CN116251097A - 奥美沙坦在治疗SARS-CoV-2包膜蛋白诱导的肾纤维化中的应用 - Google Patents
奥美沙坦在治疗SARS-CoV-2包膜蛋白诱导的肾纤维化中的应用 Download PDFInfo
- Publication number
- CN116251097A CN116251097A CN202310191777.XA CN202310191777A CN116251097A CN 116251097 A CN116251097 A CN 116251097A CN 202310191777 A CN202310191777 A CN 202310191777A CN 116251097 A CN116251097 A CN 116251097A
- Authority
- CN
- China
- Prior art keywords
- olmesartan
- cov
- envelope protein
- sars
- renal fibrosis
- 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
- 239000005480 Olmesartan Substances 0.000 title claims abstract description 48
- VTRAEEWXHOVJFV-UHFFFAOYSA-N olmesartan Chemical compound CCCC1=NC(C(C)(C)O)=C(C(O)=O)N1CC1=CC=C(C=2C(=CC=CC=2)C=2NN=NN=2)C=C1 VTRAEEWXHOVJFV-UHFFFAOYSA-N 0.000 title claims abstract description 48
- 229960005117 olmesartan Drugs 0.000 title claims abstract description 48
- 101000667982 Severe acute respiratory syndrome coronavirus 2 Envelope small membrane protein Proteins 0.000 title claims abstract description 24
- 201000002793 renal fibrosis Diseases 0.000 title claims abstract description 18
- 239000003814 drug Substances 0.000 claims abstract description 17
- 229940079593 drug Drugs 0.000 claims abstract description 6
- 239000000546 pharmaceutical excipient Substances 0.000 claims abstract description 4
- 239000004480 active ingredient Substances 0.000 claims abstract description 3
- 239000007884 disintegrant Substances 0.000 claims abstract description 3
- 239000000945 filler Substances 0.000 claims abstract description 3
- -1 glidant Substances 0.000 claims abstract description 3
- 239000000314 lubricant Substances 0.000 claims abstract description 3
- 239000004014 plasticizer Substances 0.000 claims abstract description 3
- 238000002360 preparation method Methods 0.000 claims abstract description 3
- 239000011230 binding agent Substances 0.000 claims description 2
- 238000011282 treatment Methods 0.000 abstract description 15
- 238000002347 injection Methods 0.000 abstract description 5
- 239000007924 injection Substances 0.000 abstract description 5
- 230000000144 pharmacologic effect Effects 0.000 abstract description 3
- 230000009471 action Effects 0.000 abstract description 2
- 239000000853 adhesive Substances 0.000 abstract 1
- 230000001070 adhesive effect Effects 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 108090000623 proteins and genes Proteins 0.000 description 35
- 102000004169 proteins and genes Human genes 0.000 description 35
- 210000004027 cell Anatomy 0.000 description 23
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 16
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 16
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 16
- 210000003734 kidney Anatomy 0.000 description 15
- 238000002474 experimental method Methods 0.000 description 13
- 102000016359 Fibronectins Human genes 0.000 description 12
- 108010067306 Fibronectins Proteins 0.000 description 12
- 241000699670 Mus sp. Species 0.000 description 12
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- 101710091045 Envelope protein Proteins 0.000 description 11
- 101710188315 Protein X Proteins 0.000 description 11
- 102100021696 Syncytin-1 Human genes 0.000 description 11
- 230000007705 epithelial mesenchymal transition Effects 0.000 description 11
- 238000001514 detection method Methods 0.000 description 10
- 238000001262 western blot Methods 0.000 description 10
- 241001678559 COVID-19 virus Species 0.000 description 9
- 101710143123 Mothers against decapentaplegic homolog 2 Proteins 0.000 description 9
- 102000057208 Smad2 Human genes 0.000 description 9
- 241001465754 Metazoa Species 0.000 description 8
- 229940125364 angiotensin receptor blocker Drugs 0.000 description 8
- 230000003907 kidney function Effects 0.000 description 8
- 238000000465 moulding Methods 0.000 description 8
- 238000001543 one-way ANOVA Methods 0.000 description 8
- 102000013127 Vimentin Human genes 0.000 description 7
- 108010065472 Vimentin Proteins 0.000 description 7
- 239000002333 angiotensin II receptor antagonist Substances 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 7
- 210000005048 vimentin Anatomy 0.000 description 7
- 239000005541 ACE inhibitor Substances 0.000 description 6
- 229940044094 angiotensin-converting-enzyme inhibitor Drugs 0.000 description 6
- 229940109239 creatinine Drugs 0.000 description 6
- 108020004999 messenger RNA Proteins 0.000 description 6
- 230000037361 pathway Effects 0.000 description 6
- 230000028327 secretion Effects 0.000 description 6
- 102000007469 Actins Human genes 0.000 description 5
- 108010085238 Actins Proteins 0.000 description 5
- 206010016654 Fibrosis Diseases 0.000 description 5
- 206010023421 Kidney fibrosis Diseases 0.000 description 5
- 230000004900 autophagic degradation Effects 0.000 description 5
- 230000006378 damage Effects 0.000 description 5
- 230000004761 fibrosis Effects 0.000 description 5
- 210000003205 muscle Anatomy 0.000 description 5
- 238000011321 prophylaxis Methods 0.000 description 5
- 238000011002 quantification Methods 0.000 description 5
- 210000002700 urine Anatomy 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 230000037396 body weight Effects 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 101000666405 Homo sapiens General transcription factor IIH subunit 1 Proteins 0.000 description 3
- 101000599779 Homo sapiens Insulin-like growth factor 2 mRNA-binding protein 2 Proteins 0.000 description 3
- 101001052506 Homo sapiens Microtubule-associated proteins 1A/1B light chain 3A Proteins 0.000 description 3
- 102100037919 Insulin-like growth factor 2 mRNA-binding protein 2 Human genes 0.000 description 3
- 102100024178 Microtubule-associated proteins 1A/1B light chain 3A Human genes 0.000 description 3
- 208000027418 Wounds and injury Diseases 0.000 description 3
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000010166 immunofluorescence Methods 0.000 description 3
- 208000014674 injury Diseases 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 102000003954 Autophagy-Related Proteins Human genes 0.000 description 2
- 108010082399 Autophagy-Related Proteins Proteins 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 208000025721 COVID-19 Diseases 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- 101000950671 Chelon ramada Myosin light chain 3, skeletal muscle isoform Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 238000003304 gavage Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- 208000017169 kidney disease Diseases 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000008816 organ damage Effects 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 201000001474 proteinuria Diseases 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 210000005084 renal tissue Anatomy 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000002485 urinary effect Effects 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 102100030988 Angiotensin-converting enzyme Human genes 0.000 description 1
- 102100035765 Angiotensin-converting enzyme 2 Human genes 0.000 description 1
- 108090000975 Angiotensin-converting enzyme 2 Proteins 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 206010007572 Cardiac hypertrophy Diseases 0.000 description 1
- 208000006029 Cardiomegaly Diseases 0.000 description 1
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- 101100086437 Drosophila melanogaster Rap1 gene Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 101100247326 Mucor circinelloides f. lusitanicus RAS3 gene Proteins 0.000 description 1
- 238000011887 Necropsy Methods 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 101000933967 Pseudomonas phage KPP25 Major capsid protein Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 229940078123 Ras inhibitor Drugs 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000010913 Type 1 Angiotensin Receptor Human genes 0.000 description 1
- 108010062481 Type 1 Angiotensin Receptor Proteins 0.000 description 1
- 230000002730 additional effect Effects 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 230000003276 anti-hypertensive effect Effects 0.000 description 1
- 239000002220 antihypertensive agent Substances 0.000 description 1
- 229940127088 antihypertensive drug Drugs 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 210000004957 autophagosome Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 235000003642 hunger Nutrition 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 210000001804 kidney proximal tubule epithelial cell Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 208000037890 multiple organ injury Diseases 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 238000010827 pathological analysis Methods 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000036454 renin-angiotensin system Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 230000037351 starvation Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000011285 therapeutic regimen Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 102000027257 transmembrane receptors Human genes 0.000 description 1
- 108091008578 transmembrane receptors Proteins 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000010024 tubular injury Effects 0.000 description 1
- 208000037978 tubular injury Diseases 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 230000007923 virulence factor Effects 0.000 description 1
- 239000000304 virulence factor Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4164—1,3-Diazoles
- A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
Landscapes
- Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Urology & Nephrology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明属于医药技术领域,尤其涉及奥美沙坦在治疗SARS‑CoV‑2包膜蛋白诱导的肾纤维化中的应用;公开了奥美沙坦用于制备治疗SARS‑CoV‑2包膜蛋白诱导肾纤维化的药物的应用;其中,奥美沙坦为治疗SARS‑CoV‑2包膜蛋白诱导肾纤维化药物中的主要活性成分;上述药物还包括药学上可接受的赋形剂,所述赋形剂选自粘合剂、填料、增塑剂、助流剂、崩解剂和润滑剂中的一种或者任意组合;上述药物还可为口服剂或注射剂;本发明首次公开了奥美沙坦的新用途,为奥美沙坦预防和/或治疗SARS‑CoV‑2包膜蛋白引起的肾纤维化的药理作用提供了理论依据。
Description
技术领域
本发明属于医药技术领域,尤其涉及奥美沙坦在治疗SARS-CoV-2包膜蛋白诱导的肾纤维化中的应用。
背景技术
SARS-CoV-2除了侵害呼吸系统造成炎症(COVID-19),往往伴随着其他并发症,其中有心血管疾病、高血压和糖尿病。还会损害全身多种器官。肾脏就是主要受损器官之一。SARS-CoV-2能够直接感染肾细胞,感染后的肾脏主要表现为肾小管损伤,并在患者尸检样本发现肾小管间质纤维化相关指标显著增加。临床上,SARS-CoV-2感染者的肾脏损害,不仅会增加治疗和护理的难度,还会增加患有潜在肾脏疾病的人的死亡率。包膜(Envelope,E)蛋白是SARS-CoV-2的主要结构蛋白之一,能够作为一种独立的毒力因子引起炎症和多器官损伤。而且包膜蛋白所诱导的损伤类型与SARS-CoV-2直接感染引起的损伤高度相似,能够很好地代替研究SARS-CoV-2相关的病理过程。然而,能够证明ACEIs/ARBs对SARS-CoV-2引起的器官损伤的有益作用的实验研究的证据仍然难以找到。而且临床上也没有针对这些并发症的专门治疗方案。因此,验证ACEIs/ARBs或研究某一种ACEIs/ARBs药物对于SARS-CoV-2引起的器官损伤的作用具有重要意义。
奥美沙坦是血管紧张素受体阻滞剂(ARBs)类的降压药物,是一种选择性血管紧张素Ⅱ1型受体(AT1)拮抗剂,具有剂量小、起效快、降压作用更强而持久、不良反应的发生率低等明显优点,对动脉硬化、心肌肥厚、心力衰竭、糖尿病、肾病等均具有较好作用,在临床上被广泛运用。COVID-19并发心血管疾病的患者通常使用肾素血管紧张素系统(RAS)抑制剂,特别是血管紧张素转换酶抑制剂(ACEIs)和血管紧张素受体阻滞剂(ARBs)。有趣的是,血管紧张素转换酶2(ACE2)是RAS3的关键成分之一,也是病毒侵袭的主要跨膜受体。因此,使用RAS抑制剂可能对COVID-19患者产生额外的影响。目前,从临床角度来看,ACEIs/ARBs似乎至少是无害的。
发明内容
本发明首次发现了奥美沙坦能有效抑制SARS-CoV-2包膜蛋白诱导的肾纤维化,基于该发现,将奥美沙坦应用于SARS-CoV-2包膜蛋白诱导的肾纤维化中的治疗,探究其疗效。
本发明采用的技术方案如下:
本发明首先构建了SARS-CoV-2包膜蛋白诱导的肾纤维化动物医学模型,具体构建方法为:小鼠尾静脉注射纯化的SARS-CoV-2包膜蛋白,造模28天后筛选得到发生肾纤维化的动物医学模型,并收集尿液检测尿肌酐和尿蛋白水平,监测肾功能。
之后本发明进一步验证了奥美沙坦对SARS-CoV-2包膜蛋白诱导的肾纤维化动物医学模型的药效,具体步骤为:
S1:预给药,预防组:在造模前4天给予小鼠奥美沙坦;
S2:对预防组和未经过预给药的小鼠进行造模,未经过预给药的为治疗组;(SARS-CoV-2包膜蛋白诱导的肾纤维化动物医学模型)
S3:给药,预防组和治疗组造模3天后给予奥美沙坦。
S4:给药25天后,收集预防组和治疗组小鼠肾脏组织,进行病理学分析。
在S1中,预防给药的奥美沙坦的给药剂量为10mg/kg/d,持续4天。
S3造模4天后持续给药,奥美沙坦的给药剂量为10mg/kg/d。
本发明还公开了SARS-CoV-2包膜蛋白诱导的肾上皮间充质转化细胞医学模型构建方法,以及奥美沙坦对细胞医学模型的疗效;具体步骤如下:
A1:造模,SARS-CoV-2包膜蛋白诱导的HK-2上皮间充质转化细胞模型;
A2:给药,给予SARS-CoV-2包膜蛋白诱导的HK-2上皮间充质转化细胞模型奥美沙坦,观察药效。
在A1中,HK-2细胞无血清饥饿12小时后,将包膜蛋白(2μg/mL)加入HK-2细胞培养基中继续培养24小时,HK-2细胞出现明显上皮间充质转化。
在A2中,奥美沙坦的给药剂量为20μM,处理24小时。
综上,本发明公开了奥美沙坦用于制备治疗SARS-CoV-2包膜蛋白诱导肾纤维化的药物的应用。
其中,奥美沙坦为治疗SARS-CoV-2包膜蛋白诱导肾纤维化药物中的主要活性成分。
上述药物还包括药学上可接受的赋形剂,所述赋形剂选自粘合剂、填料、增塑剂、助流剂、崩解剂和润滑剂中的一种或者任意组合。上述药物还可为口服剂或注射剂
与现有技术相比,本发明有如下优势:
本发明首次公开了奥美沙坦的新用途,为奥美沙坦预防和/或治疗SARS-CoV-2包膜蛋白引起的肾纤维化的药理作用提供了理论依据。
附图说明
图1:奥美沙坦对包膜蛋白造模后肾的病理结构的影响。(A)肾脏外观;(B)代表性肾脏样本的H&E染色(比例尺=50μm);(C)代表性肾脏样本的Masson染色(比例尺=50μm)。
图2:奥美沙坦对包膜蛋白造模后肾功能的影响。(A)通过尿蛋白与肌酐的比值来评估蛋白尿水平;(B)治疗结束时,检测血清尿素氮(BUN)评价肾功能;(C)治疗结束时,检测血清肌酐(SCr)评价肾功能。(所有值均为平均值±SD;单向方差分析。与对照组相比,###P<0.001,与2-E组相比,**P<0.01,***P<0.001,n=6)
图3:奥美沙坦对包膜蛋白造模后肾脏中纤维连接蛋白(FN)、波形蛋白和α-平滑肌肌动蛋白(α-SMA)蛋白表达的影响。(A)Western-blotting探究2-E蛋白和奥美沙坦对各组肾脏中纤维连接蛋白(FN)、波形蛋白和α-平滑肌肌动蛋白(α-SMA)蛋白表达的影响的代表性蛋白印迹分析;(B)蛋白表达水平相对定量分析。(所有值均为平均值±SD;单向方差分析。与对照组相比,###P<0.001,与2-E组相比,***P<0.001,n=6)
图4:奥美沙坦对包膜蛋白造模后肾脏中纤维连接蛋白(FN)、波形蛋白和α-平滑肌肌动蛋白(α-SMA)mRNA表达的影响。各组肾脏中FN、I型胶原、波形蛋白和α-SMA的mRNA的表达水平相对定量分析。(所有值均为平均值±SD;单向方差分析。与对照组相比,###P<0.001,与2-E组相比,***P<0.001,n=6)
图5:奥美沙坦对包膜蛋白造模后小鼠TGF-β1的分泌以及TGF-β1/Smad2/3信号通路的影响。(A)小鼠血清中TGF-β1的水平;(B)各组肾脏中TGF-β1/Smad2/3通路的激活情况和(C)相对定量;(D)TGF-β1在小鼠肾脏中的mRNA表达水平。(所有值均为平均值±SD;单向方差分析。与对照组相比,###P<0.001,与2-E组相比,***P<0.001,n=6)
图6:奥美沙坦对2-E蛋白诱导的HK-2细胞上皮间充质转化的影响。奥美沙坦以剂量依赖的方式抑制2-E蛋白诱导的HK-2细胞上皮间充质转化。(EMT)(所有值均为平均值±SD;单向方差分析。与对照组相比,###P<0.001,与2-E组相比,***P<0.001,n=5)
图7:奥美沙坦对包膜蛋白造模后HK-2的TGF-β1的分泌以及TGF-β1/Smad2/3信号通路的影响。(A)HK-2细胞培养上清中TGF-β1的水平;(B)TGF-β1在HK-2细胞中的mRNA表达水平;(C)TGF-β1/Smad2/3通路的激活情况和(D)相对定量。(所有值均为平均值±SD;单向方差分析。与对照组相比,###P<0.001,nsP>0.05,与2-E组相比,***P<0.001,nsP>0.05,n=6)
图8:奥美沙坦增强自噬缓解纤维化。(A)各组肾脏中TGF-β1的自噬降解及(B)相对定量(n=6);(C)各组细胞中TGF-β1的自噬降解及(E)相对定量(n=5);(D)用GFP-LC3B慢病毒转染的HK-2细胞在不同处理(比例尺=20μm)中自噬小体和TGF-β1表达和分布的代表性免疫荧光图像。(所有值均为平均值±SD;单向方差分析。与对照组相比,nsP>0.05,##P<0.01,###P<0.001,与2-E组相比,*P<0.05,**P<0.01,***P<0.001)
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。本发明所用试剂和原料均市售可得。
本发明进行了动物实验和细胞实验,对构建的肾纤维化动物模型和细胞模型给予奥美沙坦干预,利用组织病理染色、蛋白免疫印迹、实时荧光定量PCR和免疫荧光等实验方法进行检测。本发明的实施例中,动物实验结果显示,奥美沙坦能够减缓2-E蛋白造成的肾纤维化症状。细胞实验结果显示,奥美沙坦能够促进TGF-β1的自噬降解,从而减缓上皮间充质转化。
在本发明实施例中,具体进行了以下步骤的干预实验:
(1)小鼠尾静脉注射纯化2-E蛋白;(2)组织形态学分析;(3)离体细胞研究;(4)蛋白免疫印迹实验;(5)实时荧光定量PCR实验;(6)免疫荧光实验;(7)数据分析:实验结果以Mean±SD表示,采用单因素方差分析进行统计学比较,P<0.05认为有显著性差异。
实施例1奥美沙坦改善SARS-CoV-2包膜蛋白诱导的小鼠肾纤维化
1、动物
C57BL/6小鼠,雄性,8周,购于上海灵畅实验动物有限公司,饲养于复旦大学药学院实验动物中心SPF级动物实验室。
2、实验材料
肌酐测定试剂盒(南京建成生物工程研究所),尿素氮测定试剂盒(南京建成生物工程研究所),尿蛋白定量测试盒(南京建成生物工程研究所),多聚甲醛(国药集团化学试剂有限公司),DMEM/F-12培养基(BIOAGRIO),胎牛血清(BIOAGRIO),胰蛋白酶(BIOAGRIO),青霉素/链霉素(BIOAGRIO),RIPA裂解液(碧云天生物技术公司),BCA法蛋白定量试剂盒(碧云天生物技术公司),5×loading buffer(上海翊圣生物科技有限公司),DMSO(Sigma),化学发光超敏Western ECL(Bio-Rad),预染蛋白marker(ThermoFisher),HRP标记山羊抗兔IgG(H+L)(上海翊圣生物科技有限公司),Trizol(TAKAR),PrimeScriptTM RT Master Mix(perfect Real Time;TAKAR),SYBR Premix Ex TaqTM(Takara)。抗体:α-SMA(Abcam)、纤维连接蛋白(FN;Abcam)、GAPDH(CST)、p-Smad23(CST)、Smad23(CST)、vimentin(CST)、LC3A/B(CST)、P62(ABclonal Technology)和TGF-β1(ABclonal Technology)。
3、实验仪器
生物安全柜(Thermo Fisher),CO2细胞培养箱(Thermo Fisher)、冷冻离心机(Thermo Fisher)、微量移液器(Eppendorf)、恒温水浴锅(上海精宏实验设备有限公司)、凝胶电泳装置(Bio-Rad)、转印装置(Bio-Rad)、凝胶图像处理系统
(Bio-Rad)、倒置显微镜(Carl Zeiss)、Q-RTPCR仪(Bio-Rad,美国)、组织匀浆仪(上海净信科技)、酶标仪(Tecan Systems Inc.)。
4、实验方法
(1)SARS-CoV-2包膜蛋白诱导的肾纤维化小鼠模型的建立和给药治疗
将24只8周龄的雄性C57BL/6小鼠随机分为4组:正常对照组(Mock group):小鼠尾静脉注射与纯化蛋白等体积的TBS(纯化蛋白洗脱液)、模型组(2-E group):小鼠经尾静脉注射2-E蛋白(25mg·kg-1体重);预防组(Prophylaxis group):在注射2-E蛋白之前,通过灌胃给予小鼠奥美沙坦(10mg·kg-1·d-1体重)三天。2-E注射后三天,继续灌胃给予奥美沙坦(10mg·kg-1·d-1体重)以模拟临床给药;治疗组(Treatment group):小鼠在2-E蛋白注射后三天通过灌胃给予奥美沙坦(10mg·kg-1·d-1体重)。
(1)肾功能检测
给药期间,收集尿液样本,通过蛋白尿来评估肾功能,蛋白尿通过尿蛋白与肌酐的比率来量化。实验结束时,收集血液样本,通过血清肌酐(SCr)和尿素氮(BUN)来评估肾功能。
(2)肾病理检测
肾脏组织用4%多聚甲醛固定,石蜡包埋,并进行HE染色和Masson三色染色,检测小鼠肾病理变化。
(3)蛋白和基因水平检测纤维化相关蛋白表达水平和mRNA水平
应用Western-blotting和RT-PCR检测对照组、2-E组、治疗组和预防组中肾纤维化标志物包括纤维连接蛋白(FN)、波形蛋白和α-平滑肌肌动蛋白(α-SMA)的蛋白和mRNA表达变化。
(4)蛋白水平检测小鼠血清TGF-β1的分泌以及肾脏TGF-β1/Smad2/3通路
应用ELISA检测小鼠血清中TGF-β1的含量;应用Western-blotting检测小鼠肾脏TGF-β1的蛋白表达水平以及Smad2/3的磷酸化水平。
(5)蛋白水平检测自噬相关蛋白以及TGF-β1表达水平
应用Western-blotting检测对照组、2-E组、治疗组和预防组中LC3、P62和TGF-β1的蛋白表达变化
实验结果如图1-5以及图8所示:奥美沙坦有效减缓了治疗组和预防组2-E蛋白造成的肾功能损伤,调节TGF-β1分泌并抑制TGF-β1/Smad2/3通路激活,降低纤维化相关蛋白的表达,减轻纤维化症状。
实施例2奥美沙坦缓解SARS-CoV-2包膜蛋白诱导的肾上皮间充质转化细胞医学模型的损伤
实验方法如下:
(1)SARS-CoV-2包膜蛋白诱导的肾上皮间充质转化细胞模型构建和奥美沙坦干预
将生长状态良好的HK-2细胞(人肾近端小管上皮细胞)在RPMI1640培养基(ThermoFisher Scientific)中培养,培养基中含有10%血清(Gibco)和100U·mL-1青霉素/链霉素(Gibco)。
将5×105HK-2细胞接种在具有完整培养基的6孔板中12小时。然后,在无血清的培养基中饥饿12小时后,模型组(2-E)将包膜蛋白(2μg/mL)加入HK-2细胞培养基中继续培养24小时;给药组在加入包膜蛋白(2μg/mL)的同时加入20μM奥美沙坦,处理24小时;对照组不做处理。
(2)蛋白水平检测上皮间充质转化相关蛋白表达水平
应用Western-blotting检测3组细胞纤维连接蛋白(FN)、波形蛋白和α-平滑肌肌动蛋白(α-SMA)的表达变化。
(3)蛋白水平检测HK-2细胞TGF-β1的分泌以及肾脏TGF-β1/Smad2/3通路
应用ELISA检测HK-2细胞培养上清中TGF-β1的含量;应用Western-blotting检测HK-2细胞中TGF-β1的蛋白表达水平以及Smad2/3的磷酸化水平。
(4)蛋白水平检测自噬相关蛋白以及TGF-β1表达水平
应用Western-blotting检测HK-2细胞中LC3、P62和TGF-β1的蛋白表达变化
实验结果如图6-图8,奥美沙坦显著降低了上皮间充质转化相关蛋白表达。奥美沙坦的上述药效的作用机制是促进TGF-β1的自噬降解,从而降低了TGF-β1分泌并抑制TGF-β1/Smad2/3通路激活。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (4)
1.奥美沙坦用于制备治疗SARS-CoV-2包膜蛋白诱导肾纤维化的药物。
2.根据权利要求1所述的药物,奥美沙坦为治疗SARS-CoV-2包膜蛋白诱导肾纤维化药物中的主要活性成分。
3.根据权利要求2所述的药物,其特征在于,所述药物还包括药学上可接受的赋形剂,所述赋形剂选自粘合剂、填料、增塑剂、助流剂、崩解剂和润滑剂中的一种或者任意组合。
4.根据权利要求3所述的药物,其特征在于,所述药物还可为口服剂或注射剂。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310191777.XA CN116251097A (zh) | 2023-03-02 | 2023-03-02 | 奥美沙坦在治疗SARS-CoV-2包膜蛋白诱导的肾纤维化中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310191777.XA CN116251097A (zh) | 2023-03-02 | 2023-03-02 | 奥美沙坦在治疗SARS-CoV-2包膜蛋白诱导的肾纤维化中的应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116251097A true CN116251097A (zh) | 2023-06-13 |
Family
ID=86682239
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310191777.XA Pending CN116251097A (zh) | 2023-03-02 | 2023-03-02 | 奥美沙坦在治疗SARS-CoV-2包膜蛋白诱导的肾纤维化中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116251097A (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116158403A (zh) * | 2023-03-02 | 2023-05-26 | 复旦大学 | SARS-CoV-2包膜蛋白诱导的肾纤维化模型的建立 |
-
2023
- 2023-03-02 CN CN202310191777.XA patent/CN116251097A/zh active Pending
Non-Patent Citations (2)
Title |
---|
SANG HEON SUH等: "Olmesartan Attenuates Kidney Fibrosis in a Murine Model of Alport Syndrome by Suppressing Tubular Expression of TGF", 《INT. J. MOL. SCI.》, vol. 20, no. 15, pages 1 - 15 * |
向启蒙: "奥美沙坦对高尿酸诱导的HK-2细胞上皮间质转化和细胞外基质合成的影响及机制", 南昌大学学报(医学版)., vol. 62, no. 2, pages 1 - 13 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116158403A (zh) * | 2023-03-02 | 2023-05-26 | 复旦大学 | SARS-CoV-2包膜蛋白诱导的肾纤维化模型的建立 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2018288021B2 (en) | Use of cannabidiol in the treatment of Tuberous Sclerosis complex | |
EA024147B1 (ru) | Комбинированные композиции для лечения болезни альцгеймера и родственных заболеваний зонизамидом и акампросатом | |
EA015560B1 (ru) | Способ лечения вирусного гепатита | |
JP6151123B2 (ja) | ネコの全身性疾患の予防又は治療の為のアンジオテンシンii受容体アンタゴニスト | |
CN102105153A (zh) | 使用sglt2抑制剂和含其组合物治疗高尿酸血症的方法 | |
CN116251097A (zh) | 奥美沙坦在治疗SARS-CoV-2包膜蛋白诱导的肾纤维化中的应用 | |
Boyd et al. | Vasopressin decreases sepsis-induced pulmonary inflammation through the V2R | |
JP2011529078A (ja) | 自己免疫疾患を治療および予防するための方法および組成物 | |
KR102373721B1 (ko) | 다발성 골수종 치료 | |
Li et al. | Tiliroside is a new potential therapeutic drug for osteoporosis in mice | |
Fang et al. | Protective effect of irbesartan by inhibiting ANGPTL2 expression in diabetic kidney disease | |
Dai et al. | Astragaloside IV alleviates sepsis-induced muscle atrophy by inhibiting the TGF-β1/Smad signaling pathway | |
CN112107575B (zh) | 木脂素化合物联合呋喃香豆素化合物治疗肝纤维化 | |
CN114712379B (zh) | 黄芪甲苷iv在制备预防和治疗腹膜透析肠道并发症药物中的应用 | |
CN108948158B (zh) | 四连接素模拟肽tnp及其应用 | |
Angelova et al. | Effects of partial liquid ventilation on lipopolysaccharide-induced inflammatory responses in rats | |
SE1851333A1 (en) | Treatment of osteoarthritis | |
CN114306350B (zh) | 胆固醇硫酸盐在制备预防脓毒症的药物中的用途 | |
CN114469940B (zh) | 小分子化合物aq-390在制备抵抗细胞焦亡药物及抑制剂的应用 | |
EP4389144A1 (en) | Regenerative agent for bone tissue and lung tissue, and pharmaceutical composition for regenerating bone tissue and lung tissue | |
US20240082229A1 (en) | Methods for preventing or treating h. pylori infection | |
WO2024135719A1 (ja) | PGAM-Chk1結合阻害剤を含む肺疾患、肝疾患又は腎疾患の治療剤 | |
CN108135886A (zh) | 用于糖尿病性肾病治疗的pde4抑制剂 | |
CN107496441B (zh) | 环糊精在治疗和/预防脓毒症中的应用 | |
US20210393734A1 (en) | Therapeutic agent for arthritis |
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 |