JP2022009362A - Screening method for substance that enhances nitric oxide production in vascular endothelial cells - Google Patents
Screening method for substance that enhances nitric oxide production in vascular endothelial cells Download PDFInfo
- Publication number
- JP2022009362A JP2022009362A JP2021172441A JP2021172441A JP2022009362A JP 2022009362 A JP2022009362 A JP 2022009362A JP 2021172441 A JP2021172441 A JP 2021172441A JP 2021172441 A JP2021172441 A JP 2021172441A JP 2022009362 A JP2022009362 A JP 2022009362A
- Authority
- JP
- Japan
- Prior art keywords
- nitric oxide
- endothelial cells
- vascular endothelial
- indicator
- amount
- 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.)
- Granted
Links
- 210000003556 vascular endothelial cell Anatomy 0.000 title claims abstract description 88
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 title abstract description 361
- 238000000034 method Methods 0.000 title abstract description 81
- 239000000126 substance Substances 0.000 title abstract description 79
- 238000004519 manufacturing process Methods 0.000 title abstract description 56
- 238000012216 screening Methods 0.000 title abstract description 26
- 241000220259 Raphanus Species 0.000 claims abstract description 38
- 235000006140 Raphanus sativus var sativus Nutrition 0.000 claims abstract description 38
- 239000004480 active ingredient Substances 0.000 claims abstract description 9
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract description 69
- 239000001301 oxygen Substances 0.000 abstract description 69
- 229910052760 oxygen Inorganic materials 0.000 abstract description 69
- 210000004748 cultured cell Anatomy 0.000 abstract description 59
- 238000012360 testing method Methods 0.000 abstract description 55
- 238000006243 chemical reaction Methods 0.000 abstract description 33
- -1 feed Substances 0.000 abstract description 11
- 239000003623 enhancer Substances 0.000 abstract description 9
- 235000013305 food Nutrition 0.000 abstract description 7
- 238000000338 in vitro Methods 0.000 abstract description 7
- 239000003814 drug Substances 0.000 abstract description 4
- 239000003674 animal food additive Substances 0.000 abstract description 2
- 235000015872 dietary supplement Nutrition 0.000 abstract description 2
- 229940079593 drug Drugs 0.000 abstract description 2
- 235000013373 food additive Nutrition 0.000 abstract description 2
- 239000002778 food additive Substances 0.000 abstract description 2
- 235000013376 functional food Nutrition 0.000 abstract description 2
- 230000036541 health Effects 0.000 abstract description 2
- 239000013589 supplement Substances 0.000 abstract description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 65
- 229910001424 calcium ion Inorganic materials 0.000 description 65
- 239000000243 solution Substances 0.000 description 42
- 210000004027 cell Anatomy 0.000 description 34
- 102000008299 Nitric Oxide Synthase Human genes 0.000 description 27
- 108010021487 Nitric Oxide Synthase Proteins 0.000 description 27
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 22
- 238000005259 measurement Methods 0.000 description 19
- 239000011575 calcium Substances 0.000 description 18
- 239000003153 chemical reaction reagent Substances 0.000 description 15
- 238000001514 detection method Methods 0.000 description 15
- 235000013399 edible fruits Nutrition 0.000 description 14
- 238000011156 evaluation Methods 0.000 description 14
- 210000000805 cytoplasm Anatomy 0.000 description 12
- 230000002708 enhancing effect Effects 0.000 description 10
- 210000004204 blood vessel Anatomy 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 230000003834 intracellular effect Effects 0.000 description 9
- 230000005284 excitation Effects 0.000 description 8
- 210000000170 cell membrane Anatomy 0.000 description 7
- 230000008859 change Effects 0.000 description 7
- 210000002889 endothelial cell Anatomy 0.000 description 7
- 230000010534 mechanism of action Effects 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000007796 conventional method Methods 0.000 description 6
- 239000004519 grease Substances 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 229910052757 nitrogen Inorganic materials 0.000 description 6
- MXZACTZQSGYANA-UHFFFAOYSA-N chembl545463 Chemical compound Cl.C1=CC(OC)=CC=C1C(N=C1)=CN2C1=NC(C)=C2O MXZACTZQSGYANA-UHFFFAOYSA-N 0.000 description 5
- CYAJEMFRSQGFIG-ISWIILBPSA-N microcystin-LA Natural products CO[C@@H](Cc1ccccc1)[C@@H](C)C=C(C)C=C[C@H](NC(=O)CNC(=O)[C@@H](C)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](C)NC(=O)C(=C)N(C)C(=O)CC[C@@H](C)C(=O)O)C(=O)O)[C@H](C)C(=O)N CYAJEMFRSQGFIG-ISWIILBPSA-N 0.000 description 5
- 230000035699 permeability Effects 0.000 description 5
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 4
- 125000004172 4-methoxyphenyl group Chemical group [H]C1=C([H])C(OC([H])([H])[H])=C([H])C([H])=C1* 0.000 description 4
- 239000007995 HEPES buffer Substances 0.000 description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 230000009471 action Effects 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 239000000654 additive Substances 0.000 description 4
- 239000012298 atmosphere Substances 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- MCYGCOOQRICGCR-UHFFFAOYSA-N 2h-pyrazin-3-one;hydrochloride Chemical compound Cl.O=C1CN=CC=N1 MCYGCOOQRICGCR-UHFFFAOYSA-N 0.000 description 3
- 208000024172 Cardiovascular disease Diseases 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- 230000000996 additive effect Effects 0.000 description 3
- 210000002403 aortic endothelial cell Anatomy 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 230000036962 time dependent Effects 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- SNTCYKDOBVXFPB-UHFFFAOYSA-N 2-[3-(4,5-dimethoxy-2-nitrophenyl)sulfonyloxy-2,4,5,7-tetrafluoro-6-oxoxanthen-9-yl]benzoic acid Chemical compound COc1cc(c(cc1OC)S(=O)(=O)Oc1c(F)cc2c(-c3ccccc3C(O)=O)c3cc(F)c(=O)c(F)c3oc2c1F)[N+]([O-])=O SNTCYKDOBVXFPB-UHFFFAOYSA-N 0.000 description 2
- LAQJGAKLLZAETL-UHFFFAOYSA-N 2h-pyrazin-3-one Chemical compound O=C1CN=CC=N1 LAQJGAKLLZAETL-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 102000000584 Calmodulin Human genes 0.000 description 2
- 108010041952 Calmodulin Proteins 0.000 description 2
- 235000005956 Cosmos caudatus Nutrition 0.000 description 2
- 239000004375 Dextrin Substances 0.000 description 2
- 229920001353 Dextrin Polymers 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- BEVHTVRRVVEMEF-UHFFFAOYSA-N [6'-acetyloxy-4-amino-2',7'-difluoro-5-(methylamino)-3-oxospiro[2-benzofuran-1,9'-xanthene]-3'-yl] acetate Chemical compound C12=CC(F)=C(OC(C)=O)C=C2OC2=CC(OC(C)=O)=C(F)C=C2C21OC(=O)C1=C(N)C(NC)=CC=C21 BEVHTVRRVVEMEF-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- QOMNQGZXFYNBNG-UHFFFAOYSA-N acetyloxymethyl 2-[2-[2-[5-[3-(acetyloxymethoxy)-2,7-difluoro-6-oxoxanthen-9-yl]-2-[bis[2-(acetyloxymethoxy)-2-oxoethyl]amino]phenoxy]ethoxy]-n-[2-(acetyloxymethoxy)-2-oxoethyl]-4-methylanilino]acetate Chemical compound CC(=O)OCOC(=O)CN(CC(=O)OCOC(C)=O)C1=CC=C(C)C=C1OCCOC1=CC(C2=C3C=C(F)C(=O)C=C3OC3=CC(OCOC(C)=O)=C(F)C=C32)=CC=C1N(CC(=O)OCOC(C)=O)CC(=O)OCOC(C)=O QOMNQGZXFYNBNG-UHFFFAOYSA-N 0.000 description 2
- 208000011775 arteriosclerosis disease Diseases 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 208000026106 cerebrovascular disease Diseases 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 231100000433 cytotoxic Toxicity 0.000 description 2
- 230000001472 cytotoxic effect Effects 0.000 description 2
- 235000019425 dextrin Nutrition 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000007884 disintegrant Substances 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 238000003379 elimination reaction Methods 0.000 description 2
- 239000003995 emulsifying agent Substances 0.000 description 2
- 210000003989 endothelium vascular Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002073 fluorescence micrograph Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 208000019622 heart disease Diseases 0.000 description 2
- 238000001638 lipofection Methods 0.000 description 2
- 239000000314 lubricant Substances 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 238000000520 microinjection Methods 0.000 description 2
- 210000004925 microvascular endothelial cell Anatomy 0.000 description 2
- CMFNMSMUKZHDEY-UHFFFAOYSA-N peroxynitrous acid Chemical compound OON=O CMFNMSMUKZHDEY-UHFFFAOYSA-N 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 125000000951 phenoxy group Chemical group [H]C1=C([H])C([H])=C(O*)C([H])=C1[H] 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000002000 scavenging effect Effects 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 230000003845 vascular endothelial function Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- QCHFTSOMWOSFHM-WPRPVWTQSA-N (+)-Pilocarpine Chemical compound C1OC(=O)[C@@H](CC)[C@H]1CC1=CN=CN1C QCHFTSOMWOSFHM-WPRPVWTQSA-N 0.000 description 1
- SGTJXHYDKUXJRK-VAWYXSNFSA-N 1-[(e)-2-methoxyethenyl]pyrene Chemical compound C1=C2C(/C=C/OC)=CC=C(C=C3)C2=C2C3=CC=CC2=C1 SGTJXHYDKUXJRK-VAWYXSNFSA-N 0.000 description 1
- VBICKXHEKHSIBG-UHFFFAOYSA-N 1-monostearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)CO VBICKXHEKHSIBG-UHFFFAOYSA-N 0.000 description 1
- PXEZTIWVRVSYOK-UHFFFAOYSA-N 2-(3,6-diacetyloxy-2,7-dichloro-9h-xanthen-9-yl)benzoic acid Chemical compound C1=2C=C(Cl)C(OC(=O)C)=CC=2OC2=CC(OC(C)=O)=C(Cl)C=C2C1C1=CC=CC=C1C(O)=O PXEZTIWVRVSYOK-UHFFFAOYSA-N 0.000 description 1
- XYJODUBPWNZLML-UHFFFAOYSA-N 5-ethyl-6-phenyl-6h-phenanthridine-3,8-diamine Chemical compound C12=CC(N)=CC=C2C2=CC=C(N)C=C2N(CC)C1C1=CC=CC=C1 XYJODUBPWNZLML-UHFFFAOYSA-N 0.000 description 1
- 244000215068 Acacia senegal Species 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 241000416162 Astragalus gummifer Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 238000004435 EPR spectroscopy Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- OZLGRUXZXMRXGP-UHFFFAOYSA-N Fluo-3 Chemical compound CC1=CC=C(N(CC(O)=O)CC(O)=O)C(OCCOC=2C(=CC=C(C=2)C2=C3C=C(Cl)C(=O)C=C3OC3=CC(O)=C(Cl)C=C32)N(CC(O)=O)CC(O)=O)=C1 OZLGRUXZXMRXGP-UHFFFAOYSA-N 0.000 description 1
- OUVXYXNWSVIOSJ-UHFFFAOYSA-N Fluo-4 Chemical compound CC1=CC=C(N(CC(O)=O)CC(O)=O)C(OCCOC=2C(=CC=C(C=2)C2=C3C=C(F)C(=O)C=C3OC3=CC(O)=C(F)C=C32)N(CC(O)=O)CC(O)=O)=C1 OUVXYXNWSVIOSJ-UHFFFAOYSA-N 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 1
- 229930064664 L-arginine Natural products 0.000 description 1
- 235000014852 L-arginine Nutrition 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 239000004166 Lanolin Substances 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 229920001214 Polysorbate 60 Polymers 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 235000019774 Rice Bran oil Nutrition 0.000 description 1
- QCHFTSOMWOSFHM-UHFFFAOYSA-N SJ000285536 Natural products C1OC(=O)C(CC)C1CC1=CN=CN1C QCHFTSOMWOSFHM-UHFFFAOYSA-N 0.000 description 1
- NWGKJDSIEKMTRX-AAZCQSIUSA-N Sorbitan monooleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O NWGKJDSIEKMTRX-AAZCQSIUSA-N 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- MPFIISQEADXBEO-UHFFFAOYSA-M X-rhod-1 Chemical compound [Br-].CC(=O)OCOC(=O)CN(CC(=O)OCOC(C)=O)C1=CC=C(C)C=C1OCCOC1=CC(C=2C3=CC=4CCCN5CCCC(C=45)=C3OC3=C4C5=[N+](CCC4)CCCC5=CC3=2)=CC=C1N(CC(=O)OCOC(C)=O)CC(=O)OCOC(C)=O MPFIISQEADXBEO-UHFFFAOYSA-M 0.000 description 1
- APERIXFHHNDFQV-UHFFFAOYSA-N [2-[2-[2-[bis(carboxymethyl)amino]-5-methylphenoxy]ethoxy]-4-[3,6-bis(dimethylamino)xanthen-9-ylidene]cyclohexa-2,5-dien-1-ylidene]-bis(carboxymethyl)azanium;chloride Chemical compound [Cl-].C12=CC=C(N(C)C)C=C2OC2=CC(N(C)C)=CC=C2C1=C(C=1)C=CC(=[N+](CC(O)=O)CC(O)=O)C=1OCCOC1=CC(C)=CC=C1N(CC(O)=O)CC(O)=O APERIXFHHNDFQV-UHFFFAOYSA-N 0.000 description 1
- 239000000205 acacia gum Substances 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 1
- 229960004373 acetylcholine Drugs 0.000 description 1
- AMJRSUWJSRKGNO-UHFFFAOYSA-N acetyloxymethyl 2-[n-[2-(acetyloxymethoxy)-2-oxoethyl]-2-[2-[2-[bis[2-(acetyloxymethoxy)-2-oxoethyl]amino]-5-(2,7-dichloro-3-hydroxy-6-oxoxanthen-9-yl)phenoxy]ethoxy]-4-methylanilino]acetate Chemical compound CC(=O)OCOC(=O)CN(CC(=O)OCOC(C)=O)C1=CC=C(C)C=C1OCCOC1=CC(C2=C3C=C(Cl)C(=O)C=C3OC3=CC(O)=C(Cl)C=C32)=CC=C1N(CC(=O)OCOC(C)=O)CC(=O)OCOC(C)=O AMJRSUWJSRKGNO-UHFFFAOYSA-N 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 235000010419 agar Nutrition 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 235000010216 calcium carbonate Nutrition 0.000 description 1
- CJZGTCYPCWQAJB-UHFFFAOYSA-L calcium stearate Chemical compound [Ca+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O CJZGTCYPCWQAJB-UHFFFAOYSA-L 0.000 description 1
- 239000008116 calcium stearate Substances 0.000 description 1
- 235000013539 calcium stearate Nutrition 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 229960004484 carbachol Drugs 0.000 description 1
- AIXAANGOTKPUOY-UHFFFAOYSA-N carbachol Chemical compound [Cl-].C[N+](C)(C)CCOC(N)=O AIXAANGOTKPUOY-UHFFFAOYSA-N 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229940082500 cetostearyl alcohol Drugs 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 238000002038 chemiluminescence detection Methods 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 210000004351 coronary vessel Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 210000004696 endometrium Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000021323 fish oil Nutrition 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 1
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 1
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 1
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 description 1
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 description 1
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 description 1
- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- PNDZEEPOYCVIIY-UHFFFAOYSA-N indo-1 Chemical compound CC1=CC=C(N(CC(O)=O)CC(O)=O)C(OCCOC=2C(=CC=C(C=2)C=2N=C3[CH]C(=CC=C3C=2)C(O)=O)N(CC(O)=O)CC(O)=O)=C1 PNDZEEPOYCVIIY-UHFFFAOYSA-N 0.000 description 1
- 230000004941 influx Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 229940039717 lanolin Drugs 0.000 description 1
- 235000019388 lanolin Nutrition 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 1
- 210000004924 lung microvascular endothelial cell Anatomy 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229940126601 medicinal product Drugs 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 210000004088 microvessel Anatomy 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- XTBLDMQMUSHDEN-UHFFFAOYSA-N naphthalene-2,3-diamine Chemical compound C1=CC=C2C=C(N)C(N)=CC2=C1 XTBLDMQMUSHDEN-UHFFFAOYSA-N 0.000 description 1
- 229940005654 nitrite ion Drugs 0.000 description 1
- 231100001083 no cytotoxicity Toxicity 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- GLDOVTGHNKAZLK-UHFFFAOYSA-N octadecan-1-ol Chemical compound CCCCCCCCCCCCCCCCCCO GLDOVTGHNKAZLK-UHFFFAOYSA-N 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 229960001416 pilocarpine Drugs 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 210000001147 pulmonary artery Anatomy 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000002040 relaxant effect Effects 0.000 description 1
- 239000008165 rice bran oil Substances 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 230000000391 smoking effect Effects 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- OULAJFUGPPVRBK-UHFFFAOYSA-N tetratriacontyl alcohol Natural products CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCO OULAJFUGPPVRBK-UHFFFAOYSA-N 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 210000001644 umbilical artery Anatomy 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 229940124549 vasodilator Drugs 0.000 description 1
- 239000003071 vasodilator agent Substances 0.000 description 1
Images
Abstract
Description
本発明は、血管内皮細胞の一酸化窒素（ＮＯ）産生を亢進する物質のスクリーニング方法に関する。 The present invention relates to a method for screening a substance that enhances nitric oxide (NO) production in vascular endothelial cells.
日本人の死因別死亡率のうち、循環器疾患である心疾患と脳血管疾患が占める割合は癌に匹敵している。循環器疾患は、死亡のみならず、治療、後遺症のために、個人的にも社会的にも大きな損失が生じるため、これらを予防することが求められている。 Of the mortality rates by cause of death in Japan, the proportion of cardiovascular diseases such as heart disease and cerebrovascular disease is comparable to that of cancer. Cardiovascular diseases cause great personal and social losses not only due to death but also due to treatment and sequelae, and it is required to prevent them.
動脈硬化をはじめとした血管関連疾患は、血管の最も内側に位置している血管内皮の機能が低下してしまうことで起こる。血管は、外側から、外膜、中膜、内膜の三層で構成されている。血管の最も内側に位置する内膜には血管内皮細胞が存在しており、血管内皮細胞は一酸化窒素（ＮＯ）を放出している。ＮＯは、血管の収縮及び弛緩の調節、白血球などの血管内皮への付着による血栓形成の防御などに大きく関わっており、血管に直接働きかけることで血管を保護している。しかし、喫煙、飲酒、高血圧及び糖尿病等により血管が酸化ストレスを受けると、血管内皮細胞が傷害を受け、ＮＯの産生が抑制されてしまい、結果として動脈硬化を始めとした心疾患、脳血管疾患が引き起こされる。血管関連疾患を予防するには、血管内皮機能を改善して、血管の弛緩作用を維持させる必要がある。 Blood vessel-related diseases such as arteriosclerosis are caused by a decrease in the function of the vascular endothelium located inside the blood vessels. Blood vessels are composed of three layers, the adventitia, the media, and the intima, from the outside. Vascular endothelial cells are present in the innermost endometrium of blood vessels, and the vascular endothelial cells release nitric oxide (NO). NO is greatly involved in the regulation of contraction and relaxation of blood vessels and the protection of thrombus formation due to attachment of leukocytes to the vascular endothelium, and protects blood vessels by acting directly on the blood vessels. However, when blood vessels are subjected to oxidative stress due to smoking, drinking, hypertension, diabetes, etc., vascular endothelial cells are damaged and NO production is suppressed, resulting in heart disease such as arteriosclerosis and cerebrovascular disease. Is triggered. To prevent vascular-related diseases, it is necessary to improve vascular endothelial function and maintain the relaxing action of blood vessels.
血管内皮細胞のＮＯ産生能を測る方法として、グリース反応法（Ｇｒｉｅｓｓ法）、電極法、電子スピン共鳴法、オゾン化学発光法等のＮＯを直接又は間接的に測定する方法（例えば、非特許文献１）、ＮＯを合成する酵素である一酸化窒素合成酵素（ＮＯＳ）を専用蛍光試薬で測定する方法、ウエスタンブロットで測定する方法等が知られている。 As a method for measuring the NO-producing ability of vascular endothelial cells, a method for directly or indirectly measuring NO such as a grease reaction method (griess method), an electrode method, an electron spin resonance method, and an ozone chemiluminescence method (for example, non-patent literature). 1), a method of measuring nitric oxide synthase (NOS), which is an enzyme for synthesizing NO, with a dedicated fluorescent reagent, a method of measuring with a western blot, and the like are known.
また、血管拡張物質として、アセチルコリン、カルバコール、ピロカルピンなどが知られている（非特許文献１）。 Further, as a vasodilator, acetylcholine, carbachol, pilocarpine and the like are known (Non-Patent Document 1).
Ｇｒｉｅｓｓ法は、ＮＯの代謝物であるＮＯ２ －がＧｒｉｅｓｓ試薬と反応して生成するアゾ化合物の吸光度を測定する方法であり、ＮＯの生成量を推測するもっとも簡便な方法として知られている。Ｇｒｉｅｓｓ法は、ＮＯの半減期が短く（３～６秒程度）不安定なため、ＮＯの酸化物である亜硝酸イオンＮＯ２ －及び硝酸イオンＮＯ３ －の存在量からＮＯの存在量を算出する方法である。Ｇｒｉｅｓｓ法では、例えば、ＮＯ２ －は２，３－ジアミノナフタレンと反応して、蛍光付加体であるナフタレントリアゾールを形成するので、この蛍光付加体の生成量を蛍光で測定する。最近では生体試料中で代謝されたＮＯ２ －のほとんどはさらに酸化されてＮＯ３ －となることが明らかになったため、還元酵素を共存させて、ＮＯ３ －をＮＯ２ －に還元して、「ＮＯ２ －とＮＯ３ －」の合計量に基づいてＮＯの存在量を推測する方法が一般的になっている。 The Griess method is a method for measuring the absorbance of an azo compound produced by reacting NO 2- , which is a metabolite of NO, with a Griess reagent, and is known as the simplest method for estimating the amount of NO produced. Since the Griess method has a short half-life of NO (about 3 to 6 seconds) and is unstable, the abundance of NO is calculated from the abundance of nitrite ion NO 2- and nitrate ion NO 3- , which are oxides of NO. How to do it. In the Griess method, for example, NO 2- reacts with 2,3 - diaminonaphthalene to form naphthalenetriazole, which is a fluorescent adduct, and the amount of this fluorescent adduct produced is measured by fluorescence. Recently, it has become clear that most of the NO 2- metabolized in biological samples is further oxidized to NO 3- , so reductase was allowed to coexist to reduce NO 3- to NO 2- . A method of estimating the abundance of NO based on the total amount of "NO 2- and NO 3- " has become common.
また、ＮＯＳを専用蛍光試薬で測定する方法、ウエスタンブロットで測定する方法も広く使われている。 Further, a method of measuring NOS with a dedicated fluorescent reagent and a method of measuring NOS by Western blotting are also widely used.
しかし、これらは間接的にＮＯの産生を測定したものであり、実際のＮＯの産生量を反映しているのか否か、ＮＯ３ －を全て還元剤でＮＯ２ －に還元できているのか否か、また細胞のある一瞬の時間を切り取って得られた結果に過ぎず細胞の状態を反映できているのか否か等の問題がある。また、ウエスタンブロットは、専門的な技術が必要とされ手技による結果のばらつきがあり、また高価な抗体を必要とする。加えてＧｒｉｅｓｓ法は、多種類の試薬を添加する作業が多く、また、定量のための検量線の作成に多くの時間がかかる。蛍光によるＮＯＳ測定でも、細胞と蛍光色素のインキュベート後にウェルの培地を全部除去し、再度測定バッファを入れる等、かなり煩雑で手間がかかる問題もある。 However, these are measures of NO production indirectly, and whether or not they reflect the actual amount of NO production, and whether or not all NO 3 − can be reduced to NO 2 − with a reducing agent. There are also problems such as whether or not the state of the cell can be reflected only as a result obtained by cutting out a certain momentary time of the cell. In addition, Western blotting requires specialized techniques, the results vary depending on the procedure, and expensive antibodies are required. In addition, in the Griess method, many kinds of reagents are added, and it takes a lot of time to prepare a calibration curve for quantification. Even in the NOS measurement by fluorescence, there is a problem that it is considerably complicated and time-consuming, such as removing all the medium of the well after incubating the cells and the fluorescent dye and reinserting the measurement buffer.
直接的にＮＯを検出する方法として、電極法、ＥＳＲ法があるが、高価な装置と高度な技術が必要とされる。 As a method for directly detecting NO, there are an electrode method and an ESR method, but expensive equipment and advanced technology are required.
そこで、本発明は、血管内皮細胞の一酸化窒素（ＮＯ）産生を亢進する物質を、迅速かつ簡便に選抜できるスクリーニング方法の提供を目的とする。 Therefore, an object of the present invention is to provide a screening method capable of quickly and easily selecting a substance that promotes nitric oxide (NO) production in vascular endothelial cells.
本発明は、血管内皮細胞の一酸化窒素（ＮＯ）産生を亢進する物質のスクリーニング方法であって、インビトロで、血管内皮細胞由来の培養細胞を含む反応液に一酸化窒素指示薬及び活性酸素指示薬を添加するステップと、上記培養細胞に被験物質を接触させるステップと、上記一酸化窒素指示薬及び活性酸素指示薬を指標として、一酸化窒素量及び活性酸素量を測定するステップと、を含み、一酸化窒素量の測定値が一酸化窒素の基準値を超え、かつ活性酸素量の測定値が活性酸素の基準値以下である場合、上記被験物質が血管内皮細胞の一酸化窒素産生を亢進する物質であると評価される、スクリーニング方法に関する。 The present invention is a method for screening a substance that promotes nitric oxide (NO) production in vascular endothelial cells, in which a nitric oxide indicator and an active oxygen indicator are added to a reaction solution containing cultured cells derived from vascular endothelial cells in vitro. Nitric oxide includes a step of adding, a step of contacting the test substance with the cultured cells, and a step of measuring the amount of nitric oxide and the amount of active oxygen using the nitric oxide indicator and the active oxygen indicator as indicators. When the measured value of the amount exceeds the standard value of nitric oxide and the measured value of the amount of active oxygen is equal to or less than the standard value of active oxygen, the above-mentioned test substance is a substance that promotes nitric oxide production in vascular endothelial cells. Regarding the screening method that is evaluated as.
本発明のスクリーニング方法は、一酸化窒素指示薬及び活性酸素指示薬を指標とした測定に基づくものであるため、検出にあたり高価な装置、高度な技術、及び煩雑な操作は要求されず、迅速かつ簡便に血管内皮細胞のＮＯ産生を亢進する物質を選抜することができる。 Since the screening method of the present invention is based on measurement using a nitric oxide indicator and an active oxygen indicator as indicators, expensive equipment, advanced technology, and complicated operations are not required for detection, and it is quick and easy. Substances that enhance NO production of vascular endothelial cells can be selected.
また、血管内皮細胞は、スーパーオキシド（Ｏ２ －）を産生することがある。スーパーオキシドは、一酸化窒素との反応性が高く、一酸化窒素の消去に関与する（つまり、一酸化窒素の産生亢進効果を打ち消す）ほか、消去反応により生成するペルオキシナイトライト（ＯＮＯＯ－）は、スーパーオキシドよりも更に強い細胞傷害作用を有する。したがって、スーパーオキシド量を増加させない被験物質の方が好ましい。本発明のスクリーニング方法は、ＮＯと共にスーパーオキシドを同時にモニタすることで、被験物質のＮＯ産生亢進効果をより明確化でき、ＮＯの産生のみを亢進する物質の選別が可能となる。 In addition, vascular endothelial cells may produce superoxide ( O2- ) . Superoxide is highly reactive with nitric oxide and is involved in the scavenging of nitric oxide (that is, it counteracts the nitric oxide production enhancing effect), and the peroxynitrite ( ONOO- ) produced by the scavenging reaction is , Has a stronger cytotoxic effect than superoxide. Therefore, a test substance that does not increase the amount of superoxide is preferable. In the screening method of the present invention, by simultaneously monitoring superoxide together with NO, the effect of enhancing NO production of the test substance can be further clarified, and the substance that enhances only NO production can be selected.
本発明のスクリーニング方法において、一酸化窒素の基準値が、上記培養細胞を被験物質に接触させずに測定した一酸化窒素量の測定値であり、かつ活性酸素の基準値が、上記培養細胞を被験物質に接触させずに測定した活性酸素量の測定値であってもよい。これにより、ＮＯ及びスーパーオキシド量の増減をより正確に評価することができる。 In the screening method of the present invention, the reference value of nitric oxide is the measured value of the amount of nitric oxide measured without contacting the cultured cells with the test substance, and the reference value of active oxygen is the above-mentioned cultured cells. It may be a measured value of the amount of active oxygen measured without contacting with the test substance. This makes it possible to more accurately evaluate the increase / decrease in the amount of NO and superoxide.
本発明のスクリーニング方法において、上記活性酸素はスーパーオキシド（Ｏ２ －）であってよい。 In the screening method of the present invention, the active oxygen may be superoxide ( O2- ) .
本発明のスクリーニング方法において、上記一酸化窒素量の測定が蛍光の検出に基づくものであり、かつ上記活性酸素量の測定が化学発光の検出に基づくものであってもよい。これにより、蛍光及び化学発光の測定装置による検出が可能となり、より一層迅速かつ簡便に血管内皮細胞によるＮＯ産生を亢進する物質を選抜することができる。 In the screening method of the present invention, the measurement of the amount of nitric oxide may be based on the detection of fluorescence, and the measurement of the amount of active oxygen may be based on the detection of chemiluminescence. This enables detection by a fluorescence and chemiluminescence measuring device, and makes it possible to more quickly and easily select a substance that enhances NO production by vascular endothelial cells.
本発明のスクリーニング方法において、上記一酸化窒素指示薬が蛍光指示薬であり、かつ上記活性酸素指示薬が化学発光指示薬であってもよい。 In the screening method of the present invention, the nitric oxide indicator may be a fluorescence indicator and the active oxygen indicator may be a chemiluminescence indicator.
本発明のスクリーニング方法は、上記培養細胞に上記被験物質を接触させる前に、上記培養細胞を含む反応液にカルシウムイオン（Ｃａ２＋）指示薬を添加するステップと、上記培養細胞に上記被験物質を接触させた後に、上記カルシウムイオン指示薬を指標として、カルシウムイオン量を測定するステップと、カルシウムイオン量の測定値に基づき、上記被験物質が血管内皮細胞による一酸化窒素産生を亢進する作用機序を判定するステップと、を更に含み、カルシウムイオン量の測定値がカルシウムイオンの基準値を超える場合、上記被験物質が、カルモジュリンを介した一酸化窒素合成酵素の活性化により一酸化窒素産生を亢進すると判定する、又はカルシウムイオン量の測定値がカルシウムイオンの基準値以下の場合、上記被験物質が、一酸化窒素合成酵素を直接活性化することにより一酸化窒素産生を亢進すると判定するものであってもよい。 The screening method of the present invention comprises a step of adding a calcium ion (Ca 2+ ) indicator to a reaction solution containing the cultured cells and contacting the cultured cells with the test substance before contacting the cultured cells with the test substance. Then, based on the step of measuring the calcium ion amount using the calcium ion indicator as an index and the measured value of the calcium ion amount, the mechanism of action of the test substance to enhance the production of nitrogen monoxide by the vascular endothelial cells is determined. When the measured value of calcium ion exceeds the reference value of calcium ion, it is determined that the above-mentioned test substance enhances nitrogen monoxide production by activation of nitrogen monoxide synthase via carmodulin. Or, if the measured value of the amount of calcium ions is less than or equal to the reference value of calcium ions, even if it is determined that the above-mentioned test substance enhances the production of nitrogen monoxide by directly activating the nitrogen monoxide synthase. good.
血管内皮細胞によるＮＯ産生の作用機序の一つとして、Ｃａ２＋チャネルの開口による細胞内へのＣａ２＋流入や、Ｃａ２＋ストアからのＣａ２＋の放出により、細胞内Ｃａ２＋濃度が上昇し、それがトリガーとなってＮＯ合成酵素（Ｎｉｔｒｉｃ Ｏｘｉｄｅ Ｓｙｎｔｈａｓｅ：ＮＯＳ）が活性化し、Ｌ－アルギニンからＮＯが産生されるという作用機序が知られている。細胞質内Ｃａ２＋量（Ｃａ２＋濃度）を同時にモニタすることにより、被験物質がＮＯ産生を亢進する作用機序を同時に推定することが可能となる。 As one of the mechanisms of action of NO production by vascular endothelial cells, the intracellular Ca 2+ concentration increases due to the intracellular Ca 2+ influx due to the opening of the Ca 2+ channel and the release of Ca 2+ from the Ca 2+ store. The mechanism of action is known in which NO synthase (NOS) is activated by this as a trigger, and NO is produced from L-arginine. By simultaneously monitoring the amount of Ca 2+ in the cytoplasm (Ca 2+ concentration), it is possible to simultaneously estimate the mechanism of action of the test substance to enhance NO production.
上記カルシウムイオンの基準値が、上記培養細胞を被験物質に接触させずに測定したカルシウムイオン量の測定値であってもよい。これにより、カルシウムイオン量の増減をより正確に評価することができる。 The reference value of the calcium ion may be a measured value of the amount of calcium ion measured without contacting the cultured cells with the test substance. This makes it possible to more accurately evaluate the increase or decrease in the amount of calcium ions.
上記カルシウムイオン量の測定が蛍光の検出に基づくものであってもよく、上記カルシウムイオン指示薬が蛍光指示薬であってもよい。 The measurement of the calcium ion amount may be based on the detection of fluorescence, and the calcium ion indicator may be a fluorescence indicator.
本発明はまた、桜島大根を有効成分として含有する、血管内皮細胞の一酸化窒素産生亢進剤を提供する。本発明者らは、桜島大根が血管内皮細胞のＮＯ産生を亢進し、かつ活性酸素（スーパーオキシド）の産生には影響を与えない作用を有するという新規知見を得た。本発明に係る血管内皮細胞の一酸化窒素産生亢進剤は、この新規知見に基づくものである。 The present invention also provides a nitric oxide production enhancer for vascular endothelial cells containing Sakurajima radish as an active ingredient. The present inventors have obtained a novel finding that Sakurajima radish has an action of enhancing NO production of vascular endothelial cells and not affecting the production of active oxygen (superoxide). The nitric oxide production enhancer for vascular endothelial cells according to the present invention is based on this novel finding.
本発明によれば、血管内皮細胞の一酸化窒素（ＮＯ）産生を亢進する物質を、迅速かつ簡便に選抜できるスクリーニング方法の提供が可能となる。 INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a screening method capable of quickly and easily selecting a substance that promotes nitric oxide (NO) production in vascular endothelial cells.
本発明のスクリーニング方法によれば、従来法と比較して、ＮＯ量の測定に要する時間と経費を大幅に軽減でき、またリアルタイムで迅速かつ簡便に定量的な解析をすることが可能である。したがって、多くの被験物質を評価することができるため、スクリーニング方法として有用である。また、本発明の好ましい態様によれば、ＮＯＳの活性化に関与する細胞質内Ｃａ２＋濃度を同時にモニタすることで、作用機序の一端も解明することが可能となる。 According to the screening method of the present invention, the time and cost required for measuring the amount of NO can be significantly reduced as compared with the conventional method, and quantitative analysis can be performed quickly and easily in real time. Therefore, since many test substances can be evaluated, it is useful as a screening method. Further, according to a preferred embodiment of the present invention, it is possible to elucidate a part of the mechanism of action by simultaneously monitoring the concentration of Ca 2+ in the cytoplasm involved in the activation of NOS.
世界に先駆けて超高齢社会に突入した日本において、健康長寿を妨げる大きな原因となる循環器疾患の予防には高い関心が見込まれる。さらに、「食」による予防医療への期待も高まっている今、食への応用が可能な物質について血管内皮細胞の機能改善効果を迅速に簡便に評価し、効果の高い物質を選抜できる方法は、健康長寿社会実現に大いに貢献できるものと思われる。 In Japan, which has entered a super-aging society ahead of the rest of the world, great interest is expected in the prevention of cardiovascular diseases, which are a major obstacle to healthy longevity. Furthermore, now that expectations for preventive medicine by "food" are increasing, there is a method that can quickly and easily evaluate the function improvement effect of vascular endothelial cells for substances that can be applied to food and select highly effective substances. , It seems that it can greatly contribute to the realization of a healthy and long-lived society.
以下、本発明を実施するための形態について詳細に説明する。ただし、本発明は以下の実施形態に限定されるものではない。 Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.
〔血管内皮細胞の一酸化窒素（ＮＯ）産生を亢進する物質のスクリーニング方法〕
本実施形態に係るスクリーニング方法は、血管内皮細胞のＮＯ産生を亢進する物質を選抜するためのものである。本実施形態に係るスクリーニング方法は、インビトロで、血管内皮細胞由来の培養細胞を含む反応液に一酸化窒素指示薬及び活性酸素指示薬を添加するステップと、培養細胞に被験物質を接触させるステップと、一酸化窒素指示薬及び活性酸素指示薬を指標として、一酸化窒素量及び活性酸素量を測定するステップと、を含む。
[Screening method for substances that promote nitric oxide (NO) production in vascular endothelial cells]
The screening method according to the present embodiment is for selecting a substance that enhances NO production of vascular endothelial cells. The screening method according to the present embodiment includes, in vitro, a step of adding a nitric oxide indicator and an active oxygen indicator to a reaction solution containing cultured cells derived from vascular endothelial cells, and a step of contacting the cultured cells with a test substance. It includes a step of measuring the amount of nitric oxide and the amount of active oxygen using the nitric oxide indicator and the active oxygen indicator as indicators.
（血管内皮細胞由来の培養細胞）
血管内皮細胞由来の培養細胞は、例えば、生体から採取された血管内皮細胞から樹立された細胞株であってもよく、多能性幹細胞（例えば、ｉＰＳ細胞、ＥＳ細胞）から血管内皮細胞に分化誘導された細胞株であってもよい。
(Cultured cells derived from vascular endothelial cells)
The cultured cells derived from vascular endothelial cells may be, for example, a cell line established from vascular endothelial cells collected from a living body, and differentiate from pluripotent stem cells (eg, iPS cells, ES cells) into vascular endothelial cells. It may be an induced cell line.
生体から採取された血管内皮細胞としては、被験物質のＮＯ産生亢進効果をより正確に評価できることから、哺乳動物から採取された血管内皮細胞であることが好ましく、ブタ、ヒト、イヌ、ウシ、モルモット、ウサギ等から採取された血管内皮細胞であることがより好ましい。採取された血管内皮細胞から細胞株を樹立する方法は、特に制限されず、常法に従うことができる。 As the vascular endothelial cells collected from a living body, vascular endothelial cells collected from mammals are preferable because the NO production enhancing effect of the test substance can be evaluated more accurately, and pigs, humans, dogs, cows, and guinea pigs. , It is more preferable that it is a vascular endothelial cell collected from a rabbit or the like. The method for establishing a cell line from the collected vascular endothelial cells is not particularly limited, and a conventional method can be followed.
血管内皮細胞由来の培養細胞の具体例としては、臍帯静脈内皮細胞、臍帯動脈内皮細胞、冠状動脈内皮細胞、伏在静脈内皮細胞、肺動脈内皮細胞、大動脈内皮細胞、皮膚血管内皮細胞、皮膚微小血管内皮細胞、子宮微小血管内皮細胞、肺微小血管内皮細胞、心臓微小血管内皮細胞、皮膚微小リンパ管内皮細胞等が挙げられる。 Specific examples of cultured cells derived from vascular endothelial cells include umbilical venous endothelial cells, umbilical artery endothelial cells, coronary artery endothelial cells, saphenous venous endothelial cells, pulmonary artery endothelial cells, aortic endothelial cells, cutaneous vascular endothelial cells, and cutaneous microvessels. Examples thereof include endothelial cells, uterine microvascular endothelial cells, lung microvascular endothelial cells, cardiac microvascular endothelial cells, and cutaneous microlymphocyte endothelial cells.
血管内皮細胞由来の培養細胞は、各培養細胞に適した培養培地及び温度等の条件下で培養することで維持及び継代することができる。例えば、ブタ大動脈内皮細胞の場合、血管内皮細胞用培養メディウム（コスモ・バイオ（株）製）中、３７℃、５％ＣＯ２雰囲気下で培養することができる。 Cultured cells derived from vascular endothelial cells can be maintained and subcultured by culturing under conditions such as a culture medium and temperature suitable for each cultured cell. For example, porcine aortic endothelial cells can be cultured in a culture medium for vascular endothelial cells (manufactured by Cosmo Bio Co., Ltd.) at 37 ° C. in a 5% CO 2 atmosphere.
（反応液）
血管内皮細胞由来の培養細胞を含む反応液は、血管内皮細胞由来の培養細胞を緩衝液（例えば、ＲＨ緩衝液、ＰＢＳ（－）緩衝液、ＨＥＰＥＳ緩衝液、１ｍＭ ＣａＣｌ２を含むＨＥＰＥＳ溶液）等に懸濁させることで調製することができる。
(Reaction solution)
The reaction solution containing the cultured cells derived from the vascular endothelial cells is a buffer solution (for example, RH buffer solution, PBS (-) buffer solution, HEPES buffer solution, HEPES solution containing 1 mM CaCl 2 ) or the like. It can be prepared by suspending it in.
反応液中の培養細胞の密度は、使用する一酸化窒素指示薬及び活性酸素指示薬の種類、培養細胞の種類等に応じて適宜設定してよい。反応液中の培養細胞の密度は、通常１．０～２．０×１０５ｃｅｌｌｓ／ｍＬである。 The density of the cultured cells in the reaction solution may be appropriately set according to the type of nitric oxide indicator and active oxygen indicator used, the type of cultured cells, and the like. The density of cultured cells in the reaction solution is usually 1.0 to 2.0 × 105 cells / mL.
（一酸化窒素指示薬）
一酸化窒素指示薬としては、一酸化窒素の存在量と相関する信号を検出できる試薬であればよく、例えば、一酸化窒素と定量的に反応する蛍光指示薬、発光（例えば、化学発光）指示薬、又は吸光指示薬を用いることができる。一酸化窒素指示薬は、市販されているものを用いてもよい。そのような指示薬として、例えば、ジアミノフルオレセイン－２ジアセテート（ＤＡＦ－２ＤＡ）、ジアミノフルオレセイン－ＦＭ ジアセテート（ＤＡＦ－ＦＭ ＤＡ）、ジアミノローダミン－４Ｍ アセトキシメチルエステル（ＤＡＲ－４Ｍ ＡＭ、いずれも積水メディカル（株）製）が挙げられる。
(Nitric oxide indicator)
The nitric oxide indicator may be any reagent that can detect a signal that correlates with the abundance of nitric oxide. For example, a fluorescence indicator that quantitatively reacts with nitric oxide, a luminescence (for example, chemiluminescence) indicator, or an indicator. A chemiluminescent indicator can be used. As the nitric oxide indicator, a commercially available one may be used. Such indicators include, for example, diaminofluorescein-2 diacetate (DAF-2DA), diaminofluorescein-FM diacetate (DAF-FM DA), diaminorhodamine-4M acetoxymethyl ester (DAR-4M AM, all of which are Sekisui Medical). (Made by Co., Ltd.).
血管内皮細胞由来の培養細胞を含む反応液に一酸化窒素指示薬を添加する方法としては、例えば、上述した一酸化窒素指示薬又はその溶液等を反応液に加える方法が挙げられる。反応液に添加された一酸化窒素指示薬は、細胞膜を透過した反応液中の一酸化窒素と反応するか、又は一酸化窒素指示薬そのものの細胞膜透過性により血管内皮細胞由来の培養細胞の細胞内に取り込まれ、細胞質内の一酸化窒素と反応することになる。 Examples of the method of adding a nitric oxide indicator to a reaction solution containing cultured cells derived from vascular endothelial cells include a method of adding the above-mentioned nitric oxide indicator or a solution thereof to the reaction solution. The nitric oxide indicator added to the reaction solution either reacts with nitric oxide in the reaction solution that has permeated the cell membrane, or the cell membrane permeability of the nitric oxide indicator itself causes it to enter the cells of cultured cells derived from vascular endothelial cells. It is taken up and reacts with nitric oxide in the cytoplasm.
反応液中又は細胞質内の一酸化窒素量の測定は、一酸化窒素指示薬を指標として、常法により行われる。例えば、一酸化窒素指示薬が蛍光指示薬の場合、励起光源から出力された励起光を血管内皮細胞由来の培養細胞を含む試料容器に照射し、放出された蛍光を蛍光検出器により検出することで、一酸化窒素量を測定することができる。一酸化窒素指示薬が化学発光指示薬の場合、血管内皮細胞由来の培養細胞を含む試料容器から放出された化学発光を化学発光検出器で検出することで、一酸化窒素量を測定することができる。 The amount of nitric oxide in the reaction solution or the cytoplasm is measured by a conventional method using a nitric oxide indicator as an index. For example, when the nitric oxide indicator is a fluorescence indicator, the excitation light output from the excitation light source is applied to a sample container containing cultured cells derived from vascular endothelial cells, and the emitted fluorescence is detected by a fluorescence detector. The amount of nitric oxide can be measured. When the nitric oxide indicator is a chemiluminescence indicator, the amount of nitric oxide can be measured by detecting chemiluminescence emitted from a sample container containing cultured cells derived from vascular endothelial cells with a chemiluminescence detector.
（活性酸素指示薬）
活性酸素指示薬としては、活性酸素の存在量と相関する信号を検出できる試薬であればよく、例えば、活性酸素と定量的に反応する蛍光指示薬、発光（例えば、化学発光）指示薬、又は吸光指示薬を用いることができる。活性酸素指示薬は、市販されているものを用いてもよい。そのような指示薬として、例えば、２－メチル－６－フェニル－３，７－ジヒドロイミダゾ［１，２－ａ］ピラジン－３－オン（ＣＬＡ）、２－メチル－６－（４－メトキシフェニル）－３，７－ジヒドロイミダゾ［１，２－ａ］ピラジン－３－オン（ＭＣＬＡ）、２－メチル‐６－ｐ－メトキシフェニルエチニルイミダゾピラジノン（ＭＰＥＣ）、インドシアニン型イミダゾピラノジン化合物（ＮＩＲ－ＣＬＡ）、２－［２，４，５，７－テトラフルオロ－６－（２－ニトロ－４，５－ジメトキシフェニルスルホニルオキシ）－３－オキソ－３Ｈ－キサンテン－９－イル］安息香酸（ＢＥＳ－Ｓｏ）ジヒドロエチジウム等のスーパーオキシドと反応する指示薬、カルボキシ－Ｈ２ＤＣＦＤＡ（２’，７’－ジクロロジヒドロフルオレセインジアセテート）、ジヒドロカルセイン、ジヒドロローダミン１２３、ルミノール等の過酸化水素と反応する指示薬、ＣＭ－Ｈ２ＤＣＦＤＣ、プロキシルフルオレスカミン、ＴＥＰＯ－９－ＡＣ等のヒドロキシラジカルと反応する指示薬、トランス－１－（２’－メトキシビニル）ピレン等の一重項酸素と反応する指示薬が挙げられる。
(Active oxygen indicator)
The active oxygen indicator may be any reagent that can detect a signal that correlates with the abundance of active oxygen. Can be used. As the active oxygen indicator, a commercially available one may be used. Such indicators include, for example, 2-methyl-6-phenyl-3,7-dihydroimidazole [1,2-a] pyrazine-3-one (CLA), 2-methyl-6- (4-methoxyphenyl). -3,7-Dihydroimidazole [1,2-a] pyrazine-3-one (MCLA), 2-methyl-6-p-methoxyphenylethynyl imidazolipyrazinone (MPEC), indocyanine-type imidazole pyranodine compound ( NIR-CLA), 2- [2,4,5,7-tetrafluoro-6- (2-nitro-4,5-dimethoxyphenylsulfonyloxy) -3-oxo-3H-xanthene-9-yl] benzoic acid (BES-So) Indicator that reacts with superoxide such as dihydroethidium, indicator that reacts with hydrogen peroxide such as carboxy-H2DCFDA (2', 7'-dichlorodihydrofluorescein diacetate), dihydrocalcane, dihydrorodamine 123, luminol, etc. , CM-H2DCFDC, Proxilfluorescamine, TEPO-9-AC and other indicators that react with hydroxy radicals, and trans-1- (2'-methoxyvinyl) pyrene and other indicators that react with single-term oxygen.
血管内皮細胞由来の培養細胞を含む反応液に活性酸素指示薬を添加する方法としては、例えば、上述した活性酸素指示薬又はその溶液等を反応液に加える方法が挙げられる。反応液に添加された活性酸素指示薬は、反応液中の活性酸素と反応するか、又は活性酸素指示薬そのものの細胞膜透過性により血管内皮細胞由来の培養細胞の細胞内に取り込まれ、細胞質内の活性酸素と反応することになる。 Examples of the method of adding the active oxygen indicator to the reaction solution containing cultured cells derived from vascular endothelial cells include a method of adding the above-mentioned active oxygen indicator or a solution thereof to the reaction solution. The active oxygen indicator added to the reaction solution reacts with the active oxygen in the reaction solution, or is taken up into the cells of cultured cells derived from vascular endothelial cells by the cell membrane permeability of the active oxygen indicator itself, and the activity in the cytoplasm. It will react with oxygen.
インビトロで血管内皮細胞由来の培養細胞を含む反応液に活性酸素指示薬を添加するステップは、活性酸素量を迅速に評価できることから、インビトロで血管内皮細胞由来の培養細胞の細胞内に活性酸素指示薬を添加するステップであってもよい。 Since the step of adding the active oxygen indicator to the reaction solution containing the cultured cells derived from the vascular endothelial cells in vitro can rapidly evaluate the amount of active oxygen, the active oxygen indicator is placed in the cells of the cultured cells derived from the vascular endothelial cells in vitro. It may be a step of adding.
血管内皮細胞由来の培養細胞の細胞内に活性酸素指示薬を添加する方法としては、例えば、活性酸素指示薬そのものの細胞膜透過性を利用して反応液に活性酸素指示薬を添加することにより細胞内に移動させる方法、活性酸素指示薬と細胞内への物質導入に汎用されている試薬（例えば、リポフェクション試薬）を混合して反応液に添加することにより細胞内に導入する方法、物理的に活性酸素指示薬を細胞内に導入する方法（例えば、マイクロインジェクション法、エレクトロポレーション法）を挙げることができる。 As a method of adding an active oxygen indicator into the cells of cultured cells derived from vascular endothelial cells, for example, the active oxygen indicator moves into the cell by adding the active oxygen indicator to the reaction solution by utilizing the cell membrane permeability of the active oxygen indicator itself. A method of mixing an active oxygen indicator with a reagent commonly used for introducing a substance into a cell (for example, a lipofection reagent) and adding it to a reaction solution to introduce the active oxygen indicator into the cell, a method of physically introducing the active oxygen indicator. Examples thereof include a method of introducing into cells (for example, a microinjection method and an electroporation method).
反応液中又は細胞質内の活性酸素量の測定は、活性酸素指示薬を指標として、常法により行われる。活性酸素量の測定方法の具体例としては、一酸化窒素量の測定方法の具体例と同様の方法を例示できる。 The amount of active oxygen in the reaction solution or in the cytoplasm is measured by a conventional method using an active oxygen indicator as an index. As a specific example of the method for measuring the amount of active oxygen, the same method as the specific example of the method for measuring the amount of nitric oxide can be exemplified.
一酸化窒素指示薬と活性酸素指示薬の添加順序は任意であり、同時に添加してもよい。 The order of addition of the nitric oxide indicator and the active oxygen indicator is arbitrary, and they may be added at the same time.
一酸化窒素量及び活性酸素量の測定においては、より迅速かつより簡便に検出を可能にするという観点から、一酸化窒素指示薬が蛍光指示薬であり、かつ活性酸素指示薬が化学発光指示薬であってよい。 In the measurement of the amount of nitric oxide and the amount of active oxygen, the nitric oxide indicator may be a fluorescence indicator and the active oxygen indicator may be a chemiluminescence indicator from the viewpoint of enabling faster and easier detection. ..
一酸化窒素量及び活性酸素量は、略同時に測定してもよい。これにより、同一の条件下における一酸化窒素量及び活性酸素量を測定することができ、血管内皮細胞のＮＯ産生の亢進効果をより正確に評価することができる。 The amount of nitric oxide and the amount of active oxygen may be measured substantially at the same time. Thereby, the amount of nitric oxide and the amount of active oxygen can be measured under the same conditions, and the effect of enhancing NO production of vascular endothelial cells can be evaluated more accurately.
一酸化窒素量及び活性酸素量は、例えば、蛍光検出装置、化学発光検出装置、吸光度測定装置を用いて測定することができる。また、一酸化窒素量及び活性酸素量を蛍光と化学発光により略同時に測定する場合、例えば、特開２００４－６１４３８号公報、特開２０００－１３１２３４号公報等に記載の蛍光及び化学発光を同時に測定する装置（例えば、浜松ホトニクス株式会社製，ＣＦＬ－Ｃ２０００）を用いて測定することもできる。 The amount of nitric oxide and the amount of active oxygen can be measured using, for example, a fluorescence detection device, a chemiluminescence detection device, and an absorbance measuring device. When the amount of nitric oxide and the amount of active oxygen are measured substantially simultaneously by fluorescence and chemiluminescence, for example, the fluorescence and chemiluminescence described in JP-A-2004-61438 and JP-A-2000-131234 are simultaneously measured. It can also be measured using a device (for example, manufactured by Hamamatsu Photonics Co., Ltd., CFL-C2000).
検出する活性酸素としては、スーパーオキシド、過酸化水素、ヒドロキシラジカル、一重項酸素が挙げられる。より精度よい評価が可能になることから、活性酸素としてスーパーオキシドを採用することが好ましい。 Examples of the active oxygen to be detected include superoxide, hydrogen peroxide, hydroxyl radical, and singlet oxygen. It is preferable to use superoxide as the active oxygen because it enables more accurate evaluation.
（被験物質）
被験物質に特に制限はなく、任意の物質を用いることができる。被験物質としては、例えば、天然由来の化合物、人工合成された化合物、ペプチド、タンパク質、脂質、核酸（リボ核酸、デオキシリボ核酸等）が挙げられる。被験物質として、食品由来の物質を用いてもよい。被験物質は、単一の化合物であってもよく、抽出物（例えば、桜島大根の抽出物）等の組成物であってもよい。
(Test substance)
The test substance is not particularly limited, and any substance can be used. Examples of the test substance include naturally occurring compounds, artificially synthesized compounds, peptides, proteins, lipids, and nucleic acids (ribonucleic acid, deoxyribonucleic acid, etc.). A food-derived substance may be used as the test substance. The test substance may be a single compound or a composition such as an extract (for example, an extract of Sakurajima radish).
被験物質を血管内皮細胞由来の培養細胞に接触させる方法としては、例えば、血管内皮細胞由来の培養細胞を含む反応液に被験物質、又は被験物質の溶液を添加することにより行うことができる。 As a method for bringing the test substance into contact with the cultured cells derived from the vascular endothelial cells, for example, the test substance or a solution of the test substance can be added to the reaction solution containing the cultured cells derived from the vascular endothelial cells.
（評価ステップ）
上述のように測定された一酸化窒素量及び活性酸素量に基づき、被験物質が血管内皮細胞の一酸化窒素産生を亢進するか否かを評価することができる。すなわち、一酸化窒素量の測定値が一酸化窒素の基準値を超え、かつ活性酸素量の測定値が活性酸素の基準値以下である場合、当該被験物質は、血管内皮細胞の一酸化窒素産生を亢進する物質であると評価される。本実施形態に係るスクリーニング方法は、このような評価を行うステップを更に含むものであってもよい。
(Evaluation step)
Based on the amount of nitric oxide and the amount of active oxygen measured as described above, it is possible to evaluate whether or not the test substance enhances nitric oxide production in vascular endothelial cells. That is, when the measured value of the amount of nitric oxide exceeds the standard value of nitric oxide and the measured value of the amount of active oxygen is equal to or less than the standard value of active oxygen, the test substance produces nitric oxide in vascular endothelial cells. It is evaluated as a substance that enhances oxygen species. The screening method according to the present embodiment may further include a step of performing such an evaluation.
（一酸化窒素の基準値）
評価の際の一酸化窒素の基準値は、評価目的、求める一酸化窒素産生の亢進効果の水準の程度に応じて、随時設定することができる。一酸化窒素の基準値の一例として、例えば、血管内皮細胞由来の培養細胞を被験物質に接触させずに測定した一酸化窒素量の測定値を基準値とすることができる。このように基準値を設定することによって、被験物質との接触による一酸化窒素量の増減をより正確に評価することができる。また、例えば、血管内皮細胞由来の培養細胞を、一酸化窒素産生を亢進する（又は抑制する）ことが知られている既知物質と接触させて測定した一酸化窒素量の測定値を基準値とすることもできる。
(Reference value of nitric oxide)
The reference value of nitric oxide at the time of evaluation can be set at any time according to the purpose of evaluation and the degree of the desired level of the effect of enhancing nitric oxide production. As an example of the reference value of nitric oxide, for example, the measured value of the amount of nitric oxide measured without contacting the cultured cells derived from vascular endothelial cells with the test substance can be used as the reference value. By setting the reference value in this way, it is possible to more accurately evaluate the increase or decrease in the amount of nitric oxide due to contact with the test substance. Further, for example, the measured value of the amount of nitric oxide measured by contacting cultured cells derived from vascular endothelial cells with a known substance known to enhance (or suppress) nitric oxide production is used as a reference value. You can also do it.
（活性酸素の基準値）
評価の際の活性酸素の基準値は、評価目的、求める一酸化窒素産生の亢進効果の水準の程度に応じて、随時設定することができる。活性酸素の基準値の一例として、例えば、血管内皮細胞由来の培養細胞を被験物質に接触させずに測定した活性酸素量の測定値を基準値とすることができる。このように基準値を設定することによって、被験物質との接触による活性酸素量の増減をより正確に評価することができる。また、例えば、血管内皮細胞由来の培養細胞を、一酸化窒素産生を亢進する（又は抑制する）ことが知られている既知物質と接触させて測定した活性酸素量の測定値を基準値とすることもできる。
(Reference value of active oxygen)
The reference value of active oxygen at the time of evaluation can be set at any time according to the purpose of evaluation and the degree of the desired level of the effect of enhancing nitric oxide production. As an example of the reference value of active oxygen, for example, the measured value of the amount of active oxygen measured without contacting the cultured cells derived from vascular endothelial cells with the test substance can be used as the reference value. By setting the reference value in this way, it is possible to more accurately evaluate the increase or decrease in the amount of active oxygen due to contact with the test substance. Further, for example, the measured value of the amount of active oxygen measured by contacting cultured cells derived from vascular endothelial cells with a known substance known to enhance (or suppress) nitric oxide production is used as a reference value. You can also do it.
（カルシウムイオン量の評価）
本実施形態に係るスクリーニング方法は、一酸化窒素量及び活性酸素量に基づく評価と共に、カルシウムイオン量に基づいた評価を行ってもよい。すなわち、本実施形態に係るスクリーニング方法は、血管内皮細胞由来の培養細胞に被験物質を接触させる前に、血管内皮細胞由来の培養細胞を含む反応液にカルシウムイオン（Ｃａ２＋）指示薬を添加するステップと、血管内皮細胞由来の培養細胞に被験物質を接触させた後に、カルシウムイオン指示薬を指標として、カルシウムイオン量を測定するステップと、カルシウムイオン量の測定値に基づき、被験物質が血管内皮細胞による一酸化窒素産生を亢進する作用機序を判定するステップと、を更に含んでいてもよい。これにより、被験物質がＮＯ産生を亢進する作用機序を同時に推定することが可能となる。
(Evaluation of calcium ion amount)
In the screening method according to the present embodiment, the evaluation based on the amount of calcium ion may be performed together with the evaluation based on the amount of nitric oxide and the amount of active oxygen. That is, the screening method according to the present embodiment is a step of adding a calcium ion (Ca 2+ ) indicator to a reaction solution containing cultured cells derived from vascular endothelial cells before contacting the test substance with cultured cells derived from vascular endothelial cells. After contacting the cultured cells derived from vascular endothelial cells with the test substance, the test substance is based on the vascular endothelial cells based on the step of measuring the calcium ion amount using the calcium ion indicator as an index and the measured value of the calcium ion amount. It may further include a step of determining the mechanism of action that enhances nitrogen monoxide production. This makes it possible to simultaneously estimate the mechanism of action of the test substance to enhance NO production.
（カルシウムイオン指示薬）
カルシウムイオン指示薬としては、カルシウムイオンの存在量と相関する信号を検出できる試薬であればよく、例えば、カルシウムイオンと定量的に反応する蛍光指示薬、発光（例えば、化学発光）指示薬、又は吸光指示薬を用いることができる。カルシウムイオン指示薬は、市販されているものを用いてもよい。そのような指示薬として、例えば、Ｆｌｕｏ－３、Ｆｌｕｏ－３ ＡＭ、Ｆｌｕｏ－４、Ｆｌｕｏ－４ ＡＭ（いずれもＤＯＪＩＮＤＯ社製）、Ｉｎｄｏ－１、Ｉｎｄｏ－５Ｆ、ｂｉｓ－ｆｕｒａ－２、Ｑｕｉｎ－２、Ｑｕｉｎ－ＡＭ、Ｒｈｏｄ－２、Ｘ－Ｒｈｏｄ－１（いずれもＭｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ社製）が挙げられる。
(Calcium ion indicator)
The calcium ion indicator may be any reagent that can detect a signal that correlates with the abundance of calcium ions. Can be used. As the calcium ion indicator, a commercially available one may be used. Such indicators include, for example, Fluo-3, Fluo-3 AM, Fluo-4, Fluo-4 AM (all manufactured by DOJINDO), Indo-1, Indo-5F, bis-fura-2, Quiin-2. , Quiin-AM, Rhod-2, X-Rhod-1 (all manufactured by Molecular Probes).
血管内皮細胞由来の培養細胞を含む反応液にカルシウムイオン指示薬を添加する方法としては、例えば、上述したカルシウムイオン指示薬又はその溶液等を反応液に加える方法が挙げられる。反応液に添加されたカルシウムイオン指示薬は、細胞膜を透過した反応液中のカルシウムイオンと反応するか、又はカルシウムイオン指示薬そのものの細胞膜透過性により血管内皮細胞由来の培養細胞の細胞内に取り込まれ、細胞質内のカルシウムイオンと反応することになる。 Examples of the method of adding a calcium ion indicator to a reaction solution containing cultured cells derived from vascular endothelial cells include a method of adding the above-mentioned calcium ion indicator or a solution thereof to the reaction solution. The calcium ion indicator added to the reaction solution either reacts with calcium ions in the reaction solution that has permeated the cell membrane, or is taken up into the cells of cultured cells derived from vascular endothelial cells by the cell membrane permeability of the calcium ion indicator itself. It will react with calcium ions in the cytoplasm.
インビトロで血管内皮細胞由来の培養細胞を含む反応液にカルシウムイオン（Ｃａ２＋）指示薬を添加するステップは、細胞質内の濃度変化をより迅速に評価できることから、インビトロで血管内皮細胞由来の培養細胞の細胞内にカルシウムイオン（Ｃａ２＋）指示薬を添加するステップであることが好ましい。 The step of adding a calcium ion (Ca 2+ ) indicator to the reaction solution containing cultured cells derived from vascular endothelial cells in vitro can evaluate the change in concentration in the cytoplasm more rapidly, and thus the cultured cells derived from vascular endothelial cells in vitro. It is preferably a step of adding a calcium ion (Ca 2+ ) indicator into the cell.
血管内皮細胞由来の培養細胞の細胞内にカルシウムイオン指示薬を添加する方法としては、例えば、カルシウムイオン指示薬そのものの細胞膜透過性を利用して反応液にカルシウムイオン指示薬を添加することにより細胞内に移動させる方法、カルシウムイオン指示薬と細胞内への物質導入に汎用されている試薬（例えば、リポフェクション試薬）を混合して反応液に添加することにより細胞内に導入する方法、物理的にカルシウムイオン指示薬を細胞内に導入する方法（例えば、マイクロインジェクション法、エレクトロポレーション法）を挙げることができる。 As a method of adding a calcium ion indicator into the cells of cultured cells derived from vascular endothelial cells, for example, the calcium ion indicator moves into the cell by adding the calcium ion indicator to the reaction solution by utilizing the cell membrane permeability of the calcium ion indicator itself. A method of mixing a calcium ion indicator with a reagent commonly used for introducing a substance into a cell (for example, a lipofection reagent) and adding it to a reaction solution to introduce the substance into the cell, a method of physically introducing a calcium ion indicator. Examples thereof include a method of introducing into cells (for example, a microinjection method and an electroporation method).
反応液中又は細胞質内のカルシウムイオン量の測定は、カルシウムイオン指示薬を指標として、常法により行われる。例えば、カルシウムイオン指示薬が蛍光指示薬の場合、励起光源から出力された励起光を血管内皮細胞由来の培養細胞を含む試料容器に照射し、放出された蛍光を蛍光検出器により検出することで、カルシウムイオン量を測定することができる。カルシウムイオン指示薬が化学発光指示薬の場合、血管内皮細胞由来の培養細胞を含む試料容器から放出された化学発光を化学発光検出器で検出することで、カルシウムイオン量を測定することができる。 The amount of calcium ions in the reaction solution or in the cytoplasm is measured by a conventional method using a calcium ion indicator as an index. For example, when the calcium ion indicator is a fluorescence indicator, the excitation light output from the excitation light source is applied to a sample container containing cultured cells derived from vascular endothelial cells, and the emitted fluorescence is detected by a fluorescence detector to obtain calcium. The amount of ions can be measured. When the calcium ion indicator is a chemiluminescence indicator, the amount of calcium ions can be measured by detecting the chemiluminescence emitted from the sample container containing the cultured cells derived from vascular endothelial cells with the chemiluminescence detector.
（測定順序の例）
カルシウムイオン指示薬を添加する場合、一酸化窒素指示薬、活性酸素指示薬及びカルシウムイオン指示薬の添加順序は任意であり、同時に添加してもよい。また、各指示薬から検出される信号の種類に応じて、例えば、２種の量（例えば、一酸化窒素量及び活性酸素量）を略同時に測定したうえで、別途他の１種の量（例えば、カルシウムイオン量）を測定してもよいし（測定を２回に分けて行う）、一酸化窒素量、活性酸素量及びカルシウムイオン量の３種を略同時に測定してもよい。
(Example of measurement order)
When the calcium ion indicator is added, the order of addition of the nitric oxide indicator, the active oxygen indicator and the calcium ion indicator is arbitrary, and they may be added at the same time. Further, depending on the type of signal detected from each indicator, for example, two kinds of amounts (for example, the amount of nitrogen monoxide and the amount of active oxygen) are measured substantially simultaneously, and then another one kind of amount (for example, for example) is separately measured. , Calcium ion amount) may be measured (measurement is performed in two steps), or three types of nitrogen monoxide amount, active oxygen amount and calcium ion amount may be measured substantially at the same time.
前者の具体例としては、例えば、一酸化窒素量を蛍光指示薬（例えば、ジアミノフルオレセイン－２ジアセテート（ＤＡＦ－２ＤＡ），励起波長４９５ｎｍ，蛍光波長５１５ｎｍ）を使用し、活性酸素量を化学発光指示薬（例えば、６－（４－メトキシフェニル）－２－メチル－３，７－ジヒドロイミダゾ［１，２－ａ］ピラジン－３－オン塩酸塩（ＭＣＬＡ），発光波長４６５ｎｍ）を使用し、蛍光及び化学発光を同時に測定する装置（例えば、ＣＦＬ－Ｃ２０００，浜松ホトニクス株式会社製）を用いて略同時に測定した後、別途カルシウムイオン量を蛍光指示薬（例えば、１－［２－アミノ－５－（２，７－ジフルオロ－６－アセトキシメトキシ－３－オキソ－９－キサンテニル）フェノキシ］－２－（２－アミノ－５－メチルフェノキシ）エタン－Ｎ，Ｎ，Ｎ’，Ｎ’－テトラ酢酸（Ｆｌｕｏ－４ ＡＭ），励起波長４９５ｎｍ，蛍光波長５１８ｎｍ）を使用し、蛍光を測定する装置を用いて測定することができる。 As a specific example of the former, for example, a fluorescence indicator (for example, diaminofluoresene-2 diacetate (DAF-2DA), an excitation wavelength of 495 nm, a fluorescence wavelength of 515 nm) is used for the amount of nitrogen monoxide, and a chemiluminescence indicator is used for the amount of active oxygen. (For example, 6- (4-methoxyphenyl) -2-methyl-3,7-dihydroimidazole [1,2-a] pyrazine-3-one hydrochloride (MCLA), emission wavelength 465 nm) is used for fluorescence and fluorescence. After measuring approximately simultaneously using a device that simultaneously measures chemiluminescence (for example, CFL-C2000, manufactured by Hamamatsu Photonics Co., Ltd.), the amount of calcium ion is separately measured with a fluorescence indicator (for example, 1- [2-amino-5- (2). , 7-Difluoro-6-acetoxymethoxy-3-oxo-9-xanthenyl) phenoxy] -2- (2-amino-5-methylphenoxy) ethane-N, N, N', N'-tetraacetic acid (Fluoro-) 4 AM), excitation wavelength 495 nm, fluorescence wavelength 518 nm) can be used for measurement using a device that measures fluorescence.
後者の具体例としては、例えば、一酸化窒素量を蛍光指示薬（例えば、ジアミノフルオレセイン－２ジアセテート（ＤＡＦ－２ＤＡ），励起波長４９５ｎｍ，蛍光波長５１５ｎｍ）を使用し、活性酸素量を化学発光指示薬（例えば、６－（４－メトキシフェニル）－２－メチル－３，７－ジヒドロイミダゾ［１，２－ａ］ピラジン－３－オン塩酸塩（ＭＣＬＡ），発光波長４６５ｎｍ）を使用し、カルシウムイオン量を蛍光指示薬（例えば、１－［２－アミノ－５－（２，７－ジフルオロ－６－アセトキシメトキシ－３－オキソ－９－キサンテニル）フェノキシ］－２－（２－アミノ－５－メチルフェノキシ）エタン－Ｎ，Ｎ，Ｎ’，Ｎ’－テトラ酢酸（Ｆｌｕｏ－４ ＡＭ），励起波長４９５ｎｍ，蛍光波長５１８ｎｍ）を使用し、蛍光及び化学発光を同時に測定する装置（例えば、ＣＦＬ－Ｃ２０００，浜松ホトニクス株式会社製）を用いて略同時に測定することができる。 As a specific example of the latter, for example, a fluorescence indicator (for example, diaminofluoresene-2 diacetate (DAF-2DA), an excitation wavelength of 495 nm, a fluorescence wavelength of 515 nm) is used for the amount of nitrogen monoxide, and a chemiluminescence indicator is used for the amount of active oxygen. (For example, 6- (4-methoxyphenyl) -2-methyl-3,7-dihydroimidazole [1,2-a] pyrazine-3-one hydrochloride (MCLA), emission wavelength 465 nm) is used and the calcium ion. Fluorescence indicator (eg 1- [2-amino-5- (2,7-difluoro-6-acetoxymethoxy-3-oxo-9-xanthenyl) phenoxy] -2- (2-amino-5-methylphenoxy) -2- (2-amino-5-methylphenoxy) ) A device that simultaneously measures fluorescence and chemiluminescence using ethane-N, N, N', N'-tetraacetic acid (Fluo-4 AM), excitation wavelength 495 nm, fluorescence wavelength 518 nm) (eg, CFL-C2000, It can be measured almost simultaneously using Hamamatsu Photonics Co., Ltd.).
（カルシウムイオン量に基づく評価ステップ）
上述のように測定されたカルシウムイオン量に基づき、被験物質がＮＯ産生を亢進する作用機序を同時に推定することができる。すなわち、カルシウムイオン量の測定値がカルシウムイオンの基準値を超える場合、被験物質が、カルモジュリンを介した一酸化窒素合成酵素の活性化により一酸化窒素産生を亢進すると判定することができる。また、カルシウムイオン量の測定値がカルシウムイオンの基準値以下の場合、被験物質が、一酸化窒素合成酵素を直接活性化することにより一酸化窒素産生を亢進すると判定することができる。本実施形態に係るスクリーニング方法は、このような評価を行うステップを更に含むものであってもよい。
(Evaluation step based on the amount of calcium ions)
Based on the amount of calcium ions measured as described above, the mechanism of action of the test substance to enhance NO production can be simultaneously estimated. That is, when the measured value of the amount of calcium ions exceeds the reference value of calcium ions, it can be determined that the test substance enhances nitric oxide production by activating nitric oxide synthase via calmodulin. Further, when the measured value of the amount of calcium ions is equal to or less than the reference value of calcium ions, it can be determined that the test substance enhances nitric oxide production by directly activating nitric oxide synthase. The screening method according to the present embodiment may further include a step of performing such an evaluation.
（カルシウムイオンの基準値）
評価の際のカルシウムイオンの基準値は、例えば、血管内皮細胞由来の培養細胞を被験物質に接触させずに測定したカルシウムイオン量の測定値を基準値とすることができる。このように基準値を設定することによって、被験物質との接触によるカルシウムイオン量の増減をより正確に評価することができる。また、例えば、カルモジュリンを介した一酸化窒素合成酵素の活性化により一酸化窒素産生を亢進する（又は一酸化窒素合成酵素を直接活性化することにより一酸化窒素産生を亢進する）ことが知られている既知物質と接触させて測定したカルシウムイオン量の測定値を基準値とすることもできる。
(Reference value of calcium ion)
As the reference value of calcium ion at the time of evaluation, for example, the measured value of the amount of calcium ion measured without contacting the cultured cells derived from vascular endothelial cells with the test substance can be used as the reference value. By setting the reference value in this way, it is possible to more accurately evaluate the increase or decrease in the amount of calcium ions due to contact with the test substance. Further, for example, it is known that activation of nitric oxide synthase via calmodulin enhances nitric oxide production (or direct activation of nitric oxide synthase enhances nitric oxide production). It is also possible to use the measured value of the amount of calcium ions measured in contact with a known substance as a reference value.
〔血管内皮細胞の一酸化窒素（ＮＯ）産生亢進剤〕
本実施形態のスクリーニング方法によれば、血管内皮細胞のＮＯ産生を亢進する被験物質を選抜することができる。選抜された被験物質は、血管内皮細胞のＮＯ産生を亢進する作用に基づき、血管内皮細胞のＮＯ産生亢進剤として利用することもできる。
[Nitric oxide (NO) production enhancer for vascular endothelial cells]
According to the screening method of the present embodiment, a test substance that enhances NO production of vascular endothelial cells can be selected. The selected test substance can also be used as a NO production-enhancing agent for vascular endothelial cells based on the action of enhancing NO production of vascular endothelial cells.
例えば、後述する実施例で具体的に確認しているとおり、桜島大根は、血管内皮細胞のＮＯ産生を亢進し、活性酸素（スーパーオキシド）の産生には影響を与えない。したがって、本発明の一実施形態として、桜島大根を有効成分として含有する、血管内皮細胞の一酸化窒素（ＮＯ）産生亢進剤が提供される。当該有効成分は、桜島大根（例えば、実、葉又は皮）の抽出物であることが好ましく、桜島大根の水溶性抽出物であることがより好ましい。水溶性抽出物は、例えば、メタノール／水／酢酸溶液（９０：９．５：０．５（体積比））で桜島大根を抽出して得られる、水溶性成分を含む抽出物であってよい。 For example, as specifically confirmed in Examples described later, Sakurajima radish enhances NO production of vascular endothelial cells and does not affect the production of active oxygen (superoxide). Therefore, as one embodiment of the present invention, there is provided a nitric oxide (NO) production enhancer for vascular endothelial cells containing Sakurajima radish as an active ingredient. The active ingredient is preferably an extract of Sakurajima radish (for example, fruit, leaf or skin), and more preferably a water-soluble extract of Sakurajima radish. The water-soluble extract may be, for example, an extract containing a water-soluble component obtained by extracting Sakurajima radish with a methanol / water / acetic acid solution (90: 9.5: 0.5 (volume ratio)). ..
本実施形態の血管内皮細胞のＮＯ産生亢進剤は、固体、液体（懸濁液を含む）、ペースト等のいずれの形状であってもよい。また、剤形としては、散剤、丸剤、顆粒剤、錠剤、シロップ剤、トローチ剤、カプセル剤等のいずれであってもよい。 The NO production enhancer for vascular endothelial cells of the present embodiment may be in any form of solid, liquid (including suspension), paste and the like. The dosage form may be any of powders, pills, granules, tablets, syrups, troches, capsules and the like.
上述の各種製剤は、有効成分（本発明のスクリーニング方法により選抜された被験物質）のみからなるものであってもよく、当該有効成分と薬学的に許容される添加剤との混合物であってもよい。また、上述の製剤は、例えば、有効成分、及び必要に応じて添加剤を混和し、上記剤形に成形することによって調製することができる。添加剤を含む場合、上記有効成分の含有量は、例えば、製剤全量を基準として、０．１～９０質量％である。 The above-mentioned various preparations may consist only of an active ingredient (a test substance selected by the screening method of the present invention), or may be a mixture of the active ingredient and a pharmaceutically acceptable additive. good. In addition, the above-mentioned preparation can be prepared, for example, by mixing the active ingredient and, if necessary, an additive, and molding into the above-mentioned dosage form. When the additive is contained, the content of the active ingredient is, for example, 0.1 to 90% by mass based on the total amount of the pharmaceutical product.
薬学的に許容される添加剤としては、例えば、賦形剤、結合剤、滑沢剤、崩壊剤、乳化剤、界面活性剤、基剤を挙げることができる。 Pharmaceutically acceptable additives include, for example, excipients, binders, lubricants, disintegrants, emulsifiers, surfactants and bases.
賦形剤としては、例えば、ラクトース、スクロース、デンプン、デキストリン等が挙げられる。結合剤としては、例えば、ポリビニルアルコール、アラビアゴム、トラガント、ゼラチン、ヒドロキシプロピルメチルセルロース、ヒドロキシプロピルセルロース、カルボキシメチルセルロースナトリウム、ポリビニルピロリドン等が挙げられる。滑沢剤としては、例えば、ステアリン酸マグネシウム、ステアリン酸カルシウム、タルク等が挙げられる。崩壊剤としては、例えば、結晶セルロース、寒天、ゼラチン、炭酸カルシウム、炭酸水素ナトリウム、デキストリン等が挙げられる。乳化剤又は界面活性剤としては、例えば、Ｔｗｅｅｎ６０、Ｔｗｅｅｎ８０、Ｓｐａｎ８０、モノステアリン酸グリセリン等が挙げられる。基剤としては、例えば、セトステアリルアルコール、ラノリン、ポリエチレングリコール、米糠油、魚油（ＤＨＡ、ＥＰＡ等）、オリーブ油等が挙げられる。 Examples of the excipient include lactose, sucrose, starch, dextrin and the like. Examples of the binder include polyvinyl alcohol, gum arabic, tragant, gelatin, hydroxypropylmethyl cellulose, hydroxypropyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidone and the like. Examples of the lubricant include magnesium stearate, calcium stearate, talc and the like. Examples of the disintegrant include crystalline cellulose, agar, gelatin, calcium carbonate, sodium hydrogencarbonate, dextrin and the like. Examples of the emulsifier or surfactant include Tween60, Tween80, Span80, glycerin monostearate and the like. Examples of the base include cetostearyl alcohol, lanolin, polyethylene glycol, rice bran oil, fish oil (DHA, EPA, etc.), olive oil and the like.
本実施形態の血管内皮細胞のＮＯ産生亢進剤は、ヒトに投与しても、非ヒト哺乳動物に投与してもよい。投与量及び投与方法は、投与される個体の状態、年齢等に応じて適宜決定することができる。好適な投与方法としては、例えば、経口投与が挙げられる。 The NO production enhancer for vascular endothelial cells of the present embodiment may be administered to humans or non-human mammals. The dose and administration method can be appropriately determined according to the condition, age, etc. of the individual to be administered. Suitable administration methods include, for example, oral administration.
本実施形態の血管内皮細胞のＮＯ産生亢進剤は、医薬品、医薬部外品、一般食品、サプリメント、特定保健用食品、機能性食品、栄養補助食品、食品添加物、飼料、飼料添加物等として使用することができる。 The NO production enhancer for vascular endothelial cells of the present embodiment is used as a drug, a non-medicinal product, a general food, a supplement, a food for specified health use, a functional food, a dietary supplement, a food additive, a feed, a feed additive, etc. Can be used.
以下、本発明を実施例に基づいてより具体的に説明する。ただし、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail based on examples. However, the present invention is not limited to the following examples.
〔実施例１：一酸化窒素量及びスーパーオキシド量の測定に基づく被験物質の評価〕
＜被験物質の調製＞
鹿児島県産の桜島大根（Ｒａｐｈａｎｕｓ ｓａｔｉｖｕｓ ｃｖ． Ｓａｋｕｒａｊｉｍａ Ｄａｉｋｏｎ）を可食部（実）と非可食部（葉及び皮）に分け、それぞれホモジナイズした後、凍結乾燥により粉末化した。次に、各粉末５０ｍｇに対し、メタノール／水／酢酸溶液（９０：９．５：０．５（体積比））２ｍＬを添加して混合し、超音波処理を５分間行ってから遠心分離（１，６１０×ｇ、１０分間、４℃）して残渣を取り除いた後、抽出溶媒を除去することにより、水溶性成分からなる被験物質を得た。
[Example 1: Evaluation of test substance based on measurement of nitric oxide amount and superoxide amount]
<Preparation of test substance>
Sakurajima radish (Sakurajima radish) from Kagoshima prefecture was divided into edible parts (fruits) and non-edible parts (leaves and skins), homogenized, and then pulverized by freeze-drying. Next, 2 mL of a methanol / water / acetic acid solution (90: 9.5: 0.5 (volume ratio)) was added to 50 mg of each powder and mixed, and ultrasonic treatment was performed for 5 minutes before centrifugation ( The residue was removed at 1,610 × g for 10 minutes at 4 ° C., and then the extraction solvent was removed to obtain a test substance composed of a water-soluble component.
また、比較のために、桜島大根に代えて青首大根（Ｒａｐｈａｎｕｓ ｓａｔｉｖｕｓ ｖａｒ． Ｌｏｎｇｉｐｉｎｎａｔｕｓ）を用いて、上記と同様に被験物質を調製した。 In addition, for comparison, a test substance was prepared in the same manner as above using radish (Radhanus radish var. Longipinnatus) instead of Sakurajima radish.
＜血管内皮細胞由来の培養細胞の調製＞
ブタ血管内皮細胞由来の培養細胞（ブタ大動脈内皮細胞）は、増殖培地（血管内皮細胞用培養メディウム（コスモ・バイオ（株）製）中、３７℃、５％ＣＯ２雰囲気下で維持及び継代した。
<Preparation of cultured cells derived from vascular endothelial cells>
Cultured cells derived from porcine vascular endothelial cells (porcine aortic endothelial cells) are maintained and passaged in a growth medium (culture medium for vascular endothelial cells (manufactured by Cosmo Bio Co., Ltd.) at 37 ° C. under a 5% CO 2 atmosphere. did.
＜一酸化窒素量及びスーパーオキシド量の同時計測＞
ブタ血管内皮細胞由来の培養細胞が８０％コンフルエントになった時点で、増殖培地５ｍＬに終濃度５０μＭとなるようにＮＯ検出用蛍光試薬（ＤＡＦ－２ＤＡ（ジアミノフルオレセイン－２ジアセテート），五稜化薬（株）製）を添加した。３７℃、５％ＣＯ２雰囲気下で１時間インキュベートした後、細胞を回収した。回収した細胞に１ｍＭ ＣａＣｌ２を含むＨＥＰＥＳ溶液を添加し、細胞密度を１．０～２．０×１０５ｃｅｌｌｓ／ｍＬに調製した細胞溶液を調製した。当該細胞溶液を２ｍＬキュベット（１０ｍｍ角ディスポキュベット）に移し、これに終濃度０．５μＭとなるようにスーパーオキシド検出用化学発光試薬（ＭＣＬＡ（６－（４－メトキシフェニル）－２－メチル－３，７－ジヒドロイミダゾ［１，２－ａ］ピラジン－３－オン塩酸塩），東京化成工業（株）製）を添加した。７分間プレインキュベートした後、キュベットを蛍光及び化学発光を同時に測定する装置（ＣＦＬ－Ｃ２０００，浜松ホトニクス株式会社製）にセットし、測定を開始した。測定開始から１２００ポイント（１０分）経過後に２ｍｇ／ｍＬの被験物質を１００μＬ添加し、更に４時間測定を継続した。被験物質として、桜島大根の可食部（実）から抽出した水溶性成分、比較として青首大根の可食部（実）から抽出した水溶性成分、及び対照（コントロール）として１ｍＭ ＣａＣｌ２及び０．５μＭ ＭＣＬＡを含むＨＥＰＥＳ溶液を使用した。
<Simultaneous measurement of nitric oxide and superoxide>
When the cultured cells derived from porcine vascular endothelial cells become 80% confluent, the fluorescent reagent for NO detection (DAF-2DA (diaminofluorescein-2 diacetate), pentodelation agent so that the final concentration is 50 μM in 5 mL of the growth medium. (Manufactured by Co., Ltd.) was added. After incubating for 1 hour in a 5% CO 2 atmosphere at 37 ° C, cells were harvested. A HEPES solution containing 1 mM CaCl 2 was added to the collected cells to prepare a cell solution having a cell density of 1.0 to 2.0 × 10 5 cells / mL. Transfer the cell solution to a 2 mL cuvette (10 mm square disposable cuvette) and add a chemiluminescent reagent for superoxide detection (MCLA (6- (4-methoxyphenyl) -2-methyl-3) to a final concentration of 0.5 μM. , 7-Dihydroimidazole [1,2-a] pyrazine-3-one hydrochloride), manufactured by Tokyo Chemical Industry Co., Ltd.) was added. After pre-incubating for 7 minutes, the cuvette was set in a device (CFL-C2000, manufactured by Hamamatsu Photonics Co., Ltd.) for measuring fluorescence and chemiluminescence at the same time, and the measurement was started. After 1200 points (10 minutes) from the start of the measurement, 100 μL of the test substance of 2 mg / mL was added, and the measurement was continued for another 4 hours. Water-soluble components extracted from the edible part (fruit) of Sakurajima radish as test substances, water-soluble components extracted from the edible part (fruit) of blue-necked radish for comparison, and 1 mM CaCl 2 and 0 as controls. A HEPES solution containing .5 μM MCLA was used.
＜カルシウムイオン量の計測＞
ＮＯ検出用蛍光試薬の代わりにＣａ２＋検出用蛍光試薬（Ｆｌｕｏ－４ ＡＭ（１－［２－アミノ－５－（２，７－ジフルオロ－６－アセトキシメトキシ－３－オキソ－９－キサンテニル）フェノキシ］－２－（２－アミノ－５－メチルフェノキシ）エタン－Ｎ，Ｎ，Ｎ’，Ｎ’－テトラ酢酸），同仁化学研究所製）を終濃度３μＭとなるように添加したこと以外は、＜一酸化窒素量及びスーパーオキシド量の同時計測＞と同様の操作を行い、カルシウムイオン量を計測した。
<Measurement of calcium ion amount>
Instead of NO detection fluorescent reagent, Ca 2+ detection fluorescent reagent (Fluoro-4 AM (1- [2-amino-5 (2,7-difluoro-6-acetoxymethoxy-3-oxo-9-xanthenyl) phenoxy) ] -2- (2-Amino-5-methylphenoxy) ethane-N, N, N', N'-tetraacetic acid), manufactured by Dojin Chemical Research Institute) was added to a final concentration of 3 μM. The same operation as <Simultaneous measurement of nitric oxide amount and superoxide amount> was performed to measure the calcium ion amount.
結果を図１～図３に示す。図１は、一酸化窒素（ＮＯ）量の経時変化を示すグラフである。図２は、スーパーオキシド（Ｏ２ －）量の経時変化を示すグラフである。図３は、細胞質内カルシウムイオン（Ｃａ２＋）量の経時変化を示すグラフである。 The results are shown in FIGS. 1 to 3. FIG. 1 is a graph showing the change over time in the amount of nitric oxide (NO). FIG. 2 is a graph showing the change over time in the amount of superoxide ( O2- ) . FIG. 3 is a graph showing the change over time in the amount of calcium ions (Ca 2+ ) in the cytoplasm.
桜島大根の可食部（実）から抽出した水溶性成分は、対照（コントロール）と比較して、一酸化窒素（ＮＯ）量を有意に増加させることが確認された（図１）。この結果は、桜島大根の可食部（実）から抽出した水溶性成分が、血管内皮細胞による一酸化窒素産生を亢進することを示している。当該亢進作用は、青首大根の可食部（実）から抽出した水溶性成分には認められず（図１）、桜島大根に特有の作用であった。 It was confirmed that the water-soluble component extracted from the edible portion (fruit) of Sakurajima radish significantly increased the amount of nitric oxide (NO) as compared with the control (control) (Fig. 1). This result indicates that the water-soluble component extracted from the edible part (fruit) of Sakurajima radish enhances nitric oxide production by vascular endothelial cells. The enhancing action was not observed in the water-soluble component extracted from the edible part (fruit) of the blue-necked radish (Fig. 1), and was an action peculiar to Sakurajima radish.
また、血管内皮機能の破綻に関与するスーパーオキシド（Ｏ２ －）量の増加は、桜島大根及び青首大根共に認められず、被験物質を添加することによる細胞傷害もないことが確認された（図２）。スーパーオキシド（Ｏ２ －）は、一酸化窒素との反応性が高く、一酸化窒素の消去に関与する（つまり、一酸化窒素の産生亢進効果を打ち消す）ほか、消去反応により生成するペルオキシナイトライト（ＯＮＯＯ－）は、スーパーオキシドよりも更に強い細胞傷害作用を有する。したがって、スーパーオキシド（Ｏ２ －）量を増加させない被験物質の方が好ましい。 In addition, no increase in the amount of superoxide ( O2- ) involved in the disruption of vascular endothelial function was observed in both Sakurajima radish and Ao-neck radish, and it was confirmed that there was no cytotoxicity due to the addition of the test substance (). Figure 2). Superoxide (O 2- ) is highly reactive with nitric oxide and is involved in the elimination of nitric oxide (that is, cancels the effect of enhancing the production of nitric oxide), and peroxynitrite produced by the elimination reaction. ( ONOO- ) has a stronger cytotoxic effect than superoxide. Therefore, a test substance that does not increase the amount of superoxide ( O2- ) is preferable.
さらに、桜島大根は一酸化窒素（ＮＯ）量と同様に、対照（コントロール）と比較して、細胞質内カルシウムイオン（Ｃａ２＋）量を有意に増加させることが確認された（図３）。この結果は、桜島大根の可食部（実）から抽出した水溶性成分による一酸化窒素産生の亢進には、細胞質内カルシウムイオン（Ｃａ２＋）濃度の上昇を介した一酸化窒素合成酵素（ＮＯＳ）活性の亢進が関与していることを示唆している。 Furthermore, it was confirmed that Sakurajima radish significantly increased the amount of calcium ion (Ca 2+ ) in the cytoplasm as compared with the control (control) as well as the amount of nitric oxide (NO) (Fig. 3). The results show that nitric oxide synthase (NOS) through an increase in intracellular calcium ion (Ca 2+ ) concentration was used to enhance nitric oxide production by the water-soluble component extracted from the edible part (fruit) of Sakurajima radish. ) It is suggested that the increase in activity is involved.
〔参考例１：従来法による一酸化窒素量の測定〕
一酸化窒素量の測定に従来用いられているグリース法（Ｇｒｉｅｓｓ法）により、桜島大根による一酸化窒素産生亢進効果を確認した。
[Reference Example 1: Measurement of Nitric Oxide by Conventional Method]
The effect of Sakurajima radish on increasing nitric oxide production was confirmed by the grease method (Grease method) conventionally used for measuring the amount of nitric oxide.
ブタ血管内皮細胞由来の培養細胞が８０％コンフルエントになった時点で、増殖培地５ｍＬに終濃度０．６ｍｇ／ｍＬ、１．２ｍｇ／ｍＬ、２．４ｍｇ／ｍＬ又は４．８ｍｇ／ｍＬとなるように桜島大根の実、皮及び葉から抽出した水溶性成分を添加した。同様に、終濃度１μｇ／ｍＬ、１０μｇ／ｍＬ、５０μｇ／ｍＬ又は１００μｇ／ｍＬとなるように桜島大根の実から抽出した水溶性成分を添加した。対照（コントロール）として、桜島大根の水溶性成分を添加しない培養細胞も用意した。３７℃、５％ＣＯ２雰囲気下でコンフルエントになるまでインキュベートした後、細胞を回収した。回収した細胞に対して、Ｇｒｉｅｓｓ法を利用したキット（ＮＯ２／ＮＯ３ Ａｓｓａｙ Ｋｉｔ－ＦＸ（Ｆｌｕｏｒｏｍｅｔｒｉｃ），（株）同仁化学研究所製）により一酸化窒素量を測定した。結果を図４に示す。 When the cultured cells derived from porcine vascular endothelial cells become 80% confluent, the final concentration should be 0.6 mg / mL, 1.2 mg / mL, 2.4 mg / mL or 4.8 mg / mL in 5 mL of growth medium. A water-soluble component extracted from the fruit, skin and leaves of Sakurajima radish was added to the mixture. Similarly, a water-soluble component extracted from Sakurajima radish nuts was added so as to have a final concentration of 1 μg / mL, 10 μg / mL, 50 μg / mL or 100 μg / mL. As a control, cultured cells to which the water-soluble component of Sakurajima radish was not added were also prepared. Cells were harvested after incubation at 37 ° C. under a 5% CO 2 atmosphere until confluent. The amount of nitric oxide was measured on the collected cells by a kit using the Griess method (NO 2 / NO 3 Assay Kit-FX (Fluorometric), manufactured by Dojin Chemical Research Institute Co., Ltd.). The results are shown in FIG.
図４は、グリース法（Ｇｒｉｅｓｓ法）により測定した一酸化窒素（ＮＯ）量を示すグラフである。桜島大根の実、皮及び葉から抽出した水溶性成分のいずれも、対照（コントロール）と比較して、一酸化窒素（ＮＯ）量の増加が認められた。また、桜島大根の実から抽出した水溶性成分は、濃度依存的にＮＯ量の増加が認められ（図４（Ｂ））、５０μｇ／ｍＬ以上の濃度で有効であることが分かった。 FIG. 4 is a graph showing the amount of nitric oxide (NO) measured by the grease method. An increase in the amount of nitric oxide (NO) was observed in all of the water-soluble components extracted from the fruits, skins and leaves of Sakurajima radish as compared with the control. In addition, the water-soluble component extracted from the fruit of Sakurajima radish showed an increase in the amount of NO in a concentration-dependent manner (FIG. 4 (B)), and was found to be effective at a concentration of 50 μg / mL or more.
（一酸化窒素合成酵素（ＮＯＳ）発現量の測定）
更に、桜島大根又は青首大根の実から抽出した水溶性成分を終濃度０．６ｍｇ／ｍＬ添加した培養細胞、及び対照（コントロール）の培養細胞を使用して、一酸化窒素合成酵素（ＮＯＳ）発現量を測定した。
(Measurement of nitric oxide synthase (NOS) expression level)
Furthermore, using cultured cells to which a water-soluble component extracted from Sakurajima radish or blue-necked radish nuts was added at a final concentration of 0.6 mg / mL, and control (control) cultured cells, nitric oxide synthase (NOS) was used. The expression level was measured.
上記培養細胞に対し、蛍光顕微鏡によりＮＯＳを測定するキット（ＥＺＣｅｌｌ Ｉｎｔｒａｃｅｌｌｕｌａｒ Ｎｉｔｒｉｃ Ｏｘｉｄｅ Ｓｙｎｔｈａｓｅ（ＮＯＳ） Ｄｅｔｅｃｔｉｏｎ Ｋｉｔ，バイオビジョン製）により細胞内ＮＯＳを検出した。結果を図５に示す。 In the above cultured cells, intracellular NOS was detected by a kit for measuring NOS with a fluorescence microscope (EZCell Intracellular Nitric Oxide Synthesis (NOS) Detection Kit, manufactured by Biovision). The results are shown in FIG.
図５（Ａ）は、桜島大根又は青首大根の実から抽出した水溶性成分を添加した培養細胞、及び対照（コントロール）の培養細胞に対して細胞内ＮＯＳ検出試験を実施したときの顕微鏡像を示す写真である。図５中、「ＮＯＳ」は蛍光顕微鏡像、「血管内皮細胞」は位相差顕微鏡像、「ｍｅｒｇｅ」は蛍光顕微鏡像と位相差顕微鏡像のマージ像である。ＮＯＳは細胞内で発現しており、桜島大根の実から抽出した水溶性成分を添加したときに、対照（コントロール）と比較して、最も活性化することが示された。なお、図５（Ｂ）は、細胞内ＮＯＳ検出試験において、蛍光の輝度毎に集計した細胞数のヒストグラムである。
FIG. 5 (A) is a microscopic image when an intracellular NOS detection test was carried out on cultured cells to which a water-soluble component extracted from the fruit of Sakurajima radish or blue-necked radish was added, and a control (control) cultured cell. It is a photograph showing. In FIG. 5, "NOS" is a fluorescence microscope image, "vascular endothelial cell" is a phase-contrast microscope image, and "merge" is a merged image of a fluorescence microscope image and a phase-contrast microscope image. It was shown that NOS is expressed intracellularly and is most activated when a water-soluble component extracted from Sakurajima radish fruit is added, as compared with a control. Note that FIG. 5B is a histogram of the number of cells aggregated for each fluorescence brightness in the intracellular NOS detection test.
Claims (1)
A nitric oxide-enhancing agent for vascular endothelial cells containing Sakurajima radish as an active ingredient.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2021172441A JP7097029B2 (en) | 2017-06-07 | 2021-10-21 | Screening method for substances that enhance nitric oxide production in vascular endothelial cells |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2017112908A JP7016107B2 (en) | 2017-06-07 | 2017-06-07 | Screening method for substances that enhance nitric oxide production in vascular endothelial cells |
| JP2021172441A JP7097029B2 (en) | 2017-06-07 | 2021-10-21 | Screening method for substances that enhance nitric oxide production in vascular endothelial cells |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2017112908A Division JP7016107B2 (en) | 2017-06-07 | 2017-06-07 | Screening method for substances that enhance nitric oxide production in vascular endothelial cells |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| JP2022009362A true JP2022009362A (en) | 2022-01-14 |
| JP2022009362A5 JP2022009362A5 (en) | 2022-03-01 |
| JP7097029B2 JP7097029B2 (en) | 2022-07-07 |
Family
ID=87888651
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2021172441A Active JP7097029B2 (en) | 2017-06-07 | 2021-10-21 | Screening method for substances that enhance nitric oxide production in vascular endothelial cells |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP7097029B2 (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61112024A (en) * | 1984-11-05 | 1986-05-30 | Horiuchi:Kk | Antioxidant food |
| JPS61239855A (en) * | 1985-04-15 | 1986-10-25 | Tottori Pref Gov Kajitsu Nogyo Kyodo Kumiai Rengokai | Food for improving physical constitution |
| WO2010035253A1 (en) * | 2008-09-28 | 2010-04-01 | Joshua Waldhorn | Effective nitric oxide generating preparations |
| CN103610152A (en) * | 2013-11-30 | 2014-03-05 | 大连润扬科技发展有限公司 | Nutritional cooking soup with effect of relieving neurasthenia and preparation method thereof |
| CN103610058A (en) * | 2013-11-30 | 2014-03-05 | 大连润扬科技发展有限公司 | Winter nourishing paste and processing process thereof |
-
2021
- 2021-10-21 JP JP2021172441A patent/JP7097029B2/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61112024A (en) * | 1984-11-05 | 1986-05-30 | Horiuchi:Kk | Antioxidant food |
| JPS61239855A (en) * | 1985-04-15 | 1986-10-25 | Tottori Pref Gov Kajitsu Nogyo Kyodo Kumiai Rengokai | Food for improving physical constitution |
| WO2010035253A1 (en) * | 2008-09-28 | 2010-04-01 | Joshua Waldhorn | Effective nitric oxide generating preparations |
| CN103610152A (en) * | 2013-11-30 | 2014-03-05 | 大连润扬科技发展有限公司 | Nutritional cooking soup with effect of relieving neurasthenia and preparation method thereof |
| CN103610058A (en) * | 2013-11-30 | 2014-03-05 | 大连润扬科技发展有限公司 | Winter nourishing paste and processing process thereof |
Non-Patent Citations (3)
| Title |
|---|
| 日本内科学会雑誌 (1995) VOL.84, NO.1, P.30-35, JPN6022009222, ISSN: 0004725298 * |
| 日本農芸化学会2015年度中四国・西日本支部合同大会講演要旨集 (2015) P.41(B-1), JPN6022009220, ISSN: 0004725300 * |
| 日本農芸化学会大会講演要旨集 (2016) 3E081, JPN6022009221, ISSN: 0004725299 * |
Also Published As
| Publication number | Publication date |
|---|---|
| JP7097029B2 (en) | 2022-07-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| So et al. | Uric acid transport and disease | |
| RU2191772C2 (en) | Derivatives of dialkyl ether, (pharmaceutical composition) | |
| Tokonami et al. | α-Ketoglutarate regulates acid-base balance through an intrarenal paracrine mechanism | |
| Naito et al. | Oxidative stress markers | |
| Yeh et al. | Long-term administration of advanced glycation end-product stimulates the activation of NLRP3 inflammasome and sparking the development of renal injury | |
| Bayrasheva et al. | Uninephrectomized high-fat-fed nicotinamide-streptozotocin-induced diabetic rats: a model for the investigation of diabetic nephropathy in type 2 diabetes | |
| Katakawa et al. | Taurine and magnesium supplementation enhances the function of endothelial progenitor cells through antioxidation in healthy men and spontaneously hypertensive rats | |
| JP2017132748A (en) | Antistress active substances extracted from oyster meat | |
| Sparger et al. | Investigation of immune cell markers in feline oral squamous cell carcinoma | |
| US9446013B2 (en) | Method of lowering circulating oxidized low density lipoprotein-beta-2-glycoprotein 1 complex for treatment of atherosclerosis | |
| JP7097029B2 (en) | Screening method for substances that enhance nitric oxide production in vascular endothelial cells | |
| JP7016107B2 (en) | Screening method for substances that enhance nitric oxide production in vascular endothelial cells | |
| Williams et al. | Renal nitrate clearance in chronic kidney disease | |
| Yin et al. | Tissue concentrations of 4-HNE in the black walnut extract model of laminitis: indication of oxidant stress in affected laminae | |
| Kirby et al. | Urinary MCP1 and Microalbumin increase prior to onset of Azotemia in mice with polycystic kidney disease | |
| JP2012057990A (en) | Freshness determination method of new meat texture | |
| Makita et al. | Importance of glycation in the acceleration of low density lipoprotein (LDL) uptake into macrophages in patients with diabetes mellitus | |
| CN115141145A (en) | Fluorescence probe for detecting lysosome hypobromous acid, preparation method and application | |
| JP6249511B2 (en) | Method for the diagnostic determination of the risks posed by changes in oxidative balance | |
| Li et al. | Erythrocyte osmotic fragility increases with serum advanced glycated end products in cigarette smokers | |
| Villalpando et al. | Beneficial Effects of Spirulina aqueous extract on vasodilator function of arteries from hypertensive rats | |
| Pantea et al. | Changes of oxidative stress indices and antioxidant system in the liver tissue on the administration of some coordination compound of copper, derivatives of thiosemicarbazide | |
| Wang et al. | Plasma ascorbic acid concentrations in healthy dogs | |
| Maharani et al. | The Effect of Red Dragon Fruit (Hylocereus Polyrhizus) Juice on Total Cholesterol Level in Women | |
| Huang et al. | Impaired peroxisomal import in Drosophila hepatocyte-like cells induces cardiac dysfunction through the pro-inflammatory cytokine Upd3 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20211028 |
|
| A711 | Notification of change in applicant |
Free format text: JAPANESE INTERMEDIATE CODE: A711 Effective date: 20211221 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20211221 |
|
| RD04 | Notification of resignation of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7424 Effective date: 20220104 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20220128 |
|
| A871 | Explanation of circumstances concerning accelerated examination |
Free format text: JAPANESE INTERMEDIATE CODE: A871 Effective date: 20220128 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20220217 |
|
| RD02 | Notification of acceptance of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7422 Effective date: 20220217 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20220224 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20220311 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20220509 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20220520 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20220615 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 7097029 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |