Application of Xinjiang medicine mulberry leaf extract in angiogenesis of human umbilical vein endothelial cells

Abstract

The invention relates to the technical field of biological medicines, in particular to application of a Xinjiang medicine mulberry leaf extract in angiogenesis of human umbilical vein endothelial cells, which is technically characterized in that the Xinjiang medicine mulberry leaf extract promotes angiogenesis related gene expression and reduces oxidative stress response by promoting proliferation and migration of the umbilical vein endothelial cells and in-vitro tubule formation; further, the angiogenesis capability of human umbilical vein endothelial cells is promoted. Experiments prove that the cell activity is obviously enhanced under the concentration of 62.5, 125 or 250 [mu] g / mL of the mulberry leaf extract, and the difference has statistical significance (P is less than 0.001); compared with a blank group, the apoptosis rate of each drug group has no statistical difference, the active oxygen content of cells is reduced under the drug concentration of 62.5 mu g / mL and 125 mu g / mL, and the difference has statistical significance (P is less than 0.05); compared with a blank group, the relative horizontal migration rate, the vertical migration rate, the total in-vitro tubule formation length and angiogenesis-related mRNA and VEGF protein expression of cells in each drug group are remarkably enhanced.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of Xinjiang medicinal mulberry leaf extract in human umbilical vein endothelial cell angiogenesis. Background Art

[0002] Xinjiang medicinal mulberry belongs to the Morus genus, Moraceae, and Morus nigra species in plant taxonomy. As an ancient cultivated plant in Xinjiang, it is mainly distributed in Aksu, Hotan, and Kashgar in southern Xinjiang. In recent years, medicinal mulberry has gained wide attention as a plant variety for both medicinal and edible purposes. Modern chemical composition and pharmacological studies have shown that medicinal mulberry has a variety of pharmacological activities such as hypoglycemic, anti-oxidation, and anti-tumor due to its rich active ingredients such as flavonoids, polyphenols, and polysaccharides.

[0003] Human umbilical vein endothelial cell (HUVEC) angiogenesis refers to the formation of new blood vessels from existing capillaries or post-capillary veins, which is very important for physiological processes such as organ growth, embryonic development and wound healing.

[0004] At present, there is still no known effect of Xinjiang medicinal mulberry leaves on angiogenesis in the existing technology.

[0005] To this end, this application provides the application of Xinjiang medicinal mulberry leaf extract in human umbilical vein endothelial cell angiogenesis through research. Summary of the invention

[0006] The purpose of the present invention is to solve the prior art problems raised in the above background technology and to provide an application of Xinjiang medicinal mulberry leaf extract in human umbilical vein endothelial cell angiogenesis.

[0007] The above-mentioned purpose of the present invention is achieved like this:

[0008] The scheme of the invention provides the use of Xinjiang medicinal mulberry leaf extract in preparing a medicine for promoting angiogenesis of human umbilical vein endothelial cells.

[0009] Furthermore, the Xinjiang medicinal mulberry leaf extract promotes the proliferation, migration and in vitro tubule formation of umbilical vein endothelial cells, promotes the expression of angiogenesis-related genes, reduces oxidative stress response, and thus promotes the angiogenesis ability of human umbilical vein endothelial cells.

[0010] Furthermore, the concentration of the mulberry leaf extract is 62.5 μg / mL, 125 μg / mL or 250 μg / mL.

[0011] The difficulty and significance of the technical problem solved by the present invention are:

[0012] In recent years, medicinal mulberry has gained wide attention as a plant variety for both medicinal and edible purposes. Modern chemical composition and pharmacological studies have shown that medicinal mulberry has multiple pharmacological activities such as hypoglycemic, anti-oxidation, and anti-tumor due to its rich active ingredients such as flavonoids, polyphenols, and polysaccharides. However, the effect of Xinjiang medicinal mulberry leaves on angiogenesis is still unknown in the prior art. Human umbilical vein endothelial cells (HUVECs) are derived from the umbilical cord tissue of newborns, which is easy to obtain, has no ethical controversy, and is convenient for extracting sufficient cells. It has the ability of cells to form tubes, is easy to establish experimental models, and is the main experimental means for studying the function of vascular endothelial cells in vitro. To this end, the present invention provides a technical solution for the application of Xinjiang medicinal mulberry leaf extract in human umbilical vein endothelial cell angiogenesis, and in the experiment, different concentrations of Xinjiang medicinal mulberry leaf extract were used for intervention, confirming its effect on the angiogenic ability of human umbilical vein endothelial cells, and further confirming that Xinjiang medicinal mulberry leaf extract can promote the proliferation, migration and in vitro tubule formation of HUVECs, upregulate the expression of angiogenesis-related genes, reduce oxidative stress response, and further promote the angiogenic ability of HUVECs.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention provides a new application of Xinjiang medicinal mulberry leaf extract in human umbilical vein endothelial cell angiogenesis. The Xinjiang medicinal mulberry leaf extract can promote the proliferation, migration and in vitro tubule formation of HUVECs, upregulate the expression of angiogenesis-related genes, reduce oxidative stress response, and thus promote the angiogenesis ability of HUVECs. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The effects of different concentrations of Xinjiang medicinal mulberry leaf extracts on HUVECs cell viability in the embodiments of the present invention (ns means no statistically significant difference, **P<0.01, ***P<0.001, ****P<0.0001);

[0016] Figure 2 Flow cytometry analysis of HUVECs apoptosis in each group in the present invention;

[0017] Figure 3 The effects of different concentrations of Xinjiang medicine mulberry leaf extract on HUVECs apoptosis in the embodiments of the present invention;

[0018] Figure 4 This is a flow cytometry diagram of the reactive oxygen content of HUVECs cells in each group in the embodiment of the present invention;

[0019] Figure 5 The effect of different concentrations of Xinjiang medicinal mulberry leaf extract on the reactive oxygen content of HUVECs cells in the embodiment of the present invention (ns means no statistically significant difference, **P<0.05, **P<0.01);

[0020] Figure 6 It is the HUVECs scratch test in the embodiment of the present invention;

[0021] Figure 7 is the horizontal mobility of HUVECs at 12 and 24 h in the examples of the present invention (ns means no statistically significant difference, *P<0.05, **P<0.01);

[0022] Figure 8 It is the Transwell experiment in the embodiment of the present invention;

[0023] Fig. 9 The effect of Xinjiang medicine mulberry leaf extract on the vertical migration ability of HUVECs in the embodiment of the present invention (***P<0.001, ***P<0.0001);

[0024] Fig.10 is the tubule formation experiment in the embodiment of the present invention;

[0025] Fig.11 The effect of Xinjiang medicinal mulberry leaf extract on the tubule formation ability of HUVECs in the embodiment of the present invention (**P<0.01, ****P<0.0001);

[0026] Fig.12 The effect of Xinjiang medicine mulberry leaf extract on the mRNA expression level of HUVECs angiogenesis-related factors in the embodiment of the present invention;

[0027] Fig.13 It is the cell immunofluorescence staining in the embodiment of the present invention;

[0028] Fig.14 This is the effect of the Xinjiang medicine mulberry leaf extract on the expression of VEGF protein in HUVECs in the examples of the present invention (*P<0.05, **P<0.01). DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0030] The implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0031] As shown in the drawings, a preferred embodiment of the present invention is provided.

[0032] The embodiment of the present invention provides the use of Xinjiang medicine mulberry leaf extract in the preparation of a drug for promoting angiogenesis of human umbilical vein endothelial cells. It is also confirmed that Xinjiang medicine mulberry leaf extract can promote the proliferation, migration and in vitro tubule formation of HUVECs, upregulate the expression of angiogenesis-related genes, reduce oxidative stress response, and thus promote the angiogenesis ability of HUVECs.

[0033] The specific experiments of the embodiments of the present invention are as follows:

[0034] 1. Materials

[0035] 1.1 Cells

[0036] Human umbilical vein endothelial cells (HUVECs) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. HUVEC cells were cultured in DMEM medium containing 10% fetal bovine serum, penicillin (100 U / mL) and streptomycin (100 mg / mL) in an incubator at 37°C and 5% CO2 saturated humidity.

[0037] 1.2. Xinjiang medicinal mulberry leaves

[0038] Collected from the medicinal mulberry planting base in Aksu area of ​​Xinjiang Uygur Autonomous Region.

[0039] 1.3 Reagents

[0040] DMEM culture medium (Gbico, USA); fetal bovine serum (BI, USA); cytotoxicity detection kit CCK-8 (Proteintech, UK); apoptosis detection kit (Biyuntian Biotechnology Co., Ltd., China); reactive oxygen species detection kit (Biyuntian Biotechnology Co., Ltd., China); Matrigel (Corning, USA); Trizol RNA isolation reagent (Invitrogen, USA); reverse transcription kit (Takara, Japan); fluorescence real-time quantification kit (Takara, Japan); PCR primers (Sangon Biotechnology Co., Ltd., China); VEGF antibody (Cell Signaling Technology, USA); Alexa Fluor 488 rabbit anti-mouse polyclonal antibody (Cell Signaling Technology, USA); Transwell chamber (Corning, USA), pore size 8 μm.

[0041] 1.4 Instruments

[0042] BC-J160 carbon dioxide cell culture incubator (Boxun Medical Biological Instrument Co., Ltd., China); BS124S electronic balance (Thermo, USA); Multiskan GO full-wavelength microplate reader (Thermo, USA);

[0043] DMILLED inverted microscope (Leica, Germany); CHT210R high-speed refrigerated centrifuge (Hunan Xiangyi Experimental Instrument Co., Ltd., China); CytoFLEX flow cytometer (Beckman Coulter, USA); A51683 real-time fluorescence quantitative PCR instrument (Thermo, USA); Forma88500 -80℃ refrigerator (Thermo, USA).

[0044] 2. Methods

[0045] 2.1. Preparation of Xinjiang medicinal mulberry leaf extract

[0046] Xinjiang medicinal mulberry leaves were dried, crushed and sieved, and 70% ethanol was used as the extracting solution. The condensation reflux method was adopted, with an extraction time of 70 minutes, an extraction temperature of 80°C, a solid-liquid ratio of 1:45, vacuum filtration, rotary evaporation, freeze drying, and grinding into powder.

[0047] 2.2 Detection of cell viability by CCK-8 method HUVECs in logarithmic growth phase were divided into: blank group (complete culture medium), negative control group (complete culture medium containing cells), experimental group [Xinjiang medicine mulberry leaf extract of different concentrations (0, 15.625, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μg / ml) solution], 6 parallel wells were set up in each group, 100 μL was added to each well of 96-well plate, and after co-culture with HUVECs cells for 24 hours, the OD value was detected according to the CCK-8 kit manual method, and the cell proliferation rate was calculated. Cell proliferation rate = (OD experimental group-OD blank group) / (OD negative control group-OD blank group) × 100%.

[0048] 2.3 Experimental Grouping

[0049] The mice were divided into groups according to the experimental results under the above “2.2”: blank group (complete culture medium intervention) and drug group (62.5 μg / ml, 125 μg / ml and 250 μg / ml concentrations of mulberry leaf extract solution).

[0050] 2.4 Flow cytometry detection of cell apoptosis rate and reactive oxygen content

[0051] 2.4.1. HUVECs cells in the logarithmic growth phase were collected and counted at 1×10 4 After the cells adhered to the wall, they were grouped and intervened according to the method in "2.3". Three parallel wells were set up in each group. The cells were placed in a cell culture incubator for intervention for 2 days. The cells in each group were digested and digestion was terminated with old culture medium. The cells were transferred to centrifuge tubes and the apoptosis rate of each group was determined according to the instructions of the cell apoptosis detection kit.

[0052] 2.4.2. HUVECs cells in the logarithmic growth phase were collected and counted at 1×10 4 The cells were cultured in 6 cm culture dishes. After the cells adhered to the wall, they were grouped and intervened according to the method in "2.3". Three parallel wells were set up in each group and placed in a cell culture incubator for 2 days. After the intervention, the old culture medium was aspirated into the corresponding centrifuge tube. The cells were digested with phenol red-free trypsin, and the digestion was terminated with the old culture medium. The cells were transferred to the original centrifuge tubes, and the reactive oxygen content of each group of cells was measured according to the instructions of the reactive oxygen kit.

[0053] 2.5 Scratch test

[0054] HUVECs were grouped according to the method in "2.3" above and 1×10 5 The cells were inoculated at a density of 100 / well in a 6-well plate. When the cell fusion reached 80%, a uniform horizontal line was scratched on the bottom of the well plate with a 200 μL pipette tip. There were 3 replicate wells in each group. The scratch width was measured under an inverted microscope at 0, 12, 24, and 36 h, and the migration rate was calculated using Image J software. Mobility rate = (0 h scratch width - scratch width at each time period / 0 h scratch width) × 100%.

[0055] 2.6 Transwell experiment

[0056] According to the method in "2.3", each group has 3 replicate wells. HUVECs 2×10 4 Cells were inoculated at a density of 100 / well in the upper chamber of a Transwell 24-well plate, 200 μL of cell suspension was added, and 500 μL of complete medium containing 20% ​​serum was added to the lower chamber. After culturing in an incubator for 8 hours, methanol was fixed for 20 minutes, and the non-migrated cells in the chamber were wiped off with a wet cotton swab, and then stained with 0.2% crystal violet for 20 minutes. After staining, the residual dye was thoroughly washed off with PBS solution. The Transwell chamber was placed under an inverted microscope for observation, and the number of cell migration was recorded by taking pictures.

[0057] 2.7 Tubule formation assay

[0058] After the matrix gel was liquefied overnight at 4°C, the 96-well plate was taken out and placed on ice for precooling. 50 μL of matrix gel was added to each well. After being placed flat at 4°C for 10 min, the 96-well plate with matrix gel was placed in a 37°C, 5% CO2 incubator and waited for 30 min. The 96-well plate with solidified matrix gel was taken out and grouped according to the method under "2.3", with 3 replicates in each group. 2×10 HUVECs were plated 4 The cells were inoculated on the matrix gel at a density of 1000 / well, carefully shaken and placed in a 37°C, 5% CO2 incubator for culture. After 4 hours, the 96-well plate was taken out, and the HUVECs tubule formation was observed under an inverted microscope and photographed.

[0059] 2.8 RT-PCR experiments

[0060] HUVECs were grouped according to the method in "2.3" and inoculated into cell culture dishes, with 3 parallels in each group. After 48 hours of intervention, the cell supernatant was discarded, the cells were collected, and the total cell RNA was extracted using the Trizol method. After quantification, it was reverse transcribed into cDNA using a reverse transcription kit. Amplification was performed on a real-time quantitative fluorescence PCR instrument, with GAPDH as the internal reference. RT-PCR reaction conditions: (1) pre-denaturation at 95°C for 2 minutes; (2) denaturation at 95°C for 5 seconds; (3) annealing at 58°C for 30 seconds, for a total of 40 cycles; (4) extension at 72°C for 5 minutes. -ΔΔCT The relative quantitative method was used to detect the mRNA expressions of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet endothelial cell adhesion molecule-1 (CD31), and cell surface marker (cluster of differentiation 34, CD34). The primer sequences are shown in Table 1.

[0061] Table 1 RT-qPCR primers

[0062]

[0063] 2.9 Immunofluorescence staining

[0064] HUVECs were grouped according to the method in "2.3" and 1×10 5 After inoculation at a density of 100 cells / well in a 6-well plate and cultured for 24 hours, the cells were fixed with 4% paraformaldehyde for 30 minutes, treated with 0.5% Triton 100 for 15 minutes, and washed three times with PBS. Blocked with 5% BSA blocking solution at room temperature for 30 minutes, VEGF antibody was added, and incubated overnight at 4°C. The next day, the cells were labeled with Alexa Fluor488 and incubated at room temperature for 1 hour. After incubation, the cells were washed three times with PBS and the cell nuclei were labeled with the fluorescent dye DAPI. The whole process was carried out in the dark to prevent fluorescence quenching. Observed and photographed under a fluorescence microscope.

[0065] 3. Results

[0066] 3.1 Effects of different concentrations of Xinjiang medicinal mulberry leaf extract on HUVECs activity

[0067] The CCK-8 results showed that compared with the blank group, the concentrations of 62.5, 125, and 250 μg / mL significantly enhanced cell viability, which was statistically significant (P < 0.001); the concentrations of 1000 and 2000 μg / mL significantly inhibited cell viability, and the difference was statistically significant (P < 0.01). Therefore, 62.5, 125, and 250 μg / mL concentrations of Xinjiang medicinal mulberry leaf extract were selected for subsequent experiments.

[0068] 3.2 Effect of Xinjiang medicinal mulberry leaf extract on apoptosis of HUVECs

[0069] The results of flow cytometry showed that there was no statistical difference in the apoptosis rate among the groups. Figure 2 , Figure 3 shown.

[0070] 3.3 Effect of Xinjiang medicinal mulberry leaf extract on the content of reactive oxygen species in HUVECs

[0071] The results of flow cytometry showed that compared with the blank group, the reactive oxygen content of HUVECs cells decreased at drug concentrations of 62.5 and 125 μg / mL, and the difference was statistically significant (P < 0.05). There was no statistical difference at a drug concentration of 250 μg / mL. Figure 4 , Figure 5 .

[0072] 3.4 Effect of Xinjiang medicinal mulberry leaf extract on the migration ability of HUVECs

[0073] 3.4.1 Horizontal Migration Capability

[0074] The results of the scratch test showed that compared with the blank group, the horizontal mobility of HUVECs at drug concentrations of 62.5, 125, and 250 μg / mL was enhanced and in a dose-dependent manner (P < 0.01). Figure 6 , Figure 7 shown.

[0075] 3.4.2 Vertical Migration Ability

[0076] The results of the Transwell experiment showed that compared with the blank group, the number of HUVECs vertical migration increased significantly at drug concentrations of 62.5, 125, and 250 μg / mL, and was dose-dependent, with statistical significance (P < 0.001). Figure 8 , Fig. 9 shown.

[0077] 3.5 Effect of Xinjiang medicinal mulberry leaf extract on the angiogenesis ability of HUVECs in vitro

[0078] The results of tubule formation showed that compared with the blank group, the number of HUVECs in vitro tubule branches increased significantly at drug concentrations of 62.5, 125, and 250 μg / mL, and was dose-dependent, with statistical significance (P < 0.01). Fig.10 , Fig.11 shown.

[0079] 3.6 Effect of Xinjiang medicinal mulberry leaf extract on the expression of VEGF, FGF, CD31, and CD34 mRNA in HUVECs

[0080] RT-PCR results showed that compared with the blank group, the expression of VEGF, FGF, CD31, and CD34 in HUVECs increased at drug concentrations of 62.5, 125, and 250 μg / mL, and the difference was statistically significant (P < 0.05). The expression of VEGF, FGF, and CD34 was dose-dependent. Fig.12 abd in; CD31 was expressed at the highest concentration of 125 μg / mL. Fig.12 c in.

[0081] 3.7 Effect of Xinjiang medicinal mulberry leaf extract on VEGF protein expression in HUVECs

[0082] The results of cell immunofluorescence staining showed that compared with the blank group, the average fluorescence intensity at drug concentrations of 125 and 250 μg / mL was enhanced, and the difference was statistically significant. There was no statistical difference at a drug concentration of 62.5 μg / mL. Fig.13 , Fig.14 shown.

[0083] As mentioned above, the inventors of this application have previously shown that mulberry leaf extract can inhibit oxidative stress and inflammatory response, promote osteogenic differentiation, and has a therapeutic effect on periodontitis. The above experiment of the present invention mainly confirms the effect of Xinjiang medicinal mulberry leaf extract on the angiogenesis ability of HUVECs in vitro, in order to provide a new theoretical basis for the treatment of periodontitis with medicinal mulberry leaves.

[0084] Angiogenesis is regulated by endogenous pro-angiogenic mediators and begins to mediate angiogenesis, prompting endothelial cells to produce proteases, thereby degrading the basement membrane, endothelial cells migrate to the vascular bed, form new blood vessel buds, proliferate, elongate blood vessel buds, and finally form tubes. The above CCK-8 results showed that the activity of HUVECs was significantly enhanced at 62.5, 125, and 250 μg / mL mulberry leaf extract concentrations, and the flow cytometry apoptosis experiment also verified that the above mulberry leaf extract concentrations had no effect on HUVECs apoptosis. Reactive oxygen plays a key role in mediating oxidative stress in the body, regulating transcription factors and apoptosis. The results of flow cytometry reactive oxygen showed that 62.5 and 125 μg / mL mulberry leaf extracts can effectively inhibit the reactive oxygen content of HUVECs. In the scratch horizontal migration experiment and the Transwell chamber vertical migration experiment, both the horizontal migration and vertical migration abilities of the drug group were significantly higher than those of the blank group, indicating that mulberry leaf extract has a promoting effect on HUVECs migration. In the in vitro tubule formation experiment, the total length of the in vitro tubule structure and its branch points of the tubules in the drug group increased significantly, indicating that mulberry leaf extract has the effect of promoting the angiogenic differentiation of HUVECs. Angiogenesis is closely related to the regulation of endogenous pro-angiogenic mediators. VEGF is a highly specific pro-vascular endothelial cell growth factor that promotes vascular endothelial cell migration, proliferation and angiogenesis. FGF can promote the migration of endothelial cells and the proliferation of smooth muscle cells, and promote the formation of new blood vessels. CD31 is an endothelial cell marker that participates in biological processes such as inflammatory response, cell migration, angiogenesis and cell apoptosis. CD34 plays a role in promoting angiogenesis and maintaining vascular stability in vascular endothelial cells. In the RT-PCR experiment, the mulberry leaf extract group can upregulate the expression of VEGF, FGF, CD31, and CD34 genes to varying degrees. And in immunofluorescence staining, mulberry leaf extract promotes VEGF protein expression.

[0085] In summary, Xinjiang medicinal mulberry leaf extract can promote the proliferation, migration and in vitro tubule formation of umbilical vein endothelial cells within the corresponding concentration range, promote the expression of angiogenesis-related genes, reduce oxidative stress response, and thus promote the angiogenesis ability of human umbilical vein endothelial cells.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of Xinjiang medicinal mulberry leaf extract in the preparation of drugs for promoting angiogenesis in human umbilical vein endothelial cells.

2. The use according to claim 1, characterized in that: The Xinjiang medicinal mulberry leaf extract promotes the proliferation, migration and in vitro tubule formation of umbilical vein endothelial cells, promotes the expression of angiogenesis-related genes, reduces oxidative stress response, and thus promotes the angiogenesis ability of human umbilical vein endothelial cells.

3. The use according to claim 1 or 2, characterized in that: The concentration of the mulberry leaf extract is 62.5 μg / mL, 125 μg / mL or 250 μg / mL.

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