CN114306280B - 一种雷公藤红素纳米药物及irf1/gstm3轴在制备银屑病药物中的应用 - Google Patents
一种雷公藤红素纳米药物及irf1/gstm3轴在制备银屑病药物中的应用 Download PDFInfo
- Publication number
- CN114306280B CN114306280B CN202210062480.9A CN202210062480A CN114306280B CN 114306280 B CN114306280 B CN 114306280B CN 202210062480 A CN202210062480 A CN 202210062480A CN 114306280 B CN114306280 B CN 114306280B
- Authority
- CN
- China
- Prior art keywords
- gstm3
- psoriasis
- irf1
- tripterine
- nio
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 201000004681 Psoriasis Diseases 0.000 title claims abstract description 89
- 239000003814 drug Substances 0.000 title claims abstract description 34
- KQJSQWZMSAGSHN-JJWQIEBTSA-N celastrol Chemical compound C([C@H]1[C@]2(C)CC[C@@]34C)[C@](C)(C(O)=O)CC[C@]1(C)CC[C@]2(C)C4=CC=C1C3=CC(=O)C(O)=C1C KQJSQWZMSAGSHN-JJWQIEBTSA-N 0.000 title claims abstract description 26
- 229940079593 drug Drugs 0.000 title claims abstract description 24
- 102100036528 Glutathione S-transferase Mu 3 Human genes 0.000 title abstract description 113
- 101001071716 Homo sapiens Glutathione S-transferase Mu 3 Proteins 0.000 title abstract description 112
- 101000598002 Homo sapiens Interferon regulatory factor 1 Proteins 0.000 title abstract description 86
- 102100036981 Interferon regulatory factor 1 Human genes 0.000 title abstract description 86
- 238000002360 preparation method Methods 0.000 title description 14
- 238000000034 method Methods 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 12
- 239000000523 sample Substances 0.000 claims description 8
- 206010033661 Pancytopenia Diseases 0.000 claims description 6
- 235000012000 cholesterol Nutrition 0.000 claims description 6
- 208000024389 cytopenia Diseases 0.000 claims description 6
- 238000012545 processing Methods 0.000 claims description 6
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 claims description 5
- 238000010025 steaming Methods 0.000 claims description 5
- 238000005303 weighing Methods 0.000 claims description 5
- 230000006837 decompression Effects 0.000 claims description 3
- 230000036571 hydration Effects 0.000 claims description 3
- 238000006703 hydration reaction Methods 0.000 claims description 3
- 230000014509 gene expression Effects 0.000 abstract description 38
- 230000000694 effects Effects 0.000 abstract description 31
- 238000011282 treatment Methods 0.000 abstract description 29
- 238000002474 experimental method Methods 0.000 abstract description 25
- 238000013518 transcription Methods 0.000 abstract description 10
- 230000035897 transcription Effects 0.000 abstract description 10
- 230000005764 inhibitory process Effects 0.000 abstract description 8
- 238000004458 analytical method Methods 0.000 abstract description 7
- 238000001727 in vivo Methods 0.000 abstract description 7
- 230000008595 infiltration Effects 0.000 abstract description 7
- 238000001764 infiltration Methods 0.000 abstract description 7
- 230000002757 inflammatory effect Effects 0.000 abstract description 7
- 230000002265 prevention Effects 0.000 abstract description 6
- 125000003345 AMP group Chemical group 0.000 abstract description 5
- 206010067484 Adverse reaction Diseases 0.000 abstract description 5
- 230000006838 adverse reaction Effects 0.000 abstract description 5
- 230000004888 barrier function Effects 0.000 abstract description 5
- 229920006227 ethylene-grafted-maleic anhydride Polymers 0.000 abstract description 5
- 238000000338 in vitro Methods 0.000 abstract description 5
- 239000000203 mixture Substances 0.000 abstract description 5
- 230000037380 skin damage Effects 0.000 abstract description 5
- 230000008685 targeting Effects 0.000 abstract description 5
- 230000009471 action Effects 0.000 abstract description 4
- 238000009472 formulation Methods 0.000 abstract description 4
- 230000007246 mechanism Effects 0.000 abstract description 3
- 206010040844 Skin exfoliation Diseases 0.000 abstract 1
- 238000012795 verification Methods 0.000 abstract 1
- 230000003313 weakening effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 59
- 241000699670 Mus sp. Species 0.000 description 24
- 239000000499 gel Substances 0.000 description 22
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 22
- DOUYETYNHWVLEO-UHFFFAOYSA-N imiquimod Chemical compound C1=CC=CC2=C3N(CC(C)C)C=NC3=C(N)N=C21 DOUYETYNHWVLEO-UHFFFAOYSA-N 0.000 description 21
- 108090000623 proteins and genes Proteins 0.000 description 20
- 230000002018 overexpression Effects 0.000 description 18
- 229960002751 imiquimod Drugs 0.000 description 17
- 108020004999 messenger RNA Proteins 0.000 description 17
- 238000011529 RT qPCR Methods 0.000 description 14
- 239000006071 cream Substances 0.000 description 12
- 210000003491 skin Anatomy 0.000 description 12
- 108010024636 Glutathione Proteins 0.000 description 11
- 229960003180 glutathione Drugs 0.000 description 11
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 230000003902 lesion Effects 0.000 description 9
- 238000011002 quantification Methods 0.000 description 9
- -1 zhou Inorganic materials 0.000 description 9
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 8
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 8
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 8
- 241000282414 Homo sapiens Species 0.000 description 8
- 108020004459 Small interfering RNA Proteins 0.000 description 8
- 150000001413 amino acids Chemical group 0.000 description 8
- 230000004663 cell proliferation Effects 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 230000035755 proliferation Effects 0.000 description 8
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 8
- 206010061218 Inflammation Diseases 0.000 description 7
- 108060001084 Luciferase Proteins 0.000 description 7
- 239000005089 Luciferase Substances 0.000 description 7
- 241000830536 Tripterygium wilfordii Species 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- 230000006698 induction Effects 0.000 description 7
- 230000004054 inflammatory process Effects 0.000 description 7
- 239000003112 inhibitor Substances 0.000 description 7
- 238000010172 mouse model Methods 0.000 description 7
- 230000037361 pathway Effects 0.000 description 7
- 230000000638 stimulation Effects 0.000 description 7
- 238000001262 western blot Methods 0.000 description 7
- 101100283975 Bos taurus GSTM1 gene Proteins 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 6
- 102000004289 Interferon regulatory factor 1 Human genes 0.000 description 6
- 108090000890 Interferon regulatory factor 1 Proteins 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 6
- 230000003110 anti-inflammatory effect Effects 0.000 description 6
- 239000003086 colorant Substances 0.000 description 6
- 230000004060 metabolic process Effects 0.000 description 6
- 239000008194 pharmaceutical composition Substances 0.000 description 6
- 230000001185 psoriatic effect Effects 0.000 description 6
- 206010040882 skin lesion Diseases 0.000 description 6
- 231100000444 skin lesion Toxicity 0.000 description 6
- 101150058670 Gstm3 gene Proteins 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 239000002552 dosage form Substances 0.000 description 5
- 239000003937 drug carrier Substances 0.000 description 5
- 210000002510 keratinocyte Anatomy 0.000 description 5
- DNIAPMSPPWPWGF-UHFFFAOYSA-N monopropylene glycol Natural products CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 5
- 238000003753 real-time PCR Methods 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 4
- 102000017278 Glutaredoxin Human genes 0.000 description 4
- 108050005205 Glutaredoxin Proteins 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- HVUMOYIDDBPOLL-XWVZOOPGSA-N Sorbitan monostearate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O HVUMOYIDDBPOLL-XWVZOOPGSA-N 0.000 description 4
- 229920002472 Starch Polymers 0.000 description 4
- LWZFANDGMFTDAV-BURFUSLBSA-N [(2r)-2-[(2r,3r,4s)-3,4-dihydroxyoxolan-2-yl]-2-hydroxyethyl] dodecanoate Chemical compound CCCCCCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O LWZFANDGMFTDAV-BURFUSLBSA-N 0.000 description 4
- 229940024606 amino acid Drugs 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 230000001684 chronic effect Effects 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 230000028993 immune response Effects 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 239000002502 liposome Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 235000011067 sorbitan monolaureate Nutrition 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 239000008107 starch Substances 0.000 description 4
- 235000019698 starch Nutrition 0.000 description 4
- 235000015398 thunder god vine Nutrition 0.000 description 4
- 206010009944 Colon cancer Diseases 0.000 description 3
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 3
- 108050006400 Cyclin Proteins 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 102100039696 Glutamate-cysteine ligase catalytic subunit Human genes 0.000 description 3
- 102100033398 Glutamate-cysteine ligase regulatory subunit Human genes 0.000 description 3
- 102100036533 Glutathione S-transferase Mu 2 Human genes 0.000 description 3
- 102100023523 Glutathione S-transferase Mu 4 Human genes 0.000 description 3
- 101001034527 Homo sapiens Glutamate-cysteine ligase catalytic subunit Proteins 0.000 description 3
- 101000870644 Homo sapiens Glutamate-cysteine ligase regulatory subunit Proteins 0.000 description 3
- 101001071691 Homo sapiens Glutathione S-transferase Mu 2 Proteins 0.000 description 3
- 101000906399 Homo sapiens Glutathione S-transferase Mu 4 Proteins 0.000 description 3
- 238000012404 In vitro experiment Methods 0.000 description 3
- 102000000589 Interleukin-1 Human genes 0.000 description 3
- 108010002352 Interleukin-1 Proteins 0.000 description 3
- 102100030703 Interleukin-22 Human genes 0.000 description 3
- 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 3
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 3
- 229920000881 Modified starch Polymers 0.000 description 3
- 108091034117 Oligonucleotide Proteins 0.000 description 3
- 102000009339 Proliferating Cell Nuclear Antigen Human genes 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 239000003963 antioxidant agent Substances 0.000 description 3
- 230000003078 antioxidant effect Effects 0.000 description 3
- 235000006708 antioxidants Nutrition 0.000 description 3
- 230000006907 apoptotic process Effects 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- LWQQLNNNIPYSNX-UROSTWAQSA-N calcipotriol Chemical compound C1([C@H](O)/C=C/[C@@H](C)[C@@H]2[C@]3(CCCC(/[C@@H]3CC2)=C\C=C\2C([C@@H](O)C[C@H](O)C/2)=C)C)CC1 LWQQLNNNIPYSNX-UROSTWAQSA-N 0.000 description 3
- 229960002882 calcipotriol Drugs 0.000 description 3
- 238000010609 cell counting kit-8 assay Methods 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 230000000875 corresponding effect Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000002222 downregulating effect Effects 0.000 description 3
- 238000004520 electroporation Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 235000011187 glycerol Nutrition 0.000 description 3
- 108010074109 interleukin-22 Proteins 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000002503 metabolic effect Effects 0.000 description 3
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 3
- 239000008108 microcrystalline cellulose Substances 0.000 description 3
- 229940016286 microcrystalline cellulose Drugs 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 239000002674 ointment Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 230000008591 skin barrier function Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 238000011269 treatment regimen Methods 0.000 description 3
- 230000003827 upregulation Effects 0.000 description 3
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 description 2
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- FMMWHPNWAFZXNH-UHFFFAOYSA-N Benz[a]pyrene Chemical compound C1=C2C3=CC=CC=C3C=C(C=C3)C2=C2C3=CC=CC2=C1 FMMWHPNWAFZXNH-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
- 108091026890 Coding region Proteins 0.000 description 2
- 229920001353 Dextrin Polymers 0.000 description 2
- 239000004375 Dextrin Substances 0.000 description 2
- 206010061819 Disease recurrence Diseases 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 229930186217 Glycolipid Natural products 0.000 description 2
- 101150001766 Gstm4 gene Proteins 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 101100283988 Homo sapiens GSTM3 gene Proteins 0.000 description 2
- 102000014150 Interferons Human genes 0.000 description 2
- 108010050904 Interferons Proteins 0.000 description 2
- 108050003558 Interleukin-17 Proteins 0.000 description 2
- 102000013691 Interleukin-17 Human genes 0.000 description 2
- 208000008839 Kidney Neoplasms Diseases 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 2
- 239000004368 Modified starch Substances 0.000 description 2
- 101100393855 Mus musculus Gstm5 gene Proteins 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 206010033733 Papule Diseases 0.000 description 2
- 101100393859 Rattus norvegicus Gstm7 gene Proteins 0.000 description 2
- 206010038389 Renal cancer Diseases 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 102000001742 Tumor Suppressor Proteins Human genes 0.000 description 2
- 108010040002 Tumor Suppressor Proteins Proteins 0.000 description 2
- 208000036142 Viral infection Diseases 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 235000010443 alginic acid Nutrition 0.000 description 2
- 229920000615 alginic acid Polymers 0.000 description 2
- 230000001772 anti-angiogenic effect Effects 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 239000012298 atmosphere Substances 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 230000031018 biological processes and functions Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 239000004568 cement Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 239000006184 cosolvent Substances 0.000 description 2
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000001784 detoxification Methods 0.000 description 2
- 235000019425 dextrin Nutrition 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 230000002996 emotional effect Effects 0.000 description 2
- 239000003995 emulsifying agent Substances 0.000 description 2
- 238000010201 enrichment analysis Methods 0.000 description 2
- 239000013604 expression vector Substances 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
- 238000003209 gene knockout Methods 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 239000001341 hydroxy propyl starch Substances 0.000 description 2
- 235000013828 hydroxypropyl starch Nutrition 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 230000015788 innate immune response Effects 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 229940079322 interferon Drugs 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 201000010982 kidney cancer Diseases 0.000 description 2
- 230000003907 kidney function Effects 0.000 description 2
- 230000003908 liver function Effects 0.000 description 2
- 238000003468 luciferase reporter gene assay Methods 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
- 230000037353 metabolic pathway Effects 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- 235000019426 modified starch Nutrition 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 230000037311 normal skin Effects 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 230000036542 oxidative stress Effects 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 239000006187 pill Substances 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 2
- 230000000861 pro-apoptotic effect Effects 0.000 description 2
- 230000009323 psychological health Effects 0.000 description 2
- 230000007115 recruitment Effects 0.000 description 2
- 230000000306 recurrent effect Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000012679 serum free medium Substances 0.000 description 2
- 239000004055 small Interfering RNA Substances 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 230000035882 stress Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000003826 tablet Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 238000012085 transcriptional profiling Methods 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- 230000009278 visceral effect Effects 0.000 description 2
- NOOLISFMXDJSKH-UTLUCORTSA-N (+)-Neomenthol Chemical compound CC(C)[C@@H]1CC[C@@H](C)C[C@@H]1O NOOLISFMXDJSKH-UTLUCORTSA-N 0.000 description 1
- DTGKSKDOIYIVQL-WEDXCCLWSA-N (+)-borneol Chemical compound C1C[C@@]2(C)[C@@H](O)C[C@@H]1C2(C)C DTGKSKDOIYIVQL-WEDXCCLWSA-N 0.000 description 1
- REPVLJRCJUVQFA-UHFFFAOYSA-N (-)-isopinocampheol Natural products C1C(O)C(C)C2C(C)(C)C1C2 REPVLJRCJUVQFA-UHFFFAOYSA-N 0.000 description 1
- DNISEZBAYYIQFB-PHDIDXHHSA-N (2r,3r)-2,3-diacetyloxybutanedioic acid Chemical compound CC(=O)O[C@@H](C(O)=O)[C@H](C(O)=O)OC(C)=O DNISEZBAYYIQFB-PHDIDXHHSA-N 0.000 description 1
- KQJSQWZMSAGSHN-UHFFFAOYSA-N (9beta,13alpha,14beta,20alpha)-3-hydroxy-9,13-dimethyl-2-oxo-24,25,26-trinoroleana-1(10),3,5,7-tetraen-29-oic acid Natural products CC12CCC3(C)C4CC(C)(C(O)=O)CCC4(C)CCC3(C)C2=CC=C2C1=CC(=O)C(O)=C2C KQJSQWZMSAGSHN-UHFFFAOYSA-N 0.000 description 1
- LDVVTQMJQSCDMK-UHFFFAOYSA-N 1,3-dihydroxypropan-2-yl formate Chemical compound OCC(CO)OC=O LDVVTQMJQSCDMK-UHFFFAOYSA-N 0.000 description 1
- AXTGDCSMTYGJND-UHFFFAOYSA-N 1-dodecylazepan-2-one Chemical compound CCCCCCCCCCCCN1CCCCCC1=O AXTGDCSMTYGJND-UHFFFAOYSA-N 0.000 description 1
- VBICKXHEKHSIBG-UHFFFAOYSA-N 1-monostearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)CO VBICKXHEKHSIBG-UHFFFAOYSA-N 0.000 description 1
- 235000007173 Abies balsamea Nutrition 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 1
- 108010082126 Alanine transaminase Proteins 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
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 1
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 239000004857 Balsam Substances 0.000 description 1
- TXVHTIQJNYSSKO-UHFFFAOYSA-N BeP Natural products C1=CC=C2C3=CC=CC=C3C3=CC=CC4=CC=C1C2=C34 TXVHTIQJNYSSKO-UHFFFAOYSA-N 0.000 description 1
- 235000016068 Berberis vulgaris Nutrition 0.000 description 1
- 241000335053 Beta vulgaris Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- AQKDBFWJOPNOKZ-UHFFFAOYSA-N Celastrol Natural products CC12CCC3(C)C4CC(C)(C(O)=O)CCC4(C)CCC3(C)C2=CC=C2C1=CC(=O)C(=O)C2C AQKDBFWJOPNOKZ-UHFFFAOYSA-N 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 244000163122 Curcuma domestica Species 0.000 description 1
- 235000003392 Curcuma domestica Nutrition 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- NOOLISFMXDJSKH-UHFFFAOYSA-N DL-menthol Natural products CC(C)C1CCC(C)CC1O NOOLISFMXDJSKH-UHFFFAOYSA-N 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- 206010012438 Dermatitis atopic Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 238000003718 Dual-Luciferase Reporter Assay System Methods 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 208000019028 Epidermal thickening Diseases 0.000 description 1
- 239000001116 FEMA 4028 Substances 0.000 description 1
- 240000008168 Ficus benjamina Species 0.000 description 1
- 102100037009 Filaggrin-2 Human genes 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- 241000206672 Gelidium Species 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 102100036534 Glutathione S-transferase Mu 1 Human genes 0.000 description 1
- 101710153774 Glutathione S-transferase Mu 3 Proteins 0.000 description 1
- 229920002907 Guar gum Polymers 0.000 description 1
- 108010023302 HDL Cholesterol Proteins 0.000 description 1
- 239000004705 High-molecular-weight polyethylene Substances 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000878281 Homo sapiens Filaggrin-2 Proteins 0.000 description 1
- 101001071694 Homo sapiens Glutathione S-transferase Mu 1 Proteins 0.000 description 1
- 101100452802 Homo sapiens IRF1 gene Proteins 0.000 description 1
- 101000588302 Homo sapiens Nuclear factor erythroid 2-related factor 2 Proteins 0.000 description 1
- 101000712969 Homo sapiens Ras association domain-containing protein 5 Proteins 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- 244000018716 Impatiens biflora Species 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 102000003777 Interleukin-1 beta Human genes 0.000 description 1
- 108090000193 Interleukin-1 beta Proteins 0.000 description 1
- 101150064984 Irf1 gene Proteins 0.000 description 1
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 description 1
- 108010028554 LDL Cholesterol Proteins 0.000 description 1
- 241000254158 Lampyridae Species 0.000 description 1
- ARIWANIATODDMH-UHFFFAOYSA-N Lauric acid monoglyceride Natural products CCCCCCCCCCCC(=O)OCC(O)CO ARIWANIATODDMH-UHFFFAOYSA-N 0.000 description 1
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 1
- 240000007472 Leucaena leucocephala Species 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 229920002774 Maltodextrin Polymers 0.000 description 1
- 239000005913 Maltodextrin Substances 0.000 description 1
- 240000003183 Manihot esculenta Species 0.000 description 1
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 244000062730 Melissa officinalis Species 0.000 description 1
- 235000010654 Melissa officinalis Nutrition 0.000 description 1
- 108700011259 MicroRNAs Proteins 0.000 description 1
- 241000228347 Monascus <ascomycete fungus> Species 0.000 description 1
- 206010061309 Neoplasm progression Diseases 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 102100031701 Nuclear factor erythroid 2-related factor 2 Human genes 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 102000004140 Oncostatin M Human genes 0.000 description 1
- 108090000630 Oncostatin M Proteins 0.000 description 1
- 238000010222 PCR analysis Methods 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 241000009328 Perro Species 0.000 description 1
- 208000009565 Pharyngeal Neoplasms Diseases 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 208000003251 Pruritus Diseases 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 238000003559 RNA-seq method Methods 0.000 description 1
- 102100033239 Ras association domain-containing protein 5 Human genes 0.000 description 1
- 108010052090 Renilla Luciferases Proteins 0.000 description 1
- 102100028255 Renin Human genes 0.000 description 1
- 108090000783 Renin Proteins 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 229920001800 Shellac Polymers 0.000 description 1
- 108091027967 Small hairpin RNA Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 206010054094 Tumour necrosis Diseases 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
- SXEHKFHPFVVDIR-UHFFFAOYSA-N [4-(4-hydrazinylphenyl)phenyl]hydrazine Chemical compound C1=CC(NN)=CC=C1C1=CC=C(NN)C=C1 SXEHKFHPFVVDIR-UHFFFAOYSA-N 0.000 description 1
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000033289 adaptive immune response Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 235000010419 agar Nutrition 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 229940030999 antipsoriatics Drugs 0.000 description 1
- 201000008937 atopic dermatitis Diseases 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 102000015736 beta 2-Microglobulin Human genes 0.000 description 1
- 108010081355 beta 2-Microglobulin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical group OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 description 1
- 235000011175 beta-cyclodextrine Nutrition 0.000 description 1
- 229960004853 betadex Drugs 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 238000003766 bioinformatics method Methods 0.000 description 1
- VYLDEYYOISNGST-UHFFFAOYSA-N bissulfosuccinimidyl suberate Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)CCCCCCC(=O)ON1C(=O)C(S(O)(=O)=O)CC1=O VYLDEYYOISNGST-UHFFFAOYSA-N 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 229940116229 borneol Drugs 0.000 description 1
- CKDOCTFBFTVPSN-UHFFFAOYSA-N borneol Natural products C1CC2(C)C(C)CC1C2(C)C CKDOCTFBFTVPSN-UHFFFAOYSA-N 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 235000010216 calcium carbonate Nutrition 0.000 description 1
- FUFJGUQYACFECW-UHFFFAOYSA-L calcium hydrogenphosphate Chemical compound [Ca+2].OP([O-])([O-])=O FUFJGUQYACFECW-UHFFFAOYSA-L 0.000 description 1
- 235000010957 calcium stearoyl-2-lactylate Nutrition 0.000 description 1
- OEUVSBXAMBLPES-UHFFFAOYSA-L calcium stearoyl-2-lactylate Chemical compound [Ca+2].CCCCCCCCCCCCCCCCCC(=O)OC(C)C(=O)OC(C)C([O-])=O.CCCCCCCCCCCCCCCCCC(=O)OC(C)C(=O)OC(C)C([O-])=O OEUVSBXAMBLPES-UHFFFAOYSA-L 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 231100000357 carcinogen Toxicity 0.000 description 1
- 239000003183 carcinogenic agent Substances 0.000 description 1
- 239000004106 carminic acid Substances 0.000 description 1
- 235000012730 carminic acid Nutrition 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000004709 cell invasion Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 229930002875 chlorophyll Natural products 0.000 description 1
- 235000019804 chlorophyll Nutrition 0.000 description 1
- 229940106705 chlorophyll Drugs 0.000 description 1
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- 229940080423 cochineal Drugs 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000010219 correlation analysis Methods 0.000 description 1
- 238000002316 cosmetic surgery Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 229940109239 creatinine Drugs 0.000 description 1
- 235000003373 curcuma longa Nutrition 0.000 description 1
- 238000012303 cytoplasmic staining Methods 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000002074 deregulated effect Effects 0.000 description 1
- 235000019700 dicalcium phosphate Nutrition 0.000 description 1
- 229940095079 dicalcium phosphate anhydrous Drugs 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- DTGKSKDOIYIVQL-UHFFFAOYSA-N dl-isoborneol Natural products C1CC2(C)C(O)CC1C2(C)C DTGKSKDOIYIVQL-UHFFFAOYSA-N 0.000 description 1
- 210000004670 early embryonic cell Anatomy 0.000 description 1
- 210000005069 ears Anatomy 0.000 description 1
- 230000002681 effect on RNA Effects 0.000 description 1
- 239000012039 electrophile Substances 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 231100000317 environmental toxin Toxicity 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 210000005175 epidermal keratinocyte Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000010408 film Substances 0.000 description 1
- 239000007888 film coating Substances 0.000 description 1
- 238000009501 film coating Methods 0.000 description 1
- 239000008098 formaldehyde solution Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 235000010417 guar gum Nutrition 0.000 description 1
- 239000000665 guar gum Substances 0.000 description 1
- 229960002154 guar gum Drugs 0.000 description 1
- 235000009200 high fat diet Nutrition 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 230000000887 hydrating effect Effects 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000037451 immune surveillance Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000034435 immune system development Effects 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 229960002725 isoflurane Drugs 0.000 description 1
- 230000007803 itching Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 206010023841 laryngeal neoplasm Diseases 0.000 description 1
- 229960003639 laurocapram Drugs 0.000 description 1
- 210000003041 ligament Anatomy 0.000 description 1
- 239000000865 liniment Substances 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 238000011866 long-term treatment Methods 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 229940035034 maltodextrin Drugs 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 229940041616 menthol Drugs 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 238000010208 microarray analysis Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 208000025113 myeloid leukemia Diseases 0.000 description 1
- 230000013184 negative regulation of T cell differentiation Effects 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000012758 nuclear staining Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000006072 paste Substances 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 238000010827 pathological analysis Methods 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 125000005575 polycyclic aromatic hydrocarbon group Chemical group 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 231100000683 possible toxicity Toxicity 0.000 description 1
- 229920001592 potato starch Polymers 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000028617 response to DNA damage stimulus Effects 0.000 description 1
- 238000011076 safety test Methods 0.000 description 1
- 238000007480 sanger sequencing Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000004208 shellac Substances 0.000 description 1
- 235000013874 shellac Nutrition 0.000 description 1
- 229940113147 shellac Drugs 0.000 description 1
- ZLGIYFNHBLSMPS-ATJNOEHPSA-N shellac Chemical compound OCCCCCC(O)C(O)CCCCCCCC(O)=O.C1C23[C@H](C(O)=O)CCC2[C@](C)(CO)[C@@H]1C(C(O)=O)=C[C@@H]3O ZLGIYFNHBLSMPS-ATJNOEHPSA-N 0.000 description 1
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 238000007390 skin biopsy Methods 0.000 description 1
- 208000017520 skin disease Diseases 0.000 description 1
- 230000036620 skin dryness Effects 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 235000015424 sodium Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 229940083466 soybean lecithin Drugs 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 235000000891 standard diet Nutrition 0.000 description 1
- 229940032147 starch Drugs 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000002636 symptomatic treatment Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 231100000901 systemic toxic effect Toxicity 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 238000001757 thermogravimetry curve Methods 0.000 description 1
- 238000011200 topical administration Methods 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 230000005751 tumor progression Effects 0.000 description 1
- 235000013976 turmeric Nutrition 0.000 description 1
- 229940116269 uric acid Drugs 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 235000010493 xanthan gum Nutrition 0.000 description 1
- 239000000230 xanthan gum Substances 0.000 description 1
- 229920001285 xanthan gum Polymers 0.000 description 1
- 229940082509 xanthan gum Drugs 0.000 description 1
- 239000002676 xenobiotic agent Substances 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
Abstract
本发明涉及一种雷公藤红素纳米药物及IRF1/GSTM3轴在制备银屑病药物中的应用。本发明研制了雷公藤红素(CS)的纳米外用凝胶剂型nio‑CS,发现其可有效改善银屑病样皮损及炎症浸润,其效果较其他纳米剂型更优,并能减少复发,且无明显不良反应。本发明还利用生物信息分析并联合临床标本及体内外实验验证,探究CS在防治银屑病时的作用机制,发现CS通过减弱IRF1与GSTM3的结合亲和力,来降低IRF1对GSTM3的转录抑制作用,改善炎症浸润,减弱细胞过度增殖,恢复AMPs和屏障功能表达,改善银屑病样皮损。本发明提示nio‑CS凝胶有望成为安全有效治疗银屑病和防治其复发的临床药物,而靶向IRF1/GSTM3轴可以作为一种新的防治方法,用于未来防治银屑病。
Description
技术领域
本发明涉及生物医药领域,具体地说,涉及一种雷公藤红素纳米药物及IRF1/GSTM3轴在制备银屑病药物中的应用。
背景技术
银屑病是一种常见的慢性、复发性、免疫介导的皮肤疾病,影响约全球3%的人口。银屑病有多种临床皮损表现,但最常见的表现为慢性、对称性、红斑性、鳞屑性丘疹和斑块,斑块是角质形成细胞过度增殖和不完全分化的结果。除了影响皮肤本身外,各种并发症可能会影响患者的生活质量,对患者的情绪和心理健康产生显著的负面影响,增加死亡率。
雷公藤(Tripterygium wilfordii Hook.f.,TwHf)被认为是治疗银屑病的有效中草药,并于2018年获得中国皮肤科医生专家共识批准(皮肤科分会银屑病中医治疗专家共识,2018)。与此同时,口服雷公藤会引发几种不良反应。为了避免出现这些不良反应,本课题组前期将雷公藤药物改成外用剂型,经体内实验发现可有效防治银屑病且副作用小(Ru,Y.,Li,H.,Zhang,R.,Luo,Y.,Song,J.,Kuai,L.,Xing,M.,Hong,S.,Sun,X.,Ding,X.,Lu,Y.,Liu,L.,Na,C.,Zhou,Y.,Li,B.,Li,X.,2020.Role of keratinocytes and immunecells in the anti-inflammatory effects of Tripterygium wilfordii Hook.f.in amurine model of psoriasis.Phytomedicine 77,153299.https://doi.org/10.1016/j.phymed.2020.153299)。雷公藤红素(CS)是从药用植物雷公藤中提取的一种五环三萜类化合物,具有强大的抗炎、促凋亡、抗氧化应激和抗血管生成功能。一项研究证实,CS具有明显的抗银屑病作用,使用含CS的药膏一个月可以缓解银屑病相关的瘙痒和体征,具有良好的临床应用前景(Thouvenin,M.D.,Dalmon,S.,Theunis,J.,Lauze,C.,Coubetergues,H.,Mengeaud,V.,Calvet,B.,2020.Tolerance and efficacy of a new celastrol-containing balm as adjunct care in psoriasis.J Eur Acad Dermatol Venereol34Suppl 6,10–16.https://doi.org/10.1111/jdv.16691)。然而,目前未见如本申请所述的显著提高疗效的雷公藤红素类脂质体纳米制剂。
谷胱甘肽巯基转移酶M3(GSTM3)是谷胱甘肽巯基转移酶(GST)家族的成员,GST通常被认为是肿瘤易感性的调节因子,它可以调节肿瘤的发生,加速化疗耐药,抑制细胞增殖、迁移和侵袭,促进细胞凋亡,增强氧化应激。研究发现,GSTM3在有毒物质的代谢中起重要作用,如烟草释放出的多环芳烃、苯并(A)芘等;GSTM3在促进细胞凋亡、抗炎和改善机体代谢方面有深远的影响;GSTM3在多种癌症中表达失调,GSTM3蛋白的表达在肿瘤的进展或抑制中发挥着特殊的作用,如GSTM3rs1055259与肾癌易感性显著相关,GSTM3的上调降低了细胞的锚定非依赖性生长能力,证明了GSTM3是肾癌的肿瘤抑制因子;GSTM3使肝癌细胞对放射治疗敏感,可能是一个有效的治疗靶点。然而,GSTM3在银屑病中的生物学功能有待进一步阐明。
干扰素调节因子-1(IRF-1)是一种转录因子,除早期胚胎细胞外,在所有类型的细胞中都有所表达。IRF-1通过与特定的DNA序列相互作用,调控靶基因的转录,这些靶基因在病毒感染、肿瘤免疫监视、促炎性损伤、免疫系统发育等多种生理和病理过程中都发挥着重要作用,IRF-1被认为是连接先天免疫系统和后天免疫系统的纽带。新研究发现,IRF1能够加强病毒感染导致的先天免疫反应。IRF1缺乏明显增加合并其他可变基因发生肿瘤的几率。IRF1在结直肠癌中的表达低于正常黏膜,且其表达水平与结直肠癌转移呈负相关,IRF1促进RASSF5的表达,可能通过下调RAS-RAC1途径抑制结直肠癌的转移和增殖。然而,IRF-1在银屑病中的生物学功能有待进一步阐明。
目前,银屑病尚无特效疗法,适当的对症治疗可控制症状,但存在易反复发作的特点,且长期治疗容易导致不良反应。因此开发新的银屑病防治药物和方法是非常必要的。
发明内容
本发明的目的是针对现有技术中的不足,提供一种雷公藤红素纳米药物及IRF1/GSTM3轴在制备银屑病药物中的应用。
第一方面,本发明提供了一种雷公藤红素纳米药物,所述雷公藤红素纳米药物是按照下述方法制备的:精密称取司盘20 30mg、司盘60 10mg、胆固醇10mg、雷公藤红素1mg,加入3ml体积比为2:1的氯仿甲醇,45℃水浴减压冷凝旋蒸,容器壁形成一层膜后,加入5ml水,常压60℃旋转水合30min,于超声波细胞粉碎仪处理4分钟,超声波细胞粉碎仪处理参数为200W,6mm探头,0.1s/0.1s,最终形成所述雷公藤红素纳米药物。
第二方面,本发明提供了所述雷公藤红素纳米药物的制备方法,包括以下步骤:精密称取司盘20 30mg、司盘60 10mg、胆固醇10mg、雷公藤红素1mg,加入3ml体积比为2:1的氯仿甲醇,45℃水浴减压冷凝旋蒸,容器壁形成一层膜后,加入5ml水,常压60℃旋转水合30min,于超声波细胞粉碎仪处理4分钟,超声波细胞粉碎仪处理参数为200W,6mm探头,0.1s/0.1s,最终形成所述雷公藤红素纳米药物。
第三方面,本发明提供了所述雷公藤红素纳米药物在制备防治银屑病的药物中的应用。
第四方面,本发明提供了GSTM3或其上调剂在制备防治银屑病的药物中的应用。
作为一个优选例,所述上调剂为GSTM3的过表达载体或携带GSTM3基因的慢病毒颗粒。
第五方面,本发明提供了用于阻止或减少GSTM3基因的-4749~-4740bp位置被IRF1结合的化合物在制备防治银屑病的药物中的应用。
第六方面,本发明提供了IRF1或其抑制剂在制备防治银屑病的药物中的应用。
作为一个优选例,所述抑制剂为以IRF1蛋白或其转录本为靶序列、且能够抑制IRF1蛋白表达或基因转录的小干扰RNA、dsRNA、shRNA、微小RNA、反义核酸;或能表达或形成所述小干扰RNA、dsRNA、微小RNA、反义核酸的构建物。
第七方面,本发明提供了一种防治银屑病的药物组合物,所述的药物组合物以
a)GSTM3或其上调剂
b)IRF1或其抑制剂,和/或
c)用于阻止或减少GSTM3基因的-4749~-4740bp位置被IRF1结合的化合物
为活性成分,并进一步包含药学上可接受的载体。
作为一个优选例,所述药物组合物的剂型为外用剂型或内用剂型;或所述药物组合物的剂型为贴剂、糊剂、软膏剂、凝胶剂、涂膜剂、巴布剂、喷雾剂、胶囊剂、颗粒剂、片剂、丸剂、口服液或注射液。
本发明优点在于:
1、为减少雷公藤红素(CS)的不良反应,本发明制备了CS纳米凝胶外用剂型,即nio-CS凝胶,其治疗咪喹莫特诱导的银屑病样模型可达到明显改善皮损,且无脏腑毒性的效果,并能减少复发。表明该CS剂型有望成为治疗银屑病、改善银屑病样炎症反应及减少复发的有效临床药物。
2、本发明利用实验结合生物信息分析,并联合临床标本,验证CS在防治银屑病时的作用机制。发现CS作用于HaCaT细胞后GSTM3的mRNA表达显著升高,同时IRF1表达降低。采用慢病毒转染技术过表达HaCaT细胞中IRF1表达,细胞的增殖增加,炎症浸润加重,抗菌肽(AMPs)和屏障功能表达增加。而GSTM3的过表达逆转了IRF1过表达的影响。说明IRF1/GSTM3在HaCaT细胞的生物学过程中起着关键作用,这代表了银屑病病理中的一个新的分子特征,也是防治银屑病的一个有前途的靶点。通过生物信息学分析结合实验验证,确定了IRF1是GSTM3的转录抑制因子,CS在转录水平上减轻了IRF1对GSTM3的抑制作用,表明IRF1/GSTM3轴在难治性银屑病中具有新的作用。
总的来说,本发明结果证明,nio-CS凝胶及靶向IRF1/GSTM3轴可以作为新的治疗方法,用于未来防治银屑病及银屑病样相关炎症损伤,减少疾病复发。
附图说明
图1.转录谱分析和nio-CS凝胶特征。图1a.CCK8法检测CS对HaCaT细胞和NHEK细胞48h的细胞毒性作用。图1b.差异表达mRNAs热图分析。图1c.KEGG富集分析。图1d.GO富集分析。图1e.qPCR定量检测经CS刺激与否的HaCaT细胞中GSTM2、GSTM3、GSTM4、GCLC、GCLM和IRF1的表达。图1f.nio-CS尺寸分布图。图1g.TEM图像。标尺=0.2μm和200nm。图1h.nio-CS凝胶和正常CS水凝胶(n=4)的释放曲线。数据代表来自三个独立实验的平均值±SD。***p<0.001,**p<0.01,*p<0.05。
图2.CS刺激后GSTM3和IRF1的表达改变。图2a.差异表达mRNA的火山图。灰色表示差异不显著的mRNA;红色和蓝色表示显著差异。GSTM3和IRF1在临床组织中的表达。图2b-c.CD3、GSTM3和IRF1在正常人皮肤(n=4)和银屑病皮损(n=4)中有代表性的胞浆染色。标尺=100μm。图2d-e.Western blot检测CS诱导第8天小鼠皮肤组织中IRF1和GSTM3的表达。图2f.CCK8测定指定时间的HaCaT细胞增殖能力。图2g.qPCR检测IL-1β、CXCL-10、S100A8、FLG和PCNA的表达。图2h-i.CCK8和qPCR定量确定过表达GSTM3部分改善了由IRF1过表达促进的细胞增殖/炎症/屏障功能表型。数据代表来自三个独立实验的平均值±SD。***p<0.001,**p<0.01。
图3.IRF1、GSTM3以及相关指标变化。图3a-b.用靶向GSTM3和靶向IRF1的siRNA分别转染HaCaT细胞中GSTM3和IRF1表达的蛋白质印迹分析;siGSTM3-2和siIRF1-2是最有效的siRNA,用于后续实验。图3c-d.用0、2.5和5ng/ml浓度的M5刺激24小时的HaCaT细胞中GSTM3和IRF1的qPCR定量。图3e-h.富集指标的qPCR定量。图3f-g.具有IRF1和GSTM3过表达质粒的HaCaT细胞中IRF1和GSTM3表达的蛋白质印迹分析。数据代表来自三个独立实验的平均值±SD。***P<0.001,**P<0.01,*P<0.05。
图4.体内外实验表明CS通过IRF1/GSTM3轴发挥治疗作用。图4a-b.CCK8和qPCR定量研究表明,敲除GSTM3基因和过表达IRF1均可影响CS在M5诱导的HaCaT细胞中的作用。图4c-d.CCK8和qPCR定量检测结果表明,过表达GSTM3可部分抑制IRF1过表达导致的CS疗效减弱。图4e.荧光素酶报告基因检测转染HaCaT细胞中GSTM3启动子的荧光素酶活性。图4f.GSTM3启动子区域可能的结合位点示意图。图4g.IRF1 ChIP-qPCR检测显示GSTM3的4个启动子区域均有募集。图4h.银屑病小鼠的不同治疗方案。图4i.第8天免疫组织化学标记PCNA和NF-κB-p50。比例尺=200μm,n=5个样本/组。数据代表来自三个独立实验的平均值±SD。***P<0.001,**P<0.01,*P<0.05。
图5.指标变化和ChIP-qPCR检测情况。图5a-b.富集指标的qPCR定量。图5c.采用ChIP-PCR分析证明IRF1与GSTM3启动子结合。数据代表来自三个独立实验的平均值±SD。***P<0.001,**P<0.01,*P<0.05。
图6.nio-CS纳米凝胶改善IMQ诱导的银屑病表型效果优于cel nio gel。图6a.诱导8d后代表图像展示。图6b.总PASI评分。图6c.耳朵厚度。***P<0.001,**P<0.01,*P<0.05。
图7.nio-CS改善和预防IMQ诱导的银屑病表型。图7a.照片是在诱导后第8天拍摄的;耳朵厚度和总PASI合格。图7b.组织学染色和量化图像。标尺=200μm和500μm,n=5个样品/组。图7c.第8天小鼠皮肤中IL-17a、IL-1β、IL-22和IL-23的qPCR定量。图7d.第8天小鼠皮肤中Gstm3、IRF1、GSTM1和Gstm4的qPCR定量。图7e.银屑病复发小鼠模型的不同治疗方案。图7f.诱导8d后拍照,耳朵厚度和总PASI评分均合格。图7g.组织学染色和定量图像。标尺=200μm,n=5个样品/组。图7h.诱导30天后小鼠皮肤中IL-17a、IL-1β、Gstm3和IRF1的qPCR定量。数据代表来自三个独立实验的平均值±SD。***P<0.001,**P<0.01,*P<0.05。
图8.nio-CS凝胶应用在IMQ诱导的银屑病小鼠中的疗效和安全性测试。图8a.最后一天不同组小鼠血样中肾功能(尿素氮、肌酐、血清β2-微球蛋白和血清尿酸)、肝功能(丙氨酸氨基转移酶、天冬氨酸氨基转移酶、总含量)、胆红素、直接胆红素和间接胆红素)和糖脂代谢指标(总胆固醇、甘油三酯、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇、空腹血糖和胰岛素)的ELISA定量。n=5个样品/组。图8b.来自不同组的肝,肾和脾切片的病理分析。比例尺=100μm和250μm。数据代表来自三个独立实验的平均值±SD。***P<0.001,**P<0.01,*P<0.05。
图9.雷公藤红素防治银屑病的作用机制。
具体实施方式
本申请发明人经过广泛而深入的研究,发现IRF1/GSTM3轴是CS防治银屑病的重要作用靶标。
nio-CS
CS是从药用植物雷公藤(TwHf)中提取的一种五环三萜类化合物,分子式为C29H38O4,分子量450.61,CAS号为34157-83-0,具有强大的抗炎、促凋亡、抗氧化应激和抗血管生成功能,可以用于治疗多种自身免疫性疾病及慢性炎症,包括银屑病和特应性皮炎等。据报道,CS通过降低炎症水平来改善小鼠银屑病样皮损;同时,其强大的抗炎作用在体外实验中验证与抑制T细胞分化、炎症参数、KC增殖和中性粒细胞募集有关。此外,CS被证明是NF-κB途径阻滞剂和对抗皮肤损伤的抗氧化剂。但由于CS不溶于水,口服胃肠道吸收较差,生物利用度低,且毒性较大,口服可产生全身性的毒副作用。采用局部外用给药方式,可以降低口服给药导致的全身毒性作用,提高生物利用度,使用方便,降低耐药性。CS外用剂型防治银屑病较口服给药具有更可观前景。
本发明使用nio类脂质体包裹修饰CS,显著提高了CS的稳定性及渗透性,用于银屑病局部外用经皮防治,可达到明显改善皮损,且无脏腑毒性的效果,并能减少复发。。
IRF-1、GSTM31
谷胱甘肽巯基转移酶M3(Glutathione S-Transferase Mu 3,GSTM3)是一种蛋白质编码基因,编码一种属于Mu类的谷胱甘肽巯基转移酶。Mu类酶通过与谷胱甘肽结合,在亲电性化合物(包括致癌物、治疗药物、环境毒素和氧化应激产物)的解毒过程中起作用。与GSTM3相关的疾病有喉癌和咽癌等,其参与的通路包括谷胱甘肽代谢和NRF2途径等,相关的GO富集为微蛋白质同源二聚化活性和酶的结合等。GSTM3能够还原谷胱甘肽与大量外源性和内源性疏水性亲电试剂的偶联,可能控制睾丸和脑血屏障处内源性化合物和异种生物的摄取和解毒。
干扰素调节因子-1(Interferon Regulatory Factor 1,IRF-1)是一种蛋白质编码基因。该基因编码的蛋白质是转录调节因子和肿瘤抑制因子;是先天免疫反应和获得性免疫反应相关基因的激活因子。IRF-1的激活参与了人体对病毒和细菌的免疫反应中有关基因的转录,在细胞增殖、凋亡、免疫反应和DNA损伤反应中发挥作用。IRF-1作为一种肿瘤抑制因子,它既能抑制肿瘤细胞生长,又能刺激机体针对肿瘤细胞的免疫反应。该基因的缺陷与胃癌、髓性白血病和肺癌有关。
在本发明中,所用的IRF-1、GSTM3可以是天然存在的,比如其可以被分离或纯化自哺乳动物。此外,所述的IRF-1、GSTM3也可以是人工制备的,比如可以根据常规的基因工程重组技术来生产重组IRF-1、GSTM3。优选的,本发明可采用重组的IRF-1、GSTM3。
任何适合的IRF-1、GSTM3均可用于本发明。所述的IRF-1、GSTM3包括全长的GSTM3或其生物活性片段。优选的所述的GSTM3的氨基酸序列为NM_000849.5;优选的所述的IRF-1的氨基酸序列为NM_002198.3。
经过一个或多个氨基酸残基的取代、缺失、或添加而形成的IRF-1、GSTM3的氨基酸序列也包括在本发明中。IRF-1、GSTM3或其生物活性片段包括一部分保守氨基酸的替代序列,所述经氨基酸替换的序列并不影响其活性或保留了其部分的活性。适当替换氨基酸是本领域公知的技术,所述技术可以很容易地被实施并且确保不改变所得分子的生物活性。这些技术使本领域技术人员认识到,一般来说,在一种多肽的非必要区域改变单个氨基酸基本上不会改变生物活性。见Watson,Molecular Biology of The Gene,第四版,1987,TheBenjamin/Cummings Pub.Co.P224。
任何一种GSTM3的生物活性片段都可以应用到本发明中。在这里,IRF-1、GSTM3的生物活性片段的含义是指作为一种多肽,其仍然能保持全长的IRF-1、GSTM3的全部或部分功能。优选的,所述的生物活性片段至少保持50%的全长IRF-1、GSTM3的活性。在更优选的条件下,所述活性片段能够保持全长IRF-1、GSTM3的60%、70%、80%、90%、95%、99%、或100%的活性。
本发明也可采用经修饰或改良的IRF-1、GSTM3,比如,可采用为了促进其半衰期、有效性、代谢、和/或蛋白的效力而加以修饰或改良的IRF-1、GSTM3。所述经过修饰或改良的IRF-1、GSTM3可以是一种IRF-1、GSTM3的共轭物,或其可包含被取代的或人工的氨基酸。所述经过修饰或改良的IRF-1、GSTM3可以是与天然存在的IRF-1、GSTM3具有较小的共同点,但也能缓解银屑病或缓解银屑病样炎症,且不会带来其它不良影响或毒性。也就是说,任何不影响IRF-1、GSTM3的生物活性的变化形式都可用于本发明中。
根据IRF-1、GSTM3的氨基酸序列可以方便地得出其相应的核苷酸编码序列。
用途
本发明提供nio-CS、IRF-1或其抑制剂、GSTM3或其上调剂在制备防治银屑病的药物中的用途。
银屑病是一种常见的慢性、复发性、免疫介导的皮肤和关节性疾病,影响约全球3%的人口。银屑病具有很强的遗传性,但环境因素如感染,在银屑病的发病因素中也起着重要的作用。银屑病有多种临床皮损表现,但最常见的表现为慢性、对称性、红斑性、鳞屑性丘疹和斑块,斑块是角质细胞过度增殖和不完全分化的结果。除了影响皮肤本身外,各种并发症可能会影响患者的生活质量,对患者的情绪和心理健康产生显著的负面影响,增加死亡率。
药物组合物
本发明的药物组合物可含有本文所述的活性试剂和药学上可接受的载体。药学上可接受的载体通常是安全、无毒的,包括各种赋形剂和稀释剂等。该术语指这样一些药剂载体:它们本身并不是必要的活性成分,且施用后没有过分的毒性。合适的载体是本领域普通技术人员所熟知的。在Remington's Pharmaceutical Sciences(Mack Pub.Co.N.J.1991)中可找到关于药学上可接受的赋形剂的充分讨论。在组合物中药学上可接受的载体可含有液体,如水、盐水、甘油和乙醇。另外,这些载体中还可能存在辅助性的物质,如乳化剂、填充剂、粘合剂、润湿剂、崩解剂、促渗透剂、着色剂、助溶剂等。以上,所述的乳化剂诸如乙酰化单甘油脂肪酸酯、乙酰化双甘油脂肪酸酯、蔗糖酯、山梨糖醇脂、大豆磷脂、月桂酸单甘油酯、丙二醇脂肪酸酯、硬脂酰乳酸钙、双乙酰酒石酸、单硬脂酸甘油酯、改性大豆磷脂等。所述的赋形剂诸如硬脂酸镁、微晶纤维素、乳糖、奶糖、高分子量的聚乙二醇等。所述的填充剂诸如淀粉、甘露醇、硅酸、糊精、磷酸氢钙、纤维素等。所述的粘合剂诸如羧甲基纤维素、海藻酸盐、明胶、聚乙烯吡咯烷酮、阿拉伯胶、淀粉浆、羟丙基淀粉、改良淀粉、预胶化淀粉、糊精、微晶纤维素、聚乙烯吡咯烷酮胶浆、明胶浆。所述的润湿剂诸如甘油等。所述的崩解剂诸如琼脂、碳酸钙、马铃薯淀粉、木薯淀粉、海藻酸、羟丙基淀粉、改良淀粉、羧甲基淀粉钠、微晶纤维素、瓜耳胶、苍耳胶、黄原胶等。所述的促渗透剂诸如薄荷脑、月桂氮酮、冰片等。所述的着色剂可以是植物着色剂、动物着色剂或者微生物着色剂,诸如甜菜红、姜黄、叶绿素、紫胶红、胭脂虫红、红曲着色剂等。所述的助溶剂诸如β-环糊精、麦芽糊精、吐温、乙醇、司盘类、十二烷基硫酸钠、丙二醇、聚乙二醇、甘油等。但对于本领域技术人员而言,还知晓可用于本发明的药用载体不仅限于上述类型。
剂型
对于本发明所述药物组合物的剂型没有特别的限制,可以是任何适用于外用的剂型,包括但不限于贴剂、糊剂软膏剂、凝胶剂、涂膜剂或巴布剂。也可以是任何适用于内用的剂型,包括但不限于胶囊剂、颗粒剂、片剂、丸剂、口服液或注射液等。
治疗方法
本发明提供一种防治银屑病的方法,所述方法包括给予需要的对象nio-CS;IRF-1或其抑制剂;或GSTM3或其上调剂;给予的量为治疗有效量,可根据个体的年龄、体重、性别、疾病种类和严重程度加以确定。对象可以是哺乳动物,尤其是人、鼠、兔子、猪、羊和狗等。给药方法是本领域常规的方法,例如涂抹、敷贴等,并可针对不同的试剂进行调整。
下面结合附图对本发明提供的具体实施方式作详细说明。
实施例1
一、方法和材料
1.银屑病样皮损在体模型的建立
采用IMQ诱导的银屑病样小鼠模型进行体内研究。为进行疗效实验,将小鼠随机分为3组(IMQ乳膏62.5mg/只,6h后给予不同治疗策略,连续8d):IMQ组(4mg/kg空白nio-凝胶),CS组(4mg/kg nio-CS凝胶),CAL组(1mg/kg Cal乳膏)。在复发实验中,将小鼠随机分成3组,连续用药8d,恢复12d,再用IMQ(20.8mg/只)再次激发,连续10d。所有动物手术均经上海中医药大学伦理委员会批准(编号YYLAC-2019-064-1)。
2. 0.02%类脂质体制备
精密称取司盘20 30mg、司盘60 10mg、胆固醇10mg、CS 1mg,加入3ml氯仿甲醇(2:1),45℃水浴减压冷凝旋蒸,圆底烧瓶壁形成一层膜后,加入5ml水,常压60℃旋转水合30min,于超声波细胞粉碎仪(200W,6mm探头,0.1s/0.1s)4分钟,形成0.02%CS类脂质体(nio-CS纳米凝胶)。
精密称取司盘20 30mg、司盘60 10mg、胆固醇10mg、CS 1mg,加入3ml氯仿甲醇(2:1),45℃水浴减压冷凝旋蒸,圆底烧瓶壁形成一层膜后,加入5ml水,常压60℃旋转水合30min,于超声波细胞粉碎仪(125W,3mm探头,0.5s/0.5s)4分钟,形成0.02%cel nio gel。
3.体外试验
采用转录谱检测方法检测CS(1.332μM)或DMSO对HaCaT细胞的作用。接下来,我们进行了siRNA敲除、质粒过表达、ChIP-qPCR、荧光素酶报告基因检测、qPCR、Westernblotting和挽救实验,以确定IRF1/GSTM3轴的功能。
4.临床标本
临床样本取自出具书面知情同意书的患者,经岳阳医院伦理委员会批准(编号2019-29)。银屑病样本采集自患者的皮损皮肤,正常皮肤样本采集自整容手术期间多余的皮肤。切取组织后立即用70%福尔马林固定,石蜡包埋。
5.细胞实验
5.1细胞培养和处理
HaCaT细胞系(300493)取自细胞系服务部(Eppelheim,Germany),在含10%胎牛血清、100U/ml青霉素和100μg/ml链霉素的DMEM中生长。NHEK(正常人表皮角质形成细胞,C-12006)细胞系购自Promo Cell(Heidelberg,Germany),在角质形成细胞生长培养基2(D-39006,Heidelberg,Germany)中培养。上述细胞系于37℃培养箱中,在5%CO2的潮湿气氛中培养。雷公藤红素(CS,C0869,Sigma)以20mM的最终浓度作为治疗浓度,溶于DSMO;使用M5鸡尾酒(IL-1α,IL-17A,IL-22,抑瘤素M和肿瘤坏死因子-α,2.5ng/ml)概括模拟银屑病的特征。细胞样本内加入不同浓度的CS溶液,而未经处理的样本接受等量的DMSO。
5.2细胞计数试剂盒-8(CCK-8)检测
采用CCK8比色法检测细胞存活率和增殖能力。HaCaT细胞以2000个/孔的密度接种到96孔板上。24h后,用含不同浓度CS的培养基代替当前培养基,不含CS的培养基作为空白对照。孵育48h后,每孔加入10μl的CCK8溶液(NU678;Dojindo laboratories,Tabaru,Japan)。37℃孵育4h后,测定各孔在450nm处的吸光度。通过计算用CS和DMSO处理后孔的吸光度,来测量HaCaT细胞的存活率。
5.3质粒
将人IRF1基因编码区(RefSeq NM_002198.3)或人GSTM3基因编码区(NM_000849.5)克隆到pFLAG-CMV-4(Sigma,E1775)的HindⅢ(Fermentas,ER0505)和EcoRI(Fermentas,ER0271)位点。经Sanger测序证实克隆序列正确。
5.4转染
将IRF1表达载体PFLAG-CMV-4-IRF1或其载体电穿孔转染HaCaT细胞(BTXECM830)。30μg质粒在135v/25ms处理后,在2mm的试管中被带入70μl无血清培养液中。在基因操作后48h,对细胞进行富集,检测IRF1的过表达。
为了阐明与IRF1相关的GSTM3的作用,通过电穿孔将靶向GSTM3或IRF1(GenePharm)的siRNA寡核苷酸进一步导入HaCaT细胞。通常,在135v/25s的条件下,将4μMsiRNA或对照寡核苷酸和80μl无血清培养基的混合物在2毫米试管中引入HaCaT细胞。为了补偿电穿孔过程导致的细胞死亡,对照细胞在没有寡核苷酸的情况下进行电穿孔。电穿孔后用Western blot检测细胞裂解产物的表达。
SiRNA序列如下所示:
Human siGSTM3 Forward:5’CCU GGA CAA CUG AAA CAA UTT 3’(SEQ ID NO:1);Reverse:5’AUU GUU UCA GUU GUC CAG GTT 3’(SEQ ID NO:2)
Human siIRF1 Forward:5’CCA ACU UUC GCU GUG CCA UTT 3’(SEQ ID NO:3);Reverse:5’AUG GCA CAG CGA AAG UUG GTT 3’(SEQ ID NO:4)
6.转录图谱检测
进行mRNA微阵列分析。用1.332μM的CS或DSMO处理HaCaT细胞后,在5%CO2的潮湿气氛37℃孵箱中培养,48h后,用TRIzol裂解细胞,提取总RNA。样品立即放入液氮中,维持在-80℃。每个样本经mRNA选择、片段化、cDNA合成和文库制备后,用TruSeq链mRNA LT样品制备试剂盒(RS-122-2103,Illumina)测序。RNA测序由上海生物芯片有限公司完成。
研究CS处理的HaCaT细胞的表达谱。使用Benjamini和Hochbergs方法对所得P值进行调整,以控制假发现率(调整后的P值<0.05)。根据p值<0.05和|Log2F-C|≥2,筛选出差异表达(DE)mRNA,然后用Fisher检验后进行GO和KEGG分析。
7.小鼠模型及治疗
80只平均体重20±5g的雄性BALB/c小鼠,由上海吉辉实验动物饲养有限公司提供(合格证号:20170012008759;许可证号:SCXK(Hu)2017-0012;中国上海)。动物饲养在23±2℃的标准温度下,每笼5只小鼠,按SPF级12h光照/12h暗处理。动物可以自由获得高脂肪或标准饮食和水。银屑病模型造模前异氟醚麻醉小鼠,随后从背部取下2.5cm×2.5cm的毛发,并用化学霜脱毛,实验第一天,将小鼠随机分为4组:(i)空白组小鼠不接受任何治疗;(ii)给予IMQ组小鼠IMQ乳膏(中国四川明信制药有限公司,62.5mg/小鼠),随后在6小时后给予空白nio-gel(4mg/kg);(iii)治疗组小鼠在6小时后外用IMQ乳膏和nio-CS凝胶(4mg/kg);(iv)治疗组小鼠在6小时后外用IMQ乳膏和卡泊三醇乳膏。在8天期间,所有组的小鼠耳朵和背部的皮肤上每天施用IMQ霜,并且在6小时后连续8天施用不同的处理。最后一天,对所有小鼠实施安乐死并进行分析。
为了观察银屑病的复发情况,在实验的第一天,将小鼠随机分为4组:对照组、银屑病复发组、nio-CS治疗的银屑病复发组和Cal治疗的银屑病复发组。建立银屑病复发的小鼠模型如下:以与上述相同的分组和治疗连续8天的建模阶段,在没有任何药物应用的情况下恢复12天,然后所有小鼠用(20.8mg/小鼠)再连续每天施用IMQ霜10天。所有的小鼠都被安乐死,并在第29天取样。
银屑病的严重程度用适应的人类临床银屑病面积和严重程度指数(PASI)来测量,该指数的评分范围为0-4(无;1、轻微;2、适度;3、标注;4、非常显著)。此外,耳朵厚度值已经标准化,为治疗当天的值除以0天的值。
8.组织学分析
诱导后第8天对小鼠实施安乐死,并将样品固定在4%中性缓冲甲醛溶液中48小时。以常规方式处理组织以进行组织学评价并包埋在石蜡中,制备5微米切片,并用苏木精和伊红染色。载玻片在数字切片扫描仪(C12000-02;NanoZoomer Digital Pathology,Hamamatsu,Japan)下观察。
9.定量聚合酶链反应(q-PCR)
使用标准的TRIzol方案从皮肤活检和HaCaT细胞中提取总mRNA。通过用紫外分光光度计测量吸光度260/280比率来确定RNA浓度和纯度。使用逆转录试剂盒制备cDNA,并使用逆转录酶进行逆转录。使用2-ΔΔCT方法分析相对定量数据。
10.染色质共沉淀-定量聚合酶链反应(ChIP-qPCR)
为了探索结合GSTM3启动子的转录因子,应用了在线软件JASPAR和meme-FIMO。在CS刺激后,我们对HaCaT细胞进行了芯片定量聚合酶链反应。如前所述进行ChIP分析(Ru,Y.,Li,H.,Zhang,R.,Luo,Y.,Song,J.,Kuai,L.,Xing,M.,Hong,S.,Sun,X.,Ding,X.,Lu,Y.,Liu,L.,Na,C.,Zhou,Y.,Li,B.,Li,X.,2020.Role of keratinocytes and immunecells in the anti-inflammatory effects of Tripterygium wilfordii Hook.f.in amurine model of psoriasis.Phytomedicine 77,153299.https://doi.org/10.1016/j.phymed.2020.153299),并使用兔IgG同种型对照(稀释至1/2000;CST,3900S),抗IRF1(1:50,CST,8478)(以1/1000稀释;15214-1-AP,蛋白科技)。以相同数量的输入和目的基因为模板进行定量聚合酶链反应实验。用于芯片定量聚合酶链反应的四个IRF1结合位点列于表1。
表1.检测IRF-1结合位点的四种引物
11.Western blot分析
用于蛋白质印迹分析的主要抗体有抗GSTM3(1:1000,15214-1-AP,Proteintech)抗体和抗IRF1(1:1000,8478,Cell Signaling Technology)抗体。
12.免疫组织化学染色
部分切片用增殖细胞核抗原(以1/6400、ab29、Abcam稀释)、NF-κB-p50(以1/600、ab32360、Abcam稀释)、CD3(以1/3200、ab16669、Abcam稀释)、GSTM3(以1/1600、15214-1-AP、Proteintech稀释)和IRF1(以1/30、ab8478、Abcam稀释)抗体染色。用二氨基联苯胺对组织进行显影,并将其固定在中性香脂中。
13.荧光素酶报告实验
HaCaT细胞以每孔2.0×105细胞密度接种于24孔板中,培养24h后转染。然后,用含有不同数量结合基序的合成片段构建质粒,并用内参质粒pRL-SV40分别和pGL4.27、GSTM3-BS 1-4#、GSTM3-BS 2-4#、GSTM 3-4#、GSTM3-BS4#共转染细胞。上述各组分为接受或不接受CS治疗。转染后24小时收集细胞裂解物,然后用双荧光素酶报告分析系统(Promega,Madison,WI)在Berthold AutoLumat LB9507支架光度计上检测萤火虫和海肾素荧光素酶活性。相对荧光素酶活性的值显示萤火虫荧光素酶活性与每次测定的海肾荧光素酶活性归一化。与相应的空载体相比,CS治疗组具有显著差异。
14.统计分析
实验数据用SPSS 22.0进行分析,GraphPad Prism 5.2进行作图,所有数据均表示为平均值±标准误差(S.E.M.)。用t检验或方差分析比较组间差异。***p<0.001、**p<0.01和p≤0.05的值分别被认为有显著或非常显著的差异。
二、结果
1.体外实验发现CS治疗可升高GSTM3表达和抑制IRF1表达
为了鉴定CS的潜在毒性,采用CCK-8试验检测了CS在HaCaT和NHEK细胞中的细胞毒性。我们发现CS显著抑制了HaCaT细胞(IC50=1.332μM)和NHEK细胞(IC50=1.712μM)的生长。CS在HaCaT细胞中发挥更大的亲和力和特异性(图1a)。因此,1.332μM CS刺激的HaCaT细胞被用作后续研究的实验条件。
为了评估CS刺激改变的mRNAs,进行了转录组测序。在1036个差异表达(DE)mRNA(|log2 fold-change|≥1,调整后P值<0.05)中,372个(35.91%)上调,664个(64.09%)下调(图1b和图2a)。这一结果提示,正常细胞和CS处理的HaCaT细胞的mRNAs表达不一致,DEmRNAs的表达可能受CS刺激的调节。
为了评估基因突变的潜在机制,我们进行了关联分析。KEGG富集揭示谷胱甘肽代谢途径(路径:hsa00480)是差异最明显的途径(图1c)。此外,GO分析确定了许多与代谢相关过程和免疫/炎症相关过程相关的生物过程(图1d)。这些结果表明,CS刺激下调的DE mRNAs参与免疫反应,有利于减轻炎症浸润;而上调的DE mRNAs与代谢成分异常相关,而代谢成分异常常与银屑病合并。
GSTM3是银屑病中研究较少的谷胱甘肽相关基因,是CS诱导后谷胱甘肽代谢途径中最显著的改变的mRNA(图2a)。为了进一步探讨GSTM3的调控机制,我们利用PROMO和AliBaba对其启动子区域进行了结合元件筛选,获得了C/EBPGR、YY1和IRF-1,而IRF-1也在我们的转录组数据中得到了鉴定(图2a)。
为了确定CS作用后相应基因的变化,我们检测了HaCaT细胞中GSTM2、GSTM3、GSTM4、GCLC、GCLM和IRF1的表达水平。如(图1e)所示,CS显著上调GSTM2、GSTM3、GSTM4、GCLC和GCLM的表达,而下调IRF1的表达。
总的来说,CS在体外和体内显著上调谷胱甘肽相关基因,同时下调IRF1。
2.银屑病皮损中GSTM3的低表达和IRF1的高表达
结合临床,对人体标本中GSTM3和IRF1的表型特征进行了染色。首先,在银屑病皮损中发现CD3+染色浸润(图2b和图2c)。接下来,我们检测了银屑病皮损和非皮损皮肤中GSTM3和IRF1的激活状态。值得注意的是,与正常皮肤相比,银屑病皮损中GSTM3的胞浆染色减少,而IRF1的核染色增加(图2b和图2c)。综上所述,这些数据支持IRF1和GSTM3在人类银屑病进展中起关键作用。
3.IRF1通过GSTM3加重银屑病样缺陷
为了确定GSTM3和IRF1在M5诱导的细胞模型中的作用,我们在实验前用空白、GSTM3或IRF1小干扰RNA(siRNA)预处理HaCaT细胞。SiGSTM3-2和siIRF1-2对RNA的表达有很强的抑制作用,降幅超过90%,并用于后续的实验(图3a和图3b)。M5(包含IL-1α、IL-17A、IL-22、抑瘤素M和肿瘤坏死因子-α)已被证明可以诱导细胞增殖和炎症,在体外概括了银屑病的许多特征。为了确定刺激的最佳M5浓度,将HaCaT细胞与不同浓度的M5(0、2.5和5ng/ml)培养,2.5ng/ml的M5对GSTM3和IRF1有明显的修饰作用(图3c和图3d)。CCK8实验显示,M5在72h时促进细胞增殖,而siGSTM3 HaCaT细胞的增殖能力增强,siIRF1细胞的增殖能力下降(图2f)。M5刺激后炎症、AMPs和增殖标记物加重,而GSTM3基因敲除后升高更明显,IRF1沉默后与M5刺激的细胞相比下调(图2g和图3e)。提示GSTM3和IRF1可能影响银屑病发病过程中的细胞增殖、免疫浸润、AMPs和屏障功能标志物的表达,可能影响银屑病发病的核心途径。
为了研究IRF1和GSTM3之间的相互作用,我们用空白、GSTM3或IRF1过表达质粒预处理HaCaT细胞(图3f和图3g)。在IRF1过表达的细胞中观察到增殖增加,而GSTM3过表达在48h和72h逆转了这种过度增殖(图2h)。同时检测免疫浸润、AMPs、屏障标志物、增殖等指标,与我们的预期一致,GSTM3的过表达逆转了IRF1过表达导致的细胞表型(图2i和图3h)。这些数据表明IRF1和GSTM3之间的显著负相关关系。
4.CS通过IRF1/GSTM3轴发挥防治作用
为了进一步探讨CS刺激HaCaT细胞对IRF1/GSTM3轴的影响,我们接下来进行了功能实验。抑制GSTM3和IRF1的过度表达可逆转CS的防治作用(图4a、4b和图5a)。因此,我们提供了GSTM3和IRF1表达改变影响CS疗效的证据。为了阐明CS是如何通过IRF1/GSTM3轴调节下游进程的,我们发现GSTM3的过表达消除了IRF1过表达导致的CS的疗效减弱(图4c、4d和图5b)。因此,我们证实CS通过IRF1/GSTM3轴改善表型损伤。
5.CS直接作用于结合位点(BSS)以减弱IRF1介导的GSTM3抑制作用
利用在线软件PROMOO和AliBaba,在人GSTM3基因转录起始点的启动子元件中预测了4个潜在的IRF1结合位点(图4f)。为了证实GSTM3是IRF1的转录靶点,将GSTM3启动子区域克隆到pGL4.27荧光素酶报告基因中,构建了4个BS质粒。4个报道的质粒分别含有GSTM3,BS1#:-4907bp至4230bp(共9138bp),BS2#:-1200bp至4230bp(共5431bp),BS3#:从3457bp至4230bp(共774bp),BS4#:从3913bp至4230bp(共318bp)。与对照组相比,IRF1的相对荧光素酶活性显著增强,CS诱导后,IRF1的相对荧光素酶活性降低(图4e)。结果表明,IRF1可能是GSTM3的转录抑制因子,CS的引入在转录水平上减轻了GSTM3的抑制作用。
为了进一步检测4种BSS的结合强度,进行了ChIP-qPCR分析。我们发现IRF1在GSTM3启动子区域的BS1#处有很强的结合,CS显著减弱了结合强度(图4g和图5c)。因此,CS诱导将IRF1从GSTM3启动子区域逐出,而BS1#在体外对CS诱导的GSTM3表达显示出最高的活性。
6.nio-CS凝胶改善咪喹莫特(IMQ)诱导的银屑病皮肤炎症及银屑病复发模型表型
为了验证以上发现,我们运用IMQ诱导的银屑病样小鼠模型进行体内实验(图4h,8天),我们制备得到CS纳米凝胶nio-CS和cel nio gel。nio-CS能够促进CS溶解度的同时,还能保持CS持续释放(图1f、1g和图1h)。在治疗过程中,nio-CS凝胶的应用可缓解皮肤干燥粗糙,降低PASI评分(银屑病面积和严重程度指数)和耳朵厚度,且效果较cel nio gel更为显著(图6,PASI评分day7时p<0.01,day8 p<0.05;耳朵厚度day8时p<0.01);甚至与卡泊三醇(CAL,阳性组)相似(图7a)。组织学上,nio-CS凝胶治疗可防止异常表皮增厚、网纹延长、角化过度和表皮炎症过度浸润,疗效与卡泊三醇相似甚至更好(图4i、7b和图7c)。此外,Gstm3、GSTM1和Gstm4 mRNA在银屑病样皮损中的表达与对照组相比降低,但在nio-CS治疗后表达增强,而在IRF1中则表现出相反的趋势(图2d和2e)。这些结果表明,nio-CS凝胶可以抑制银屑病皮肤过度炎症,改变IRF1/GSTM3轴相关基因。
为评价nio-CS凝胶的安全性,在实验结束时检测糖脂代谢和肝肾功能、肝、肾、脾组织病理学相关指标(图8a和图8b)。与IMQ组相比,nio-CS治疗组的大多数指标没有改变,显示出可接受的安全性。
另一方面,为了进一步验证CS对皮肤屏障功能和疾病复发的保护作用,我们扩展了银屑病复发模型。用或不用CS/Cal乳膏治疗小鼠8天,休息12天,然后再用1/3剂量的IMQ乳膏刺激10天(图7e)。
在二次刺激中,先前使用IMQ和Cal乳膏的小鼠在给药的最后一天出现严重的银屑病样皮肤损害,而先前的nio-CS凝胶治疗显示轻微的皮肤损害(图7f、7g和图7h)。此外,我们计数了最终皮损中的部分基因表达,发现银屑病样皮损中IL-17a和IL-1β的表达比对照组增加,而nio-CS治疗可以持续抑制这些表达。IMQ组皮肤屏障受损与FLG、Flg2表达下降有关,nio-CS治疗可保护皮肤屏障功能。GSTM3在银屑病样皮损中与对照组相比降低,但在nio-CS治疗后增加,而在IRF1中表现出相反的趋势(图7d和图7h)。
以上结果提示nio-CS凝胶具有预防银屑病复发的作用。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
SEQUENCE LISTING
<110> 上海市皮肤病医院
<120> 一种雷公藤红素纳米药物及IRF1/GSTM3轴在制备银屑病药物中的应用
<130> /
<160> 12
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> RNA
<213> 人工序列
<400> 1
ccuggacaac ugaaacaau 19
<210> 2
<211> 19
<212> RNA
<213> 人工序列
<400> 2
auuguuucag uuguccagg 19
<210> 3
<211> 19
<212> RNA
<213> 人工序列
<400> 3
ccaacuuucg cugugccau 19
<210> 4
<211> 19
<212> RNA
<213> 人工序列
<400> 4
auggcacagc gaaaguugg 19
<210> 5
<211> 24
<212> DNA
<213> 人工序列
<400> 5
aatttatagc actaaatgcc caca 24
<210> 6
<211> 24
<212> DNA
<213> 人工序列
<400> 6
ttgcgtctat ttgattcttc tctc 24
<210> 7
<211> 23
<212> DNA
<213> 人工序列
<400> 7
gctctcattt ttccctaacc ttt 23
<210> 8
<211> 23
<212> DNA
<213> 人工序列
<400> 8
agccttctca gaatcaccct cta 23
<210> 9
<211> 23
<212> DNA
<213> 人工序列
<400> 9
ggaggcactg tctacaaaaa aat 23
<210> 10
<211> 23
<212> DNA
<213> 人工序列
<400> 10
cctgaaagga aaatacacca aat 23
<210> 11
<211> 22
<212> DNA
<213> 人工序列
<400> 11
cttgttttgt ttcatcctgt cc 22
<210> 12
<211> 22
<212> DNA
<213> 人工序列
<400> 12
cctttttccc ttttagcctt ta 22
Claims (1)
1.雷公藤红素纳米药物在制备防治银屑病的药物中的应用,其特征在于,所述雷公藤红素纳米药物是按照下述方法制备的:精密称取司盘2030mg、司盘6010mg、胆固醇10mg、雷公藤红素1mg,加入3ml体积比为2:1的氯仿甲醇,45℃水浴减压冷凝旋蒸,容器壁形成一层膜后,加入5ml水,常压60℃旋转水合30min,于超声波细胞粉碎仪处理4分钟,超声波细胞粉碎仪处理参数为200W,6mm探头,0.1s/0.1s,最终形成所述雷公藤红素纳米药物。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210062480.9A CN114306280B (zh) | 2022-01-19 | 2022-01-19 | 一种雷公藤红素纳米药物及irf1/gstm3轴在制备银屑病药物中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210062480.9A CN114306280B (zh) | 2022-01-19 | 2022-01-19 | 一种雷公藤红素纳米药物及irf1/gstm3轴在制备银屑病药物中的应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114306280A CN114306280A (zh) | 2022-04-12 |
CN114306280B true CN114306280B (zh) | 2023-11-21 |
Family
ID=81029686
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210062480.9A Active CN114306280B (zh) | 2022-01-19 | 2022-01-19 | 一种雷公藤红素纳米药物及irf1/gstm3轴在制备银屑病药物中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114306280B (zh) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102225205A (zh) * | 2011-06-17 | 2011-10-26 | 江苏省中医药研究院 | 一种雷公藤红素纳米结构脂质载体及其制备方法和用途 |
CN106880638A (zh) * | 2017-02-23 | 2017-06-23 | 中国人民解放军第四军医大学 | 可抑制角质形成细胞的过度增殖的抑制剂、抑制剂组合物及应用 |
CN108743534A (zh) * | 2018-06-20 | 2018-11-06 | 澳门大学 | 一种雷公藤红素或雷公藤红素衍生物囊泡及其制备方法 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017201448A1 (en) * | 2016-05-20 | 2017-11-23 | U.S. Government As Represented By The Department Of Veterans Affairs | Animal models for psoriasis and screening methods |
-
2022
- 2022-01-19 CN CN202210062480.9A patent/CN114306280B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102225205A (zh) * | 2011-06-17 | 2011-10-26 | 江苏省中医药研究院 | 一种雷公藤红素纳米结构脂质载体及其制备方法和用途 |
CN106880638A (zh) * | 2017-02-23 | 2017-06-23 | 中国人民解放军第四军医大学 | 可抑制角质形成细胞的过度增殖的抑制剂、抑制剂组合物及应用 |
CN108743534A (zh) * | 2018-06-20 | 2018-11-06 | 澳门大学 | 一种雷公藤红素或雷公藤红素衍生物囊泡及其制备方法 |
Also Published As
Publication number | Publication date |
---|---|
CN114306280A (zh) | 2022-04-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10202601B2 (en) | C/EBPα short activating RNA compositions and methods of use | |
Liu et al. | MicroRNA-129-5p inhibits the development of autoimmune encephalomyelitis-related epilepsy by targeting HMGB1 through the TLR4/NF-kB signaling pathway | |
Fu et al. | Suppressing long noncoding RNA OGRU ameliorates diabetic retinopathy by inhibition of oxidative stress and inflammation via miR-320/USP14 axis | |
Hu et al. | Chlorogenic acid alleviates acetaminophen-induced liver injury in mice via regulating Nrf2-mediated HSP60-initiated liver inflammation | |
Hu et al. | Genistein protects epilepsy-induced brain injury through regulating the JAK2/STAT3 and Keap1/Nrf2 signaling pathways in the developing rats | |
Fu et al. | Exploring a novel triptolide derivative possess anti-colitis effect via regulating T cell differentiation | |
Mou et al. | Dopamine receptor agonists ameliorate bleomycin-induced pulmonary fibrosis by repressing fibroblast differentiation and proliferation | |
CN110064045A (zh) | 微肽cip2a-bp在治疗癌症中的应用 | |
CN114306280B (zh) | 一种雷公藤红素纳米药物及irf1/gstm3轴在制备银屑病药物中的应用 | |
CN113398246B (zh) | eIF3I-PDL1-IRS4轴在制备难治性溃疡药物中的应用 | |
Chen et al. | Activation of mitochondrial DNA-mediated cGAS-STING pathway contributes to chronic postsurgical pain by inducing type I interferons and A1 reactive astrocytes in the spinal cord | |
WO2022095976A1 (zh) | 小分子sr9009在抗衰老以及减轻衰老引起的慢性炎症中的用途 | |
Ding et al. | RETRACTED: Sevoflurane improves nerve regeneration and repair of neurological deficit in brain damage rats via microRNA-490-5p/CDK1 axis | |
CN116019916A (zh) | 非小细胞肺癌靶点arid1a及其抑制剂在制备肺癌治疗药物中的用途 | |
KR20230012596A (ko) | 췌장염의 치료 및 췌장 암의 예방 방법 | |
KR20160093637A (ko) | 종양에 대한 직접적 억제 효과를 갖는 인터페론을 결정하는 방법 및 그의 용도 | |
CN108403711B (zh) | 一种用于检测和治疗炎性肠病的microRNA | |
Yu et al. | Atractylodin alleviates cancer anorexia-cachexia syndrome by regulating NPY through hypothalamic Sirt1/AMPK axis-induced autophagy | |
CN115414485B (zh) | uN2CpolyG蛋白抑制剂的用途 | |
Bu et al. | JunB-EGFR axis is critical for TGF-β1/P38 MAPK signaling-mediated hepatic stellate cells proliferation in liver fibrosis | |
Qin et al. | Protective effect of fluoxetine against oxidative stress induced by renal ischemia-reperfusion injury via the regulation of miR-450b-5p/Nrf2 axis | |
CN113337594B (zh) | Lpcat1基因在制备治疗肝脏炎症药物及诊断试剂盒中的应用 | |
Kim et al. | Alleviation of preeclampsia-like symptoms through PlGF and eNOS regulation by hypoxia-and NF-κB-responsive miR-214-3p deletion | |
Jia et al. | Inhibition of spinal BRD4 alleviates pyroptosis and M1 microglia polarization via STING-IRF3 pathway in morphine-tolerant rats | |
CN116688124A (zh) | 雌激素响应相关通路的抑制及其用途 |
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 |