Application of antisense oligonucleotide small molecule in preparation of medicine for improving fatty liver
Through the targeted delivery technology of GalNAC-APJ ASO, APJ expression is inhibited, and the treatment problems of non-alcoholic fatty liver are solved, and the problems of liver lipid accumulation and insulin tolerance are significantly improved. It provides a novel treatment method with smaller toxic side effects.
Patent Information
- Application Number
- CN202411551745.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-01
- Publication Date
- 2025-01-10
AI Technical Summary
There is a lack of product use of antisense oligonucleotide small molecules that inhibit APJ in the prior art for the treatment of liver diseases such as non-alcoholic fatty liver, and existing treatment methods are ineffective against non-alcoholic fatty liver.
GalNAC-APJ ASO is used as an antisense oligonucleotide small molecule targeting hepatocytes, and the specific delivery and inhibition of APJ is achieved through N-acetylgalactosamine coupling modification, which significantly reduces the APJ expression level in mice. This improves glucose tolerance, insulin tolerance, hepatic steatosis and inhibits adipocyte hypertrophy.
GalNAC-APJ ASO significantly improves non-alcoholic fatty liver symptoms, reduces liver TG levels, inhibits adipocyte hypertrophy, and provides a novel treatment for non-alcoholic fatty liver, which is smaller than the prior art. toxic side effects.
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Figure CN119280262A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of medicine and disease treatment, and specifically relates to the application of antisense oligonucleotide small molecules in the preparation of medicines for improving fatty liver. Background Art
[0002] Nonalcoholic fatty liver disease is the liver component of metabolic syndrome and the most common chronic liver disease worldwide. It is a highly prevalent dynamic disease that occurs in all age groups. Its main sign is that triglycerides accumulate to more than 5% to 10% of the liver weight, and it occurs in the absence of secondary factors such as excessive alcohol consumption, viral or autoimmune hepatitis, or congenital liver disease. Nonalcoholic fatty liver disease can rapidly develop into cirrhosis and liver malignancies. It is estimated that the global prevalence of nonalcoholic fatty liver disease is 25%, which increases to 55.5% in patients with T2DM. Not only is there no effective treatment for metabolic syndrome, but existing treatments for nonalcoholic fatty liver disease are ineffective against the progression of the disease to nonalcoholic hepatitis and fibrosis. There is currently no treatment for nonalcoholic fatty liver disease approved by the US Food and Drug Administration. Among them, the English name of nonalcoholic fatty liver disease is Nonalcoholic fatty liver disease, NAFLD.
[0003] APJ contains 7 transmembrane units and 308 amino acids and belongs to the family of G protein-coupled receptors. Apelin and ELABELA are endogenous ligands of APJ receptors. APJ is widely distributed in the human central nervous system and cardiovascular tissues. A large number of studies have shown that the activation of APJ has the effects of enhancing myocardial contractility, lowering blood pressure, promoting angiogenesis, regulating the body's immune response and pituitary-related hormone release, regulating insulin secretion, and participating in the pathological and physiological processes of diabetic vascular lesions, heart failure, atrial fibrillation, ischemia-reperfusion injury, etc. However, the effect of APJ on the occurrence and development of liver diseases including non-alcoholic fatty liver disease is not clear. However, there are no reports on the use of antisense oligonucleotide small molecules that inhibit APJ in the treatment of liver diseases including non-alcoholic fatty liver disease. Among them, the full English name of APJ is Apelin Peptide Jejunum. The English name of G protein-coupled receptors is G protein coupled receptors, GPCRs. Summary of the invention
[0004] In order to solve the problem that there is no report on the use of antisense oligonucleotide small molecules that inhibit APJ in the prior art for treating liver diseases including non-alcoholic fatty liver disease, the present invention finds that the application of APJ antisense oligonucleotide small molecules targeting hepatocytes GalNAC-APJ ASO to treat high-fat-fed mice in vivo can significantly reduce the expression level of APJ in the liver of mice and inhibit the phenotype of non-alcoholic fatty liver disease in mice. Therefore, the present invention provides the use of antisense oligonucleotide small molecules in the preparation of drugs for improving fatty liver.
[0005] To achieve the above purpose, the present invention adopts the following technical solution.
[0006] The present invention provides the use of antisense oligonucleotide small molecules in the preparation of drugs for improving fatty liver. The present invention provides the use of antisense oligonucleotide small molecules in the preparation of drugs for improving fatty liver, which solves the problem of the current reports on the use of antisense oligonucleotide small molecules that inhibit APJ in the treatment of liver diseases including non-alcoholic fatty liver.
[0007] The antisense oligonucleotide small molecule is GalNAC-APJ ASO, and the GalNAC-APJ ASO is obtained by modifying the 3′ end of APJ ASO by GalNAC coupling.
[0008] The base sequence of the APJ ASO is shown in SEQ ID NO.1: TTTTCGCGGCTGGTTCGAAA.
[0009] The Chinese name of GalNAC is N-acetylgalactosamine. N-acetylgalactosamine coupling modification is currently the most commonly used small nucleic acid drug delivery system. N-acetylated galactosamine is covalently conjugated to the 3′ end of the sense chain of siRNA of different sequences in a trivalent state to form a polysaccharide-siRNA monoconjugate, thereby achieving specific delivery to hepatocytes, and allowing the drug to enter the cell to function through cellular endocytosis. The present invention also provides the use of the antisense oligonucleotide small molecule in the preparation of a drug for improving the disease caused by non-alcoholic fatty liver or alleviating its symptoms.
[0010] The present invention has been found through research that the GalNAC-APJ ASO has the following effects:
[0011] The GalNAC-APJ ASO can improve glucose tolerance and insulin resistance.
[0012] The GalNAC-APJ ASO can improve hepatic steatosis.
[0013] The GalNAC-APJ ASO is able to inhibit hepatic lipid accumulation.
[0014] The GalNAC-APJ ASO was able to reduce liver TG levels.
[0015] The GalNAC-APJ ASO is able to inhibit adipocyte hypertrophy.
[0016] Preferably, the drug contains the GalNAC-APJ ASO as an active ingredient and is supplemented with a pharmaceutically acceptable excipient or carrier.
[0017] The drug also has the following functions:
[0018] (1) The medicament is used for improving glucose tolerance and insulin resistance.
[0019] (2) The drug is used to improve hepatic fatty degeneration.
[0020] (3) The drug is used to inhibit hepatic lipid accumulation.
[0021] (3) The drug is used to reduce liver TG levels.
[0022] (4) The drug is used to inhibit fat cell hypertrophy.
[0023] Preferably, the administration of the drug includes any one of parenteral administration and intravenous administration. Parenteral administration includes subcutaneous administration, intradermal administration, intramuscular injection and intraperitoneal injection, which is convenient for administration; intravenous administration has a rapid onset of effect.
[0024] Preferably, the medicine includes any one of tablets, capsules, granules, pills, emulsions, liquids, patches, solutions, ointments and injections.
[0025] Preferably, the drug is a tablet, and GalNAC-APJ ASO is mixed with an excipient to obtain a mixed powder; the mixed powder is mixed with starch slurry to obtain a soft material, which is then granulated, dried and granulated in sequence, and the obtained granules are mixed with talcum powder and then tableted to obtain the tablet.
[0026] Preferably, the drug is a capsule, and the mixed powder of GalNAC-APJ ASO and excipients is sieved and then filled into a soft capsule material made of gelatin to obtain the capsule.
[0027] Preferably, the drug is a granule, and GalNAC-APJ ASO is mixed with an excipient to obtain a mixed powder; the mixed powder is mixed with an ethanol aqueous solution to obtain a soft material, which is then granulated, dried and granulated in sequence to obtain the granule.
[0028] Preferably, the excipients include any one or more of starch, sucrose, trehalose, mannitol and sorbitol. The above excipients combine with GalNAC-APJ ASO to form a stable inclusion complex, increase the stability of GalNAC-APJ ASO, protect GalNAC-APJ ASO from environmental factors, and improve the stability and bioavailability of GalNAC-APJ ASO.
[0029] Preferably, the carrier includes any one or more of nanoparticles, liposomes, transfersomes, microspheres, and hydrogels. The above carriers all have good biocompatibility, biodegradability, and renewability, thereby improving the utilization rate, safety, and effectiveness of the drug. Among them, nanoparticles can adjust the release rate of drugs in the human body, change the biomembrane permeability and in vivo distribution of drug molecules, and can deliver drugs to targeted organs; liposomes, transfersomes, microspheres, and hydrogels can improve the ability of drugs to penetrate the skin barrier, improve the bioavailability of drugs, and reduce the frequency of drug administration.
[0030] The present invention can prepare GalNAC-APJ ASO into the following specific pharmaceutical dosage forms, but is not limited thereto: any pharmaceutical dosage form that can be used to prevent and treat fatty liver can be prepared by using the field of pharmaceutical preparations.
[0031] Preferably, the drug is a solution.
[0032] The carrier used in the solution includes a solvent.
[0033] The solvent is any one of DMSO, water, ethanol, glycerol and physiological saline.
[0034] Preferably, the solution is prepared by mixing the GalNAC-APJ ASO with physiological saline.
[0035] The concentration of the GalNAC-APJ ASO in the solution is 0.1 mg / mL to 1 mg / mL. The solution drug has a large dispersion, fast absorption, rapid action, and good bioavailability; the active ingredients are evenly dispersed and can be accurately measured and used, especially suitable for children and elderly patients; the dosage size is easy to adjust and control; it can reduce the local irritation of certain soluble drugs; and it can increase the stability and safety of certain drugs. Therefore, the dosage form of the above-mentioned drug provided by the present invention is a solution.
[0036] Compared with the prior art, the present invention has the following beneficial effects:
[0037] The present invention provides the use of antisense oligonucleotide small molecules in the preparation of drugs for improving fatty liver, and provides a new method for treating non-alcoholic fatty liver. The present invention proves the positive therapeutic effect of GalNAC-APJ ASO in non-alcoholic fatty liver through in vivo experiments (non-alcoholic fatty liver model induced by high-fat diet in mice). Specifically, GalNAC-APJ ASO significantly improves the symptoms of non-alcoholic fatty liver in mice, improves glucose tolerance and insulin tolerance in mice, improves liver steatosis, reduces liver lipid accumulation, reduces liver TG levels, inhibits fat cell hypertrophy, and provides candidate drugs for the treatment of non-alcoholic fatty liver and its related symptoms. In addition, GalNAC-APJ ASO specifically targets liver tissue, has less toxic side effects than existing drugs for treating non-alcoholic fatty liver, and has made significant progress compared with the prior art. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] Figure 1 This is an in vitro validation experiment of APJ ASO knocking down APJ in liver AML-12 cells in the present invention; wherein, Figure 1 Figure A in the figure is the base sequence of APJ ASO; Figure 1 Figure B shows APJ ASO modified with GalNAC; Figure 1 Figure C in the figure is a normal light microscopic picture of the fluorescent positive control of cell transfection efficiency; Figure 1 Figure D in the figure is a green fluorescence microscopic picture of the fluorescent positive control of cell transfection efficiency; Figure 1 Figure E shows the expression level of AplnrmRNA in AML-12 cells after transfection of APJ ASO by RT-qPCR.
[0039] Figure 2 The experimental process of the animal experiment in the present invention and the RT-qPCR verification of knocking down liver APJ; wherein, Figure 2 Figure A in the figure is the experimental process of the animal experiment in the present invention; Figure 2 Panel B shows the expression level of Aplnr mRNA in liver.
[0040] Figure 3 The weight of mice in the present invention is as follows: Figure 3 Figure A shows the weight changes of mice; Figure 3 Figure B shows the average daily food intake of mice; Figure 3 Figure C shows abdominal fat in mice.
[0041] Figure 4 The results of the mouse GTT and ITT experiments in the present invention, wherein: Figure 4 Figure A in the figure is the fasting blood glucose of mice in the present invention; Figure 4Figure B in the figure is the blood glucose level-time curve of mice in each group of GTT experiment; Figure 4 Figure C in the figure is the AUC of the blood glucose level-time curve of each group of mice in the GTT experiment of the present invention; Figure 4 Figure D in the figure is the blood glucose level-time curve of mice in each group of ITT experiment; Figure 4 Figure E in the figure is the AUC of the blood glucose level-time curve of each group of mice in the ITT experiment. GTT refers to glucose tolerance test, ITT refers to insulin tolerance test, and AUC refers to area under the curve.
[0042] Figure 5 The non-alcoholic fatty liver phenotype of mice in the present invention: wherein, Figure 5 Figure A shows the liver weight of mice; Figure 5 Figure B is a physical picture of the liver of mice in the Control group; Figure 5 Figure C in the figure is a physical picture of the liver of mice in the HFD group; Figure 5 Figure D is a physical picture of the liver of mice in the HFD+ELA-21 group; Figure 5 Figure E shows the HE staining results of liver tissue of mice in the Control group; Figure 5 Figure F shows the HE staining results of liver tissues of mice in the HFD group; Figure 5 Figure G shows the HE staining results of liver tissues of mice in the HFD+ELA-21 group; Figure 5 Figure H shows the Oil Red O staining results of liver tissue of mice in the Control group; Figure 5 Figure I in the figure shows the Oil Red O staining results of liver tissues of mice in the HFD group; Figure 5 Figure J shows the Oil Red O staining results of liver tissue of mice in the HFD+ELA-21 group.
[0043] Figure 6 The HE staining results of white and brown adipose tissue of mice in the present invention.
[0044] Figure 7 is the serum lipid level of mice in the present invention: wherein, Figure 7 Panel A shows the serum high-density lipoprotein level in mice; Figure 7 Panel B shows the serum low-density lipoprotein level in mice; Figure 7 Figure C shows the total cholesterol level in mouse serum; Figure 7 Figure D shows the serum triglyceride level in mice.
[0045] Figure 8 is the lipid level in mouse liver in the present invention: wherein, Figure 8 Panel A shows the level of high-density lipoprotein in mouse liver; Figure 8 Panel B shows the level of low-density lipoprotein in mouse liver; Figure 8 Figure C shows the total cholesterol level in mouse liver; Figure 8 Panel D shows triglyceride levels in mouse liver. DETAILED DESCRIPTION
[0046] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.
[0047] The mice used in the embodiments of the present invention are all of the C57BL / 6J strain and are raised in the Experimental Animal Center of Jiangxi University of Traditional Chinese Medicine in a specific pathogen-free animal room. Experimental Animal Use License No.: SCXK(Gan)2023-0001.
[0048] Among them, the English name of specific pathogens is specific pathogen free, SPF.
[0049] All mice used in the experiment were 8-week-old male mice, and all mouse experiments complied with the "Regulations on the Use of Experimental Animals" formulated by the National Ethics Committee and the Jiangxi University of Traditional Chinese Medicine. The mice used in the experiment are referred to as C57BL / 6J mice below.
[0050] Example 1
[0051] 1. In vitro validation of APJ ASO knockdown in AML-12 cells
[0052] In vitro cultured liver AML-12 cells were transfected with Lipofectamine TM 3000 transfection reagents were used to transfect APJASO 284 、ASO 427 、ASO 528 、ASO 837 and ASO 1358 , using an empty plasmid with GFP-tagged protein as a positive control, the transfection efficiency was observed under a fluorescence microscope. Total RNA was extracted 24 hours after transfection, and RT-qPCR was performed to detect the expression level of Aplnr mRNA in cells.
[0053] Among them, the base sequence of APJ ASO is as follows Figure 1 Figure A and as shown in SEQ ID NO.1: TTTTCGCGGCTGGTTCGAAA.
[0054] Fluorescence images of transfection efficiency Figure 1 As shown in Figure B; the expression level of AplnrmRNA in cells is shown in Figure 1 As shown in Figure C. Liver AML-12 cells transfected with ASO 427、ASO 528 、ASO 837 and ASO 1358 After that, the expression level of AplnrmRNA in cells was significantly reduced. We selected ASO 427 Conduct subsequent animal experiments.
[0055] 2. Fatty liver modeling, grouping, and drug administration
[0056] (1) Fatty liver model
[0057] After C57BL / 6J mice were fed a high-fat diet (45 kcal% fat, HFD) for 12 weeks, the lipid level (total cholesterol) in the serum increased, indicating that the model was successful, and the C57BL / 6J mouse model group mice were obtained.
[0058] (2) Grouping
[0059] Seventeen mice were randomly divided into three groups: control group, 5 mice; HFD group, 6 mice; HFD+GalNAC-APJ ASO group, 6 mice. The mice in the HFD+GalNAC-APJ ASO group were intraperitoneally injected with GalNAC-APJ ASO once a day starting from the 13th week, and samples were collected at the 16th week for subsequent experiments.
[0060] Among them, the control group is also called the Control group.
[0061] The experimental process of animal experiments is as follows Figure 2 shown.
[0062] The specific grouping and dosing methods are as follows:
[0063] Control group: The mice were fed with normal mouse diet and given an intraperitoneal injection of an equal volume of saline. Here, "equal volume" means that the volume of the injected saline is the same as the volume of the injected ELA-21 solution.
[0064] HFD group: fed with a high-fat diet, and given an intraperitoneal injection of an equal volume of saline. Here, "equal volume" means that the volume of the injected saline is the same as the volume of the injected ELA-21 solution.
[0065] HFD+GalNAC-APJ ASO group: mice were fed with a high-fat diet and given an intraperitoneal injection of GalNAC-APJ ASO solution; the amount of GalNAC-APJ ASO injected into mice was 3 mg / kg / day.
[0066] Among them, GalNAC-APJ ASO was synthesized by Nanjing GenScript Biotechnology Co., Ltd. with a purity of >98% and stored at -20°C. Before use, it was dissolved in physiological saline and sterilized with a 0.22μm filter to obtain a GalNAC-APJ ASO solution with a concentration of 0.3mg / mL.
[0067] Depend on Figure 2 It can be seen that the experimental process of animal experiments and RT-qPCR verified the effect of GalNAC-APJ ASO in knocking down liver APJ. The results showed that GalNAC-APJ ASO can significantly reduce the expression level of Aplnr mRNA in mouse liver tissue.
[0068] 2. Glucose tolerance and insulin tolerance test
[0069] After overnight fasting, the mice in the above groups were intraperitoneally injected with 1g / kg of glucose, and their blood glucose levels were tested at 0, 15 minutes, 30 minutes, 60 minutes, 90 minutes and 120 minutes. The mice in the above groups in the fasting state were intraperitoneally injected with 0.75U / kg of insulin, and their blood glucose levels were tested at 0, 15 minutes, 30 minutes, 60 minutes, 90 minutes and 120 minutes. The results are as follows: Figure 4 shown.
[0070] Among them, the method for detecting blood sugar level is: cut 2mm from the tip of the mouse tail, discard the first drop of blood, and use Sinocare GA-3 blood sugar meter and corresponding blood sugar test strips to detect the blood sugar level of the second drop of blood.
[0071] Depend on Figure 4 It can be seen that the fasting blood glucose of mice Figure 4 As shown in Figure A; The blood glucose level-time curves of mice in each group of GTT experiment are as follows Figure 4 As shown in Figure B; AUC of the blood glucose level-time curve of each group of mice in the GTT experiment Figure 4 As shown in Figure C; The blood glucose level-time curves of mice in each group of ITT experiment are as follows Figure 4 As shown in Figure D; the AUC of the blood glucose level-time curve of each group of mice in the ITT experiment is as follows Figure 4 As shown in Figure E. GTT refers to glucose tolerance test, ITT refers to insulin tolerance test, and AUC refers to area under the curve. There was no significant difference in the fasting blood glucose level of each group of mice. Compared with the control group, the blood glucose level and AUC value of the HFD group mice at each time point were significantly increased after oral glucose gavage or intraperitoneal injection of insulin, and the blood glucose level and AUC value at each time point were significantly reduced after GalNAC-APJ ASO intervention.
[0072] The above results indicate that GalNAC-APJ ASO treatment significantly improved glucose tolerance and insulin resistance induced by a high-fat diet in mice.
[0073] 3. Sample collection and processing
[0074] The mice in each group obtained at the end of the experiment were fasted for 12 hours before sampling, but not water deprivation, weighed, and blood was collected by removing the eyeballs. The liver tissue was removed and the body was photographed with a macrophotographer.
[0075] The weight changes of mice were as follows Figure 3 As shown in Figure A, the average daily food intake of mice is Figure 3 As shown in Figure B, the abdominal fat of mice Figure 3 As shown in Figure C. Compared with the control group, the weight and abdominal fat of the high-fat-fed mice increased significantly; after the intervention of GalNAC-APJ ASO, there was no significant change in the weight, abdominal fat and diet of the mice.
[0076] Depend on Figure 3 It can be seen that GalNAC-APJ ASO treatment did not affect the body weight and body fat content of mice.
[0077] The 2cm×1.5cm×0.3cm livers and 1.5cm×1.0cm×0.2cm adipose tissues of mice in the control group, HFD group, and HFD+GalNAC-APJ ASO group were fixed in 4% by mass paraformaldehyde, and the remaining livers and adipose tissues were quick-frozen in liquid nitrogen for 30 minutes and then stored at -80°C for later use.
[0078] The mouse blood collected from each group was placed in a 1.5 mL centrifuge tube, allowed to stand at room temperature for 1 hour, centrifuged at 4°C and 3500 r / min for 10 minutes, the supernatant was collected to obtain plasma samples, and stored at -80°C for later use.
[0079] 4. HE staining and Oil Red O staining of liver and adipose tissue
[0080] The liver and adipose tissue in step 2 were fixed in a 4% by mass paraformaldehyde solution at 4°C for 24 hours, and then the tissues were removed and rinsed with running water. After automatic dehydration and wax immersion, they were embedded in paraffin and sliced with a paraffin slicer with a thickness of 4 μm. The slices were baked in an oven at 65°C for 2 hours and stored at room temperature for later use to obtain paraffin sections.
[0081] Paraffin sections were dewaxed to water by an automatic stainer, and then stained with hematoxylin for 5 minutes, washed with distilled water, differentiated with differentiation solution, washed with distilled water, and blued with blueing solution until the cytoplasm was dark blue and the nucleus was not stained, rinsed with running water, and the sections were sequentially dehydrated with 85% ethanol and 95% ethanol for 5 minutes each, and stained with eosin solution for 5 minutes. The sections were then sequentially placed in 100% ethanol I for 3 minutes, 100% ethanol II for 3 minutes, 100% ethanol III for 3 minutes, xylene I for 3 minutes, and xylene II for 3 minutes; the sections were sealed with neutral gum and photographed under a microscope.
[0082] After the liver in step 2 was fixed in a 4% by volume paraformaldehyde solution at 4°C for 24 hours, the tissue was removed and rinsed with running water overnight, precipitated with a 30% by mass sucrose solution, embedded in OCT, sliced with a freezing microtome, with a slice thickness of 8 μm, incubated with 100% by volume isopropanol for 5 minutes, incubated with 0.5% by volume Oil Red O solution at 60°C for 7 minutes, washed with 85% by volume isopropanol solution for 3 minutes, washed with distilled water, stained with hematoxylin for 1 minute, washed with distilled water, and sealed with a sealing agent. The lipids were red or orange-yellow, and the cell nuclei were light blue. The slices were photographed under a microscope.
[0083] Results of liver tissue Figure 5 The results for adipose tissue are shown in Figure 6 shown.
[0084] Depend on Figure 5 It is known that under the condition of high-fat diet, mice can develop fatty liver, and the pathological manifestations are diffuse enlargement of the liver, yellowing of the color, typical increase in the level of liver fatty degeneration and excessive fat accumulation. Figure 5 The results of Figure C are HE staining. Figure 5 The results in Figure D are Oil Red staining.
[0085] However, the symptoms were significantly improved after GalNAC-APJ ASO treatment, and the level of fatty degeneration and fat accumulation in the liver of mice were significantly reduced. Figure 5 The results of Figure C are HE staining. Figure 5 The results in Figure D are Oil Red staining.
[0086] The above experimental results show that GalNAC-APJ ASO can effectively alleviate the progression of this type of fatty liver disease.
[0087] Depend on Figure 6 It can be seen that compared with the control group mice, the size of white fat and brown fat cells in high-fat diet mice was significantly increased, while GalNAC-APJ ASO intervention significantly reduced the size of fat cells.
[0088] The above results indicate that GalNAC-APJ ASO treatment significantly inhibited the hypertrophy of white fat and brown adipocytes in mice.
[0089] 5. Blood lipid level detection
[0090] The plasma samples prepared above were taken and the levels of LDL-C, HDL-C, TC and TG in mouse plasma were detected using LDL-C kit, HDL-C kit, TC kit and TG kit respectively. The results are as follows: Figure 7 shown.
[0091] Among them, the Chinese name of LDL-C is low-density lipoprotein cholesterol; the Chinese name of HDL-C is high-density lipoprotein cholesterol; the Chinese name of TC is serum total cholesterol; and the Chinese name of TG is triglyceride.
[0092] LDL-C kit, HDL-C kit, TC kit, and TG kit were all purchased from Nanjing Jiancheng Bioengineering Institute.
[0093] The catalog number of the LDL-C kit is A113-1-1.
[0094] The catalog number of the HDL-C kit is A112-1-1.
[0095] The catalog number of the TC kit is A111-1-1.
[0096] The catalog number of the TG kit is A110-1-1.
[0097] Depend on Figure 7 It can be seen that a high-fat diet significantly increased the levels of plasma LDL-C, HDL-C and TC, and decreased the level of TG. Although GalNAC-APJ ASO treatment did not affect plasma LDL-C, it significantly decreased the levels of HDL-C and TC, and increased the level of TG.
[0098] 6. Detection of liver lipid levels
[0099] The liver tissues retained above were taken, and the weight of the liver tissues was accurately weighed. The tissues were homogenized with anhydrous ethanol, and the levels of LDL-C, HDL-C, TC and TG in the extracts were detected using LDL-C kit, HDL-C kit, TC kit and TG kit, respectively. The results were as follows: Figure 8 shown.
[0100] Among them, the Chinese name of LDL-C is low-density lipoprotein cholesterol; the Chinese name of HDL-C is high-density lipoprotein cholesterol; the Chinese name of TC is serum total cholesterol; and the Chinese name of TG is triglyceride.
[0101] LDL-C kit, HDL-C kit, TC kit, and TG kit were all purchased from Nanjing Jiancheng Bioengineering Institute.
[0102] The catalog number of the LDL-C kit is A113-1-1.
[0103] The catalog number of the HDL-C kit is A112-1-1.
[0104] The catalog number of the TC kit is A111-1-1.
[0105] The catalog number of the TG kit is A110-1-1.
[0106] Depend on Figure 8 It can be seen that a high-fat diet significantly increased the levels of LDL-C, HDL-C, TC and TG in liver tissue. Although GalNAC-APJ ASO treatment increased the levels of LDL-C, HDL-C and TC in the liver, it significantly reduced the level of TG in liver tissue, indicating that GalNAC-APJ ASO can significantly alleviate the progression of fatty liver disease.
[0107] From the above experimental results, it can be seen that in non-alcoholic fatty liver caused by a high-fat diet, GalNAC-APJ ASO significantly improves liver fatty degeneration, reduces liver lipid accumulation, and reduces liver TG levels, which can effectively alleviate the progression of this type of fatty liver disease.
[0108] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.
[0109] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.
Claims
1. The use of antisense oligonucleotide small molecules in the preparation of drugs for improving fatty liver, characterized in that: The antisense oligonucleotide small molecule is GalNAC-APJASO; The GalNAC-APJASO is obtained by modifying the 3′ end of APJASO by GalNAC coupling; The base sequence of the APJASO is shown in SEQ ID NO.
1.
2. Use of the antisense oligonucleotide small molecule according to claim 1 in the preparation of a drug for improving symptoms caused by non-alcoholic fatty liver disease.
3. The use according to any one of claims 1 to 2, characterized in that: The drug contains the GalNAC-APJASO as an active ingredient and is supplemented with a pharmaceutically acceptable excipient or carrier.
4. The use according to claim 3, characterized in that: The medicine includes any one of tablets, capsules, granules, pills, emulsions, liquids, patches, solutions, ointments and injections.
5. The use according to claim 3, characterized in that: The excipients include any one or more of starch, sucrose, trehalose, mannitol and sorbitol.
6. The use according to claim 3, characterized in that: The carrier includes any one or more of nanoparticles, liposomes, transfersomes, microspheres and hydrogels.
7. The use according to claim 3, characterized in that: The drug is a solution; The carrier aided by the solution includes a solvent; The solvent is any one or more of DMSO, water, ethanol, glycerol and physiological saline.
8. The use according to claim 7, characterized in that: The solution is prepared by compounding the GalNAC-APJASO and the solvent; The concentration of the GalNAC-APJASO in the solution is 0.1 mg / mL to 1 mg / mL.