CN114522157A - Application of calcium ion chelating agent in preparation of preparation for improving phagocytic capacity of vascular endothelial cells - Google Patents
Application of calcium ion chelating agent in preparation of preparation for improving phagocytic capacity of vascular endothelial cells Download PDFInfo
- Publication number
- CN114522157A CN114522157A CN202210167310.7A CN202210167310A CN114522157A CN 114522157 A CN114522157 A CN 114522157A CN 202210167310 A CN202210167310 A CN 202210167310A CN 114522157 A CN114522157 A CN 114522157A
- Authority
- CN
- China
- Prior art keywords
- calcium ion
- derivatives
- chelating agent
- ion chelating
- endothelial cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 title claims abstract description 36
- 229910001424 calcium ion Inorganic materials 0.000 title claims abstract description 36
- 239000002738 chelating agent Substances 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 210000003556 vascular endothelial cell Anatomy 0.000 title claims abstract description 12
- 230000000242 pagocytic effect Effects 0.000 title claims abstract description 7
- 210000002889 endothelial cell Anatomy 0.000 claims abstract description 36
- 239000003814 drug Substances 0.000 claims abstract description 29
- 229940079593 drug Drugs 0.000 claims abstract description 23
- 201000001320 Atherosclerosis Diseases 0.000 claims description 10
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 8
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 claims description 8
- 239000002245 particle Substances 0.000 claims description 8
- FTEDXVNDVHYDQW-UHFFFAOYSA-N BAPTA Chemical compound OC(=O)CN(CC(O)=O)C1=CC=CC=C1OCCOC1=CC=CC=C1N(CC(O)=O)CC(O)=O FTEDXVNDVHYDQW-UHFFFAOYSA-N 0.000 claims description 6
- 230000001737 promoting effect Effects 0.000 claims description 5
- 210000001808 exosome Anatomy 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 238000009472 formulation Methods 0.000 claims 3
- 239000000203 mixture Substances 0.000 claims 3
- FYIHPNCKLYPALH-UHFFFAOYSA-N 2-[2-(2-aminophenoxy)ethenoxy]aniline Chemical compound NC1=CC=CC=C1OC=COC1=CC=CC=C1N FYIHPNCKLYPALH-UHFFFAOYSA-N 0.000 claims 2
- BDDLHHRCDSJVKV-UHFFFAOYSA-N 7028-40-2 Chemical compound CC(O)=O.CC(O)=O.CC(O)=O.CC(O)=O BDDLHHRCDSJVKV-UHFFFAOYSA-N 0.000 claims 2
- 238000004519 manufacturing process Methods 0.000 claims 2
- 238000010521 absorption reaction Methods 0.000 claims 1
- 239000003352 sequestering agent Substances 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 22
- 206010057249 Phagocytosis Diseases 0.000 abstract description 19
- 230000008782 phagocytosis Effects 0.000 abstract description 19
- 239000012528 membrane Substances 0.000 abstract description 12
- 238000002474 experimental method Methods 0.000 abstract description 5
- 208000024172 Cardiovascular disease Diseases 0.000 abstract description 4
- 241001465754 Metazoa Species 0.000 abstract description 3
- 208000026106 cerebrovascular disease Diseases 0.000 abstract description 3
- 230000002526 effect on cardiovascular system Effects 0.000 abstract description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 12
- 239000001110 calcium chloride Substances 0.000 description 12
- 229910001628 calcium chloride Inorganic materials 0.000 description 12
- 239000011575 calcium Substances 0.000 description 10
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 229910052791 calcium Inorganic materials 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 230000010287 polarization Effects 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 230000017531 blood circulation Effects 0.000 description 4
- 239000003715 calcium chelating agent Substances 0.000 description 4
- 229960001484 edetic acid Drugs 0.000 description 4
- 230000003511 endothelial effect Effects 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 210000003462 vein Anatomy 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004204 blood vessel Anatomy 0.000 description 3
- 210000001715 carotid artery Anatomy 0.000 description 3
- 210000001168 carotid artery common Anatomy 0.000 description 3
- 210000000269 carotid artery external Anatomy 0.000 description 3
- 210000004004 carotid artery internal Anatomy 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 210000003437 trachea Anatomy 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 210000001367 artery Anatomy 0.000 description 2
- 230000003143 atherosclerotic effect Effects 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 230000002792 vascular Effects 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- YJIYWYAMZFVECX-UHFFFAOYSA-N 2-[N-[2-(acetyloxymethoxy)-2-oxoethyl]-2-[2-[2-[bis[2-(acetyloxymethoxy)-2-oxoethyl]amino]phenoxy]ethoxy]anilino]acetic acid acetyloxymethyl ester Chemical compound CC(=O)OCOC(=O)CN(CC(=O)OCOC(C)=O)C1=CC=CC=C1OCCOC1=CC=CC=C1N(CC(=O)OCOC(C)=O)CC(=O)OCOC(C)=O YJIYWYAMZFVECX-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- 231100000070 MTS assay Toxicity 0.000 description 1
- 238000000719 MTS assay Methods 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000006793 arrhythmia Effects 0.000 description 1
- 206010003119 arrhythmia Diseases 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000007541 cellular toxicity Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 210000003038 endothelium Anatomy 0.000 description 1
- 210000003989 endothelium vascular Anatomy 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 210000003195 fascia Anatomy 0.000 description 1
- 238000002875 fluorescence polarization Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000036543 hypotension Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 210000001186 vagus nerve Anatomy 0.000 description 1
- 238000005406 washing Methods 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
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
- A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- 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)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Urology & Nephrology (AREA)
- Vascular Medicine (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention belongs to the technical field of medicines for treating cardiovascular and cerebrovascular diseases, and particularly relates to application of a calcium ion chelating agent in preparation of a preparation for improving phagocytic capacity of vascular endothelial cells. According to the embodiment of the invention, compared with the prior art, the calcium ion chelating agent is proved to reduce the viscosity of the endothelial cell membrane and improve the fluidity of the endothelial cell membrane through the cell and animal experiment level, so that the phagocytosis of the endothelial cell membrane on the nano-medicament is promoted, and the utilization rate of the nano-medicament can be obviously improved.
Description
Technical Field
The invention belongs to the technical field of medicines for treating cardiovascular and cerebrovascular diseases, and particularly relates to application of a calcium ion chelating agent in preparation of a preparation for improving phagocytic capacity of vascular endothelial cells.
Background
Cardiovascular and cerebrovascular diseases seriously endanger human health, and Atherosclerosis (AS) is considered AS the main pathological basis of cardiovascular diseases. Numerous studies have shown that atherosclerotic lesions may well originate in certain areas of vascular blood flow disturbances, such as areas of narrowing and bending of blood vessel branches, which may form reverse flow, turbulence, etc. In the vascular lumen, endothelial cells are in direct contact with the blood stream and have the ability to sense changes in the shear stress of the blood stream. The disturbed flow region at the atherosclerotic lesion causes cell debris, nano-materials, bacteria, etc. to accumulate in the region near the inner wall of the blood vessel due to flow field changes, and the vascular endothelial cells can phagocytose these materials by phagocytosis independent of specific protein receptors. At present, the development of endothelial phagocytosis and atherosclerosis is still under investigation.
At present, no relevant literature reports substances relevant to promotion of phagocytosis of vascular endothelial cells or treatment of atherosclerosis by promotion of phagocytosis of vascular endothelial cells.
Disclosure of Invention
In view of the above, the present invention aims to provide an application of a calcium ion chelating agent in preparation of an agent for improving phagocytic ability of vascular endothelial cells.
Further, the calcium ion chelating agent is selected from one or more of ethylene diamine tetraacetic acid and derivatives thereof, ethylene glycol bis (2-amino ethyl ether) tetraacetic acid and derivatives thereof, and 1,2 bis (2-amino phenoxy) ethane N, N, N 'N' tetraacetic acid and derivatives thereof.
Further, the calcium ion chelating agent can also play the same role in endothelial cells in other calcium environments.
The invention also aims to provide the application of the calcium ion chelating agent in preparing a preparation for treating atherosclerosis.
Further, the calcium ion chelating agent is selected from one or more of ethylene diamine tetraacetic acid and derivatives thereof, ethylene glycol bis (2-amino ethyl ether) tetraacetic acid and derivatives thereof, and 1,2 bis (2-amino phenoxy) ethane N, N, N 'N' tetraacetic acid and derivatives thereof.
In particular, when nano-drugs enter blood vessels and are administered through endothelium, endothelial cell phagocytosis is a new mechanism for promoting atherosclerosis by abnormal shear stress. Therefore, a molecular mechanism influencing the formation and development of atherosclerosis by phagocytosis of endothelial cells under the condition of low oscillation flow is deeply discussed, and a new target and a clinical strategy are provided for the effective prevention and treatment of atherosclerosis. The calcium ion chelating agent is used for partially removing or completely removing calcium around or in the endothelial cells, so that the phagocytic capacity of the endothelial cells on the nano-drugs is effectively improved.
The invention also aims to provide application of the calcium ion chelating agent in preparing a preparation for promoting vascular endothelial cells to absorb medicaments.
Further, the calcium ion chelating agent is selected from one or more of ethylene diamine tetraacetic acid and derivatives thereof, ethylene glycol bis (2-amino ethyl ether) tetraacetic acid and derivatives thereof, and 1,2 bis (2-amino phenoxy) ethane N, N, N 'N' tetraacetic acid and derivatives thereof.
Further, the medicine comprises one or more of extracellular vesicles, exosomes, artificial vesicles, organic nano-medicine particles and inorganic nano-medicine particles.
Specifically, after the calcium ion chelating agent rapidly chelates calcium ions of vascular endothelium, the phagocytosis of endothelial cells is increased rapidly by the nano-drug or the bio-vesicle drug.
In certain embodiments, the concentration of the calcium ion chelating agent is selected to be 0.5-1.5mM, preferably 1 mM. More preferably, the mass ratio of the calcium ion chelating agent to the drug is 0.5-1: 0.5-1.
In certain embodiments, the calcium chelating agent is administered to C57BL/6 mice by tail vein injection, and it was found that when the calcium chelating agent depletes the calcium ions in the endothelial cells, i.e. in a calcium-deficient environment, the phagocytosis of the endothelial cells to the RBCEVs is enhanced, and it is clear that the calcium chelating agent can increase the fluidity of the endothelial cell membranes by reducing the intracellular calcium, thereby promoting the endothelial cells to phagocytize the RBCEVs and further improving the availability of the RBCEVs to the RBCEVs.
The invention also provides a method for improving the utilization rate of an endothelial drug, which is characterized in that the endothelial drug is combined with a calcium ion chelating agent, and the endothelial drug comprises the following components: one or more of extracellular vesicles, exosomes, artificial vesicles, organic nano-drug particles or inorganic nano-drug particles.
Further, the calcium ion chelating agent is selected from one or more of ethylene diamine tetraacetic acid and derivatives thereof, ethylene glycol bis (2-aminoethylether) tetraacetic acid and derivatives thereof, and 1,2 bis (2-aminophenoxy) ethane N, N, N 'N' tetraacetic acid and derivatives thereof.
The invention has the beneficial effects that
Compared with the prior art, the embodiment of the invention proves that the calcium ion chelating agent can reduce the viscosity of the endothelial cell membrane and improve the fluidity of the endothelial cell membrane through the level of cell and animal experiments, thereby promoting the phagocytosis of the nano-drug, namely obviously improving the utilization rate of the nano-drug.
Drawings
FIG. 1 shows the effect of calcium chelators and calcium chloride, respectively, on phagocytosis of RBCEVs by endothelial cells, en face staining (scale bar 20 μm).
Fig. 2 is a statistical plot of the effect of calcium chelators and calcium chloride, respectively, on endothelial cell phagocytosis of RBCEVs (n is 5, P <0.05 or P < 0.001).
Fig. 3 shows en face staining of mice injected with calcium chloride and calcium chelator into tail vein of mice on RBCEVs phagocytosis by left carotid LCA and right carotid RCA endothelial cells (scale bar 10 μm).
Fig. 4 is a statistical plot of the effect of the injection of calcium chloride and calcium chelator into the tail vein of mice on left carotid LCA and right carotid RCA endothelial cell phagocytosis of RBCEVs, respectively (n is 5, P is 0.01 or P is 0.001).
Fig. 5 shows the different cell viscosities under calcium ion treatment ((n ═ 3,. times.p <0.05 or. times.p <0.01,. times.p < 0.001).
Figure 6 shows the different cell viscosities under treatment with calcium chelators (n 3, P <0.05 or P <0.01, P < 0.001).
Figure 7 is a graph of the effect of different concentrations of BAPTAAM on endothelial cell survival (n-3).
Detailed Description
The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
Example 1 cell experiments
In the embodiment of the invention, the cell culture medium is a common culture medium, and no calcium ion or calcium ion chelating agent is additionally added.
In the embodiment of the invention, endothelial cells are inoculated in a cell pore plate, after the cells grow full, the cells are pretreated by a calcium ion chelating agent for 30 minutes, then the cells are washed by a fresh cell culture medium for at least 3 times, and when the cells are treated by BAPTA-AM, the cells are firstly dissolved in 10% DMSO and then dissolved in 90% normal saline (containing 20% SBE-beta-CD); the concentration of calcium chloride-treated cells was 10. mu.g/mL. Results as shown in fig. 1 and 2, endothelial cells were treated with calcium chloride, which inhibits endothelial cell phagocytosis of erythrocyte extracellular vesicles RBCEVs, and with a calcium ion chelator, BAPTAAM, which promotes endothelial cell phagocytosis of erythrocyte extracellular vesicles, respectively.
Example 2 animal experiments
In the present example, when injecting BAPTAAM into mice, it is dissolved in 10% DMSO and then dissolved in 90% physiological saline (containing 20% SBE beta CD).
In the embodiment of the invention, the concentration of the calcium chloride injection injected into a mouse is 10 mug/mL, the calcium chloride injection needs to be taken slowly during injection, otherwise, hypotension or arrhythmia of the mouse is easily caused.
In the embodiment of the invention, 15 male C57BL/6 mice with the weight of 15-20g and the age of 6 weeks are selected and subjected to carotid artery ligation operation to construct a mouse OSS carotid artery ligation model. The ligation is specifically performed as follows:
(1) anaesthetizing the mouse, fixing the mouse on an operating table in a supine position, cutting the skin from the position right above the trachea, and separating the muscles on the left side and the right side of the trachea respectively; first, forceps are used for peeling off muscles along the left trachea layer by layer to find a left carotid sheath, and then the forceps are used for carefully separating fascia close to the left carotid and vagus nerve to separate a left common carotid artery (LCA); dissection was continued up the common carotid artery, and the External Carotid Artery (ECA), Internal Carotid Artery (ICA), Occipital Artery (OA) and suprathyroid artery (STA) were found.
(2) Three branch vessels (ECA, ICA and OA) except STA were tied with 90-grade medical sutures and the blood flow was secured to STA.
(3) A right common carotid artery (RCA) was isolated and found 4 branch vessels, but not ligated, as a control group.
(4) The mouse was sutured layer by layer with 50 sutures, the wound was wiped with iodophor after suturing, and the mouse was placed on a 37 ℃ thermostatic board and transferred to the corresponding mouse cage after waiting for awakening.
In the embodiment of the invention, in the constructed OSS model, the blood flow of LCA is OSS, and the blood flow at RCA is NSS. From the results of the en face staining and data statistics shown in fig. 3 and 4, it can be seen that the LCA group had a clear red fluorescence signal of RBCEVs co-localized with endothelial cells, compared to the RCA group; when calcium chloride was injected intravenously through the tail of mice (LCA + Ca)2+),LCA+Ca2+The RBCEVs red fluorescence signal of the group was significantly lower than that of the LCA group, i.e., calcium chloride inhibited endothelial cell phagocytosis of the LCA group; in contrast, when BAPTAAM was injected (LCA + BAPTAAM), the RBCEVs red fluorescence signal was significantly stronger than the LCA group. Namely, the tail vein of the mouse is injected with calcium chloride and BAPTAAM which is a calcium chelating agent respectively, and the BAPTAAM obviously promotes the phagocytosis of the vascular endothelial cells in the area of the right carotid artery ligation OSS to the erythrocyte extracellular vesicle RBCEVs.
The results are consistent with the results of the cells of example 1, BAPTAAM inhibits calcium ion concentration in blood, promotes endothelial cells to phagocytose RBCEVs, and increases the availability of RBCEVs.
EXAMPLE 3 detection of endothelial cell Membrane fluidity by DPH Probe
The method for detecting the fluidity of the endothelial cell membrane by using the DPH probe comprises the following specific steps:
(1) preparing a working solution: and (3) diluting the DPH probe by using a diluent in the kit according to an instruction, wherein the dilution range can be up to 10000 times according to the experiment requirement, and preparing the DPH dyeing working solution.
(2) Endothelial cells are inoculated to a 12-well plate, staining treatment can be carried out after the cells are overgrown under a microscope, a culture medium is sucked away by a pipette, a staining working solution is added, and the cells are incubated in a cell incubator for at least 0.5 hour.
(3) The cells were resuspended after washing 1 time with pH 7.4HBSS or 20mM HEPES or PBS.
(4) The cells were detected using a spectrophotometer with a bias light. The setting parameters are as follows: the excitation wavelength is 355nm, and the emission wavelength is 430 nm.
The fluorescence polarization degree P is calculated according to the following formula: p ═ (IVV-GIVHIHV)/(IVV + GIVH), where the correction factor G ═ IHV/IHH, where: IVV fluorescence intensity measured when the optical axes of the polarization and analyzer are vertical; IVH, measuring fluorescence intensity when optical axes of the polarization analyzer and the polarization analyzer are respectively in vertical and horizontal directions; IHV, measured fluorescence intensity when the optical axes of the polarization analyzer and the polarization analyzer are respectively in the horizontal direction and the vertical direction; IHH fluorescence intensity measured when the optical axes of the polarization and analyzer are in the same horizontal direction; p reflects the viscosity (. eta.) of the membrane lipid domain, whereas η is inversely proportional to membrane fluidity.
As shown in FIGS. 5 and 6, the DPH probe detects BAPTAAM to reduce the viscosity of endothelial cell membrane, improve the fluidity of endothelial cell membrane, and promote the phagocytosis of RBCEVs, and the opposite is true to calcium chloride.
Example 4 cytotoxicity assay
The effect of different concentrations of BAPTAAM on endothelial cell viability was studied by MTS assay, cells were tested at BAPTAAM recommended concentrations and times (1mM, 30 min) and the following experimental steps were performed:
(1) HUVEC cells were cultured to logarithmic growth phase.
(2) Taking 96-well cell culture plate, adding 0.1ml of 1 × 10-contained solution per well4~2×1041640 medium (10% calf serum) of one of the above cells.
(3) Adding 20 mu L MTS into each well, and continuing culturing for 3-4 hours for color development.
(4) The plates were shaken for 10 seconds before detection and the color was mixed. The absorbance (OD) of each well was measured at a wavelength of 570nm (or 490nm, or 570nm and 690nm) on an enzyme-linked detector. Dose-response curves were plotted with sample dilution versus OD value (OD570, OD490, or OD570/OD690), and the amount of cytokine in the sample was calculated from the standard curve.
The results are shown in figure 7, according to BAPTAAM suggested concentration and time (1mM, 30 minutes) on cell toxicity, has better safety.
Finally, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. Use of a calcium ion chelator for the preparation of a formulation for increasing phagocytic capacity of vascular endothelial cells.
2. The use according to claim 1, wherein the calcium ion chelating agent is selected from one or more of ethylenediaminetetraacetic acid and derivatives thereof, ethylene glycol bis (2-aminoethylether) tetraacetic acid and derivatives thereof, 1,2 bis (2-aminophenoxy) ethane N, N' tetraacetic acid and derivatives thereof.
3. Use of a calcium ion chelating agent in the manufacture of a formulation for the treatment of atherosclerosis.
4. The use according to claim 3, wherein the calcium ion chelating agent is selected from one or more of ethylenediaminetetraacetic acid and derivatives thereof, ethylene glycol bis (2-aminoethylether) tetraacetic acid and derivatives thereof, 1,2 bis (2-aminophenoxy) ethane N, N, N 'N' tetraacetic acid and derivatives thereof.
5. Use of a calcium ion chelating agent in the manufacture of a formulation for promoting the absorption of a drug by endothelial cells.
6. The use according to claim 5, wherein the calcium ion chelating agent is selected from one or more of ethylenediaminetetraacetic acid and derivatives thereof, ethylene glycol bis (2-aminoethylether) tetraacetic acid and derivatives thereof, 1,2 bis (2-aminophenoxy) ethane N, N, N 'N' tetraacetic acid and derivatives thereof.
7. The use of claim 5, wherein the drug comprises one or more of extracellular vesicles, exosomes, artificial vesicles, organic nano-drug particles, inorganic nano-drug particles in admixture.
8. The use of any one of claims 5-7, wherein said endothelial cells comprise vascular endothelial cells.
9. A method of increasing the availability of an endothelially administered drug wherein said endothelially administered drug is combined with a calcium ion sequestering agent and wherein said endothelially administered drug comprises: one or more of extracellular vesicles, exosomes, artificial vesicles, organic nano-drug particles or inorganic nano-drug particles.
10. The method of claim 9, wherein the calcium ion chelating agent is selected from one or more of ethylenediaminetetraacetic acid and derivatives thereof, ethylene glycol bis (2-aminoethylether) tetraacetic acid and derivatives thereof, 1,2 bis (2-aminophenoxy) ethane N, N' tetraacetic acid and derivatives thereof.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210167310.7A CN114522157B (en) | 2022-02-23 | 2022-02-23 | Application of calcium ion chelating agent in preparation of preparation for improving phagocytic capacity of vascular endothelial cells |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210167310.7A CN114522157B (en) | 2022-02-23 | 2022-02-23 | Application of calcium ion chelating agent in preparation of preparation for improving phagocytic capacity of vascular endothelial cells |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114522157A true CN114522157A (en) | 2022-05-24 |
CN114522157B CN114522157B (en) | 2023-08-15 |
Family
ID=81625351
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210167310.7A Active CN114522157B (en) | 2022-02-23 | 2022-02-23 | Application of calcium ion chelating agent in preparation of preparation for improving phagocytic capacity of vascular endothelial cells |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114522157B (en) |
Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH01229048A (en) * | 1988-03-09 | 1989-09-12 | Kibun Kk | Production of gel |
US6015834A (en) * | 1992-10-20 | 2000-01-18 | Toronto Neuroprotection Group | In vivo treatment of mammalian cells with a cell membrane permeant calcium buffer |
US20060121128A1 (en) * | 2003-01-17 | 2006-06-08 | Erik Schwiebert | Methods and compositions for p2x receptor calcium entry channels and other calcium entry mechanisms |
CN103397017A (en) * | 2013-08-16 | 2013-11-20 | 泸州医学院 | Formation method of inside-out patch in patch clamp experiment |
CN105477637A (en) * | 2015-12-16 | 2016-04-13 | 孙荣距 | Application of Orail gene silencing to vascular endothelial cell calcium overload injury |
US20160354411A1 (en) * | 2015-06-05 | 2016-12-08 | Darlene E. McCord | Olivamine-induced improvement in endothelial cells viability and function |
CN107530333A (en) * | 2015-02-27 | 2018-01-02 | 钙医学公司 | Pancreatitis treatment |
CN111184700A (en) * | 2019-04-10 | 2020-05-22 | 重庆大学 | Erythrocyte membrane encapsulated rapamycin nanoparticle, preparation method and application thereof |
EP3795178A1 (en) * | 2019-09-19 | 2021-03-24 | Nh Theraguix | Methods for triggering m1 macrophage polarization |
-
2022
- 2022-02-23 CN CN202210167310.7A patent/CN114522157B/en active Active
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH01229048A (en) * | 1988-03-09 | 1989-09-12 | Kibun Kk | Production of gel |
US6015834A (en) * | 1992-10-20 | 2000-01-18 | Toronto Neuroprotection Group | In vivo treatment of mammalian cells with a cell membrane permeant calcium buffer |
US20060121128A1 (en) * | 2003-01-17 | 2006-06-08 | Erik Schwiebert | Methods and compositions for p2x receptor calcium entry channels and other calcium entry mechanisms |
CN103397017A (en) * | 2013-08-16 | 2013-11-20 | 泸州医学院 | Formation method of inside-out patch in patch clamp experiment |
CN107530333A (en) * | 2015-02-27 | 2018-01-02 | 钙医学公司 | Pancreatitis treatment |
US20160354411A1 (en) * | 2015-06-05 | 2016-12-08 | Darlene E. McCord | Olivamine-induced improvement in endothelial cells viability and function |
CN105477637A (en) * | 2015-12-16 | 2016-04-13 | 孙荣距 | Application of Orail gene silencing to vascular endothelial cell calcium overload injury |
CN111184700A (en) * | 2019-04-10 | 2020-05-22 | 重庆大学 | Erythrocyte membrane encapsulated rapamycin nanoparticle, preparation method and application thereof |
EP3795178A1 (en) * | 2019-09-19 | 2021-03-24 | Nh Theraguix | Methods for triggering m1 macrophage polarization |
Non-Patent Citations (5)
Title |
---|
YA-TING CHEN,等: "Microparticles (Exosomes) and Atherosclerosis", CURRENTS ATHEROSCLEROSIS, vol. 22, no. 6, pages 1 - 9, XP037157625, DOI: 10.1007/s11883-020-00841-z * |
ZAILIN FU,等: "A Promising Therapeutic Agent for Acute Liver Failure", ACS APPLLIED MATERIALS & INTERFACES, vol. 11, no. 43, pages 39574 - 39585 * |
宋必卫,等: "BAPTA-AM的研究现状", 中国药理学通报, vol. 25, no. 7, pages 851 - 853 * |
张林,等: "芍药苷、薄荷脑对葛根素在Calu-3细胞模型转运过程中膜流动性、钠-钾离子ATP酶和钙离子ATP酶的影响", 中国中药杂志, vol. 43, no. 4, pages 731 - 735 * |
覃弦,等: "氧化应激介导低切和扰动流促内皮细胞吞噬胞外囊泡", 医用生物力学, vol. 36, no. 1, pages 394 * |
Also Published As
Publication number | Publication date |
---|---|
CN114522157B (en) | 2023-08-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Sun et al. | Bone microenvironment regulative hydrogels with ROS scavenging and prolonged oxygen-generating for enhancing bone repair | |
DE69333286T2 (en) | A THROMBIN BLOOD FRACTION FOR USE IN A MEDICAL PROCEDURE | |
JPH06508835A (en) | trauma cleansing agent | |
Boissonneault et al. | Oxysterols, cholesterol biosynthesis, and vascular endothelial cell monolayer barrier function | |
CN102438635B (en) | Activated leukocyte composition | |
JPS63501718A (en) | How to enhance the healing of mammalian epithelial wounds | |
US4374830A (en) | Platelet aggregating material from equine arterial tissue | |
FR2491475A1 (en) | PROCESS FOR THE PREPARATION OF CHONDROITI SULFATE AND PHARMACEUTICAL COMPOSITION CONTAINING THE SAME | |
US6569838B1 (en) | Facilitation of keloid healing with CM101/GBS toxin | |
August et al. | Histological and ultrastructural findings in chloroquine-induced cardiomyopathy | |
CN114522157A (en) | Application of calcium ion chelating agent in preparation of preparation for improving phagocytic capacity of vascular endothelial cells | |
CA2742345A1 (en) | Hyperbranched polyglycerol for improving heart function | |
JP2011516572A (en) | Actin cytoskeletal rearrangement and method of regulation of intercellular gap formation | |
CN101569608B (en) | Oral solid lipid nano-particle preparation of calcitonin and preparation method thereof | |
CN104220083B (en) | For treating the method and composition of arteriosclerotic vascular diseases | |
CN114642733B (en) | Composition for treating androgenetic alopecia and preparation method thereof | |
Behera et al. | In vivo and in vitro effects of alloxan on collagen characteristics of bone, skin and tendon of Swiss mice | |
WO2016094415A1 (en) | Methods and drug therapies for patency of occluded blood vessels following angioplasty | |
Pappas et al. | Pancreatitis-associated ascitic fluid: effect on the oxygen consumption of liver cells | |
CN113929735B (en) | Gly-Pro-Arg-Pro-NHCH 2 CH 2 NH-warfarin, its synthesis, activity and use | |
Bagdade et al. | Atherosclerosis and oral contraceptive use. Serum from oral contraceptive users stimulates growth of arterial smooth muscle cells. | |
JPH08508916A (en) | Method for removing residual microbicides from percutaneous medical devices | |
TW201717992A (en) | Composition for promoting hair growth or promoting hair cell growth, and applications thereof comprising a polypeptide composition and a fat-soluble compound to solve the problem in treating Alopecia adopted by prior art | |
Pregosin et al. | Formulating Customizable Extracellular Matrix Scaffolds From Decellularized Mouse Kidneys: SA-PO017 | |
Saggau et al. | Changes of coagulation and fat-metabolism following pulmonary microembolism after trauma and hemorrhage |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |