red dye shows a large number of fat particles, indicating that there is a large amount of neutral fat accumulation in the liver. The red staining area of the (R,R)-59 injection group is reduced, indicating that the lipid accumulation is significantly reduced compared with the adjuvant group, indicating that (R,R)-59 has the effect of reducing liver fat accumulation. BODIPY is a fluorescent dye that specifically labels intracellular fat. From Figure 3 the comparison of BODIPY staining group in B, it can be seen that in the adjuvant injection group, green fluorescent labeling shows that there are a large number of lipid droplets in the liver cells, indicating that the lipid accumulation is obvious. In the (R,R)-59 injection group, the green fluorescence intensity is low and the number of lipid droplets is significantly reduced, further proving the potential effect of (R,R)-59 in reducing liver lipid accumulation and alleviating lipid toxicity.

[0165] Figure 3 C is the determination of liver triglyceride (TG) level.Figure 3 The results of the determination of TG concentration are shown in Table C. From Table C, it can be seen that the TG level of the adjuvant injection group is higher, with an average of close to 20 mM / mg, indicating that the lipid in the liver is significantly accumulated. Figure 4 From the analysis in Table C, it can be seen that the TG level of the adjuvant injection group is higher, with an average of close to 20 mM / mg, indicating that the lipid in the liver is significantly accumulated. The TG level of the (R,R)-59 injection group is significantly reduced, with an average of 10-15 mM / mg, indicating that the lipid in the liver is significantly reduced, and statistical analysis shows that there is a significant difference between the two groups.

[0166] Four, experiment of the effect of (R,R)-59 on promoting autophagy of liver lipid

[0167] The specific operation steps are as follows:

[0168] (I) Section preparation

[0169] Take the liver tissue out from -80℃ and slice it on a freezing microtome (thickness 5 μm).

[0170] (II) Fixation and permeation

[0171] (1) Fixation: Put the section into 4% PFA for 10-15 minutes (room temperature). Wash the section with PBS for 3 times, 5 minutes each time.

[0172] (2) Permeate the cell membrane with 0.1%-0.3% Triton X-100 (prepared in PBS), incubate for 5-10 minutes. Wash with PBS for 2 times, 5 minutes each time.

[0173] (III) Blocking

[0174] Block non-specific binding sites: Add blocking solution (5%-10% BSA or normal goat serum) on the section, incubate at room temperature for 1 hour. Wash with PBS once to remove excess blocking solution.

[0175] (IV) Incubation of primary antibody

[0176] Prepare the working solution of primary antibody: Dilute the primary antibody with 1%-3% BSA according to the recommended concentration of the antibody (such as 1:100-1:500). Add the working solution of primary antibody (50-100 μL) to the section. Incubate overnight at 4℃ in a wet box to ensure that the antibody is fully combined with the target protein. After the end, wash the section: wash with PBS for 3 times, 5 minutes each time, to remove the unbound primary antibody.

[0177] (V) Incubation of secondary antibody

[0178] Dilute the secondary antibody (labeled with fluorescent dye) with 1%-3% BSA according to the recommended concentration (such as 1:200-1:500) to prepare the working solution. Add the working solution of secondary antibody to the section, incubate at room temperature for 1 hour, and avoid light treatment. Wash with PBS for 3 times, 5 minutes each time, to remove the unbound secondary antibody.

[0179] (vi) DAPI staining

[0180] Prepare DAPI working solution (1 pg / mL). Drop on the section, incubate at room temperature for 5 minutes. Wash with PBS twice to remove excess dye.

[0181] (vii) Mounting

[0182] Drop anti-fade mounting medium on the section, gently cover the coverslip. Squeeze out the air bubbles, seal the edge to avoid evaporation of the mounting medium.

[0183] (viii) Microscopy

[0184] Use fluorescence microscope to observe. Choose excitation wavelength according to the fluorescence label (e.g. FITC: 488 nm, DAPI: 360 nm). Target signal: fluorescence intensity represents the expression level of target protein. Results are shown in Figure 4 .

[0185] Figure 5 Immunofluorescence images of liver frozen sections of control group, adjuvant group (DMSO: corn oil = 1:9) and experimental group ((R,R)-59). Mainly focus on the expression and distribution of different proteins (LAMP2A, LC3B and PLIN2). The left half of the image is the colocalization experiment of LAMP2A and PLIN2. The right half: colocalization experiment of LC3B and PLIN2. The green fluorescence part shows LAMP2A and LC3B respectively. Red fluorescent protein represents PLIN2 (lipid droplet associated protein). Combine the above three labels to show the colocalization relationship.

[0186] In the control group: the distribution of LAMP2A (green) and PLIN2 (red) is less, and there is no obvious colocalization phenomenon. The distribution of LC3B (green) and PLIN2 (red) is relatively scattered, indicating that the distribution relationship between autophagy-related markers and lipid droplets is weak under control conditions. Experimental observation results: after compound treatment ((R,R)-59), the green (LAMP2A) signal is significantly enhanced compared to the adjuvant injection group, and there is partial colocalization (yellow area) with red (PLIN2), showing that compound treatment enhances the association between lysosomes and lipid droplets. LC3B and PLIN2: LC3B (green) signal is significantly enhanced, and more green signal is wrapped around the red (PLIN2), forming obvious colocalization (yellow area). This indicates that (R,R)-59 treatment promotes the association between lipid droplets and autophagosomes.

[0187] After treatment with compound (R-R-59), the LAMP2A signal increased, indicating that lysosome activity was enhanced or the number increased. The LC3B signal increased, indicating that autophagy activity was significantly enhanced. The co-localization of PLIN2 with the above two markers increased, suggesting that the compound might participate in the degradation or metabolism of lipid droplets by promoting lysosome and autophagy-related pathways.

[0188] The above experimental results prove that in the (R,R)-59 treatment group, the co-localization signal between lipid droplets (PLIN2) in hepatocytes and autophagy-related markers (LC3B) and lysosome markers (LAMP2A) is significantly enhanced, indicating that (R,R)-59 can promote the degradation of lipid droplets by activating the autophagy-lysosome pathway.

[0189] Five, (R,R)-59 protects the liver function of acute liver injury mouse model test

[0190] Experimental animals: 8-week-old C57BL / 6 male mice, randomly divided into 2 groups (control group and experimental group), 4 mice in each group. After fasting the mice for 12 hours, the control group (APAP+adjuvant (DMSO: corn oil = 1:9)): after intraperitoneal injection of 300 mg / kg APAP solution (dissolved in warm saline), 2 hours and 4 hours later, inject adjuvant (10 mg / kg). Experimental group (APAP+(R,R)-59): after intraperitoneal injection of 300 mg / kg APAP solution (dissolved in warm saline), 2 hours and 4 hours later, intraperitoneal injection of (R,R)-59 solution (10 mg / kg). 4 mice in each group.

[0191] Serum detection (ALT and AST) to detect mouse liver function.

[0192] After dissecting the mice to take whole blood samples 24 hours after APAP administration, centrifugation to separate serum, and detecting the levels of ALT (glutamic-pyruvic transaminase) and AST (glutamic-oxaloacetic transaminase) in serum by automatic biochemical analyzer.

[0193] The serum test results are shown in Figure 5 .

[0194] From Figure 6 analysis, the ALT and AST levels of the control group mice increased significantly. The ALT and AST levels of the experimental group mice decreased significantly, thereby proving that the compound (R,R)-59 has a significant liver protection effect.

[0195] Six, morphological test of (R,R)-59 affecting acute liver injury

[0196] The experimental animals were divided into two groups (control group and experimental group), the control group (APAP + adjuvant (DMSO: corn oil = 1:9)): after intraperitoneal injection of 300 mg / kg APAP solution (dissolved in warm saline), 2 hours and 4 hours later, 10 mg / kg of adjuvant was injected. The experimental group (APAP + (R,R)-59): after intraperitoneal injection of 300 mg / kg APAP solution (dissolved in warm saline), 2 hours and 4 hours later, 10 mg / kg of (R,R)-59 solution was injected. The mouse liver was taken 24 hours after administration, fixed in 4% paraformaldehyde, paraffin-embedded, sectioned and stained with HE, and the histopathological changes were observed, and the results are shown in Figure 6 .

[0197] From the analysis in Figure 7 , it can be seen that the liver cells of the mice in the control group showed large area of hepatocyte necrosis, inflammatory infiltration and vacuolar degeneration. While the liver cells of the mice in the experimental group recovered well, with only a small amount of inflammatory infiltration. This indicates that the compound (R,R)-59 has a liver protection effect on the liver damage caused by APAP.

[0198] Seven, (R,R)-59 affects the level of inflammation in liver tissue test

[0199] Similarly, paraffin sections were used as experimental materials for immunofluorescence experiments, and the specific operation steps were as described above. The marker proteins of immune cells were stained, and the inflammatory response in the tissue was observed, and the results are shown in Figure 7 .

[0200] From the analysis in Figure 8 , it can be seen that APAP treatment plays a role in tissue damage, which can cause significant infiltration of neutrophils (Ly6G marker) and increase with time. Macrophages (F4 / 80 marker) have the same phenotype in APAP treatment, and the level of inflammation in the tissue is significantly restored after injection of SETDB1 activator in the time window of damage. Thus, it is proved that the treatment of (R,R)-59 regulates the recruitment and activation of inflammatory cells, and plays a corresponding role in tissue repair or alleviating inflammatory response.

[0201] Eight, (R,R)-59 affects the apoptosis detection test in acute liver injury model in liver tissue

[0202] The expression of apoptosis proteins cleaved-PARP and cleaved-caspase3 in liver tissue was detected using IF staining experiment. The experimental animals were divided into 2 groups (control group and experimental group), the control group (APAP + adjuvant (DMSO: corn oil = 1:9)): after intraperitoneal injection of 300 mg / kg APAP solution (dissolved in warm saline), 2 hours and 4 hours later, adjuvant (10 mg / kg) was injected. The experimental group (APAP + (R,R)-59): after intraperitoneal injection of 300 mg / kg APAP solution (dissolved in warm saline), 2 hours and 4 hours later, (R,R)-59 solution (10 mg / kg) was injected. The specific operation steps are as described above, and the results are shown in Figure 8 .

[0203] From Figure 9 analysis, the proportion of apoptotic cells in the control group of mice increased significantly. The proportion of apoptotic cells in the experimental group of mice decreased significantly, and the expression of apoptosis proteins in the liver tissue of the experimental group of mice injected with (R,R)-59 after the establishment of the APAP liver injury model was significantly down-regulated compared with the control group, thereby proving that the treatment of (R,R)-59 has obvious alleviating effect on cell apoptosis of damaged liver tissue, and plays a role in tissue repair.

[0204] Nine, (R,R)-59 affects cell apoptosis in liver tissue in acute liver injury model Western blot experiment

[0205] The experimental animals in this project were divided into 2 groups (control group and experimental group), the control group (APAP + adjuvant (DMSO: corn oil = 1:9)): after intraperitoneal injection of 300 mg / kg APAP solution (dissolved in warm saline), 2 hours and 4 hours later, adjuvant (10 mg / kg) was injected. The experimental group (APAP + (R,R)-59): after intraperitoneal injection of 300 mg / kg APAP solution (dissolved in warm saline), 2 hours and 4 hours later, (R,R)-59 solution (10 mg / kg) was injected. The target proteins detected by Western blot are SETDB1, Bcl-2, cleaved-Caspase3. The specific operation steps are as described above, and the results are shown in ​ .

[0206] The Western blot detection results show that the expression of SETDB1 gene in the experimental group of mice is significantly activated, the anti-apoptotic protein Bcl-2 is significantly up-regulated, and the expression of apoptosis protein cleaved-caspase3 is significantly down-regulated.

[0207] In summary, the application of the SETDB1 activator in the organ detoxification drug, (R,R)-59 can be used as a SETDB1 activator, by regulating the expression of genes, so that hepatocytes can more effectively process fatty acids, reduce the accumulation of fat in hepatocytes, and at the same time, inhibit the accumulation of lipid droplets and triglycerides in hepatocytes, reduce the level of lipotoxic substances, thereby reducing liver cell damage and inflammatory response, and regulating lipotoxicity and promoting autophagy of lipids to effectively alleviate liver toxicity caused by starvation or other factors, therefore, (R,R)-59 is expected to become a new drug for treating lipotoxicity and related metabolic diseases, and is particularly suitable for patients with non-alcoholic fatty liver disease and fatty liver disease caused by malnutrition, and the SETDB1 activator (R,R)-59 can alleviate the inflammatory response and hepatocyte apoptosis caused by APAP overdose, and promote liver repair, providing a new drug for treating liver damage. In the field of biological medicine technology, it has popularization and application value.

[0208] The above examples are only preferred embodiments for fully illustrating the present application, and the protection scope of the present application is not limited thereto. Any equivalent replacement or transformation made by those skilled in the art on the basis of the present application is within the protection scope of the present application.

Claims

1. The use of SETDB1 activator (R,R)-59 in the preparation of drugs for non-alcoholic fatty liver disease (NAFLD), characterized in that, The structural formula of (R,R)-59 is shown in formula (1); 2. The use according to claim 1, characterized in that, The dosage range of (R,R)-59 is 0.6 to 0.7 mg / kg.

3. The use according to claim 1, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

4. The use according to claim 3, characterized in that, The carrier includes a diluent and / or a pH adjuster.

Images