Application of SETDB1 activator in medicine for treating organ injury and medicine

Abstract

The invention relates to an application of an SETDB1 activator in a medicine for treating organ injury and the medicine. According to the application of the SETDB1 activator in the medicine for treating the organ injury, the SETDB1 activator is (R, R)-59. The invention also provides a medicine for treating organ injury, and the medicine comprises the SETDB1 activating agent (R, R)-59. The invention provides a new application of the SETDB1 activator (R, R)-59, the SETDB1 activator (R, R)-59 can effectively relieve hepatotoxicity caused by hunger or other factors by adjusting lipid toxicity and promoting lipid autophagy, the SETDB1 activator (R, R)-59 can inhibit inflammatory response and hepatocyte apoptosis caused by excessive APAP, liver repair is promoted, and the SETDB1 activator (R, R)-59 can be used for preventing and treating liver diseases. And a new medicine is provided for treating the medicine-induced liver injury.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an application of a SETDB1 activator in a drug for treating organ damage and a drug. Background Art

[0002] The liver plays a vital role in the human body. It is a multifunctional metabolic organ that is 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 lifestyles, liver health is facing unprecedented threats.

[0003] The first type of liver damage is that under the influence of bad living habits such as hunger, malnutrition, metabolic disorders and drug abuse, the toxicity level of the liver increases significantly, posing a serious threat to liver health. Studies have shown that lipotoxicity is a major factor leading to liver damage. This toxicity is mainly caused by the excessive accumulation of fatty acids and their metabolites in liver cells. In addition, lipophagy is a key physiological process for clearing excess lipids in cells, and its dysfunction has also been found to be closely related to liver toxicity.

[0004] In current medical practice, the treatment of liver toxicity mentioned above mainly focuses on symptomatic treatment, such as using drugs to relieve liver inflammation or reduce liver burden. Although these methods can relieve symptoms to a certain 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, overdose may cause severe acute liver injury (ALI) and become one of the main causes of liver failure. Existing treatments mainly rely on N-acetylcysteine ​​(NAC), but its efficacy is limited by the time window of use (usually within 8 hours of overdose) and has limited therapeutic effect on existing liver cell damage.

[0006] Therefore, there is an urgent need to develop therapeutic drugs that can target liver toxicity and treat acute liver injury in order to achieve more effective treatment effects and improve liver health. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide an application of a SETDB1 activator in a drug for treating organ damage and a drug, so as to provide a new use of a SETDB1 activator, and a 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 a SETDB1 activator is used to treat acute liver damage caused by acetaminophen overdose, so as to effectively improve APAP-induced liver damage, providing a new drug for acute liver damage caused by acetaminophen overdose.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] A use of a SETDB1 activator in a drug for treating 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] Experiments have found that (R,R)-59 can act as a SETDB1 activator. By regulating gene expression, it can effectively reduce the level of lipotoxic substances, thereby alleviating liver cell damage and inflammatory response. At the same time, (R,R)-59 can act as a SETDB1 activator, effectively promoting liver repair and reducing liver cell apoptosis.

[0012] Preferably, the SETDB1 activator is used in a drug for organ detoxification.

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

[0014] (R,R)-59 can act as a SETDB1 activator. By regulating gene expression, it enables hepatocytes to process fatty acids more effectively and reduce fat accumulation in hepatocytes. At the same time, it can inhibit the accumulation of free fatty acids and triglycerides in hepatocytes and reduce the level of lipotoxic substances, thereby alleviating hepatocyte damage and inflammatory response. It can also regulate lipotoxicity and promote lipid autophagy to effectively alleviate liver toxicity caused by starvation or other factors.

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

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

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

[0018] (R,R)-59 can act as a SETDB1 activator, significantly upregulating the downstream anti-apoptotic protein Bcl-2 and downregulating the pro-apoptotic protein Cleaved-caspase3, thereby effectively inhibiting the oxidative stress, inflammatory response and hepatocyte apoptosis caused by excessive APAP and promoting liver repair.

[0019] Preferably, when used as a drug for treating acute liver damage caused by acetaminophen overdose, the dosage range of (R,R)-59 is 0.6 to 0.7 mg / kg.

[0020] Preferably, the (R,R)-59 is used in a drug for processing fatty acids, wherein the (R,R)-59 activates SETDB1 to increase lipid autophagy, allowing cells to break down lipids, thereby reducing the accumulation of intracellular fat.

[0021] Experimental studies have found that (R,R)-59 upregulates gene expression through methylation and upregulates the lipid autophagy pathway to promote lipid decomposition in cells. This dual mechanism of action makes (R,R)-59 significantly superior in regulating lipid metabolism.

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

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

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

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

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

[0027] Preferably, the (R,R)-59 is used in a drug for inducing activation of the lipid autophagy pathway. The (R,R)-59 can induce activation of the lipid autophagy pathway and promote lipid degradation in hepatocytes by enhancing the formation and function of lipid autophagosomes.

[0028] Among them, (R,R)-59 can significantly increase the expression levels of lipid autophagy-related genes in the liver, such as microtubule-associated protein light chain 3 (LC3) and lysosomal associated protein 1 (LAMP1). The upregulation of these genes helps promote the decomposition and clearance of intracellular lipids, thereby effectively alleviating fat accumulation in the liver.

[0029] The present invention also provides a drug for treating organ damage, wherein the drug comprises the SETDB1 activator (R, R)-59.

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

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

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

[0033] Preferably, the drug 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 damage caused by fat accumulation. The dosage of the drug can be adjusted according to the patient's weight, age, gender, health status and severity of the disease.

[0034] Preferably, the drug can be administered in the form of oral administration or injection, such as tablets, capsules, suspensions or injections. In addition, the drug 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 invention are as follows:

[0036] The application of the SETDB1 activator of the present invention in the medicine for treating organ damage, with (R, R)-59 as a SETDB1 activator, can regulate the expression of genes, so that liver cells can more effectively process fatty acids, reduce the accumulation of fat in liver cells, and 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 lipid autophagy 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 to activate the downstream anti-apoptotic protein Bcl-2 to significantly upregulate and the pro-apoptotic protein Cleaved-caspase to downregulate, thereby effectively inhibiting the oxidative stress, inflammatory response and hepatocyte 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 hepatocyte apoptosis caused by excessive APAP, and acute liver injury caused by malnutrition, and has promotion and application value in the field of biomedical technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the result diagram of the effect of SETDB1 activator (R,R)-59 on SETDB1 expression level;

[0038] Figure 2 This is a comparison of the morphological observations of the effects of (R,R)-59 on lipid accumulation in the liver;

[0039] Figure 3 The figure shows the pathological observation of the effect of (R,R)-59 on the accumulation of lipids in the liver and the test results of triglycerides in the liver tissue;

[0040] Figure 4 This is the experimental result diagram of the effect of (R,R)-59 on promoting liver lipid autophagy;

[0041] Figure 5 This is a graph showing the effect of serum (R,R)-59 on liver function in an acute liver injury model;

[0042] Figure 6 This is the morphological observation result of pathological detection of (R,R)-59 affecting acute liver injury;

[0043] Figure 7 This is a graph showing the effect of (R,R)-59 on the level of inflammation in liver tissue;

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

[0045] Fig. 9 The results are from Western blot. DETAILED DESCRIPTION

[0046] The following will describe the embodiments of the present invention with reference to the accompanying drawings and preferred embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are only for illustrating the present invention, not for limiting the scope of protection of the present invention.

[0047] The present invention intends to disclose the use of a SETDB1 activator in a drug for treating organ damage, so as 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, so as to effectively improve APAP-induced liver damage, providing a new drug for acute liver damage caused by acetaminophen overdose.

[0048] Wherein, the use of a SETDB1 activator in a drug for treating organ damage, the SETDB1 activator is (R,R)-59, and the structural formula of (R,R)-59 is shown in formula (1);

[0049]

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

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

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

[0053] In some embodiments, the organ comprises at least one of the liver, kidney, heart, lung and intestine, in particular the liver.

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

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

[0056] In some embodiments, the use of the SETDB1 activator (R,R)-59 in drugs for processing fatty acids, (R,R)-59 activates SETDB1 to increase lipid autophagy, allowing cells to break down lipids and reduce the accumulation of intracellular fat.

[0057] In some embodiments, the SETDB1 activator (R,R)-59 is used as 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 cells, so that (R,R)-59 inhibits the accumulation of free fatty acids and triglycerides in hepatocytes, reduces the level of lipotoxic substances, and thus reduces hepatocyte damage and inflammatory response.

[0060] In some embodiments, the SETDB1 activator (R,R)-59 is used in drugs that induce activation of the lipid autophagy pathway. Thus, (R,R)-59 can induce activation of the autophagy pathway, promote lipid degradation in hepatocytes by enhancing the formation and function of lipid autophagosomes, and help the liver clear excess lipids, thereby maintaining the metabolic homeostasis and functional health of the liver.

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

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

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

[0064] Specific experimental detection and analysis

[0065] 1. Experiment on the effect of SETDB1 activator (R,R)-59 on SETDB1 expression level

[0066] The experiment of this project was divided into three groups: normal control group, adjuvant (DMSO: corn oil = 1:9) injection group and (R,R)-59 injection group. (R,R)-59 was treated as a SETDB1 activator. The mice in the normal control group were not treated in any way. The mice in the adjuvant (DMSO: corn oil = 1:9) injection group and the (R,R)-59 injection group were starved mice. Specifically, the non-alcoholic fatty liver model was starved for 48 hours, and then the adjuvant and (R,R)-59 were intraperitoneally injected, respectively, with an injection dose of 10 mg / Kg. After 24 hours, the mice were killed by dislocation of the neck and dissected to obtain liver tissue samples. The liver tissue was fixed with 4% paraformaldehyde for 24 hours, and then paraffin-embedded to make paraffin sections and frozen sections. Some were directly frozen with liquid nitrogen and stored in a -80℃ refrigerator for use as samples for other index detection.

[0067] The specific operation steps are as follows: Protein immunoblotting (western blot)

[0068] 1. Protein sample extraction

[0069] (1) RIPA lysis buffer (Radio Immunoprecipitation Assay Lysis buffer) uses surfactants to lyse cell membranes (including nuclear membranes) and extract soluble proteins from tissues or cells. Mix various inhibitors and lysis buffer evenly and place on ice for later use. The lysis buffer should be prepared and used immediately.

[0070] (2) Weigh out 20 mg of liver tissue stored in a -80°C refrigerator and place it into a 1.5 mL EP tube. Add 400 μL of the above lysis solution.

[0071] (3) Place a magnetic bead in the EP tube and physically break it in a grinder at 60 Hz for 3 min (to prevent protein sample degradation during processing, the entire operation should be performed on an ice brick taken out from a low temperature).

[0072] (4) Precool the low-temperature centrifuge to 4°C, place the protein sample that has completed the grinding process into the low-temperature centrifuge, set the speed to 8000g, and centrifuge for 10 minutes. During centrifugation, the protein will precipitate at the bottom of the tube and the supernatant will be separated.

[0073] (5) After centrifugation, remove the supernatant, transfer it to a new EP tube, and store the sample in a -80°C refrigerator.

[0074] (II) Determination of protein concentration

[0075] (1) The protein concentration was determined using the BCA protein concentration detection kit. Before determining the concentration, a standard curve was determined using a BSA solution of standard concentration.

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

[0077] (3) The colorimetric reagent in the kit should be prepared according to the ratio of solution A: solution B = 50:1 as specified in the instructions. Losses will occur during the addition of samples, so prepare a little more reagent than the required volume to allow for losses during the process.

[0078] (4) Add 100 μL of working solution to each well of the microplate, place in a 37°C incubator and incubate for 30 min. Measure the absorbance at 570 nm using an ELISA reader. Draw a standard curve based on the OD value and the standard protein concentration (statistical value R2 ≥ 0.99).

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

[0080] (1) The collected protein samples were quantified using a BCA kit, and the method was prepared according to the protein concentration standard curve. To reduce experimental errors, three replicate wells were set for each sample. The protein OD value was measured at 570 nm and the protein concentration was calculated by inserting it into the standard curve.

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

[0082] (3) Protein denaturation: Centrifuge the prepared protein sample in a centrifuge to ensure that the liquid is at the bottom of the EP tube. Heat the tube in a 95°C metal bath for 10 min to denature the protein. During the process, care must be taken to prevent the EP tube cap from breaking open due to heating, causing the liquid to splash out and affect the experimental results. After heating, cool and centrifuge for later use.

[0083] (4) Prepare SDS-PAGE gel of appropriate experimental concentration: According to the molecular weight of different proteins, select gels of different concentrations. It consists of two parts: concentrating gel and separating gel. According to the sample volume and the number of experimental samples, select the thickness of the gel plate and add plastic combs of different specifications to reserve sample holes. A sample with a volume of 10 μL generally requires a gel plate with a thickness of 1.0 mm, while a sample with a volume of 20 μL requires a gel plate with a thickness of 1.5 mm.

[0084] (5) Sample loading: After the gel plates solidify, place the two gel plates in the electrophoresis tank and fix them at the same time. Add SDS electrophoresis buffer to the electrophoresis tank and slowly add the sample to the gel wells reserved for gel making. Avoid bubbles during the process that affect the sample volume and cause errors in the results. When loading the sample, a protein marker should also be added to one of the sample wells to facilitate the subsequent differentiation of the target protein bands.

[0085] (6) Electrophoresis: The electrophoresis instrument is set to a constant voltage. The upper layer of concentrated gel is electrophoresed at a voltage of 70 V. The voltage of the lower layer of separation gel can be adjusted appropriately, but it should not exceed 120 V. Too high a voltage will affect the uniformity of the protein bands.

[0086] (IV) Electrophoresis transfer

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

[0088] (2) Use the wet transfer method to transfer the membrane. Add the transfer electrophoresis buffer, transfer clip, sponge, filter paper and PVDF membrane into the transfer container. Expel the bubbles in the arranged transfer filter paper and PVDF membrane and carefully close the transfer clip. Place the transfer clip in the transfer electrophoresis tank in the correct electrode order.

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

[0090] (4) Treatment and blocking of target bands: After the electrotransfer is completed, remove the transfer clip. The protein on the PVDF membrane has no obvious characteristics. If the target bands are to be distinguished, they can be marked with Ponceau protein dye. First, remove the PVDF membrane and activate it in methanol for 15-20 seconds, then place the membrane in Ponceau dye. After the protein bands are colored, cut the membrane according to the size of the target protein in the experiment. Then use TBST working solution to wash the Ponceau until the PVDF membrane turns white again. Block with 5% skim milk. The purpose is to bind the protein in the blank area of ​​the PVDF membrane in advance to avoid the background of blurred bands caused by the nonspecific binding of the subsequent antibodies. Place the incubation box on a shaker at a speed of 20-25rpm and block at room temperature for 1h.

[0091] (V) Antibody incubation:

[0092] (1) Use primary antibody diluent to dilute the protein primary antibody according to the properties of the antibody. The general ratio range is 1:2000-1:10000. After preparation, place it on ice for later use.

[0093] (2) Wash the blocked protein bands twice with TBST to remove the remaining milk and place them in an antibody incubation box. Add the prepared primary antibody solution and place them on a shaker at 4°C for slow shaking. The incubation time should be more than 8 hours, and can generally be overnight.

[0094] (3) After the primary antibody incubation is completed, it is recovered (stored in a 4°C refrigerator and can be reused), and the residual primary antibody is washed with TBST solution, shaken at 60-80 rpm, and washed 4X8min. New TBST solution needs to be replaced each time.

[0095] (4) After the primary antibody is completed, the secondary antibody should be incubated according to the resistance of the primary antibody. The secondary antibody is diluted with 2.5% skim milk. The blocked and recovered milk can be diluted in half to prepare different concentrations of secondary antibodies according to the protein type. The general concentration range is 1:5000-1:15000. Add the secondary antibody to the incubation box, shake gently on a shaker, and incubate at room temperature for 2 hours.

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

[0097] (VI) Chemiluminescence imaging

[0098] (1) Using a chemiluminescent development kit, mix luminescent solution A and solution B in a ratio of 1:1, vortex mix and set aside (because the luminescence principle is an enzyme-catalyzed reaction, it should be prepared and used immediately to ensure the luminescence effect).

[0099] (2) Spread a smooth plastic film on a flat surface, use a pipette to draw up an appropriate amount of luminescent liquid and place it evenly on the plastic film, remove the PVDF membrane from the TBST solution, use enzyme-free paper to absorb as much water as possible from the protein-free side, then contact the protein side with the luminescent liquid, gently lift and lower the membrane several times to ensure even contact with the luminescent liquid, incubate for 2 minutes, move it into a dark folder, and then put it into a light-proof developing room for fluorescent exposure.

[0100] (3) After entering the darkroom, adjust the exposure time according to the brightness of the band until the protein band displays an 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 the figure. Figure 1 shown.

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

[0102] from Figure 1The analysis showed that in the adjuvant injection group, the expression level of SETDB1 was increased compared with the normal control group. In the (R,R)-59 injection group, the expression of SETDB1 was further significantly enhanced, which proved that (R,R)-59 could effectively activate the expression of SETDB1. H3K9me3 is a histone modification mark catalyzed by SETDB1. In the (R,R)-59 injection group, the level of H3K9me3 was significantly increased, which was consistent with the enhancement of SETDB1 expression, thus proving that the activity of SETDB1 was also significantly enhanced. Comprehensive analysis showed that (R,R)-59, as a SETDB1 activator, significantly increased the expression of SETDB1 and the level of H3K9me3 catalyzed by it. This shows that (R,R)-59 has a significant role in the activation of SETDB1 and the regulation of epigenetic modification.

[0103] 2. Morphological observation experiment on the effect of (R,R)-59 on lipid accumulation in the liver

[0104] The specific steps are:

[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 were starved mice. Specifically, the non-alcoholic fatty liver model was established by starvation for 48 hours, and then the adjuvant and (R,R)-59 were intraperitoneally injected, respectively, with an injection dose of 10 mg / Kg. After 24 hours, the mice were killed by dislocation of the neck and the liver characteristics were observed after dissection. The experiment was divided into an adjuvant intraperitoneal injection group and a (R,R)-59 intraperitoneal injection group. The results are shown in the figure. Figure 2 shown.

[0106] Figure 2 A shows the liver of the adjuvant intraperitoneal injection group. Figure 2 A. It can be seen that the liver is larger in size and lighter in color. This appearance is usually associated with hepatic fatty degeneration, indicating that 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 size, darker in color, and healthier in appearance, indicating that lipid accumulation in the liver is reduced, thus proving that the SETDB1 activator (R,R)-59 plays an effective role in reducing lipid accumulation.

[0108] 3. Pathological observation of the effect of (R,R)-59 on liver lipid accumulation and the detection of triglycerides in liver tissue The specific operation steps are as follows:

[0109] 1. HE staining

[0110] (I) Sample preparation: Paraffin section: The section thickness is 5 μm.

[0111] (2) Dyeing

[0112] (1) Conventional dewaxing

[0113] Remove paraffin: Soak sections in xylene solution twice for 5 minutes each to remove paraffin.

[0114] (2) Gradient hydration: The concentration of the solvent is reduced each time it is replaced, from xylene to different concentrations of ethanol, and finally to distilled water. The sequence is as follows:

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

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

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

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

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

[0120] (3) Dyeing

[0121] 1) Hematoxylin staining: The sections were stained with hematoxylin dye.

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

[0123] 2) Differentiation and blueing:

[0124] Stain sections with hematoxylin, usually for 10–20 seconds, until the tissue takes on an appropriate blue color.

[0125] Subsequently, the sections were rinsed in running water for 2-3 minutes for anti-blueing.

[0126] Staining (Oil Red O)

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

[0128] 2) Heat the solution in a 45°C water bath for 15 minutes. The incompletely dissolved Oil Red O can be removed by filtering with filter paper. Immerse the sections in the Oil Red O staining solution. The staining time is usually 10–15 minutes. Gently shake the sections to ensure that the Oil Red O dye is evenly distributed.

[0129] 3) Rinse: Rinse the sections with 70% isopropanol (or 70% ethanol) to gently remove excess dye, being careful not to wipe the sections. Allow the sections to air dry.

[0130] 4) Hydration and sealing: Transfer the sections to distilled water for rinsing to remove excess isopropanol.

[0131] (4) Dehydration and sealing

[0132] Stepwise dehydration: Soak the sections in 70%, 85%, 95%, and 100% ethanol for 2 minutes each. Soak in xylene solution for 5 minutes.

[0133] (5) Sealing: Use an appropriate amount of neutral gum sealing liquid and drop the sealing liquid onto the surface of the slice. Gently cover with a coverslip to remove bubbles.

[0134] (III) Final inspection

[0135] Use a microscope to observe the staining effect and ensure that the cell nucleus is blue-purple and the cytoplasm is pink. And observe the pathological changes.

[0136] 2. Bodipy staining experiment:

[0137] Bodipy staining is a small molecule fluorescent probe used to detect lipids, and is often used to label lipids in tissue sections or cell cultures. Bodipy dye can bind to specific parts of lipids in cells, especially interact with fatty acids, triglycerides, and phospholipids, and emit fluorescence at specific wavelengths.

[0138] (I) Sample preparation

[0139] Tissue sections: Tissue sections prepared using a cryostat, usually 5 μm thick.

[0140] (II) Slice preparation and fixation

[0141] (1) Fixation: Frozen sections can be fixed with a fixative, and the commonly used fixative is 4% paraformaldehyde (PFA). The fixation time is 10-15 minutes (at room temperature). Avoid over-fixation to avoid affecting lipid staining.

[0142] (2) Rinse: Wash the sections twice with PBS (phosphate buffered saline), 5 minutes each time, to remove the fixative and residual impurities.

[0143] (III) Infiltration treatment

[0144] (1) Permeabilization solution: Use 0.2% Triton X-100 as the permeabilization solution to properly permeate the cell membrane and help BODIPY dye better enter the tissue. The time is 5-10 minutes (room temperature).

[0145] (IV) BODIPY staining

[0146] (1) BODIPY dye solution: Prepare BODIPY dye solution at a concentration of 2 ng / mL. Dissolve the BODIPY dye solution in PBS at a ratio of 1:4000 and stain in the dark. Place the sections in the BODIPY dye solution for 15 minutes.

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

[0148] (3) DAPI staining (optional). If you need to stain the cell nucleus, you can choose DAPI staining. DAPI can bind to DNA and display the cell nucleus. DAPI staining solution: 1 μg / mL DAPI solution, staining for 5–10 minutes.

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

[0150] (V) Sealing: Sealing solution: Use a sealing solution suitable for fluorescent staining, such as a sealing solution containing anti-fluorescence attenuation. Add an appropriate amount of sealing solution to the surface of the slice and gently cover it with a coverslip to avoid bubbles.

[0151] (VI) Observation of results: Microscope observation: Use a fluorescence microscope to observe the sections. The lipid part stained with BODIPY usually shows green fluorescence (the emission wavelength is usually 510-520nm) and is observed under 488nm excitation. If DAPI staining is performed, the cell nucleus shows blue fluorescence. The results are as follows Figure 3 shown.

[0152] Figure 3 A is hematoxylin-eosin staining (H&E staining). Figure 3 The analysis in A shows that the adjuvant injection group showed a large accumulation of lipid droplets in hepatocytes, and obvious vacuolar structures in the liver, indicating excessive lipid accumulation in the liver. In contrast, the (R,R)-59 injection group showed a significant decrease in the number of lipid droplets in hepatocytes, showing fewer vacuolar structures, indicating less lipid accumulation.

[0153] 3. TG detection experiment

[0154] Steps:

[0155] (I) Sample preparation

[0156] Experimental materials: Mouse liver tissue: The weight is generally 50–100 mg.

[0157] (II) Lipid Extraction

[0158] (1) Tissue homogenization

[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 saline. Use a homogenizer or ultrasonic disruptor to fully homogenize the tissue until it is completely homogenized.

[0160] (2) Lipid extraction

[0161] Organic solvent extraction method: Add chloroform-methanol (2:1, v / v) mixed solution to the tissue homogenate at a ratio of 2:1 (for example: add 1 mL of chloroform-methanol solution to 500 μL of homogenate). Vortex mix for 10 minutes. Let stand at room temperature until the lipids are fully dissolved. Add 0.2 times the volume of deionized water (such as 400 μL), vortex mix, let stand for 10 minutes and then separate the phases. Centrifuge at low speed (1500 rpm, 10 minutes), and take the lower organic phase (chloroform phase), which is the lipid extract.

[0162] (3) Triglyceride (TG) detection was performed using a commercially available triglyceride detection kit (brand: Solarbio). The enzymatic method was used to determine the triglyceride content by glycerol oxidase reaction. The procedure was performed according to the kit instructions: enzyme reaction reagents (including glycerol ester hydrolase and oxidase, etc.) were added, and the reaction product generated a colored substance. The reaction was carried out at a constant temperature of 37°C for 10–30 minutes.

[0163] (4) Colorimetric determination: After the reaction is completed, use a spectrophotometer or microplate reader to measure the absorbance value at a specified wavelength (generally 490-550nm). Calculate the TG content in the sample based on the standard curve. The results are as follows: Figure 3 shown.

[0164] Figure 3 B is Oil Red O staining and BODIPY staining, among which Oil Red O staining is specifically used to detect neutral lipids in tissues. Figure 3 Comparative analysis of the Oil Red O staining group in B shows that in the liver sections of the adjuvant injection group, the red dye showed a large number of fat particles, indicating that a large amount of neutral fat accumulated in the liver. The red staining area in the (R,R)-59 injection group decreased, indicating that lipid accumulation was significantly reduced compared to the adjuvant group, indicating that (R,R)-59 has the effect of reducing liver fat accumulation. BODIPY is a fluorescent dye that specifically marks intracellular fat. Figure 3 Comparative analysis of the BODIPY staining group in B shows that in the adjuvant injection group, green fluorescence labeling showed a large number of lipid droplets in the hepatocytes, indicating significant lipid accumulation. In the (R,R)-59 injection group, the green fluorescence intensity was low and the number of lipid droplets was significantly reduced, further demonstrating the potential role of (R,R)-59 in reducing hepatic lipid accumulation and alleviating lipotoxicity.

[0165] Figure 3 C is the determination of liver triglyceride (TG) level. Figure 3 C shows the results of TG concentration measurement. Figure 3 Analysis in C shows that the TG level in the adjuvant injection group was higher, averaging close to 20mM / mg, indicating significant lipid accumulation in the liver. The TG level in the (R,R)-59 injection group was significantly reduced, averaging 10-15mM / mg, indicating a significant reduction in lipids in the liver. Statistical analysis showed significant differences between the two groups.

[0166] IV. Experiment on the effect of (R,R)-59 on promoting liver lipid autophagy

[0167] The specific steps are:

[0168] (I) Slice preparation

[0169] The liver tissue was taken out from -80°C and sliced ​​on a freezing microtome (thickness 5 μm).

[0170] (II) Fixation and penetration

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

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

[0173] (III) Closure

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

[0175] (IV) Primary antibody incubation

[0176] Prepare primary antibody working solution: dilute primary antibody with 1%-3% BSA according to the recommended concentration of antibody (e.g. 1:100–1:500). Add primary antibody working solution (50-100 μL) to the slice. Incubate overnight at 4°C in a humidified chamber to ensure that the antibody is fully bound to the target protein. Wash the slices after completion: wash 3 times with PBS for 5 minutes each time to remove unbound primary antibody.

[0177] (V) Secondary Antibody Incubation

[0178] Dilute the secondary antibody (labeled with fluorescent dye) with 1%-3% BSA and prepare the working solution according to the recommended concentration (e.g. 1:200-1:500). Add the secondary antibody working solution to the slices and incubate at room temperature for 1 hour in the dark. Wash with PBS 3 times, 5 minutes each time, to remove unbound secondary antibody.

[0179] (VI) DAPI staining

[0180] Prepare DAPI working solution (1 μg / mL). Add it dropwise on the slices and incubate at room temperature for 5 minutes. Wash twice with PBS to remove excess dye.

[0181] (VII) Sealing

[0182] Add anti-fluorescence attenuation mounting solution to the slice and gently cover with a coverslip. Squeeze out bubbles and seal the edges to prevent the mounting solution from evaporating.

[0183] (VIII) Microscope observation

[0184] Observe using a fluorescence microscope. Select the excitation wavelength according to the fluorescent marker (such as 488nm for FITC and 360nm for DAPI). Target signal: The fluorescence intensity represents the expression level of the target protein. The results are as follows Figure 4 shown.

[0185] Figure 4 Immunofluorescence images of frozen liver sections of the control group, adjuvant group (DMSO: corn oil = 1:9) and experimental group ((R,R)-59), focusing on the expression and distribution of different proteins (LAMP2A, LC3B and PLIN2). The left half of the image is the co-localization experiment of LAMP2A and PLIN2. The right half: the co-localization experiment of LC3B and PLIN2. The green fluorescent part shows LAMP2A and LC3B respectively. The red fluorescent protein represents PLIN2 (lipid droplet-associated protein). The above three markers were merged to show the co-localization relationship.

[0186] In the control group: LAMP2A (green) and PLIN2 (red) were less distributed and there was no obvious co-localization phenomenon. The distribution of LC3B (green) and PLIN2 (red) was relatively scattered, indicating that the relationship between autophagy-related markers and lipid droplet distribution was weak under control conditions. Experimental observation results: After compound treatment ((R,R)-59), the green (LAMP2A) signal was significantly enhanced compared with the adjuvant injection group, and there was partial co-localization with red (PLIN2) (yellow area), indicating that compound treatment enhanced the association between lysosomes and lipid droplets. LC3B and PLIN2: The LC3B (green) signal was significantly enhanced, and more green signals appeared around the red (PLIN2), forming an obvious co-localization (yellow area). This shows that (R,R)-59 treatment promotes the association between lipid droplets and autophagosomes.

[0187] After treatment with the compound (RR-59), the LAMP2A signal increased, indicating that lysosomal 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 may participate in the degradation or metabolism of lipid droplets by promoting lysosomal and autophagy-related pathways.

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

[0189] V. Experiment on the protection of liver function in acute liver injury mouse model by (R,R)-59

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

[0191] Serum tests (ALT and AST) were used to detect the liver function of mice.

[0192] 24 hours after APAP administration, the mice were dissected to obtain whole blood samples, which were then centrifuged to separate the serum. The levels of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) in the serum were detected using an automatic biochemical analyzer.

[0193] Serum test results such as Figure 5 shown.

[0194] from Figure 5 The analysis showed that the ALT and AST levels of the mice in the control group were significantly increased, while the ALT and AST levels of the mice in the experimental group were significantly decreased, which proved that compound (R,R)-59 has a significant liver protective effect.

[0195] 6. Pathological Detection Morphological Test of the Effect of (R,R)-59 on Acute Liver Injury

[0196] 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), the adjuvant (10 mg / kg) was injected 2 hours and 4 hours later. The experimental group (APAP+(R,R)-59): after intraperitoneal injection of 300 mg / kg APAP solution (dissolved in warm saline), the (R,R)-59 solution (10 mg / kg) was injected intraperitoneally 2 hours and 4 hours later. The mouse liver was taken 24 hours after administration, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned and HE stained to observe the tissue pathological changes. The results are as follows. Figure 6 shown.

[0197] from Figure 6 The analysis showed that the liver cells of mice in the control group showed large-area liver cell necrosis, inflammatory infiltration and vacuolar degeneration. The liver cell structure of mice in the experimental group recovered well, with only a small amount of inflammatory infiltration. This shows that the compound (R,R)-59 has a protective effect on liver damage caused by APAP.

[0198] VII. Experiment on the effect of (R,R)-59 on the level of inflammation in liver tissue

[0199] The immunofluorescence experiment was also carried out using paraffin sections as experimental materials. The specific operation steps were as mentioned above. The marker proteins of immune cells were stained and the inflammatory response in the tissue was observed. The results are as follows: Figure 7 shown.

[0200] from Figure 7 The analysis showed that APAP treatment plays a role in tissue damage, which causes significant infiltration of neutrophils (Ly6G marker), which increases over time. Macrophages (F4 / 80 marker) have the same phenotype after APAP treatment, and the level of inflammation in the tissue is significantly restored after injection of SETDB1 activator within the time window of damage. This proves that (R,R)-59 treatment has a regulatory effect on the recruitment and activation of inflammatory cells, and plays a corresponding role in tissue repair or reducing inflammatory response.

[0201] 8. Effect of (R,R)-59 on apoptosis in liver tissue in acute liver injury model

[0202] IF staining was used to detect the expression of apoptotic proteins cleaved-PARP and cleaved-caspase3 in liver tissue. 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), the adjuvant (10 mg / kg) was injected 2 hours and 4 hours later. The experimental group (APAP+(R,R)-59): after intraperitoneal injection of 300 mg / kg APAP solution (dissolved in warm saline), the (R,R)-59 solution (10 mg / kg) was injected intraperitoneally 2 hours and 4 hours later. The specific operation steps are as mentioned above, and the results are shown in the figure. Figure 8 shown.

[0203] from Figure 8 The analysis showed that the proportion of apoptotic cells in the control group mice increased significantly, while the proportion of apoptotic cells in the experimental group mice decreased significantly. After the APAP liver injury model was established, the expression of apoptotic proteins in the liver tissue of the experimental group mice injected with (R,R)-59 was significantly downregulated compared with the control group, which proved that the treatment of (R,R)-59 had a significant effect on alleviating the apoptosis of damaged liver tissue and played a role in tissue repair.

[0204] IX. Effect of (R,R)-59 on apoptosis in liver tissue in acute liver injury model (Western blot study)

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

[0206] Western blot results showed that the expression of SETDB1 gene in the experimental group of mice was significantly activated, the anti-apoptotic protein Bcl-2 was significantly upregulated, and the expression of apoptotic protein cleaved-caspase3 was significantly downregulated.

[0207] In summary, the application of the SETDB1 activator of the present invention in the organ detoxification drug, with (R, R)-59 as a SETDB1 activator, can regulate the expression of genes, so that liver cells can more effectively process fatty acids, reduce the accumulation of fat in liver cells, and inhibit the accumulation of fat droplets 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. 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. The SETDB1 activator (R, R)-59 can alleviate the inflammatory response and hepatocyte apoptosis caused by excessive APAP, promote liver repair, and provide a new drug for treating liver damage. In the field of biomedical technology, it has promotion and application value.

[0208] The above embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Any equivalent substitution or change made by a person skilled in the art based on the present invention is within the protection scope of the present invention.

Claims

1. Use of a SETDB1 activator in a drug for treating organ damage, characterized in that: The SETDB1 activator is (R,R)-59, and the structural formula of (R,R)-59 is shown in formula (1); (1)。 2. The use according to claim 1, characterized in that: Use of the SETDB1 activator in drugs for organ detoxification.

3. The use according to claim 2, characterized in that: When used as a liver detoxification drug, the dosage range of the (R,R)-59 is 0.6 to 0.7 mg / kg.

4. The use according to claim 1, characterized in that: The use of the SETDB1 activator in a drug for treating acute liver injury caused by acetaminophen overdose, when used as a drug for treating acute liver injury caused by acetaminophen overdose, the dosage range of the (R,R)-59 is 0.6 to 0.7 mg / kg.

5. The use according to claim 1, characterized in that: The organ includes at least one of a liver, a kidney, a heart, a lung, and an intestinal tract.

6. The use according to claim 1, characterized in that: The use of the (R,R)-59 in drugs for processing fatty acids, wherein the (R,R)-59 activates SETDB1 to increase lipid autophagy, thereby enabling cells to decompose lipids and reduce the accumulation of intracellular fat; And / or, use of the (R,R)-59 in a drug for reducing serum alanine aminotransferase (ALT); And / or, use of the (R,R)-59 in a drug for reducing serum aspartate aminotransferase (AST).

7. The use according to claim 1, characterized in that: Use of the (R,R)-59 in drugs for inhibiting the accumulation of free fatty acids and triglycerides in cells; And / or, use of the (R,R)-59 in a drug for inducing activation of the lipid autophagy pathway.

8. A drug for treating organ damage, characterized in that: The drugs include the SETDB1 activator (R,R)-59.

9. The drug according to claim 8, characterized in that The medicament further includes a pharmaceutically acceptable carrier.

10. The drug according to claim 9, characterized in that The carrier includes a diluent and / or a pH adjuster.

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