Application of 3, 5-dihydroxy-4-methoxybenzyl alcohol in preparation of medicine for treating H / R injury of vascular endothelial cells

Abstract

The invention discloses application of 3, 5-dihydroxy-4-methoxybenzyl alcohol or pharmaceutical salt thereof in preparation of drugs for treating vascular endothelial cell H / R injury, in-vitro antioxidant experiments confirm that the compound has antioxidant ability, which indicates that the compound has potential application value in the aspect of inhibiting reactive oxygen species (ROS) as an antioxidant agent. Furthermore, it is found that 3, 5-dihydroxy-4-methoxybenzyl alcohol has oxidative stress vascular endothelial cell H / R damage, and 3, 5-dihydroxy-4-methoxybenzyl alcohol can be used for preparing the medicine for treating vascular endothelial cell H / R damage.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to application of 3,5-dihydroxy-4-methoxybenzyl alcohol in preparing a drug for treating H / R injury of vascular endothelial cells. Background Art

[0002] Ischemia / reperfusion (I / R) refers to the process in which tissues and organs receive blood supply again after blood is blocked. Ischemia leads to insufficient oxygen and nutrient supply to tissues or organs, resulting in cell damage and death. When blood flows back to the ischemic area again, the metabolic abnormalities and free radical accumulation caused by ischemia may further cause cell and tissue damage, which is called I / R injury. This kind of injury occurs in various organ diseases or clinical treatment processes in the human body, including myocardial infarction, thrombolytic therapy for stroke, and transplantation of organs such as heart, kidney, and liver. I / R injury is generally manifested as tissue and organ necrosis, hemorrhage, edema, inflammatory response, organ dysfunction, etc., among which the injury mechanism includes oxidative stress caused by increased production of reactive oxygen species (ROS), inflammatory response, cell death (programmed apoptosis, cell necrosis, spontaneous death), endothelial cell dysfunction, etc. The endothelial cell hypoxia / reoxygenation (H / R) model is a classic experimental cell model used to simulate ischemia / reperfusion (I / R) injury and related disease processes of endothelial cells. This model has the characteristics of simple operation, intuitive results, and good repeatability, and can accurately reflect the characteristics of ischemia-reperfusion injury at the cellular level.

[0003] The giant oyster (Crassostrea gigas) is a sessile marine mollusk that lives in the intertidal zone. It has strong adaptability due to its wide tolerance to the environment and is generally considered to be an ideal model for studying stress resistance biological patterns. The latest research has found that there is a new type of antioxidant molecule in oysters, namely 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), which shows excellent antioxidant properties. The application of DHMBA in the preparation of drugs for the treatment of vascular endothelial cells suffering from hypoxia / reoxygenation (H / R) injury has not been reported yet. Summary of the invention

[0004] The purpose of the present invention is to provide the use of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) in preparing a drug for treating H / R injury of vascular endothelial cells.

[0005] The present invention is achieved through the following technical solutions: Application of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) or its pharmaceutically acceptable salt in preparing a drug for treating H / R injury of vascular endothelial cells.

[0006] The structural formula of 3,5-dihydroxy-4-methoxybenzyl alcohol is as follows:

[0007] The medicine contains pharmaceutically usable auxiliary materials and is made into various dosage forms, including liquid and solid dosage forms.

[0008] The dosage forms include tablets, capsules, oral liquids, lozenges, granules, granules, pills, powders, ointments, pills, suspensions, powders, injections, suppositories, sprays, drops or patches.

[0009] Preferably, the concentration of the drug 3,5-dihydroxy-4-methoxybenzyl alcohol is 125-1000 μM, more preferably 250-1000 μM, and most preferably 500 μM.

[0010] A medicine for treating H / R injury of vascular endothelial cells contains 3,5-dihydroxy-4-methoxybenzyl alcohol or a pharmaceutically acceptable salt thereof as an active ingredient.

[0011] The beneficial effects of the present invention are as follows: In the in vitro antioxidant experiment, the present invention determined that 3,5-dihydroxy-4-methoxybenzyl alcohol DHMBA has antioxidant ability through the DPPH experiment; in the oxygen free radical absorption capacity (ORAC) experiment, it was found that the oxygen free radical absorption capacity of 3,5-dihydroxy-4-methoxybenzyl alcohol DHMBA was higher than Trolox and L-ascorbic acid, and only slightly weaker than chlorogenic acid; DHMBA had a dose-dependent inhibitory effect on copper-mediated LDL oxidation in the concentration range of 180 ~ 540 μM. Although there are only in vitro antioxidant results, it shows that 3,5-dihydroxy-4-methoxybenzyl alcohol DHMBA has potential application value in inhibiting reactive oxygen species (ROS) as an antioxidant. Further, the present invention found that 3,5-dihydroxy-4-methoxybenzyl alcohol can treat H / R damage of vascular endothelial cells in the presence of oxidative stress, and can be used to prepare drugs for treating H / R damage of vascular endothelial cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is the experimental grouping and the administration steps of DHMBA and signal pathway regulators.

[0013] Figure 2 The CCK-8 method in Example 2 was used to detect the effect of DHMBA on the survival rate of EA·hy926 cells after H / R. The results are expressed as mean ± SD, n = 3, * Indicates comparison with ControlP <0.05; # Compared with H / R group P <0.05.

[0014] Figure 3 The effect of DHMBA on LDH leakage after H / R in EA·hy926 cells in Example 3. The results are expressed as mean ± SD, n = 5, * Compared with the Control group P <0.05; # Compared with H / R group P <0.05; ▲ Compared with H / R group +125 μM P <0.05; & Compared with H / R group + 250 μM group P <0.05.

[0015] Figure 4 The effect of DHMBA on the changes of total glutathione (left) and GSH / GSSG ratio (right) in EA·hy926 cells induced by H / R was detected by UV spectrophotometry in Example 4. The results are expressed as mean ± SD, n = 3; * Compared with the Control group P <0.05; # Compared with H / R group P <0.05.

[0016] Figure 5 The ELISA method in Example 5 was used to detect the effect of DHMBA on the content of inflammatory factors in EA·hy926 cells after H / R. (A left) TNF-α content, n = 4; (B) IL-6 content, n = 3. The results are expressed as mean ± SD. * Compared with the Control group P <0.05; # Compared with H / R group P <0.05.

[0017] Figure 6 The effect of DHMBA on the migration ability of H / R EA·hy926 cells was detected by the cell scratch test in Example 6. The results are expressed as mean ± SD, n = 3; * Comparison with Control P <0.05; # Compared with H / R group P <0.05 (×100, Bar = 500 μm).

[0018] Figure 7 The effect of DHMBA on the angiogenesis ability of EA·hy926 cells after H / R was detected by the tube formation experiment in Example 7. The first row of left pictures shows the number of cell branches, the first row of right pictures shows the length of cell branches, the second row of left pictures shows the number of meshes, and the second row of right pictures shows the number of nodes. The results are expressed as mean ± SD, n = 5. * Comparison with Control P <0.05; # Compared with H / R group P <0.05 (×100, Bar = 500 μm).

[0019] Figure 8 The effect of DHMBA on the level of ROS in EA·hy926 cells after H / R was detected by flow cytometry in Example 8. The results are expressed as mean ± SD, n = 6, * Compared with the Control group P <0.05; # Compared with H / R group P <0.05.

[0020] Fig. 9 DHMBA in Example 9 antagonizes H / R injury and dysfunction of EA·hy926 cells by reducing ROS levels. (A) Flow cytometry was used to detect ROS levels; (B) Cell scratch assay was used to detect impaired endothelial migration function; (C) Spectrophotometry was used to detect LDH leakage. The results are expressed as mean ± SD, n = 5. * Compared with the Control group P <0.05, # Compared with H / R group P <0.05, & Compared with H / R + 500 μM DHMBA group P <0.05 (×100, Bar = 500 μm). DETAILED DESCRIPTION

[0021] The following is a further description of the present invention, rather than a limitation of the present invention.

[0022] Example 1: Preparation before experiment The anoxic solution was prepared as follows: 8.0063 g NaCl, 0.8946 g KCl, 0.1 g MgCl2·6H2O, 0.1 g CaCl 2、 Mix 0.9532 g HEPES and 3.735 mL C3H5O3Na, and add ultrapure water to 1 L.

[0023] EA·hy926 cells (human umbilical vein cell fusion cells) are grown for 2-3 days until the bottom of the dish is covered by 80%-90%, and then they can be used to construct the H / R model; Figure 1 As shown, cells were divided into the following groups: (1) Control group: The cells did not undergo H / R modeling. During the drug incubation period and the entire modeling process, the cells were replaced with DMEM culture medium containing 1% fetal bovine serum (FBS) and placed in a cell culture incubator.

[0024] (2) H / R group: The hypoxic solution was pre-filled with nitrogen for 20 min to exhaust the dissolved oxygen, and the culture medium was replaced with the hypoxic solution. After being placed in an anaerobic workstation (37°C, 1% O2, 5% CO2) for 3 h, the reoxygenation solution (DMEM culture medium containing 1% FBS) was replaced and the cells were returned to the cell culture incubator for another 1 h. The H / R model was then established.

[0025] (3) H / R + DHMBA group: DHMBA (3,5-dihydroxy-4-methoxybenzyl alcohol) was diluted to the required concentration (125 μM, 250 μM, 500 μM, 1000 μM) in DMEM medium containing 1% FBS and pre-incubated for 12 h. When the H / R model was constructed later (see step (2)), DHMBA of corresponding concentrations was also added to the hypoxic and reoxygenated solutions.

[0026] (4) H / R + edaravone (Eda) group: Eda was diluted to 200 μM in DMEM medium containing 1% FBS, and the cells were pre-treated and incubated for 24 h before the H / R model was established.

[0027] (5) H / R + DHMBA + oligomycin A (OA) group: DHMBA was first diluted to 500 μM in DMEM medium containing 1% FBS, and the cells were pre-incubated for 12 h. OA was then diluted to 10 μM in DMEM medium containing 1% FBS, and the cells were pre-incubated for 30 min before the H / R model was constructed.

[0028] DHMBA preparation and storage: DHMBA is dissolved in DMSO, the stock solution concentration is 1M, and it is stored at -80 ℃ in the dark. It is diluted to the final concentration when used, and the experiment is kept in the dark. The stock solution for temporary use is stored in a -20 ℃ refrigerator.

[0029] Embodiment 2: EA·hy926 cells were seeded in 96-well plates at a seeding density of 5×10 4 / mL, 100 μL per well, placed in the incubator, and proceed to the next experimental operation after growing for 24 hours; The experiment was divided into Control group, H / R group, and H / R + DHMBA group. The treatment of each group was as shown in Example 1. Five replicate wells were set up for each group, with 100 μL in each well, and the cells were returned to the incubator for incubation for 12 h. After the H / R model was established, a mixture of CCK-8 reagent and serum-free DMEM at a ratio of 1:10 was added, incubated in the dark, and then tested according to the above aspects.

[0030] The experimental results are as follows Figure 2 As shown, from Figure 2 It can be seen that H / R significantly reduced the viability of cells, while DHMBA could improve the decreased cell activity caused by H / R in a dose-dependent manner, and showed significant improvement at a concentration of 250-1000 μM.

[0031] Example 3 The composition of LDH detection working solution is as follows:

[0032] Reagents must be prepared immediately before use and must be kept away from light.

[0033] Experimental steps: (1) EA·hy926 cells were seeded in a 96-well plate at a seeding density of 5×10 4 / mL, cultured for 24 h, and when the cells grew to 80% confluence, they were divided into experimental groups: Control group, H / R group, H / R + DHMBA group. The treatment of each group is shown in Example 1.

[0034] (2) After the H / R model is completed, 120 μL of the culture solution from each well is transferred to a new 96-well plate, and 60 μL of LDH detection working solution is added to each well; (3) Mix well, incubate at room temperature in the dark for 30 min, and measure the absorbance at 490 nm. Use 600 nm as the reference wavelength for dual-wavelength measurement, and use the difference in absorbance to compare the release of LDH in each group of cells.

[0035] The experimental results are as follows Figure 3 As shown, from Figure 3 It can be seen that H / R can lead to a significant release of LDH, while DHMBA can inhibit the release of LDH caused by H / R in a dose-dependent manner, and shows significant inhibitory ability at a concentration of 250-1000 μM.

[0036] Example 4 Reagent configuration

[0037] Preparation of oxidized glutathione (GSSG) stock solution (10 mM): Dissolve 5 g GSSG in 816 μL deionized water and mix well. Aliquot and store at -20 °C.

[0038] Preparation of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) stock solution: Dissolve 4.5 mg DTNB and 1.5 mL DMSO and mix well. Aliquot and store at -20 °C.

[0039] Preparation of Protein Removal Reagent M (5%): Mix 0.2 g of Protein Removal Reagent with 4 mL of Total Glutathione Assay Buffer, prepare fresh and use on the same day.

[0040] Preparation of NADPH stock solution (40 mg / mL): Dissolve 4 mg NADPH in 100 μL deionized water, mix well, and then divide into aliquots and store at -80 ℃.

[0041] Preparation of 1× glutathione reductase: 5× glutathione reductase 50 μL and total glutathione assay buffer 200 μL are freshly prepared and used on the same day.

[0042] Preparation of total glutathione detection working solution: Mix 132 μL of 1× glutathione reductase, 132 μL of DTNB stock solution and 3 mL of total glutathione detection buffer.

[0043] Preparation of 0.5 mg / mL NADPH: Mix 10 μL of NADPH stock solution with 790 μL of total glutathione detection buffer. Preparation of GSH scavenging auxiliary solution: Mix 53 μL of GSH scavenging auxiliary stock solution with 47 μL of deionized water.

[0044] Preparation of GSH scavenging reagent working solution: 10.8 μL of GSH scavenging reagent and 89.2 μL of anhydrous ethanol were freshly prepared and used on the same day.

[0045] GSSG standards (10 mM GSSG stock solution diluted with protein removal reagent M), numbered AF, see the table below:

[0046] Experimental procedures 1. Inoculate cells in a 7 cm dish at a density of 1×10 5 / mL, cultured for 24 h until the cells grew to 80% confluence; experimental groups: Control group, H / R group, H / R + DHMBA group, the treatment of each group is shown in Example 1.

[0047] 2. After the cells were treated accordingly, they were digested and collected using 0.125% trypsin-EDTA, washed once with PBS, and centrifuged to collect the cells (1000 rpm, 3 min), and the supernatant was discarded; 3. Add protein removal reagent M 3 times the volume of cell sediment and vortex thoroughly to mix; 4. The samples were rapidly frozen and thawed twice using liquid nitrogen and a 37°C water bath, placed in an ice bath for 5 min, and centrifuged (4°C, 10,000 g, 10 min). The supernatant was collected for the determination of total glutathione and GSSG content. 5. Sample preparation for determination of GSSG content: Add 1 / 5 volume of GSH scavenging auxiliary solution to the sample to be tested, vortex mix, then add 1 / 25 volume of GSH scavenging reagent working solution, vortex mix. Incubate at 25 °C for 60 min; 6. Add samples for total glutathione determination, samples for GSSG content determination and standards to the 96-well plate, add 150 μL of total glutathione detection working solution, mix well, and incubate at room temperature for 5 min; 7. Add 0.5% mg / mL NADPH solution, mix well, incubate at room temperature for 1 h, and then measure the absorbance at 412 nm using an ELISA reader; 8. Based on the total glutathione content and GSSG content obtained by measurement, calculate the GSH content and then calculate the GSSG / GSH value.

[0048] The experimental results are as follows Figure 4 As shown, from Figure 4 It can be seen that H / R does not lead to changes in the total amount of Glutathione, but can lead to a significant decrease in the ratio of GSH / GSSG. DHMBA at a concentration of 500 μM can significantly increase the ratio of GSH / GSSG.

[0049] Example 5 Reagent preparation

[0050] Preparation of TNF-α Standard

[0051] Prepare the standard within 2 hours before use. The diluted standard (10000 pg / mL) should be used within 12 hours. If stored at -20 ℃, it can be used within 2 days, but it should not be repeatedly frozen and thawed.

[0052] Preparation of biotin-labeled anti-human TNF-α antibody working solution: 10 μL biotin-labeled anti-human TNF-α and 990 μL antibody diluent should be prepared within 2 hours before use.

[0053] Preparation of avidin-peroxidase complex (ABC) working solution: 10 μL ABC and 990 μL ABC diluent should be prepared within 1 hour before use. Pre-warm at 37°C for at least 30 min before adding to the ELISA plate wells.

[0054] Preparation of 1× buffered washing solution: Mix 2 mL of 25× buffered washing solution and 48 mL of deionized water and use it immediately.

[0055] Experimental procedures 1. The cells were seeded in a 7 cm dish at a density of 1×10 5 cells / mL, 4 mL per dish, culture for 24 h until the cells grow to 80% confluence; 2. Experimental groups: Control group, H / R group, H / R + DHMBA group. The treatment of each group is shown in Example 1. 3. After the cells are treated accordingly, cell lysate is obtained according to the method described in 2.2.6, and the concentration of each group of proteins is measured; 4. Add 100 μL of the standard samples of each concentration prepared in the above steps to the wells in turn. The last well with only the sample diluent is used as the zero well, and an additional well is used as a blank color development well. Add 100 μL of the sample to be tested, cover with the sealing film, and incubate at 37 °C for 90 min. 5. Shake off the liquid in the ELISA plate, turn it upside down on absorbent paper, and tap it a few times to completely remove the residual liquid; add 100 μL of the prepared biotin anti-human TNF-α antibody working solution to each well (except the TMB blank color development well), cover with a sealing film, and incubate at 37 °C for 60 min; 6. Wash three times with 1× washing buffer, 300 μL per well, 1 min each time; add 100 μL of ABC working solution to each well (except TMB blank color well), cover with sealing film, and incubate at 37°C for 30 min; 7. Wash 5 times with 1× washing buffer, add 300 μL of washing buffer to each well, each time for 1 min; add 90 μL of TMB colorimetric solution to each well, and incubate at 37°C in the dark for 15 min; 8. Add 100 μL of stop solution to each well, and the blue color will turn yellow immediately. Use an enzyme-labeled instrument to measure the absorbance at a wavelength of 450 nm.

[0056] 9. Set the TMB blank color development well (add only TMB color development solution and stop solution) as the control. After subtracting the absorbance value of the TMB blank color development well from the absorbance values ​​of all standards and samples, draw a standard curve and calculate the TNF-α concentration of the corresponding sample based on the absorbance value of the sample.

[0057] ELISA method to detect the content of inflammatory factor IL-6 The experimental principle and experimental steps are basically the same as those for TNF-α detection, except that the TNF-α standard is replaced by the IL-6 standard; the biotin-labeled anti-human TNF-α antibody working solution is replaced by the biotin-labeled anti-human IL-6 antibody working solution.

[0058] The experimental results are as follows Figure 5 As shown, from Figure 5 It can be seen that H / R can significantly induce the expression of inflammatory factors TNF-α and IL-6, but DHMBA at a concentration of 500 μM can effectively reduce the induction effect of H / R on the two inflammatory factors TNF-α and IL-6.

[0059] Example 6 1. Count the cells using a cell counting plate and dilute the EA·hy926 cells to 1×10 5 / mL, inoculated in a 6-well plate, 2 mL in each well, placed in a cell culture incubator and cultured for 36 h. When the cells grew to 90%-95% confluence, they were divided into experimental groups: Control group, H / R group, H / R + DHMBA group. The treatment of each group is shown in Example 1.

[0060] 2. After the H / R model is completed, use a sterile 300 μL glass pipette tip to vertically scratch the same thickness in each well; 3. Wash off the cells detached during streaking with PBS and add DMEM medium containing 10% FBS; 4. Take a photo and mark the detailed position of the streaking line, record as 0 h. Place the cells in the incubator and continue to culture. After 24 h, observe the cell migration status at the streaking line, record as 24 h; 5. Use ImageJ software to calculate the cell scratch area and analyze the cell migration rate. Migration rate = (0 h cell scratch area - 24 h cell scratch area) / 0 h cell scratch area × 100%.

[0061] The experimental results are as follows Figure 6 As shown, from Figure 6 It can be seen that H / R can significantly reduce the migration distance of cells. DHMBA at a concentration of 500 μM can effectively enhance the inhibitory effect of H / R on cell migration, but there is no obvious improvement at a low concentration of 250 μM.

[0062] Example 7 1. Count the cells using a cell counting plate and dilute the EA·hy926 cells to 1×10 5100 μg / mL, inoculated in 3.5 cm dishes, 2 mL in each well, placed in an incubator and cultured for 24 h; experimental groups: Control group, H / R group, H / R + DHMBA group. The treatment of each group is shown in Example 1.

[0063] 2. Matrix gel plating: First, rinse the 24-well plate with pre-cooled PBS, add 200 μL of matrix gel to each well in a low-temperature environment, gently shake the plate to spread it evenly, and place it in a 37 ℃ environment. The matrix gel plating can be completed in 30 minutes; 3. After the H / R model was completed, the cells were digested and collected and inoculated on the pre-fixed matrix gel surface with an inoculation density of 5×10 4 / mL, 200 μL per well, shake evenly, and place in a cell culture incubator for 24 h; 4. Observe the cell tube formation and take photos for storage. Use ImageJ software to analyze the number of nodes, branches, meshes, total length of tubes, and total length of branches.

[0064] The experimental results are as follows Figure 7 As shown, from Figure 7 It can be seen that H / R can significantly reduce the number of cell branches, total branch length, number of meshes and number of nodes. DHMBA has a concentration-dependent inhibitory effect of H / R on the number of cell branches, total branch length, number of meshes and number of nodes, all of which are significantly improved at a concentration of 500μM.

[0065] 1. EA·hy926 cells were seeded in six-well plates at a seeding density of 1×10 5 / mL, culture for 24 h until the cells grow to 80% confluence; 2. Experimental groups: Control group, H / R group, H / R + DHMBA group. The treatment of each group is shown in Example 1.

[0066] 3. After the H / R model, wash twice with PBS and add 1 mL of serum-free 1640 medium containing 5 μM DCFH-DA probe to each well, and then incubate at 37 °C for 30 min. 4. After incubation, wash twice with PBS, digest the cells with 0.25% trypsin digestion solution and collect them into pre-cooled 1.5 mL centrifuge tubes and store them in an ice box; 5. Centrifuge each group of cell suspensions at 1000 rpm for 3 min, then wash with pre-cooled PBS to remove residual probes; 6. Continue centrifugation, discard the agar, add 400 μL PBS to the cell pellet, mix well, and use flow cytometry to detect the average fluorescence intensity under the FITC channel. If the cells cannot be detected in time, they need to be stored in an ice box and protected from light.

[0067] The experimental results are as follows Figure 8 As shown, from Figure 8 It can be seen that H / R can significantly activate the production of cellular ROS. DHMBA at a concentration of 500 μM can effectively inhibit the production of cellular ROS induced by H / R, but there is no obvious inhibitory effect at a low concentration of 250 μM.

[0068] Example 9 Streaming

[0069] (1) EA·hy926 cells were seeded in a six-well plate at a seeding density of 1×10 5 / mL, culture for 24 h, until the cells grow to 80% confluence; (2) Experimental groups: Control group, H / R group, H / R + DHMBA group, H / R + Eda group, H / R + DHMBA + OA group. The treatment of each group is shown in Example 1.

[0070] (3) After the H / R model, wash twice with PBS and add 1 mL of serum-free 1640 medium containing 5 μM DCFH-DA probe to each well, and then incubate at 37 °C for 30 min. (4) After incubation, wash twice with PBS, digest the cells with 0.25% trypsin digestion solution and collect them into pre-cooled 1.5 mL centrifuge tubes and store them in an ice box; (5) Centrifuge each group of cell suspensions at 1000 rpm for 3 min, then wash with pre-cooled PBS to remove residual probes; (6) Continue centrifugation, discard the agar, add 400 μL PBS to the cell pellet, mix well, and use flow cytometry to detect the average fluorescence intensity under the FITC channel. If the cells cannot be detected in time, they need to be stored in an ice box and protected from light.

[0071] Scratches (1) Count the cells using a cell counting plate and dilute the EA·hy926 cells to 1×10 5 / mL, inoculated in a 6-well plate, 2 mL in each well, placed in a cell culture incubator and cultured for 36 h. When the cells grew to 90%~95% confluence, they were divided into experimental groups: Control group, H / R group, H / R + DHMBA group, H / R + Eda group, H / R + DHMBA + OA group. The treatment of each group is shown in Example 1.

[0072] (2) After the H / R model is completed, use a sterile 300 μL glass pipette tip to vertically scratch the same thickness in each well; (3) Wash off the cells detached during streaking with PBS and add DMEM medium containing 10% FBS; (4) Take a photo and mark the detailed position of the streaking line, and record it as 0 h. Place the cells in the incubator and continue to culture. After 24 h, observe the cell migration status at the streaking line, and record it as 24 h. (5) The scratch area of ​​cells was calculated using ImageJ software and the cell migration rate was analyzed. Migration rate = (cell scratch area at 0 h - cell scratch area at 24 h) / cell scratch area at 0 h × 100%.

[0073] LDH The composition of LDH detection working solution is as follows:

[0074] Reagents must be prepared immediately before use and must be kept away from light.

[0075] Experimental procedures (1) EA·hy926 cells were seeded in a 96-well plate at a seeding density of 5×10 4 / mL, cultured for 24 h, and when the cells grew to 80% confluence, they were divided into experimental groups: Control group, H / R group, H / R + DHMBA group, H / R + Eda group, and H / R + DHMBA + OA group. The treatment of each group is shown in Example 1.

[0076] (2) After the H / R model is completed, 120 μL of the culture solution from each well is taken to a new 96-well plate, and 60 μL of LDH detection working solution is added to each well; mix well, incubate at room temperature in the dark for 30 min, and measure the absorbance at 490 nm. Use 600 nm as the reference wavelength for dual-wavelength measurement, and use the difference in absorbance to compare the release of LDH in each group of cells.

[0077] The experimental results are as follows Fig. 9 As shown, from Fig. 9 It can be seen that through comparative experiments, it can be found that 500 μM DHMBA and 200 μM Eda have similar effects in improving H / R-dependent LDH release, migration inhibition and ROS production, but this improvement effect of DHMBA can be blocked by 10 μM OA.

Claims

1. Use of 3,5-dihydroxy-4-methoxybenzyl alcohol or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating H / R injury of vascular endothelial cells.

2. The use according to claim 1, characterized in that: The medicine contains pharmaceutically usable auxiliary materials and is made into various dosage forms, including liquid and solid dosage forms.

3. The use according to claim 2, characterized in that: The dosage forms include tablets, capsules, oral liquids, lozenges, granules, granules, pills, powders, ointments, pills, suspensions, powders, injections, suppositories, sprays, drops or patches.

4. The use according to claim 1, characterized in that: The concentration of 3,5-dihydroxy-4-methoxybenzyl alcohol in the drug is 125-1000 μM.

5. The use according to claim 1, characterized in that: The concentration of 3,5-dihydroxy-4-methoxybenzyl alcohol in the drug is 250-1000 μM.

6. The use according to claim 1, characterized in that: The concentration of 3,5-dihydroxy-4-methoxybenzyl alcohol in the drug is 500 μM.

Images