Application of amitriptyline in preparation of medicine for improving endothelial cell function
阅读说明：本技术 阿米替林在制备改善内皮细胞功能的药物中的应用 (Application of amitriptyline in preparation of medicine for improving endothelial cell function ) 是由 钟赟 季杨 陈静 叶锦豪 陈昌浓 陈换珍 于 2021-09-01 设计创作，主要内容包括：本发明公开了阿米替林在制备改善内皮细胞功能的药物中的应用,属于医药领域。所述改善内皮细胞功能的药物包括如下任一项：促进内皮细胞中eNOS磷酸化和NO释放的药物；抑制TNF-α诱导的内皮细胞炎症反应的药物；抑制TNF-α诱导的内皮细胞活性氧产生的药物；通过抑制MAPK信号通路以及NF-κB的活化,减轻内皮细胞炎症的药物。本发明首次公开指出阿米替林在血管内皮损伤和炎症反应中的作用机制,通过试验验证阿米替林可以抑制血管内皮损伤和炎症反应,以改善内皮细胞功能,这为预防由内皮细胞炎症以及内皮细胞功能障碍引起的心血管疾病有很好的应用前景,为心血管疾病的预防和治疗开辟新的医药途径。(The invention discloses application of amitriptyline in preparation of a medicine for improving endothelial cell functions, and belongs to the field of medicines. The medicine for improving the endothelial cell function comprises any one of the following medicines: a drug that promotes eNOS phosphorylation and NO release in endothelial cells; drugs that inhibit TNF-alpha induced inflammatory responses of endothelial cells; a drug that inhibits TNF- α -induced reactive oxygen species production by endothelial cells; the medicine can reduce the inflammation of endothelial cells by inhibiting MAPK signal path and NF-kB activation. The invention discloses the action mechanism of the amitriptyline in vascular endothelial injury and inflammatory reaction for the first time, and tests prove that the amitriptyline can inhibit the vascular endothelial injury and inflammatory reaction to improve the function of endothelial cells, so that the invention has good application prospect for preventing cardiovascular diseases caused by endothelial cell inflammation and endothelial cell dysfunction and opens up a new medical approach for preventing and treating the cardiovascular diseases.)
1. Application of amitriptyline in preparing medicine for improving endothelial cell function.
2. The use of claim 1, wherein the agent that improves endothelial cell function comprises any one of:
(1) a drug that promotes eNOS phosphorylation and NO release in endothelial cells;
(2) drugs that inhibit TNF-alpha induced inflammatory responses of endothelial cells;
(3) a drug that inhibits TNF- α -induced reactive oxygen species production by endothelial cells;
(4) the medicine can reduce the inflammation of endothelial cells by inhibiting MAPK signal path and NF-kB activation.
3. The application of amitriptyline in preparing a medicament for inhibiting endothelial cell inflammation is characterized in that the amitriptyline inhibits the adhesion of monocytes to endothelial cells by inhibiting the expression of inflammatory related molecules induced by TNF-alpha, so as to improve the functions of the endothelial cells.
4. The use of claim 3, wherein the inflammation-associated molecule comprises ICAM-1, VCAM-1, MCP-1.
5. The application of amitriptyline in preparing the medicine for inhibiting TNF-alpha induced endothelial cell injury is characterized in that the amitriptyline can relieve the endothelial cell injury and improve the endothelial cell function by promoting the proliferation, migration and lumen formation of endothelial cells.
Technical Field
The invention relates to the field of medicines, in particular to application of amitriptyline in preparing a medicine for improving endothelial cell functions.
Background
Atherosclerosis (AS) is the leading cause of cardiovascular disease (CVD). AS is a chronic inflammatory disease of the intima of arteries, and its pathological changes can be divided into three stages, namely fatty streak formation, fibrous plaque and atheromatous plaque. At various stages, blood components, blood vessel wall constituent cells and extracellular matrix interact, wherein endothelial insufficiency, lipoprotein invasion and modification, leukocyte aggregation, vascular smooth muscle cell proliferation and migration, foam cell formation, extracellular matrix deposition and the like cause thickening and hardening of arterial walls, and lumen stenosis or occlusion, so that the blood supply function of arteries is reduced, and the occurrence and the development of cardiovascular disease CVD are promoted.
The pathogenesis of AS is quite complex and has not yet been fully elucidated, and the "theory of endothelial inflammatory response" is the most widely recognized important theory explaining the mechanism of AS at present. The theory suggests that AS is a chronic inflammatory response in which plasma lipoproteins, vascular endothelial cells, and blood components interact with each other through the interaction of various cytokines. When the Endothelial Cells (ECs) are continuously damaged by being stimulated by factors such as ischemia, hypoxia, blood flow shearing force and self-metabolism disorder, the morphological and structural changes of the ECs occur in order to adapt to the changes of internal and external environments, the intercellular connection is broken, the local vascular endothelial structure is incomplete, the permeability is increased, the ECs secrete a large amount of adhesion molecules such as vascular cell adhesion factor-1 (VCAM-1), intercellular adhesion molecule-1 (intercellular adhesion molecule-1, ICAM-1), the adhesion and aggregation of monocytes and inflammatory cells are promoted, the adhesion and aggregation are promoted under the action of monocyte chemotactic protein-1 (MCP-1), the adhesion molecules migrate to the ECs and are differentiated into macrophages, the macrophages take up a large amount of Oxidized Low Density Lipoprotein (Oxidized Low Density Lipoprotein) through the scavenger receptors indicated by the macrophages, ox-LDL), into foam cells; after ox-LDL is taken up, macrophages are stimulated to express a plurality of inflammatory cytokines to enhance inflammatory reaction and oxidative stress, and the formation of AS plaques is promoted and the development of AS is accelerated through the change of MAPK, NF-kappa b, PI3K-AKT and other channels. Therefore, reducing endothelial cell inflammation and improving endothelial dysfunction may be effective methods for preventing and treating AS, which is extremely important for reducing the risk of cardiovascular diseases.
Amitriptyline hydrochloride, the english name is amitriptyline hydrochloride. Has a chemical formula of C20H23N & HCl is a common tricyclic antidepressant in clinic and has anti-inflammatory and anti-apoptosis effects. In recent years, researches show that AMP can inhibit reuptake of 5-hydroxytryptamine and norepinephrine, can be accumulated in lysosomes, and can interfere combination of acid sphingomyelinase (ASMASE) and cell membranes, so that ASMASE is exfoliated, and further the function of the ASMASE is inhibited. ASMase, an important member of the sphingomyelin family, is an important component of the cell membrane structure and hydrolyzes sphingomyelin to Ceramide (Ceramide) in cells. Ceramides, as key molecules of sphingomyelin metabolism, play a regulatory role in many cellular activities, such as inflammation, growth, differentiation, senescence, apoptosis, and the like. In recent years, it has been found that the accumulation of Ceramide causes the increase of Reactive Oxygen Species (ROS), inhibits the activation of eNOS, and causes the decrease of NO release and activity, and in some small coronary arteries, Ceramide signals impair the endothelium-dependent diastolic function by reducing the bioavailability of NO in the coronary arteries. Therefore, the inhibition of the activity of ASMase in AS process and the reduction of release of Ceramide to improve the function of blood vessels ECs can provide a new target for the treatment of CVD.
Although studies have reported that amitriptyline exerts a protective effect in a variety of diseases, to date, amitriptyline has not been reported to be able to reduce endothelial cell inflammation or improve endothelial function and its mechanism of action.
Disclosure of Invention
The invention aims to provide application of amitriptyline in preparing a medicine for improving endothelial cell functions, solve the problems in the prior art, discover a new application way of the amitriptyline, and provide a new medicine for treating endothelial inflammation.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides application of amitriptyline in preparing a medicine for improving endothelial cell functions.
Preferably, the drug for improving endothelial cell function comprises any one of:
(1) a drug that promotes eNOS phosphorylation and NO release in endothelial cells;
(2) drugs that inhibit TNF-alpha induced inflammatory responses of endothelial cells;
(3) a drug that inhibits TNF- α -induced reactive oxygen species production by endothelial cells;
(4) the medicine can reduce the inflammation of endothelial cells by inhibiting MAPK signal path and NF-kB activation.
The invention also provides application of the amitriptyline in preparing a medicament for inhibiting endothelial cell inflammation, wherein the amitriptyline inhibits the adhesion effect of monocytes to endothelial cells by inhibiting the expression of inflammatory related molecules induced by TNF-alpha, so as to realize the improvement of the functions of the endothelial cells.
Preferably, the inflammation-associated molecule includes ICAM-1, VCAM-1, MCP-1.
The invention also provides application of the amitriptyline in preparation of a medicine for inhibiting TNF-alpha induced endothelial cell injury, wherein the amitriptyline can relieve endothelial cell injury and improve endothelial cell function by promoting endothelial cell proliferation, migration and lumen formation.
The invention discloses the following technical effects:
the invention discloses that the amitriptyline can obviously inhibit MAPK-NF-kB signal channels to reduce TNF-alpha induced human umbilical vein endothelial cell inflammatory responses, such as protein expression of NF-k B, ICAM-1, VCAM-1, MCP-1 and the like, and the adhesion quantity of mononuclear cells; amitriptyline can relieve TNF-alpha induced endothelial cell dysfunction of human umbilical vein, such as promotion of endothelial cell proliferation and migration, and restoration of lumen formation ability. The invention finds the new application of amitriptyline, provides a new medicine for treating endothelial cell inflammation, and has good application prospect for preventing and treating cardiovascular diseases.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a graph of amitriptyline promoting endothelial cell function; a: western blotting to detect the effect of AMP (0-5. mu.M, 24hours) on the expression level of p-eNOS protein in HUVEC; b: effect of AMP (0-5. mu.M, 24hours) on NO release in HUVEC; c: EdU measures the effect of AMP (2.5 μ M, 24hours) on HUVEC proliferation, scale: 400 μm; d: statistical results of HUVEC proliferation; e: CCK-8 measures the effect of AMP (0-5. mu.M, 24hours) on HUVEC cell viability;*P＜0.05，**P＜0.01，***P＜0.001vs.Con，Mean±SEM，n＝5；
FIG. 2 is a graph showing that amitriptyline inhibits TNF- α -induced endothelial cell adhesion-related molecule expression and monocyte adhesion; a: western blotting detection AMP to TNF-alpha induced HUVEC VCAM-1, ICAM-1 and MCP-1 protein expression level influence; B-C: ElISA measures the effect of AMP on the release of sVCAM-1 and sICAM-1 from TNF-alpha-induced HUVEC supernatant; d: effect of AMP on TNF- α induced monocyte adhesion, scale: 400 μm;*P＜0.05，**P＜0.01，***P＜0.001vs.Con，#P＜0.05，##P＜0.01，###P＜0.001vs.TNF-α，Mean±SEM，n＝3；
FIG. 3 is a graph of amitriptyline alleviating TNF- α -induced endothelial cell dysfunction; a: transwell assay examined the effect of AMP on TNF- α induced HUVEC migration, scale: 4X 200. mu.m, 10X 200. mu.m, 20X 100. mu.m; b: number of cells that HUVEC migrated into the lower chamber; c: EdU measures the effect of AMP on TNF- α induced HUVEC proliferation, scale: 400 μm; d: (iv) EdU positive rate; e: tube format measures the effect of AMP on TNF- α induced HUVEC tubule formation, scale: 200 mu m; f: measuring the total length of the finished pipe; p <0.05, P < 0.01, P < 0.001vs. Con,#P＜0.05，##P＜0.01，###P＜0.001vs.TNF-α,Mean±SEM，n＝5；
FIG. 4 is a graph showing that amitriptyline inhibits TNF- α -induced reactive oxygen species production in endothelial cells; a: dihydroethydium (DHE) measures the fluorescence of AMP on TNF- α -induced reactive oxygen species, scale: 400 μm; b: counting the fluorescence intensity of the active oxygen;*P＜0.05，**P＜0.01，***P＜0.001vs.Con，#P＜0.05，##P＜0.01，###P＜0.001vs.TNF-α,Mean±SEM，n＝5；
FIG. 5 is a graph showing that amitriptyline inhibits TNF- α -induced ASMase activation and Ceramide release; a: the amount of Ceramide in the supernatant of HUVEC; B-C: activity of ASMase from HUVEC supernatant and release of Ceramide; p <0.05, P < 0.01, P < 0.001,##P＜0.01，###P＜0.001；
FIG. 6 is a graph showing that amitriptyline inhibits TNF- α -induced activation of endothelial MAPK; a: western blotting detection AMP on TNF-alpha induced P-NF-kB, P-ERK1/2, P-P38 and P-JNK protein expression level influence; b: the statistical results of the expression levels of P-NF-kB, P-ERK1/2, P-P38 and P-JNK proteins;*P＜0.05，**P＜0.01，***P＜0.001vs.Con，#P＜0.05，##P＜0.01，###P＜0.001vs.TNF-α,Mean±SEM；n＝3。
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1
1. Experimental Material
Amitriptyline hydrochloride was purchased from sigma, usa; human recombinant TNF- α was purchased from Beijing Sino Biological, Inc.; EBM-2 medium was purchased from Lonza, 1640 medium was purchased from Gibco, USA; EdU kit was purchased from sharp bo, guangzhou; matrigel was purchased from corning, usa; ASMase antibodies were purchased from ibotex, china; antibodies such as ICAM-1, VCAM-1, MCP-1, MAPK, p-NF- κ B and the like were purchased from CST corporation, USA; ICAM-1 and VCAM-1Elisa kits were purchased from David, China, Inc.
2. Experimental cell culture and model establishment
Human Umbilical Vein Endothelial Cells (HUVEC) were purchased from Cell Applications, Inc. of America.
Cells were cultured in a medium containing 95% air and 5% CO2At 37 ℃ in a sterile incubator. When the cells are in good growth state and grow to 80% density, the cells are divided into 4 groups according to the following treatment:
control group: a control group;
TNF- α group alone: TNF- α (20 ng/ml);
TNF-. alpha. + AMP group: TNF-. alpha. (20ng/ml) + AMP (2.5. mu.M);
AMP group: AMP (2.5. mu.M).
3. Experimental methods
(1) Detection of CCK-8 cell viability
HUVECs were plated in 96-well plates (8X 10) in groups according to experimental design3cells/well), and carrying out subsequent experiments after the cell state is stable; preparing a CCK-8 reaction solution: according to EBM-2 (serum-free): CCK-8 solution ═ 10: 1, fully and uniformly mixing, and keeping out of the sun in the whole process; discarding the old culture medium in a 96-well plate, washing the cells once with preheated PBS, adding 110 μ l of CCK-8 reaction solution into each sample well to be detected, placing at 37 ℃, and adding 5% CO2Incubating in an incubator for 3 hours; reading OD values in the holes at the wavelength of 450nm by using an enzyme-labeling instrument;
(2) EdU detection of cell proliferation
1) EdU labeling: preparing an EdU A liquid culture medium: cell culture medium containing serum was run at 1000: 1, diluting the EdU A solution with the final concentration of 50 mu M; incubating the cells in a cell culture box for 4hours by using a diluted EdU A solution (50 mu M) culture medium; thirdly, discarding the old culture medium, and washing each experimental hole for 5 minutes by PBS (phosphate buffer solution);
2) cell fixation: adding 4% paraformaldehyde (100 mu l) into each hole of a 48-hole plate, incubating for 30 minutes at 18-25 ℃, and discarding a stationary liquid; adding 2mg/ml glycine (100 mu l) into each hole to neutralize redundant aldehyde groups, incubating for 5 minutes by using a shaking table, absorbing and removing solution in the holes, and washing for 5 minutes by using PBS; ③ abandoning PBS, respectively adding 0.5 percent TritonX-100 penetrant (100 mu l) to each experimental hole, incubating for 10 minutes at the temperature of 18-25 ℃ by a shaker, washing once by PBS for 5 minutes;
3) appllo staining: preparing 1 XApollo dyeing reaction liquid according to needs; keeping the whole process away from light, adding 1 XApollo staining reaction solution (100 μ l) into each hole, incubating for 30 minutes at 18-25 ℃ by a dehydration shaking table, and absorbing and removing the staining reaction solution; adding 0.5 percent TritonX-100 penetrant (100 mu l) into each hole, slowly shaking the decoloring shaker for 10 minutes, repeating for 2 times, and discarding the penetrant; ③ adding 100 mul of absolute methanol into each hole to reduce the dyeing background, and standing for 5 minutes; washing with PBS for 5 min;
4) DNA staining: use ddH2O is according to 100: 1, diluting a reagent F according to the proportion of 1, preparing 1 × Hoechest staining solution, using the solution as it is, and keeping away from light for later use; secondly, keeping the whole process away from light, adding 1 XHoechest staining solution (100 mu l) into each hole, incubating for 30 minutes at the temperature of 18-25 ℃ by a dehydration shaking table, and absorbing and removing liquid in the holes; ③ washing the PBS for 5 minutes once; adding PBS (200 mu l) into each hole to keep the sample moist, observing and photographing under a fluorescence microscope or storing at 4 ℃ in a dark place, and photographing within 3 days.
(3) Nitric acid reductase method for detecting content of nitric oxide
Cell supernatants were collected and assayed for nitric oxide levels according to kit instructions and OD values were measured for each sample at 550nm using a microplate reader.
(4) Elisa method for measuring content of VCAM-1, ICAM-1 and Ceramide in cell supernatant
The treated cell supernatants were collected and the levels of VCAM-1, ICAM-1 and Ceramide were determined according to the kit protocol, and the OD of each sample was measured at a wavelength of 450nm using a microplate reader.
(5) Monocyte adhesion assay
Spreading HUVEC to 12-hole plates, and performing grouping treatment according to experimental design; sucking a certain volume of THP-1 cell suspension, centrifuging at 950rpm for 3 minutes; discardSupernatant, 1ml EBM-2 medium heavy suspension, and cell count, required THP-1 cell count, EBM-2 medium dilution cell suspension to 4 x 105cells/ml; adding calcein into the cell suspension in a dark place, gently mixing the calcein and the cell suspension to obtain a mixture, and incubating the mixture at room temperature for 15 minutes; taking out the processed 12-hole plate from the incubator, discarding the old culture medium, adding THP-1 cell suspension, shaking up by 1ml/well method in the shape of '8', and putting the cell into the incubator to incubate for 45 minutes; discarding the culture medium, washing the non-adhered mononuclear cells with PBS, and repeating for 3 times; fresh medium was added, pictures were taken under a fluorescent microscope and ImageJ software was used for data analysis.
(6) Transwell detection of cell migration
Taking HUVEC with P4 in logarithmic growth phase and good state, spreading the HUVEC to a 6-hole plate, and processing the HUVEC in groups according to experimental design; after the cells are treated, digesting, centrifuging and collecting the cells, resuspending the cells in serum-free EBM-2 medium, counting the cells, and preparing the cells into a concentration of 4 multiplied by 104cell/ml cell suspension; the prepared Transwell chamber (8 μm) was placed in a 24-well plate, 150 μ l of cell suspension was added to the upper chamber, and 700 μ l of EBM-2 medium containing 5% FBS was added to the lower chamber; putting the 24-hole plate provided with the Transwell chamber into a cell culture box for incubation for 12-16 hours; taking out the 24-well plate from the incubator, carefully discarding the culture medium in the upper chamber and the lower chamber, washing the chamber for 1 time by PBS, and fixing the chamber in 4% paraformaldehyde for 30 minutes; washing with PBS for 1 time, placing the cell in a crystal violet solution for dyeing for 45 minutes, discarding the residual crystal violet solution in the cell, and washing with PBS for 3 times; cells that did not migrate from the upper chamber to the lower chamber were carefully wiped off with a cotton swab, dried, photographed under an inverted microscope, and the data saved.
(7) Detection of endothelial cell luminal formation ability
According to the experimental design, the HUVEC with P4 in a logarithmic growth phase and a good state is paved on a 6-hole plate, and the next step of treatment is carried out after the cell state is stable; putting a 96-well plate and a sterile gun head into a refrigerator for precooling one day in advance, and dissolving Matrigel in advance; taking out the precooled 96-well plate and the aseptic gun head from the refrigerator, adding the prepared Matrigel into the 96-well plate, performing 50 mul/well operation on ice in the whole process, and coating for 1 hour at 37 ℃; separately digesting the cells in each well with pancreatin and separatingDiscarding supernatant after heart, resuspending cells in serum-free EBM-2 medium, counting cells, and preparing to 3 × 105cell/ml cell suspension; adding 100 mu l/hole of the cell suspension into a Matrigel-coated 96-well plate, and placing the 96-well plate in a cell culture box for incubation for 6 hours at 37 ℃; the photographs were taken under an inverted microscope and ImageJ software was used for data analysis.
(8) Cellular ROS detection
Spreading HUVEC to 12-hole plates, and performing grouping treatment according to experimental design; dissolving a superoxide anion fluorescent probe (DHE) in a serum-free EBM-2 culture medium under the condition of keeping out of the sun to obtain a final concentration of 20 mu M; taking out the treated 12-well plate from the incubator, discarding the old culture medium, washing with PBS for 2 times, adding EBM-2 culture medium containing DHE (20 μ M), and incubating for 45 min in a cell incubator; old culture medium in the wells was discarded, washed with PBS 2 times, fresh culture medium was added, pictures were taken under a fluorescent microscope, and ImageJ software was used for data analysis.
(9) ASMase Activity assay
1) Taking the ASMase activity kit out of the refrigerator in advance, and returning to the room temperature;
2) preparing a working solution: (ii) AbRed indicator: dissolving AbRed indicator (powder) in 80 μ l DMSO to obtain 200 × AbRed indicator, subpackaging, storing in refrigerator at-20 deg.C in the dark, and balancing to room temperature before use; ② sphingomyelin working solution: the sphingomyelin solution and the sphingomyelinase reaction mixture were mixed as required in a ratio of 1: 100, and the components are mixed and used as the mixture at present; ③ Enzyme Mix: adding 5ml of detection buffer solution into the whole Enzyme Mix (powder), fully mixing and dissolving, subpackaging and storing in a refrigerator at the temperature of-20 ℃; sphingomyelinase reaction solution: enzyme Mix and AbRed indicator were mixed as required at 200: 1, mixing the components, using the mixture as a preparation, and storing the mixture in dark;
3) taking out the treated cells from the incubator, discarding the old culture medium, washing the cells once with PBS, and completely sucking out the residual PBS;
4) add 1 × Mammalian Lysis Buffer (300 μ l) to 6-well plate, incubate for 15min at room temperature (18-25 ℃); collecting cell lysate in 1.5ml EP tube, centrifuging at 1500rpm for 5min, transferring supernatant to new EP tube, and performing ice operation;
5) adding a sample to be detected (50 mu l) into a black 96-well plate, and adding a detection buffer solution (50 mu l) into a blank well;
6) adding sphingomyelin (50 mu l) working solution into each sample to be detected, and incubating for 2 hours at 37 ℃;
7) adding 50 μ l of sphingomyelinase reaction liquid into the mixed reaction liquid to be detected;
8) the cells were incubated under light-shielding conditions at room temperature (18-25 ℃) for 2 hours, and the absorbance was read with a fluorescence microplate reader set at Ex/Em ═ 540/590 nm.
(10) Western blot method for detecting protein expression of VCAM-1, ICAM-1, MCP-1, p-eNOS, p-JNK, p-ERK1/2, p-p38 and p-NF-kappa B
After the endothelial cell medicament is processed, adding cell lysate to lyse cells on ice, centrifuging for 15min at 4 ℃ at 12000g, and transferring supernatant to a new EP tube, namely a protein sample stock solution; protein concentration was determined using BCA kit; the 5 × loading buffer and protein sample were mixed according to 1: 4, mixing in proportion, and carrying out water bath at 100 ℃ for 10 min; cooling on ice for 1 min; proteins were separated by electrophoresis on a 10% SDS-PAGE gel, followed by membrane transfer, blocking, incubation of the relevant primary antibody overnight, next day, membrane washing, secondary antibody incubation, image acquisition on PVDF membrane in chemiluminescence apparatus and quantitative analysis using ImageJ software, GAPDH as internal control.
4. Statistical analysis
Statistical analysis is carried out on the data by adopting SPSS 21.0 software, each group of experiments are repeated at least more than 3 times, and all the data are calculated by mean +/-standard errorAnd (4) showing. The Mann-Whitney U test in the nonparametric test was used for the test of the significance of the differences between two or more groups of varying variances, and the Kruskal-Wallis H test in the nonparametric test was used for the test of the significance of the differences between the groups or subgroups of varying variances. For the comparison between two sets of data with uniform variance, an independent sample t test was used. P<0.05 the difference was considered statistically significant.
5. Analysis of results
(1) Amitriptyline can promote the function of umbilical vein endothelial cells
The drug provided by the invention directly acts on HUVEC for 24h at different concentrations, and the result shows that the phosphorylation degree of eNOS is increased in a concentration-dependent manner within the range of 0-5 mu M of amitriptyline drug concentration (figure 1A), and the amitriptyline promotes the release of NO in the supernatant of HUVEC (figure 1B), wherein the effect is most obvious at the concentration of 2.5 mu M.
To further study the effect of amitriptyline on HUVEC function, we examined the viability and proliferative function of cells (fig. 1D), and the results showed that, compared to the control group, after amitriptyline treatment, HUVEC activity was significantly enhanced, EdU positive cell number was significantly increased (fig. 1C), and the difference was statistically significant (P <0.05), indicating that amitriptyline promoted endothelial cell function.
(2) Amitriptyline can inhibit TNF-alpha induced endothelial cell adhesion related molecule expression and monocyte adhesion
The drug provided by the invention acts on HUVEC for 1h, and then TNF-alpha (20ng/ml) acts for 23h, as shown in figure 2A, amitriptyline treated alone has no difference compared with a control group; after TNF-alpha (20ng/ml) treatment, the expression of adhesion molecules VCAM-1, ICAM-1 and MCP-1 is obviously increased compared with that of a control group; pretreatment of amitriptyline significantly inhibited the expression of these molecules, and the Elisa results are consistent with Western blot (FIG. 2B, C).
We further studied the role of amitriptyline in TNF- α induced monocyte adhesion, and the results showed that, after TNF- α treatment for 24h, THP-1 adhered to HUVEC was significantly increased compared to the control group, while the intervention of amitriptyline significantly inhibited the adhesion of THP-1; amitriptyline alone treated HUVECs with no significant statistical difference compared to the control group (fig. 2D); these results demonstrate that amitriptyline blocks monocyte adhesion to endothelial cells in inflammatory states by inhibiting the expression of endothelial cell adhesion molecules.
(3) Amitriptyline can alleviate TNF-alpha induced endothelial cell dysfunction
Endothelial cell function plays an important role in the development and progression of AS. Tanswell examined the migration ability of HUVEC, as shown in FIGS. 3A and B, TNF- α inhibited endothelial cell migration compared to the control group, whereas the number of cells migrating under the Transwell chamber was significantly increased after the drug provided by the present invention; the EdU experiment detects the cell proliferation capacity, as shown in fig. 3C and D, the EdU positive rate of the TNF- α group is significantly lower than that of the Con group, the cell proliferation capacity is reduced, and the EdU positive rate after the amitriptyline is added is higher than that of the TNF- α group, indicating the recovery of the cell proliferation capacity; as shown in FIGS. 3E and F, the results of Tube formation show that the total Tube length of HUVEC was decreased after TNF- α treatment, and was significantly increased after the advanced addition of amitriptyline; the above results suggest that amitriptyline can alleviate TNF-alpha induced endothelial dysfunction.
(4) Amitriptyline can inhibit TNF-alpha induced endothelial cell reactive oxygen species production
Oxidative stress plays an important role in endothelial cell activation and the development and progression of atherosclerosis. We then examined the production of reactive oxygen species in endothelial cells by DHE.
As shown in FIGS. 4A and B, TNF- α can promote the production of reactive oxygen species in HUVEC, and amitriptyline provided by the present invention can down-regulate the production of reactive oxygen species in HUVEC compared to TNF- α group, and amitriptyline alone has less effect on the production of reactive oxygen species in HUVEC compared to Con group, which indicates that amitriptyline can inhibit TNF- α -induced production of reactive oxygen species in HUVEC.
(5) Amitriptyline inhibits TNF-alpha induced ASMase activation and Ceramide release
To investigate the effect of amitriptyline, a drug provided by the present invention, on TNF- α -induced endothelial ASMase, we examined the activity of ASMase and the amount of Ceramide, a downstream product, in the cell supernatant. In the presence of TNF- α (5-30ng/ml), the amount of Ceramide in the supernatant of HUVEC was increased in a concentration-dependent manner as compared to the control group (FIG. 5A), and amitriptyline was able to inhibit the activity of ASMASe and the release of Ceramide in a concentration-dependent manner (FIGS. 5B, C), and thus, amitriptyline was a potent inhibitor of ASMASe activity.
(6) Effect of amitriptyline on TNF-alpha induced activation of MAPK pathway in endothelial cells
TNF-alpha can activate MAPK pathway by combining with TNFR, and the study on whether the drug amitriptyline provided by the invention can reduce the proinflammatory effect induced by TNF-alpha by inhibiting MAPK signal transduction pathway or not is carried out.
As shown in FIGS. 6A and B, Western blotting results show that TNF-alpha can significantly up-regulate the expression of p-NF-kappa B, p-JNK, p-ERK1/2 and p-p38 in HUVEC, when amitriptyline is pretreated, the expression of p-NF-kappa B, p-JNK, p-ERK and p-p38 is reduced compared with the TNF-alpha group, and the expression of the proteins is not significantly changed compared with the Con group after the amitriptyline is singly treated. These results indicate that amitriptyline may exert an anti-inflammatory effect by inhibiting the MAPK pathway, thereby improving endothelial function.
The experimental results prove that the amitriptyline can reduce the generation of adhesion molecules and active oxygen secreted by endothelial cells in an inflammatory state, inhibit the adhesion of monocytes to the endothelial cells and relieve further injury and dysfunction of the endothelial cells by effectively inhibiting MAPK-NF-kB signal transduction pathways.
Endothelial cells, which are important components of blood vessels, have the ability to resist thrombosis, resist inflammation, and produce vasoactive substances to regulate vascular tone, and play an important role in the homeostasis of vascular function. Chronic vascular endothelial injury can cause imbalance of vasoconstriction and relaxation functions, active oxygen is increased, the generation and secretion of inflammatory factors and inflammatory mediators can not only damage the endothelium, so that the bioavailability of NO is insufficient, but also promote the proliferation and migration of vascular smooth muscle cells, further cause vascular lesions, and accelerate the development process of atherosclerosis. The medicine provided by the invention has the characteristics of inhibiting endothelial cell inflammation and protecting endothelial cell functions well in the endothelial cell injury experiment. Therefore, the medicine provided by the invention has good application prospect in preventing cardiovascular diseases caused by endothelial cell inflammation and endothelial cell dysfunction, and opens up a new medical approach for preventing and treating the cardiovascular diseases.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.