Tea polysaccharide / selenium nano-particles for improving hypoxia tolerance of cells and application of tea polysaccharide / selenium nano-particles

Abstract

The invention discloses tea polysaccharide / selenium nanoparticles for improving cell hypoxia tolerance and application, and belongs to the technical field of cytobiology and medicine. According to the invention, tea polysaccharide / selenium nanoparticles are adopted, and the tea polysaccharide / selenium nanoparticles are at least one of tea polysaccharide, tea polysaccharide nanoparticles and tea polysaccharide selenium nanoparticles; the tea polysaccharide / selenium nanoparticles are prepared through electrostatic interaction or redox reaction. The tea polysaccharide / selenium nanoparticles have the effects of improving the RLE-6TN hypoxia tolerance of cells, including: reducing cell apoptosis caused by hypoxia; the generation of cell active oxygen caused by oxygen deficit is reduced; the reduction of respective enzyme activity in cells caused by oxygen deficit is reduced; and the expression of related genes and proteins of an intracellular hypoxia stress pathway caused by hypoxia is down-regulated.

Description

Technical Field

[0001] The present invention relates to the technical fields of cell biology and medical technology, and particularly relates to a tea polysaccharide / selenium nanoparticle for improving the hypoxia tolerance of cells and its application. Background Art

[0002] RLE-6TN cells, as a classic lung epithelial cell model, play an important role in simulating and studying the pathogenesis of lung diseases and pharmaceutical methods. Such cells not only possess the typical characteristics of lung epithelial cells, such as expressing specific cell surface molecules and cytokines, but also can maintain a relatively stable cell morphology and function in an in vitro environment. Therefore, they have become an important tool for the study of lung diseases. However, under physiological and pathological conditions, lung tissues often face the challenge of hypoxia. The hypoxic environment is a huge threat to lung epithelial cells, which can lead to an exacerbation of the intracellular oxidative stress response, abnormal energy metabolism, and disorders of a series of signaling pathways, thereby significantly reducing the viability and adaptability of cells. Under extreme hypoxia conditions, RLE-6TN cells even undergo cell damage and apoptosis, seriously affecting the normal function of the lungs.

[0003] Therefore, preparing a drug for improving the hypoxia tolerance of RLE-6TN cells, that is, enhancing the viability and adaptability of RLE-6TN cells in a hypoxic environment, has become a technical problem urgently to be solved in the prior art. Summary of the Invention

[0004] Based on the above technical problems to be solved by the present invention, the present invention provides a tea polysaccharide / selenium nanoparticle for improving the hypoxia tolerance of cells and its application.

[0005] One object of the present invention is to provide a tea polysaccharide / selenium nanoparticle for improving the hypoxia tolerance of cells, and the tea polysaccharide / selenium nanoparticle is at least one of tea polysaccharide, tea polysaccharide nanoparticle, and tea polysaccharide selenium nanoparticle.

[0006] Further, the tea polysaccharide includes moisture, protein, total sugar, uronic acid, and tea polyphenols;

[0007] Further, the tea polysaccharide is extracted by hot water extraction method, including: weighing tea powder at 3% - 7% in water, extracting with hot water at 60°C - 80°C for 2h - 4h to obtain the extracted tea solution; centrifuging the extracted tea solution at 3000rpm - 5000rpm for 5min - 30min to obtain the supernatant; adding sevage solution to the supernatant, oscillating and separating to obtain tea polysaccharide, protein, and sevage solution which are divided into 3 layers from top to bottom; the sevage solution is chloroform: n-butanol = 4:1; centrifuging the upper layer of the tea polysaccharide solution at 6000rpm - 10000rpm for 10min - 30min to remove the residual protein, obtaining the tea polysaccharide solution without protein; adding 3 - 5 times the volume of absolute ethanol to the tea polysaccharide solution without protein, refrigerating and standing for 6h - 24h, centrifuging at 3000rpm - 5000rpm to obtain the precipitate, dissolving in water, concentrating, and freeze-drying to obtain the tea polysaccharide;

[0008] Further, the preparation of the tea polysaccharide selenium nanoparticles includes: preparing a solution by mixing the tea polysaccharide solution and sodium selenite solution at a mass-to-volume ratio of (0 - 0.6):1, and mixing evenly to obtain a tea polysaccharide-sodium selenite mixture; freshly preparing ascorbic acid solution, dropping the 30mM - 50mM ascorbic acid solution in equal volume into the tea polysaccharide-sodium selenite mixture, stirring in the dark at 30°C - 50°C for 2h - 6h; dialyzing with deionized water to obtain the tea polysaccharide selenium nanoparticles after removing the excess ascorbic acid and sodium selenite.

[0009] Further, the tea polysaccharide / selenium nanoparticles are prepared by electrostatic interaction or redox reaction.

[0010] Further, the concentration range of the tea polysaccharide / selenium nanoparticles is 0.1μg / mL - 100μg / mL.

[0011] Further, the particle size range of the tea polysaccharide / selenium nanoparticles is 80nm - 1000nm; preferably, the particle size range of the tea polysaccharide / selenium nanoparticles is 100nm - 500nm.

[0012] Further, the tea polysaccharide / selenium nanoparticles are tea polysaccharide / selenium nanoparticles in which the selenium nanoparticles are encapsulated by the tea polysaccharide.

[0013] The second object of the present invention is to provide an application of the tea polysaccharide / selenium nanoparticles for improving the hypoxia tolerance of cells, and the application is the application of the tea polysaccharide / selenium nanoparticles for improving the hypoxia tolerance of cells.

[0014] Further, the application includes exposing RLE-6TN cells to a culture medium containing the tea polysaccharide / selenium nanoparticles.

[0015] Further, the application includes subjecting the RLE-6TN cells to hypoxia treatment for 8 h to 12 h after being exposed to tea polysaccharide / selenium nanoparticles for 0.5 h to 2 h, so as to provide the viability and adaptability of the cells in a hypoxic environment.

[0016] Further, the tea polysaccharide / selenium nanoparticles can alleviate cell apoptosis caused by hypoxia.

[0017] Further, the tea polysaccharide / selenium nanoparticles can significantly alleviate the change in enzyme activity caused by hypoxia.

[0018] Further, the tea polysaccharide / selenium nanoparticles can significantly alleviate the expression of genes and proteins related to the hypoxia pathway triggered by hypoxia.

[0019] Compared with the prior art, the present invention provides a tea polysaccharide / selenium nanoparticle and an application for improving the hypoxia tolerance of cells, and has the following beneficial effects:

[0020] 1. By intervening in hypoxic cells with tea polysaccharide, tea polysaccharide nanoparticles and tea polysaccharide selenium nanoparticles, the present invention obtains tea polysaccharide / selenium nanoparticles that can rapidly relieve the damage caused by hypoxia in RLE-6TN cells; meanwhile, the addition of tea polysaccharide / selenium nanoparticles improves the cell viability and antioxidant capacity of hypoxic RLE-6TN cells; reduces the activities of lactate dehydrogenase (LDH) and the content of malondialdehyde (MDA) in hypoxic RLE-6TN cells, and at the same time reduces the expression levels of genes and proteins of PI3K, AKT, mTOR, p70S6K and HIF-1α in hypoxic RLE-6TN cells. The prepared nanoparticles have the characteristics of small particle size and strong antioxidant capacity, and can better reach the inside of cells to improve oxygen tolerance.

[0021] 2. The tea polysaccharide, its nanoparticles and tea polysaccharide selenium nanoparticles provided by the present invention have the characteristics of improving the hypoxia tolerance of RLE-6TN cells and being simple and easy to operate.

[0022] 3. The drug for improving the hypoxia tolerance of RLE-6TN cells prepared by the present invention can significantly improve the viability and adaptability of RLE-6TN cells in a hypoxic environment, and provides a new solution for alleviating cell hypoxia stress and acute hypoxia, and preparing drugs related to cell hypoxia and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The characteristic diagrams of the tea polysaccharide / selenium nanoparticles implemented by the present invention are shown, wherein, FIGS. A-F are transmission electron microscope diagrams of nano selenium (SeNPs), FIGS. G-L are transmission electron microscope diagrams of tea polysaccharide / selenium nanoparticles (TPS-SeNPs), FIGS. D-F are EDS energy spectra of nano selenium (SeNPs), and FIGS. J-L are EDS energy spectra of tea polysaccharide / selenium nanoparticles (TPS-SeNPs).

[0024] Figure 2 Shows the toxicity graph of a tea polysaccharide / selenium nanoparticle of an embodiment of the present invention against RLE-6TN cells. Among them, Figure A is the TPS hypoxia intervention group; Figure B is the TPS1-NPs hypoxia intervention group; Figure C is the TPS2-NPs hypoxia intervention group; Figure D is the SeNPs hypoxia intervention group; Figure E is the TPS-SeNPs hypoxia intervention group;

[0025] Figure 3 Shows the morphological influence graph of a tea polysaccharide / selenium nanoparticle of an embodiment of the present invention on the RLE-6TN hypoxia model;

[0026] Figure 4 Shows the apoptosis situation and apoptosis ratio graph of a tea polysaccharide / selenium nanoparticle of an embodiment of the present invention against RLE-6TN cells under hypoxic conditions. Among them, Figure A is the normoxia group; Figure B is the hypoxia group; Figure C is the TPS hypoxia intervention group; Figure D is the TPS1-NPs hypoxia intervention group; Figure E is the TPS2-NPs hypoxia intervention group; Figure F is the SeNPs hypoxia intervention group; Figure G is the TPS-SeNPs hypoxia intervention group.

[0027] Figure 5 Shows the influence graph of the fluorescence intensity of intracellular ROS staining of a tea polysaccharide / selenium nanoparticle of an embodiment of the present invention against RLE-6TN cells under hypoxic conditions;

[0028] Figure 6 Shows the influence graph of a tea polysaccharide / selenium nanoparticle of an embodiment of the present invention on antioxidant-related indexes of RLE-6TN cells under hypoxic conditions. Among them, Figure A is the influence on GSH-Px activity, Figure B is the influence on SOD activity, Figure C is the influence on LDH activity, and Figure D is the influence on MDA activity;

[0029] Figure 7 Shows the expression level graph of PI3K, AKT, mTOR, p70S6K, and HIF-1α of hypoxia-related genes in RLE-6TN cells treated with a tea polysaccharide / selenium nanoparticle of an embodiment of the present invention under hypoxic conditions;

[0030] Figure 8 Shows the expression level of hypoxia-related proteins in RLE-6TN cells treated with a tea polysaccharide / selenium nanoparticle of an embodiment of the present invention under hypoxic conditions. Among them, Figure A is the protein band graph of PI3K, AKT, mTOR, p70S6K, and HIF-1α; Figure B is the statistical graph of the gray value of the PI3K protein band; Figure C is the statistical graph of the gray value of the AKT protein band; Figure D is the statistical graph of the gray value of the mTOR protein band; Figure E is the statistical graph of the gray value of the p70S6K protein band; Figure F is the statistical graph of the gray value of the HIF-1α protein band. Detailed implementation manners

[0031] Exemplary embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present application are shown in the drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided so that the present application can be more thoroughly understood and the scope of the present application can be fully conveyed to those skilled in the art.

[0032] In the following embodiments, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art, and the reagents and materials in the present invention are obtained from the market or other public channels.

[0033] The experimental raw materials and equipment involved in the present invention mainly include but are not limited to:

[0034] (1) Reagents:

[0035] Tea polysaccharide (TPS) is extracted by hot water extraction method; the specific extraction method is: weigh 5.0 g of tea powder and add 100 mL of water, and extract with hot water at 70 °C for 3 h. After the extraction is completed, centrifuge at 4000 rpm for 15 min to obtain the supernatant. Then add 300 mL of sevage solution and shake for 20 min, pour it into a separating funnel for liquid separation, which is mainly divided into three layers from top to bottom: tea polysaccharide, protein, and sevage solution. Take the upper layer of tea polysaccharide solution and centrifuge at 8000 rpm for 20 min to remove the residual protein. Add four times the volume of absolute ethanol to the obtained solution, and let it stand in a refrigerator at 4 °C for 12 h. Then centrifuge at 4000 rpm for 15 min to obtain a precipitate, dissolve it with a small amount of water, and concentrate it by rotary evaporation. Finally, freeze-dry to obtain tea polysaccharide (TPS), please refer to Figure 1 . Among them, the sevage solution is chloroform: n-butanol = 4:1.

[0036] The source of the tea powder is Hubei Enshi Lanbei Tea Co., Ltd.; the specific variety is Chunbo Green.

[0037] The internal components of the tea polysaccharide are 7.60% water, 5.82% protein, 31.02% total sugar, 14.21% uronic acid, 1.98% tea polyphenols, etc.

[0038] Preparation of tea polysaccharide nano-selenium: Prepared by the redox method; specifically, the method is as follows: Mix tea polysaccharide solutions (0, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL) with equal amounts of sodium selenite solutions (2.5 mM, 5 mM, 10 mM, 20 mM, 40 mM, 80 mM) under magnetic stirring. Then, drop an equal amount of freshly prepared 40 mM ascorbic acid solution into the mixture under vigorous stirring, and stir for 4 h in the dark at 40 °C. Finally, dialyze (3500 Da) with deionized water in the dark for 4 days to remove excess ascorbic acid and sodium selenite.

[0039] Ascorbic acid was purchased from Aladdin.

[0040] The cells used were rat type II alveolar epithelial cells (RLE-6TN), purchased from Procell.

[0041] (2) Instruments:

[0042] DF-101S magnetic stirrer: Yuhua Instrument Co., Ltd.; A580 AOELAB ultraviolet spectrophotometer: Aoyi Instrument Shanghai Co., Ltd.; neofuge 15R refrigerated centrifuge: heal force; zeta sizer Nano WD-9405A decolorizing shaker: Beijing Liuyi Instrument Factory; DYY-6C electrophoresis apparatus: Beijing Liuyi Instrument Factory; AX-II dark box: Guangdong Yuehua Medical Instrument Factory Co., Ltd.; Tanon-4800 exposure instrument: Tianneng; MCO-15AC CO2 incubator: SANYO; IX51 inverted microscope: OLYMPUS; MULTISKAN MK3 microplate reader: Thermo; FACSCalibur flow cytometer: BD Biosciences; FI3 imaging system: Nikon, Japan; SCI1000-G PCR gradient gene amplifier: SCILOGEX.

[0043] The preparation method and application of a tea polysaccharide / selenium nanoparticle for improving the hypoxia tolerance of cells according to the present invention can significantly improve the viability and adaptability of RLE-6TN cells in a hypoxic environment through specific treatment means, providing a new solution for alleviating cell hypoxic stress and acute hypoxia, and preparing drugs related to cell hypoxia and other fields.

[0044] The technical principle of the present invention mainly includes:

[0045] Hypoxia induced apoptosis of RLE-6TN cells, increased the activities of lactate dehydrogenase (LDH) and the content of malondialdehyde (MDA), enhanced the generation of reactive oxygen species (ROS), decreased the activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), and significantly increased the gene and protein expression levels of the hypoxia stress pathway (PI3K, AKT, mTOR, p70S6K, and HIF-1α). The mechanism by which tea polysaccharide / selenium nanoparticles improve the hypoxia tolerance of RLE-6TN cells includes reducing hypoxia-induced apoptosis, slowing down the generation of reactive oxygen species (ROS) caused by hypoxia, alleviating the decrease in the activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) caused by hypoxia, and reducing the gene and protein expression of the hypoxia stress pathway (PI3K, AKT, mTOR, p70S6K, and HIF-1α) triggered by hypoxia.

[0046] Based on the above principle, the present invention provides a tea polysaccharide / selenium nanoparticle for improving the hypoxia tolerance of cells and its application, including: after exposing RLE-6TN cells to tea polysaccharide / selenium nanoparticles for 0.5 h to 2 h, subjecting them to hypoxia treatment for 8 h to 12 h. The tea polysaccharide / selenium nanoparticles of the present invention are prepared by physical or chemical methods; further, they are prepared by electrostatic interaction or redox reaction. The RLE-6TN cells are type II alveolar epithelial cells of rats identified by SP-C immunofluorescence, with a purity of over 90% and free of HIV-1, HBV, HCV, mycoplasma, bacteria, yeast, and fungi, etc.;

[0047] The present invention provides a verification of the improvement of the hypoxia tolerance of RLE-6TN cells by tea polysaccharide / selenium nanoparticles, including: (1) treating RLE-6TN cells with tea polysaccharide / selenium nanoparticles at gradient concentrations; (2) detecting the viability of RLE-6TN cells treated with tea polysaccharide / selenium nanoparticles by the CCK-8 assay to obtain the concentration of tea polysaccharide / selenium nanoparticle intervention. Further, the present invention uses an inverted microscope to observe cell growth and morphology under normoxia, hypoxia, and tea polysaccharide / selenium nanoparticle intervention. Further, ROS staining, DAPI counterstaining of cell nuclei, mounting, and observation under a fluorescence microscope are performed on the cell treatments of the normoxia, hypoxia, and tea polysaccharide / selenium nanoparticle intervention groups to explore the ROS scavenging effect of TPS-NPs and TPS-SeNPs on cells. Further, the activities of glutathione peroxidase (GSH-Px), lactate dehydrogenase (LDH), superoxide dismutase (SOD), and the content of malondialdehyde (MDA) in the cells of the normoxia, hypoxia, and tea polysaccharide / selenium nanoparticle intervention groups are detected. Further, the gene and protein expression levels of PI3K, AKT, mTOR, p70S6K, and HIF-1α in the cells of the normoxia, hypoxia, and tea polysaccharide / selenium nanoparticle intervention groups are detected.

[0048] Tea polysaccharide is a kind of acidic polysaccharide or acidic glycoprotein combined with protein, which is an active compound polysaccharide in tea. Tea polysaccharide is composed of carbohydrates, pectin, protein, etc. The polysaccharide part includes water-soluble polysaccharides such as arabinose, xylose, fucose, glucose, and galactose. The protein part mainly consists of 20 common amino acids. In addition, it also includes a large number of mineral elements, including calcium, magnesium, iron, manganese, etc., and a small amount of trace elements. The application of tea polysaccharide in improving the hypoxia tolerance of RLE-6TN cells, that is, enhancing its survival ability and adaptability in a hypoxic environment, is of great significance for understanding and treating lung diseases related to cell hypoxia.

[0049] The tea polysaccharide / selenium nanoparticles of the present invention have the effect of improving the hypoxia tolerance of RLE-6TN cells, including: (1) reducing cell apoptosis caused by hypoxia; (2) reducing the generation of intracellular reactive oxygen species caused by hypoxia; (3) reducing the decrease of intracellular enzyme activities caused by hypoxia; (4) down-regulating the expression of related genes and proteins in the intracellular hypoxia stress pathway caused by hypoxia.

[0050] Example 1

[0051] The present invention proposes the modeling conditions of hypoxic RLE-6TN cell groups and RLE-6TN cells intervened by tea polysaccharide / selenium nanoparticles under hypoxia.

[0052] It mainly includes:

[0053] (1) Screening of the concentration of tea polysaccharide / selenium nanoparticle intervention: Use the CCK-8 cell proliferation and cytotoxicity detection kit to explore the cytotoxicity of tea polysaccharide (TPS), tea polysaccharide nanoparticles-1 (TPS1-NPs, 400nm), tea polysaccharide nanoparticles-2 (TPS2-NPs, 200nm), nano-selenium (SeNPs) and tea polysaccharide nano-selenium (TPS-SeNPs) at concentrations of 0, 10, 25, 50, 100, and 150 μg / mL;

[0054] (2) Conditions of the hypoxic modeling group: Cultivate RLE-6TN cells in an incubator containing 95% N2 and 5% CO2 for 12h; the cell viability is reduced to 50% compared with the control, and the number of cells per measurement unit is about 5000, and the difference between the hypoxic treatment group and the control group is the largest;

[0055] (3) Modeling conditions for tea polysaccharide / selenium nanoparticles to intervene in hypoxic RLE-6TN cells: First, add tea polysaccharide (TPS), tea polysaccharide nanoparticles-1 (TPS1-NPs, 400 nm), tea polysaccharide nanoparticles-2 (TPS2-NPs, 200 nm), nano-selenium (SeNPs), and tea polysaccharide nano-selenium (TPS-SeNPs) respectively and treat for 1 h, then place the cells in an incubator containing 95% N2 and 5% CO2 and culture for 12 h.

[0056] Results:

[0057] Please refer to Figure 2 A-E. The safe doses of the above-mentioned TPS, TPS1-NPs, TPS2-NPs, SeNPs, and TPS-SeNPs at different concentrations of 10, 25, 50, 100, and 150 μg / mL on RLE-6TN cells were explored by the CCK-8 method. It was found that there was no significant difference in cell growth between the concentration of 10 μg / mL and the blank group. The concentrations of TPS, TPS1-NPs, TPS2-NPs, SeNPs, and TPS-SeNPs were screened to be 10 μg / mL. It shows that the final intervention concentrations of TPS, TPS1-NPs, TPS2-NPs, SeNPs, and TPS-SeNPs were screened to be 10 μg / mL.

[0058] Example 2

[0059] The present invention proposes a cell morphology observation experiment.

[0060] It mainly includes:

[0061] An inverted microscope was used to observe the differences in cell growth and morphology under normoxic and hypoxic environments, and photos were taken for recording.

[0062] The tea polysaccharide selenium nanoparticles prepared from the above-mentioned tea polysaccharide solutions with different concentrations were measured for particle size and total antioxidant capacity, and the tea polysaccharide with the smallest particle size and the strongest total antioxidant capacity of the prepared tea polysaccharide selenium nanoparticles was screened as the optimal concentration.

[0063] Results:

[0064] Please refer to Figure 3 , in the normoxic group, the cells adhered and grew into a continuous sheet, and the cells showed a polygonal shape. In the hypoxic group, it was obvious that the adherent cells were significantly reduced, the cell gaps were widened, and a small amount of cell debris could be observed. Compared with the hypoxic group, the number of cells in the hypoxic intervention group treated with the sample was significantly increased, the adherent cells were increased, and the cell gaps were narrowed. This indicates that the hypoxic intervention group can significantly improve the changes brought about by hypoxia and has the effect of resisting hypoxic stress.

[0065] Please refer toFigure 2 E. Among the hypoxia intervention groups, the effect of tea polysaccharide selenium nanoparticles (TPS-SeNPs) on cell morphology was the least, and the cells grew most densely. The concentration of tea polysaccharide / selenium nanoparticles when it did not affect the growth of RLE-6TN cells was 10 μg / mL. Among the cell morphologies of RLE-6TN cells intervened with hypoxia by tea polysaccharide / selenium nanoparticles, the effect of TPS-SeNPs on cell morphology was the least, and the cells grew most densely.

[0066] Example 3

[0067] The present invention provides an experiment for detecting the apoptosis rate of cells.

[0068] It mainly includes:

[0069] The present invention measures through the operation instructions of a cell apoptosis detection kit and a flow cytometer to explore the apoptosis conditions of RLE-6TN cells in the normoxia group, hypoxia group, and hypoxia intervention group.

[0070] For the RLE-6TN cells in the above different treatment groups, digest the cells with 0.25% trypsin without EDTA. After terminating the digestion, collect the RLE-6TN cells, centrifuge at 1500 rpm for 5 min, discard the supernatant, resuspend with PBS, and repeatedly wash the cells with PBS 2 times, 1500 rpm, 5 min; then add 500 μL of Binding Buffer, resuspend the cells, mix 5 μL of AnnexinV-FITC and then add 5 μL of PI, mix well, and react at room temperature in the dark for 5 - 15 min (at the same time, set negative controls, that is, normal cells without adding Annexin and PI; positive control 1, use the solvent group with the most obvious apoptosis effect as the positive control, only add 5 μL of AnnexinV single label; positive control 2, use the solvent group with the most obvious apoptosis effect as the positive control, only add 5 μL of PI single label), and detect the apoptosis conditions of RLE-6TN cells by using a flow cytometer.

[0071] Results:

[0072] Cell apoptosis is a process of programmed active cell death regulated by genes. Different from necrosis, it is another form of cell death. Cell apoptosis is a basic phenomenon of life and plays a crucial role in maintaining normal development, growth of the body, and ensuring the stability of the internal environment. Apoptotic cells can show cell shrinkage and vacuole formation, chromatin condensation and margination, cytoplasmic condensation, nuclear fragmentation, formation of apoptotic bodies, etc. in morphology.

[0073] Please refer to Figure 4, the first quadrant represents dead cells, the second quadrant represents late apoptotic cells, the third quadrant represents normal cells, and the fourth quadrant represents early apoptotic cells. It was found that the apoptosis rate of the normoxia group was 5.45%, and the apoptosis rate of the hypoxia group increased by 16.96% compared with that of the normoxia group. According to the apoptosis data, it was found that compared with the hypoxia group, the apoptosis rates of the TPS group, the TPS1-NPs group, and the TPS2-NPs group decreased by 8.92%, 8.22%, and 9.10% respectively. In particular, the apoptosis rate of the TPS-SeNPs group was the lowest, showing a better effect of slowing down cell apoptosis. The above results indicate that the hypoxia intervention group can well alleviate the damage caused by hypoxia.

[0074] Example 4

[0075] The present invention provides a detection experiment for cellular ROS.

[0076] It mainly includes:

[0077] The present invention uses the method of ROS staining to explore the scavenging effect on cellular ROS. Among them, DAPI stains the cell nucleus and shows blue under ultraviolet excitation light, while the ROS staining solution shows red. Single-cell suspensions of RLE-6TN cells in the normoxia group, hypoxia group, and hypoxia intervention group were prepared. According to 5 2×10 cells per well, the cells were evenly inoculated into 6-well plates and cultured overnight at 37°C and 5% CO2 saturated humidity; the cells were digested with 0.25% trypsin without EDTA, collected after digestion was terminated, centrifuged at 1500 rpm for 5 min, the supernatant was removed, resuspended with PBS, rinsed twice with PBS repeatedly, 1 mL of DCFH diluted with PBS was added, incubated in a 37°C incubator for 20 min, mixed every 3 min, the cells were washed three times with serum-free medium, and after being stimulated with a reactive oxygen positive control for 20 min, the intracellular ROS content was detected by flow cytometry.

[0078] Results:

[0079] Please refer to Figure 5 , after hypoxia treatment, the fluorescence intensity of the hypoxia group was 8 times that of the normoxia group. At the same time, compared with the hypoxia group, the fluorescence intensity of the TPS-SeNPs group decreased by 59.09%, the TPS group decreased by 42.05%, the TPS1-NPs group decreased by 36.36%, and the TPS2-NPs group decreased by 40.91%. There was no significant difference among the four groups. The results indicate that hypoxia treatment can significantly promote the generation of ROS, and the ROS scavenging effect of other hypoxia intervention groups except the SeNPs group is significant.

[0080] Example 5

[0081] The present invention provides a detection experiment for cell oxidation-related markers.

[0082] It mainly includes:

[0083] Determine the contents of glutathione peroxidase (GSH-Px), lactate dehydrogenase (LDH), superoxide dismutase (SOD) and malondialdehyde (MDA) in the normoxia group, hypoxia group and hypoxia intervention group according to the operation instructions marked on the kit, and conduct inter-group comparisons to evaluate the alleviating effect of the sample on cells after hypoxia treatment. Among them, the activity of GSH-Px is the effect of deducting the non-enzymatic reaction per minute per milligram of protein, and reducing the GSH-Px concentration by 1 μmol / L is one enzyme activity unit. One SOD activity unit U represents the amount of SOD corresponding to 50% inhibition rate of SOD in 1 mL reaction solution per milligram of tissue protein. One LDH activity unit is to catalyze the production of 1 nmol / L pyruvate per minute.

[0084] Results:

[0085] Please refer to Figure 6 A. The GSH-Px activity in the normoxia group reached 240.12 U / mgprot, which was 181.71 U / mgprot higher than that in the hypoxia group. It shows that after 12 hours of hypoxia, the activity of glutathione peroxidase will be significantly inhibited, thereby inhibiting the decomposition of peroxides and damaging and interfering with the structure and function of cells. Compared with the hypoxia group, the hypoxia intervention group can significantly increase the activity of GSH-Px. Especially in the TPS-SeNPs group, the activity reached 170.59 U / mgprot, which was 112.18 U / mgprot higher than that in the hypoxia group. Although the GSH-Px activity in the SeNPs group was only 23.23 U / mgprot higher than that in the hypoxia group, there was also a significant difference. The GSH-Px activity in the TPS group reached 141.20 U / mgprot, the GSH-Px activity in the TPS1-NPs group reached 133.20 U / mgprot, and the GSH-Px activity in the TPS2-NPs group reached 102.58 U / mgprot, and there were significant differences among them.

[0086] Please refer to Figure 6B. The SOD activity of the normoxia group reached 17.92 U / mgprot, while that of the hypoxia group decreased by 10.72 U / mgprot compared with the normoxia group, indicating that the SOD activity was inhibited under hypoxic conditions. Among the hypoxia intervention groups, the SOD activity of the TPS-SeNPs group was the highest, significantly higher than that of the other four groups, reaching 13.99 U / mgprot. The SOD activity of the SeNPs group was the lowest among the hypoxia intervention groups, with an activity of 8.38 U / mgprot. This may be because the SeNPs sample aggregated and transformed into gray elemental selenium, which had no biological activity. The SOD activity of the TPS2-NPs group reached 9.63 U / mgprot, while the TPS group and the TPS1-NPs group were significantly higher than the TPS2-NPs group, and there was no significant difference between them.

[0087] Please refer to Figure 6 C. The LDH value of the normoxia group was 115.28 U / L, while that of the hypoxia group increased significantly by 853.32 U / L, indicating that under hypoxic conditions, cells underwent glycolysis, carried out incomplete oxidative decomposition, released energy, and produced lactic acid at the same time. Compared with the hypoxia group, the LDH activity of the hypoxia intervention group decreased significantly, indicating that it could effectively alleviate the glycolysis caused by hypoxia. The LDH activity of the SeNPs group was the highest among the hypoxia intervention groups, reaching 801.37 U / L. Followed by the TPS group, with an LDH activity of 686.46 U / L.

[0088] Please refer to Figure 6 D. The MDA content of the normoxia group was 2.37 nmol / mgprot, while that of the hypoxia group increased by 21.46 nmol / mgprot. This indicates that hypoxia would increase the MDA content, damage the cell structure, cause damage to its function, and affect the normal physiological and biochemical reactions of organisms. Among the hypoxia intervention groups, the MDA content of the TPS-SeNPs group was the lowest, decreasing by 15.47 nmol / mgprot compared with the hypoxia group. The MDA content of the SeNPs group was the highest among the hypoxia intervention groups, reaching 18.66 nmol / mgprot. This indicates that the combination of tea polysaccharide and selenium nanoparticles could effectively reduce the MDA content caused by hypoxia. The MDA contents of the TPS1-NPs group and the TPS2-NPs group were 12.25 and 11.80 nmol / mgprot respectively, with no significant difference.

[0089] Both TPS-NPs and TPS-SeNPs can significantly alleviate the damage caused by hypoxia, and the effect of the TPS-SeNPs group is the most obvious. Compared with the hypoxia group, the LDH activity, MDA content and ROS in the TPS-SeNPs group decreased by 508.10 U / L, 15.48 nmol / mgprot and 59.09% respectively, while the GSH-Px activity and SOD activity increased by 112.19 U / mgprot and 6.79 U / mgprot respectively. This indicates that the present tea polysaccharide / selenium nanoparticles reduce the damage of reactive oxygen species to cells by enhancing the activity of antioxidant enzymes and increasing the ability to scavenge reactive oxygen species.

[0090] Example 6

[0091] The present invention provides an experimental method for detecting the expression of genes and proteins related to the cell hypoxia pathway.

[0092] It mainly includes:

[0093] Detect the expression levels of PI3K, AKT, mTOR, p70S6K, and HIF-1α genes in the mTOR pathway by RT-PCR and detect the protein expression of PI3K, AKT, mTOR, p70S6K, and HIF-1α by WB. Extract the RNA of cells using the Trizol method, that is, add 1 ml of Trizol reagent into the cells, pipette and mix well, transfer to a 1.5 mL RNase-free EP tube, and lyse for 10 min. Add 200 μL of chloroform, vigorously invert and mix several times, and let stand at room temperature for 5 minutes; centrifuge at 12,000 rpm at 4°C for 15 min, and it can be seen that it is divided into three phases: upper (RNA), middle (protein), and lower (DNA); transfer the upper aqueous phase (about 400 μL) to another new 1.5 mL RNase-free EP tube, add 400 μL of isopropanol, mix well and let stand at room temperature for 10 min; centrifuge at 12,000 rpm at 4°C for 10 min, and a white RNA precipitate can be seen at the bottom of the tube. Discard the supernatant, add 1 mL of RNase-free 75% ethanol, vortex and mix well, centrifuge at 10,000 rpm at 4°C for 5 min, and repeat this step once; discard the supernatant, air-dry the RNA precipitate for 5 - 10 min, dissolve the precipitate in 20 μL of DEPC water, and take 2 μL of the dissolved RNA to measure the concentration and purity of the sample with a micro-spectrophotometer. Use a reverse transcription kit to reverse transcribe the extracted mRNA into cDNA. According to the method and system in the fluorescence quantitative kit, add the corresponding primers, fluorescent dye, and cDNA, and then react at 50°C for 2 min, 95°C for 10 min; 95°C for 30 sec, 60°C for 30 sec, for 40 cycles, and draw a melting curve. The amplification reaction of the target gene is carried out for each batch of samples, and the ΔCt of PI3K, AKT, mTOR, p70S6K, and HIF-1α is calculated. The relative expression level of the target gene is calculated using 2 -ΔΔCt for calculation and analysis.

[0094] Detection of protein expression: First, wash the cells three times repeatedly with pre-cooled PBS (0.01M, pH 7.2 - 7.3) at 4°C. Then add RIPA lysis buffer, adding 10 μL of PMSF (100 mM) per 1 mL of lysis buffer, shake well and place on ice for 30 min for lysis. After lysis, centrifuge at 12,000 rpm for 5 min at 4°C, and take the supernatant, which is the total protein of the cells. Then use the BCA method to detect the concentration of the extracted total protein solution. For the sample whose protein concentration has been determined, mix it with 5× protein loading buffer, place it in boiling water for a boiling water bath for 10 min, check if the sample is transparent and then use a pipette to aspirate to see if it is viscous. If it is not transparent or viscous, the boiling time of the sample needs to be extended. After denaturation, cool to room temperature and perform SDS-PAGE electrophoresis. After electrophoresis, cut the target band according to the Marker from the gel, rinse it with distilled water, cut a PVDF membrane and filter paper of the same size as the PAGE gel. Soak the PVDF membrane in methanol for a few seconds and then soak it in the electrotransfer buffer together with the filter paper. Place them in order: black plate - fiber pad - filter paper - gel - PVDF membrane - filter paper - fiber pad - white plate, clamp the plates and put them into the electrotransfer instrument, with the side of the black plate facing the black negative electrode. Fill the electrotransfer tank with electrotransfer liquid and start electrotransfer; Immunoreaction and color development, that is, first perform blocking, soak the PVDF membrane with TBST (blocking solution) containing 5% skim milk powder, and block it on a shaker at room temperature for 2 h. Phosphorylated proteins are blocked with 1% - 3% BSA; Dilute the corresponding primary antibody with the blocking solution, soak the PVDF membrane in the primary antibody incubation solution, and incubate overnight at 4°C. Wash the PVDF membrane 5 - 6 times thoroughly with TBST for 5 min each time to wash away the excess primary antibody; Dilute the corresponding HRP-labeled secondary antibody with the blocking solution --- diluted 1:50000, soak the PVDF membrane in the secondary antibody incubation solution, and incubate on a shaker at 37°C for 2 h; Wash the PVDF membrane 5 - 6 times thoroughly with TBST for 5 min each time to wash away the excess secondary antibody. Mix the enhancer solution and the stable peroxidase solution in the ECL reagent in a 1:1 ratio, drop the working solution onto the PVDF membrane, react for several minutes until the fluorescent band is obvious, then use filter paper to absorb the excess substrate solution, cover with a plastic wrap, expose the X-ray film, develop it in the developer, fix it in the fixer, and wash the film. Air-dry the film, scan the film, then use PhotoShop to organize and adjust the gel image analysis, and use BandScan to analyze the gray value of the film.

[0095] Results:

[0096] Please refer to Figure 7 A - E and Figure 8Compared with the normoxia group, it was found that the expression levels of PI3K, AKT, mTOR, p70S6K, and HIF-1α genes in the hypoxia group increased significantly, by 8.82-fold, 11.68-fold, 5.67-fold, 9.36-fold, and 20.05-fold, respectively. Compared with the hypoxia group, the hypoxia intervention group could significantly reduce the expression levels of PI3K, AKT, mTOR, p70S6K, and HIF-1α genes, and effectively alleviate the impact of hypoxia, especially the TPS-SeNPs group.

[0097] Compared with the normoxia group, the proteins of PI3K, AKT, mTOR, p70S6K, and HIF-1α in the hypoxia group showed an up-regulated state. Compared with the hypoxia group, the hypoxia intervention group showed a down-regulated state for all proteins. For the PI3K protein, the hypoxia group was up-regulated 1.96-fold compared with the normoxia group. Compared with the hypoxia group, the SeNPs group was only down-regulated by 7.35%, and there was no significant difference from the hypoxia group. Similarly, neither the TPS group nor the TPS1-NPs group had a significant difference from the hypoxia group. The TPS2-NPs group was down-regulated by 23.53% compared with the hypoxia group. Among the hypoxia intervention groups, the expression level of PI3K protein in the TPS-SeNPs group was the lowest and there was no significant difference from the normoxia group.

[0098] This invention explored the effects of TPS-NPs and TPS-SeNPs on hypoxia tolerance. The results showed that hypoxia treatment promoted the apoptosis of RLE-6TN cells, increased the activities of lactate dehydrogenase (LDH) and malondialdehyde (MDA), enhanced the generation of reactive oxygen species (ROS), decreased the activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), and significantly increased the expression levels of genes and proteins in the hypoxia stress pathway (PI3K, AKT, mTOR, p70S6K, and HIF-1α). Both TPS-NPs and TPS-SeNPs could significantly alleviate the damage caused by hypoxia, and the effect of the TPS-SeNPs group was the most obvious. Compared with the hypoxia group, the LDH activity, MDA content, and ROS in the TPS-SeNPs group decreased by 508.10 U / L, 15.48 nmol / mgprot, and 59.09% respectively, and the activities of GSH-Px and SOD increased by 112.19 U / mgprot and 6.79 U / mgprot respectively. At the same time, the study also found that the TPS-SeNPs group mainly improved the hypoxia tolerance of RLE-6TN by down-regulating the expression of related genes and proteins in the hypoxia stress pathway.

[0099] Comparative Example

[0100] This invention proposed a comparative experiment on the improvement of cell hypoxia tolerance by tea polysaccharide / selenium nanoparticles compared with other methods.

[0101] It mainly includes:

[0102] According to the relevant methods of the above embodiments, the relevant indicators of tea polysaccharide / selenium nanoparticles, selenium nanoparticles, and tea polysaccharide nanoparticles on cell hypoxia were compared and detected.

[0103] Results:

[0104] Please refer to Figure 4 the column chart of. The apoptosis rate of cells in the hypoxia group was 22.41%, while the apoptosis rate of cells intervened by tea polysaccharide / selenium nanoparticles was 13.49%, lower than 16.48% of the SeNPs hypoxia intervention group and also lower than 14.19% of the tea polysaccharide nanoparticle hypoxia intervention group, indicating that tea polysaccharide / selenium nanoparticles can better alleviate cell apoptosis caused by hypoxia. Please refer to Figure 5 , compared with the hypoxia group, the fluorescence intensity of the TPS-SeNPs group decreased by 59.09%, while the fluorescence intensity of the SeNPs group only decreased by 10.23%, the TPS1-NPs group decreased by 36.36%, and the TPS2-NPs group decreased by 40.91%, indicating that S tea polysaccharide / selenium nanoparticles have a better inhibitory effect on ROS generation caused by hypoxia. Please refer to Figure 6A-D, in the TPS-SeNPs group, the activity of GSH-Px reached 170.59 U / mgprot, which was 112.18 U / mgprot higher than that in the hypoxia group. The GSH-Px activity in the SeNPs group was only 23.23 U / mgprot higher than that in the hypoxia group. The GSH-Px activity in the TPS group reached 141.20 U / mgprot, the GSH-Px activity in the TPS1-NPs group reached 133.20 U / mgprot, and the GSH-Px activity in the TPS2-NPs group reached 102.58 U / mgprot, all of which were lower than the activation degree of GSH-Px activity in the TPS-SeNPs group; in the hypoxia intervention group, the SOD activity in the TPS-SeNPs group was the highest, significantly higher than that in other intervention groups, and its activity reached 13.99 U / mgprot. The SOD activity in the SeNPs group was the lowest in the hypoxia intervention group, with an activity of 8.38 U / mgprot. The SOD activity in the TPS2-NPs group was also lower than that in the TPS-SeNPs group, with an activity of 9.63 U / mgprot, indicating that the TPS-SeNPs group could better trigger the activity of antioxidant and other related enzymes, play a role in balancing the intracellular redox, and thus alleviate the damage of ROS to cells. Some studies have found that in a hypoxic environment, the body will up-regulate the level of HIF-1α through signal pathways such as mTOR, and promote the expression of hypoxia-related genes. The results showed that the expression levels of PI3K, AKT, mTOR, p70S6K, and HIF-1α genes in the hypoxia group increased significantly, while the expression levels of PI3K, AKT, mTOR, p70S6K, and HIF-1α genes in the TPS-SeNPs group decreased by 87.26%, 85.19%, 80.07%, 85.41, and 87.41%; the expression levels of hypoxia-related genes in the TPS-SeNPs group were lower than those in other hypoxia intervention groups and showed no significant difference from the normoxia group. In the hypoxia intervention group, the expression level of PI3K protein in the TPS-SeNPs group was the lowest and showed no significant difference from the normoxia group. The SeNPs group only down-regulated by 7.35% and showed no significant difference from the hypoxia group. Similarly, both the TPS group and the TPS1-NPs group showed no significant difference from the hypoxia group. The TPS1-NPs group was down-regulated by 23.53% compared with the hypoxia group, indicating that tea polysaccharide / selenium nanoparticles can reduce the expression of hypoxia pathway-related genes and proteins, and thus improve the hypoxia tolerance of cells.

[0105] In summary, a tea polysaccharide / selenium nanoparticle for improving the hypoxia tolerance of cells and its application according to the present invention provide a new method for cell protection and treatment.

[0106] It should be noted that the term "including" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "including..." does not exclude the presence of additional identical elements in the process, method, article, or apparatus that includes the said element.

[0107] The above are only embodiments of the present application and are not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included within the scope of the claims of the present application.

Claims

1. A tea polysaccharide / selenium nanoparticle for improving the hypoxia tolerance of cells, characterized in that, The tea polysaccharide / selenium nanoparticles are at least one of tea polysaccharide, tea polysaccharide nanoparticles, and tea polysaccharide-selenium nanoparticles; The tea polysaccharide includes moisture, protein, total sugar, uronic acid, and tea polyphenols; The tea polysaccharide is extracted by the hot water extraction method, including: S11. Weigh tea powder at 3% - 7% in water, and extract it with hot water at 60°C - 80°C for 2h - 4h to obtain the extracted tea solution; S12. Centrifuge the extracted tea solution at 3000rpm - 5000rpm for 5min - 30min to obtain the supernatant; S13. Add the sevage solution to the supernatant, shake it, and separate the layers to obtain tea polysaccharide, protein, and sevage solution that are divided into 3 layers from top to bottom; the sevage solution is chloroform: n-butanol = 4:1; S14. Centrifuge the upper layer of the tea polysaccharide solution at 6000rpm - 10000rpm for 10min - 30min to remove the residual protein and obtain the tea polysaccharide solution without protein; S15. Add 3 - 5 times the volume of absolute ethanol to the tea polysaccharide solution without protein, refrigerate and let it stand for 6h - 24h, centrifuge at 3000rpm - 5000rpm to obtain the precipitate, dissolve it in water, concentrate it, and freeze-dry it to obtain the tea polysaccharide; The preparation of the tea polysaccharide-selenium nanoparticles includes: S21. Prepare a solution by mixing the tea polysaccharide solution and sodium selenite solution at a mass-to-volume ratio of (0 - 0.6):1, and mix well to obtain a tea polysaccharide-sodium selenite mixture; S22. Freshly prepare an ascorbic acid solution, drop an equal volume of 30mM - 50mM of the ascorbic acid solution into the tea polysaccharide-sodium selenite mixture, stir it in the dark at 30°C - 50°C for 2h - 6h; dialyze with deionized water to obtain the tea polysaccharide-selenium nanoparticles that have removed excess ascorbic acid and sodium selenite.

2. The tea polysaccharide / selenium nanoparticles according to claim 1, characterized in that, The tea polysaccharide / selenium nanoparticles are prepared by electrostatic interaction or redox reaction.

3. The tea polysaccharide / selenium nanoparticles according to claim 1, wherein The concentration range of the tea polysaccharide / selenium nanoparticles is 0.1μg / mL - 100μg / mL.

4. The tea polysaccharide / selenium nanoparticles according to claim 1, wherein The particle size range of the tea polysaccharide / selenium nanoparticles is 80nm - 1000nm.

5. Use of tea polysaccharide / selenium nanoparticles for improving the hypoxia tolerance of cells, characterized in that, The application is the application of the tea polysaccharide / selenium nanoparticles described in any one of claims 1 - 4; The application is the application of the tea polysaccharide / selenium nanoparticles for improving cell hypoxia tolerance.

6. The application according to claim 5, characterized in that, The application includes exposing RLE-6TN cells to a culture medium containing the tea polysaccharide / selenium nanoparticles.

7. The application according to claim 5, wherein The application includes subjecting the RLE-6TN cells to hypoxia treatment for 8h - 12h after exposing them to the tea polysaccharide / selenium nanoparticles for 0.5h - 2h.

8. The application according to claim 5, wherein The tea polysaccharide / selenium nanoparticles can alleviate cell apoptosis caused by hypoxia.

9. The application according to claim 5, characterized in that The tea polysaccharide / selenium nanoparticles can significantly alleviate the changes in enzyme activity caused by hypoxia.

10. The application according to claim 5, wherein The tea polysaccharide / selenium nanoparticles can alleviate the expression of hypoxia pathway-related genes and proteins triggered by hypoxia.

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