CN117551649B - 一种用于治疗对乙酰氨基酚急性肝损伤的miRNA及其应用 - Google Patents
一种用于治疗对乙酰氨基酚急性肝损伤的miRNA及其应用 Download PDFInfo
- Publication number
- CN117551649B CN117551649B CN202410044847.3A CN202410044847A CN117551649B CN 117551649 B CN117551649 B CN 117551649B CN 202410044847 A CN202410044847 A CN 202410044847A CN 117551649 B CN117551649 B CN 117551649B
- Authority
- CN
- China
- Prior art keywords
- mir
- liver injury
- apap
- mirna
- acute liver
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 title claims abstract description 65
- 206010067125 Liver injury Diseases 0.000 title claims abstract description 36
- 231100000439 acute liver injury Toxicity 0.000 title claims abstract description 29
- 108091070501 miRNA Proteins 0.000 title claims abstract description 26
- 239000002679 microRNA Substances 0.000 title claims abstract description 25
- 229960005489 paracetamol Drugs 0.000 title claims abstract description 11
- 208000029618 autoimmune pulmonary alveolar proteinosis Diseases 0.000 claims abstract description 42
- 108091047337 miR-4319 stem-loop Proteins 0.000 claims abstract description 34
- 102100023332 Dual specificity mitogen-activated protein kinase kinase 7 Human genes 0.000 claims abstract description 17
- 101000624594 Homo sapiens Dual specificity mitogen-activated protein kinase kinase 7 Proteins 0.000 claims abstract description 17
- 239000003814 drug Substances 0.000 claims abstract description 9
- 238000002360 preparation method Methods 0.000 claims abstract description 7
- 230000000692 anti-sense effect Effects 0.000 claims description 5
- 239000003112 inhibitor Substances 0.000 claims description 5
- 231100000753 hepatic injury Toxicity 0.000 abstract description 10
- 230000001225 therapeutic effect Effects 0.000 abstract description 4
- 230000008506 pathogenesis Effects 0.000 abstract description 3
- 201000010099 disease Diseases 0.000 abstract description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 2
- 238000010172 mouse model Methods 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 241000699670 Mus sp. Species 0.000 description 30
- 230000000694 effects Effects 0.000 description 11
- 230000004913 activation Effects 0.000 description 9
- 210000005228 liver tissue Anatomy 0.000 description 9
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 230000004048 modification Effects 0.000 description 8
- 238000012986 modification Methods 0.000 description 8
- 210000004185 liver Anatomy 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 7
- 210000003954 umbilical cord Anatomy 0.000 description 7
- 210000001808 exosome Anatomy 0.000 description 6
- 108700011259 MicroRNAs Proteins 0.000 description 5
- 210000003470 mitochondria Anatomy 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 4
- URNSECGXFRDEDC-UHFFFAOYSA-N N-acetyl-1,4-benzoquinone imine Chemical compound CC(=O)N=C1C=CC(=O)C=C1 URNSECGXFRDEDC-UHFFFAOYSA-N 0.000 description 4
- 229960004308 acetylcysteine Drugs 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 4
- 239000007928 intraperitoneal injection Substances 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 239000003642 reactive oxygen metabolite Substances 0.000 description 4
- 230000019491 signal transduction Effects 0.000 description 4
- 108091006146 Channels Proteins 0.000 description 3
- 208000008964 Chemical and Drug Induced Liver Injury Diseases 0.000 description 3
- 108010051975 Glycogen Synthase Kinase 3 beta Proteins 0.000 description 3
- 102100038104 Glycogen synthase kinase-3 beta Human genes 0.000 description 3
- 230000002159 abnormal effect Effects 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 3
- 210000003494 hepatocyte Anatomy 0.000 description 3
- 210000005229 liver cell Anatomy 0.000 description 3
- 230000002438 mitochondrial effect Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 2
- 108010082126 Alanine transaminase Proteins 0.000 description 2
- 102100023274 Dual specificity mitogen-activated protein kinase kinase 4 Human genes 0.000 description 2
- 108010024636 Glutathione Proteins 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 101001115395 Homo sapiens Dual specificity mitogen-activated protein kinase kinase 4 Proteins 0.000 description 2
- 101000963987 Homo sapiens SH3 domain-binding protein 5 Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108010067028 Mitochondrial Permeability Transition Pore Proteins 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 206010028851 Necrosis Diseases 0.000 description 2
- 208000005374 Poisoning Diseases 0.000 description 2
- 102100040119 SH3 domain-binding protein 5 Human genes 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 230000003908 liver function Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 108091059786 miR-128-1 stem-loop Proteins 0.000 description 2
- 108091084874 miR-128-2 stem-loop Proteins 0.000 description 2
- 108091078862 miR-128-3 stem-loop Proteins 0.000 description 2
- 108091073005 miR-128-4 stem-loop Proteins 0.000 description 2
- 210000001700 mitochondrial membrane Anatomy 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 231100000572 poisoning Toxicity 0.000 description 2
- 230000000607 poisoning effect Effects 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- PLRACCBDVIHHLZ-UHFFFAOYSA-N 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine Chemical compound C1N(C)CCC(C=2C=CC=CC=2)=C1 PLRACCBDVIHHLZ-UHFFFAOYSA-N 0.000 description 1
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 1
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 1
- 238000009020 BCA Protein Assay Kit Methods 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 101710167800 Capsid assembly scaffolding protein Proteins 0.000 description 1
- 206010072268 Drug-induced liver injury Diseases 0.000 description 1
- 102100021008 Endonuclease G, mitochondrial Human genes 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 206010019670 Hepatic function abnormal Diseases 0.000 description 1
- 206010019851 Hepatotoxicity Diseases 0.000 description 1
- 101001059454 Homo sapiens Serine/threonine-protein kinase MARK2 Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- 108010058682 Mitochondrial Proteins Proteins 0.000 description 1
- 102000006404 Mitochondrial Proteins Human genes 0.000 description 1
- 108090000744 Mitogen-Activated Protein Kinase Kinases Proteins 0.000 description 1
- 102000004232 Mitogen-Activated Protein Kinase Kinases Human genes 0.000 description 1
- 101001135571 Mus musculus Tyrosine-protein phosphatase non-receptor type 2 Proteins 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 108091093105 Nuclear DNA Proteins 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 101710130420 Probable capsid assembly scaffolding protein Proteins 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 101710204410 Scaffold protein Proteins 0.000 description 1
- 102100028904 Serine/threonine-protein kinase MARK2 Human genes 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 102000002933 Thioredoxin Human genes 0.000 description 1
- 102100034795 Thioredoxin, mitochondrial Human genes 0.000 description 1
- 101710188853 Thioredoxin, mitochondrial Proteins 0.000 description 1
- 108090000340 Transaminases Proteins 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000001754 anti-pyretic effect Effects 0.000 description 1
- 239000002221 antipyretic Substances 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 210000000013 bile duct Anatomy 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 210000000172 cytosol Anatomy 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000002222 downregulating effect Effects 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 108010047964 endonuclease G Proteins 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 231100000304 hepatotoxicity Toxicity 0.000 description 1
- 230000007686 hepatotoxicity Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 108091070038 miR-338 stem-loop Proteins 0.000 description 1
- 108091074070 miR-338-3 stem-loop Proteins 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 239000000041 non-steroidal anti-inflammatory agent Substances 0.000 description 1
- 229940021182 non-steroidal anti-inflammatory drug Drugs 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000027756 respiratory electron transport chain Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 108060008226 thioredoxin Proteins 0.000 description 1
- 229940094937 thioredoxin Drugs 0.000 description 1
- 102000014898 transaminase activity proteins Human genes 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000009750 upstream signaling Effects 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
- C12N2310/141—MicroRNAs, miRNAs
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/12—Dual-specificity kinases (2.7.12)
- C12Y207/12002—Mitogen-activated protein kinase kinase (2.7.12.2), i.e. MAPKK or MEK1 or MEK2
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plant Pathology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Virology (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明适用于生物医药技术领域，提供了一种用于治疗对乙酰氨基酚急性肝损伤的miRNA及其应用，所述miRNA为miR‑4319，其序列为：UCCCUGAGCAAAGCCAC，并将miR‑4319序列的修饰产物miR‑4319 agomir应用于制备治疗APAP致急性肝损伤的药物中。通过构建APAP致肝损伤小鼠模型，首次公开了miR‑4319的修饰产物miR‑4319 agomir可以抑制MKK7的表达，进而通过该方式对APAP致肝损伤模型起到治疗作用，有助于加深人们对MMK7/JNK信号通路参与APAP致肝损伤发病机制的认识；并为该病的治疗提供了新的治疗靶点。
Description
技术领域
本发明属于生物医药技术领域，尤其涉及一种用于治疗对乙酰氨基酚急性肝损伤的miRNA及其应用。
背景技术
对乙酰氨基酚（Acetaminophen, APAP）是一种广泛使用的具有解热和镇痛作用的非甾体抗炎药,但使用不当会导致药物性肝损伤。在欧美，APAP是药物致急性肝损伤（Drug-induced actue liver injury, DILI）的主要原因。N-乙酰半胱氨酸（N-acetyl cysteine,NAC）是唯一被临床认可的特异性治疗APAP急性肝损伤的药物，但该药物仅在APAP中毒早期有效，在APAP中毒8小时后疗效显著降低，且NAC治疗窗窄，毒副作用显著，限制了其在临床的应用。针对这一治疗困境，临床亟待有效保肝药物的开发。
细胞内JNK信号通路持续激活被认为是APAP致肝损伤的分子机制之一。N-乙酰-对苯醌亚胺（N-Acetyl-p-benzoquinone imine，NAPQI）是APAP的毒性代谢产物，大量NAPQI耗竭细胞内还原型谷胱甘肽（Reduced Glutathione， GSH），导致线粒体超氧化物生成增多，进而氧化线粒体内的硫氧还蛋白，使得硫氧还蛋白脱离ASK1（Apoptosis Signal-Regulating Kinase 1，即氧化还原敏感性丝氨酸/苏氨酸激酶），触发ASK1的自我激活。除了氧化硫氧还蛋白，线粒体氧化应激还能激活GSK-3β（糖原合成酶激酶-3β），活化的GSK-3β进一步激活MLK3（混合谱系酶3）。活化的ASK1和MLK3激活蛋白激酶MKK4/7（丝裂原活化蛋白激酶激酶的一种），MKK4/7又通过磷酸化激活JNK（应激活化蛋白激酶），活化的JNK由细胞质转位到线粒体，与线粒体膜上Sab蛋白（也被称为SH3BP5蛋白，是一种具有调控线粒体生理功能的线粒体外膜支架蛋白）结合，引起线粒体内Src（酪氨酸激酶的一种）失活，最终导致电子传递链功能障碍，ROS（Reactive Oxygen Species，活性氧）释放增加。ROS继续激活JNK上游分子ASK1和MLK3，导致JNK持续激活，线粒体持续生成ROS，形成了一个自我维持的激活回路。磷酸化JNK向线粒体转移的同时，引起胞浆内Bax激活并转位到线粒体上，触发了线粒体通透性转变孔（mitochondrial permeability transition pore，MPTP）的开启，导致线粒体膜电位缺失和ATP耗竭。线粒体膜通透性的改变，最终导致大量线粒体蛋白释放到胞浆中，包括凋亡诱导因子（apoptosis-inducing factor，AIF）和核酸内切酶G，它们移位至细胞核，导致细胞核DNA断裂，最终引起细胞死亡。因此，抑制JNK信号通路活化能有效预防或缓解APAP导致的肝毒性。
脐带间充质干细胞（Mesenchymal Stem Cells，MSCs）是指存在于新生儿脐带组织中的一种多功能干细胞，它能分化成许多种组织细胞，具有广阔的临床应用前景。有研究人员将脐带间充质干细胞用于APAP致急性肝损伤动物模型的治疗，获得了良好的治疗效果。外泌体是由细胞分泌的直径在40-100nm的盘状囊泡，内含复杂 RNA 和蛋白质。研究发现，脐带间充质干细胞能够分泌外泌体，其内富含的miRNA分子被认为是脐带间充质干细胞发挥治疗作用核心效应分子。miRNAs（microRNAs）是一组细胞内广泛表达的小分子RNA（其长度约为22个核苷酸），他们不编码任何蛋白质，但可在转录后通过干扰mRNA的翻译和/或稳定性来抑制靶基因蛋白质的表达。像miR-338-3p通过靶向CAMK IIα的mRNA，抑制下游炎症信号通路异常活化，减轻APAP诱导的肝细胞损伤。如前所述，JNK信号通路异常活化是APAP致肝损伤的发病机制之一，MKK7作为活化JNK的上游信号分子，下调MKK7的蛋白表达水平能够抑制JNK信号通路的活化，进而减弱APAP对肝细胞的损伤。
通过研究首次发现miR-4319能够靶向MKK7的mRNA分子，下调MKK7的蛋白表达水平，进而干扰JNK信号通路活化，发挥对APAP致肝损伤的保护效应。而现有技术中并无相关记载。
发明内容
本发明实施例的目的在于提供一种用于治疗对乙酰氨基酚急性肝损伤的miRNA及其应用，旨在解决上述背景技术中提出的问题。
本发明实施例是这样实现的，在前期研究中，发现脐带间充质干细胞分泌的外泌体对APAP诱导的肝损伤具有保护效应。为寻找治疗APAP致急性肝损伤的有效治疗药物，从脐带间充质干细胞外泌体miRNA分子集合中，筛选到MKK7的抑制剂miR-4319分子，该miRNA序列的修饰产物miR-4319 agomir可有效治疗APAP导致的急性肝损伤。具体的：
所述miR-4319的序列为UCCCUGAGCAAAGCCAC。
进一步的技术方案，miR-4319 agomir的序列为：
Sense: 5’-UCCCUGAGCAAAGCCAC- 3’；
Antisense:5’-GsUsGGCUUUGCUCAGsGsGsAs-Chol- 3’。
本发明实施例的另一目的在于，一种用于治疗对乙酰氨基酚急性肝损伤的miRNA的应用，基于上述的用于治疗对乙酰氨基酚急性肝损伤的miRNA，将miRNA序列的修饰产物miR-4319 agomir用于制备MKK7抑制剂。
进一步的技术方案，将所述MKK7抑制剂应用于制备治疗APAP致急性肝损伤的药物中。
本发明实施例提供的一种用于治疗对乙酰氨基酚急性肝损伤的miRNA及其应用，通过构建APAP致肝损伤小鼠模型，首次公开了miR-4319的修饰产物miR-4319 agomir可以抑制MKK7的表达，进而通过该方式对APAP致肝损伤模型起到治疗作用，有助于加深人们对MMK7/JNK信号通路参与APAP致肝损伤发病机制的认识，并为该病的治疗提供了新的治疗靶点。
附图说明
图1为不同来源脐带间充质干细胞外泌体miRNA分子集的韦恩图；
图2为3个在线工具预测靶向MKK7 mRNA的miRNA分子集与图1中的miRNA分子集交集形成的韦恩图；
图3为miR-4319 agomir的位点修饰示意图；
图4为不同实验组小鼠血清ALT活性比较；
图5为不同实验组小鼠血清AST活性比较；
图6为不同实验组小鼠肝组织HE染色结果；
图7为不同实验组小鼠肝组织Anti-MKK7的免疫印迹结果。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
以下结合具体实施例对本发明的具体实现进行详细描述。
实施例1、筛选靶向MKK7的miRNA：
从Pubmed的GEO数据库中下载人脐带间充质干细胞外泌体miRNA的分子集：GSE159814、GSE211008、GSE209966和GSE69909。取4个miRNA分子集的交集，获得160个miRNA分子，如图1（GSE编号代表Pubmed GEO数据库中不同的数据来源，Pubmed GEO数据库网址为https://www.ncbi.nlm.nih.gov/geo/）所示。利用TargetScan、miRDB以及RNAInter数据库对靶向MKK7的miRNA进行预测。TargetScan取种属间位点保守的分子，miRDB取TargetScore在60以上的分子，RNAInter取Score在0.3以上的分子。然后利用韦恩图将图1交集中的160个miRNA分子与这3个预测分子集之间取交集，共获得2个miRNAs，即miR-128-3p和miR-4319，如图2所示。由于miR-128-3p与MKK7的相关性被报道过，故选择miR-4319进行后续研究。miR-4319的序列为：UCCCUGAGCAAAGCCAC。
为保证肝细胞摄取miRNA分子的效率以及miRNA分子在体内外实验中的稳定性，以miR-4319序列为模板，合成miR-4319的agomir（修饰位点如图3所示）用于后续实验研究。miR-4319 agomir的序列为：
Sense: 5’-UCCCUGAGCAAAGCCAC- 3’；
Antisense:5’-GsUsGGCUUUGCUCAGsGsGsAs-Chol- 3’。
要求对miR-4319 agomir的反义链进行化学修饰，具体修饰细节为：3’端胆固醇修饰，5’端两个硫代骨架修饰，3’端四个硫代骨架修饰，反义链全碱基甲基化修饰。
实施例2 、APAP致小鼠急性肝损伤模型的构建：
1、APAP致小鼠急性肝损伤模型的构建：
按照本领域现有技术中公开的方法，通过腹腔注射APAP的方式来构建小鼠急性肝损伤模型：
选择品种为C57BL/6的6 周龄清洁级雄性鼠30只，体重15-20g，标准饲料喂养，自由进食和饮水，室温18-22℃。随机分为对照组（Control组）、APAP致急性肝损伤模型组（Model组） 和miR-4319 agomir治疗组（Treatment组），每组各10只小鼠。
使用上海阿拉丁生化科技股份有限公司的APAP诱发小鼠急性肝损伤。全部实验小鼠实验前一晚禁食，但不禁水；次日模型组和治疗组小鼠按照APAP 300 mg/kg的剂量腹腔注射APAP水溶液，对照组小鼠腹腔注射等体积生理盐水；治疗组小鼠在APAP给药后，立即尾静脉注射miR-4319 agomir，剂量为20nmol/只，剩余对照组和模型组小鼠用等体积无菌生理盐水做替代。APAP给药24 h后，腹腔麻醉小鼠，摘除眼球取血，再处死动物，摘取肝脏。使用南京建成生物工程研究所的试剂盒检测小鼠血清ALT和AST酶活性：天门冬氨酸氨基转移酶 （谷草转氨酶/AST/GOT） 测试盒（微板法）和丙氨酸氨基转移酶（谷丙转氨酶/ALT/GPT）测试盒（赖氏法）微板法。
2、肝组织HE染色：
将小鼠肝脏置于40g/L多聚甲醛固定24h，梯度乙醇脱水，石蜡包埋切片。置50℃烤箱6h。二甲苯脱蜡，梯度酒精由高到低，苏木素染色，伊红染色，梯度酒精脱水，二甲苯透明，各15min，中性树胶封片。
3、肝组织免疫印迹：
用含蛋白酶抑制剂的冷RIPA（强）（碧云天）提取小鼠肝脏总蛋白，10000g离心，10分钟后收集上清液。使用BCA蛋白质测定试剂盒测定上清液的蛋白质浓度。总蛋白（50μg）进行凝胶浓度为10%的SDS-PAGE电泳，并转移到硝酸纤维素膜上。在4℃温度下，与anti-MKK7一抗（1:500稀释）孵育过夜，然后与辣根过氧化物酶偶联的二抗（1:2000稀释）室温孵育2小时。通过增强型化学发光检测系统进行可视化。
4、统计学方法：
应用GraphPad Prism 8自带统计软件分析：采用t检验（Independent-Samples tTest）进行两样本均数的比较；采用单因素方差分析（0ne-Way ANOVA）进行多组样本的比较。
实施例3、miR-4319 agomir减轻APAP诱导的小鼠急性肝损伤：
1、miR-4319 agomir降低APAP致急性肝损伤小鼠血清中ALT和AST的酶活性：
ALT和AST是表征肝功能正常与否的生化指标，在肝功能受损时，血清ALT和AST活性升高。如图4和5所示，APAP损伤了小鼠肝功能，在小鼠腹腔注射APAP 24小时后，小鼠血清中ALT和AST活性显著增高（p<0.01）；而miR-4319 agomir的治疗能显著减轻APAP导致的急性肝损伤，与模型组小鼠相比，治疗组小鼠血清中ALT和AST活性显著降低（p <0.01）。
2、miR-4319 agomir减轻了APAP致急性肝损伤小鼠肝组织的坏死范围：
对各实验组小鼠肝组织切片进行HE染色，实验结果如图6所示，对照组肝小叶结构完整，清晰易辨认，肝细胞以中央静脉为中心呈放射状排列，小叶间静、动脉及小叶间胆管结构完整（图6A）；围绕模型组小鼠的肝小叶的中央静脉周边，可见广泛的肝细胞凝固性坏死，肝脏细胞间质及肝小叶中央静脉内存在大量红细胞淤积（图6B）；治疗组小鼠肝小叶组织间隙充血程度及肝细胞凝固性坏死范围要显著轻于模型组小鼠（图6C）。
3、miR-4319 agomir减轻了APAP致急性肝损伤小鼠肝脏组织内MKK7的蛋白质表达水平：
如图7所示，APAP不改变小鼠肝组织中MKK7蛋白质的表达水平，但急性肝损伤小鼠经过miR-4319 agomir治疗后，能有效降低小鼠肝组织中MKK7蛋白质表达水平。由此可见，miR-4319 agomir通过抑制MKK7蛋白质的表达，从而对APAP诱导的急性肝损伤发挥治疗作用。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
Claims (2)
1.一种用于治疗对乙酰氨基酚急性肝损伤的miRNA的应用，所述miRNA为miR-4319，其序列为：UCCCUGAGCAAAGCCAC，其特征在于，将miR-4319序列的修饰产物miR-4319 agomir应用于制备治疗APAP致急性肝损伤的药物中，所述miR-4319 agomir的序列为：
Sense: 5’-UCCCUGAGCAAAGCCAC- 3’；
Antisense:5’-GsUsGGCUUUGCUCAGsGsGsAs-Chol- 3’。
2.根据权利要求1所述的用于治疗对乙酰氨基酚急性肝损伤的miRNA的应用，其特征在于，将miR-4319 agomir应用于制备MKK7抑制剂中，并将制备的MKK7抑制剂应用于制备治疗APAP致急性肝损伤的药物中。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410044847.3A CN117551649B (zh) | 2024-01-12 | 2024-01-12 | 一种用于治疗对乙酰氨基酚急性肝损伤的miRNA及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410044847.3A CN117551649B (zh) | 2024-01-12 | 2024-01-12 | 一种用于治疗对乙酰氨基酚急性肝损伤的miRNA及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117551649A CN117551649A (zh) | 2024-02-13 |
| CN117551649B true CN117551649B (zh) | 2024-03-19 |
Family
ID=89823677
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410044847.3A Active CN117551649B (zh) | 2024-01-12 | 2024-01-12 | 一种用于治疗对乙酰氨基酚急性肝损伤的miRNA及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117551649B (zh) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111748543A (zh) * | 2020-07-01 | 2020-10-09 | 吉林大学 | 免疫调节蛋白突变体及其核苷酸序列、重组质粒载体、工程菌、构建方法与应用 |
| CN112816707A (zh) * | 2021-01-26 | 2021-05-18 | 吉林大学 | 一种细胞的膜受体蛋白鉴别方法以及验证方法 |
| CN113403690A (zh) * | 2021-06-21 | 2021-09-17 | 吉林大学 | 一种dna编码化合物库药物分子垂钓方法 |
-
2024
- 2024-01-12 CN CN202410044847.3A patent/CN117551649B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111748543A (zh) * | 2020-07-01 | 2020-10-09 | 吉林大学 | 免疫调节蛋白突变体及其核苷酸序列、重组质粒载体、工程菌、构建方法与应用 |
| CN112816707A (zh) * | 2021-01-26 | 2021-05-18 | 吉林大学 | 一种细胞的膜受体蛋白鉴别方法以及验证方法 |
| CN113403690A (zh) * | 2021-06-21 | 2021-09-17 | 吉林大学 | 一种dna编码化合物库药物分子垂钓方法 |
Non-Patent Citations (3)
| Title |
|---|
| A review on the role of DANCR in the carcinogenesis;Soudeh Ghafouri‑Fard等;Cancer Cell International;20221231;第22卷;1-19 * |
| lncＲNA DNAJC3-AS1 靶向miＲ-4319 调控皮肤鳞状细胞癌增 殖、迁移和侵袭;周钰等;遵义医科大学学报;20230430;第46卷(第4期);381-388 * |
| MiR-4319 Suppress the Malignancy of Triple-Negative Breast Cancer by Regulating Self-Renewal and Tumorigenesis of Stem Cells;Jiahui Chu等;Cellular Physiology and Biochemistry;20180718;第48卷;第593-604页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117551649A (zh) | 2024-02-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ma et al. | The oncogenic microRNA miR-21 promotes regulated necrosis in mice | |
| Liu et al. | Effect of NF-κB and p38 MAPK in activated monocytes/macrophages on pro-inflammatory cytokines of rats with acute pancreatitis | |
| Han et al. | Hypouricemic effects of extracts from Urtica hyperborea Jacq. ex Wedd. in hyperuricemia mice through XOD, URAT1, and OAT1 | |
| Fang et al. | Actinidia chinensis Planch root extract attenuates proliferation and metastasis of hepatocellular carcinoma by inhibiting epithelial-mesenchymal transition | |
| Nie et al. | miR‐331‐3p inhibits inflammatory response after intracerebral hemorrhage by directly targeting NLRP6 | |
| Bao et al. | Berberine regulates GPX4 to inhibit ferroptosis of islet β cells | |
| Huang et al. | [Retracted] miR‐92b‐3p Exerts Neuroprotective Effects on Ischemia/Reperfusion‐Induced Cerebral Injury via Targeting NOX4 in a Rat Model | |
| Zhang et al. | Tubeimoside I Ameliorates Doxorubicin‐Induced Cardiotoxicity by Upregulating SIRT3 | |
| Hu et al. | Baicalin inhibits airway smooth muscle cells proliferation through the RAS signaling pathway in murine asthmatic airway remodeling model | |
| Chang et al. | Total glucosides of paeony inhibited autophagy and improved acute kidney injury induced by ischemia-reperfusion via the lncRNA TUG1/miR-29a/PTEN Axis | |
| CN117551649B (zh) | 一种用于治疗对乙酰氨基酚急性肝损伤的miRNA及其应用 | |
| Wang et al. | Therapeutic Mechanism and Effect of Camptothecin on Dextran Sodium Sulfate‐Induced Ulcerative Colitis in Mice | |
| Qian et al. | Plastrum testudinis induces γ-globin gene expression through epigenetic histone modifications within the γ-globin gene promoter via activation of the p38 MAPK signaling pathway | |
| Li et al. | Schisandrol A, a bioactive constituent from Schisandrae Chinensis Fructus, alleviates drug-induced liver injury by autophagy activation via exosomes | |
| CN114480256B (zh) | 甘草次酸诱导肝细胞氧化应激和炎症反应及其应用 | |
| CN105567690A (zh) | 一种抑制ido1表达的抑制剂及其应用 | |
| El Agaty | Triiodothyronine attenuates the progression of renal injury in a rat model of chronic kidney disease | |
| Zhao et al. | Huaier extract attenuates acute kidney injury to chronic kidney disease transition by inhibiting endoplasmic reticulum stress and apoptosis via miR‐1271 upregulation | |
| Zhu et al. | Qingre lishi decoction ameliorates imiquimod-induced psoriasis-like skin lesions in SKH-1 mice by regulating the Treg-DC-Th17 axis and inhibiting MAPK-mediated DC maturation | |
| Zhao et al. | Retracted: Alpinumisoflavone Exhibits Anticancer Activities in Glioblastoma Multiforme by Suppressing Glycolysis | |
| Liu et al. | Ethanol induces necroptosis in gastric epithelial cells in vitro | |
| Zhou et al. | MicroRNA miR-155-5p knockdown attenuates Angiostrongylus cantonensis-induced eosinophilic meningitis by downregulating MMP9 and TSLP proteins | |
| Bai et al. | miR‐302a‐3p targets FMR1 to regulate pyroptosis of renal tubular epithelial cells induced by hypoxia–reoxygenation injury | |
| Wang et al. | Effects of Long Noncoding RNA HOTAIR Targeting miR‐138 on Inflammatory Response and Oxidative Stress in Rat Cardiomyocytes Induced by Hypoxia and Reoxygenation | |
| Li et al. | Tenuifolin in the prevention of Alzheimer's disease‐like phenotypes: investigation of the mechanisms from the perspectives of calpain system, ferroptosis, and apoptosis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |