CN114522157B - 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
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- CN114522157B CN114522157B CN202210167310.7A CN202210167310A CN114522157B CN 114522157 B CN114522157 B CN 114522157B CN 202210167310 A CN202210167310 A CN 202210167310A CN 114522157 B CN114522157 B CN 114522157B
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- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 title claims abstract description 34
- 229910001424 calcium ion Inorganic materials 0.000 title claims abstract description 34
- 239000002738 chelating agent Substances 0.000 title claims abstract description 31
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 210000003556 vascular endothelial cell Anatomy 0.000 title claims abstract description 11
- 230000000242 pagocytic effect Effects 0.000 title abstract description 8
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- 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 claims description 15
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- 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 description 1
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- 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]
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- 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
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- 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
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- 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
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Abstract
The invention belongs to the technical field of medicaments 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. Compared with the prior art, the embodiment of the invention shows that the calcium ion chelating agent can reduce the viscosity of endothelial cell membranes and improve the fluidity of the endothelial cell membranes through the cell and animal experimental level, thereby promoting the phagocytosis of the nano-drug, and obviously improving the utilization rate of the nano-drug.
Description
Technical Field
The invention belongs to the technical field of medicaments 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 are serious hazards to human health, and Atherosclerosis (AS) is considered to be the main pathological basis of cardiovascular disease. Numerous studies have shown that atherosclerotic lesions develop in specific areas of vascular flow disturbance, such as areas of vascular branch stenosis and tortuosity, where reverse flow, oscillating flow, turbulence, etc. occur. Inside the lumen of the blood vessel, endothelial cells are in direct contact with the blood flow and have the ability to sense changes in shear stress in the blood flow. The turbulent flow region at the atherosclerotic lesion causes cell debris, nano-substances, bacteria, etc. to accumulate in the region near the inner wall of the blood vessel due to the change of the flow field, and the vascular endothelial cells can phagocytose these substances by phagocytosis independent of specific protein receptors. Currently, the development and progression of endocytosis and atherosclerosis are still under investigation.
At present, no relevant literature reports on substances related to promotion of vascular endothelial cell phagocytosis or treatment of atherosclerosis by promotion of vascular endothelial cell phagocytosis.
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 a preparation for improving phagocytic capacity of vascular endothelial cells.
Further, the calcium ion chelating agent is selected from one or more of ethylenediamine tetraacetic acid and derivatives thereof, ethylene glycol bis (2 amino diethyl ether) tetraacetic acid and derivatives thereof, 1,2 bis (2 amino phenoxy) ethane N, N' tetraacetic acid and derivatives thereof.
Further, the calcium ion chelating agent may also serve the same function in endothelial cells of other calcium environments.
The invention also aims to provide the application of the calcium ion chelating agent in preparation of preparations for treating atherosclerosis.
Further, the calcium ion chelating agent is selected from one or more of ethylenediamine tetraacetic acid and derivatives thereof, ethylene glycol bis (2 amino diethyl ether) tetraacetic acid and derivatives thereof, 1,2 bis (2 amino phenoxy) ethane N, N' tetraacetic acid and derivatives thereof.
In particular, when the nano-drug is administered through the endothelium after entering the blood vessel, endothelial cell phagocytosis is a new mechanism for abnormal shear stress to drive atherosclerosis. Therefore, the molecular mechanism of endothelial cell phagocytosis affecting the formation and development of atherosclerosis under low oscillatory flow conditions is being studied in depth, and will provide new targets and clinical strategies for effective prevention and treatment of atherosclerosis. The calcium ion chelating agent is used for partially or completely removing calcium around or in the endothelial cells, so that the phagocytic capacity of the endothelial cells to the nano-drugs is effectively improved.
The invention also aims to provide an application of the calcium ion chelating agent in preparation of a preparation for promoting vascular endothelial cells to absorb medicines.
Further, the calcium ion chelating agent is selected from one or more of ethylenediamine tetraacetic acid and derivatives thereof, ethylene glycol bis (2 amino diethyl ether) tetraacetic acid and derivatives thereof, 1,2 bis (2 amino phenoxy) ethane N, N' tetraacetic acid and derivatives thereof.
Further, the drug comprises one or more of extracellular vesicles, exosomes, artificial vesicles, organic nano-drug particles, inorganic nano-drug particles.
Specifically, after the calcium ion chelating agent quickly chelates calcium ions of vascular endothelium, the phagocytosis of endothelial cells is rapidly increased by the nano-drug or the biological vesicle drug.
In certain embodiments, the concentration of the calcium ion chelating agent is selected to be 0.5 to 1.5mM, preferably 1mM. 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 administration of the calcium ion chelating agent to C57BL/6 mice by tail vein injection, the detection shows that when the calcium ion chelating agent depletes the calcium ions in the endothelial cells, i.e., in a calcium deficient environment, the phagocytic extracellular vesicles RBCEVs of the endothelial cells are enhanced, which means that the calcium chelating agent can increase the fluidity of the endothelial cell membranes by reducing the intracellular calcium, thereby promoting the phagocytic RBCEVs of the endothelial cells and further improving the availability of the erythrocyte extracellular vesicles RBCEVs.
The present invention also provides a method for improving the availability of an endothelial drug, characterized in that the endothelial drug is used in combination with a calcium ion chelating agent, the endothelial drug comprising: 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 ethylenediamine tetraacetic acid and derivatives thereof, ethylene glycol bis (2-aminoethyl ether) tetraacetic acid and derivatives thereof, 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 shows that the calcium ion chelating agent can reduce the viscosity of endothelial cell membranes and improve the fluidity of the endothelial cell membranes through the cell and animal experimental level, thereby promoting the phagocytosis of the nano-drug, and obviously improving the utilization rate of the nano-drug.
Drawings
Fig. 1 shows the effect of calcium ion chelator and calcium chloride on endothelial phagocytosis of RBCEVs, respectively, on en face staining (scale = 20 μm).
Fig. 2 is a statistical plot of the effect of calcium ion chelator and calcium chloride on endothelial phagocytosis of RBCEVs, respectively (n=5, P <0.05 or P < 0.001).
Fig. 3 is an en face staining (scale = 10 μm) of the effect of calcium chloride and calcium chelator injections, respectively, into the tail vein of the mice on phagocytosis of RBCEVs by left and right carotid RCA endothelial cells.
Fig. 4 is a statistical plot of the effect of calcium chloride and calcium chelator on phagocytosis of RBCEVs by left and right carotid LCA endothelial cells, respectively, by tail vein of mice (n=5, P <0.01 or P < 0.001).
Fig. 5 shows the different cell viscosities under calcium ion treatment ((n=3, < P0.05 or P <0.01, < P < 0.001).
Fig. 6 shows the viscosity of cells under treatment with a calcium ion chelating agent (n=3, P <0.05 or P <0.01, P < 0.001).
Fig. 7 is the effect of different concentrations of BAPTA-AM on endothelial cell viability (n=3).
Detailed Description
The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.
Example 1 cell experiment
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, the cells are pretreated by a calcium ion chelating agent for 30 minutes, then the cells are washed for at least 3 times by a fresh cell culture medium, and when the cells are treated by BAPTA-AM, the cells are firstly dissolved in 10% DMSO and then dissolved in 90% physiological saline (containing 20% SBE-beta-CD); the concentration of calcium chloride treated cells was 10. Mu.g/mL. As shown in fig. 1 and 2, the endothelial cells were treated with calcium chloride and the calcium ion chelating agent BAPTA-AM, respectively, with calcium chloride inhibiting the endothelial cells from phagocytizing the extracellular vesicles of erythrocytes RBCEVs, while BAPTA-AM promotes the endothelial cells from phagocytizing the extracellular vesicles of erythrocytes.
Example 2 animal experiments
In the examples of the present invention, BAPTA-AM was injected into mice by dissolving in 10% DMSO and then in 90% physiological saline (20% SBE. Beta. CD).
In the embodiment of the invention, the concentration of the calcium chloride injection injected into the mice is 10 mug/mL, and the injection needs to be slowly taken, otherwise, the hypotension or arrhythmia of the mice are easy to cause.
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 carotid artery ligation operation is performed on the mice, so as to construct a mouse OSS carotid artery ligation model. The specific steps of ligation are as follows:
(1) After the mice are anesthetized, the mice are fixed on an operating table in a supine mode, skin is cut from the right upper portion of the trachea, and muscles on the left side and the right side of the trachea are separated respectively; firstly, using forceps to strip muscles layer by layer along a left trachea to find a left carotid sheath, and then using the forceps to carefully separate fascia close to the left carotid artery and vagus nerve, and separating out a left common carotid artery (LCA); continuing with the dissection up the common carotid artery, the External Carotid Artery (ECA), internal Carotid Artery (ICA), occipital Artery (OA) and suprathyroidial artery (STA) were found.
(2) Three branch vessels (ECA, ICA and OA) other than STA were ligated with 90 medical sutures, and the blood flow of STA was ensured to be smooth.
(3) The right common carotid artery (RCA) was isolated and 4 branch vessels were found, but not ligated, as a control group.
(4) The wound was wiped with iodophor after suturing with 50 sutures layer by layer and the mice were placed on a 37 ℃ thermostatic plate waiting to wake up and transfer to the corresponding squirrel cage.
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. Results from en face staining and data statistics are shown in fig. 3 and 4, it can be seen that LCA group has obvious rbc evs red fluorescent signal co-localized with endothelial cells compared to RCA group; when calcium chloride was intravenously injected via the tail of the mice (LCA+Ca) 2+ )，LCA+Ca 2+ The red fluorescence signal of the RBCEVs of the group is obviously lower than that of the LCA group, namely, calcium chloride inhibits endothelial cell phagocytosis of the LCA group; in contrast, when BAPTA-AM was injected (LCA+BAPTA-AM), RBCEVs red fluorescence signal was significantly stronger than that of LCA group. Namely, the vein of the tail of the mouse is respectively injected with calcium chloride and a calcium chelating agent BAPTA-AM, and the BAPTA-AM obviously promotes the phagocytosis of the red blood cell extracellular vesicle RBCEVs by vascular endothelial cells in the right carotid artery ligation OSS area.
This result is consistent with the cell results of example 1, and BAPTA-AM can inhibit calcium ion concentration in blood, promote endothelial cells to phagocytose RBCIVs, and improve the availability of RBCIVs.
EXAMPLE 3DPH Probe for detecting endothelial cell membrane fluidity
The DPH probe is used for detecting the endothelial cell membrane fluidity, and the specific steps are as follows:
(1) Preparing a working solution: and diluting the DPH probe by using a diluent in the kit according to the use instruction, wherein the dilution range can be at most 10000 times according to the experimental requirement, and preparing the DPH dyeing working solution.
(2) Endothelial cells were seeded into 12-well plates and stained after confluence of cells was observed under a microscope, medium was aspirated with a pipette, staining broth was added, and incubated in a cell incubator for at least 0.5 hours.
(3) Cells were washed 1 time with pH 7.4HBSS or 20mM HEPES or PBS and resuspended.
(4) Cells were detected with a spectrophotometer with polarized light. The setting parameters are as follows: excitation wavelength=355 nm, emission wavelength=430 nm.
The fluorescence polarization degree P was calculated according to the following formula: p= (IVV-GIVHIHV)/(IVV +givh), wherein correction factor g=ihv/IHH, where: IVV fluorescence intensity measured when the polarization and analyzer optical axes are in the vertical direction; IVH, the fluorescence intensity measured when the optical axes of the polarizer and the analyzer are vertical and horizontal directions respectively; IHV is fluorescence intensity measured when the optical axes of the polarizer and the analyzer are respectively in the horizontal direction and the vertical direction; IHH fluorescence intensity measured when the polarization and analyzer optical axes are in the same horizontal direction; p can reflect the viscosity (η) of the membrane lipid region, with η being inversely proportional to the membrane fluidity.
As shown in FIGS. 5 and 6, the DPH probe detected BAPTA-AM reduced the viscosity of endothelial cell membrane and improved the fluidity of endothelial cell membrane, thereby promoting the phagocytosis of RBCEVs, whereas calcium chloride was the opposite.
Example 4 cytotoxicity test
The effect of different concentrations of BAPTA-AM on endothelial cell viability was studied by MTS assay, cells were assayed at the suggested concentration and time of BAPTA-AM (1 mM,30 minutes) as follows:
(1) HUVEC cells were cultured to log phase.
(2) 96-well cell culture plates were prepared, and 0.1ml of 1X 10-containing medium was added to each well 4 ～2×10 4 1640 medium (10% calf serum) of one of the above cells.
(3) mu.L MTS was added to each well, and the culture was continued for 3 to 4 hours to develop color.
(4) The plates were shaken for 10 seconds before detection and the color was mixed. The absorbance (OD) of each well was measured on an ELISA at 570nm (or 490nm, or 570nm and 690 nm). Dose-response curves were plotted against OD values (OD 570, OD490 or OD570/OD 690) using sample dilutions, and the amount of cytokine in the samples was calculated from the standard curves.
As shown in FIG. 7, the BAPTA-AM recommended concentration and time (1 mM,30 minutes) have no significant toxicity to cells and have better safety.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and 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 modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (2)
1. Use of a calcium ion chelating agent, said calcium ion chelating agent being BAPTA-AM, for the preparation of a formulation for promoting the absorption of a drug by endothelial cells, said drug being erythrocyte extracellular vesicles RBCIVs.
2. The use of claim 1, wherein the endothelial cells comprise vascular endothelial cells.
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