Application of OLA1 in preparation of medicine for inhibiting vascular endothelial penetration

Abstract

The invention discloses an application of OLA1 in preparation of a medicine for inhibiting vascular endothelial penetration, and belongs to the technical field of biological medicine, in a primary HUVECs cell knockout or overexpression OLA1 stable transfection cell line, through an immunofluorescence experiment, a Transwell endothelial permeability determination experiment and a fluorescein scratch experiment, it is found that when OLA1 is inhibited, connection between cells is tighter; in-vivo experiments show that through caudal vein injection of Evans Blue and collection of mouse tissues, it is found that the vascular permeability of an Ola1 knockout mouse is weakened compared with a control group, and Ola1 gene knockout can inhibit the vascular endothelial permeability in the mouse body; in primary HUVECs (human umbilical vein endothelial cells) with OLA1 knocked out, intercellular chained protein VE-Cadherin is detected through immunofluorescence, and the situation that the permeability of thrombin-induced endothelial cells is reduced by knocking out the OLA1 is found.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application of OLA1 in the preparation of drugs for inhibiting vascular endothelial permeability. Background Art

[0002] Endothelial cells are closely arranged on the inner surface of blood vessels to form the endothelium, which is a natural barrier between the blood vessel wall and the blood, maintaining homeostasis within tissues and blood vessels and regulating many physiological and pathological processes. The integrity of the vascular barrier can be disrupted by a variety of soluble permeability factors, and changes in barrier function can exacerbate tissue damage during disease progression. Understanding endothelial barrier function is crucial for vascular homeostasis. Many signaling pathways that promote vascular permeability can also be triggered during diseases, leading to prolonged or uncontrolled vascular leakage. Therefore, finding proteins that can effectively inhibit vascular leakage will provide feasible solutions for new drug research and development and applications, and will provide new strategies for the treatment of diseases related to vascular leakage.

[0003] Human OLA1 (Human Obg-like ATPase1) is a protein named in 2007. It is a widely expressed gene, expressed in a variety of tumor and normal cell lines, and has both GTPase and ATPase activities. The gene is highly conserved and has important physiological functions. Current research has found that human OLA1 plays an intrinsic regulatory role in cellular stress responses, such as oxidative stress and heat shock, suggesting that OLA1 may have important pathophysiological functions. However, whether it plays a role in vascular endothelial leakage has not been studied. Summary of the Invention

[0004] Technical problems to be solved:

[0005] In view of the deficiencies of the prior art, the present application solves the technical problems that many signaling pathways that promote vascular permeability can also be triggered during diseases, leading to prolonged or uncontrolled vascular leakage, and whether human OLA1 plays a role in vascular endothelial leakage has not been studied. The present application provides the application of OLA1 in the preparation of drugs for inhibiting vascular endothelial permeability, providing a feasible strategy for the preparation of drugs to solve vascular permeability diseases.

[0006] Technical solutions:

[0007] To achieve the above object, the present application is realized through the following technical solutions:

[0008] The application of OLA1 in the preparation of drugs for inhibiting vascular endothelial permeability.

[0009] Further, the drug for inhibiting vascular endothelial permeability is an injection form or a non-injection form.

[0010] Furthermore, the drug for inhibiting vascular endothelial permeability is an injection, tablet, capsule, powder, pill, granule, solution, suspension, syrup, suppository or inhalant.

[0011] Furthermore, the drug for inhibiting vascular endothelial permeability includes an OLA1 inhibitor and pharmaceutically acceptable excipients.

[0012] Furthermore, the excipients include one or several of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants.

[0013] Principle explanation: In the Ola1 gene knockout mouse model of this application, the role of OLA1 in vascular leakage was studied: In the established Ola1 gene knockout mouse model, Evans Blue was administered by tail vein injection to Ola1 gene knockout mice and normal mice, and the permeability of tissues such as the heart, liver, spleen, lungs, kidneys, brain, and intestine was compared with that of normal mice to draw a conclusion and determine whether the role of OLA1 in vascular endothelial permeability is inhibitory or promoting under physiological conditions; in primary HUVECs cells, OLA1 overexpression and knockout plasmids were infected by virus methods to construct knockout / overexpression OLA1 cell lines, and immunofluorescence, fluorescein scratch, and chamber migration experiments were used to detect endothelial cell permeability; the effect of knocking out OLA1 on thrombin-induced permeability was detected by cell immunofluorescence experiments.

[0014] Beneficial effects:

[0015] This application provides the use of OLA1 in the preparation of a drug for inhibiting vascular endothelial permeability. Compared with the prior art, it has the following beneficial effects:

[0016] 1. By tail vein injection of Evans Blue and collecting mouse tissues, it was found that the vascular permeability of Ola1 knockout mice was weakened compared with the control group;

[0017] 2. In the primary HUVECs cell knockout or overexpression OLA1 stable cell line, through immunofluorescence experiments, Transwell endothelial permeability measurement experiments, and fluorescein scratch experiments, it was found that in OLA1 knockout cells, the cell junctions were tighter, and in cells overexpressing OLA1, the intercellular permeability was higher;

[0018] 3. In vivo results showed that the vascular endothelial permeability of Ola1 gene knockout mice was significantly weakened;

[0019] 4. Through immunofluorescence experiments, it was found that knocking out OLA1 could reduce thrombin-induced permeability. Description of the Drawings

[0020] Figure 1 This is a figure showing that ola1 was knocked out in this application, resulting in weakened vascular permeability in the major organs of mice.

[0021] Figure 2 This is a figure showing the effect of knocking out OLA1 in primary HUVECs of this application on permeability; among them, A is a figure showing the transfection efficiency of knocking out OLA1 detected in primary HUVECs, and B is a figure of cell immunofluorescence assay.

[0022] Figure 3 This is a figure showing the effect of knocking out OLA1 in primary HUVECs of this application on permeability; among them, A is a figure of cell scratch fluorescence dye experiment, and B is a statistical chart describing the change in the diffusion area of the fluorescence dye.

[0023] Figure 4 This is a figure showing the effect of knocking out OLA1 in primary HUVECs of this application on permeability; among them, A is a schematic diagram of Transwell FITC-Dextran cell permeability experiment after knocking out OLA1 in primary HUVECs, and B is a figure of measuring the fluorescence value of PBS in the lower chamber with an enzyme-labeled instrument.

[0024] Figure 5 This is a figure showing the effect of overexpressing OLA1 in primary HUVECs of this application on permeability; among them, A is a figure showing the transfection efficiency of overexpressing OLA1 detected in primary HUVECs, and B is a figure of cell immunofluorescence assay.

[0025] Figure 6 This is a figure showing the effect of overexpressing OLA1 in primary HUVECs of this application on permeability; among them, A is a figure of cell scratch fluorescence dye experiment, and B is a statistical chart describing the change in the diffusion area of the fluorescence dye.

[0026] Figure 7 This is a figure showing the effect of overexpressing OLA1 in primary HUVECs of this application on permeability; among them, A is a schematic diagram of Transwell FITC-Dextran cell permeability experiment after overexpressing OLA1 in primary HUVECs, and B is a figure of measuring the fluorescence value of PBS in the lower chamber with an enzyme-labeled instrument.

[0027] Figure 8 This is a figure showing the effect of knocking out the OLA1 gene in primary HUVECs of this application on thrombin-mediated permeability, specifically a figure of cell immunofluorescence staining. Detailed implementation mode

[0028] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and equivalent changes or modifications also fall within the scope defined by the claims of this application.

[0029] Example 1:

[0030] An application of OLA1 in the preparation of drugs for inhibiting vascular endothelial permeability, preparation of experimental materials:

[0031] 1. The electrophoresis instrument was purchased from Liuyi Instrument Factory in Beijing, China;

[0032] 2. FITC-dextran (70 kDa) was purchased from Sigma Company;

[0033] 3. ECM medium was purchased from Sciencell Company;

[0034] 4. DMEM was purchased from HyClone Company;

[0035] 5. RNA isolater was purchased from Novizan Biotech Co., Ltd. in Nanjing;

[0036] 6. VE-cadherin was purchased from Santa Cruz Company;

[0037] 7. Flag antibody was purchased from sigma company;

[0038] 8. OLA1 antibody was purchased from Santa Cruz Company;

[0039] Implementation method:

[0040] 1. Evans blue dye (EBD) was used to detect tissue permeability

[0041] (1) Prepare a 0.5% sterile Evans blue solution with PBS;

[0042] (2) Draw 200 μL of Evans blue solution into a syringe, avoiding air bubbles entering the syringe;

[0043] (3) After weighing 4 pairs of male Tek Cre Ola1 fl / fl and Ola1 fl / fl mice at 8-12 weeks old, place them in a restraint device so that the animals cannot move freely, and only the tails are exposed outside;

[0044] (4) Expose the mouse tail under a 150 W infrared heating lamp for about 1 min to dilate the tail vein;

[0045] (5) Hold the mouse tail with the left thumb and index finger, and insert the needle parallel to the mouse tail into the tail vein with the right hand, and slowly inject 200 μL of Evans blue solution;

[0046] (6) After the injection, put the mouse back into the cage and observe for 30 min;

[0047] (7) Sacrifice the mouse by cervical dislocation, place the mouse dorsal side down, and nail the feet to the whiteboard;

[0048] (8) Open the abdominal cavity (liver, spleen, kidney, brain, intestine) and thoracic cavity (heart, lung) to expose the thoracic and abdominal organs;

[0049] (9) Take photos of the organs with differential changes, collect the heart, liver, spleen, lung, kidney, brain, and intestine of the mouse and place them in a 1.5 mL EPP tube, and weigh the tissues;

[0050] (10) Cut the tissues into pieces and add 1 mL of formamide to each tissue organ;

[0051] (11) Put all the tissues into a 55 °C water bath and incubate for 48 h to extract Evans blue from the tissues;

[0052] (12) Centrifuge the formamide / Evans blue mixture to precipitate the tissue fragments;

[0053] (13) Use 1 mL of formamide as the blank group and measure the absorbance at 610 nm;

[0054] (14) Calculate the extravasated Evans Blue per mg of tissue.

[0055] Figure 1 This is the figure showing that the function of ola1 is impaired by knockout in this application, and the vascular permeability of the main organs in the mouse body is weakened, and the vascular permeability of the mouse tissue organs. Select Ola1 male mice aged 8 - 12 weeks fl / fl and Tek Cre Ola1 fl / fl 4 male mice each, and then inject Evans blue into the tail vein to observe the permeability of the lung, liver, and kidney.

[0056] Example 2:

[0057] Establishment of primary HUVEC overexpression and knockout OLA1 stable cell lines:

[0058] Implementation method:

[0059] Day1:

[0060] Seed 293T cells into a 6-well plate and wait until the cell density reaches 70% - 80% for transfection.

[0061] Before transfection, replace the old medium of 293T cells with 1 mL of opti-MEM serum-free medium;

[0062] Prepare Mix1: Take corresponding 1.5 mL Eppendorf tubes according to the number of plasmids. Add 100 μL of opti-MEM serum-free medium to each tube, and add 2 μg of plasmid DNA, 1.5 μg of A, and 0.5 μg of G (4:3:1) respectively. Mix well and let stand at room temperature for 5 min;

[0063] Prepare Mix2: Take corresponding 1.5 mL Eppendorf tubes according to the number of plasmids. Add 100 μL of opti-MEM serum-free medium to each tube, and add

[0064] 3 μL of lipo8000. Mix well and let stand at room temperature for 5 min;

[0065] Mix Mix1 and Mix2, and let stand at room temperature for 20 min;

[0066] Add the above mixture to the 293T medium and gently shake to mix;

[0067] After 6 h, replace it with complete medium;

[0068] Day2:

[0069] Seed the cells to be transfected in a six-well plate. When the density is about 50%, prepare for transfection;

[0070] Day3:

[0071] Take out the primary HUVECs cells to be infected, replace with 1 mL of ECM complete medium, and add 10 μL (2 mg / mL)

[0072] polybrene;

[0073] Aspirate the 48 h virus supernatant into a centrifuge tube, centrifuge at 3500 rpm for 5 min, then filter out the cell debris in the supernatant with a 0.45 μm filter. Add 1.5 mL of virus stock solution to the primary HUVECs cells, gently shake and place in the incubator;

[0074] After 16 h of infection, replace with fresh ECM medium;

[0075] After culturing for 72 h, add puro (1 μg / ml) to screen the cells until the untransfected cells die (about

[0076] 7 days);

[0077] When the transfection efficiency of the cells is relatively high after screening, extract cell proteins and detect the infection efficiency by WB.

[0078] Example 3:

[0079] Implementation method:

[0080] Cell immunofluorescence experiment:

[0081] Day1: Preparation of coverslips:

[0082] (1) After disinfecting the small round coverslips, place them in a 24-well plate;

[0083] (2) After digesting primary HUVECs cells, transfer them into a 15 mL centrifuge tube. After centrifugation, discard the supernatant, resuspend with 1 mL of medium, and take 10 μL of the cell suspension for counting;

[0084] (3) Count the density of primary HUVECs cells as 5x10 4 cells / well, plate them in the 24-well plate with small round coverslips, and incubate in an incubator at 37°C with 5% CO2.

[0085] Day2: Fixation of cells and incubation with primary antibody:

[0086] (4) After the cells adhere and grow confluently, take the 24-well plate to the outer laboratory;

[0087] (5) Washing: Discard the medium, and gently wash away the residual culture medium and dead cells with PBS;

[0088] (6) Fixation: Add 500 μL of 4% paraformaldehyde to each well and fix in the dark for 20 min;

[0089] (7) Washing: Discard the paraformaldehyde, wash three times with PBS, and shake on a shaker at room temperature for 10 min each time;

[0090] (8) Blocking: Block with 5% bovine serum albumin (BSA) at room temperature for 1 - 2 h;

[0091] (9) Incubation with primary antibody: Stick a sealing film in a wet box, select the primary antibody VE-cadherin (1:1000), dilute it with 5% BSA, drop 20 μL of the antibody on the sealing film for each well, gently take out the coverslip with forceps, invert it on the antibody, make the antibody cover the coverslip completely without generating bubbles. Finally, place the wet box in the refrigerator at 4°C overnight;

[0092] Day3: Incubation with secondary antibody and mounting:

[0093] (10) Washing: Recover the primary antibody, gently place the coverslip in the 24-well plate, wash 3 times with PBS on a shaker at room temperature for 10 min each time;

[0094] (11) Incubate with secondary antibody: Start the operation in the dark from this step. In a dark environment, in a humid box covered with sealing film, add 20 μL of the secondary antibody corresponding to the primary antibody. Invert the slide on the sealing film with the added secondary antibody and incubate in the dark for 1 h;

[0095] (12) Wash: Recover the secondary antibody under light-proof conditions. Gently place the slide in a 24-well plate and wash it 3 times with PBS on a shaker at room temperature for 10 min each time;

[0096] (13) Mount the slide: Add 10 μL of the mounting medium on the glass slide. Take out the slide, gently blot the moisture with filter paper, invert it on the glass slide, and finally apply nail polish around the cover glass to fix it.

[0097] After drying, take fluorescence images with a laser confocal microscope.

[0098] Example 4:

[0099] Implementation method:

[0100] Cell fluorescence scratch assay:

[0101] (1) First, culture primary HUVECs cells in a 35 mm dish until the cell density reaches 75 - 85%;

[0102] (2) Take out the cells from the incubator, gently pour out the culture medium or siphon it with a pipette into a waste container, discard the culture medium, and wash 3 times with PBS to wash away the residual culture medium;

[0103] (3) Under light-proof conditions, add 1 mL of preheated (37 °C) 0.05% fluorescein yellow dye and cut three times with a surgical blade;

[0104] (4) Let it stand at room temperature in the dark for 3 min;

[0105] (5) Recover the fluorescein yellow dye (it can be reused about ten times), wash 3 times with PBS to wash away the residual fluorescein yellow dye;

[0106] (6) Add 1 mL of 4% paraformaldehyde and fix it in the dark for 10 min;

[0107] (7) Take pictures of the diffusion degree of the fluorescein yellow dye and take photos;

[0108] (8) Use Image J software to statistically calculate the taken photos and finally analyze them with GraphPad Prism 9.

[0109] Example 5:

[0110] Implementation method:

[0111] Transwell endothelial permeability assay:

[0112] (1) Select a filter membrane of a polyester (PET) transparent insert chamber in a 24-well Transwell plate (the diameter of the insert chamber is 6.5 mm, and the diameter of the filter membrane pores is 0.4 μm). Count the primary HUVECs cells at 3x10 4 and seed them on the filter membrane of the insert chamber. After adding 200 μL of complete medium to the upper chamber and 750 μL of complete medium to the lower chamber, place it in a 37°C, 5% CO2 incubator and grow for 2 - 3 days to ensure that the cells fuse into a single cell layer;

[0113] (2) After the cells grow and fuse into a single cell layer, take the cells out of the incubator, wash the single cell layer with PBS, then add 200 μL of fresh ECM medium to the upper chamber and add FITC dextran at a final concentration of 1 mg / mL;

[0114] (3) Replace the lower chamber with 700 μL of PBS;

[0115] (4) Incubate in the dark at 37°C, 5% CO2 incubator for 2 h;

[0116] (5) After about 2 h, take the PBS from the lower chamber and add it to a black 96-well plate in the dark (three replicates for each sample), and then put it into a microplate reader to measure the fluorescence value. The excitation wavelength is 492 nm and the emission wavelength is 520 nm.

[0117] As Figure 1 shown, Figure 1 This is a graph showing that knocking out the ola1 gene in this application impairs the OLA1 function, and the vascular permeability of the lungs, liver, kidneys, and skin of the mice is weakened;

[0118] As Figure 2 shown, Figure 2 This is a graph showing the effect of knocking out OLA1 in primary HUVECs in this application on permeability; among them Figure 2 A in it is a graph showing the transfection efficiency of knocking out OLA1 detected in primary HUVECs cells. The expression level of OLA1 is detected by extracting cell proteins through Western Blot, Figure 2 B in it is a graph of cell immunofluorescence assay. In the cells with OLA1 knocked out in primary HUVECs, immunofluorescence staining is performed using 1:1000 VE-cadherin to observe the changes in cell-cell junctions.

[0119] As Figure 3 shown, Figure 3 This is a graph showing the effect of knocking out OLA1 in primary HUVECs in this application on permeability; among them Figure 3In Figure A, it is the experimental diagram of cell scratch fluorescence dye. The scratch fluorescence dye experiment was carried out in primary HUVECs cells with OLA1 knocked out. The diffusion area of the fluorescence dye was recorded 3 minutes after scratching. There were three pictures in each group, and Image J was used to analyze the change in the diffusion area of the fluorescence dye. In Figure B, it is the statistical chart of the change in the diffusion area of the fluorescence dye analyzed by ImageJ. The statistical software was GraphPad Prism 9, the statistical method was the T-test, and the data were expressed as mean ± standard deviation. *P < 0.05, **P < 0.01.

[0120] As Figure 4 shown, Figure 4 This is the figure showing the effect of knocking out OLA1 in primary HUVECs of this application on permeability; among them Figure 4 In Figure A, it is a schematic diagram of the Transwell FITC-Dextran cell permeability experiment after knocking out OLA1 in primary HUVECs cells. Figure 4 In Figure B, it is the figure of measuring the fluorescence value of PBS in the lower chamber by an enzyme-linked immunosorbent assay. Take 100 μL in a 96-well plate, with two replicates in each group. The permeability of the cells was measured by measuring the fluorescence value of FITC-Dextran that penetrated into the lower chamber in an enzyme-linked immunosorbent assay. (*P < 0.05, ***P < 0.001).

[0121] As Figure 5 shown, Figure 5 This is the figure showing the effect of overexpressing OLA1 in primary HUVECs of this application on permeability; among them Figure 5 In Figure A, it is the figure of detecting the transfection efficiency of overexpressing OLA1 in primary HUVECs cells. The expression level of OLA1 was detected by extracting cell proteins through Western Blot. Figure 5 In Figure B, it is the figure of cell immunofluorescence assay. In primary HUVECs cells with overexpressed OLA1, immunofluorescence staining was performed using 1:1000 VE-cadherin to observe the change in cell-cell junctions.

[0122] As Figure 6 shown, Figure 6 This is the figure showing the effect of overexpressing OLA1 in primary HUVECs of this application on permeability; among them Figure 6 In Figure A, it is the experimental diagram of cell scratch fluorescence dye. The scratch fluorescence dye experiment was carried out in primary HUVECs cells with overexpressed OLA1. The diffusion area of the fluorescence dye was recorded 3 minutes after scratching. There were three pictures in each group, and Image J was used to analyze the change in the diffusion area of the fluorescence dye. Figure 6 In Figure B, it is the statistical chart describing the change in the diffusion area of the fluorescence dye. The statistical software was Prism 9, the statistical method was the T-test, and the data were expressed as mean ± standard deviation. *P < 0.05, **P < 0.01.

[0123] As Figure 7 shown, Figure 7 This is a diagram showing the effect of overexpressing OLA1 in primary HUVECs of this application on permeability; among them Figure 7 A in it is a schematic diagram of the Transwell FITC-Dextran cell permeability experiment after overexpressing OLA1 in primary HUVECs cells. Figure 7 B in it is a diagram showing the measurement of fluorescence value of PBS in the lower chamber by an enzyme-linked immunosorbent assay. Take 100 μL in a 96-well plate, with two replicates in each group, and measure the cell permeability by measuring the fluorescence value of FITC-Dextran that has permeated into the lower chamber in an enzyme-linked immunosorbent assay. (*P < 0.05, ***P < 0.001).

[0124] As Figure 8 shown, Figure 8 This is a diagram showing the effect of knocking out the OLA1 gene in primary HUVECs of this application on thrombin-mediated permeability. Specifically, it is an immunofluorescence staining diagram. In the cells of primary HUVECs with OLA1 knocked out, immunofluorescence staining is performed using 1:1000 VE-cadherin to observe the changes in cell-cell junctions.

[0125] The above has made an exemplary description of the present invention. It should be noted that without departing from the core of the present invention, any simple deformation, modification, or equivalent replacement that can be made by those skilled in the art without creative labor falls within the protection scope of the present invention.

Claims

1. Use of OLA1 in the preparation of a drug for inhibiting vascular endothelial permeability.

2. Use of OLA1 according to claim 1 in the preparation of a drug for inhibiting vascular endothelial permeability, characterized in that: The drug for inhibiting vascular endothelial permeability is in injection form or non-injection form.

3. Use of an OLA1 according to claim 1 in the preparation of a drug for inhibiting vascular endothelial permeability, characterized in that: The drug for inhibiting vascular endothelial permeability is an injection, tablet, capsule, powder, pill, granule, solution, suspension, syrup, suppository or inhalant.

4. Use of an OLA1 as claimed in claim 1 in the preparation of a drug for inhibiting vascular endothelial permeability, characterized in that: The drug for inhibiting vascular endothelial permeability comprises an OLA1 inhibitor and a pharmaceutically acceptable excipient.

5. Use of an OLA1 in the preparation of a drug for inhibiting vascular endothelial permeability according to claim 1, characterized in that: The excipient comprises one or more of a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, surfactant, adsorbent carrier, lubricant.

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