CN117771259A - 银椴甙在制备用于减轻急性肾损伤的药物中的应用 - Google Patents
银椴甙在制备用于减轻急性肾损伤的药物中的应用 Download PDFInfo
- Publication number
- CN117771259A CN117771259A CN202410017828.1A CN202410017828A CN117771259A CN 117771259 A CN117771259 A CN 117771259A CN 202410017828 A CN202410017828 A CN 202410017828A CN 117771259 A CN117771259 A CN 117771259A
- Authority
- CN
- China
- Prior art keywords
- kidney injury
- cisplatin
- acute kidney
- induced
- injury
- 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
- 201000011040 acute kidney failure Diseases 0.000 title claims abstract description 59
- 208000009304 Acute Kidney Injury Diseases 0.000 title claims abstract description 54
- 208000033626 Renal failure acute Diseases 0.000 title claims abstract description 52
- 239000003814 drug Substances 0.000 title claims abstract description 21
- DVGGLGXQSFURLP-FIZCXTQCSA-N potengriffioside A Natural products O[C@@H]1[C@@H](COC(=O)C=Cc2ccc(O)cc2)O[C@@H](Oc2c(oc3cc(O)cc(O)c3c2=O)-c2ccc(O)cc2)[C@H](O)[C@H]1O DVGGLGXQSFURLP-FIZCXTQCSA-N 0.000 title claims abstract description 14
- LTRRTGCXRIMDTF-UHFFFAOYSA-N tiliroside Natural products OC1C(COC(=O)C=Cc2ccc(O)cc2)OC(OC3=C(Oc4cc(O)cc(O)c4C3)c5ccc(O)c(O)c5)C(O)C1O LTRRTGCXRIMDTF-UHFFFAOYSA-N 0.000 title claims abstract description 14
- DVGGLGXQSFURLP-VWMSDXGPSA-N tribuloside Chemical compound C([C@@H]1[C@H]([C@@H]([C@@H](O)[C@H](OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=CC(O)=CC=2)=O)O1)O)O)OC(=O)\C=C\C1=CC=C(O)C=C1 DVGGLGXQSFURLP-VWMSDXGPSA-N 0.000 title claims abstract description 14
- 238000002360 preparation method Methods 0.000 title description 3
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 claims abstract description 51
- 229960004316 cisplatin Drugs 0.000 claims abstract description 51
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims abstract description 50
- 229910052742 iron Inorganic materials 0.000 claims abstract description 27
- 230000034994 death Effects 0.000 claims abstract description 26
- 208000028867 ischemia Diseases 0.000 claims abstract description 19
- 230000010410 reperfusion Effects 0.000 claims abstract description 18
- 210000003734 kidney Anatomy 0.000 claims abstract description 13
- 230000003907 kidney function Effects 0.000 claims description 13
- 230000006378 damage Effects 0.000 claims description 8
- 206010061481 Renal injury Diseases 0.000 claims description 7
- 208000037806 kidney injury Diseases 0.000 claims description 7
- 238000011282 treatment Methods 0.000 abstract description 29
- 229940079593 drug Drugs 0.000 abstract description 16
- 238000000338 in vitro Methods 0.000 abstract description 7
- 238000010172 mouse model Methods 0.000 abstract description 4
- 210000004027 cell Anatomy 0.000 description 21
- 241000699670 Mus sp. Species 0.000 description 16
- 101000588302 Homo sapiens Nuclear factor erythroid 2-related factor 2 Proteins 0.000 description 15
- 102100031701 Nuclear factor erythroid 2-related factor 2 Human genes 0.000 description 15
- 241000699666 Mus <mouse, genus> Species 0.000 description 14
- 210000004926 tubular epithelial cell Anatomy 0.000 description 14
- 238000001514 detection method Methods 0.000 description 11
- 210000005084 renal tissue Anatomy 0.000 description 11
- 108090000623 proteins and genes Proteins 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 102000004034 Kelch-Like ECH-Associated Protein 1 Human genes 0.000 description 7
- 108090000484 Kelch-Like ECH-Associated Protein 1 Proteins 0.000 description 7
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 7
- 230000003859 lipid peroxidation Effects 0.000 description 7
- 229940118019 malondialdehyde Drugs 0.000 description 7
- 230000001575 pathological effect Effects 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 7
- 230000010024 tubular injury Effects 0.000 description 7
- 208000037978 tubular injury Diseases 0.000 description 7
- 238000001262 western blot Methods 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 229940105442 cisplatin injection Drugs 0.000 description 6
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 6
- 229930182470 glycoside Natural products 0.000 description 6
- -1 glycoside flavonoid Chemical class 0.000 description 6
- JVJFIQYAHPMBBX-FNORWQNLSA-N (E)-4-hydroxynon-2-enal Chemical compound CCCCCC(O)\C=C\C=O JVJFIQYAHPMBBX-FNORWQNLSA-N 0.000 description 5
- JVJFIQYAHPMBBX-UHFFFAOYSA-N HNE Natural products CCCCCC(O)C=CC=O JVJFIQYAHPMBBX-UHFFFAOYSA-N 0.000 description 5
- 238000011529 RT qPCR Methods 0.000 description 5
- 208000027418 Wounds and injury Diseases 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 208000014674 injury Diseases 0.000 description 5
- 210000002254 renal artery Anatomy 0.000 description 5
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 4
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 4
- 101000829725 Homo sapiens Phospholipid hydroperoxide glutathione peroxidase Proteins 0.000 description 4
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 4
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 4
- 241000328973 Tilia miqueliana Species 0.000 description 4
- 210000000683 abdominal cavity Anatomy 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- 230000002438 mitochondrial effect Effects 0.000 description 4
- 230000001105 regulatory 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
- 108010024636 Glutathione Proteins 0.000 description 3
- 102100034459 Hepatitis A virus cellular receptor 1 Human genes 0.000 description 3
- 101710185991 Hepatitis A virus cellular receptor 1 homolog Proteins 0.000 description 3
- 102000013519 Lipocalin-2 Human genes 0.000 description 3
- 108010051335 Lipocalin-2 Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 102100023410 Phospholipid hydroperoxide glutathione peroxidase Human genes 0.000 description 3
- PFYWPQMAWCYNGW-UHFFFAOYSA-M [6-(dimethylamino)-9-(2-methoxycarbonylphenyl)xanthen-3-ylidene]-dimethylazanium;perchlorate Chemical compound [O-]Cl(=O)(=O)=O.COC(=O)C1=CC=CC=C1C1=C2C=CC(=[N+](C)C)C=C2OC2=CC(N(C)C)=CC=C21 PFYWPQMAWCYNGW-UHFFFAOYSA-M 0.000 description 3
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 229940109239 creatinine Drugs 0.000 description 3
- 230000008034 disappearance Effects 0.000 description 3
- 229930003935 flavonoid Natural products 0.000 description 3
- 235000017173 flavonoids Nutrition 0.000 description 3
- 150000002338 glycosides Chemical class 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 231100000417 nephrotoxicity Toxicity 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000010798 ubiquitination Methods 0.000 description 3
- 230000034512 ubiquitination Effects 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 206010021143 Hypoxia Diseases 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 210000002469 basement membrane Anatomy 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 208000020832 chronic kidney disease Diseases 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000003828 downregulation Effects 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 229960003180 glutathione Drugs 0.000 description 2
- 229920003063 hydroxymethyl cellulose Polymers 0.000 description 2
- 229940031574 hydroxymethyl cellulose Drugs 0.000 description 2
- 238000003125 immunofluorescent labeling Methods 0.000 description 2
- 230000002055 immunohistochemical effect Effects 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 210000000110 microvilli Anatomy 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000036542 oxidative stress Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 230000008961 swelling Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000003827 upregulation Effects 0.000 description 2
- 230000002477 vacuolizing effect Effects 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 206010002660 Anoxia Diseases 0.000 description 1
- 241000976983 Anoxia Species 0.000 description 1
- 238000000116 DAPI staining Methods 0.000 description 1
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 1
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 108010018924 Heme Oxygenase-1 Proteins 0.000 description 1
- 102100028006 Heme oxygenase 1 Human genes 0.000 description 1
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 241001531051 Potentilla chinensis Species 0.000 description 1
- 206010048988 Renal artery occlusion Diseases 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 102000006275 Ubiquitin-Protein Ligases Human genes 0.000 description 1
- 108010083111 Ubiquitin-Protein Ligases Proteins 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000007953 anoxia Effects 0.000 description 1
- 230000003178 anti-diabetic effect Effects 0.000 description 1
- 239000003472 antidiabetic agent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 208000035850 clinical syndrome Diseases 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000002872 contrast media Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 235000018927 edible plant Nutrition 0.000 description 1
- 208000028208 end stage renal disease Diseases 0.000 description 1
- 201000000523 end stage renal failure Diseases 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 229910001447 ferric ion Inorganic materials 0.000 description 1
- 229910001448 ferrous ion Inorganic materials 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 238000003304 gavage Methods 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229960003284 iron Drugs 0.000 description 1
- 229960002725 isoflurane Drugs 0.000 description 1
- 210000000231 kidney cortex Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000028161 membrane depolarization Effects 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 230000006676 mitochondrial damage Effects 0.000 description 1
- 210000001700 mitochondrial membrane Anatomy 0.000 description 1
- 230000008965 mitochondrial swelling Effects 0.000 description 1
- 238000003032 molecular docking Methods 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 230000003589 nefrotoxic effect Effects 0.000 description 1
- 231100000381 nephrotoxic Toxicity 0.000 description 1
- 230000000324 neuroprotective effect Effects 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 230000004850 protein–protein interaction Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开银椴甙在制备用于减轻急性肾损伤药物中的应用，通过在顺铂或缺血再灌注诱导的小鼠模型及体外细胞上使用银椴甙进行干预治疗，可显著改善急性肾损伤肾结构和功能、肾小管细胞铁死亡等，本申请的发现将极有可能为防治急性肾损伤提供有效的临床药物。
Description
技术领域
本发明属于生物医药领域，具体涉及银椴甙在制备用于减轻急性肾损伤的药物中的用途。
背景技术
急性肾损伤(Acute kidney injury,AKI)是一类常见的以肾功能迅速下降为特征的临床综合征。流行病学研究表明，我国每年住院病人AKI检出率超过2％，大约有290万AKI病人住院治疗，其中约有70万的患者死于AKI，存活的患者也较易进展至慢性肾病甚至终末期肾病[1]。在临床上，诸多因素如脓毒症、造影剂、缺血缺氧以及肾毒性药物等都可导致AKI的发生，并且由于AKI的病因及发病机制的复杂性，导致临床上缺乏治疗AKI的特效药物[2,3]，因而开发新的有效的能够改善急性肾损伤的相关药物具备重要的临床意义。
近年来，系列研究表明，各种病理因素诱导的肾小管上皮细胞发生铁死亡促进了急性肾损伤的发生发展，靶向肾小管上皮细胞铁死亡为临床治疗急性肾损伤提供了有效的策略[4]。NRF2是一种核转录因子，被认为是细胞内的最重要的抗氧化应激调节因子，在细胞内发挥抗铁死亡作用。正常生理水平下，E3连接酶接头蛋白KEAP1介导NRF2发生泛素化降解，因而靶向抑制KEAP1-NRF2蛋白质互相作用，进而激活NRF2具备抗细胞铁死亡的作用[5]，进而具备治疗急性肾损伤的潜力。
Tiliroside(银椴甙)是一种天然糖苷类黄酮，存在于多种可食用植物例如：委陵菜及金花莲等的的果实、叶子或根中[6]，其分子式如下所示：
在不同的体内和体外模型中，tiliroside已显示出抗糖尿病和神经保护作用。目前尚未有报道tiliroside是否能够保护急性肾损伤。
参考文献：
1.Yang,L.,et al.,Acute kidney injury in China:a cross-sectionalsurvey.Lancet,2015.
386(10002):p.1465-71.
2.Gameiro,J.,et al.,Acute Kidney Injury:From Diagnosis to PreventionandTreatment Strategies.J Clin Med,2020.9(6).
3.Makris,K.and L.Spanou,Acute Kidney Injury:Definition,Pathophysiology andClinical Phenotypes.Clin Biochem Rev,2016.37(2):p.85-98.
4.Hu,Z.,et al.,Emerging Role of Ferroptosis in Acute KidneyInjury.Oxid Med CellLongev,2019.2019:p.8010614.
5.Crisman,E.,et al.,KEAP1-NRF2 protein-protein interactioninhibitors:Design,pharmacological properties and therapeutic potential.MedRes Rev,2023.43(1):p.237-287.
6.Ninomiya,K.,et al.,Potent anti-obese principle from Rosa canina:structuralrequirements and mode of action of trans-tiliroside.Bioorg Med ChemLett,2007.
17(11):p.3059-64.
发明内容
发明目的：本发明提供了一种天然糖苷类黄酮药物tiliroside在制备用于减轻急性肾损伤药物中的应用，该药物在制备减轻肾毒性药物或者缺血再灌注诱导的急性肾损伤相关病症的药物中的用途，解决了现有技术中没有合适的药物用于治疗急性肾损伤的技术问题。
具体地，所述急性肾损伤为顺铂诱导的急性肾损伤。银椴甙改善顺铂诱导的肾脏损伤和肾脏功能，同时，银椴甙改善顺铂诱导的铁死亡。
另一方面，所述急性肾损伤为缺血再灌注诱导的急性肾损伤。银椴甙改善缺血再灌注诱导的肾脏损伤和肾功能。
本发明提供了一种天然糖苷类黄酮药物tiliroside在制备治疗急性肾损伤药物中的用途，并首次研究发现tiliroside通过结合KEAP1进而抑制KEAP1-NRF2蛋白质互相结合，上调NRF2蛋白表达激活进而改善病理因素诱导的肾小管上皮细胞铁死亡，进而改善急性肾损伤。
本发明通过在顺铂或双侧肾动脉夹闭诱导急性肾损伤小鼠模型及体外细胞模型上使用tiliroside，提出了将tiliroside作为一种天然小分子化合物在减轻肾毒性药物或缺血缺氧诱导的急性肾损伤相关病症的药物中的用途。结果发现，在急性肾损伤小鼠模型及体外细胞上利用tiliroside进行干预治疗，可显著改善急性肾损伤肾结构和功能、肾小管细胞铁死亡等；本发明的发现将有可能为防治急性肾损伤提供有效的临床药物。
附图说明
图1显示tiliroside通过与KEAP1结合在小鼠肾组织和体外培养的人源肾小管上皮细胞中激活NRF2的表达(Til:tiliroside)；
图2显示在顺铂诱导急性肾损伤模型中，tiliroside治疗改善顺铂诱导的肾脏损伤和肾脏功能(Til:tiliroside；CDDP:顺铂)；
图3显示在顺铂诱导急性肾损伤模型中，tiliroside治疗改善顺铂诱导的肾小管上皮细胞的铁死亡；
图4显示在体外肾小管上皮细胞的顺铂模型中，tiliroside治疗改善顺铂诱导的铁死亡；
图5显示在肾动脉缺血再灌注诱导的急性肾损伤模型中，tiliroside治疗改善缺血再灌注诱导的肾脏损伤和肾功能(IRI:缺血再灌注诱导急性肾损伤)。
具体实施方式
下面结合附图和具体实施方式对本发明做更进一步的具体说明，本发明的上述和/或其他方面的优点将会变得更加清楚。
材料和方法
1)材料和试剂
Tiliroside化合物购于美国Apexbio公司，NGAL抗体购于Abcam公司，4-HNE、KIM-1及MPO抗体购于美国R&D Systems公司，β-actin、NRF2、KEAP1、HO-1及GPX4抗体均购于武汉三鹰公司。脂质过氧化探针BODIPY 581/591C11，MitoSOX及TMRM检测试剂盒购于赛默飞世尔公司。MDA，GSH，铁离子检测试剂盒及CCK8检测试剂购于北京索莱宝公司。实验所用小鼠为6-8周雄性C57BL/6J小黑鼠，购买自江苏集萃药康生物科技股份有限公司。
2)顺铂诱导急性肾损伤细胞模型及实验分组
人源肾小管上皮细胞(HK2)使用含有10％胎牛血清的DMEM/F12培养基培养，培养条件为37℃，5％二氧化碳和95％空气。在HK2生长至70％密度时，换成含1％胎牛血清的DMEM/F12培养基培养，并加入10μM tiliroside(溶于DMSO)预处理1h或用DMSO(作为对照组)相同处理，1h后加入10μg/ml顺铂刺激，将细胞在培养箱中孵育24h后，最后收集细胞进行qRT-PCR、Western Blot检测以及CCK8等试验。
3)顺铂诱导及缺血再灌注诱导的急性肾损伤小鼠模型及实验分组
将小鼠随机分为四组，每组6只，分别为空白对照组(vehicle)、tiliroside组、顺铂模型组(cisplatin)和tiliroside治疗组(cisplatin+tiliroside)。Tiliroside溶于0.5％羟甲基纤维素钠溶液中，以15mg/kg(低剂量)或者30mg/kg(高剂量)剂量通过灌胃给药。
具体地：
空白对照组：小鼠从顺铂注射前48h开始至处死小鼠每天灌胃一次溶剂，溶剂为0.5％羟甲基纤维素钠溶液；
tiliroside组：小鼠从顺铂注射前48h开始至处死小鼠每天灌胃一次30mg/kg(高剂量)tiliroside；
顺铂模型组(cisplatin)：小鼠从顺铂注射前48h开始至处死小鼠每天灌胃一次溶剂，溶剂处理48h之后通过腹腔一次性注射20mg/kg的顺铂，小鼠于顺铂注射72h后处死并取材；
tiliroside治疗组(cisplatin+tiliroside)：小鼠从顺铂注射前48h开始每天分别灌胃一次15mg/kg(低剂量)或者30mg/kg(高剂量)tiliroside，tiliroside处理48h之后通过腹腔一次性注射20mg/kg的顺铂，小鼠于顺铂注射72h后处死并取材；
对于缺血再灌注诱导的小鼠急性肾损伤模型，利用异氟烷麻醉小鼠并消毒打开小鼠腹腔，利用无损伤微型动脉夹迅速将两侧肾动脉夹闭，小鼠肾脏由红色变为紫黑色提示夹闭成功，小鼠在恒温台上持续夹闭30min，松开动脉夹，肾脏颜色会由紫黑色变为红色，对照假手术组小鼠同样打开腹腔找到肾动脉，但不夹闭，再灌注24h即可成功造模，动物组别及处理与顺铂组一致。所有动物实验均遵守中国实验动物管理和使用条例。
4)肾功能检测
小鼠血液标本经离心后收集血清成分，于南京市儿童医院检验科全自动生化仪上检测血清肌酐和血清尿素氮指标。
5)肾小管损伤评分
肾脏糖原PAS病理染色中观察小管损伤程度，并根据半定量损伤评分标准进行小管损伤评估，正常肾小管组织为0分，肾小管损伤程度小于30％的为1分，肾小管损伤程度在30％至60％之间的为2分，大于60％的为3分，大于70％的为4分。
6)组织学和免疫荧光染色
肾脏组织经多聚甲醛固定、石蜡包理，组织切片后进行免疫组织化学染色。4-HNE一抗浓度为1:100，NRF2一抗浓度为1:100。DAPI染色根据标作说明进行。
7)蛋白质免疫印迹(Western blot)
肾脏组织按照文献方法提取蛋白。蛋白质免疫印迹(Western blot)结果用ImageJ软件进行灰度分析。
8)实时定量PCR(qRT-PCR)
肾脏组织按照文献方法提取RNA，逆转录以及实时定量PCR(qRT-PCR)。
9)CCK8、LDH、GSH、TMRM、BODIPY 581/591C11、铁离子及MDA检测
按照检测试剂盒说明书操作。
10)透射电镜
肾脏皮质透射电镜检测委托广州金域医学检验中心完成
11)统计分析
细胞实验重复三次并统计，使用均值士SD表示数据。多组间比较用单因素方差分析(ANOVA)，两组间数据比较用T检验。以P<0.05为具有统计学意义。
实施例1Tliroside通过破坏KEAP1-NRF2相互结合，上调NRF2的表达活化。
鉴于NRF2在细胞抗铁死亡中关键作用，我们有目的的从天然化合物库中通过分子对接软件Autodock vina筛选KEAP1-NRF2蛋白-蛋白相互作用的可能抑制剂，我们的对接结果显示，tiliroside能够竞争性与KEAP1蛋白直接结合(图1A)，进而抑制KEAP1-NRF2相互结合；进一步的细胞蛋白热稳定性实验证实了tiliroside与KEAP1蛋白直接结合(图1B)。QRT-PCR、western blotting、泛素化降解实验及免疫荧光染色(图1C-1F)显示，在HK2细胞中，tiliroside能够通过抑制KEAP1介导的NRF2泛素化降解进而显著上调NRF2的表达活化。进一步的实验显示，tiliroside处理能够显著上调小鼠肾组织中NRF2的表达及活化(图1G-1I)。
实施例2Tiliroside改善顺铂诱导急性肾损伤模型中的肾脏损伤和肾功能。
为了评估检测tiliroside在顺铂诱导急性肾损伤中的作用，我们检测了小鼠血清中肾脏相关生化指标，腹腔注射顺铂72小时后，尿素氮和血清肌酐指标明显升高，肾小管管腔扩张、肾小管上皮细胞肿胀，出现空泡状改变或细胞脱落、基底膜裸露、微绒毛结构消失等肾脏病理损伤表现也较为严重，低剂量15mg/kg/d tiliroside治疗后，小鼠肾功能轻微改善，而在高剂量30mg/kg/d tiliroside治疗后，小鼠肾功能指标显著改善，并且高剂量tiliroside处理不会造成小鼠肾脏的毒副作用(图2A,2B)。此外，肾小管损伤评分也提示tiliroside治疗可显著改善顺铂引起的肾脏病理损伤(图2C)。这些结果表明tiliroside可以保护顺铂诱导的急性肾损伤，并且其本身未见显著的肾毒性。
为了进一步证明tiliroside在保护顺铂诱导急性肾损伤中的作用，我们检测了急性肾损伤早期特异性生物指标，NGAL和KIM-1。Western blot检测结果显示，KIM-1以及NGAL在顺铂诱导的急性肾损伤小鼠的肾脏中高表达，在tiliroside治疗后，其表达水平显著下降(图2D)，说明tiliroside能够改善顺铂诱导的急性肾损伤。
实施例3在顺铂诱导急性肾损伤模型中，tiliroside治疗改善顺铂诱导的铁死亡。
我们的实验结果提示tiliroside是一种KEAP1-NRF2蛋白互相作用抑制剂，能够激活NRF2的表达，而NRF2在细胞铁死亡中发挥关键调控作用，为了进一步证明tiliroside在保护顺铂诱导急性肾损伤中对肾小管上皮细胞铁死亡的调控作用，我们检测了一系列细胞铁死亡的指标，包括肾组织二价铁离子含量(图3A)、脂质过氧化产物丙二醛MDA(图3B)、还原性谷胱甘肽GSH(图3C)及铁死亡通路相关基因GPX4等的表达(图3D)，我们的结果显示tiliroside处理显著改善了顺铂诱导的铁死亡指标。此外，Western blot结果显示顺铂诱导肾组织中过氧化物酶(MPO)表达显著上调，而tiliroside处理显著抑制了MPO蛋白水平上调(图3E)；同时，tiliroside能显著恢复抗铁死亡蛋白GPX4的表达，而GPX4蛋白水平在顺铂诱导小鼠肾组织表达显著下调(图3E)。
顺铂可引起氧化应激并产生过度的ROS，过量的ROS可能导致脂质过氧化并产生反应性脂质，如4-HNE，进而导致细胞铁死亡，因而我们检测了肾组织中4-HNE的表达水平。免疫组化结果显示，tiliroside治疗后，肾脏4-HNE蛋白水平明显降低(图3F)。线粒体破坏是细胞铁死亡另一关键特征，因而我们利用透射电镜下观察小鼠肾小管细胞中线粒体超微结构，腹腔注射顺铂可导致小鼠肾脏线粒体肿胀、嵴消失、空泡状改变等，tiliroside治疗后线粒体结构损伤减轻(图3G)，提示tiliroside治疗可保护顺铂诱导急性肾损伤的线粒体结构。综上，在顺铂诱导急性肾损伤模型中，tiliroside治疗改善顺铂诱导的肾小管上皮细胞铁死亡。
实施例4在体外肾小管上皮细胞的顺铂模型中，tiliroside治疗改善顺铂诱导的铁死亡。
利用体外培养的肾小管上皮细胞，tiliroside预处理1h，之后加入10μg/ml的顺铂继续处理24h，使用CCK8检测细胞活性，检测乳酸脱氢酶(LDH)释放，以及丙二醛(MDA)和还原性谷胱甘肽水平，利用脂质过氧化探针BODIPY 581/591C11染色检测细胞脂质过氧化水平，同时利用MitoSox和TMRM染色检测观察线粒体损伤。我们的数据显示，顺铂刺激导致细胞活性显著下调，促进LDH释放，以及脂质过氧化上调和还原性谷胱甘肽下调，并导致线粒体中超氧化物的产生增加以及线粒体膜电位去极化严重，以上指标提示顺铂诱导肾小管上皮细胞发生了显著的铁死亡，而tiliroside处理显著改善了顺铂诱导的肾小管上皮细胞铁死亡(图4A-G所示)。
实施例5Tiliroside改善缺血再灌注(IRI)诱导急性肾损伤模型中的肾脏损伤和肾功能。
为了评估检测tiliroside在缺血再灌注诱导急性肾损伤中的作用，我们检测了小鼠血清中肾脏相关生化指标，小鼠行肾动脉缺血再灌注24小时后，尿素氮和血清肌酐指标明显升高，肾小管管腔扩张、肾小管上皮细胞肿胀，出现空泡状改变或细胞脱落、基底膜裸露、微绒毛结构消失等肾脏病理损伤表现也较为严重，而tiliroside治疗后，其相应肾功能指标显著下降(图5A,5B)。此外，肾小管损伤评分也提示tiliroside治疗可显著改善缺血再灌注引起的肾脏病理损伤(图5C)。进一步，我们检测了肾组织丙二醛(MDA)的水平，发现tiliroside治疗显著下调缺血再灌注导致的肾组织脂质过氧化(图5D)。以上结果提示tiliroside能够改善缺血再灌注诱导的急性肾损伤。
综上所述，本发明提供了一种通过抑制KEAP1-NRF2蛋白与蛋白互相作用的天然小分子药物tiliroside在制备用于减轻急性肾损伤相关病症的药物中的用途，该药通过灌胃的方式给药，剂量为每天30mg/kg，通过上调NRF2的表达和激活，进而减少氧化应激及细胞铁死亡，改善急性肾损伤。
以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本发明的具体实施例而已，并不用于限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (6)
1.银椴甙在制备用于减轻急性肾损伤药物中的应用。
2.根据权利要求1所述的应用，其特征在于，所述急性肾损伤为顺铂诱导的急性肾损伤。
3.根据权利要求1或2所述的应用，其特征在于，银椴甙改善顺铂诱导的肾脏损伤和肾脏功能。
4.根据权利要求1或2所述的应用，其特征在于，银椴甙改善顺铂诱导的铁死亡。
5.根据权利要求1所述的应用，其特征在于，所述急性肾损伤为缺血再灌注诱导的急性肾损伤。
6.根据权利要求1所述的应用，其特征在于，银椴甙改善缺血再灌注诱导的肾脏损伤和肾功能。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410017828.1A CN117771259A (zh) | 2024-01-05 | 2024-01-05 | 银椴甙在制备用于减轻急性肾损伤的药物中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410017828.1A CN117771259A (zh) | 2024-01-05 | 2024-01-05 | 银椴甙在制备用于减轻急性肾损伤的药物中的应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117771259A true CN117771259A (zh) | 2024-03-29 |
Family
ID=90381657
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410017828.1A Pending CN117771259A (zh) | 2024-01-05 | 2024-01-05 | 银椴甙在制备用于减轻急性肾损伤的药物中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117771259A (zh) |
-
2024
- 2024-01-05 CN CN202410017828.1A patent/CN117771259A/zh active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chen et al. | Ginkgo biloba extract-761 protects myocardium by regulating Akt/Nrf2 signal pathway | |
| Yu et al. | Danshensu attenuates cisplatin-induced nephrotoxicity through activation of Nrf2 pathway and inhibition of NF-κB | |
| CN111358790B (zh) | Nsc228155在制备防治急性肾损伤的药物中的用途 | |
| CN111358789B (zh) | Nsc228155在制备防治慢性肾纤维化药物中的用途 | |
| Zhang et al. | Hirudin protects against isoproternol-induced myocardial infraction by alleviating oxidative via an Nrf2 dependent manner | |
| Wang et al. | Renal protective effect of probucol in rats with contrast-induced nephropathy and its underlying mechanism | |
| CN108434139B (zh) | 缺氧诱导因子脯氨酰羟化酶活性抑制剂在制备防治急性肾损伤药物中的应用 | |
| Li et al. | Baicalin alleviates contrast-induced acute kidney injury through ROS/NLRP3/Caspase-1/GSDMD pathway-mediated proptosis in vitro | |
| Zeng et al. | Aggravated intestinal ischemia‑reperfusion injury is associated with activated mitochondrial autophagy in a mouse model of diabetes | |
| Chen et al. | Astragaloside IV inhibits NLRP3 inflammasome-mediated pyroptosis via activation of Nrf-2/HO-1 signaling pathway and protects against doxorubicin-induced cardiac dysfunction | |
| Zhang et al. | Liensinine pretreatment reduces inflammation, oxidative stress, apoptosis, and autophagy to alleviate sepsis acute kidney injury | |
| Peng et al. | Dehydrocostus lactone inhibits the proliferation of esophageal cancer cells in vivo and in vitro through ROS-mediated apoptosis and autophagy | |
| Zhang et al. | Carbon monoxide attenuates LPS-induced myocardial dysfunction in rats by regulating the mitochondrial dynamic equilibrium | |
| Jeon et al. | Inhibitory activity of aromadendrin from prickly pear (Opuntia ficus-indica) root on aldose reductase and the formation of advanced glycation end products | |
| CN117771259A (zh) | 银椴甙在制备用于减轻急性肾损伤的药物中的应用 | |
| WO2017054640A1 (zh) | 延胡索乙素在制备抗顺铂毒性药物中的应用 | |
| CN115590914B (zh) | 一种红大戟提取物及其在制备抗乳腺癌药物中的应用 | |
| Li et al. | Lugrandoside attenuates LPS-induced acute respiratory distress syndrome by anti-inflammation and anti-apoptosis in mice | |
| CN113876840A (zh) | 苦荞黄酮在制备治疗胰腺癌药物中的应用 | |
| CN113244237B (zh) | Bi8622在制备用于减轻顺铂诱导的急性肾损伤的药物中的应用 | |
| Huang et al. | Soluble thrombomodulin alleviates Diquat-induced acute kidney injury by inhibiting the HMGB1/IκBα/NF-κB signalling pathway | |
| CN113855684B (zh) | 奥利索西在制备用于减轻或改善顺铂诱导的急性肾损伤的药物中的用途 | |
| Tian et al. | Effects of the PI3K/Akt/HO-1 pathway on autophagy in a sepsis-induced acute lung injury mouse model | |
| CN115778965B (zh) | 女贞苷g13在制备抗心肌纤维化药物中的新应用 | |
| Wang et al. | Ethanol extract of Nepeta angustifoia CY Wu ameliorates hyperuricemia in fructose-induced mice. |
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 |