Application of aloe-emodin in targeting VEGFR2 to inhibit breast cancer cell angiogenesis and drugs

Abstract

This application relates to the field of biomedical technology, specifically to the application and drug of aloe-emodin in inhibiting angiogenesis around breast cancer cells by targeting VEGFR2. On one hand, this application mainly provides an application of aloe-emodin in inhibiting angiogenesis around breast cancer cells, clarifying that aloe-emodin can inhibit angiogenesis around breast cancer cells by targeting the VEGFR2 signaling pathway, thereby inhibiting breast cancer cell metastasis and ultimately playing an inhibitory role in breast cancer. On the other hand, this application provides a breast cancer therapeutic drug containing aloe-emodin. Tumor development and progression depend on the regeneration and growth of blood vessels around cells, which plays a significant role in promoting metastasis. Metastatic breast cancer is a major cause of death in patients, and the discovery and research of anti-angiogenic targeted drugs are of great significance for the treatment of breast cancer.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to the application and drug of aloe-emodin in targeting VEGFR2 to inhibit angiogenesis around breast cancer cells. Background Technology

[0002] Breast cancer (BC) is one of the most common malignant tumors in the world today. Currently, the main clinical treatments for breast cancer include radiotherapy, chemotherapy, and surgery. However, while killing tumor cells, these treatments also pose certain safety risks to organs throughout the body. Therefore, actively exploring drugs and technologies for breast cancer treatment is of great significance in alleviating the suffering caused by breast cancer.

[0003] Angiogenesis refers to the formation of new blood vessels from existing capillaries or postcapillary veins. Cancer-related death is associated with the invasion and migration of tumor cells, and angiogenesis is an important mechanism mediating cancer cell metastasis. Therefore, anti-angiogenesis may be a promising cancer treatment technique.

[0004] Traditional Chinese medicine believes that malignant tumors often arise from qi stagnation, blood stasis, phlegm accumulation, and pathogenic toxins. Clinically, clearing heat and detoxifying, and promoting blood circulation and removing blood stasis can effectively treat breast cancer. Aloe-emodin is a natural anthraquinone compound found in traditional Chinese medicines such as aloe, rhubarb, and hyacinth bean. Studies have shown that it has the effects of clearing heat, detoxifying, and removing blood stasis. In contrast, traditional chemotherapy drugs have significant side effects. Aloe-emodin has advantages such as fewer side effects and natural safety compared to traditional chemotherapy drugs. However, currently, no relevant technology has disclosed the mechanism of action of aloe-emodin on breast cancer. Summary of the Invention

[0005] The problem this application aims to solve is to provide the application of aloe-emodin in inhibiting angiogenesis around breast cancer cells. It clarifies that aloe-emodin can inhibit angiogenesis around breast cancer cells by targeting the VEGFR2 signaling pathway, thereby inhibiting the metastasis of breast cancer cells and ultimately playing an inhibitory role in breast cancer.

[0006] To solve the above-mentioned technical problems, this application adopts the following technical solution:

[0007] On the one hand, this application provides the application of aloe-emodin in inhibiting angiogenesis around breast cancer cells.

[0008] In some embodiments, the aloe-emodin described above inhibits angiogenesis around breast cancer cells by targeting the VEGFR2 signaling pathway.

[0009] VEGFR2, or vascular endothelial growth factor receptor 2, plays a crucial role in the development and progression of tumors, which depend on the regeneration and growth of blood vessels surrounding cells and promote metastasis. Metastatic breast cancer is a leading cause of death in patients, and the discovery and research of anti-angiogenic targeted drugs are of great significance for the treatment of breast cancer. Aloe vera extract can inhibit the formation of blood vessels around breast cancer cells by targeting the VEGFR2 signaling pathway, thereby inhibiting the metastasis of breast cancer cells and ultimately playing an inhibitory role in breast cancer.

[0010] On the one hand, this application provides the application of the above-mentioned aloe-emodin in inhibiting the proliferation of breast cancer cells.

[0011] On the one hand, this application provides the application of the above-mentioned aloe-emodin in inhibiting the in vitro migration ability of breast cancer cells.

[0012] On the one hand, this application provides the application of the above-mentioned aloe-emodin in inhibiting the in vitro invasion ability of breast cancer cells.

[0013] On the one hand, this application provides a breast cancer treatment drug containing the aforementioned aloe-emodin.

[0014] In some embodiments, the mechanism of action of the breast cancer treatment drug includes:

[0015] Inhibits angiogenesis around breast cancer cells;

[0016] And / or, inhibit the proliferation of breast cancer cells;

[0017] And / or, inhibit the ability of breast cancer cells to migrate in vitro;

[0018] And / or, inhibit the ability of breast cancer cells to invade in vitro.

[0019] This application has the following beneficial effects:

[0020] (1) The aloe-emodin provided in this application can inhibit the formation of blood vessels around breast cancer cells by targeting the VEGFR2 signaling pathway, thereby inhibiting the metastasis of breast cancer cells and ultimately playing an inhibitory role in breast cancer.

[0021] (2) The aloe-emodin provided in this application can not only inhibit the formation of blood vessels around breast cancer cells, but also control the development of breast cancer by inhibiting the proliferation of breast cancer cells, inhibiting the in vitro migration ability of breast cancer cells, and inhibiting the in vitro invasion ability of breast cancer cells.

[0022] (3) The drug provided in this application can directly target VEGFR2, thereby regulating the VEGFR2 signaling pathway, inhibiting angiogenesis around breast cancer cells, and also has the functions of inhibiting breast cancer cell proliferation, inhibiting breast cancer cell migration in vitro, and inhibiting breast cancer cell invasion in vitro, which is of great significance for the treatment of breast cancer. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in this disclosure, the accompanying drawings used in some embodiments of this disclosure will be briefly described below. Obviously, the drawings described below are merely drawings of some embodiments of this disclosure, and those skilled in the art can obtain other drawings based on these drawings. Furthermore, the drawings described below can be regarded as schematic diagrams and are not intended to limit the actual process of the methods involved in the embodiments of this disclosure.

[0024] Figure 1 This is a statistical chart showing the experimental results of aloe-emodin on the viability of MDA-MB-231 and MDA-MB-468 CCK8 breast cancer cells in this embodiment of the invention.

[0025] Figure 2 The results of the plate colony experiment and statistical graphs of the effect of aloe-emodin on the proliferation ability of breast cancer cells MDA-MB-231 and MDA-MB-468 in this embodiment of the invention (*p<0.05, **p<0.01, ***p<0.001, ****p<0.001);

[0026] Figure 3 The scratch assay results and statistical graphs of the effect of aloe-emodin on the migration ability of MDA-MB-231 and MDA-MB-468 breast cancer cells in this embodiment of the invention (*p<0.05, **p<0.01, ***p<0.001, ****p<0.001);

[0027] Figure 4 The transwell experiment results and statistical graphs showing the effect of aloe-emodin on the invasive ability of MDA-MB-231 and MDA-MB-468 breast cancer cells in this embodiment of the invention (*p<0.05, **p<0.01, ***p<0.001, ****p<0.001);

[0028] Figure 5 The following are the results and statistical graphs of the in vitro lumen formation experiment on the effect of aloe-emodin on the angiogenesis ability of MDA-MB-231 and MDA-MB-468 breast cancer cells in the embodiments of the present invention (*p<0.05, **p<0.01, ***p<0.001, ****p<0.001).

[0029] Figure 6 These are the biotin-labeled map of aloe vera emodin, the protein targeting protein network map, and the KEGG pathway enrichment analysis map in the embodiments of the present invention.

[0030] Figure 7 This is a schematic diagram of the molecular docking between aloe-emodin and VEGFR2 in an embodiment of the present invention;

[0031] Figure 8 The following is a statistical chart of tumor volume changes in nude mice after administration of aloe-emodin in an example of the present invention, and a comparative analysis of tumor mass and volume at different administration concentrations (*p<0.05, **p<0.01, ***p<0.001, ****p<0.001).

[0032] Figure 9 The images show HE staining, CD31 immunohistochemical staining, and CD31 statistical plots of tumor tissue in this invention example (*p<0.05, **p<0.01, ***p<0.001, ****p<0.001).

[0033] Figure 10 This is an electrophoresis result of the effect of aloe-emodin on the expression of VEGFR2 and its downstream proteins PLCG1, p-PLCG1, ERK, P38 and p-P38 in MDA-MB-231 and MDA-MB-468 breast cancer cells in this embodiment of the invention. Detailed Implementation

[0034] The technical solutions in some embodiments of this disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. Based on the embodiments provided in this disclosure, all other embodiments obtained by those skilled in the art are within the scope of protection of this disclosure.

[0035] Cell lines and related materials: Human breast normal epithelial cells MCF-10A, breast cancer MDA-MB-231, and MDA-MB-468 cells were originally in our laboratory. Fetal bovine serum (Wuhan Pronosei Life Sciences Co., Ltd.), DMEM (Wuhan Sewell Biotechnology Co., Ltd.), penicillin-streptomycin mixture (Wuhan Sewell Biotechnology Co., Ltd.), trypsin (Wuhan Sewell Biotechnology Co., Ltd.), PBS buffer (Wuhan Sewell Biotechnology Co., Ltd.), serum-free rapid cryopreservation solution (Suzhou Xinsaimei Biotechnology Co., Ltd.), tissue fixative (Lanjieke Technology Co., Ltd.), crystal violet (Wuhan Sewell Biotechnology Co., Ltd.), 25cm aerated slant-top culture flasks (Wuhan Sewell Biotechnology Co., Ltd.), 6-well plates, 96-well plates (Wuhan Sewell Biotechnology Co., Ltd.), 1.5mL sterile enzyme-free EP centrifuge tubes (Wuhan Sewell Biotechnology Co., Ltd.), and 2mL Kelvin internal spiral cryopreservation tubes (Thermo Fisher Scientific).

[0036] Main reagents and kits: CCK8 reagent (Beijing Solarbio Biotechnology Co., Ltd.), enhanced RIPA lysis buffer (Shanghai Beyotime Biotechnology Co., Ltd.), PMSF protease inhibitor (Shanghai Aomor Biotechnology Co., Ltd.), phosphatase inhibitor (Shanghai Aomor Biotechnology Co., Ltd.), SDS-PAGE protein loading buffer (Wuhan Sewell Biotechnology Co., Ltd.), marker (Wuhan Sewell Biotechnology Co., Ltd.), gel preparation kit (Shanghai Yamei Biomedical Technology Co., Ltd.), Super ECL Plus ultrasensitive chemiluminescence kit (Suzhou Youyilandi Biotechnology Co., Ltd.), Tris-base (BioFROXX), Glycine (BioFROXX), Sodium Dodecyl Sulfate SDS (BioFROXX), Tris buffered Saline (BioFROXX), Tween-20 (BioFROXX), protein-free rapid blocking buffer (Shanghai Yamei Biomedical Technology Co., Ltd.), primary antibody, secondary antibody (Shanghai Aomor Biotechnology Co., Ltd.), PVDF membrane (Merck Millipore, USA), Pullsown kit (Thermo), aloe-emodin (MedChemExpress, MCE).

[0037] Related instruments: Biosafety cabinet (Haier Biomedical), low-speed centrifuge (Thermo Fisher Scientific), CO2 incubator (Thermo Fisher Scientific), ultra-high-speed centrifuge (Thermo Fisher Scientific), vertical electrophoresis apparatus (Bio-rad, USA), wet transfer apparatus (Bio-rad, USA), chemiluminescence imaging system (Bio-rad, USA).

[0038] Example 1: Effects of aloe-emodin on breast cancer cell viability

[0039] Cell suspensions of 1000-2000 cells / well for breast cancer MDA-MB-231 cells, breast cancer MDA-MB-468 cells, and normal human breast epithelial MCF-10A cells were seeded into 96-well plates, with 5 replicates per group. 100 μL of PBS was added to the outer edge of the culture plate to prevent evaporation. After cell attachment, cells were treated with aloe-emodin at different concentration gradients (0-400 μM) for 24 h and 48 h, respectively. Afterward, the culture medium was removed, and the cells were gently washed once with PBS. 100 μL of complete culture medium (basal medium + 10% fetal bovine serum + 1% penicillin-streptomycin mixture) containing 10% CCK-8 was added to each well. A negative control well containing only CCK-8 was included to eliminate background interference. After incubation for 1.5 h, the absorbance was measured at 450 nm using a microplate reader, and the cell proliferation inhibition rate was calculated at 24 h and 48 h. The formula for calculating the cell proliferation inhibition rate is (experimental group absorbance - blank control absorbance) / (control group absorbance - blank control absorbance) × 100%. Figure 1 As shown in the figure. The results indicate that aloe-emodin can effectively inhibit the activity of breast cancer cells.

[0040] Example 2: Effects of aloe-emodin on the proliferation of breast cancer cells

[0041] After treating breast cancer MDA-MB-231 and MDA-MB-468 cells with 0, 10, 20, and 40 μM aloe-emodin for 48 h, respectively, the cells were digested, centrifuged, and the cell concentrations of each group were counted. 900 cells were taken from each sample and seeded into six-well plates. The cells were then observed under a microscope to ensure that each cell was individually distributed. The plates were then cultured in an incubator, with the culture medium changed every 3 days. Cell status was observed and contamination was prevented. The size of the cell colonies at the bottom of the culture dishes was observed using refractive index. When appropriate, the culture medium was discarded, and the cells were washed twice with PBS. Then, 1 mL of 4% paraformaldehyde solution was added to fix the cells at the bottom of the wells for 20 min. The cells were washed again with PBS, and finally, 1 mL of 0.1% crystal violet was injected for staining for 15 min. The cells were then rinsed with water until the background of the culture dishes became clear. The plates were air-dried and photographed. The cell colony count was calculated using ImageJ software. The experimental results are shown below. Figure 2 As shown in the figure. The results indicate that aloe-emodin can effectively inhibit the proliferation of breast cancer cells.

[0042] Example 3: Effect of aloe-emodin on the in vitro migration ability of breast cancer cells

[0043] MDA-MB-231 and MDA-MB-468 breast cancer cells were seeded in 6-well plates and treated with 0, 10, 20, and 40 μM aloe-emodin. Cells were cultured until they formed a monolayer. A vertical line was then drawn in the center of the culture dish using a sterile 200 μL pipette tip. The pipette tip was held against a ruler, and the pressure was moderate and the speed was uniform, ensuring a consistent line thickness. The culture dish was then gently shaken to suspend detached cells in the original culture medium. The original medium was then discarded and serum-free medium was added. This was recorded as 0h. Subsequently, at 12h, 24h, and 48h intervals after the first scratch, cell migration images were taken at the same location using a microscope, with scale bars clearly marked to ensure consistent image capture parameters. After the experiment, the migration rate was calculated using ImageJ software. The formula was: Migration rate = (0h scratch area - 48h scratch area) / 0h scratch area × 100%. The experimental results are shown below. Figure 3 As shown in the figure. The results indicate that aloe-emodin can effectively inhibit the in vitro migration ability of breast cancer cells.

[0044] Example 4: Effect of aloe-emodin on the in vitro invasive ability of breast cancer cells

[0045] Pre-chill the Matrigel, pipette tips, EP tubes, and other materials at 4°C one day in advance. Perform the entire experiment on ice. Mix the serum-free cell culture medium with the diluted Matrigel, add the mixture to the Transwell chambers, avoiding air bubbles, and incubate the chambers in an incubator for 2 hours. After incubation, aspirate the culture medium from the chambers, add 100 μL of serum-free medium to each well, and incubate for another 30 minutes. Inject 600 μL of complete culture medium containing 30% FBS into a 24-well plate, and insert the microchambers into the wells. Breast cancer cells MDA-MB-468 and MDA-MB-231, treated with 0, 10, 20, and 40 μM aloe-emodin respectively, were digested and centrifuged. They were washed twice with PBS buffer, the supernatant was discarded, and the cells were resuspended in serum-free medium to adjust the cell count to 200 cells / μL. 100 μL of cell suspension was added to the upper chamber, and the cells were incubated for approximately 36 hours (until attached cells appeared at the bottom of the wells in group Si-3). Culture was then stopped, and cell staining, photography, and cell counting were performed. The experimental results are shown below. Figure 4 As shown in the figure. The results indicate that aloe-emodin can effectively inhibit the in vitro invasive ability of breast cancer cells.

[0046] Example 5: In vitro experiment on the effect of aloe-emodin on angiogenesis in breast cancer cells

[0047] Pre-cooling materials such as matrix gel, pipette tips, and EP tubes were used. 50 μL of matrix gel was added to each well of a 96-well plate. Three auxiliary wells were set up for each of the four aloe-emodin treatment concentrations (0, 10, 20, and 40 μM). The entire experiment was conducted on ice. The 96-well plates were placed in an incubator for 1 hour to allow gel formation. HUVEC cells of passages 3-5 were selected for the experiment. When cell confluence reached 80%, cells were digested, centrifuged, and counted. Cells were seeded at 10,000 per well in the gel, and 100 μL of conditioned medium was added for culture. After 5 hours, images were taken and the tube-forming ability of each group was calculated using ImageJ software. The experimental results are shown below. Figure 5 As shown.

[0048] The results showed that aloe-emodin had a significant inhibitory effect on angiogenesis in breast cancer cells.

[0049] Example 6: Verifying the affinity of aloe-emodin for VEGFR2

[0050] Pull-down assay: First, biotin was used to label the aloe-emodin monomer for the pull-down experiment. Successful biotin labeling was confirmed by mass spectrometry and ¹H NMR spectroscopy. MDA-MB-231 breast cancer cells were treated with protein lysis buffer (ripa:protease inhibitor:phosphatase inhibitor = 100:1:1). After collecting the lysis buffer, it was centrifuged at 10000×g for 10-15 min at 4℃ to remove cell debris. The supernatant was collected as the lysis buffer containing total intracellular protein. 50 μL of streptavidin gel was added to the spin column for equilibration. 300 μL of biotin-labeled small molecules and biotin (5 mM) were added to the spin column. Another spin column was fitted with 300 μL of TBS buffer, the top and bottom caps were tightened, and the column was incubated on a shaker at 4℃ for 1 h. After incubation, discard unbound biotin, add 250 μL of biotin blocking buffer, incubate at room temperature for 5 min, centrifuge and discard the blocking buffer, add 300 μL of protein lysis buffer, tighten the top and bottom caps, and incubate overnight on a shaker at 4°C. After incubation, centrifuge and discard the unbound protein lysis buffer. Add 250 μL of wash buffer to the tube, centrifuge and discard the wash buffer, repeating three times. Add 250 μL of elution buffer, mix by pipetting, and transfer the bound protein to an EP tube. Add DTT to the EP tube, mix at room temperature, centrifuge and discard the solution. Add IAA to the EP tube, mix at room temperature in the dark, centrifuge and discard the solution. Add NH4HCO3 to the EP tube, mix at room temperature, centrifuge and discard the solution, and perform reductive alkylation. Add Trypsin enzyme solution, mix, centrifuge, collect the supernatant, and perform proteolytic digestion. Add NH4HCO3, shake to mix, centrifuge and collect the supernatant. Combine the two supernatants and desalt and extract the enzymatically digested peptides using a Monospin micro-desalting column.

[0051] MS experiments: After concentration and drying using a vacuum concentrator, the sample was reconstituted with 0.1% FA, and an equal volume of sample was loaded onto the spectrometer. Separation was performed using an EASY-nLC 1000 (Thermo Scientific, USA) with an analytical column (C18, 1.9 μm, 75 μm × 20 cm) and a flow rate of 500 nL / min. The mass spectrometer was an Orbitrap Fusion Lumos (Thermo Scientific, USA). Tandem mass spectrometry was performed in Data Dependent Acquisition (DDA) mode. The full scan resolution was 60,000 (FWHM), the mass-to-charge ratio range was set to m / z = 350-1800, the automatic gain control (AGC) was set to 4e5, the scan window was 0.7 m / z, and the maximum ion implantation time was 50 ms. In HCD fragmentation mode, the collision energy was set to 30%. The secondary mass spectrometry resolution was set to 15,000, the AGC was set to 1e5, and the maximum ion implantation time was 22 ms.

[0052] Based on the mass spectrometry results, the target protein of aloe-emodin was identified and molecular docking was performed, with the results as follows: Figure 6 As shown.

[0053] Molecular docking: Maestral was used to predict the affinity of the small molecule target VEGFR2 for aloe-emodin, and PyMOL was used to visualize and analyze the results, such as... Figure 7 As shown in the figure, the results indicate that VEGFR2 has a high affinity for aloe-emodin.

[0054] Example 7: Nude Mouse Proliferative Tumor Experiment

[0055] Culture MDA-MB-231 breast cancer cells. Using a 1mL syringe, aspirate the cell suspension and slowly inject 0.1-0.2mL (approximately 1×10⁻⁶ cells) subcutaneously into the right axilla of a nude mouse. 6 -2×10 6 (1 cell), after inoculation, feed normally, and after 7-10 days, when tumor nodules are palpable, begin measuring tumor size. When the tumor grows to 50-100mm... 3 Nude mice were randomly divided into a control group (receiving an equal volume of solvent) and low-, medium-, and high-dose aloe-emodin groups (corresponding doses of 10 mg / kg, 25 mg / kg, and 50 mg / kg, administered intraperitoneally once daily for 2-3 weeks), with 6 mice in each group. During the administration period, the length (a) and width (b) of the tumor were measured every 2-3 days using calipers, and calculated using the formula V = 1 / 2 × a × b. 2 Calculate tumor volume, record changes, and simultaneously observe changes in body weight. Experimental results are as follows: Figure 8 As shown in the figure. The results indicate that aloe-emodin can inhibit the growth of breast cancer cells.

[0056] Example 8: Immunohistochemical staining to detect the effect of aloe-emodin on angiogenesis in breast cancer cells

[0057] Sample preparation:

[0058] The breast cancer MDA-MB-231 tumor tissue obtained in Example 7 was embedded in paraffin and cut into 4 μm thick sections. The sections were dewaxed twice in xylene, 10 min each time. Then, the sections were washed with anhydrous ethanol for 10 min to remove the xylene. After that, the sections were sequentially immersed in 95%, 90%, 80%, and 70% ethanol, 2 min each time.

[0059] Perform antigen retrieval on tissue sections:

[0060] Place the slides in a container containing citrate buffer (working solution) and microwave at 92-98°C for 13 minutes. Remove the container and allow to cool to room temperature for 15 minutes. Then, wash with PBS three times for 5 minutes each time.

[0061] Antibody incubation:

[0062] Non-specific binding sites were blocked by adding 1% BSA blocking solution to the container, followed by incubation at 4°C overnight with CD31 primary antibody. The next day, the slides were incubated with the corresponding secondary antibody, and nuclear counterstaining was performed using DAB. Finally, the slides were observed under a microscope.

[0063] calculate:

[0064] A single endothelial cell or cluster of endothelial cells stained brownish-yellow was considered as one counting unit, while vessels with a lumen larger than eight red blood cells and a thick muscular layer were not counted. First, five areas with high MVD were selected under low magnification, and the highest vascular density areas were determined by observing CD31-positive cells (brown-stained endothelial cells). Under high magnification (200×), isolated endothelial cells or cell clusters were considered as one counting unit, and the number of microvessels in the five fields was recorded, and the average value was taken. Counting criteria: a single positively stained vascular endothelial cell; a single positively stained vascular endothelial cell cluster; branching structures on the vascular trunk, as shown in the results. Figure 9 As shown in the figure. The results indicate that it can effectively inhibit angiogenesis in breast cancer cells.

[0065] Example 9: Western blot detection of expression of downstream proteins related to the VEGFR2 signaling pathway

[0066] Protein extraction:

[0067] The lysis buffer was prepared as follows: (NaF: 1:100, Na2P2O7: 1:10, Na3VO4: 1:200, PMSF: 1:100, RIPA to make up the difference).

[0068] Obtain breast cancer cells MDA-MB-231 and MDA-MB-468 cells after treatment with 40 μM aloe-emodin:

[0069] Discard the original culture medium, add 2 mL of PBS to the pipette and slowly rotate to wash, repeat twice, discard the PBS, add 1000 μL of protein lysis buffer to each bottle, incubate on ice for 5 min to allow the protein to lyse, scrape the desired cells from the culture flask with a cell brush into a new 1.5 mL EP tube and label it.

[0070] Place on ice for 10 minutes, shake vigorously for 15 seconds, and repeat three times. After the third shake, set the centrifuge speed to 12000 r / min, 4℃, and centrifuge for 20 minutes. Take an appropriate amount of supernatant.

[0071] The protein sample concentration was determined by BCA method and adjusted to be consistent.

[0072] After adding 5× Loading buffer, the protein was denatured at high temperature in a metal bath for 5 minutes.

[0073] Western blot experiment:

[0074] Rubber Mixture:

[0075] Clean the glass plates thoroughly with tap water, rinse three times with distilled water, wipe them dry with clean paper, and reassemble them. Pour distilled water to check for leaks. Use a 10% (w / w) SDS-PAGE discontinuous gel system for electrophoresis separation. Prepare the separating gel according to the kit instructions. Mix the reagents and add them to the glass plate, filling it to approximately 3 cm from the top edge. Add isopropanol to flatten the lower gel layer. Let it stand for 30 minutes until a clear boundary appears between the lower gel and the isopropanol. Recover the isopropanol and rinse with distilled water. Prepare the stacking gel. Mix the reagents and slowly add them to the glass plate. Once full, immediately insert a comb rinsed with distilled water vertically to prevent air bubbles. Wait for the upper gel to solidify.

[0076] Electrophoresis:

[0077] After solidification, install the gel in the vertical electrophoresis tank, add freshly prepared electrophoresis buffer, vertically remove the comb, and select the appropriate sample volume according to the protein to be measured. Turn on the electrophoresis apparatus and select 80V constant voltage. After about 30 minutes, when the markers disperse and bands of different molecular weights are clearly visible, increase the pressure to 120V constant voltage. After about 1 hour, the indicator in the protein sample reaches the bottom of the gel, and stop electrophoresis.

[0078] Transfer and development:

[0079] Remove the gel and cut it according to the marker band corresponding to the desired protein molecular weight. Soak the gel in the electroporation buffer. Cut a PVDF membrane of the same size as the cut gel, and use dry tweezers to soak it in methanol for activation. After 5 minutes, place it in the prepared electroporation buffer, along with the transfer sponge pad, filter paper, etc., which are also soaked in the electroporation buffer for equilibration.

[0080] Using the sandwich method, sandwich the soaked sponge pad, two layers of filter paper, the cut gel, the PVDF membrane, two more layers of filter paper, and the sponge pad in sequence, while using a glass rod to remove air bubbles to avoid affecting the membrane transfer effect.

[0081] Connect the power supply. Connect the electroporation apparatus using the "black gel, white membrane" method, with the gel at the negative electrode and the PVDF membrane at the anode. Simultaneously, cover the electroporation apparatus with ice packs and plenty of ice to prevent overheating. Select a constant current of 200mA for 90 minutes to transfer the target protein from the gel to the PVDF membrane. After electroporation, place the transferred membrane in 5% skim milk for 2 hours to seal.

[0082] After blocking, the membrane was washed with TBST for 10 min and then incubated with the corresponding antibody diluted 1:1000 by volume at 4°C overnight; β-actin was selected as the internal control protein.

[0083] After primary antibody treatment, the membrane was washed twice with TBST, the first time for 10 minutes and the second time for 20 minutes.

[0084] After washing the membrane, add the corresponding species of secondary antibody according to the primary antibody and incubate at low speed for 1 hour.

[0085] After the secondary antibody incubation was completed, the membrane was washed twice with TBST, the first time for 10 minutes and the second time for 20 minutes.

[0086] The experimental results were obtained by developing with ECL and recording the results as follows: Figure 10 As shown, according to Figure 10 The results showed that the expression levels of VEGFR2, p-PLCG1, p-P38, and ERK were all decreased, indicating that aloe-emodin can inhibit the expression of VEGFR2 and regulate its downstream signaling pathways.

[0087] The above description is merely a preferred embodiment of this disclosure. It should be understood that this disclosure is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or techniques or knowledge in related fields. Any modifications and variations made by those skilled in the art that do not depart from the spirit and scope of this disclosure should be within the protection scope of the appended claims.

Claims

1. Application of aloe-emodin in inhibiting angiogenesis around breast cancer cells.

2. Use according to claim 1, characterized in that, The aloe-emodin inhibits angiogenesis around breast cancer cells by targeting VEGFR2.

3. The application of aloe-emodin as described in claim 1 in inhibiting the proliferation of breast cancer cells.

4. The application of aloe-emodin as described in claim 1 in inhibiting the in vitro migration of breast cancer cells.

5. The application of aloe-emodin as described in claim 1 in inhibiting the in vitro invasion of breast cancer cells.

6. A breast cancer treatment drug containing aloe-emodin as described in claim 1.

7. The breast cancer therapeutic drug according to claim 6, characterized by, The mechanisms of action of the breast cancer treatment drugs include: Inhibits angiogenesis around breast cancer cells; And / or, inhibit the proliferation of breast cancer cells; And / or, inhibit the ability of breast cancer cells to migrate in vitro; And / or, inhibit the ability of breast cancer cells to invade in vitro.

Images