CN113545316A - 血根碱在制备trpa1通道激动剂中的应用 - Google Patents
血根碱在制备trpa1通道激动剂中的应用 Download PDFInfo
- Publication number
- CN113545316A CN113545316A CN202010332195.5A CN202010332195A CN113545316A CN 113545316 A CN113545316 A CN 113545316A CN 202010332195 A CN202010332195 A CN 202010332195A CN 113545316 A CN113545316 A CN 113545316A
- Authority
- CN
- China
- Prior art keywords
- sanguinarine
- trpa1
- channel
- trpa1 channel
- agonist
- 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
- INVGWHRKADIJHF-UHFFFAOYSA-N Sanguinarin Chemical compound C1=C2OCOC2=CC2=C3[N+](C)=CC4=C(OCO5)C5=CC=C4C3=CC=C21 INVGWHRKADIJHF-UHFFFAOYSA-N 0.000 title claims abstract description 203
- FCEXWTOTHXCQCQ-UHFFFAOYSA-N Ethoxydihydrosanguinarine Natural products C12=CC=C3OCOC3=C2C(OCC)N(C)C(C2=C3)=C1C=CC2=CC1=C3OCO1 FCEXWTOTHXCQCQ-UHFFFAOYSA-N 0.000 title claims abstract description 101
- 229940084560 sanguinarine Drugs 0.000 title claims abstract description 101
- YZRQUTZNTDAYPJ-UHFFFAOYSA-N sanguinarine pseudobase Natural products C1=C2OCOC2=CC2=C3N(C)C(O)C4=C(OCO5)C5=CC=C4C3=CC=C21 YZRQUTZNTDAYPJ-UHFFFAOYSA-N 0.000 title claims abstract description 101
- 108010036769 TRPA1 Cation Channel Proteins 0.000 title claims abstract description 71
- 102000012253 TRPA1 Cation Channel Human genes 0.000 title claims abstract description 71
- 239000000556 agonist Substances 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title description 5
- 208000002193 Pain Diseases 0.000 claims abstract description 30
- 108091006146 Channels Proteins 0.000 claims abstract description 29
- 230000036407 pain Effects 0.000 claims abstract description 27
- 238000011156 evaluation Methods 0.000 claims abstract description 13
- 230000003285 pharmacodynamic effect Effects 0.000 claims abstract description 12
- 238000012216 screening Methods 0.000 claims abstract description 11
- 229940035676 analgesics Drugs 0.000 claims abstract description 10
- 239000000730 antalgic agent Substances 0.000 claims abstract description 10
- 238000010171 animal model Methods 0.000 claims abstract description 6
- 238000010276 construction Methods 0.000 claims description 7
- 239000003814 drug Substances 0.000 claims description 7
- 241001465754 Metazoa Species 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 6
- 230000008685 targeting Effects 0.000 claims description 6
- 201000010099 disease Diseases 0.000 claims description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 210000002569 neuron Anatomy 0.000 abstract description 45
- 230000004044 response Effects 0.000 abstract description 13
- 230000001270 agonistic effect Effects 0.000 abstract description 10
- 101150092793 Trpa1 gene Proteins 0.000 abstract description 6
- 238000003209 gene knockout Methods 0.000 abstract description 6
- 230000003213 activating effect Effects 0.000 abstract description 5
- 230000000144 pharmacologic effect Effects 0.000 abstract description 5
- 230000001419 dependent effect Effects 0.000 abstract description 4
- 101000764872 Homo sapiens Transient receptor potential cation channel subfamily A member 1 Proteins 0.000 abstract description 3
- 230000000903 blocking effect Effects 0.000 abstract description 3
- 230000008587 neuronal excitability Effects 0.000 abstract description 2
- 102100026186 Transient receptor potential cation channel subfamily A member 1 Human genes 0.000 abstract 2
- ZOJBYZNEUISWFT-UHFFFAOYSA-N allyl isothiocyanate Chemical compound C=CCN=C=S ZOJBYZNEUISWFT-UHFFFAOYSA-N 0.000 description 30
- 210000004027 cell Anatomy 0.000 description 25
- 241000699670 Mus sp. Species 0.000 description 17
- 235000016720 allyl isothiocyanate Nutrition 0.000 description 15
- 230000000694 effects Effects 0.000 description 14
- 230000036982 action potential Effects 0.000 description 12
- 239000011575 calcium Substances 0.000 description 12
- 239000012528 membrane Substances 0.000 description 12
- 241000699666 Mus <mouse, genus> Species 0.000 description 11
- 239000011521 glass Substances 0.000 description 11
- 239000000243 solution Substances 0.000 description 10
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 229910001424 calcium ion Inorganic materials 0.000 description 9
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 8
- 229910052791 calcium Inorganic materials 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 230000035772 mutation Effects 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 206010001497 Agitation Diseases 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 241000894007 species Species 0.000 description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- 101100482465 Caenorhabditis elegans trpa-1 gene Proteins 0.000 description 5
- 230000004913 activation Effects 0.000 description 5
- 239000012091 fetal bovine serum Substances 0.000 description 5
- 238000003384 imaging method Methods 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- JVJFIQYAHPMBBX-UHFFFAOYSA-N 4-hydroxynonenal Chemical compound CCCCCC(O)C=CC=O JVJFIQYAHPMBBX-UHFFFAOYSA-N 0.000 description 4
- HGINCPLSRVDWNT-UHFFFAOYSA-N Acrolein Chemical compound C=CC=O HGINCPLSRVDWNT-UHFFFAOYSA-N 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 230000004941 influx Effects 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- 230000003204 osmotic effect Effects 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 102000005962 receptors Human genes 0.000 description 4
- 108020003175 receptors Proteins 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 210000003594 spinal ganglia Anatomy 0.000 description 4
- 102000042565 transient receptor (TC 1.A.4) family Human genes 0.000 description 4
- 108091053409 transient receptor (TC 1.A.4) family Proteins 0.000 description 4
- KDXKERNSBIXSRK-RXMQYKEDSA-N D-lysine Chemical compound NCCCC[C@@H](N)C(O)=O KDXKERNSBIXSRK-RXMQYKEDSA-N 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 3
- 230000008484 agonism Effects 0.000 description 3
- 230000000202 analgesic effect Effects 0.000 description 3
- 238000007664 blowing Methods 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 230000002708 enhancing effect Effects 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000004054 inflammatory process Effects 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 230000001537 neural effect Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 230000001953 sensory effect Effects 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 102000008102 Ankyrins Human genes 0.000 description 2
- 108010049777 Ankyrins Proteins 0.000 description 2
- GRRNUXAQVGOGFE-UHFFFAOYSA-N Hygromycin-B Natural products OC1C(NC)CC(N)C(O)C1OC1C2OC3(C(C(O)C(O)C(C(N)CO)O3)O)OC2C(O)C(CO)O1 GRRNUXAQVGOGFE-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 102000003610 TRPM8 Human genes 0.000 description 2
- 102000003566 TRPV1 Human genes 0.000 description 2
- 102000003565 TRPV2 Human genes 0.000 description 2
- 102000003568 TRPV3 Human genes 0.000 description 2
- 102000003567 TRPV4 Human genes 0.000 description 2
- 101150098315 TRPV4 gene Proteins 0.000 description 2
- 101150111302 Trpm8 gene Proteins 0.000 description 2
- 101150016206 Trpv1 gene Proteins 0.000 description 2
- 101150077905 Trpv2 gene Proteins 0.000 description 2
- 101150043371 Trpv3 gene Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000010009 beating Methods 0.000 description 2
- 230000006399 behavior Effects 0.000 description 2
- HUCVOHYBFXVBRW-UHFFFAOYSA-M caesium hydroxide Chemical compound [OH-].[Cs+] HUCVOHYBFXVBRW-UHFFFAOYSA-M 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000012377 drug delivery Methods 0.000 description 2
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000010304 firing Methods 0.000 description 2
- LPEPZBJOKDYZAD-UHFFFAOYSA-N flufenamic acid Chemical compound OC(=O)C1=CC=CC=C1NC1=CC=CC(C(F)(F)F)=C1 LPEPZBJOKDYZAD-UHFFFAOYSA-N 0.000 description 2
- 229960004369 flufenamic acid Drugs 0.000 description 2
- YFHXZQPUBCBNIP-UHFFFAOYSA-N fura-2 Chemical compound CC1=CC=C(N(CC(O)=O)CC(O)=O)C(OCCOC=2C(=CC=3OC(=CC=3C=2)C=2OC(=CN=2)C(O)=O)N(CC(O)=O)CC(O)=O)=C1 YFHXZQPUBCBNIP-UHFFFAOYSA-N 0.000 description 2
- 230000005484 gravity Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- GRRNUXAQVGOGFE-NZSRVPFOSA-N hygromycin B Chemical compound O[C@@H]1[C@@H](NC)C[C@@H](N)[C@H](O)[C@H]1O[C@H]1[C@H]2O[C@@]3([C@@H]([C@@H](O)[C@@H](O)[C@@H](C(N)CO)O3)O)O[C@H]2[C@@H](O)[C@@H](CO)O1 GRRNUXAQVGOGFE-NZSRVPFOSA-N 0.000 description 2
- 229940097277 hygromycin b Drugs 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 210000005036 nerve Anatomy 0.000 description 2
- 210000000653 nervous system Anatomy 0.000 description 2
- 239000002547 new drug Substances 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 210000001428 peripheral nervous system Anatomy 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 208000017520 skin disease Diseases 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- ODLHGICHYURWBS-LKONHMLTSA-N trappsol cyclo Chemical compound CC(O)COC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)COCC(O)C)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1COCC(C)O ODLHGICHYURWBS-LKONHMLTSA-N 0.000 description 2
- 238000011870 unpaired t-test Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- YQXYQOXRCNEATG-ZAYJLJTISA-N (2s,3s,6r)-3-[[(3r)-3-amino-5-[carbamimidoyl(methyl)amino]pentanoyl]amino]-6-(4-amino-2-oxopyrimidin-1-yl)-3,6-dihydro-2h-pyran-2-carboxylic acid;hydrochloride Chemical compound Cl.O1[C@H](C(O)=O)[C@@H](NC(=O)C[C@H](N)CCN(C)C(N)=N)C=C[C@@H]1N1C(=O)N=C(N)C=C1 YQXYQOXRCNEATG-ZAYJLJTISA-N 0.000 description 1
- UHHHTIKWXBRCLT-VDBOFHIQSA-N (4s,4ar,5s,5ar,6r,12ar)-4-(dimethylamino)-1,5,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4a,5,5a,6-tetrahydro-4h-tetracene-2-carboxamide;ethanol;hydrate;dihydrochloride Chemical compound O.Cl.Cl.CCO.C1=CC=C2[C@H](C)[C@@H]([C@H](O)[C@@H]3[C@](C(O)=C(C(N)=O)C(=O)[C@H]3N(C)C)(O)C3=O)C3=C(O)C2=C1O.C1=CC=C2[C@H](C)[C@@H]([C@H](O)[C@@H]3[C@](C(O)=C(C(N)=O)C(=O)[C@H]3N(C)C)(O)C3=O)C3=C(O)C2=C1O UHHHTIKWXBRCLT-VDBOFHIQSA-N 0.000 description 1
- 241000284466 Antarctothoa delta Species 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108091005462 Cation channels Proteins 0.000 description 1
- 102000010831 Cytoskeletal Proteins Human genes 0.000 description 1
- 108010037414 Cytoskeletal Proteins Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 206010065390 Inflammatory pain Diseases 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 101100482469 Mus musculus Trpa1 gene Proteins 0.000 description 1
- 208000028389 Nerve injury Diseases 0.000 description 1
- 208000007920 Neurogenic Inflammation Diseases 0.000 description 1
- 208000001294 Nociceptive Pain Diseases 0.000 description 1
- 102000016979 Other receptors Human genes 0.000 description 1
- 206010033425 Pain in extremity Diseases 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 241000218180 Papaveraceae Species 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 101100482470 Rattus norvegicus Trpa1 gene Proteins 0.000 description 1
- 241001093501 Rutaceae Species 0.000 description 1
- 229940124639 Selective inhibitor Drugs 0.000 description 1
- 241001223864 Sphyraena barracuda Species 0.000 description 1
- 229940123223 TRPA1 agonist Drugs 0.000 description 1
- 102000003563 TRPV Human genes 0.000 description 1
- 108060008564 TRPV Proteins 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 241001106476 Violaceae Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 208000005298 acute pain Diseases 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000036592 analgesia Effects 0.000 description 1
- 230000001088 anti-asthma Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 239000000924 antiasthmatic agent Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 238000012865 aseptic processing Methods 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 238000009227 behaviour therapy Methods 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- 229930015421 benzophenanthridine alkaloid Natural products 0.000 description 1
- 229930189065 blasticidin Natural products 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000005388 borosilicate glass Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000009460 calcium influx Effects 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 208000037893 chronic inflammatory disorder Diseases 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002999 depolarising effect Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229960001172 doxycycline hyclate Drugs 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 210000003194 forelimb Anatomy 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000003304 gavage Methods 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 102000052408 human TRPA1 Human genes 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 210000002977 intracellular fluid Anatomy 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 210000003141 lower extremity Anatomy 0.000 description 1
- 210000004705 lumbosacral region Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000008764 nerve damage Effects 0.000 description 1
- 210000004126 nerve fiber Anatomy 0.000 description 1
- 230000001272 neurogenic effect Effects 0.000 description 1
- 230000003955 neuronal function Effects 0.000 description 1
- 230000000269 nucleophilic effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000002263 peptidergic effect Effects 0.000 description 1
- 210000002951 peptidergic neuron Anatomy 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 230000001681 protective effect Effects 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
- 230000004648 relaxation of smooth muscle Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 210000001170 unmyelinated nerve fiber Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4741—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having oxygen as a ring hetero atom, e.g. tubocuraran derivatives, noscapine, bicuculline
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/15—Humanized animals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/30—Animals modified by surgical methods
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/03—Animals modified by random mutagenesis, e.g. using ENU, chemicals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Environmental Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Animal Husbandry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Public Health (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明提供了血根碱在制备TRPA1通道激动剂中的应用。血根碱可作为工具分子用于构建疼痛动物模型，应用于镇痛药物的临床前药效学评价。本发明显示了血根碱具有强效的TRPA1激动活性和选择性，可作为TRPA1通道激动剂工具分子应用于以TRPA1通道为靶点的通道调制剂的筛选中；DRG神经元电生理记录显示，血根碱可以通过特异性地激活DRG神经元上的TRPA1通道增强神经元的兴奋性；在C57小鼠上通过足底注射血根碱可以计量依赖性诱导产生疼痛反应，而TRPA1基因敲除和药理学阻断显著缓解疼痛反应，实现了血根碱在以TRPA1为靶点的镇痛药物临床前药效学评价中的应用。
Description
技术领域
本发明属于药物研发领域，具体地涉及血根碱在制备TRPA1通道激动剂中的应用，其中，血根碱可作为TRPA1通道激动剂工具分子应用于以TRPA1通道为靶点的通道调节剂的筛选。此外，血根碱可作为工具分子构建动物疼痛模型，应用于镇痛药物的临床前药效学评价。
背景技术
血根碱(Sanguinarine，SA)是一种主要存在于罂粟科、蓝堇科及芸香科植物中的苯并菲啶类生物碱，其化学名为13-methyl[1.3]benzodioxolo[5,6,-c]-1,3-dioxolo[4,5-i]phenanthridinium，分子式为C20H14NO4，相对分子质量为332.33，化学结构式为：
临床上，血根碱在治疗皮肤病、肿瘤、妇科疾病等方面具有重要作用。国内外诸多药理学研究表明，血根碱具有杀虫、抗炎、抗菌、抗氧化、抗肿瘤、调节平滑肌舒张、改善肝功能、增强免疫力等作用(Choi WY.等，Anticancer Res.2009Nov；29(11):4457-65.)。
瞬时受体电位通道(Transient receptor potential，TRP)是由存在于细胞膜上的一类重要阳离子通道构成的蛋白超家族，受到诸多不同机制的调控，包括环境中的物理、化学刺激，细胞内的信号转导通路等。TRP通道在机体内作为细胞感受器分子介导对环境中各种刺激的响应，通过对细胞内钙离子浓度的调节参与一系列的生理病理进程。TRPA1(Transient receptor potential ankyrin subtype 1)是TRP通道超家族中重要的一员。TRPA1通道为四个同源亚基形成的四聚体，每个亚基具有6次跨膜结构S1-S6，N端和C端均位于胞内，其中S5-S6结构域中的亲水区域形成离子通透性孔道；N端具有16个串联的锚蛋白重复序列，序列中的半胱氨酸和赖氨酸残基为亲电性激动剂和氧化剂靶向通道提供活性位点(Macpherson LJ等，J Neurosci.2007Oct17；27(42):11412-5.；Wang L等，J BiolChem.2012Feb 24；287(9):6169-76.)；同时，锚蛋白重复序列可能通过与细胞内骨架蛋白的相互作用影响通道嵌入细胞膜及其在膜上的定位并参与通道对机械刺激的感知(Niliusand Owsianik,Genome Biol.2011；12(3):218.)。哺乳动物的TRPA1通道在多组织器官中分布广泛，如外周感觉神经系统、胃肠道系统和呼吸系统等。TRPA1在外周感觉神经系统中主要表达在Aδ和C纤维初级传入的肽能和非肽能神经元中(Andrade EL等，PharmacolTher.2012Feb；133(2):189-204.)，如背根神经节(DRG)、三叉神经节(TG)和结状神经节(NG)神经元，且呈现与TRPV1通道共表达的特点(La JH等，Neuroscience.2011Jul 14；186:179-87.)。
TRPA1作为细胞膜上的多模态“感受器分子”，受到多重因素的调控，包括pH、O2水平、亲电性和亲核性分子、温度、膜脂、电压、细胞内Ca2+、痕量重金属等。TRPA1存在着多种激活途径，包括直接的配体激活和其他受体介导的间接激活，几乎可以对每一个潜在的有害外部刺激和与损伤相关的内源性信号产生响应，其激活会向外周神经系统传递感知觉信号，进而触发保护性行为反应和启动组织修复机制。在多种疾病动物模型中获得的数据有力地表明，TRPA1与疼痛和炎症有关，并且在慢性炎症性疾病和组织损伤的发生、发展和维持中发挥重要作用，包括哮喘、糖尿病、关节炎和皮肤病等。在人体中，TRPA1通道主要分布于外周，在中枢神经系统和心脏中表达丰度较低，因此降低了该靶点的中枢和心脏副作用风险(Story GM等，Cell.2003Mar 21；112(6):819-29.)，在新药研发中具有极大的前景。TRPA1通道已经成为镇痛和抗哮喘的新型治疗靶点，靶向TRPA1的药物开发符合当前该领域对新药的医疗需求(Nilius B等，Pharmacol Rev.2014Jul；66(3):676-814.)。
目前，使用不同类型的TRPA1激动剂作为工具分子深入探索TRPA1通道的功能，评价此通道在多种病理性疾病中作为治疗靶点的可行性已经成为此领域的重要研究内容。TRPA1通道小分子激动剂异硫氰酸烯丙酯(Allyl isothiocyanate,AITC)、丙烯醛(Acrolein)和4-羟基壬烯醛(4-Hydroxynonenal,HNE)等的发现，推动了TRPA1通道参与机体各种生理和病理性过程的机制研究，为相应的药物研发提供了坚实的理论基础和铺垫。然而，研究中发现已有的经典激动剂存在多种问题，包括分子选择性欠佳，水溶性差，易挥发，具有刺激性气味等。如AITC在科学实验中被广泛用作TRPA1的特异性激动剂诱导急性疼痛和神经源性炎症的发生，但是最近的研究表明TRPA1并不是AITC在体内的唯一靶点，在体内给与AITC后产生的炎症和伤害性疼痛反应可能部分由TRPV1介导。同时，以其作为阳性激动剂进行TRPA1的抑制剂筛选时，其分子不稳定、易挥发给化合物筛选工作造成诸多不便。因此，无论是为了探索新的TRPA1功能，还是克服已有经典激动剂的理化性质缺陷，都亟需寻找新型的TRPA1激动剂。
本研究应用基于电流检测的IonWorks Barracuda(IWB)高通量筛选系统，结合手动膜片钳电生理记录等研究手段，发现血根碱具有强效的TRPA1通道激动活性，能够显著增加DRG神经元的兴奋性，诱导动作电位的发放；同时，在C57小鼠动物模型上，足底注射血根碱可以诱导产生TRPA1依赖的疼痛反应。研究结果显示了血根碱作为TRPA1激动剂在TRPA1通道调制剂的筛选、构建动物疼痛模型并进行镇痛药物临床前药效学评价中的应用前景。
发明内容
本发明提供了血根碱在制备TRPA1通道激动剂中的应用。
在上述应用中，血根碱作为TRPA1通道阳性激动剂工具分子，可应用于以TRPA1通道为靶点的通道调制剂的筛选。
此外，在上述应用中，所述TRPA1通道激动剂是用于构建动物疼痛模型的药剂，其中所述模型可用于镇痛药物的临床前药效学评价。
其中，所述TRPA1通道激动剂是用于构建TRPA1通道相关疾病模型的药剂，并对以TRPA1通道为靶点的化合物进行临床前药效学评价。
根据本发明，提供了血根碱作为TRPA1通道阳性激动剂工具分子用于以TRPA1通道为靶点筛选通道调制剂的应用或用于制备以TRPA1通道为靶点筛选通道调制剂的药剂的应用。
根据本发明，提供了血根碱作为工具分子用于制备构建动物疼痛模型的药剂的应用，其中，所述模型可用于进行镇痛药物的临床前药效学评价。
本发明显示了血根碱具有强效的TRPA1激动效应；通道选择性表明血根碱的激动活性在TRP通道超家族中具有高度选择性；在DRG神经元上，血根碱通过选择性地激活TRPA1通道引起神经元胞外钙离子的内流、诱导动作电位的发放；在C57小鼠足底注射血根碱可以诱导产生显著的剂量依赖性疼痛反应，而TRPA1基因敲除和药理学阻断显著缓解疼痛反应，实现了血根碱在诱发的动物疼痛模型的构建并在该模型上进行镇痛药物的临床前药效学评价中的应用。
附图说明
图1为血根碱(Sanguinarine，SA)对TRPA1通道的激动效应，显示了血根碱是TRPA1通道选择性激动剂，其中：A-C：上部分别为血根碱激活人TRPA1(hTRPA1)、小鼠TRPA1(mTRPA1)和大鼠TRPA1(rTRPA1)通道电流的时程图(统计均为+100mV处通道电流值)；下部分别为相应的I-V曲线；D为血根碱激活三个物种TRPA1通道的电流密度统计；E为血根碱激活mTRPA1的DRC曲线；F为给予1μM血根碱后TRPA1、TRPV1、TRPV2、TRPV3、TRPV4、TRPM8通道的电流密度统计；
图2为血根碱显著增强DRG神经元兴奋性，其中：A为给予血根碱或AITC时，野生型小鼠DRG神经元细胞内钙离子浓度变化；B为给予血根碱或AITC时，TRPA1基因敲除小鼠的DRG神经元细胞内钙离子浓度变化；C为野生型小鼠DRG神经元中，血根碱和AITC引起的神经元细胞内钙离子浓度变化统计；D为野生型小鼠的DRG神经元中，血根碱或AITC对神经元细胞膜电位和动作电位发放的影响；E为TRPA1基因敲除小鼠的DRG神经元中，血根碱或AITC对神经元细胞膜电位和动作电位发放的影响；
图3为TRPA1通道N端活性位点突变对血根碱激动效应的影响，其中：A-G分别为血根碱诱导的野生型TRPA1(WT)及携带单个位点突变的TRPA1通道代表性电流时程图；H为血根碱激活野生型和突变体TRPA1通道的电流密度统计，其中FA为TRPA1通道非亲电性激动剂氟灭酸(Flufenamic acid)；
图4为血根碱在小鼠中通过激活TRPA1诱导产生疼痛反应。其中：A为不同剂量的血根碱在小鼠上诱导产生舔足时间的时程统计；B为不同剂量的血根碱在60min内小鼠上诱导产生的总的舔足时间；C-D为0.1％血根碱诱导的足部疼痛模型中，HC030031(HC)的镇痛效应统计；E-F为在野生型(TRPA1+/+)和TRPA1基因敲除(TRPA1-/-)小鼠中，足底注射0.1％血根碱后60分钟内小鼠的舔足时间统计。
附图中显示的实验数据是采用非配对t检验进行统计分析的，组间存在差异的显著性以ns(P＞0.05)，*(P＜0.05)，**(P＜0.01)，***(P＜0.001)表示。
具体实施方式
以下结合技术方案和附图详细说明本发明的具体实施例。
实施例1血根碱强效激动TRPA1通道电流
1.TRPA1稳转株细胞培养
TRPA1-HEK293稳转细胞(上海药物研究所国际科学家工作站)
培养基配方：DMEM basic(Gibco)，加入10％胎牛血清(Fetal bovine Fetalbovine serum,FBS)，1％双抗(青霉素-链霉素)，潮霉素B(Hygromycin B，50μg/ml)和杀稻瘟菌素(BlasticidinS HCl，5μg/ml)。
细胞培养在37℃，5％CO2，具有饱和湿度的无菌环境中进行，使用0.25％胰酶消化传代和铺板。TRPA1稳转株细胞使用多四环素(Doxycycline hyclate，0.3μg/ml)诱导TRPA1的表达，24h后进行手动膜片钳电生理记录。
按照5×105个/ml的密度将培养的HEK293细胞接种到6孔板中，细胞密度生长到80％左右时按照Lipo3000的标准将EGFP质粒与目的质粒按照1:9的比例进行共同转染，转染6h后换液，再培养至24h后进行手动膜片钳电生理记录。
2.HEK293细胞培养和转染
HEK293细胞使用加入10％胎牛血清(Fetal bovine Fetal bovine serum,FBS)和1％双抗(青霉素-链霉素)的DMEM basic(Gibco)培养基培养。细胞培养在37℃，含有5％CO2、湿度饱和的恒温无菌细胞培养箱中。按照5×105个/ml的密度将培养的HEK293细胞接种到6孔板中，细胞密度生长到80％左右时按照Lipo3000的标准将EGFP质粒与目的质粒按照1:9的比例进行共同转染，转染6h后换液，再培养至24h后进行手动膜片钳电生理记录。
3.全细胞电生理记录
电生理记录采用HEKA EPC-10膜片钳放大器(德国HEKA公司)，信号采集使用pClamp10.0软件，采样频率为10kHz，滤波为2.9kHz。硼硅酸盐玻璃毛细管拉制成电极，内部灌注细胞內液后电极电阻为2-3MΩ。记录在室温下进行，化合物是由DAD-12快速给药系统(美国ALA公司)给予细胞，依赖重力提供驱动力。电生理记录使用内外液配方如下表：
TRPA1全细胞记录使用细胞外液
用NaOH调节pH至7.4，渗透压调至300-310mOsm，保存于4℃备用
TRPA1全细胞记录使用细胞內液
用CsOH调节pH至7.2，渗透压调至295-300mOsm，保存于4℃备用
使用DMSO溶解配置10mM浓度血根碱母液，-20℃冷藏，实验中稀释使用(测试浓度中DMSO体积百分比量为0.1％)。电生理记录中细胞钳制在0mV，给予斜坡电压(-100mV～+100mV 300ms)刺激，电流稳定后通过快速给药系统给予1μM血根碱，记录+100mV时TRPA1通道电流。
4.实验结果分析
电生理数据分析利用Clampfit 10.2(Molecular Devices,Sunnyvale,CA)，再利用GraphPad Prim 5(GraphPad Sofware,San Diego,CA)，剂量效应曲线用希尔等式(Hillequation)拟合。采用非配对t检验对数据进行统计分析，组间存在差异的显著性以ns(P＞0.05)，*(P＜0.05)，**(P＜0.01)，***(P＜0.001)表示。
如图1所示，血根碱激活hTRPA1通道的EC50为0.09(0.04-0.13)μM，起效浓度在0.01μM左右。同时，血根碱激活的通道电流可被TRPA1的特异性阻断剂HC030031(HC)几乎完全抑制，说明膜片钳记录的为特异性TRPA1电流，而不是血根碱诱导的其他非特异性效应。除了hTRPA1通道，还检测了血根碱对其他物种TRPA1通道的活性，如小鼠和大鼠，以确证血根碱的激动活性是否有物种特异性。如图1所示，在hTRPA1、mTRPA1和rTRPA1稳转细胞中，1μM血根碱均能激动出很强的通道电流，且电流呈现典型的TRPA1通道特性：①三种物种TRPA1通道电流均能被HC030031抑制；②I-V曲线得出的反转电位(Reversal potential)均在0mV。电流密度统计结果显示(图1的D)，三种物种TRPA1通道均呈现很高的电流密度，且没有显著性差异，表明血根碱对TRPA1通道的激动无物种特异性。
在机体的外周神经系统中，除了TRPA1通道表达外，还分布有其他多种神经性TRP通道。本申请中采用各TRP通道稳转细胞株，结合手动膜片钳检测进一步考察血根碱是否对TRP通道超家族其他成员同样具有激动。如图1的F所示，血根碱只对TRPA1通道具有激动效应，对检测的其TRPV1,TRPV2,TRPV3,TRPV4,和TRPM8通道没有明显的激动效应。同时，这些TRP通道却可以被各自的阳性激动剂显著激活，诱导出很强的通道电流，且通道电流能被对应的抑制剂显著阻断。这表明血根碱对TRPA1通道的激动效应具有特异性。
已有研究表明，TRPA1通道N端存在着一系列活性半胱氨酸残基和赖氨酸残基与通道激活开放相关，如Cys414、Cys421、Cys621、Cys641、Cys665和Lys710等。本申请中对各个氨基酸位点进行单突变处理，如将半胱氨酸单突变为丙氨酸(C414A,C421A,C621A,C641A和C665A)，将赖氨酸突变为精氨酸(K710R)。手动膜片钳电生理记录显示(如图3)，单突变C414A和C621A几乎可完全消除血根碱激活的TRPA1通道电流，其余单突变通道虽然仍有活性，但与野生型(wild type,WT)相比，血根碱激动的全细胞电流显著降低。这表明C414和C621两个位点对于血根碱诱导的TRPA1通道开放至关重要；而C421，C641，C665和K710单突变处理后通道电流显著降低，表明这些位点参与血根碱的激动过程，但并不是核心位点。
以上实验结果表明血根碱是TRPA1通道的选择性激动剂，因此可作为TRPA1通道的阳性激动剂工具分子应用于TRPA1通道调制剂的筛选。
实施例2血根碱增强DRG神经元兴奋性、诱导疼痛模型
目前认为钙离子大量流入(钙负载)和神经兴奋性升高是机体神经损伤的两个重要诱因。钙成像检测和手动膜片钳实验都证明血根碱对异源性表达的TRPA1通道具有强烈的激动活性，提示血根碱可能增大神经元内源性的TRPA1活性，增强神经元的兴奋性。发明人分离培养了C57小鼠的背根神经节神经元(DRG)，检测了血根碱对相应DRG神经元的钙流入的影响，以及对神经元膜电位和动作电位发放的影响。
背根神经节神经元(DRG)神经元的分离与培养：
(1)试剂和器械PBS缓冲液，DMEM：F12+10％FBS，Neurobasal+B27，Poly-D-lysine，酶溶液，溶液都经过无菌处理；小剪刀，镊子，眼科剪，所有器械用75％酒精消毒处理。
(2)包被玻片，8mm玻片彻底清洗后浸泡在95％酒精中，置于摇床上48h，用镊子逐个取出置于火焰上燃去酒精，将玻片放在适当大小的Dish中，用Poly-D-lysine(0.01％)包被玻片，直到神经元分离实验开始前洗去Poly-D-lysine，包被时间约为1h，用无菌PBS缓冲液快速清洗玻片3遍，敞开置于超净台中，晾干后可使用。
(3)分离DRG神经元，取C57小鼠1只，脱颈处死；小鼠表面皮肤用75％酒精消毒后带入细胞间；剪取小鼠脊柱的腰椎部分(前肢到后肢段)，纵向剪开，置于冰冷的PBS溶液中；去除脊柱中的脊髓和膜，将椎间孔中的神经核团暴露出来，用精细镊子挑出DRG神经核团，去除附着的神经纤维、结缔组织膜和血块；将DRG神经核团剪碎，悬液吸入15ml离心管中，稍离心后小心吸去上清，加入酶溶液(1mg/ml Colleagenase+0.25mg/ml Trypsin，DMEM：F12溶解之后过滤除菌)，稍微晃动离心管使组织完全散开，置于37℃培养箱中消化30min(消化过程中应最大限度地保证组织小块均匀接触酶溶液，同时拧松离心管盖，保证消化环境的CO2浓度和渗透压)。
(4)吹打，消化时间到时，轻轻吸出上层清液，剩余的组织块悬浊液用2ml含有血清的培养液清洗3遍；将酶溶液清洗干净后，加入1ml含血清培养液轻轻吹打几次，吸出肉眼可见的组织块到一个新的离心管中，加入1ml含血清培养液轻轻吹打几次，重复上述步骤2-3次，可收集到3-4ml神经细胞悬液。
(5)培养得到的细胞悬液用70μm的滤膜进行过滤，滤液接种在预先包被的玻片上，置于培养箱中培养(37℃，5％CO2)；培养24h后将培养基更换成Neurobasal+B27培养基，之后两天换液一次，培养的DRG神经元在5天内使用。
钙成像检测
去除培养的DRG神经元的培养基，用含2μM Fura-2/AM(Ca2+的一种比率荧光染料)的外液(含有2mM CaCl2)在37℃环境下孵育30min，之后，未能进入神经细胞的染料分子用外液洗去；Fura-2在结合Ca2+后，其最大的激发波长由380nm变为340nm，而钙成像检测系统运行时不断地在两种波长之间快速切换，因此荧光强度比值F340/F380能够实时反映胞内自由Ca2+的变化；血根碱溶液由灌流系统供给细胞(重力驱动)。DRG神经元钙成像实验和电生理记录使用外液配方如下：
DRG神经元全细胞记录用外液
用NaOH调节pH至7.4，渗透压调至300-310mOsm，保存于4℃备用
钙显像结果分析显示(图2)，在WT DRG神经元中，100μM AITC和1μM血根碱可使胞外Ca2+大量流入，导致神经元的荧光强度大幅增强；而在TRPA1敲除(Trpa1-/-)小鼠DRG神经元中，AITC和血根碱则无此效应。为了确证这是由于TRPA1敲除，而非神经元功能损伤或神经元选择错误等因素造成，发明人在AITC和血根碱之后加入30mM K+刺激DRG神经元，通过使神经元去极化导致胞外Ca2+大量进入胞内，以此来验证DRG神经元的功能完整性。如图2所示高钾溶液在TRPA1敲除的DRG中能够诱导明显的钙信号增强，因此证实了血根碱诱导DRG神经元钙信号增强的TRPA1通道依赖性。
发明人通过电生理技术进一步研究了血根碱对DRG神经元兴奋性的影响。结果显示血根碱可以显著上调DRG神经元的膜电位水平，由-60～-70mV上调至-40mV左右并诱导神经元动作电位(Action potential)的发放，当化合物刺激移除后膜电位逐渐恢复至-60～-70mV左右。同时，对血根碱有响应的神经元在给予100μM AITC后同样会引起膜电位的升高和动作电位的发放，但是在给予TRPA1的选择性阻断剂30μM HC030031进行孵育后血根碱诱导的膜电位升高和动作电位的发放被抑制。从TRPA1基因敲除小鼠的背根神经节中分离培养的神经元，在给予血根碱和AITC后没有出现显著的膜电位变化和动作电位的发放。同时在每个神经元记录的后期，通过电流钳向钳制的小DRG神经元注入50ms的电流刺激(100pA)时，在记录的神经元中可以检测到高频的动作电位发放，说明检测的DRG神经元功能是完好的。
在上述工作中发明人系统性研究了血根碱对TRPA1通道的激动活性，发现血根碱能够激活小鼠DRG神经元上的TRPA1通道引起显著的钙离子内流，增强神经元的兴奋性，诱导高频率动作电位的发放。因此血根碱可能通过增强小鼠体内的TRPA1活性导致机体神经损伤，诱导炎症和疼痛反应。发明人在小鼠上尝试构建相应的疼痛模型，20μl质量浓度分别为0.3％、0.1％、0.033％、0.011％和0.0037％的血根碱(Gi/V，使用ddH2O配置质量浓度为20％的羟丙基-β-环糊精作为溶媒)分别通过足底皮下注射的方式诱导小鼠疼痛。注射血根碱后监测时间60min，查看并统计小鼠的舔足时间(Licking Time)，通过不同组别小鼠的舔足时间评价疼痛的强度。由数据统计可以看出(图4)，不同浓度的血根碱可导致各组小鼠出现舔足时间的明显差异，且呈现剂量依赖效应。统计学数据显示，0.0037％血根碱即可引起明显的疼痛行为。
发明人应用药理学阻断和TRPA1敲除两种方法进行行为学检测验证血根碱在小鼠上诱导的疼痛反应是否是TRPA1依赖的，同时检验TRPA1的选择性抑制剂在该疼痛模型上的镇痛效应。HC030031作为TRPA1通道的特异性抑制剂，发明人通过灌胃给药的方式提前30min给予实验组小鼠300mg/kg的剂量，然后使用0.1％血根碱足底注射进行造模。在此实验中，HC030031使用溶媒5％DMSO+95％(20％羟丙基-β-环糊精)进行溶解，充分震荡后呈现白色乳浊液，完全满足灌胃给药的要求。行为学实验结果显示HC030031能够显著抑制血根碱导致的疼痛，且其镇痛效应在监测统计的60min内始终存在。发明人还使用TRPA1敲除和同窝对照小鼠验证血根碱的致痛特性是否与TRPA1直接相关。如图4所示，TRPA1基因敲除使得0.1％血根碱的致痛效应显著减弱。综上结果表明，血根碱可通过激活TRPA1通道使机体出现明显的炎症和疼痛反应，而TRPA1敲除或预给予TRPA1特异性抑制剂进行药理学阻断均能显著缓解血根碱注射导致的疼痛。
如本发明所表明的，血根碱能够高效、选择性地激活TRPA1通道，可作为TRPA1通道的激动剂工具分子应用于TRPA1通道调制剂的筛选。此外，通过对背根神经节的电生理记录和疼痛动物模型的构建表明，血根碱可通过激活神经元上的TRPA1通道在动物模型上诱导疼痛反应的发生，这表明血根碱作为工具分子可用于构建疼痛模型并进行镇痛药物的临床前药效学评价。
Claims (9)
1.血根碱在制备TRPA1通道激动剂中的应用。
2.根据权利要求1所述的应用，其中，血根碱作为TRPA1通道激动剂工具分子用于以TRPA1通道为靶点的通道调制剂的筛选。
3.根据权利要求1所述的应用，其中，所述TRPA1通道激动剂是用于构建动物疼痛模型的药剂。
4.根据权利要求3所述的应用，其中，所述模型用于镇痛药物的临床前药效学评价。
5.根据权利要求1所述的应用，其中，所述TRPA1通道激动剂是用于构建TRPA1通道相关疾病模型的药剂。
6.根据权利要求5所述的应用，其中，所述模型用于对以TRPA1通道为靶点的化合物进行临床前药效学评价。
7.血根碱作为TRPA1通道阳性激动剂工具分子用于制备以TRPA1通道为靶点筛选通道调制剂的药剂的应用。
8.血根碱作为工具分子用于制备构建动物疼痛模型的药剂的应用。
9.根据权利要求8所述的应用，其中，所述模型用于进行镇痛药物的临床前药效学评价。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010332195.5A CN113545316B (zh) | 2020-04-24 | 2020-04-24 | 血根碱在制备trpa1通道激动剂中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010332195.5A CN113545316B (zh) | 2020-04-24 | 2020-04-24 | 血根碱在制备trpa1通道激动剂中的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113545316A true CN113545316A (zh) | 2021-10-26 |
| CN113545316B CN113545316B (zh) | 2022-05-17 |
Family
ID=78129659
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010332195.5A Active CN113545316B (zh) | 2020-04-24 | 2020-04-24 | 血根碱在制备trpa1通道激动剂中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113545316B (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023174116A1 (zh) * | 2022-03-15 | 2023-09-21 | 浙江大学 | Wnt5a调节剂及其应用 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1980649A (zh) * | 2004-05-17 | 2007-06-13 | 康宾纳特克斯公司 | 治疗免疫炎性疾病的方法和试药 |
| CN101983188A (zh) * | 2008-04-01 | 2011-03-02 | 安斯泰来制药有限公司 | 吲哚啉酮化合物 |
| CN102850431A (zh) * | 2012-04-06 | 2013-01-02 | 兰州大学 | 基于阿片肽Biphalin和神经肽FF的嵌合肽及其合成和应用 |
| AU2014100614A4 (en) * | 2014-04-22 | 2014-07-24 | Macau University Of Science And Technology | Methods for novel drug discovery, treatment and selective targeting for gefitinib-resistant non-small-cell lung cancer harboring t790m mutation |
| CN104997786A (zh) * | 2015-07-14 | 2015-10-28 | 华中科技大学同济医学院附属同济医院 | 贝美前列腺素作为trpa1通道激动剂在制备药品中的应用 |
| CN107007605A (zh) * | 2017-06-07 | 2017-08-04 | 淮安市第人民医院 | 血根碱在制备抑制心肌梗死后心室重构药物中的应用 |
-
2020
- 2020-04-24 CN CN202010332195.5A patent/CN113545316B/zh active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1980649A (zh) * | 2004-05-17 | 2007-06-13 | 康宾纳特克斯公司 | 治疗免疫炎性疾病的方法和试药 |
| CN101983188A (zh) * | 2008-04-01 | 2011-03-02 | 安斯泰来制药有限公司 | 吲哚啉酮化合物 |
| CN102850431A (zh) * | 2012-04-06 | 2013-01-02 | 兰州大学 | 基于阿片肽Biphalin和神经肽FF的嵌合肽及其合成和应用 |
| AU2014100614A4 (en) * | 2014-04-22 | 2014-07-24 | Macau University Of Science And Technology | Methods for novel drug discovery, treatment and selective targeting for gefitinib-resistant non-small-cell lung cancer harboring t790m mutation |
| CN104997786A (zh) * | 2015-07-14 | 2015-10-28 | 华中科技大学同济医学院附属同济医院 | 贝美前列腺素作为trpa1通道激动剂在制备药品中的应用 |
| CN107007605A (zh) * | 2017-06-07 | 2017-08-04 | 淮安市第人民医院 | 血根碱在制备抑制心肌梗死后心室重构药物中的应用 |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023174116A1 (zh) * | 2022-03-15 | 2023-09-21 | 浙江大学 | Wnt5a调节剂及其应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113545316B (zh) | 2022-05-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Leo et al. | Taar1-mediated modulation of presynaptic dopaminergic neurotransmission: role of D2 dopamine autoreceptors | |
| Harris et al. | The neurotoxicity induced by ethanol withdrawal in mature organotypic hippocampal slices might involve cross‐talk between metabotropic glutamate type 5 receptors and N‐methyl‐D‐aspartate receptors | |
| Li et al. | Activation of GABA B receptors ameliorates cognitive impairment via restoring the balance of HCN1/HCN2 surface expression in the hippocampal CA1 area in rats with chronic cerebral hypoperfusion | |
| Vaillend et al. | Spatial discrimination learning and CA1 hippocampal synaptic plasticity in mdx and mdx3cv mice lacking dystrophin gene products | |
| Andoh et al. | Assessing microglial dynamics by live imaging | |
| Yang et al. | Paradoxical effects of VEGF on synaptic activity partially involved in notch1 signaling in the mouse hippocampus | |
| CN113545316B (zh) | 血根碱在制备trpa1通道激动剂中的应用 | |
| KR20030031466A (ko) | 포유동물 세포 증식을 조절하기 위한 ａ２ｂ 아데노신수용체 길항 물질의 동정 및 사용 방법 | |
| Hone et al. | Expression of α3β2β4 nicotinic acetylcholine receptors by rat adrenal chromaffin cells determined using novel conopeptide antagonists | |
| US20220213141A1 (en) | Dynamics within supramolecuar ikvav matrices enhance functional maturation of human ipscs-derived neurons and regeneration | |
| Shimono et al. | Long‐term recording of LTP in cultured hippocampal slices | |
| Malakooti et al. | The long isoform of intersectin-1 has a role in learning and memory | |
| CN1447690A (zh) | 对囊性纤维化跨膜传导调节蛋白氯离子通道的抑制 | |
| Kimura et al. | Early exposure to environmental enrichment protects male rats against neuropathic pain development after nerve injury | |
| JP2002526775A (ja) | ＧＤＮＦについての新規Ｒｅｔ非依存性シグナリング経路 | |
| Fuentealba et al. | A choline-evoked [Ca2+] c signal causes catecholamine release and hyperpolarization of chromaffin cells | |
| Weng et al. | Recording synaptic plasticity in acute hippocampal slices maintained in a small-volume recycling-, perfusion-, and submersion-type chamber system | |
| Wu et al. | Sensitivity of spinal neurons to GABA and glycine during voluntary movement in behaving monkeys | |
| KR100538425B1 (ko) | 안정 피로 모델, 그 제작방법, 그 모델을 사용하는평가방법 및 그 평가방법을 이용함으로써 선택된 약제 | |
| US20240270745A1 (en) | Isoquinoline alkaloid compound, and preparation method therefor and use thereof | |
| WO2023174116A1 (zh) | Wnt5a调节剂及其应用 | |
| Kumro et al. | α7 nicotinic acetylcholine receptors are necessary for basal forebrain activation to increase expression of the nerve growth factor receptor TrkA | |
| CN118845996A (zh) | 胡蜂毒肽作为trpv1拮抗剂在治疗神经病理性疼痛中的应用 | |
| WO2006000464A2 (de) | Verfahren zum auffinden schmerzrelevanter substanzen unter verwendung schmerzrelevanter proteine | |
| Ong | Protocadherin-gamma (Pcdh-γ) and its role in sensory axons and epidermal reinnervation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |