Method for inhibiting endothelial cell apoptosis induced by docosahexaenoic acid

Abstract

The invention provides a method for blocking endothelial cell apoptosis induced by docosahexaenoic acid (DHA) through a p38MAPK inhibitor, and relates to the technical field of biology. According to the method, endothelial cells serve as a research model, and cell apoptosis induced by high-concentration docosahexaenoic acid (DHA) is remarkably reduced by adding a p38MAPK specific inhibitor. The method comprises the following specific steps: cell culture, DHA and inhibitor treatment, and detection of the apoptosis rate by using flow cytometry. Experimental results show that the p38MAPK inhibitor can effectively reduce cell apoptosis caused by DHA (docosahexaenoic acid). The method has the advantages that the method is simple and efficient, the effect of the p38MAPK signal channel in DHA-induced endothelial cell apoptosis is further disclosed, and a theoretical basis and a practical basis are provided for developing novel medicines for treating endothelial dysfunction related diseases.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for significantly reducing endothelial cell apoptosis induced by docosahexaenoic acid (DHA) by inhibiting p38 MAPK pathway and its application. Background Art

[0002] Endothelial cells play a key role in maintaining vascular function and integrity, but under certain conditions, such as high concentrations of DHA exposure, endothelial cells may be subject to significant oxidative stress stimulation, thereby inducing cell apoptosis. As an important ω-3 polyunsaturated fatty acid, DHA has a variety of biological effects, but at high concentrations it can have cytotoxic effects on endothelial cells, manifested as increased generation of reactive oxygen species (ROS), mitochondrial dysfunction, and increased apoptosis rate.

[0003] p38 MAPK is a mitogen-activated protein kinase that plays an important role in cellular stress and inflammatory responses. Studies have shown that the activation of p38 MAPK is closely related to cell apoptosis, especially in the process of DHA-induced cell apoptosis. By inhibiting the p38 MAPK signaling pathway, it is possible to reduce DHA-induced endothelial cell apoptosis. However, there is currently a lack of systematic research on the protective effect of p38 MAPK inhibitors in DHA-induced cell apoptosis.

[0004] p38 MAPK inhibitors include a variety of selective drugs. Such small molecule inhibitors are widely used in cellular studies to explore the specific functions and molecular mechanisms of p38 MAPK in various pathological processes (such as oxidative stress, inflammation and apoptosis).

[0005] Based on previous experimental studies, the present invention found that the addition of a p38 MAPK specific inhibitor can significantly reduce DHA-induced endothelial cell apoptosis, further providing a theoretical basis and practical methods for studying the endothelial cell protection mechanism and the prevention and treatment of related diseases. Summary of the invention

[0006] The purpose of the present invention is to provide a method for significantly reducing DHA-induced endothelial cell apoptosis by inhibiting the p38 MAPK signaling pathway, and to further explore its molecular mechanism.

[0007] The technical solution adopted by the present invention to solve the above technical problem is to explore the inhibitory effect on cell apoptosis by adding p38 MAPK inhibitor small molecules on the basis of inducing endothelial cell apoptosis with high concentration of DHA.

[0008] In order to achieve the above object, the present invention provides the following technical solution, and the specific operation includes the following steps: Endothelial cells were used as a research model and were seeded at a density of 9,000 cells per square centimeter. The experimental cells were used within 20 generations: (1) P2 endothelial cells with a growth density of 70-80% were taken, the culture medium was discarded, and the cells were washed once with PBS buffer to remove the residual culture medium; (2) Add 0.3 ml of 0.05% trypsin and digest the cells for about 2 min until the edges of the cells float. Use a pipette to blow the cells, aspirate the cell suspension, and transfer it to a 1.5 ml centrifuge tube containing 1 ml of DMEM serum culture medium. Continue to blow and mix to terminate the digestion. (3) After centrifugation at 1000 rpm for 3 min, the supernatant was discarded, 1 ml of DMEM culture medium containing 10% FBS was added to resuspend the cells, and the cells were counted using a cell counting plate to calculate the cell density; (4) Add 1 ml of DMEM culture medium containing 10% FBS to each well of the culture plate; (5) Add 9.0×10 3 cells, shake them horizontally in a cross shape to make the cells evenly distributed, and place them in an incubator for 24 hours until the cells are completely attached to the wall; (6) Docosahexaenoic acid (DHA; Cayman Chemical) was dissolved in ethanol and then mixed with phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8mMKH 2 PO 4 ) mixed. DHA was added to the cells at confluence (ensuring that the cells were not disturbed) for up to 24 hours; (7) Dissolve the p38 MAPK inhibitor in DMSO. Add the p38 MAPK inhibitor to the culture system 30 minutes before adding DHA; (8) Place the cell culture dish at 37°C and 5% CO 2 The cells were cultured in a cell culture incubator at a concentration of 1:1. (9) After 24 h, remove the cells and observe the cell condition under a microscope. Collect the cells from the culture plate and count them. Suspend the cells in binding buffer and adjust the cell concentration to 1 × 10 6cells / mL. Then, FITC-labeled Annexin V reagent was added, gently mixed, and incubated in the dark for 15 minutes at room temperature. After incubation, the cells were resuspended in binding buffer, and propidium iodide (PI) was added as a dead cell dye and incubated in the dark for 5 minutes. Finally, flow cytometry was immediately used for detection and data collection to distinguish early apoptotic cells (Annexin V-FITC positive, PI negative) from late apoptotic or necrotic cells (Annexin V-FITC positive, PI positive).

[0009] Apoptosis status detection Under the apoptosis condition induced by high concentration of DHA (125 μM), 5 μM p38 MAPK inhibitor was added, and the apoptosis rate was detected by flow cytometry; Observe whether the addition of p38 MAPK inhibitor can significantly inhibit DHA-induced cell apoptosis.

[0010] The advantages and innovations of the present invention include: (1) It provides a simple and efficient cell protection method, which significantly reduces DHA-induced endothelial cell apoptosis through p38 MAPK inhibitor; (2). In-depth revelation of the role of p38 MAPK signaling pathway in DHA-induced endothelial cell apoptosis; (3) It provides new ideas for the development of new drugs to treat diseases related to endothelial dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The figure shows the cell morphology observation of endothelial cell P2 cells treated under different conditions. Each cell was cultured under different conditions, which were ① ethanol + 5% BSA + 2μl DMSO ② 125μM DHA, ③ 125μM DHA + 5μM p38 MAPK inhibitor, and ④ 5μM p38 MAPK inhibitor. It can be found that when the culture condition is 125μM DHA, the cells appear to shrink and float without adhering to the wall, while under the condition of 125μM DHA + 5μM p38 MAPK inhibitor, the cells adhere to the wall normally. Therefore, we assume that inhibiting p38 MAPK can block DHA-induced cell apoptosis.

[0012] Figure 2The figure shows the flow cytometry of endothelial cell P2 cells treated for 24 hours under different conditions. The different treatment groups are ① ethanol + 5% BSA + 2μl DMSO ② 125μM DHA, ③ 125μM DHA + 5μM p38 MAPK inhibitor, and ④ 5μM p38 MAPK inhibitor. It can be found that when the culture condition is 125μM DHA, 81.1% of cells die, while under the condition of 125μM DHA + 5μM p38 MAPK inhibitor, 17.8% of cells die. Therefore, we believe that inhibiting p38MAPK can block DHA-induced cell apoptosis. DETAILED DESCRIPTION

[0013] Example Endothelial cells were purchased from Starfish Biotech and proliferated according to the manufacturer's protocol.

[0014] A method to block DHA-induced endothelial cell apoptosis by small molecules of p38 MAPK inhibitors, and its effect was verified by microscopic observation and flow cytometry: (1) P2 endothelial cells with a growth density of 70-80% were taken, the culture medium was discarded, and the cells were washed once with PBS buffer to remove the residual culture medium; (2) Add 0.3 ml of 0.05% trypsin and digest the cells for about 2 min until the edges of the cells float. Use a pipette to blow the cells, aspirate the cell suspension, and transfer it to a 1.5 ml centrifuge tube containing 1 ml of DMEM serum culture medium. Continue to blow and mix to terminate the digestion. (3) After centrifugation at 1000 rpm for 3 min, the supernatant was discarded, 1 ml of DMEM culture medium containing 10% FBS was added to resuspend the cells, and the cells were counted using a cell counting plate to calculate the cell density; (4) Add 1 ml of DMEM culture medium containing 10% FBS to each well of the culture plate; (5) Add 9.0 × 103 cells to each culture well, shake horizontally in a cross shape to evenly distribute the cells, and place in an incubator for 24 h until the cells are completely attached to the wall; (6) Docosahexaenoic acid (DHA; Cayman Chemical) was dissolved in ethanol and then mixed with phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8mMKH 2 PO 4 ) mixed. DHA was added to the cells at confluence (ensuring that the cells were not disturbed) for up to 24 hours; (7) Dissolve the p38 MAPK inhibitor in DMSO. Add the p38 MAPK inhibitor to the culture system 30 minutes before adding DHA; (8) Place the cell culture dish at 37°C and 5% CO 2 The cells were cultured in a cell culture incubator at a concentration of 1:1. (9) After 24 h, remove the cells and observe the cell condition under a microscope. Collect the cells from the culture plate and count them. Suspend the cells in binding buffer and adjust the cell concentration to 1 × 10 6 cells / mL. Then, FITC-labeled Annexin V reagent was added, gently mixed, and incubated in the dark for 15 minutes at room temperature. After incubation, the cells were resuspended in binding buffer, and propidium iodide (PI) was added as a dead cell dye and incubated in the dark for 5 minutes. Finally, flow cytometry was immediately used for detection and data collection to distinguish early apoptotic cells (Annexin V-FITC positive, PI negative) from late apoptotic or necrotic cells (Annexin V-FITC positive, PI positive).

[0015] Cell apoptosis status detection: (1) Morphological observation: The cells under the above culture conditions were observed using a Leica DMIL inverted microscope. The results showed that when ethanol + 5% BSA + 2 μl DMSO, 125 μM DHA + 5 μM p38 MAPK inhibitor, and 5 μM p38 MAPK inhibitor were added to the culture conditions, the cells still adhered to the wall and showed certain activity. Figure 1 As shown; (2) Collect the cells after 24 h of treatment and add FITC-labeled Annexin V reagent, mix gently, and incubate at room temperature in the dark for 15 minutes. After incubation, resuspend the cells in binding buffer and add propidium iodide (PI) as a dead cell dye, incubate in the dark for 5 minutes, and immediately put them on the machine for detection and data collection. The results showed that 81.1% of the cells treated with 125 μM DHA died, while 17.8% of the cells died under the condition of 125 μM DHA + 5 μM p38 MAPK inhibitor, as shown in Figure 2. Figure 2 shown.

Claims

1. A method for inhibiting endothelial cell apoptosis induced by docosahexaenoic acid (DHA), characterized in that: Adding p38 MAPK inhibitors (including SB202190, SB203580, etc.) to endothelial cells treated with 125 μM DHA can block endothelial cell apoptosis induced by DHA.

2. The method according to claim 1, characterized in that DHA is used at a concentration of 125 μM and has a maximum duration of action of 24 hours: (1) Endothelial cells were seeded at a density of 9000 cells per square centimeter to ensure that the experimental cells were used within 20 generations; (2) P2 endothelial cells with a growth density of 70%-80% were taken, the culture medium was discarded, and the cells were washed once with PBS buffer; (3) Add 0.05% trypsin and digest the cells for about 2 minutes. After the cell edges float, blow the cells and aspirate the cell suspension and transfer it to DMEM culture medium containing 10% FBS to terminate the digestion. (4) After centrifugation (1000 rpm, 3 minutes), the supernatant was discarded, the cells were resuspended and counted, and then inoculated into the wells of the culture plate. 9.0×10 3 cells; (5) Before cell culture, docosahexaenoic acid (DHA) was dissolved in PBS containing 5% fatty acid-free bovine serum albumin, and the p38 MAPK inhibitor was added to the culture system 30 minutes before the addition of DHA; (6) After 24 hours of culture, cell apoptosis was detected using Annexin V-FITC / PI staining combined with flow cytometry.

3. The method according to claim 2, characterized in that After adding p38 MAPK inhibitor, the apoptotic effect was detected by flow cytometry.

4. The method according to claim 2, characterized in that: This method was used to study the mechanism by which DHA induces endothelial cell apoptosis and to develop therapeutic strategies for diseases associated with endothelial dysfunction.

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