Application of 3,5-dihydroxy-4-methoxybenzyl alcohol in preparing a drug for treating vascular endothelial cell H / R injury

Abstract

The present invention discloses the use of 3,5-dihydroxy-4-methoxybenzyl alcohol or its medicinal salt in the preparation of a drug for treating vascular endothelial cell H / R injury. In an in vitro antioxidant experiment, it was determined that this compound has antioxidant ability, indicating that this compound has potential application value as an antioxidant agent in inhibiting reactive oxygen species (ROS). Further, the present invention discovers that 3,5-dihydroxy-4-methoxybenzyl alcohol is present in the H / R injury of vascular endothelial cells with oxidative stress and can be used to prepare a drug for treating vascular endothelial cell H / R injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically to the application of 3,5-dihydroxy-4-methoxybenzyl alcohol in the preparation of a medicament for treating hypoxia / reoxygenation (H / R) injury of vascular endothelial cells. Background Art

[0002] Ischemia / reperfusion (I / R) refers to the process in which tissues and organs are reperfused after blood blockage. Ischemia results in insufficient oxygen and nutrient supply to tissues or organs, leading to cell damage and death. When blood flows back to the ischemic area again, due to metabolic abnormalities and free radical accumulation caused by ischemia, it 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 of the human body, including myocardial infarction, thrombolytic therapy for stroke, and transplantation of organs such as the heart, kidney, and liver. The general manifestations of I / R injury are tissue and organ necrosis, bleeding, edema, inflammatory response, organ dysfunction, etc. The injury mechanisms include oxidative stress, inflammatory response, cell death (programmed apoptosis, necrosis, autophagy) caused by an increase in the production of reactive oxygen species (ROS), and vascular endothelial cell dysfunction. The hypoxia / reoxygenation (H / R) model of vascular endothelial cells is a classic experimental cell model used to simulate the process of ischemia / reperfusion (I / R) injury of vascular endothelial cells and related diseases. 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 Pacific oyster (Crassostrea gigas) is a sessile marine mollusk living in the intertidal zone. It has extremely strong adaptability due to its wide tolerance to the environment and is generally considered an ideal model for studying anti-stress biological patterns. The latest research found that there is a new 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 a medicament for treating hypoxia / reoxygenation (H / R) injury of vascular endothelial cells has not been reported yet. Summary of the Invention

[0004] The object of the present invention is to provide the application of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) in the preparation of a medicament for treating hypoxia / reoxygenation (H / R) injury of vascular endothelial cells.

[0005] The present invention is achieved by the following technical solutions:

[0006] Use of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating hypoxia / reoxygenation (H / R) injury of vascular endothelial cells.

[0007] The structural formula of 3,5-dihydroxy-4-methoxybenzyl alcohol is as follows:

[0008]

[0009] The medicament contains pharmaceutically acceptable excipients and is made into various dosage forms, including liquid and solid dosage forms.

[0010] The dosage forms include tablets, capsules, oral liquids, buccal tablets, granules, infusion granules, pills, powders, ointments, pills, suspensions, powders, injections, suppositories, sprays, drops or patches.

[0011] Preferably, the concentration of 3,5-dihydroxy-4-methoxybenzyl alcohol in the medicament is 125 - 1000 μM, more preferably 250 - 1000 μM, and most preferably 500 μM.

[0012] A medicament for treating hypoxia / reoxygenation (H / R) injury of vascular endothelial cells, containing 3,5-dihydroxy-4-methoxybenzyl alcohol or a pharmaceutically acceptable salt thereof as an active ingredient.

[0013] The beneficial effects of the present invention are as follows: In the in vitro antioxidant experiment of the present invention, it was determined through the DPPH experiment that 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) has antioxidant ability; in the oxygen radical absorbance capacity (ORAC) experiment, it was found that the oxygen radical absorbance capacity of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) is higher than that of Trolox and L-ascorbic acid, and is only slightly weaker than that of chlorogenic acid; DHMBA has 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 as an antioxidant agent in inhibiting reactive oxygen species (ROS). Further, the present invention found that 3,5-dihydroxy-4-methoxybenzyl alcohol can treat hypoxia / reoxygenation (H / R) injury of vascular endothelial cells with oxidative stress and can be used to prepare a medicament for treating hypoxia / reoxygenation (H / R) injury of vascular endothelial cells. Description of the Drawings

[0014] Figure 1 It is the experimental grouping and the administration steps of DHMBA and signal pathway regulators.

[0015] Figure 2 It is the effect of CCK-8 method in Example 2 to detect the survival rate of EA.hy926 cells after H / R by DHMBA. The results are expressed as mean ± SD, n = 3,* Indicates comparison with Control P <0.05; # Indicates comparison with the H / R group P <0.05.

[0016] Figure 3 This is the effect of DHMBA on LDH leakage in EA.hy926 cells after H / R in Example 3. The results are expressed as mean ± SD, n = 5, * Indicates comparison with the Control group P <0.05; # Indicates comparison with the H / R group P <0.05; ▲ Indicates comparison with the H / R group + 125 μM P <0.05; & Indicates comparison with the H / R group + 250 μM group P <0.05.

[0017] Figure 4 This is the effect of DHMBA on the changes in total glutathione (left) and GSH / GSSG ratio (right) in EA.hy926 cells induced by H / R detected by ultraviolet spectrophotometry in Example 4. The results are expressed as mean ± SD, n = 3; * Indicates comparison with the Control group P <0.05; # Indicates comparison with the H / R group P <0.05.

[0018] Figure 5 This is the effect of DHMBA on the content of inflammatory factors in EA.hy926 cells after H / R detected by ELISA method in Example 5. (A left) Content of TNF-α, n = 4; (B) Content of IL-6, n = 3. The results are expressed as mean ± SD, * Indicates comparison with the Control group P <0.05; # Indicates comparison with the H / R group P <0.05.

[0019] Figure 6 This is the effect of DHMBA on the migration ability of H / R-treated EA.hy926 cells detected by the cell scratch assay in Example 6. The results are expressed as mean ± SD, n = 3; * Compared with Control P <0.05; # Compared with the H / R group P <0.05 (×100, Bar = 500 μm).

[0020] Figure 7 This is the experiment of detecting the effect of DHMBA on the angiogenesis ability of EA·hy926 cells after H / R in Example 7. Among them, the left figure in the first row is the number of cell branches, the right figure in the first row is the length of cell branches, the left figure in the second row is the number of meshes, and the right figure in the second row is the number of nodes. The results are expressed as mean ± SD, n = 5, * Compared with Control P <0.05; # Compared with the H / R group P <0.05 (×100, Bar = 500 μm).

[0021] Figure 8 This is the experiment of detecting the effect of DHMBA on the intracellular ROS level of EA·hy926 cells after H / R 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 the H / R group P <0.05.

[0022] Figure 9 This is the figure of DHMBA antagonizing H / R injury and dysfunction of EA·hy926 cells by reducing the ROS level in Example 9. (A) Detection of ROS level by flow cytometry; (B) Detection of impaired endothelial migration function by cell scratch assay; (C) Detection of LDH leakage by spectrophotometry. The results are expressed as mean ± SD, n = 5, * Indicates comparison with the Control group P <0.05, # Indicates comparison with the H / R group P <0.05, & Indicates comparison with the H / R + 500 μM DHMBA group P <0.05 (×100, Bar = 500 μm). Detailed implementation mode

[0023] The following is a further description of the present invention, rather than a limitation of the present invention.

[0024] Example 1: Preparation before experiment

[0025] The preparation of the hypoxic solution is as follows: 8.0063 g of NaCl, 0.8946 g of KCl, 0.1 g of MgCl2·6H2O, 0.1 g of CaCl 2、 0.9532 g of HEPES, 3.735 mL of C3H5O3Na are mixed, and ultrapure water is added to 1 L.

[0026] EA·hy926 cells (human umbilical vein cell fusion cells) are grown for 2 - 3 days until 80% - 90% of the bottom of the dish is covered, and then they can be used for the construction of the H / R model. As Figure 1 shown, the cells are divided into the following groups:

[0027] (1) Control group: The cells are not subjected to H / R modeling. During the incubation of the drug in the dosing group and throughout the modeling process, the medium is replaced with DMEM medium containing 1% fetal bovine serum (FBS), and they are placed in a cell culture incubator.

[0028] (2) H / R group: The hypoxia solution is pre - filled with nitrogen for 20 min to exhaust the dissolved oxygen in it. The original medium is replaced with the hypoxia solution, and after being placed in an anaerobic workstation (37 °C, 1% O2, 5% CO2) for 3 h, the re - oxygenation solution (DMEM medium containing 1% FBS) is replaced and then cultured in the cell culture incubator for another 1 h. Thus, the H / R model is completed.

[0029] (3) H / R + DHMBA group: DHMBA (3,5 - dihydroxy - 4 - methoxybenzyl alcohol) is diluted to the required concentrations (125 μM, 250 μM, 500 μM, 1000 μM) with DMEM medium containing 1% FBS and pre - incubated for 12 h. When constructing the H / R model later (see step (2)), the corresponding concentrations of DHMBA are also added to the hypoxia solution and the re - oxygenation solution.

[0030] (4) H / R + edaravone (Eda) group: Eda is diluted to 200 μM with DMEM medium containing 1% FBS, and after pre - dosing and incubating the cells for 24 h, the H / R model is constructed.

[0031] (5) H / R + DHMBA + oligomycin A (OA) group: First, DHMBA is diluted to 500 μM with DMEM medium containing 1% FBS and the cells are pre - incubated for 12 h; then OA is diluted to 10 μM with DMEM medium containing 1% FBS, and after pre - incubating the cells for 30 min, the H / R model is constructed.

[0032] Preparation and storage of DHMBA: DHMBA is dissolved in DMSO, the stock solution concentration is 1 M, stored in the dark at - 80 °C, diluted to the final concentration when used, and kept in the dark throughout the experimental operation. The stock solution for temporary use is stored in a - 20 °C refrigerator.

[0033] Example 2:

[0034] EA·hy926 cells are seeded in a 96 - well plate at an inoculation density of 5×10 4Cells / mL, 100 μL per well, placed in an incubator, and after growing for 24 h, the next experimental operation was carried out;

[0035] The experiment was divided into a Control group, an H / R group, and an H / R + DHMBA group. The treatments for each group are shown in Example 1. Each group was set with 5 replicate wells, 100 μL per well, and returned to the incubator for incubation for 12 h;

[0036] After the H / R model was established, a mixed solution of CCK-8 reagent and serum-free DMEM at a ratio of 1:10 was added. After incubation in the dark, detection was carried out according to the foregoing method.

[0037] The experimental results are as Figure 2 shown. It can be seen from Figure 2 that H / R significantly reduced the cell viability, while DHMBA could improve the cell viability reduction caused by H / R in a dose-dependent manner, and significant improvement ability appeared at a concentration of 250 - 1000 μM.

[0038] Example 3

[0039] The composition of the LDH detection working solution is as follows:

[0040]

[0041] The reagent must be prepared immediately before use and protected from light.

[0042] Experimental procedure:

[0043] (1) EA·hy926 cells were seeded in a 96-well plate at a seeding density of 5×10 4 cells / mL. After culturing for 24 h, when the cells grew to 80% confluence, the experimental grouping could be carried out: Control group, H / R group, H / R + DHMBA group. The treatments for each group are shown in Example 1.

[0044] After the H / R model was completed, 120 μL of the culture solution from each well was taken into a new 96-well plate, and 60 μL of the LDH detection working solution was added to each well;

[0045] (3) Mix well, incubate in the dark at room temperature for 30 min, and measure the absorbance value at 490 nm. Use 600 nm as the reference wavelength for dual-wavelength measurement, and use the difference in absorbance values to compare the release of LDH in each group of cells.

[0046] The experimental results are as Figure 3 shown. It can be seen from Figure 3 that H / R could cause obvious release of LDH, while DHMBA could inhibit the LDH release caused by H / R in a dose-dependent manner, and significant inhibitory ability appeared at a concentration of 250 - 1000 μM.

[0047] Example 4

[0048] Reagent preparation

[0049] Preparation of oxidized glutathione (GSSG) stock solution (10 mM): Dissolve 5 g of GSSG and 816 μL of deionized water, mix well, aliquot and store at -20 °C.

[0050] Preparation of 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) stock solution: Dissolve 4.5 mg of DTNB and 1.5 mL of DMSO, mix well, aliquot and store at -20 °C.

[0051] Preparation of protein removal reagent M (5%): Mix 0.2 g of protein removal reagent and 4 mL of total glutathione detection buffer, freshly prepared and used on the same day.

[0052] Preparation of NADPH stock solution (40 mg / mL): Dissolve 4 mg of NADPH and 100 μL of deionized water, mix well, aliquot and store at -80 °C.

[0053] Preparation of 1× glutathione reductase: Freshly prepare 50 μL of 5× glutathione reductase and 200 μL of total glutathione detection buffer, used on the same day.

[0054] 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.

[0055] Preparation of 0.5 mg / mL NADPH: Mix 10 μL of NADPH stock solution and 790 μL of total glutathione detection buffer. Preparation of GSH scavenging auxiliary solution: Mix 53 μL of GSH scavenging auxiliary stock solution and 47 μL of deionized water.

[0056] Preparation of GSH scavenging reagent working solution: Freshly prepare 10.8 μL of GSH scavenging reagent and 89.2 μL of absolute ethanol, used on the same day.

[0057] GSSG standards (diluted from 10 mM GSSG stock solution with protein removal reagent M), numbered A - F, see the following table:

[0058]

[0059] Experimental procedure

[0060] 1. Seed cells in 7 cm dishes at a seeding density of 1×10 5cells / mL, cultured for 24 h until the cell confluence reached 80%; Experimental grouping: Control group, H / R group, H / R + DHMBA group, and the treatments for each group are shown in Example 1.

[0061] 2. After the cells were treated accordingly, 0.125% trypsin-EDTA was used to digest and collect the cells, washed once with PBS, and the cells were collected by centrifugation (1000 rpm, 3 min), and the supernatant was aspirated.

[0062] 3. Add protein removal reagent M at 3 times the volume of the cell pellet, and vortex thoroughly to mix.

[0063] 4. Use liquid nitrogen and a 37 °C water bath to perform two rapid freeze-thaw cycles on the sample, ice-bath for 5 min, centrifuge (4 °C, 10000 g, 10 min), and collect the supernatant for the determination of total glutathione and GSSG content.

[0064] 5. Preparation of samples for GSSG content determination: Add 1 / 5 volume of GSH scavenging auxiliary solution to the sample to be tested, vortex to mix, and then add 1 / 25 volume of GSH scavenging reagent working solution, vortex to mix. Incubate at 25 °C for 60 min.

[0065] 6. Add the samples for total glutathione determination, samples for GSSG content determination, and standards to a 96-well plate, add 150 μL of total glutathione detection working solution, mix well, and incubate at room temperature for 5 min.

[0066] 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 enzyme-linked immunosorbent assay reader.

[0067] 8. Calculate the content of GSH based on the determined content of total glutathione and GSSG, and then calculate the value of GSSG / GSH.

[0068] The experimental results are as Figure 4 shown. It can be seen from Figure 4 that H / R does not cause a change in the total amount of Glutathione, but can cause a significant decrease in the ratio of GSH / GSSG. DHMBA at a concentration of 500 μM can significantly increase the ratio of GSH / GSSG.

[0069] Example 5

[0070] Reagent preparation

[0071] Preparation of TNF-α standard

[0072]

[0073] The standard product should be prepared within 2 hours before use. The diluted standard product (10000 pg / mL) should be used within 12 hours. Under the condition of frozen storage at -20 °C, it can be used within 2 days, but it should not be repeatedly frozen and thawed.

[0074] Configuration of the biotin-labeled anti-human TNF-α antibody working solution: 10 μL of biotin-labeled anti-human TNF-α and 990 μL of antibody diluent should be configured within 2 hours before use.

[0075] Configuration of the avidin-peroxidase complex (ABC) working solution: 10 μL of ABC and 990 μL of ABC diluent should be configured within 1 hour before use. Warm it at 37 °C for at least 30 min before adding it to the enzyme-labeled plate wells.

[0076] Configuration of 1× wash buffer: Mix 2 mL of 25× wash buffer and 48 mL of deionized water evenly and use it immediately after preparation.

[0077] Experimental procedures

[0078] 1. Inoculate cells in a 7 cm dish at an inoculation density of 1×10 5 cells / mL, 4 mL per dish, and culture for 24 h until the cells grow to 80% confluence;

[0079] 2. Experimental grouping: Control group, H / R group, H / R + DHMBA group. The treatments for each group are shown in Example 1;

[0080] 3. After the cells are treated accordingly, obtain cell lysates according to the method described in 2.2.6, and measure the protein concentrations of each group;

[0081] 4. Add 100 μL of each concentration of the standard product prepared in the above steps to the wells in sequence. The last well only adds the sample diluent as the zero well, and add one more well as the blank color development well. Add 100 μL of the sample to be tested as well, cover with a sealing plate film, and incubate at 37 °C for 90 min;

[0082] 5. Discard the liquid in the enzyme-labeled plate, invert it on the absorbent paper, and tap it gently to completely remove the residual liquid; Add 100 μL of the prepared biotin anti-human TNF-α antibody working solution to each well in sequence (except for the TMB blank color development well), cover with a sealing plate film, and incubate at 37 °C for 60 min;

[0083] 6. Wash 3 times with 1× wash buffer, 300 μL of washing solution per well, 1 min each time; Add 100 μL of the ABC working solution to each well in sequence (except for the TMB blank color development well), cover with a sealing plate film, and incubate at 37 °C for 30 min;

[0084] 7. Wash 5 times with 1× washing buffer, adding 300 μL of washing solution to each well for 1 min each time; add TMB chromogenic solution to each well successively at 90 μL per well, and incubate at 37 °C in the dark for 15 min;

[0085] 8. Add 100 μL of stop solution to each well, and at this time the blue color will immediately turn yellow. Measure the absorbance value with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm.

[0086] 9. Set the TMB blank chromogenic well (only adding TMB chromogenic solution and stop solution) as the control. After subtracting the absorbance value of the TMB blank chromogenic well from the absorbance values of all standards and samples, plot the standard curve, and calculate the TNF-α concentration of the corresponding sample according to the absorbance value of the sample.

[0087] Detection of the content of inflammatory factor IL-6 by ELISA

[0088] The experimental principle and experimental steps are basically the same as those for the detection of TNF-α, except that: the TNF-α standard is replaced with the IL-6 standard; the working solution of biotin-labeled anti-human TNF-α antibody is replaced with the working solution of biotin-labeled anti-human IL-6 antibody.

[0089] The experimental results are as Figure 5 shown, and it can be seen from Figure 5 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 of H / R on the two inflammatory factors TNF-α and IL-6.

[0090] Example 6

[0091] 1. Use a cell counting chamber to count and dilute EA·hy926 cells to 1×10 5 cells / mL, inoculate them into a 6-well plate at 2 mL per well, and place them in a cell culture incubator for 36 h. When the cells grow to 90% - 95% confluence, the experimental grouping can be carried out: Control group, H / R group, H / R + DHMBA group, and the treatments of each group are shown in Example 1.

[0092] 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;

[0093] 3. Wash away the cells shed during scratching with PBS, and add DMEM medium containing 10% FBS;

[0094] 4. Take a photo and mark the detailed position of the scratch, record it as 0 h. Place the cells in the incubator and continue to culture. Observe the cell migration status at the scratch after 24 h and record it as 24 h;

[0095] 5. Use the software ImageJ to calculate the cell scratch area and analyze the cell migration rate. The migration rate = (cell scratch area at 0 h - cell scratch area at 24 h) / cell scratch area at 0 h × 100%.

[0096] The experimental results are as Figure 6 shown. From Figure 6 it can be seen that H / R can significantly reduce the cell migration distance. 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.

[0097] Example 7

[0098] 1. Use a cell counting chamber to count and dilute EA·hy926 cells to 1×10 5 cells / mL, inoculate them into 3.5 cm dishes, 2 mL per well, and place them in an incubator for 24 h. Experimental grouping: Control group, H / R group, H / R + DHMBA group. The treatments for each group are shown in Example 1.

[0099] 2. Plating of Matrigel: First, rinse the 24-well plate with pre-cooled PBS. Add 200 μL of Matrigel to each well under low-temperature conditions, gently shake the plate to spread it evenly, and place it in an environment at 37 °C for 30 min to complete the plating of Matrigel.

[0100] 3. After the H / R model is completed, digest and collect the cells, and inoculate them on the surface of the pre-fixed Matrigel. The inoculation density is 5×10 4 cells / mL, 200 μL per well, shake evenly, and place it in a cell incubator to continue culturing for 24 h.

[0101] 4. Observe the cell tube formation situation and take pictures for preservation. Use ImageJ software to analyze the number of nodes, the number of branches, the number of meshes, the total length of the tubes, and the total length of the branches.

[0102] The experimental results are as Figure 7 shown. From Figure 7 it can be seen that H / R can significantly reduce the number of cell branches, the total length of the branches, the number of meshes, and the number of nodes. DHMBA has a concentration-dependent enhancing effect on the inhibitory effect of H / R on the number of cell branches, the total length of the branches, the number of meshes, and the number of nodes, and there is a significant increase at a concentration of 500 μM.

[0103] 1. Inoculate EA·hy926 cells into a six-well plate at an inoculation density of 1×10 5 cells / mL and culture for 24 h until the cells grow to 80% confluence.

[0104] 2. Experimental grouping: Control group, H / R group, H / R + DHMBA group. The treatments for each group are shown in Example 1.

[0105] 3. After the H / R model was completed, the cells were washed twice with PBS, and then 1 mL of serum-free 1640 medium containing 5 μM DCFH-DA probe was added to each well. Then, the cells were incubated in an environment at 37 °C for 30 min.

[0106] 4. After incubation, the cells were washed twice with PBS, and then digested with 0.25% trypsin digestion solution and collected into pre-cooled 1.5 mL centrifuge tubes, and stored in an ice box.

[0107] 5. The cell suspensions of each group were centrifuged at 1000 rpm for 3 min, and then washed with pre-cooled PBS to remove the residual probe.

[0108] 6. Centrifugation was continued, and the supernatant was discarded. 400 μL of PBS was added to the cell pellet and mixed well, and then the mean fluorescence intensity under the FITC pathway was detected by flow cytometry. If the cells could not be detected in time, they needed to be stored in an ice box and protected from light.

[0109] The experimental results are as Figure 8 shown. It can be seen from Figure 8 that H / R can significantly activate the production of cellular ROS, and DHMBA at a concentration of 500 μM can effectively inhibit the production of cellular ROS by H / R, but there is no obvious inhibitory effect at a low concentration of 250 μM.

[0110] Example 9

[0111] Flow cytometry

[0112] (1) EA·hy926 cells were seeded in a six-well plate at a seeding density of 1×10 5 cells / mL and cultured for 24 h until the cell confluence reached 80%.

[0113] (2) Experimental grouping: Control group, H / R group, H / R + DHMBA group, H / R + Eda group, H / R + DHMBA + OA group. The treatments for each group are shown in Example 1.

[0114] (3) After the H / R model was completed, the cells were washed twice with PBS, and then 1 mL of serum-free 1640 medium containing 5 μM DCFH-DA probe was added to each well. Then, the cells were incubated in an environment at 37 °C for 30 min.

[0115] (4) After incubation, the cells were washed twice with PBS, and then digested with 0.25% trypsin digestion solution and collected into pre-cooled 1.5 mL centrifuge tubes, and stored in an ice box.

[0116] (5) Centrifuge each group of cell suspensions at 1000 rpm for 3 min, and then wash with pre-cooled PBS to remove residual probes.

[0117] (6) Continue centrifugation, discard the supernatant, add 400 μL of PBS to the cell pellet, mix well, and then detect the mean fluorescence intensity under the FITC pathway using a flow cytometer. If the cells cannot be detected in a timely manner, they need to be stored in an ice box and protected from light.

[0118] Scratch

[0119] (1) Count the EA·hy926 cells using a hemocytometer and dilute them to 1×10 5 cells / mL, inoculate them into 6-well plates, 2 mL per well, and place them in a cell culture incubator for 36 h. When the cells reach 90% - 95% confluence, the experimental groups can be divided as follows: Control group, H / R group, H / R + DHMBA group, H / R + Eda group, H / R + DHMBA + OA group. The treatments for each group are shown in Example 1.

[0120] (2) After the H / R model is completed, use a sterile 300 μL glass pipette tip to make scratches of the same thickness vertically in each well.

[0121] (3) Wash away the detached cells during scratching with PBS, and add DMEM medium containing 10% FBS.

[0122] (4) Take a photo and mark the detailed position of the scratch, record it as 0 h. Place the cells in the incubator and continue to culture. Observe the cell migration at the scratch site after 24 h and record it as 24 h.

[0123] (5) Use the software ImageJ to calculate the cell scratch area and analyze the cell migration rate. Migration rate = (Cell scratch area at 0 h - Cell scratch area at 24 h) / Cell scratch area at 0 h × 100%.

[0124] LDH

[0125] The components for preparing the LDH detection working solution are as follows:

[0126]

[0127] The reagents must be prepared immediately before use and protected from light.

[0128] Experimental procedures

[0129] (1) Inoculate EA·hy926 cells into 96-well plates at an inoculation density of 5×10 4cells / mL, cultured for 24 h. When the cell growth reached 80% confluence, the experiment could be grouped: Control group, H / R group, H / R + DHMBA group, H / R + Eda group, H / R + DHMBA + OA group. The treatments of each group are shown in Example 1.

[0130] (2) After the H / R model was completed, 120 μL of the culture solution in each well was taken into a new 96-well plate, and 60 μL of the LDH detection working solution was added to each well; mixed well and incubated at room temperature in the dark for 30 min, and the absorbance value was measured at 490 nm. A dual-wavelength measurement was performed using 600 nm as the reference wavelength, and the difference in absorbance values was used to compare the release of LDH in cells of each group.

[0131] The experimental results are as Figure 9 shown. It can be seen from Figure 9 the comparison experiment that 500 μM DHMBA and 200 μM Eda have similar effects in improving H / R-dependent LDH release, migration inhibition, and ROS production. However, this improvement effect of DHMBA can be blocked by 10 μM OA.

Claims

1. Use of 3,5-dihydroxy-4-methoxybenzyl alcohol or its pharmaceutically acceptable salt in the preparation of a drug for treating ischemia-reperfusion injury.

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 medicine 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 medicine 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