CN117143351A - Zn-MOF for treating spinal cord injury and preparation method and application thereof - Google Patents
Zn-MOF for treating spinal cord injury and preparation method and application thereof Download PDFInfo
- Publication number
- CN117143351A CN117143351A CN202311271851.5A CN202311271851A CN117143351A CN 117143351 A CN117143351 A CN 117143351A CN 202311271851 A CN202311271851 A CN 202311271851A CN 117143351 A CN117143351 A CN 117143351A
- Authority
- CN
- China
- Prior art keywords
- mof
- spinal cord
- injury
- cord injury
- mice
- 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.)
- Pending
Links
- 239000013094 zinc-based metal-organic framework Substances 0.000 title claims abstract description 151
- 229910007566 Zn-MOF Inorganic materials 0.000 title claims abstract description 149
- 208000020431 spinal cord injury Diseases 0.000 title claims abstract description 91
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 230000036542 oxidative stress Effects 0.000 claims abstract description 29
- 239000003814 drug Substances 0.000 claims abstract description 14
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 10
- 201000010099 disease Diseases 0.000 claims abstract description 6
- 150000001875 compounds Chemical class 0.000 claims description 30
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
- 238000005119 centrifugation Methods 0.000 claims description 15
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 13
- 238000010438 heat treatment Methods 0.000 claims description 12
- 239000007787 solid Substances 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 229940074391 gallic acid Drugs 0.000 claims description 7
- 235000004515 gallic acid Nutrition 0.000 claims description 7
- 239000003937 drug carrier Substances 0.000 claims description 5
- 208000035475 disorder Diseases 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- 238000010992 reflux Methods 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 239000008194 pharmaceutical composition Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 230000006378 damage Effects 0.000 abstract description 81
- 208000027418 Wounds and injury Diseases 0.000 abstract description 76
- 208000014674 injury Diseases 0.000 abstract description 76
- 210000002569 neuron Anatomy 0.000 abstract description 27
- 238000000034 method Methods 0.000 abstract description 23
- 101000990902 Homo sapiens Matrix metalloproteinase-9 Proteins 0.000 abstract description 22
- 102100030412 Matrix metalloproteinase-9 Human genes 0.000 abstract description 22
- 229910052760 oxygen Inorganic materials 0.000 abstract description 21
- 230000008439 repair process Effects 0.000 abstract description 21
- 230000000694 effects Effects 0.000 abstract description 20
- 238000011084 recovery Methods 0.000 abstract description 18
- 108090001005 Interleukin-6 Proteins 0.000 abstract description 15
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract description 15
- 239000001301 oxygen Substances 0.000 abstract description 15
- 230000014509 gene expression Effects 0.000 abstract description 14
- 230000007659 motor function Effects 0.000 abstract description 13
- 210000004295 hippocampal neuron Anatomy 0.000 abstract description 12
- 230000003078 antioxidant effect Effects 0.000 abstract description 11
- 239000003963 antioxidant agent Substances 0.000 abstract description 8
- 230000015572 biosynthetic process Effects 0.000 abstract description 8
- 230000003834 intracellular effect Effects 0.000 abstract description 7
- 231100000331 toxic Toxicity 0.000 abstract description 7
- 230000002588 toxic effect Effects 0.000 abstract description 7
- 102000003777 Interleukin-1 beta Human genes 0.000 abstract description 6
- 108090000193 Interleukin-1 beta Proteins 0.000 abstract description 6
- 210000000056 organ Anatomy 0.000 abstract description 6
- 238000011160 research Methods 0.000 abstract description 6
- 230000014511 neuron projection development Effects 0.000 abstract description 3
- 241000699670 Mus sp. Species 0.000 description 107
- 210000004027 cell Anatomy 0.000 description 50
- 210000000278 spinal cord Anatomy 0.000 description 42
- 239000003642 reactive oxygen metabolite Substances 0.000 description 32
- 241000699666 Mus <mouse, genus> Species 0.000 description 29
- 210000003141 lower extremity Anatomy 0.000 description 26
- 239000000243 solution Substances 0.000 description 23
- 238000002474 experimental method Methods 0.000 description 21
- 210000001519 tissue Anatomy 0.000 description 19
- 230000001965 increasing effect Effects 0.000 description 18
- 239000012621 metal-organic framework Substances 0.000 description 16
- 102000004889 Interleukin-6 Human genes 0.000 description 14
- 230000006870 function Effects 0.000 description 13
- 238000004458 analytical method Methods 0.000 description 12
- 230000002829 reductive effect Effects 0.000 description 12
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 11
- 229940079593 drug Drugs 0.000 description 9
- 210000002683 foot Anatomy 0.000 description 9
- 230000033001 locomotion Effects 0.000 description 9
- 230000000971 hippocampal effect Effects 0.000 description 8
- 230000028709 inflammatory response Effects 0.000 description 8
- 230000001537 neural effect Effects 0.000 description 8
- 150000003254 radicals Chemical class 0.000 description 8
- 208000034486 Multi-organ failure Diseases 0.000 description 7
- 210000003050 axon Anatomy 0.000 description 7
- 229910002091 carbon monoxide Inorganic materials 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 230000005021 gait Effects 0.000 description 7
- -1 superoxide anions Chemical class 0.000 description 7
- 210000004556 brain Anatomy 0.000 description 6
- 230000003833 cell viability Effects 0.000 description 6
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 230000003137 locomotive effect Effects 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 5
- 230000001154 acute effect Effects 0.000 description 5
- 230000029918 bioluminescence Effects 0.000 description 5
- 238000005415 bioluminescence Methods 0.000 description 5
- 238000000684 flow cytometry Methods 0.000 description 5
- 230000007760 free radical scavenging Effects 0.000 description 5
- 210000002216 heart Anatomy 0.000 description 5
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 210000003734 kidney Anatomy 0.000 description 5
- 210000004185 liver Anatomy 0.000 description 5
- 210000004072 lung Anatomy 0.000 description 5
- 210000000952 spleen Anatomy 0.000 description 5
- 239000011550 stock solution Substances 0.000 description 5
- 206010002091 Anaesthesia Diseases 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 102000002274 Matrix Metalloproteinases Human genes 0.000 description 4
- 108010000684 Matrix Metalloproteinases Proteins 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 239000004365 Protease Substances 0.000 description 4
- 108010087230 Sincalide Proteins 0.000 description 4
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 4
- 230000037005 anaesthesia Effects 0.000 description 4
- 238000010609 cell counting kit-8 assay Methods 0.000 description 4
- 230000005779 cell damage Effects 0.000 description 4
- 208000037887 cell injury Diseases 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- 210000003414 extremity Anatomy 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 238000003384 imaging method Methods 0.000 description 4
- 230000002757 inflammatory effect Effects 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 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 4
- 230000004083 survival effect Effects 0.000 description 4
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 206010018341 Gliosis Diseases 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 3
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 3
- 241000700159 Rattus Species 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 230000021164 cell adhesion Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 229910052804 chromium Inorganic materials 0.000 description 3
- 239000011651 chromium Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 230000007387 gliosis Effects 0.000 description 3
- 229930195712 glutamate Natural products 0.000 description 3
- 229940049906 glutamate Drugs 0.000 description 3
- 230000004054 inflammatory process Effects 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 210000003205 muscle Anatomy 0.000 description 3
- 230000007830 nerve conduction Effects 0.000 description 3
- 230000008506 pathogenesis Effects 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 238000012453 sprague-dawley rat model Methods 0.000 description 3
- YFDSDPIBEUFTMI-UHFFFAOYSA-N tribromoethanol Chemical compound OCC(Br)(Br)Br YFDSDPIBEUFTMI-UHFFFAOYSA-N 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- 102000000589 Interleukin-1 Human genes 0.000 description 2
- 108010002352 Interleukin-1 Proteins 0.000 description 2
- 108090000526 Papain Proteins 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 206010053649 Vascular rupture Diseases 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000003064 anti-oxidating effect Effects 0.000 description 2
- 210000001130 astrocyte Anatomy 0.000 description 2
- 230000003376 axonal effect Effects 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 230000003542 behavioural effect Effects 0.000 description 2
- 230000002146 bilateral effect Effects 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- 210000003710 cerebral cortex Anatomy 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 210000003618 cortical neuron Anatomy 0.000 description 2
- 230000009193 crawling Effects 0.000 description 2
- 231100000433 cytotoxic Toxicity 0.000 description 2
- 230000001472 cytotoxic effect Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000012377 drug delivery Methods 0.000 description 2
- 230000004064 dysfunction Effects 0.000 description 2
- 230000000763 evoking effect Effects 0.000 description 2
- 230000002964 excitative effect Effects 0.000 description 2
- 230000003492 excitotoxic effect Effects 0.000 description 2
- 231100000063 excitotoxicity Toxicity 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 210000004209 hair Anatomy 0.000 description 2
- 210000001320 hippocampus Anatomy 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000011503 in vivo imaging Methods 0.000 description 2
- 210000004969 inflammatory cell Anatomy 0.000 description 2
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000010534 mechanism of action Effects 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 230000004770 neurodegeneration Effects 0.000 description 2
- 208000015122 neurodegenerative disease Diseases 0.000 description 2
- 230000004693 neuron damage Effects 0.000 description 2
- 210000000440 neutrophil Anatomy 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 238000010899 nucleation Methods 0.000 description 2
- 229940055729 papain Drugs 0.000 description 2
- 235000019834 papain Nutrition 0.000 description 2
- 230000004796 pathophysiological change Effects 0.000 description 2
- 230000008289 pathophysiological mechanism Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 235000019419 proteases Nutrition 0.000 description 2
- 230000011514 reflex Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 210000002027 skeletal muscle Anatomy 0.000 description 2
- 229940073490 sodium glutamate Drugs 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000000451 tissue damage Effects 0.000 description 2
- 231100000827 tissue damage Toxicity 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 229950004616 tribromoethanol Drugs 0.000 description 2
- 210000000689 upper leg Anatomy 0.000 description 2
- 239000011701 zinc Substances 0.000 description 2
- 229910052725 zinc Inorganic materials 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- KISWVXRQTGLFGD-UHFFFAOYSA-N 2-[[2-[[6-amino-2-[[2-[[2-[[5-amino-2-[[2-[[1-[2-[[6-amino-2-[(2,5-diamino-5-oxopentanoyl)amino]hexanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]-5-(diaminomethylideneamino)p Chemical compound C1CCN(C(=O)C(CCCN=C(N)N)NC(=O)C(CCCCN)NC(=O)C(N)CCC(N)=O)C1C(=O)NC(CO)C(=O)NC(CCC(N)=O)C(=O)NC(CCCN=C(N)N)C(=O)NC(CO)C(=O)NC(CCCCN)C(=O)NC(C(=O)NC(CC(C)C)C(O)=O)CC1=CC=C(O)C=C1 KISWVXRQTGLFGD-UHFFFAOYSA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 208000010392 Bone Fractures Diseases 0.000 description 1
- 102000004266 Collagen Type IV Human genes 0.000 description 1
- 108010042086 Collagen Type IV Proteins 0.000 description 1
- 102000002734 Collagen Type VI Human genes 0.000 description 1
- 108010043741 Collagen Type VI Proteins 0.000 description 1
- 102000009736 Collagen Type XI Human genes 0.000 description 1
- 108010034789 Collagen Type XI Proteins 0.000 description 1
- 206010010254 Concussion Diseases 0.000 description 1
- 208000034656 Contusions Diseases 0.000 description 1
- 208000016192 Demyelinating disease Diseases 0.000 description 1
- 206010012305 Demyelination Diseases 0.000 description 1
- 102000016942 Elastin Human genes 0.000 description 1
- 108010014258 Elastin Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 235000009161 Espostoa lanata Nutrition 0.000 description 1
- 240000001624 Espostoa lanata Species 0.000 description 1
- 206010017076 Fracture Diseases 0.000 description 1
- 229940123457 Free radical scavenger Drugs 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 102000013382 Gelatinases Human genes 0.000 description 1
- 108010026132 Gelatinases Proteins 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 1
- 102000001776 Matrix metalloproteinase-9 Human genes 0.000 description 1
- 108010015302 Matrix metalloproteinase-9 Proteins 0.000 description 1
- 102000047918 Myelin Basic Human genes 0.000 description 1
- 102000006386 Myelin Proteins Human genes 0.000 description 1
- 108010083674 Myelin Proteins Proteins 0.000 description 1
- 101710107068 Myelin basic protein Proteins 0.000 description 1
- 108010025020 Nerve Growth Factor Proteins 0.000 description 1
- 102000007072 Nerve Growth Factors Human genes 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 206010029350 Neurotoxicity Diseases 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 206010033799 Paralysis Diseases 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 230000002292 Radical scavenging effect Effects 0.000 description 1
- 206010039203 Road traffic accident Diseases 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 208000020339 Spinal injury Diseases 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 239000006180 TBST buffer Substances 0.000 description 1
- 206010044221 Toxic encephalopathy Diseases 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 208000032594 Vascular Remodeling Diseases 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000010398 acute inflammatory response Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 206010002026 amyotrophic lateral sclerosis Diseases 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000012984 biological imaging Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000000546 chi-square test Methods 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 230000009519 contusion Effects 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 239000002178 crystalline material Substances 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 229920002549 elastin Polymers 0.000 description 1
- 238000010894 electron beam technology Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 210000002165 glioblast Anatomy 0.000 description 1
- 210000000020 growth cone Anatomy 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 208000010726 hind limb paralysis Diseases 0.000 description 1
- 210000000548 hind-foot Anatomy 0.000 description 1
- 210000004394 hip joint Anatomy 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000003960 inflammatory cascade Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000009916 joint effect Effects 0.000 description 1
- 210000000629 knee joint Anatomy 0.000 description 1
- 238000002684 laminectomy Methods 0.000 description 1
- 230000003859 lipid peroxidation Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 229910052748 manganese Inorganic materials 0.000 description 1
- 239000011572 manganese Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 210000000274 microglia Anatomy 0.000 description 1
- 230000027939 micturition Effects 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 210000005012 myelin Anatomy 0.000 description 1
- 230000023105 myelination Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 210000002241 neurite Anatomy 0.000 description 1
- 230000007472 neurodevelopment Effects 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 230000000926 neurological effect Effects 0.000 description 1
- 230000016273 neuron death Effects 0.000 description 1
- 230000006764 neuronal dysfunction Effects 0.000 description 1
- 230000003961 neuronal insult Effects 0.000 description 1
- 231100000189 neurotoxic Toxicity 0.000 description 1
- 230000002887 neurotoxic effect Effects 0.000 description 1
- 230000007135 neurotoxicity Effects 0.000 description 1
- 231100000228 neurotoxicity Toxicity 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 239000003900 neurotrophic factor Substances 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 239000013110 organic ligand Substances 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 238000012261 overproduction Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000036407 pain Effects 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000013259 porous coordination polymer Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 206010041569 spinal fracture Diseases 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000002660 stem cell treatment Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000006557 surface reaction Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000003956 synaptic plasticity Effects 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 210000000115 thoracic cavity Anatomy 0.000 description 1
- 208000037816 tissue injury Diseases 0.000 description 1
- 231100000041 toxicology testing Toxicity 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229910052723 transition metal Inorganic materials 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 210000003932 urinary bladder Anatomy 0.000 description 1
- 230000008728 vascular permeability Effects 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
- C08G83/008—Supramolecular polymers
-
- 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
- A61K31/74—Synthetic polymeric materials
- A61K31/80—Polymers containing hetero atoms not provided for in groups A61K31/755 - A61K31/795
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Organic Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Epidemiology (AREA)
- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to Zn-MOF for treating spinal cord injury and a preparation method and application thereof. The Zn-MOF provided by the invention can effectively reduce the intracellular active oxygen level, reduce the IL-6 and IL-1 beta level after spinal cord injury, inhibit the expression of MMP9, realize remarkable antioxidant capacity, repair hippocampal neuron cells, resist injury induced by oxidative stress, and promote neuron branch formation and neurite growth. Meanwhile, the Zn-MOF can obviously promote oxygen free radicals in the body of a spinal cord injury mouse and promote the repair of spinal cord injury and the recovery of motor functions. The Zn-MO prepared by the method has no obvious toxic or side effect on main tissues and organs of an organism, and the safety is obviously improved, so that a new research thought and a medicine source are provided for treating diseases related to oxidative stress including spinal cord injury, and the Zn-MO has obvious clinical application prospect and social value.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to Zn-MOF for treating spinal cord injury and a preparation method and application thereof.
Background
Spinal cord injury (Spinal cord injury, SCI) is a serious complication of spinal injury, often secondary to spinal fractures caused by direct or indirect external forces, such as direct violence, exercise, falls, traffic accidents, overhead falls, and the like. Spinal cord injuries can lead to dysfunction of the patient's extremities, intestines, bladder, and sex, etc., causing serious physical and mental injuries to the patient, greatly reducing the quality of life of the patient. It is counted that there are about 2000 or more tens of thousands of spinal cord injured patients worldwide, and the number of about 70 tens of thousands of patients per year is increasing, with a male to female ratio of about 2:1, and the number of adults is greater than that of children. At present, the treatment of spinal cord injury is mainly focused on surgical treatment, drug treatment, stem cell treatment and the like, and no definite and effective treatment method exists. Therefore, there is an urgent need for more intensive studies on spinal cord injury. .
Spinal cord injury has extremely complex pathophysiological mechanisms, such as cell depletion and death, lack of neurotrophic factors, inflammatory response, axon rupture, demyelination, and gliosis, which inhibit regeneration and recovery of damaged nerves following spinal cord injury. After spinal cord injury occurs, a series of complex pathophysiological changes occur in the injured area, and the changes can be classified into primary injury and secondary injury according to time and pathophysiological changes. Primary injury is the initial stage of injury, and occurs immediately after injury, including spinal cord concussion, contusion and fracture at the time of injury, and spinal cord vascular rupture, erythrocyte shedding in the damaged area, axonal rupture, myelin rupture, etc., and the severity of injury is mainly determined by the degree of initial injury and the duration of compression of the spinal cord. Secondary injury occurs after primary injury, which triggers a complex cascade of secondary injuries that produce further chemical and mechanical injury to spinal cord tissue, including a series of inflammatory cascades that result from the aggregation of various activated inflammatory cells to the injured area, lipid peroxidation, free radical formation, apoptosis, waller degeneration, tissue edema, gliosis, and the like. Spinal cord injury is divided into acute, subacute and chronic phases. The spinal cord injury belongs to the acute phase within 48h, the injury 48h to 14d belongs to the subacute phase, the injury 14d to 6 months belongs to the medium phase, and the injury more than 6 months belongs to the chronic phase. In the acute phase of spinal cord injury, severe bleeding due to spinal cord vascular rupture, and significant increases in the levels of many inflammatory factors such as IL-6, IL-1. Beta. And TNF-alpha, can lead to cellular dysfunction and death. In the acute and subacute phases of spinal cord injury, further injury to the spinal cord can be exacerbated by disruption of the blood-spinal cord barrier, severe inflammatory reactions, and many cytotoxic substances released by inflammatory cells, including free radicals (such as superoxide anions and hydrogen peroxide, etc.). With the resolution of the acute inflammatory response, i.e., the mid-chronic phase of spinal cord injury, vascular remodeling, myelination, neural circuit remodeling, etc., may occur during this phase.
Metal-organic framework Materials (MOFs), also known as porous coordination polymers, are a class of porous crystalline materials assembled from metal ions/clusters and functional organic ligands through strong coordination bonds. MOFs have various structures, have the advantages of simple synthesis, easiness in surface functionalization, high porosity, low density, high specific surface area and loading capacity, regular pore channels, adjustable pore diameter, topological structure diversity and tailorability, good biocompatibility and biodegradability and the like, and have been widely applied to the fields of drug delivery, biological imaging and sensing, gas adsorption/separation/storage, sea water desalination, biomedical application and the like. The preparation method has good drug carrying and release controlling capacity in the fields of MOFs drug transportation, release and the like. However, the conventionally carried metals are manganese and chromium, which have great toxicity and are therefore not suitable for biomedical applications. In order to successfully apply MOFs to biological fields, such as intracellular imaging, drug carriers, etc., the non-toxicity of MOFs must be ensured.
Currently, there is still a very limited treatment for spinal cord injury, and thus, intensive studies on the pathogenesis of spinal cord injury are critical for the treatment of spinal cord injury. Studies have shown that oxidative stress mediated inflammatory responses are one of the important pathogenesis of spinal cord injury and that secondary injury during spinal cord injury treatment can be reduced by relieving oxidative stress. In addition, many studies have shown that metal-organic framework Materials (MOFs) can encapsulate drugs stably, non-toxic within their pores, and release drugs slowly. Thus, MOFs can be used as effective drug carriers for drug delivery. Zinc is a low toxicity transition metal cation and has become an ideal candidate for preparing Zn-MOF, which can be used in biological system applications, especially as a carrier for drugs. However, zn-MOF is currently less studied in terms of spinal cord injury, and there is no article reporting the repair effect of Zn-MOF on spinal cord injury. The research aims at discussing whether Zn-MOF has antioxidant capacity and can promote the repair of spinal cord injury, further researching the mechanism of Zn-MOF for promoting the repair of spinal cord injury, and providing a certain reference basis for clinically treating spinal cord injury.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a Zn-MOF for treating spinal cord injury, and a preparation method and application thereof. The Zn-MOF compound provided by the invention can effectively reduce the intracellular active oxygen level, realize remarkable antioxidation capability and promote the growth of neurons and the recovery of motor functions after spinal cord injury. Meanwhile, compared with the conventional metal framework compounds such as manganese, chromium and the like, the Zn-MOF compound has better safety, has no obvious toxic or side effect on neuron cells and main organ tissues of organisms, and provides a new medicine source for the auxiliary treatment of spinal cord injury medicines.
In order to solve the technical problems, the invention is realized by the following technical scheme.
The first aspect of the invention provides a Zn-MOF compound, which is prepared by the following preparation method:
(1) ZnCl 2 Placing gallic acid and water into a round bottom flask, refluxing and stirring uniformly;
(2) Adjusting the pH value to 7-9 by using NaOH solution, and heating in a muffle furnace to obtain a mixture;
(3) Centrifuging the mixture obtained in the step (2), and discarding the supernatant to obtain a light gray solid;
(4) And (3) washing the light gray solid obtained in the step (3) with water and drying to obtain the light gray solid.
Preferably, the ZnCl in step (1) 2 And gallic acid in a mass ratio of 1:2-6.
Preferably, the concentration of NaOH in the step (2) is 10mol/L.
Preferably, the heating temperature in the step (2) is 110-170 ℃ and the heating time is 12-36h; most preferably, the heating temperature is 140 ℃ and the heating time is 24 hours.
Preferably, the rotational speed of the centrifugation in the step (3) is 10000-15000rpm, the centrifugation time is 10-20min, and the centrifugation temperature is 2-6 ℃; most preferably, the rotational speed of the centrifugation is 10000-15000rpm, the centrifugation time is 15min, and the centrifugation temperature is 4 ℃.
Preferably, the number of times of washing in step (4) is 1 to 3; most preferably, the number of washes is 2.
The second aspect of the invention provides a method for preparing a Zn-MOF compound, comprising the steps of:
(1) ZnCl 2 Placing gallic acid and water into a round bottom flask, refluxing and stirring uniformly;
(2) Adjusting the pH value to 7-9 by using NaOH solution, and heating in a muffle furnace to obtain a mixture;
(3) Centrifuging the mixture obtained in the step (2), and discarding the supernatant to obtain a light gray solid;
(4) And (3) washing the light gray solid obtained in the step (3) with water and drying to obtain the light gray solid.
Preferably, the ZnCl in step (1) 2 And gallic acid in a mass ratio of 1:2-6.
Preferably, the concentration of NaOH in the step (2) is 10mol/L.
Preferably, the heating temperature in the step (2) is 110-170 ℃ and the heating time is 12-36h; most preferably, the heating temperature is 140 ℃ and the heating time is 24 hours.
Preferably, the rotational speed of the centrifugation in the step (3) is 10000-15000rpm, the centrifugation time is 10-20min, and the centrifugation temperature is 2-6 ℃; most preferably, the rotational speed of the centrifugation is 10000-15000rpm, the centrifugation time is 15min, and the centrifugation temperature is 4 ℃.
Preferably, the number of times of washing in step (4) is 1 to 3; most preferably, the number of washes is 2.
In a third aspect, the invention provides the use of a Zn-MOF compound as described above or a Zn-MOF compound prepared according to the preparation method described above in the preparation of a medicament for the treatment of a disease associated with oxidative stress.
Preferably, the disorder associated with oxidative stress is selected from spinal cord injury.
In a fourth aspect, the invention provides a pharmaceutical composition for the treatment of a disease associated with oxidative stress comprising a Zn-MOF compound as described above or a Zn-MOF compound prepared according to the preparation method described above, and a pharmaceutically acceptable carrier.
Preferably, the disorder associated with oxidative stress is selected from spinal cord injury.
Preferably, the pharmaceutically acceptable carrier is selected from one or more of filler, disintegrant, lubricant, adhesive, antioxidant, antibacterial agent, correctant, aromatic agent and chelating agent.
Spinal cord injury involves a complex inflammatory response process, coordinated by a number of inflammatory factors including IL-1 beta, IL-6. The expression of IL-1 beta, IL-6 is dramatically increased after spinal cord injury. It has been found that overexpression of IL-6 promotes glioblast proliferation and, therefore, lowering IL-6 levels during the acute phase of spinal cord injury can inhibit gliosis. In addition, high levels of IL-6 after spinal cord injury inhibit the growth of neuronal axons. IL-1β is one of the most widely studied pro-inflammatory cytokines, which is produced mainly by microglia, and in addition, neurons, astrocytes, etc. can also produce IL-1β. In the injured central nervous system, the expression of IL-1β is dramatically increased. IL-1β can activate the immune system, promote the production of inflammatory factors and other cytokines, and also activate neurotoxic mediators. IL-1β has a great negative impact on the formation of glial cells after spinal cord injury, plasticity of axons, etc., and is associated with a severe functional prognosis. Studies have shown that IL-1β knockout can reduce astrocyte proliferation and inhibit inflammatory responses following spinal cord injury.
Matrix metalloproteinase 9 (MMP 9), one of the most widely studied members of the family of matrix metalloproteinases (Matrix Metalloproteinases, MMPs), is a gelatinase. MMP9 is involved in degradation of extracellular matrix and can promote degradation of gelatin, type IV, type V and type XI collagen, myelin basic protein, elastin, etc. Death. After spinal cord injury, the expression of MMP9 is increased, which is associated with inflammation after spinal cord injury and disruption of the blood-spinal cord barrier. Neutrophils, macrophages and monocytes involved in the spinal cord injury inflammatory response can all express MMP9.MMP9 is upregulated and reaches maximum activity 12-24h after spinal cord injury, and neutrophils at the site of spinal cord injury are the primary source of MMP9. Inhibiting MMP9 activity can reduce vascular permeability and reduce inflammation in the first 3 days after spinal cord injury. During the development of the entire nervous system, there is expression of MMP9 and it is involved in the growth of axons during development.
The synthesized Zn-MOF can effectively reduce the intracellular active oxygen level, realize remarkable antioxidation capability, reduce the levels of IL-6 and IL-1 beta after spinal cord injury, and inhibit the expression of MMP9. The Zn-MOF treatment can promote branch formation and neurite growth, which indicates that the Zn-MOF can repair neuron cells and resist oxidative stress induced injury. In addition, in vivo studies show that the recovery of motor functions of spinal cord injured mice can be significantly promoted after Zn-MOF treatment, and BMS score improvement is obvious. Meanwhile, the ROS bioluminescence intensity in the spinal cord injury mice treated by Zn-MOF is obviously reduced, and the Zn-MOF has obvious oxygen free radical scavenging and spinal cord injury repairing effects in vivo. In addition, the methods of a foot-to-air experiment, a rod rotating experiment, a foot print experiment, a Catwalk gait analysis, an electrophysiological evaluation and the like show that the hind limb movement function of the mice after Zn-MOF treatment is obviously superior to that of the SCI model group, and further prove that the Zn-MOF can obviously improve the repair of spinal cord injury and promote the recovery of limb functions.
Compared with the prior art, the invention has the following beneficial effects:
(1) The Zn-MOF compound provided by the invention can effectively reduce the intracellular active oxygen level, reduce the IL-6 and IL-1 beta level after spinal cord injury, inhibit the expression of MMP9, realize remarkable antioxidant capacity, repair hippocampal neuron cells, resist injury induced by oxidative stress, and promote neuron branch formation and neurite growth. Meanwhile, the Zn-MOF can obviously promote oxygen free radicals in the body of a spinal cord injury mouse and promote the repair of spinal cord injury and the recovery of motor functions.
(2) The Zn-MOF compound prepared by the invention has no obvious toxic or side effect on HT22 cells, hippocampal neurons and main tissues and organs of organisms, and compared with the common metal framework compounds such as manganese, chromium and the like in the prior art, the safety is obviously improved, thereby providing a new research thought and medicine source for treating oxidative stress related diseases including spinal cord injury, and having obvious clinical application prospect and social value.
Drawings
FIG. 1 is a schematic representation of transmission electron microscopy characterization of Zn-MOF.
FIG. 2 is a schematic diagram showing the distribution of C, O, zn elements in Zn-MOF.
FIG. 3 is a schematic representation of the results of Zn-MOF analysis by a Fourier infrared spectrometer and a nanoparticle size analyzer.
FIG. 4 is a graph showing the result of Zn-MOF radical scavenging ability.
FIG. 5 is a schematic representation of the effect of DCFH-DA staining analysis of Zn-MOF at different concentrations on HT22 cell ROS levels.
FIG. 6 is a graph showing the results of flow cytometry analysis of the effect of different concentrations of Zn-MOF on HT22 cell ROS levels.
FIG. 7 is a graph showing the effect of different Zn-MOF concentrations on HT22 cell viability.
FIG. 8 is a graph showing the effect of different Zn-MOF concentrations on hippocampal neuronal cell viability.
FIG. 9 is a graph showing the effect of Zn-MOF on primary hippocampal neurite extension.
Fig. 10 is a schematic diagram of BMS scoring results for different groups.
Figure 11 is a morphological image of different groups of mice 60 days after spinal cord injury.
FIG. 12 is a graph showing the results of reactive oxygen species levels in different groups of mice.
FIG. 13 is a graph showing the results of foot fault rate for different groups of mice.
Fig. 14 is a graph showing drop latency results for different groups of mice.
Figure 15 is a graph showing the results of step size and step width for different groups of mice,
fig. 16 is a graph showing the results of the average of the maximum contact area of the left hind limb and the maximum contact average intensity of the left hind limb of the mice of different groups.
Fig. 17 is a graph showing the results of the average of the maximum contact area of the right hind limb and the maximum contact average intensity of the right hind limb of the mice of different groups.
FIG. 18 is a graph showing the results of the normal index of the step sequences of mice of different groups.
FIG. 19 is a graph showing the results of exercise-induced potentials of mice of different groups.
FIG. 20 is a graphical representation of HE staining of spinal cord tissue of different groups of mice 60 days after spinal cord injury.
FIG. 21 is a graph showing the results of HE staining of the main organs of different groups of mice.
FIG. 22 is a graph showing the results of serum IL-6 and IL-1. Beta. Content detection in mice of different groups.
FIG. 23 is a graph showing MMP9 content detection results of different groups of mice.
Detailed Description
In order to make the objects, technical solutions and effects of the present invention more clear and clear, the present invention will be described in further detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
All reagents used in the context of the present invention are commercially available, unless otherwise specified. For animal experiments, the related procedures and methods meet the medical ethics requirements. The experimental methods used in the present invention are all conventional methods and techniques in the art.
Representative results of selection from the biological experimental replicates are presented in the context figures, and data are presented as mean±sd and mean±sem as specified in the figures. All experiments were repeated at least three times. Data were analyzed using GraphPad Prism 5.0 or SPSS 22.0 software. And comparing the average value difference of two or more groups by adopting conventional medical statistical methods such as t-test, chi-square test, analysis of variance and the like.pA difference of < 0.05 was considered significant.
Example 1 preparation and characterization of Zn-MOF
(1) 1.0g ZnCl 2 3.8g of gallic acid and 50g of water are placed in a round bottom flask and are stirred under reflux.
(2) The pH was adjusted to 8 using 10mol/L NaOH solution, and the mixture was heated in a muffle furnace (Sxi-8-10, new Korean instrument Co., ltd.) at 140℃for 24 hours.
(3) Centrifuging the mixture obtained in the step (2) at the temperature of 4 ℃ and the speed of 12000rpm for 15min, and discarding the supernatant to obtain a light gray solid.
(4) And (3) washing the light gray solid obtained in the step (3) with water for 2 times and drying to obtain the Zn-MOF compound.
The Zn-MOF compound prepared above is taken, the morphology and structure of the Zn-MOF compound are observed by a transmission electron microscope, and the morphology and structure information of the sample is obtained by carrying out amplified imaging on an electron beam carrying the internal structure information of the sample, and the result is shown in figure 1. The elements on the surface of the Zn-MOF compound were then characterized using an X-ray photoelectron spectrometer, and the results are shown in FIG. 2. The results show that the Zn-MOF compound prepared exhibits a sphere-like shape with a diameter of about 100nm, and the distribution of Zn, C, O elements therein is shown in FIG. 2. The Zn-MOF compounds were further examined by a nano-particle size analyzer, respectively, and the results are shown in FIG. 3. The test result of the nanometer particle size analyzer shows that: the Zn-MOF diameter was approximately equal to 100nm, consistent with the transmission electron microscope measurements described above. The above test results indicate successful synthesis of Zn-MOF compounds.
Example 2 investigation of the Effect of Zn-MOF on oxidative stress in vitro
The Zn-MOF compound prepared in example 1 was taken and examined for its free radical scavenging ability by the ABTS method, and the specific procedure was as follows:
(1) 20mL of PBS solution was placed in a 50mL centrifuge tube, 54.87mg of ABTS was weighed and added to the centrifuge tube containing the PBS solution, and the ABTS stock solution was prepared by thoroughly mixing the solution.
(2) Preparing a piece of filter paper and placing a sufficient amount of MnO on the filter paper 2 The filter paper is then combined with MnO 2 Transfer to the top of the centrifuge tube, then to MnO 2 And (3) dropwise adding 10mL of the ABTS stock solution prepared in the step (1), filtering for 5min in a dark place, continuing to react for 12h in a dark place to form an ABTS free radical mixed solution containing ABTS free radicals (ABTS+), and storing at the temperature of-20 ℃ for standby.
(3) 100. Mu.L of Zn-MOF (6. Mu.g/mL, 12.5. Mu.g/mL, 25. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL) at various concentrations were mixed with 100. Mu.L of the ABTS free radical mixture solution, respectively; a blank and a control were also provided, wherein only 200. Mu.L of PBS solution was added to the blank, and 100. Mu.L of PBS solution and 100. Mu.L of ABTS free radical mixture solution were added to the control.
(3) Absorbance at 734nm was measured using a cell imaging multimode reader (station 5,BioTek Instruments Inc.) at 10min, 20min, 40min, 60min, 90min and 120min, respectively.
The detection results are shown in FIG. 4. The results show that Zn-MOF can obviously inhibit the formation of ABTS free radicals, has time and dose dependence, can obviously inhibit the ABTS free radicals at the concentration of 6 mug/mL, and can reach the inhibition rate (in x) of the ABTS free radicals close to 100% at the concentration of 12.5 mug/mLp< 0.001, vs 0. Mu.g/mL group (control)). The experiment shows that Zn-MOF has higher activity of eliminating ABTS, can effectively eliminate free radicals outside cells to play an antioxidant role, and can be used as an effective free radical scavenger to prevent ROS from damaging neurons.
Subsequently, HT22 cells were examined by using an active oxygen fluorescent probe (DCFH-DA) and flow cytometry to verify whether Zn-MOF also had significant free radical scavenging activity in the cells, wherein the DCFH-DA experiment was performed as follows:
(1) 9mL of DMEM culture medium without serum is absorbed and placed in a 15mL centrifuge tube wrapped by tinfoil and protected from light, 9 mu L of DCFH-DA stock solution is added, and DCFH-DA solution is prepared after fully and uniformly mixing and placed in the protected from light environment for storage.
(2) The cell damage model was constructed by adding 120mmol/L of sodium L-glutamate to HT22 cells, followed by exposure to 37℃and 5% CO 2 Is cultured in a cell culture incubator for 12 hours.
(3) After the cell damage model is successfully constructed, the cells are treated for 12 hours by adopting Zn-MOFs (12.5 mug/mL, 25 mug/mL, 50 mug/mL and 100 mug/mL) with different concentrations respectively; a blank (Control) was also set up, wherein the blank was normal HT22 cells, and a Control was HT22 cells damaged with sodium L-glutamate and added with Zn-MOF at 0. Mu.g/mL.
(4) Taking HT22 cells (cultured in 24-well plate) out of cell incubator, removing original culture medium, gently adding 200 μl of DCFH-DA solution prepared in step (1) into each well along the wall of the well, wrapping 24-well plate with tinfoil to avoid light transmission, and thenIt was placed at 37℃with 5% CO 2 Is cultured in a cell culture tank for 30min.
(5) Taking out the cells in an incubator to a light-proof room, removing DCFH-DA solution, washing twice with PBS, and then adding 500 mu L of TBST (containing Triton X-100) solution into each well to punch 3 times for 5min each time; and then sealing the film by ICC technology, and taking pictures under a laser confocal microscope.
The results are shown in FIG. 5. The results showed that the fluorescence intensity of the Control group (injured without dosing) was significantly increased compared to the blank group (Control group), indicating that the construction of the sodium L-glutamate injured HT22 cell model was successful. And, the fluorescence intensity of the Zn-MOF group was significantly reduced compared to the control group, indicating that Zn-MOF effectively inhibited intracellular ROS and alleviated oxidative stress.
The specific steps for detecting active oxygen by flow cytometry are as follows:
(1) Taking HT22 cells in logarithmic growth phase, removing original culture medium, adding 120mmol/L sodium L-glutamate solution, and then placing at 37deg.C and 5% CO 2 Culturing for 12h in a cell culture box, and constructing a cell damage model.
(2) After the cell damage model is successfully constructed, the cells are treated for 12 hours by adopting Zn-MOFs (12.5 mug/mL, 25 mug/mL, 50 mug/mL and 100 mug/mL) with different concentrations respectively; a blank (Control) was also set up, wherein the blank was normal HT22 cells, and a Control was HT22 cells damaged with sodium L-glutamate and added with Zn-MOF at 0. Mu.g/mL.
(3) The cell supernatant was removed, the cells were digested with pancreatin and resuspended in culture plates, then the cell resuspension and cell supernatant were mixed and placed in a 5mL centrifuge tube, centrifuged at 2000rpm for 5min, and the supernatant was discarded. Cells were resuspended in 500. Mu.L of PBS and transferred to a 1.5mL centrifuge tube and centrifuged at 2000rpm for 5min.
(7) Adding 300 mu L of active oxygen DCFH-DA dye solution (Biyun day), gently mixing, and incubating for 20min in dark place; centrifuging at 2000rpm for 5min, discarding supernatant, adding 500 μl PBS solution, gently mixing, centrifuging at 2000rpm for 5min again, and discarding supernatant; 300. Mu.L of PBS solution was added, and the mixture was gently mixed and transferred to a flow tube for detection.
The results are shown in FIG. 6. The results are similar to the DCFH-DA experiment, i.e. ROS levels are significantly increased in the Control group (injured without dosing) compared to the blank group (Control group); the ROS levels were significantly reduced in the Zn-MOF group compared to the control group, indicating that Zn-MOF effectively inhibited intracellular ROS and alleviated oxidative stress. The ROS level detection of HT22 cells by the reactive oxygen fluorescent probe and the flow cytometry fully proves that Zn-MOF can effectively inhibit ROS in cells to play a role in relieving oxidative stress.
Further, HT22 cells were selected for toxicity testing of Zn-MOF, as follows:
(1) HT22 cells were seeded in 96-well plates at a cell number of 5000 cells/100. Mu.L/well using a cell counter.
(2) After cell adhesion, zn-MOF was added at various concentrations (1.57,3.13,6.25, 12.5, 25, 50, 100, units: μg/mL) to each well and incubated for an additional 12 hours. A blank and a Control group were additionally provided, wherein the blank was only added with an equal volume of medium without seeding cells, and the Control group (Control group) was seeded with an equal volume of cells without Zn-MOF treatment.
(3) After 12h, 110. Mu.L of the prepared CCK-8 solution (10. Mu.L of MEM solution+10. Mu.L of CCK-8 stock solution) was added, and after mixing, the 96-well plate was wrapped with tin foil to protect from light, and then incubation was continued for 1h.
(4) After incubation, the OD (absorbance) value (absorbance) at 450nm wavelength of each group of cells was measured with a microplate reader, and then the cell viability was calculated according to the formula: cell viability (%) = (dosing group-blank)/(Control group-blank) ×100%.
The results are shown in FIG. 7. The results showed that at each concentration of 1.57-100. Mu.g/mL studied, the survival rate of the hippocampal neurons after Zn-MOF treatment was close to 100%, and the survival rate was not significantly different from that of the Control group (Control group)p> 0.05). From this, it was confirmed that Zn-MOF was excellent in safety and not significantly cytotoxic to HT 22.
Subsequently, rat hippocampal tissue cells were selected to further verify the toxicity of Zn-MOF, specifically as follows:
(1) Brain hippocampus tissue from 1 day old Sprague-Dawley (SD) rats was minced with ophthalmic scissors and added to a 15mL centrifuge tube (Corning), papain (Sigma, cat#P 4762) was added and digested at 37℃for 25 minutes.
(2) Tissue is washed after stopping the protease with bovine serum albumin.
(3) The isolated hippocampal neurons were seeded at 20000 cells/mL in 96-well plates (Corning) at 200. Mu.L per well in DMEM medium (glutamine, gibco, carlsbad, calif., cat # 12430054) containing 10% fetal bovine serum, 10% nutrient mix F-12, at 37℃at 95% O 2 、5% CO 2 Is cultured in an incubator for 6 hours.
(4) Replacing original culture medium with Neurobasal medium containing 5% B-27 (Gibco), placing at 37deg.C and 95% O 2 、5% CO 2 Is cultured in an incubator for 12 hours.
(5) After cell adhesion, zn-MOF was added at various concentrations (0.40,0.79,1.57,3.13,6.25, 12.5, 25, 50, 100, units: μg/mL) to each well and incubated for an additional 24 hours. A blank and a Control group were additionally provided, wherein the blank was only added with an equal volume of medium without seeding cells, and the Control group (Control group) was seeded with an equal volume of cells without Zn-MOF treatment.
(6) After 12h, 110. Mu.L of the prepared CCK-8 solution (10. Mu.L of MEM solution+10. Mu.L of CCK-8 stock solution) was added, and after mixing, the 96-well plate was wrapped with tin foil to protect from light, and then incubation was continued for 1h.
(7) After incubation, the OD (absorbance) value (absorbance) at 450nm wavelength of each group of cells was measured with a microplate reader, and then the cell viability was calculated according to the formula: cell viability (%) = (dosing group-blank)/(Control group-blank) ×100%.
The results are shown in FIG. 8. The results show that at each concentration of 0.40-100 mug/mL, the survival rate of the hippocampal neuron cells after Zn-MOF treatment is close to 100%, and the survival rate is not significantly different from that of a blank control group p> 0.05). Thereby proceeding toThe Zn-MOF has excellent safety and no obvious cytotoxicity to the hippocampal neuron cells.
EXAMPLE 3 repair of neurons by Zn-MOF
The method selects rat hippocampal tissue for Zn-MOF neuron damage repair detection, and comprises the following specific steps:
(1) Rat brain hippocampus tissue of 1 day old Sprague-Dawley (SD) was cut with an ophthalmic scissors and added to a 15mL centrifuge tube (Corning), papain (Sigma, cat#P 4762) was added and digested at 37℃for 25 minutes; tissue is washed after stopping the protease with bovine serum albumin.
(2) The isolated hippocampal neurons were seeded at 20000 cells/mL in 24-well plates (Corning) at 500. Mu.L per well in DMEM medium (glutamine, gibco, carlsbad, calif., cat # 12430054) containing 10% fetal bovine serum, 10% nutritional mix F-12, at 37deg.C, 95% O 2 、5% CO 2 Culturing in an incubator for 24 hours;
(3) Replacing original culture medium with 120 mu M L-sodium glutamate-containing culture medium, placing at 37deg.C and 95% O 2 、5% CO 2 Culturing for 12 hours in an incubator of (2) to form a hippocampal neuron damage model;
(4) After cell adhesion, zn-MOF with different concentrations (12.5, 25, 50, 100, unit: μg/mL) was added to each well, and incubation was continued for 24 hours, wherein Control group was a blank Control group, and hippocampal neuronal cells were not subjected to damage treatment; a blank (Control) and a Control (Injury) were additionally provided, wherein the blank was normal hippocampal neuronal cells and the Control was damage-modelled hippocampal neuronal cells, without Zn-MOF treatment. Hippocampal neuronal cells were stained and mounted using the ICC technique, the mounted slides were photographed using a laser confocal microscope, and finally the number of neuronal primary projections, secondary projections, and total projections and their length were delineated and counted using Image J software.
The effect of Zn-MOF on neuronal damage repair was evaluated by quantifying the length and total number of neuronal projections, and the results are shown in FIG. 9. The results showed that neurons of the Injury group were primary prominent, secondary, compared to the Control groupThe number and length of projections, as well as the total projections, were significantly reduced, indicating that the construction of the primary hippocampal neuron model for sodium L-glutamate injury was successful. In the Zn-MOF treated group, the length of the primary and secondary branches of the neurons and the total length of the projections were significantly greater than in the model group (x)p＜0.05，**p＜0.01，***p< 0.001, vs Injury group (0. Mu.g/mL group)), and after Zn-MOF treatment, the primary branch length of the neuron and the total length of the protrusion have no significant difference from the blank Control group (Control group). It was also found that the number of primary and secondary branches and the total number of projections were significantly greater than the model group (x) in neurons treated with Zn-MOFp＜0.01，***p< 0.001, vs Injury group (0. Mu.g/mL group)), and after Zn-MOF treatment, the number of primary branches of neurons and the total number of projections can be restored to have no significant difference from the blank group. In conclusion, the Zn-MOF compound prepared by the invention can obviously promote the growth of injured neuron protrusion after being added, and has obvious protection and recovery promotion effects on the ROS-induced neuron oxidative stress injury by inhibiting the generation of ROS to promote the axon extension of injured neurons.
Example 4 in vivo functional study of Zn-MOF
(1) 24 female mice of 6 weeks of age were selected and divided into four groups of 6, including a blank Control group (Control group, 6, without any treatment), a sham Control group (sham group, 6, with only the lamina excised, without damaging the spinal cord), a model group (Injury group, 6), a Zn-MOF group (injury+zn-MOF, 6), and the following day after spinal cord Injury, each drug was injected intraperitoneally at a volume of 100 μl/dose of 50 μg, with sham group and Injury group injected with equal volumes of PBS, 1 time daily, once daily, for three consecutive days. The method for constructing the spinal cord injury mouse model is as follows:
a. hair on the back of the mice was removed, and skin on the back of the mice was exposed.
b. Anesthesia: anesthesia was successfully indicated by intraperitoneal injection of 1.25% tribromoethanol at a dose of 200 μg/20g, smooth breathing, significant weakening of limb muscle strength, and disappearance of pain reflex and cornea reflex.
c. Exposing spinal cord: wiping the skin for 2 times by using an iodophor cotton ball to disinfect the skin; the level of thoracic vertebrae at paragraphs 9-11 (T9-T11) was determined and a longitudinal incision of 2.5cm length was made centrally on the back at the level of T9-T11. The paraspinal muscles were blunt stripped, spinous processes and laminae were removed with rongeurs, the spinal cord of the T9-T11 segment was thoroughly exposed, the T10 plane was fixed with a fixator, and the T10 lamina was excised, taking care to avoid additional tissue damage during the procedure. And then distracted with an anchor to fully expose the spinal cord.
d. The mice were fixed to a lewis-wirl-injury system device (Louisville Injury System Apparatus, LISA), spinal cord was adjusted to under the impactor, and the impact position was confirmed by monitoring the laser beam thereof, and the extent of spinal cord injury was determined by the depth of impact, in this example, the impact tip was set to 18psi, the depth of impact was 0.8mm, and the injury time was 0.5s; after the collision is completed, the mice are removed from the injury device and removed from the holder. After sufficient hemostasis was given, the mice were sutured to the muscle and skin using sutures. Mice in the sham group were T10 laminectomy and did not undergo spinal cord impact.
e. The incision was closed, gentamicin 2000U was subcutaneously injected post-operatively, 1 time daily, 3 days in succession, and 1 time of artificial bladder emptying was performed every 8 hours until spontaneous urination.
(2) Monitoring: starting from post-molding, each group of mice was monitored daily for motor status and BMS scored every 3 days, with the results shown in fig. 10. The results show that initially, all SCI mice, except the blank, had complete loss of hind limb locomotion. Subsequently, hindlimb motor function began to gradually recover over time in each group of SCI mice, with the recovery rate of Zn-MOF treated groups at day 6 of Injury being already significantly higher than in the model group (Injury group), with a gradual improvement in BMS scores. The BMS scores of Control and Sham mice continued to be 9 points throughout the duration of the experiment, while the BMS scores of Injury mice tended to increase slowly, but by day 60 the scores did not exceed 1 point, and the rate of score increase was very slow. However, the BMS score increased more rapidly and scored higher for Zn-MOF group mice than for Injury group, and the score was approximately 4 up to day 60, indicating that Zn-MOF can promote recovery of motor function in spinal cord injured mice. The morphology of each group of mice after 60 days post Injury is shown in FIG. 11, wherein the hind limbs of the Control group and the Sham group mice are normal, the hind limbs of the Injury group mice are paralyzed, and the hind limb morphology of the Zn-MOF group mice is improved compared with the Injury group mice. The results show that Zn-MOF treatment significantly enhanced behavioral functional recovery after SCI in mice.
(3) In vivo imaging of small animals: mice were imaged in vivo on day 3 post injury. First, 0.1mL/20g L-012 (chemiluminescent (CHL) probe) was injected intraperitoneally, and the concentration of L-012 was 4mg/mL. After 5 minutes, the anesthesia is injected into the abdominal cavity by using 1.25% tribromoethanol at the dosage of 200 mug/20 g, and after the anesthesia is successful, the living animal imaging is carried out.
The results are shown in FIG. 12. The results show that the bioluminescence of ROS is rarely seen in normal mouse spinal cord, whereas the bioluminescence intensity of ROS in spinal cord of spinal cord injured mice is significantly increased and increases with time. After Zn-MOF treatment, the bioluminescence intensity of ROS was significantly reduced, significantly lower than that of the model group (Injury group), even lower than that of Sham group (x)p＜0.05，**p< 0.01, vs Injury group (0. Mu.g/mL group)). The quantitative value of the ROS bioluminescence intensity indicates that the Zn-MOF promotes the repair of spinal cord injury by reducing ROS at the spinal cord of a mouse, and further proves that the Zn-MOF has obvious oxygen free radical scavenging in vivo and can reduce the high active oxygen level in the mouse after spinal cord injury, thereby playing an antioxidant role and promoting the repair after spinal cord injury.
On day 60 after spinal cord injury, mice were subjected to a foot-to-air test and a stick-turning test. The pedal-air experiment comprises the following specific steps: the mice were placed on parallel bars, framed with a square rack, allowed to move freely within a defined range, and then video-imaged for 3-5 min. In the crawling process of the mice, the lower limbs are not stepped on the parallel rods, the sensing device can automatically record the mice as foot errors once, record the total number of foot errors of 60 steps of crawling of the mice on the parallel rods, and then calculate the foot error rate of each mouse. Foot error rate = number of times the parallel bars were dropped from the lower limbs/total number of steps (60 steps).
The experimental results are shown in FIG. 13. The results showed that the foot error rate was significantly higher in the Injury group of mice compared to the Control and Sham groups, indicating that the hindlimb locomotor function was poor in the Injury group of mice. However, the foot error rate was significantly reduced in the Zn-MOF group compared to the Injury group, indicating that Zn-MOF can promote recovery of hindlimb motor function in spinal cord injured mice.
The rotating rod experiment comprises the following specific steps: turning on the rotating rod switch to make it rotate continuously; grabbing 1 mouse and placing the mouse on a rotating rod, enabling the movement direction of the mouse to be opposite to the rotation direction of the rotating rod, starting timing when the mouse is just placed on the rotating rod, and recording the movement time of each mouse on the rotating rod when the mouse falls off the rotating rod; data were collected and analyzed.
The experimental results are shown in FIG. 14. The results show that the drop latency of the Injury group mice is significantly shorter than that of the Control and Sham groups. Whereas the drop latency of the Zn-MOF group mice was significantly longer than that of the Injury group, further demonstrating that Zn-MOF can promote recovery of motor function in spinal cord injured mice.
Right hind limb paralysis of the half-cut injured mice at the early stage of injury (7 days) presents trawling, and joint movement is less and cannot be evaluated. And then, the lower limb functions are gradually recovered after 7 to 14 days, the dragging is reduced, and the knee joint and hip joint activities are obviously increased. Thus, footprint analysis was performed 14 days after injury, and the evaluation parameters in the footprint analysis experiment included step size and step width. The method comprises the following specific steps:
The experimental mice were randomly divided into 4 groups, designated as group 1-group 4, and spinal cord injury model preparation was performed according to the method described above. Wherein group 1 is Control, group 2 is Sham, group 3 is model (Injury), group 4 is Zn-MOF, right spinal cord half-cut in row T10, and Zn-MOF treatment is given simultaneously in the same manner as described above. Before the experiment, a long and narrow channel is prepared, white paper is paved in the channel, the hind feet of each group of mice are dyed by nontoxic red ink, then the mice are put into the channel prepared in advance, the mice walk freely along the channel, the hind paw footprints of the mice are visible on the white paper, and the step size or the step width is analyzed by calculating the average value of continuous 3 footprints.
The experimental results are shown in fig. 15, and the results show that the spinal cord half-cut model can cause partial loss of hindlimb locomotor function of the mice, and compared with Sham group (group 2), the hindlimb locomotor function of the mice in the Injury group (group 3) is dragged (fig. 15A), and the hindlimb locomotor function of the mice after Zn-MOF treatment (group 4) is significantly better than that of the Injury group. The footprint quantitative statistical analysis results show that the stride and the stride width of the mice in the experimental group at 14 days after operation are better than those of the SCI group (fig. 15B and 15C) (n=6 in all groups p<0.05，**p<0.01，***p<0.001, ns stands for insignificant). Thus, it is further demonstrated that Zn-MOF can significantly improve spinal cord injury repair and promote recovery of limb functions.
Subsequently, catWalk gait analysis was performed on different groups of mice, specifically as follows: placing the mice in a darkened room for a moment; then the mouse is grabbed and placed at the tail end of the organic glass floor of the CatWalk less analysis system, a cover is covered, the mouse freely passes through one end of the floor to the other end, meanwhile, the process that the mouse freely passes through the organic glass floor is observed on a computer, the mouse cannot stay in the process that the mouse passes through the organic glass floor, otherwise, the mouse must be repeated once, and after the mouse passes through the organic glass floor very smoothly, the computer automatically saves the motion video of the mouse; repeating the steps until gait related data of all experimental mice are acquired.
The experimental results are shown in FIGS. 16-18. The results showed that the average of the maximum contact area of the double hindlimb and the average of the maximum contact strength of the double hindlimb were very small for the Injury mice compared to the Control group and the Sham group, indicating that the double hindlimb locomotor function was poor for the Injury mice. However, both the mean of the maximum contact area and the mean of the maximum contact intensity of the double hind limbs were significantly increased in the Zn-MOF group mice compared to the Injury group (fig. 16-17). The normative index of the gait in the Injury mice was very small compared to the Control and Sham mice, indicating that the hindlimb locomotor function was poor in the Injury mice. Whereas the step normal index was significantly increased in the Zn-MOF group compared to the Injury group (fig. 18). The CatWalk gait analysis result shows that Zn-MOF can promote the recovery of the motor function of the spinal cord injury mice.
The above mouse behavioural tests demonstrate that Zn-MOF can promote the recovery of motor function in spinal cord injured mice. Electrophysiological testing was then performed to verify whether Zn-MOF could promote recovery of nerve conduction function in spinal cord injured mice. The method comprises the following specific steps: 1.25% 2, 2-tribromoethanol is adopted to anesthetize the mice (the dosage is 0.2mL/10 g), the hairs of the head and the thigh of the mice are removed, and the cerebral cortex movement area and the bilateral thigh gastrocnemius muscle groups of the mice are dissected; turning on the neurophysiological monitor, and setting parameters as follows: the voltage is 0.05mV, and the current is 0.05mA. All signal processing procedures were performed on BL-420N biosignal acquisition and analysis system (Chengdu Union); inserting a stimulating electrode into a cerebral cortex exercise area of a mouse, inserting an electrode for receiving electric signals into a bilateral gastrocnemius muscle group of the mouse, then collecting signals of the gastrocnemius exercise evoked potentials of the mouse, and repeating the collection of the exercise evoked potentials of each mouse for 3 times; after all mice were collected, they were analyzed.
The experimental results are shown in FIG. 19. The results show that the amplitude of the movement-induced potential of the Injury group mice is obviously reduced compared with that of the Control group and the Sham group, and the amplitude of the movement-induced potential of the Zn-MOF group mice is obviously increased compared with that of the Injury group, which proves that the Zn-MOF can effectively promote the recovery of the nerve conduction function of the spinal cord injured mice.
To more intuitively evaluate the repair of Zn-MOF to the damaged spinal cord of mice, HE staining was performed on spinal cord tissue of each group of mice on day 60 after spinal cord injury to observe the morphology of spinal cord tissue. The results show that the spinal cord tissues of the Control group and the Sham group are not obviously damaged; the spinal cord tissue of the Injury group can see obvious structural damage; the significantly reduced extent of spinal tissue Injury in the Zn-MOF group compared to the Injury group, suggesting that Zn-MOF may indeed promote repair of injured spinal cord in the mice (see figure 20).
In the previous experiments, it was verified that Zn-MOF has no obvious toxic or side effects on HT22 cells and hippocampal neurons in vitro, and to further evaluate the safety, HE staining was performed on major organs of each group of mice on day 60 after spinal cord injury to observe pathological changes of alarm, heart, liver, spleen, lung, kidney, and the results are shown in fig. 21. The results show that the brain, heart, liver, spleen, lung and kidney of different groups of mice have no obvious difference, which indicates that Zn-MOF has no obvious toxic and side effects on the brain, heart, liver, spleen, lung and kidney of mice, and further proves that Zn-MOF has higher safety.
Example 5 study of the mechanism of action of Zn-MOF
To clarify the mechanism of action of Zn-MOF and its relationship with inflammatory response after spinal cord injury, zn-MOF was injected in spinal cord injured mice for three consecutive days. The sera of the mice of the different groups were then subjected to enzyme-linked immunosorbent assay (ELISA) to detect the amounts of inflammatory factors IL-6 and IL-1β in the sera of the mice of the different groups, the results are shown in FIG. 22. The results show that the levels of IL-6 and IL-1β in the mice of the Injury group were significantly increased compared to the Control and Sham groups, while the levels of IL-6 and IL-1β in the mice of the Zn-MOF group were significantly decreased compared to the Injury group, indicating that Zn-MOF can inhibit IL-6 and IL-1β and thereby reduce the inflammatory response after spinal cord Injury.
Since the level of expression of MMP9 is generally increased after spinal cord injury, MMP9 acts as an extracellular enzyme that degrades a range of extracellular matrix proteins and participates in the spinal cord injury process. In this regard, protein extraction and immunoblotting (Western blot) were further performed on cortical neurons and spinal cord tissues of different groups of spinal cord injured mice, and then detection was performed using MMP9 antibodies, and the results are shown in fig. 23. The results show that the content of MMP9 in different groups of cortical neurons is compared: MMP9 expression was increased in the Injury group compared to the Control group, while MMP9 expression was decreased in the Zn-MOF group compared to the Injury group (FIG. 23A). Comparison of the content of MMP9 in spinal cord tissue of mice of different groups: MMP9 expression was increased in the Injury group compared to the Control and Sham groups, while MMP9 expression was significantly decreased in the Zn-MOF group compared to the Injury group (FIG. 23B). It was demonstrated that Zn-MOF could reduce MMP9 expression following spinal cord injury.
Spinal cord injury involves very complex pathophysiological mechanisms, one of which is the oxidative stress-mediated inflammatory response. The invention starts from the relation between oxidative stress and spinal cord injury, discusses the relation between Zn-MOF and oxidative stress, researches the influence of Zn-MOF on the axon extension of injured neurons at the cellular level and the influence of Zn-MOF on the spinal cord injury repair of mice at the animal level, and researches the action mechanism of Zn-MOF in the spinal cord injury repair.
Firstly, the invention verifies that Zn-MOF has antioxidant effect outside cells through ABTS+ free radical scavenging experiments, and verifies that Zn-MOF can inhibit ROS in cells at the cellular level to play an antioxidant role and promote the axon extension of injured neurons. Then, by constructing a mouse spinal cord injury model and researching the action of Zn-MOF after the mouse spinal cord injury, the Zn-MOF is found to have an antioxidant action in a spinal cord injury mouse body, the level of IL-6 and IL-1 beta after the spinal cord injury and the expression of MMP9 can be reduced, and the restoration of the motor function and the nerve conduction function of the spinal cord injury mouse is promoted, so that the restoration of the injured spinal cord of the mouse is promoted.
Studies have shown that the pathogenesis of neurodegenerative diseases may be related to oxidative stress and excitatory toxins. Glutamate, an excitatory neurotransmitter, may cause certain neurological diseases that may mediate oxidative stress damage and excitotoxicity, a phenomenon known as glutamate neurotoxicity. It can cause damage to many cellular components, including mitochondria, ultimately leading to cell death. During cell death, large amounts of Reactive Oxygen Species (ROS) such as superoxide anions (O2. Cndot.) are produced - ) Hydrogen peroxide (H) 2 O 2 ) Hydroxyl radicals (OH.) and the like, the overproduction of ROS mediates the excitotoxicity of glutamate. The invention damages HT22 cells by using L-sodium glutamate, so that excessive ROS are generated, and then DCFH-DA staining and flow cytometry are used for detecting the ROS level in HT22 cells, so that the Zn-MOF can inhibit the ROS in the cells to play a role in relieving oxidative stress.
Neurons are the most susceptible cell types in the central nervous system to oxidative stress, and are particularly susceptible to ROS. ROS can damage nucleic acids, proteins, lipids, etc. of cells, leading to neuronal dysfunction and even neuronal death. ROS are involved in the development of many neurodegenerative diseases such as alzheimer's disease, amyotrophic lateral sclerosis, parkinson's disease, etc. It has been thought that endogenously produced ROS can act as signaling molecules to regulate a range of neurological physiological processes, such as neuronal development, neuronal polarity, synaptic plasticity, growth cone routing, and regulation of neural circuits. However, excessive ROS production can lead to an imbalance in the oxidation and antioxidant mechanisms, leading to oxidative stress. The present invention verifies whether Zn-MOF promotes repair of damaged neurons by inhibiting ROS by counting the number and length of neuronal projections. The results show that the number and length of the primary and secondary projections, as well as the total projections, of neurons of the Zn-MOF group are significantly increased compared to the Injury group. It is demonstrated that Zn-MOF can promote axonal extension of injured neurons by inhibiting ROS. Further, it was confirmed whether Zn-MOF can exert its antioxidant effect in spinal cord injured mice by detecting the level of active oxygen in mice using L-012 and in vivo imaging of mice. The results show that Zn-MOF can reduce the high active oxygen level in mice after spinal cord injury, thereby playing a role in relieving oxidative stress.
To investigate whether Zn-MOF could promote repair of spinal cord injury in mice, mice of different groups were tested by BMS scoring, foot-to-foot experiments, rod rotation experiments, foot print experiments, catWalk gait analysis, electrophysiological evaluation, etc. The result shows that the hindlimb motor function of the Zn-MOF group mice is recovered faster 60 days after spinal cord Injury, the BMS score is close to 4 minutes, and the foot error rate is obviously reduced compared with the Injury group; the results of the rod rotating experiment show that the drop latency of the Zn-MOF group mice is obviously prolonged compared with that of the Injury group; the footprint experiment result shows that the step size and the step width of the Zn-MOF group mice are obviously increased compared with those of the Injury group; the CatWalk gait analysis result shows that the average value of the maximum contact area of the double hind limbs, the average strength of the maximum contact of the double hind limbs and the normal index of the walking sequence of the Zn-MOF group mice are improved by the harness compared with the Injury group. In addition, at day 60 after spinal cord injury, the morphology of spinal cord tissue was observed by HE staining of spinal cord tissue of different groups of mice. The results show a significant reduction in the extent of spinal tissue damage in the Zn-MOF group compared to the Injury group. Together, the above results demonstrate that Zn-MOF can promote recovery of motor function in spinal cord injured mice.
In addition, the invention has also conducted intensive research on the safety of Zn-MOF. As a result, zn-MOF was found to be non-significantly toxic to both HT22 cells and primary hippocampal neurons. And after 60 days of spinal cord injury, the main organs of different mice are detected and analyzed, and the brain, heart, liver, spleen, lung and kidney of the mice in different groups have no obvious difference, so that the Zn-MOF has no obvious toxic and side effects on the brain, heart, liver, spleen, lung and kidney of the mice, and the Zn-MOF has higher safety.
The above detailed description describes the analysis method according to the present invention. It should be noted that the above description is only intended to help those skilled in the art to better understand the method and idea of the present invention, and is not intended to limit the related content. Those skilled in the art may make appropriate adjustments or modifications to the present invention without departing from the principle of the present invention, and such adjustments and modifications should also fall within the scope of the present invention.
Claims (10)
1. A Zn-MOF compound characterized in that it is prepared by the following preparation method:
(1) ZnCl 2 Placing gallic acid and water into a round bottom flask, refluxing and stirring uniformly;
(2) Adjusting the pH value to 7-9 by using NaOH solution, and heating in a muffle furnace to obtain a mixture;
(3) Centrifuging the mixture obtained in the step (2), and discarding the supernatant to obtain a light gray solid;
(4) And (3) washing the light gray solid obtained in the step (3) with water and drying to obtain the light gray solid.
2. The Zn-MOF compound as defined in claim 1 wherein in step (1) the ZnCl 2 And gallic acid in a mass ratio of 1:2-6.
3. The Zn-MOF compound of claim 1, wherein the concentration of NaOH in step (2) is 10mol/L.
4. The Zn-MOF compound of claim 1, wherein the heating in step (2) is at a temperature of 110 to 170 ℃ for a time of 12 to 36 hours.
5. The Zn-MOF compound as defined in claim 1 wherein the rotational speed of centrifugation in step (3) is 10000-15000rpm, the time of centrifugation is 10-20min and the temperature of centrifugation is 2-6 ℃.
6. The Zn-MOF compound as defined in claim 1 wherein the number of times of washing in step (4) is 1 to 3.
7. Use of a Zn-MOF compound as defined in any one of claims 1 to 6 in the manufacture of a medicament for the treatment of a disease associated with oxidative stress.
8. The use according to claim 8, wherein the disorder associated with oxidative stress is selected from spinal cord injury.
9. A pharmaceutical composition for treating a disease associated with oxidative stress, comprising a Zn-MOF compound as described above or a Zn-MOF compound prepared according to the above preparation method, and a pharmaceutically acceptable carrier.
10. The pharmaceutical composition of claim 9, wherein the disorder associated with oxidative stress is selected from spinal cord injury.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311271851.5A CN117143351A (en) | 2023-09-28 | 2023-09-28 | Zn-MOF for treating spinal cord injury and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311271851.5A CN117143351A (en) | 2023-09-28 | 2023-09-28 | Zn-MOF for treating spinal cord injury and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117143351A true CN117143351A (en) | 2023-12-01 |
Family
ID=88912190
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311271851.5A Pending CN117143351A (en) | 2023-09-28 | 2023-09-28 | Zn-MOF for treating spinal cord injury and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117143351A (en) |
-
2023
- 2023-09-28 CN CN202311271851.5A patent/CN117143351A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Deng et al. | Collagen scaffold combined with human umbilical cord-mesenchymal stem cells transplantation for acute complete spinal cord injury | |
| Gao et al. | An anti-inflammatory and neuroprotective biomimetic nanoplatform for repairing spinal cord injury | |
| AU2008292407B2 (en) | Prophylactic or alleviating agent for peripheral nerve disorder induced by anti-cancer agent | |
| Parikh et al. | Curcumin-loaded self-nanomicellizing solid dispersion system: Part II: In vivo safety and efficacy assessment against behavior deficit in Alzheimer disease | |
| Tian et al. | Exogenous CGRP regulates apoptosis and autophagy to alleviate traumatic brain injury through Akt/mTOR signalling pathway | |
| Azizi et al. | Improvement of functional recovery of transected peripheral nerve by means of chitosan grafts filled with vitamin E, pyrroloquinoline quinone and their combination | |
| KR101329936B1 (en) | External preparation for skin containing flavanone derivative | |
| WO2023202477A1 (en) | Dihydromyricetin tetrahydropyrrole complex and preparation method therefor, and application | |
| Zhang et al. | Berberine enhances survival and axonal regeneration of motoneurons following spinal root avulsion and re-implantation in rats | |
| Li et al. | Chitosan-modified hollow manganese dioxide nanoparticles loaded with resveratrol for the treatment of spinal cord injury | |
| Huang et al. | Localised delivery of quercetin by thermo-sensitive PLGA-PEG-PLGA hydrogels for the treatment of brachial plexus avulsion | |
| CN117653739B (en) | Preparation method of Ce@UCNP-BCH and application thereof in spinal cord injury treatment | |
| Puniak et al. | Comparison of a serotonin antagonist, opioid antagonist, and TRH analog for the acute treatment of experimental spinal trauma | |
| Ladol et al. | The effects of Hippophae rhamnoides in neuroprotection and behavioral alterations against iron-induced epilepsy | |
| Zhu et al. | Schwann cell-derived exosomes and methylprednisolone composite patch for spinal cord injury repair | |
| RU2545734C1 (en) | Drug preparation for parkinson disease | |
| RU2275924C2 (en) | Method for preparing complex of biologically active polypeptides for normalization of brain function and pharmaceutical agent based on thereof | |
| CN117143351A (en) | Zn-MOF for treating spinal cord injury and preparation method and application thereof | |
| CN111281927A (en) | Traditional Chinese medicine composition for treating Parkinson's disease and preparation method and application thereof | |
| CN115671147B (en) | Auricularia auricula extract and application thereof | |
| KR101289220B1 (en) | Peptide substance stimulating regeneration of central nervous system neurons, pharmaceutical composition on its base, and the method of its application | |
| CN116904571A (en) | Use of CPNE1 gene or CPNE1 protein in the preparation of a medicament for preventing or treating neurological disorders | |
| Chen et al. | Bexarotene enhances astrocyte phagocytosis via ABCA1-mediated pathways in a mouse model of subarachnoid hemorrhage | |
| RU2766083C1 (en) | Biologically active substance in the form of a bioregulatory composition, exhibiting a supportive and restorative effect on tissues and organs, and pharmacological composition | |
| CN117304501A (en) | AIE-N for the treatment of spinal cord injury 3 -Zn-MOF and preparation method and application thereof |
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 |