CN117815243B - Application of a compound in the preparation of a drug for treating liver fibrosis and a NAMPT inhibitor - Google Patents
Application of a compound in the preparation of a drug for treating liver fibrosis and a NAMPT inhibitor Download PDFInfo
- Publication number
- CN117815243B CN117815243B CN202311872412.XA CN202311872412A CN117815243B CN 117815243 B CN117815243 B CN 117815243B CN 202311872412 A CN202311872412 A CN 202311872412A CN 117815243 B CN117815243 B CN 117815243B
- Authority
- CN
- China
- Prior art keywords
- nampt
- cells
- liver
- fibrosis
- hbv
- 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
- 208000019425 cirrhosis of liver Diseases 0.000 title claims abstract description 77
- 239000003814 drug Substances 0.000 title claims abstract description 32
- 229940079593 drug Drugs 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 108010064862 Nicotinamide phosphoribosyltransferase Proteins 0.000 title abstract description 126
- 102000015532 Nicotinamide phosphoribosyltransferase Human genes 0.000 title abstract description 124
- 239000003112 inhibitor Substances 0.000 title abstract description 25
- 150000001875 compounds Chemical class 0.000 title abstract description 6
- OHUHVTCQTUDPIJ-JYCIKRDWSA-N ceralasertib Chemical compound C[C@@H]1COCCN1C1=CC(C2(CC2)[S@](C)(=N)=O)=NC(C=2C=3C=CNC=3N=CC=2)=N1 OHUHVTCQTUDPIJ-JYCIKRDWSA-N 0.000 claims abstract description 39
- 241000700721 Hepatitis B virus Species 0.000 claims description 12
- 208000015181 infectious disease Diseases 0.000 claims description 9
- 210000004027 cell Anatomy 0.000 abstract description 142
- 210000004024 hepatic stellate cell Anatomy 0.000 abstract description 86
- 210000005229 liver cell Anatomy 0.000 abstract description 22
- 230000000694 effects Effects 0.000 abstract description 21
- 230000037361 pathway Effects 0.000 abstract description 20
- 206010019668 Hepatic fibrosis Diseases 0.000 abstract description 18
- 230000002401 inhibitory effect Effects 0.000 abstract description 16
- 238000011160 research Methods 0.000 abstract description 15
- 230000020411 cell activation Effects 0.000 abstract description 9
- 230000033616 DNA repair Effects 0.000 abstract description 8
- 230000009471 action Effects 0.000 abstract description 8
- 230000001105 regulatory effect Effects 0.000 abstract description 7
- 230000014509 gene expression Effects 0.000 description 66
- 210000003494 hepatocyte Anatomy 0.000 description 55
- 201000007270 liver cancer Diseases 0.000 description 39
- 208000014018 liver neoplasm Diseases 0.000 description 39
- 102000015087 Poly (ADP-Ribose) Polymerase-1 Human genes 0.000 description 36
- 108010064218 Poly (ADP-Ribose) Polymerase-1 Proteins 0.000 description 36
- 238000013518 transcription Methods 0.000 description 32
- 230000035897 transcription Effects 0.000 description 32
- 230000004913 activation Effects 0.000 description 25
- 108090000623 proteins and genes Proteins 0.000 description 25
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 24
- 101000852815 Homo sapiens Insulin receptor Proteins 0.000 description 22
- 102100036721 Insulin receptor Human genes 0.000 description 22
- 210000000130 stem cell Anatomy 0.000 description 21
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 20
- 210000002220 organoid Anatomy 0.000 description 16
- 206010028980 Neoplasm Diseases 0.000 description 15
- 238000004458 analytical method Methods 0.000 description 15
- 238000003501 co-culture Methods 0.000 description 15
- 230000015572 biosynthetic process Effects 0.000 description 14
- 210000004185 liver Anatomy 0.000 description 14
- 102100033601 Collagen alpha-1(I) chain Human genes 0.000 description 12
- 241000699670 Mus sp. Species 0.000 description 12
- 108010029483 alpha 1 Chain Collagen Type I Proteins 0.000 description 12
- 230000003993 interaction Effects 0.000 description 12
- 238000000034 method Methods 0.000 description 12
- 108091035539 telomere Proteins 0.000 description 12
- 210000003411 telomere Anatomy 0.000 description 12
- 102000055501 telomere Human genes 0.000 description 12
- 241000699666 Mus <mouse, genus> Species 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 11
- 238000003119 immunoblot Methods 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 11
- 238000001514 detection method Methods 0.000 description 10
- 230000002829 reductive effect Effects 0.000 description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 102100036732 Actin, aortic smooth muscle Human genes 0.000 description 8
- 101000929319 Homo sapiens Actin, aortic smooth muscle Proteins 0.000 description 8
- 230000034659 glycolysis Effects 0.000 description 8
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 8
- 230000007246 mechanism Effects 0.000 description 8
- 230000019491 signal transduction Effects 0.000 description 8
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 7
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 7
- 206010016654 Fibrosis Diseases 0.000 description 7
- 230000006907 apoptotic process Effects 0.000 description 7
- 210000002744 extracellular matrix Anatomy 0.000 description 7
- 230000004761 fibrosis Effects 0.000 description 7
- 239000003446 ligand Substances 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 230000035755 proliferation Effects 0.000 description 7
- 150000003384 small molecules Chemical class 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 102000000872 ATM Human genes 0.000 description 6
- 108010004586 Ataxia Telangiectasia Mutated Proteins Proteins 0.000 description 6
- 108091027967 Small hairpin RNA Proteins 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 238000001114 immunoprecipitation Methods 0.000 description 6
- 210000005228 liver tissue Anatomy 0.000 description 6
- 102000005962 receptors Human genes 0.000 description 6
- 108020003175 receptors Proteins 0.000 description 6
- 239000004055 small Interfering RNA Substances 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 230000033863 telomere maintenance Effects 0.000 description 6
- 101150084967 EPCAM gene Proteins 0.000 description 5
- 101000635938 Homo sapiens Transforming growth factor beta-1 proprotein Proteins 0.000 description 5
- 102100030742 Transforming growth factor beta-1 proprotein Human genes 0.000 description 5
- 230000007882 cirrhosis Effects 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- 230000002018 overexpression Effects 0.000 description 5
- 230000026731 phosphorylation Effects 0.000 description 5
- 238000006366 phosphorylation reaction Methods 0.000 description 5
- 230000001737 promoting effect Effects 0.000 description 5
- 230000010076 replication Effects 0.000 description 5
- 238000007920 subcutaneous administration Methods 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- 238000001262 western blot Methods 0.000 description 5
- 108010014064 CCCTC-Binding Factor Proteins 0.000 description 4
- 102000016897 CCCTC-Binding Factor Human genes 0.000 description 4
- 206010061218 Inflammation Diseases 0.000 description 4
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 description 4
- 108010033710 Telomeric Repeat Binding Protein 2 Proteins 0.000 description 4
- 102000007316 Telomeric Repeat Binding Protein 2 Human genes 0.000 description 4
- 230000005757 colony formation Effects 0.000 description 4
- 230000001332 colony forming effect Effects 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 238000003125 immunofluorescent labeling Methods 0.000 description 4
- 230000004054 inflammatory process Effects 0.000 description 4
- 238000011580 nude mouse model Methods 0.000 description 4
- -1 ATR Proteins 0.000 description 3
- 101150026109 INSR gene Proteins 0.000 description 3
- 102100034671 L-lactate dehydrogenase A chain Human genes 0.000 description 3
- 108010088350 Lactate Dehydrogenase 5 Proteins 0.000 description 3
- 206010027476 Metastases Diseases 0.000 description 3
- 241000699660 Mus musculus Species 0.000 description 3
- 208000037273 Pathologic Processes Diseases 0.000 description 3
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000009368 gene silencing by RNA Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 230000009401 metastasis Effects 0.000 description 3
- 210000000651 myofibroblast Anatomy 0.000 description 3
- 230000009054 pathological process Effects 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 230000000284 resting effect Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 230000004614 tumor growth Effects 0.000 description 3
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 108091033409 CRISPR Proteins 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 230000005778 DNA damage Effects 0.000 description 2
- 231100000277 DNA damage Toxicity 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241001559542 Hippocampus hippocampus Species 0.000 description 2
- 101001108364 Homo sapiens Neuronal cell adhesion molecule Proteins 0.000 description 2
- 101000777277 Homo sapiens Serine/threonine-protein kinase Chk2 Proteins 0.000 description 2
- 101000708766 Homo sapiens Structural maintenance of chromosomes protein 3 Proteins 0.000 description 2
- 206010061598 Immunodeficiency Diseases 0.000 description 2
- 208000029462 Immunodeficiency disease Diseases 0.000 description 2
- 206010064912 Malignant transformation Diseases 0.000 description 2
- 102100021852 Neuronal cell adhesion molecule Human genes 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 229920000776 Poly(Adenosine diphosphate-ribose) polymerase Polymers 0.000 description 2
- 102100031075 Serine/threonine-protein kinase Chk2 Human genes 0.000 description 2
- 102100032723 Structural maintenance of chromosomes protein 3 Human genes 0.000 description 2
- 102000004357 Transferases Human genes 0.000 description 2
- 108090000992 Transferases Proteins 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 239000000556 agonist Substances 0.000 description 2
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 description 2
- 238000003782 apoptosis assay Methods 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 238000004891 communication Methods 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 210000002889 endothelial cell Anatomy 0.000 description 2
- 238000010195 expression analysis Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000002440 hepatic effect Effects 0.000 description 2
- 208000002672 hepatitis B Diseases 0.000 description 2
- 230000007813 immunodeficiency Effects 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000007273 lactonization reaction Methods 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 230000036212 malign transformation Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 230000029279 positive regulation of transcription, DNA-dependent Effects 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 230000002062 proliferating effect Effects 0.000 description 2
- 239000003531 protein hydrolysate Substances 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 230000003362 replicative effect Effects 0.000 description 2
- 229930002330 retinoic acid Natural products 0.000 description 2
- 238000012174 single-cell RNA sequencing Methods 0.000 description 2
- 210000004500 stellate cell Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000009261 transgenic effect Effects 0.000 description 2
- 230000003827 upregulation Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 101100394734 Aspergillus oryzae (strain ATCC 42149 / RIB 40) hepG gene Proteins 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- 101100042788 Caenorhabditis elegans him-1 gene Proteins 0.000 description 1
- 208000008964 Chemical and Drug Induced Liver Injury Diseases 0.000 description 1
- 102100029952 Double-strand-break repair protein rad21 homolog Human genes 0.000 description 1
- 206010072268 Drug-induced liver injury Diseases 0.000 description 1
- 208000031448 Genomic Instability Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 208000027761 Hepatic autoimmune disease Diseases 0.000 description 1
- 206010019670 Hepatic function abnormal Diseases 0.000 description 1
- 208000005176 Hepatitis C Diseases 0.000 description 1
- 206010019728 Hepatitis alcoholic Diseases 0.000 description 1
- 206010019837 Hepatocellular injury Diseases 0.000 description 1
- 101000584942 Homo sapiens Double-strand-break repair protein rad21 homolog Proteins 0.000 description 1
- 101001132495 Homo sapiens GTP-binding protein RAD Proteins 0.000 description 1
- 101000840551 Homo sapiens Hexokinase-2 Proteins 0.000 description 1
- 101001050577 Homo sapiens Kinesin-like protein KIF2A Proteins 0.000 description 1
- 101001026214 Homo sapiens Potassium voltage-gated channel subfamily A member 5 Proteins 0.000 description 1
- 101001048456 Homo sapiens Protein Hook homolog 2 Proteins 0.000 description 1
- 101000785063 Homo sapiens Serine-protein kinase ATM Proteins 0.000 description 1
- 102000003746 Insulin Receptor Human genes 0.000 description 1
- 108010001127 Insulin Receptor Proteins 0.000 description 1
- 102100023426 Kinesin-like protein KIF2A Human genes 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 101150086808 NAMPT gene Proteins 0.000 description 1
- 102100033223 Nicotinamide phosphoribosyltransferase Human genes 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 102000012338 Poly(ADP-ribose) Polymerases Human genes 0.000 description 1
- 108010061844 Poly(ADP-ribose) Polymerases Proteins 0.000 description 1
- 108091006296 SLC2A1 Proteins 0.000 description 1
- 108091006298 SLC2A3 Proteins 0.000 description 1
- 102100023536 Solute carrier family 2, facilitated glucose transporter member 1 Human genes 0.000 description 1
- 102100022722 Solute carrier family 2, facilitated glucose transporter member 3 Human genes 0.000 description 1
- 102100029538 Structural maintenance of chromosomes protein 1A Human genes 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 208000002353 alcoholic hepatitis Diseases 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 238000011316 allogeneic transplantation Methods 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000035578 autophosphorylation Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 229940121422 ceralasertib Drugs 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000008045 co-localization Effects 0.000 description 1
- 238000010293 colony formation assay Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000009274 differential gene expression Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 230000003480 fibrinolytic effect Effects 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 238000010185 immunofluorescence analysis Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 208000018191 liver inflammation Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 229960003966 nicotinamide Drugs 0.000 description 1
- 235000005152 nicotinamide Nutrition 0.000 description 1
- 239000011570 nicotinamide Substances 0.000 description 1
- 235000020956 nicotinamide riboside Nutrition 0.000 description 1
- 239000011618 nicotinamide riboside Substances 0.000 description 1
- DFPAKSUCGFBDDF-UHFFFAOYSA-N nicotinic acid amide Natural products NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 1
- 208000008338 non-alcoholic fatty liver disease Diseases 0.000 description 1
- 206010053219 non-alcoholic steatohepatitis Diseases 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 210000004738 parenchymal cell Anatomy 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000002206 pro-fibrotic effect Effects 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 229960002633 ramucirumab Drugs 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- WWDMJSSVVPXVSV-YCNIQYBTSA-N retinyl ester Chemical compound CC1CCCC(C)(C)C1\C=C\C(\C)=C\C=C\C(\C)=C\C(O)=O WWDMJSSVVPXVSV-YCNIQYBTSA-N 0.000 description 1
- 201000004409 schistosomiasis Diseases 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 238000013517 stratification Methods 0.000 description 1
- 108010004731 structural maintenance of chromosome protein 1 Proteins 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 229960001727 tretinoin Drugs 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000005748 tumor development Effects 0.000 description 1
- 230000005740 tumor formation Effects 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 230000006490 viral transcription Effects 0.000 description 1
- 229940045997 vitamin a Drugs 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5375—1,4-Oxazines, e.g. morpholine
- A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
- G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Public Health (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- Analytical Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Epidemiology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to the technical field of medicines for treating hepatic fibrosis, in particular to application of a compound in preparation of a medicine for treating hepatic fibrosis and an NAMPT inhibitor. Through single cell transcriptome research, the DNA repair pathway is found to be obviously activated in HBV high-level liver cells, and then NAMPT-INSR pathway is found to play an important role in regulating hepatic stellate cell activation, so as to influence hepatic fibrosis progress. NAMPT can be used as drug action target spot to screen drugs for treating hepatic fibrosis, and then AZD6738 can be screened out to be used as NAMPT inhibitor, so as to realize the effects of inhibiting hepatic stellate cell activation and inhibiting hepatic fibrosis and liver cirrhosis. The technical scheme can solve the technical problem that medicines for treating liver fibrosis are lacking in the prior art, has important significance for developing effective medicines for treating liver fibrosis, and has ideal popularization and application values.
Description
Technical Field
The invention relates to the technical field of medicines for treating hepatic fibrosis, in particular to application of a compound in preparation of a medicine for treating hepatic fibrosis and an NAMPT inhibitor.
Background
Liver fibrosis is a pathological process that refers to abnormal proliferation of connective tissue in the liver caused by various pathogenic factors. Any liver injury has a certain liver fibrosis process in the liver repairing and healing process, and if injury factors cannot be removed for a long time, the fibrosis process can be continuously developed into liver cirrhosis for a long time, and the liver fibrosis is an important pathological process for developing the liver cirrhosis. Liver fibrosis and cirrhosis are mainly long-term damage to liver cells, which causes diffuse fibrous proliferation of intrahepatic tissues, resulting in stiffening of liver texture and impaired liver function. Various chronic liver diseases (such as hepatitis B and C, alcoholic hepatitis, nonalcoholic steatohepatitis, schistosomiasis, autoimmune liver diseases, and drug induced liver injury) can lead to the formation of liver fibrosis. Related studies have been vigorously conducted for many years, and complex mechanisms of formation of liver fibrosis have been revealed from various aspects, with Hepatic Stellate Cells (HSCs) still being a hotspot and focus of current liver fibrosis studies. The cells are located in the Disse gap between the liver sinusoidal endothelial cells and the hepatocytes, dispersed throughout the liver, accounting for about 10% of all hepatic parenchymal cells. In the physiological state, HSCs exhibit a non-proliferative resting phenotype, enriched with retinyl ester (vitamin a or retinoic acid) lipid droplets in the cytoplasm surrounding the nucleus. Under the influence of external stimuli, HSCs change from a resting state to an activated state, lipid droplets in the cytoplasm gradually disappear, collagen and other extracellular matrix (ECM) are formed in large quantities, this change being critical for hepatic fibrosis formation, HSCs being the primary cellular source of hepatic myofibroblasts. Blocking or inactivating activated HSCs, promoting enhancement of fibrinolytic activity, is a potential anti-hepatic fibrosis target. Research on new targets and new paths related to HSCs activation is of great significance to the development of effective anti-hepatic fibrosis drugs.
Hepatitis b virus (HEPATITIS B VIRUS, HBV) infection is one of the causes of liver fibrosis and liver cancer. However, liver cancer and liver fibrosis are two relatively independent pathological processes. Liver cancer is mainly caused by malignant transformation of liver cells and formation of solid tumors. Liver fibrosis is mainly caused by abnormal activation of hepatic stellate cells, and most of liver fibrosis and liver cirrhosis are benign lesions, and progress is slow, so that the early treatment prognosis effect is generally good. However, liver cancer is a malignant tumor lesion, the liver is necrotized, the functions of the liver are mostly unable to be recovered, the development is rapid, the disease condition is difficult to control, and the prognosis is relatively poor. Only a part of liver cirrhosis patients may develop liver cancer, and liver tissues of some liver cancer patients may show a certain liver fibrosis symptom. The therapeutic targets for liver cancer and liver fibrosis are quite different, the former needs to inhibit proliferation and metastasis of liver cancer cells, and the latter needs to inhibit activation of hepatic stellate cells. At present, researchers have developed a large number of medicaments for treating liver cancer, such as PD-1, latiffany, ramucirumab and the like, but the medicaments for treating liver cancer have no obvious treatment effect on liver fibrosis, and some medicaments even trigger liver inflammation to promote the generation of liver fibrosis.
Disclosure of Invention
The invention aims to provide application of AZD6738 in preparation of medicines for treating hepatic fibrosis, so as to solve the technical problem that medicines for treating hepatic fibrosis are lacking in the prior art.
In order to achieve the above purpose, the invention adopts the following technical scheme:
use of AZD6738 in the manufacture of a medicament for the treatment of liver fibrosis.
Further, the structural formula of AZD6738 is shown as formula (1):
Further, the liver fibrosis is liver fibrosis due to infection with hepatitis b virus.
Further, AZD6738 is used for inhibiting the expression amount of NAMPT protein in normal liver cells or liver cancer cells.
The principle adopting the technical scheme has the beneficial effects that:
AZD6738 (Ceralasertib) can inhibit NAMPT protein expression in normal liver cells or liver cancer cells, and AZD6738 can further inhibit hepatic stellate cell activation, thereby inhibiting liver fibrosis and liver cirrhosis progression, as NAMPT-INSR signaling pathway plays an important role in HSCs cell activation. In normal hepatocytes or hepatoma cells infected with hepatitis b virus, the expression of NAMPT protein is up-regulated significantly, so AZD6738 can be used for hepatic stellate cell activation and hepatic fibrosis caused by infection with hepatitis b virus.
FIG. 2C of example 2 shows that infection of HBV in normal hepatocytes L02 or HepG2 liver cancer cells can result in up-regulation of NAMPT protein expression. FIGS. 3F and 3G of example 3 show that both HBV-infected normal and hepatoma cells co-cultured with hepatic stellate cells overexpressing NAMPT, both lead to upregulation of activation markers for hepatic stellate cell HSCs, which means liver fibrosis progression, NAMPT is positively correlated with liver fibrosis. After AZD6738 was applied to normal hepatocytes and hepatoma cells over-expressing NAMPT, the expression level of NAMPT was significantly reduced (fig. 2D-F of example 2), thereby resisting hepatic fibrosis progression. In addition, in vivo experiments also demonstrated the inhibitory effect of administration of AZD6738 on liver fibrosis (fig. 8 of example 6).
The scheme also provides application of NAMPT protein in screening medicines for treating liver fibrosis.
Further, the drug is used for inhibiting the expression level of NAMPT protein.
Further, the liver fibrosis is caused by infection of liver cells with hepatitis b virus and activation of hepatic stellate cells.
The principle adopting the technical scheme has the beneficial effects that:
The inventor finds that DNA repair pathway and glycolysis are obviously activated in HBV high-level liver cells through single cell transcriptome study, and are the characteristics of the liver cells with high-level HBV transcription. HBV high-level hepatocytes were found to be enriched for 50 genes associated with ligand-receptor interactions produced by Hepatic Stellate Cells (HSCs), including NAMPT (nicotinamide phosphoribosyl transferase, ligand expressed in hepatocytes) and INSR (receptor on hepatic stellate cells HSCs). NAMPT-INSR signaling pathway plays a significant role in HSCs activation caused by HBV transcription, relative to signaling pathway between hepatocytes and hepatic stellate cells formed by other receptor-ligands, and is a key pathway affecting HBV-induced liver fibrosis. By utilizing the findings, the pathological mechanism of liver fibrosis and liver cirrhosis caused by HBV can be deeply known, and NAMPT-INSR signal paths, especially NAMPT, are used as relevant disease treatment targets to screen drugs for treating liver fibrosis and liver cirrhosis caused by HBV. If the drug is capable of inhibiting NAMPT transcription level or protein level, it has potential as a drug for liver fibrosis and cirrhosis. As an example, the scheme screens out a small molecule medicine AZD6738 capable of treating liver fibrosis by taking NAMPT as a medicine action target.
The scheme also provides application of AZD6738 in preparation of NAMPT inhibitor.
Further, the NAMPT inhibitor is used for reducing the expression amount of NAMPT protein in normal liver cells or liver cancer cells.
Further, normal hepatocytes or hepatoma cells are infected with hepatitis b virus.
The principle adopting the technical scheme has the beneficial effects that:
Normal liver cells or liver cancer cells infected by HBV virus, NAMPT expression level is improved (thereby leading to HSCs to activate and promote liver fibrosis progress), but NAMPT expression level is obviously reduced under the action of AZD6738, thereby avoiding HSCs to activate and liver fibrosis progress caused by HBV. These findings indicate that AZD6738 may be used as an inhibitor of NAMPT, for use in the treatment of liver fibrosis, or for basic research (non-therapeutic) use. In basic research, the action mechanism, upstream and downstream signal molecules and the like of NAMPT can be further researched by regulating and controlling the expression quantity of NAMPT. The most intuitive means to regulate the expression level of NAMPT is to directly control the expression of the gene, for example, by achieving overexpression through transgenesis and reducing the expression of the target gene through RNA interference. However, the above-described procedure is very complicated, requires designing related vectors and performing transgenic operations, and increases the cost of experimental study. If small molecule substances can be used, NAMPT expression can be affected by direct administration and culture and incubation, and the research flow is greatly shortened. For each gene or protein, a proper small molecular inhibitor does not necessarily exist, but in the research, the inventor discovers that AZD6738 can effectively inhibit the expression quantity of NAMPT, is a novel small molecular compound which can be used as a potential NAMPT inhibitor, obtains unexpected technical effects and creates conditions for basic research related to NAMPT. In fact, commercial small molecule inhibitors and agonists are very widely used as basic research tools, and the discovery of new NAMPT inhibitors has other non-medical uses in addition to medical uses, and can be used as basic tool reagents in the course of research. In this embodiment, the inventors have studied the effect of NAMPT expression on poly ADP-ribose polymerase 1 (PARP 1) by controlling NAMPT protein expression with or without AZD6738 (e.g., FIG. 8 of example 6). Therefore, the AZD6738 not only has medical application, but also can be used as a basic scientific research reagent for application.
Drawings
FIG. 1 shows the results of single cell analysis and HBV transcription detection experiments in HCCs in example 1.
FIG. 2 is a study result of the interaction between hepatocytes and HSCs promoted by HBV transcription activation NAMPT-INSR pathway of example 2.
FIG. 3 is the results of the study of the inhibitor screening, NAMPT overexpression, interaction between hepatocytes and HSCs of example 3.
FIG. 4 is a result of examining the effect of NAMPT of example 4 on liver cancer stem cells.
Fig. 5 is a Masson-stained image of example 4.
FIG. 6 is a study of NAMPT ligand-INSR receptor signaling pathway mediated crosstalk between hepatocytes and HSCs of example 5.
FIG. 7 is a study of the over-activation of PARP1 lactogenesis and telomere maintenance initiated by NAMPT-INSR signaling pathway of example 5.
Fig. 8 is an immunoblot image of liver tissue, H & E and Masson stained pictures, ATR phosphorylation, NAMPT expression and PARP1 lactonization of example 6 (AZD 6738 treatment, AG14361 treatment).
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless otherwise indicated, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents and the like used are all commercially available. The technical means used in the following examples are conventional means well known to those skilled in the art unless otherwise indicated.
In order to elucidate the mechanism of action of drugs, the cell model used in the present solution includes:
HBV replicating cell models are L02-pHBV4.1 (normal liver cells infected with HBV), hepG2.2.15 (liver cancer cells infected with HBV), MIG-MYC-pHBV4.1 (liver cancer stem cells infected with HBV), MIG-MYC (liver cancer stem cells over-express MYC formation), MIG-MYC-NAMPT (liver cancer stem cells over-express NAMPT), L02-NAMPT (normal liver cells over-express NAMPT), hepG2-NAMPT (liver cancer cells over-express NAMPT), MIG-MYC-pHBV4.1-shNampt (liver cancer stem cells infected with HBV and shRNA inhibiting NAMPT is expressed). The cell lines and models described above can be obtained commercially or prepared by conventional means of the prior art. The cell model is prepared for evaluating and testing the functions of the drug or reagent to be tested, and can be obtained by conventional means of transfecting related plasmids, the technical means used are conventional means well known to those skilled in the art, and the experimental methods used are all conventional methods and can be carried out according to the described recombination technology (see molecular cloning, laboratory Manual, 2 nd edition, cold spring harbor laboratory Press, cold spring harbor, new York). See also the relevant literature and patents previously published by the inventors:
Tang H,McLachlan A.A pregenomic RNA sequence adjacent to DR1 and complementary to epsilon influences hepatitis B virus replication efficiency.Virology.2002Nov10;303(1):199-210.doi:10.1006/viro.2002.1645.PMID:12482672.
Zhou LY,Chen EQ,Wang ML,Chen LL,Liu CP,Zeng F,Tang H.Biological characteristics comparison of HBV rtA181T mutants with truncated or substituted HBsAg expression in vitro and in vivo model systems.Sci Rep.2016Dec 15;6:39260.doi:10.1038/srep39260.PMID:27976732;PMCID:PMC5157016.
Application of CN114917348B nerve adhesion factor in preparing reagent for inhibiting and marking liver cancer cell metastasis.
The abbreviations and meanings involved in the technical scheme are as follows:
HCCs, hepatocellular carcinoma, HSCs, HBV, hepatitis B virus, NAMPT, nicotinamide ribosyl transferase, INSR, insulin receptor, ATM, ataxia telangiectasia mutated gene, ATR, ATM-related protein, aHSCs, activated HSCs, HSCs highly expressing COL1A1, sHSCs, stable HSCs with low transcription level of COL1A1, myHSCs, myofibroblast, LCSCs, liver cancer stem cell, PARP1, poly ADP ribosyl polymerase 1, TMMs, telomere maintenance mechanism.
EXAMPLE 1 Single cell transcriptomics study
Single cell transcriptome sequencing (scRNA-seq) was performed on tumor tissue of 10 hepatocellular carcinoma (HCCs) patients to study the biological characteristics and interactions between hepatocytes and Hepatic Stellate Cells (HSCs) during HCCs progression. Differential gene expression analysis was performed using transcriptome data from 71,083 single cells to determine cluster-specific markers. Pseudo-time analysis revealed the effect of HBV transcription on hepatocyte development trajectories. 9 different cell states (stained according to states 1-9) were identified in the potential hepatocyte trajectories, and the red arrows show the potential evolution direction. The experimental results are shown in FIG. 1 (single cell analysis and HBV transcription detection in HCCs), where FIG. 1A shows ten single cell RNA sequencing datasets generated from HCCs tumor tissue providing transcriptome information for 71083 individual cells and violin shows normalized expression of cell type specific marker genes. FIG. 1B is a result of assigning cell types to clusters. FIG. 1C shows a hepatocyte trajectory analysis (arrows indicate potential evolution) of the potential trajectories of all hepatocytes, with nine different cell states (stained according to states 1-9) found along the potential hepatocyte trajectories, while showing HBV transcription in hepatocytes, with hepatocytes with high HBV transcription levels being predominantly located in state 1 of the trajectories. FIG. 1D is a graph showing the classification of hepatocytes into HBV low and HBV high clusters according to HBV transcription levels. FIG. 1E is a heat map showing the change in expression of 20 highly mutated pathways associated with low or high HBV transcription, wherein DNA repair and glycolytic pathways are activated in HBV highly expressed hepatocytes.
Analytical experimental data found that hepatocytes with high levels of HBV transcription (HBV high level hepatocytes) were mainly located in the 1 st state of the trace, whereas hepatocytes with low levels of HBV transcription (HBV low level hepatocytes) were present in the 2 nd to 9 th states (fig. 1C). These results indicate that HBV transcriptional activity plays a role in the developmental pathway of hepatocytes in HCCs progression. Differential expression analysis of samples based on HBV transcription (high or low) stratification found differential expression of 20 highly variant pathways (fig. 1d, e). Notably, genes associated with DNA repair pathways (p=0.014) and glycolysis (p= < 0.001) were significantly activated in HBV high-level hepatocytes. These results indicate that activation of the DNA repair pathway and glycolysis may be characteristic of hepatocytes with high levels of HBV transcription. Experimental results show that HBV transcription affects the development track of hepatocytes.
Example 2 study of the Effect of HBV transcriptional activation on the interaction of hepatocytes with hepatic stellate cells
Further analysis of the test results of example 1 found that in HBV high-level hepatocytes, 50 genes associated with ligand-receptor interaction produced by Hepatic Stellate Cells (HSCs), including NAMPT (nicotinamide riboside transferase, ligand expressed in hepatocytes) and INSR (receptor on HSCs), were enriched. The experimental results are specifically shown in FIG. 2 (HBV transcriptional activation NAMPT-INSR pathway promotes the interaction between hepatocytes and HSC results). Among other things, figure 2A is a possible ligand-receptor interaction between high HBV transcribed hepatocytes and HSCs. FIG. 2B shows NAMPT expression in HBV high or low expressing hepatocytes, HSC marker COL1A1, receptor INSR expression in aHSCs and sHSCs. HSCs that highly express COL1A1 are defined as activated HSCs (ahcs), HSCs with low COL1A1 transcript levels are defined as stable HSCs (sHSCs), and as seen from statistics, INSR is highly expressed in aHSC clusters.
And further performing immunofluorescence analysis on the co-localization of NAMPT and INSR in HCCs cells and adjacent normal cells. FIG. 1C shows immunofluorescent staining to identify NAMPT (green) and INSR (red) expression in tumor and adjacent normal tissues. Fig. 1D shows the results of western blot analysis of NAMPT on HBV replication cell models (including L02-phbv4.1, hepg2.215, and MIG-MYC-phbv4.1) (P <0.01:, P <0.001;, P < 0.0001:). The HBV replication cell model is an HBV infection model constructed for human normal liver cells, liver cancer cells and liver cancer stem cells.
From the above experimental results, NAMPT (ligand expressed in hepatocytes) and INSR (receptor on HSC) were shown to be abundantly expressed in HBV high-level hepatocytes and Hepatic Stellate Cells (HSC) (FIG. 2A). NAMPT is an important factor in the DNA repair pathway, and is highly expressed in HBV high-level hepatocytes. Based on the expression of COL1A1, HSCs were classified as activated HSCs (ahcs) and stable HSCs (scs), INSR receptors were expressed only in aHSC (fig. 2B). These findings indicate that high levels of HBV transcription in hepatocytes may activate DNA damage and repair pathways and promote interactions with HSCs via NAMPT. Although hepatocytes with high HBV transcription levels are more in the types of ligands involved and the types of receptors on HSC cells that respond to the ligand signals are more in the types, through the transcriptomics study and data analysis, NAMPT-INSR signaling pathway can be found to play a significant role in HSC cell activation caused by HBV transcription relative to signaling pathway between hepatocytes and hepatic stellate cells formed by other receptor-ligands, and is a key pathway affecting HBV-induced hepatic fibrosis. Among these, HSC cell activation is involved in the progression of liver fibrosis and cirrhosis by proliferation and secretion of extracellular matrix. By utilizing the findings, the pathological mechanism of liver fibrosis and liver cirrhosis caused by HBV can be deeply known, and NAMPT-INSR signal paths, especially NAMPT, are used as relevant disease treatment targets to screen drugs for treating liver fibrosis and liver cirrhosis caused by HBV. If the drug is capable of inhibiting NAMPT transcription level or protein level, it has potential as a drug for liver fibrosis and cirrhosis.
Further experiments studied the expression of NAMPT and INSR, NAMPT was activated in all HBV replicating cell models (including L02-pHBV4.1, hepG2.2.15 and MIG-MYC-pHBV4.1 organoids), further demonstrating the positive correlation between HBV transcriptionally activated NAMPT and HSC activation promoted by INSR receptor (FIG. 2C, where L02 cells are normal human hepatocytes and HepG2 are human hepatoma cells). After the normal liver cell L02 is transfected with HBV to form HBV replication cell model, NAMPT protein expression level is raised, which shows that NAMPT protein expression and HBV transcription level are positively correlated. The above-described positive correlation pattern is also present in hepatoma cell HepG2. There is no necessary link between HBV infection and hepatocellular carcinoma (whether hepatocytes are transformed into cancer cells) and normal hepatocytes, after HBV infection, will show some symptoms of hepatocellular damage, but will not necessarily transform hepatocytes into hepatocellular carcinoma cells. The expression level of NAMPT protein is also mainly related to whether cells are infected with HBV or not, and has no necessary relation to whether cells are cancerous or not.
The above results indicate that HBV transcription can activate NAMPT-INSR pathway to promote the interaction of liver cell and hepatic stellate cell. Hepatic stellate cells are a resident population of fibroblasts located in the gap between the liver sinusoidal endothelial cells and the liver cells, and are the primary effector cells in the liver fibrosis process. Once the liver is damaged, hepatic stellate cells in the resting stage are activated and transformed into proliferative, pro-fibrotic, contractile myofibroblasts, which secrete a large amount of extracellular matrix components, particularly collagen, thereby promoting the progression of liver fibrosis. NAMPT-INSR pathway plays a key role in liver fibrosis or cirrhosis caused by HBV transcription, which is the first finding by the inventor that NAMPT can be used as an action target of a potential drug for treating liver fibrosis caused by HBV transcription to perform a series of drug screening.
Example 3:
(1) Investigation of hepatocytes overexpressing NAMPT
Since example 2 finds NAMPT as a potential therapeutic target, this example further investigated NAMPT overexpressing hepatocytes in order to investigate the significance of NAMPT-INSR pathway in the interaction between hepatocytes and HSCs. More specifically, this example establishes cell lines L02-NAMPT and HepG2-NAMPT (NAMPT NCBI ID 10135) that overexpress NAMPT. In normal human liver cell L02 and human liver cancer cell HepG2, NAMPT gene is over-expressed in L02 cell and HepG2 by conventional transgenic means, and the above process can be entrusted to relevant mechanism to obtain by conventional means in the prior art, and is not described in detail here. L02 cells transformed with the empty vector were designated as L02-Vec cells, and L02 cells transformed with the vector integrated with NAMPT were designated as L02-NAMPT cells. HepG2 cells transformed by the empty vector were designated HepG2-Vec cells, and HepG2 cells transformed by the NAMPT-integrated vector were designated HepG2-NAMPT cells. In L02-Vec cells, L02-NAMPT cells, hepG2-Vec cells, hepG2-NAMPT cells, NAMPT transcription (mRNA) is shown in FIG. 3A, NAMPT and protein expression is shown in FIG. 3B.
It was observed in the experimental results that overexpression of NAMPT did not directly affect colony forming ability of L02 and HepG2 cells (L0265.67 ±6.01vs. L02-NAMPT 66.67±4.10, p= 0.897;HepG2 32.33 ±5.93vs. HepG2-NAMPT 43.67±5.46, p=0.232) or apoptosis rate (L02 14.27±0.56vs. L02-NAMPT 13.77±0.71, p=0.611; hepG 2.23±0.24vs. HepG2-NAMPT 9.11±0.95, p=0.906) (fig. 3C, D), nor did it promote subcutaneous tumor growth in nude mice (fig. 3E).
The L02-NAMPT/HepG-NAMPT and the human hepatic stellate cells LX2 are subjected to cell co-culture by using a conventional co-culture method, and proliferation and apoptosis conditions of the L02-NAMPT/HepG-NAMPT cells and surface marker conditions of the human hepatic stellate cells LX2 are respectively studied. After co-cultivation with LX2, the colony forming ability of L02-NAMPT (57.67.+ -. 4.81vs. 81.33.+ -. 2.19, P=0.011) and HepG-NAMPT (43.67.+ -. 11.62vs. 81.33.+ -. 4.98, P=0.041) was enhanced (FIG. 3H), and the apoptosis rate of L02-NAMPT (14.17.+ -. 1.02vs. 4.28.+ -. 0.45, P < 0.001) and HepG-NAMPT (9.25.+ -. 1.07vs. 3.13.+ -. 0.50, P=0.007) was significantly decreased (FIG. 3I). And increased expression of ACTA2 (α -SMA) and COL1A1 in LX2 was observed, TGFB1 (TGF- β) was up-regulated, showing the characteristics of myHSC (fig. 3f, g). Hepatic stellate cells have two subpopulations cyHSCs and myHSCs, the associated molecular aggregation of extracellular matrix in myHSCs (myofibroblastic HSCs), HSCs are highly activated. Cells exhibit myHSC characteristics, suggesting that HSCs are activated and that the cells are involved in the formation of liver fibrosis and in the reconstitution of intrahepatic structures by proliferation and secretion of extracellular matrix. By increasing NAMPT expression level in normal liver cells and liver cancer cells, the activation level of stellate cell HSC can be increased, and further liver fibrosis progress can be promoted. Further illustrates that NAMPT is an action target for treating liver fibrosis, and improving NAMPT expression level can promote liver fibrosis progress, and reducing NAMPT expression level, reducing activation level of stellate cell HSC, avoiding proliferation and secretion of extracellular matrix, and inhibiting liver fibrosis.
(2) Screening of NAMPT inhibitors
In order to inhibit NAMPT expression, the means commonly used in the prior art are the use of RNA interference to inhibit gene transcription and expression, or the use of some inhibitors to inhibit NAMPT transcription and expression. The former is complicated in use process, shRNA and the like are required to be constructed and cells are required to be transfected, and medicines can be directly applied by using some small molecule inhibitors, so that effects are rapidly generated. The inventors have studied small molecule inhibitors that inhibit NAMPT.
The inventor analyzes and detects activation conditions of various proteins related to DNA damage in L02 (human normal liver cells) and L02-pHBV4.1 cells (HBV replication cell model) by Western blot, and screens potential small molecule compounds with NAMPT inhibition effect.
The results showed that the ATR phosphorylation level and NAMPT expression level were significantly increased in L02-phbv4.1 cells compared to L02 cells. At the same time, there was no significant difference in ATM phosphorylation levels of the two cell lines (fig. 2D). HBV transcription in L02-pHBV4.1 cells is at an activated level, and the expression or activation level of proteins associated with DNA repair, including NAMPT, is changed to some extent relative to normal liver cells not infected with HBV. Next, NAMPT expression was observed using KU60019 and AZD6738 on L02-pHBV4.1 cells. AZD6738 significantly reduced ATR autophosphorylation and NAMPT expression, but did not inhibit ATM-mediated CHEK2 phosphorylation using KU60019, but did not significantly affect NAMPT expression (FIG. 2E). In addition, AZD6738 also effectively reduced the increase in NAMPT expression caused by HBV replication in hepg2.2.15 cells (fig. 2F). Normal liver cells or liver cancer cells infected by HBV virus, NAMPT expression level is improved (HSC cells are further activated to promote liver fibrosis progress), but under the action of AZD6738, the activation of NAMPT by HBV is blocked, NAMPT expression level is obviously reduced, and HSC cell activation and liver fibrosis progress caused by HBV are avoided. These findings demonstrate that AZD6738 can be used as an inhibitor of NAMPT, in the treatment of liver fibrosis, or in basic research applications. In basic research, NAMPT expression is regulated, and the action mechanism, upstream and downstream signal molecules can be further researched. The most intuitive means to regulate the expression level of NAMPT is to directly control the expression of the gene, for example, by achieving overexpression through transgenesis and reducing the expression of the target gene through RNA interference. However, the above-described process is very complicated, and increases the cost of experimental study. If small molecule substances can be used, NAMPT expression can be affected by direct administration and culture and incubation, and the research flow is greatly shortened. While for each gene or protein, a proper small molecular inhibitor does not necessarily exist, in the research, the inventor discovers that AZD6738 can effectively inhibit the expression quantity of NAMPT, is a novel small molecular compound which can be used as a potential NAMPT inhibitor, obtains unexpected technical effects and creates conditions for the basic research related to NAMPT. In fact, commercial small molecule inhibitors and agonists have found very wide application as fundamental scientific tools, and new NAMPT inhibitors have other non-medical uses in addition to medical uses. In the prior art, the expression of a target gene is generally down-regulated by adopting a shRNA method, and the inventor also tries to inhibit the transcription of NAMPT genes by using a conventional shRNA method. The effect produced using the approach with shRNA was similar to that using AZD6738, a NAMPT inhibitor, and in WB experiments, similar experimental results to those of fig. 2E, F were presented.
Detailed experimental results of the above experiments referring to fig. 2 and 3, each experimental setup in fig. 2 and 3 is specifically as follows:
FIG. 2D shows immunoblotting results of the levels of ATM, P-ATM, ATR, P-ATR and NAMPT in the L02 and L02-pHBV4.1 cell lines, with ACTB (. Beta. -action) as an internal control. FIG. 2E shows the protein levels of ATM, P-ATM, ATR, P-ATR, CHEK2, P-CHEK2 and NAMPT of L02-pHBV4.1 cells treated with KU60019 (10 μm) or AZD6738 (2 μm) for 24 hours using ACTB (. Beta. -action) as an internal control. FIG. 2F shows protein levels of ATR, P-ATR and NAMPT by immunoblotting of HepG2, hepG2.215 cells treated by AZD6738 (2. Mu.M) for 24 hours, with ACTB (. Beta. -action) as an internal control. FIGS. 3A and B show the results of immunoblotting analysis against NAMPT for L02 cells and HepG2 cells overexpressing NAMPT or transformed with empty cells, with ACTB (. Beta. -action) as an internal control. FIGS. 3C and 3D show cell colony formation and apoptosis analysis of L02-Vec cells, L02-NAMPT cells, hepG2-Vec cells and HepG2-NAMPT cells. FIG. 3E shows the construction of a subcutaneous tumor model of nude mice with L02-Vec/L02-NAMPT or HepG2-Vec/HepG2-NAMPT, and the detection of the growth of subcutaneous tumors after cell injection. The experimental results show that whether NAMPT is overexpressed has no significant effect on tumor growth caused by L02 cells and HepG2 cells (mainly the latter). FIG. 3F shows immunoblotting results of ACTA2 (α -SMA), COL1A1, TGFB (TGF-. Beta.) levels in LX2 cells co-cultured with or without L02-NAMPT/HepG 2-NAMPT. FIG. 3G shows immunofluorescence detection of relevant markers in LX2 cells co-cultured with or without L02-NAMPT. This experiment was used to identify immunofluorescent staining for INSR (red) and COL1A1 (green). FIG. 3H is colony formation of L02-NAMPT/HepG2-NAMPT cells with or without LX2 co-culture. FIG. 3I is an apoptosis assay of L02-NAMPT/HepG2-NAMPT cells with or without LX2 co-culture. The data in the bar graph referred to in the above figures are expressed as mean ± standard deviation (n=3), P >0.05 ns, P <0.05, P <0.01, P < 0.001.
EXAMPLE 4 Effect of NAMPT on liver cancer Stem cells
To investigate the effect of NAMPT on Liver Cancer Stem Cells (LCSCs) and on liver fibrosis, the inventors constructed LCSCs (MIG-MYC-NAMPT cells) over-expressing NAMPT and performed three-dimensional (3D) culture. The construction process of MIG-MYC-NAMPT cells can be seen in the inventor's prior patent (application of CN114917348B neural adhesion factor in the preparation of reagent for inhibiting and labeling liver cancer cell metastasis, "(2) construction" part of NRCAM inhibiting expression and overexpressing LCSCs cells ", in this patent), except that the overexpressed target gene is changed from NRCAM to NAMPT, which is a conventional operation means in the prior art, and will not be described here. MIG-MYC-NAMPT cells overexpressing NAMPT and MIG-MYC cells overexpressing NAMPT were obtained by the above methods. The liver stem cells over-expressing MYC can be used as liver cancer stem cells.
Stem cell marker expression, such as Epcam and Afp, in MIG-MYC-NAMPT organoids (cells were cultured to form organoids by 3D) were examined using immunofluorescence confocal microscopy and found to be similar to MIG-MYC organoids (FIG. 4A). After co-culturing MIG-MYC-NAMPT organoids with LX2, MIG-MYC-NAMPT organoids were found to activate ACTA2, COL1A1 and TGFB1 expression in HSC (FIG. 4B). Epcam in MIG-MYC-NAMPT organoids also up-regulated expression (fig. 4C). In addition, NAMPT did not directly affect LCSCs colony forming ability (MIG-MYC 77.67 ±2.52vs. MIG-MYC-NAMPT 78.33±4.04, p=0.820) or apoptosis rate (MIG-MYC 5.69±0.23vs. MIG-MYC-NAMPT 5.56±0.33, p=0.684) (fig. 4D), nor promoted nude mouse subcutaneous tumor growth (fig. 4E). Whereas after co-culture with LX2, the colony forming ability of MIG-MYC-NAMPT organoids was enhanced (80.67 ±3.51vs.93.00±2.00, p=0.006), the apoptosis rate of MIG-MYC-NAMPT organoids was significantly reduced (5.24±0.36vs.3.34±0.35, p= < 0.001) (fig. 4F). These results indicate that NAMPT overexpression does not directly affect MIG-MYC stem cell properties. MIG-MYC-NAMPT organoids interact with HSCs, promoting differentiation of mouse HSCs to myHSC phenotype, and promoting the progression of liver fibrosis.
MIG-MYC and MIG-MYC-NAMPT organoids were transplanted into radiation-induced short-lived immunodeficiency C57BL/6 mice by intrahepatic injection, forming mouse tumor gene driven HCCs models. Mice were sacrificed 28 days after allogeneic transplantation, their livers were fixed and H & E and Masson stained. Adjacent liver tissues of MIG-MYC-NAMPT groups and MIG-MYC groups were examined by Masson staining. We also found pseudo-leaflet formation and severe fibrosis in tissues of MIG-MYC-NAMPT group (fig. 5). These results indicate that HBV-induced NAMPT expression elevation does not directly promote malignant transformation of hepatocytes, but may promote progression of liver fibrosis through interaction with myHSC.
The detailed experimental results of this example are shown in fig. 4 and 5. FIG. 4 shows the effect of NAMPT-INSR pathway on the characteristics of LCSCs stem cells in the mouse oncogene-driven allograft HCCs model. FIG. 4A shows the detection of Afp and Epcam by immunofluorescent staining in cell colonies of MIG-MYC-Vec and MIG-MYC-NAMPT organoids. FIG. 4B shows the results of immunoblot analysis of ACTA2, COL1A1 and TGFB levels in LX2 cells co-cultured with or without MIG-MYC-NAMPT. FIG. 4C is an experimental image of detection of Afp and Epcam by immunofluorescent staining in MIG-MYC-NAMPT cell colonies with or without LX2 co-culture. FIG. 4D is the results of colony formation and apoptosis assays for MIG-MYC-Vec and MIG-MYC-NAMPT cell colonies. FIG. 4E is a graph showing the formation of cell-derived subcutaneous tumors in nude mice. FIG. 4F is the results of colony formation and apoptosis analysis of MIG-MYC-NAMPT cell colonies with or without LX2 co-culture. FIG. 5 is Masson staining of the MIG-MYC-Vec (Mouse: 1-1) and MIG-MYC-NAMPT cell colony (Mouse: 2-1) groups.
Example 5 study of liver cell (NAMPT) -HSCs (INSR) communication initiated excessive activation of PARP1 lactogenesis and telomere maintenance
The inventors studied the receptor INSR of NAMPT in knockdown HSCs and found that activation of HSCs by hepatocytes was lost (fig. 6A-B), and in mice, after INSR in liver HSCs was knocked down, activation of HSCs by mouse tumor stem cells was also lost. Mouse tumor stem cells can continue to form liver cancer, but will not regenerate liver fibrosis (6C-H).
Among other things, fig. 6 shows that NAMPT ligand-INSR receptor signaling pathways mediate crosstalk between hepatocytes and HSCs. FIG. 6A shows the expression of INSR in LX2, LX2 -INSR-/- (INSR knockout in LX2 cells) and LX2 -INSR-/- INSR cells (rescue experiments). FIG. 6B shows Western blot results of INSR, ACTA2, COL1A1, TGFB levels in LX2, LX2 -INSR-/- and LX2 -INSR-/- -cells after co-culture with L02-NAMPT or HepG 2-NAMPT. FIG. 6C shows the Insr fl/fl; lrat-Cre mouse model construction workflow. FIG. 6D shows the results of IF staining of Cre (green) and Insr (red) in Lrat-P2A-iCre mice and ins fl/fl; lrat-Cre mouse HSCs. FIG. 6E shows the statistical results of INSR expression levels in Lrat-P2A-iCre mice and ins fl/fl, lrat-Cre mouse HSCs. FIG. 6F shows Western blot results of Insr, acta2, col1a1, tgfb levels in mouse HSCs and HSCs-Insr -/- cells after co-culture with MIG-MYC-NAMPT organoids. FIG. 6G shows the results of IF staining of Afp and Epcam in MIG-MYC-NAMPT organoids after co-culture with HSCs and HSCs-Insr -/- cells. FIG. 6H shows Lrat-P2A-iCre mice and ins fl/fl, lrat-Cre mice were evaluated for tumor formation and liver fibrosis using MIG-MYC-NAMPT organoid drive in an allogeneic liver cancer model (6 per group), H & E and Masson staining. Wherein, the statistical result is expressed as that P is less than or equal to 0.001 (x) and P is less than or equal to 0.0001 (x).
Telomeres are repetitive nucleotide sequences located at the ends of chromosomes that are critical to maintaining genomic stability and cell longevity. In certain cell types, including stem cells and cancer cells, telomere length is maintained by a special Telomere Maintenance Mechanism (TMMs). To investigate the effect of communication between hepatocytes (NAMPT) and hepatic stellate cells (INSR) on telomere length, L02-NAMPT cells were co-cultured with LX2 cells and telomere length was measured using FISH. A significant increase in telomere length was observed in L02-NAMPT cells (fig. 7A), indicating activation of TMMs in L02-NAMPT cells.
Maintenance of the T-ring structure is a critical aspect of TMMs. The Cohesion complex plays an important role in solving the T-ring structure to prevent telomere dysfunction, ultimately promoting telomere maintenance and cell life regulation. To confirm activation of T-ring structural maintenance, L02-NAMPT cells were co-cultured with LX2 cells and Western blotting analysis was performed. The major Cohesion complex components SMC1, SMC3 and RAD21 were significantly increased in activation, increased CTCF levels, and decreased TRF2 levels (FIG. 7B), indicating TMMs activation and inhibition of telomere shortening.
NAMPT is an enzyme that limits the rate of nad+ synthesis and increases the tolerance of cells to extracellular glucose depletion. INSR can induce metabolic shift to glycolysis, suggesting that the NAMPT-INSR pathway may affect the metabolic pattern of hepatocytes and HSCs. Changes in L02-NAMPT metabolism after co-culture with LX2 cells were observed using a Seahorse cell energy metabolism analyzer, and it was found that glycolysis of L02-NAMPT was significantly enhanced (FIG. 7C) and lactate dehydrogenase A (LDHA), a key enzyme for glycolysis and lactate production, was produced (FIG. 7D).
In addition to its role in DNA repair, PARP1 is also related to telomere length and T-loop. However, it was found that co-culturing L02-NAMPT cells with LX2 cells had no effect on PARP1 expression in L02-NAMPT cells (as shown in FIG. 7B). Specific sites of PARP1 can undergo lactate, and studies have shown that total lactate levels in L02-NAMPT cells are significantly increased after co-culture with LX2 (fig. 7E). To investigate the effect of co-culture on PARP1 lactate, we performed PARP1 Immunoprecipitation (IP) on L02-NAMPT Protein solutions using PARP1 antibodies and Protein A/G magnetic beads (FIG. 7F). Then, the lactate level of PARP1 was measured using a hololactamation antibody, and it was found that co-culture with LX2 promoted lactate of PARP1 (fig. 7G).
To further investigate the effect of PARP1 lactate on hepatocyte telomere maintenance, CRISPR/Cas9 was used to knock out PARP1 in L02. In addition, PARP1-7KR (K498/505/506/508/518/521/524R) mutant plasmids were constructed to completely abolish the lactate formation of PARP1. L02-PARP 1-/-cells heterologously express wild-type PARP1 and PARP1-7KR by transfection of PARP1 and PARP1-7KR plasmids. To induce lactogenesis, cells were treated with L-lactic acid (25 mM) (FIG. 7H). We then measured the level of lactate formation in PARP1, and the results showed that PARP1-7KR completely abolished the lactate formation in PARP1 (FIGS. 7I, J). IF and FISH analysis showed that telomeres were significantly shorter in cells expressing PARP1-7KR mutant compared to L02 cells expressing wild type PARP1 (fig. 7K). Furthermore, the Cohesion complex and CTCF of L02 cells expressing PARP1-7KR mutant were significantly lower and TRF2 levels were higher compared to cells expressing wild-type PARP1 (fig. 7L), suggesting that PARP1 lactate plays a key role in activation of hepatocytes TMMs.
The detailed test results are shown in fig. 7, wherein fig. 7A is a telomere FISH experiment to measure telomere length. Fig. 7B is an immunoblot analysis of key Cohesion complex factors (SMC 1, SMC3 and RAD 21), CTCF, TRF2 and PARP 1. FIG. 6C is a SeaHorse XF glycolysis assay. FIG. 7D shows the expression of GLUT1, GLUT3, HK2, LDHA, and PFK. FIG. 6E shows the results of lactate level detection. FIG. 7F shows the results of Immunoprecipitation (IP) study in PARP1 protein lysates. FIG. 6G shows PARP1 lactate level detection of L02-NAMPT with or without LX2 co-culture. FIG. 7H shows the results of lactate level detection. FIG. 7I shows the Immunoprecipitation (IP) results of PARP1 in protein lysates. Fig. 7J shows the results of PARP1 lactate level detection. FIG. 7K is a telomere FISH experiment. FIG. 7L is an immunoblot analysis of key factors of Cohesion complex, CTCF, TRF2 and PARP1 (after treatment of L-lactic acid (25 mM) for L02 cells expressing wild type PARP1 or PARP1-7 KR). Wherein, statistics P >0.05 (NS), P <0.05 (×), P <0.01 (×).
EXAMPLE 6 in vivo experimental study of methods of treating hepatitis B-associated liver fibrosis
To investigate the involvement of the ATR-NAMPT pathway and PARP1 lactate in the progression of liver fibrosis in vivo, organoids obtained by cell culture of MIG-MYC cells, MIG-MYC-phbv4.1 cells and MIG-MYC-phbv4.1-shNampt cells were transplanted into C57BL/6 mice by intrahepatic injection, which were transiently radiation-induced to develop immunodeficiency. And studied the role of the inhibitor AZD6738 in inhibiting liver fibrosis in vivo. Seven days after molding, mice were given (ig) AZD6738 (50 mg/Kg) or DMSO (control) by intragastric administration. After 28 days, all mice were sacrificed and their liver tissues were stained by immunoblotting, H & E and Masson. MIG-MYC cells are mouse liver stem cells expressing MYC (which is a mode of liver cancer stem cells), MIG-MYC-pHBV4.1 cells are liver cancer stem cells with HBV transcription being promoted, MIG-MYC-pHBV4.1-shNampt cells are liver cancer stem cells with HBV transcription being promoted, which are expressed by NAMPT and are inhibited by shRNA.
Masson staining showed that, whether AZD6738 was applied or not, the MIG-MYC-pHBV4.1-shNampt groups had reduced inflammation and fibrosis due to NAMPT inhibition relative to the MIG-MYC-pHBV4.1 groups, and that the MIG-MYC-pHBV4.1 groups had increased inflammation and fibrosis due to HBV viral transcription relative to the MIG-MYC groups. Comparing the cases with or without AZD6738, the MIG-MYC-pHBV4.1-shNampt group, the MIG-MYC-pHBV4.1 group and the MIG-MYC group all showed reduced inflammation and fibrosis, no false lobules or significant fibrosis in the AZD6738 treated group (FIG. 8A). In FIG. 8B, tissue clearance and image analysis were performed on representative MIG-MYC-pHBV4.1 mice, MIG-MYC-pHBV4.1-shnampt mice treated with DMSO or AZD6738, respectively. MYC-labeled tumor cells were stained green, while ACTA2 positive cells were stained red. shNampt and inhibition of Nampt with AZD6738 reduced ACTA2 activation around liver cancer, wherein ACTA2 activation represented HSCs activation and hepatic fibrosis progression (fig. 8B). Furthermore, immunoblot analysis showed complete inhibition of ATR phosphorylation, NAMPT expression and PARP1 lactonization in hepatocellular carcinoma tissues from MIG-MYC, MIG-MYC-phbv4.1 and MIG-MYC-phbv4.1-shNampt groups under AZD6738 treatment (fig. 8C).
PARP1 lactate was further investigated in the MIG-MYC-pHBV4.1 group by using the PARP1 specific inhibitor AG 14361. In the liver of mice, after AG14361 treatment, tumor xenografts were associated with significantly reduced tumor development (DMSO: 6/6 hepatocellular carcinoma vs. AG14361:2/6 hepatocellular carcinoma). AG14361 can realize a certain antitumor effect. But there was no significant reduction in inflammation and fibrosis in the tumor adjacent normal liver tissue (fig. 8D). Furthermore, immunoblot analysis showed that PARP1 expression and its lactate formation was completely inhibited in liver tissue of mice from group MIG-MYC-phbv4.1 treated with AG 14361. The results of the above experiments provided further evidence that PARP1 lactate plays a key role in hepatocellular carcinoma formation, as well as no significant changes in NAMPT expression and total cellular lactate levels following AG14361 treatment (fig. 8E). However, AG14361 can inhibit tumors to some extent, but has no inhibitory effect on liver fibrosis. This study showed that only AZD6738 can inhibit NAMPT expression, and thus hepatic stellate cell activation, among the small molecule compounds tested by the inventors, thereby inhibiting hepatic fibrosis progression.
The foregoing is merely exemplary of the present application, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present application, and these should also be regarded as the protection scope of the present application, which does not affect the effect of the implementation of the present application and the practical applicability of the patent. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.
Claims (1)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311872412.XA CN117815243B (en) | 2023-12-29 | 2023-12-29 | Application of a compound in the preparation of a drug for treating liver fibrosis and a NAMPT inhibitor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311872412.XA CN117815243B (en) | 2023-12-29 | 2023-12-29 | Application of a compound in the preparation of a drug for treating liver fibrosis and a NAMPT inhibitor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117815243A CN117815243A (en) | 2024-04-05 |
| CN117815243B true CN117815243B (en) | 2025-01-24 |
Family
ID=90518719
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311872412.XA Active CN117815243B (en) | 2023-12-29 | 2023-12-29 | Application of a compound in the preparation of a drug for treating liver fibrosis and a NAMPT inhibitor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117815243B (en) |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108686211A (en) * | 2017-04-12 | 2018-10-23 | 成军 | A kind of drug and therapy for treating liver fibrosis |
| WO2019084055A1 (en) * | 2017-10-23 | 2019-05-02 | Massachusetts Institute Of Technology | Calling genetic variation from single-cell transcriptomes |
| CN112043702A (en) * | 2019-06-05 | 2020-12-08 | 正大天晴药业集团股份有限公司 | Quinolines for the combined treatment of colorectal cancer |
| EP3986420A1 (en) * | 2019-06-18 | 2022-04-27 | Mitopower LLC | Nicotinyl riboside compounds and their uses |
| JP2022543658A (en) * | 2019-08-07 | 2022-10-13 | アクアラン セラピューティクス コーポレーション | Anti-NAMPT antibody and use thereof |
| WO2021102420A1 (en) * | 2019-11-22 | 2021-05-27 | The Regents Of The University Of California | Interferon signaling as a cancer biomarker |
| CN112725292B (en) * | 2021-01-11 | 2022-05-31 | 武汉大学 | A kind of establishment method and application of AAV-HBV recombinant virus and hepatitis B virus mouse model based on S gene fragmentation |
| CN114796255A (en) * | 2022-03-30 | 2022-07-29 | 深圳市旷逸生物科技有限公司 | Application of nicotinamide mononucleotide in protecting liver from being damaged |
-
2023
- 2023-12-29 CN CN202311872412.XA patent/CN117815243B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN117815243A (en) | 2024-04-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Emran et al. | Multidrug resistance in cancer: understanding molecular mechanisms, immunoprevention and therapeutic approaches | |
| Jiang et al. | Cancer exosome-derived miR-9 and miR-181a promote the development of early-stage MDSCs via interfering with SOCS3 and PIAS3 respectively in breast cancer | |
| McMahon et al. | A single H/ACA small nucleolar RNA mediates tumor suppression downstream of oncogenic RAS | |
| Qin et al. | Long noncoding RNA p53‐stabilizing and activating RNA promotes p53 signaling by inhibiting heterogeneous nuclear ribonucleoprotein K deSUMOylation and suppresses hepatocellular carcinoma | |
| Zhang et al. | The application of lncRNAs in cancer treatment and diagnosis | |
| Nieland et al. | Extracellular vesicle-mediated bilateral communication between glioblastoma and astrocytes | |
| Guo et al. | The role of exosomal microRNA in cancer drug resistance | |
| Li et al. | miR-17-5p promotes human breast cancer cell migration and invasion through suppression of HBP1 | |
| Poletto et al. | Nucleophosmin modulates stability, activity, and nucleolar accumulation of base excision repair proteins | |
| Martínez-Diez et al. | Biogenesis and dynamics of mitochondria during the cell cycle: significance of 3′ UTRs | |
| Zheng et al. | Activation of hepatic stellate cells is suppressed by microRNA-150 | |
| Guo et al. | Changes in microRNAs associated with hepatic stellate cell activation status identify signaling pathways | |
| Liu et al. | MicroRNA-708-3p as a potential therapeutic target via the ADAM17-GATA/STAT3 axis in idiopathic pulmonary fibrosis | |
| Yu et al. | N4-acetylcytidine modification of lncRNA CTC-490G23. 2 promotes cancer metastasis through interacting with PTBP1 to increase CD44 alternative splicing | |
| Litviakov et al. | Amplifications of stemness genes and the capacity of breast tumors for metastasis | |
| CN101981206A (en) | Methods and compositions related to cancer stem cells | |
| Kyjacova et al. | IER2-induced senescence drives melanoma invasion through osteopontin | |
| Zong et al. | AIF1+ CSF1R+ MSCs, induced by TNF‐α, act to generate an inflammatory microenvironment and promote hepatocarcinogenesis | |
| Andriani et al. | MiR-16 regulates the pro-tumorigenic potential of lung fibroblasts through the inhibition of HGF production in an FGFR-1-and MEK1-dependent manner | |
| Horii et al. | MicroRNA‐10a Impairs Liver Metabolism in Hepatitis C Virus‐Related Cirrhosis Through Deregulation of the Circadian Clock Gene Brain and Muscle Aryl Hydrocarbon Receptor Nuclear Translocator‐Like 1 | |
| Fu et al. | Biogenesis, cellular effects, and biomarker value of circHIPK3 | |
| Morozova et al. | System-level analysis of neuroblastoma tumor–initiating cells implicates AURKB as a novel drug target for neuroblastoma | |
| Yang et al. | RAP80 is an independent prognosis biomarker for the outcome of patients with esophageal squamous cell carcinoma | |
| Xiong et al. | Mechanism research of miR‐34a regulates Axl in non‐small‐cell lung cancer with gefitinib‐acquired resistance | |
| Ma et al. | Upregulation of miR‐335‐5p Contributes to Right Ventricular Remodeling via Calumenin in Pulmonary Arterial Hypertension |
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 |