Application of Agaricus bisporus peptide in preeclampsia

Abstract

The present invention belongs to the field of biomedicine and relates to the application of Agaricus bisporus polypeptides in preeclampsia. The present invention relates to the use of Agaricus bisporus polypeptides in the preparation of products for promoting the invasion and migration of placental trophoblastic cells or preventing and treating preeclampsia in subjects. The present invention also relates to products containing Agaricus bisporus polypeptides that promote the invasion and migration of placental trophoblastic cells or prevent and treat preeclampsia in subjects. The present invention has found that Agaricus bisporus polypeptides have the effect of promoting the proliferation and migration of human chorionic trophoblastic cells, and the promotion of the proliferation and migration of human chorionic trophoblastic cells reflects the therapeutic effect of preeclampsia, so Agaricus bisporus polypeptides can be used to treat and prevent preeclampsia in subjects.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to the application of Agaricus bisporus polypeptide in preeclampsia. Background Art

[0002] Pre-eclampsia (PE) is a disease specific to pregnancy. It occurs after 20 weeks of pregnancy. The current global incidence rate is 3-8%. The number of maternal deaths caused by eclampsia is about 7,600 to 8,200 each year, ranking second among the causes of maternal death. The intrauterine fetal death rate of pre-eclampsia before 32 weeks of pregnancy is as high as 33.3%. Clinical practice shows that PE not only endangers maternal health, but is also closely related to intrauterine growth restriction, premature birth, and low birth weight. It is estimated that 76,000 pregnant women and 500,000 fetuses die each year worldwide. In addition, pregnant women with PE also have an increased risk of cardiovascular disease and end-stage renal disease in the future. Children born to mothers with pre-eclampsia are also more likely to develop hypertension, stroke, cognitive impairment, and depression in adulthood. Therefore, early prediction, diagnosis, and timely intervention of PE are crucial to improving patient prognosis and pregnancy outcomes. The prediction of PE based solely on high-risk factors such as patient race, age, history of eclampsia, and obesity is limited, so more research focuses on identifying high-risk predictors. Currently, related biological research is mainly based on the pathophysiological changes of PE.

[0003] Researchers have studied blood biomarkers of patients with preeclampsia, including vascular endothelial growth factor, placental growth factor and soluble vascular endothelial growth factor receptor-1, placental protein-13 (PP-13), disintegrin metalloproteinase 12, fetal hemoglobin, α-1 microglobulin, core protein glycan, copeptin, inhibin A and its combined biochemical screening, thyroid transporter, etc. However, due to low specificity, lack of large-scale clinical statistical data, high testing costs, etc., it is difficult to popularize and cannot be used for prediction and diagnosis, so it has not yet been transformed into clinical applications. Based on this, the development of safe, effective, and universal functional foods for the prevention of preeclampsia may solve the urgent problem.

[0004] At present, more and more researchers are engaged in the prevention of preeclampsia, but a universal application plan has not yet been formed. For example, oxidative stress is related to the onset of preeclampsia, so some people have proposed that antioxidant vitamins C and E can prevent the occurrence of preeclampsia. Some randomized controlled trials and placebo-controlled trials have found that oral vitamins C and E do not reduce the risk of preeclampsia. A recently published randomized controlled study found that the incidence of PE in pregnant women who supplemented with calcium, magnesium, zinc, and vitamin D was lower than that in the placebo group, and the difference was statistically significant, while the incidence of PE in pregnant women who supplemented with vitamins C and E was not statistically significant. For people with low calcium intake (<600 mg / d), oral calcium supplementation of at least 1 g / d is recommended to reduce the risk of preeclampsia to a certain extent, especially for pregnant women with insufficient basal calcium intake. Some studies have also suspected that vitamin D deficiency is one of the risk factors for preeclampsia. However, it is still unknown whether vitamin D supplementation is effective. For obese pregnant women without other high-risk factors such as miscarriage and premature birth, after comprehensive evaluation, weight control can be considered by adjusting diet and exercising moderately. Foreign studies have shown that the preventive use of low molecular weight heparin and low-dose aspirin by pregnant women with a history of preeclampsia before 16 weeks of pregnancy can reduce the incidence of early preeclampsia and fetal growth restriction compared with aspirin alone. Taking folic acid supplements before and during pregnancy and dietary folic acid intake can reduce the risk of preeclampsia, but this protective effect is more statistically significant when the pre-pregnancy BMI is <24.0kg / m2.

[0005] Agaricus bisporus belongs to the class of Basidiomycetes, the order of Agaricales, the family of Agaricaceae, and the genus Agaricus. It has extremely high edible and medicinal value. The health care effects of Agaricus bisporus include lowering blood pressure. Among them, Lau et al. extracted 8 peptides from Agaricus bisporus, 3 of which are highly active angiotensin I converting enzyme inhibitors, and angiotensin I converting enzyme inhibitors can reduce the allergy rate of people and relieve the symptoms of high blood pressure in pregnant women. The present invention attempts to explore whether the active peptides in the edible mushroom Agaricus bisporus can become a highly safe preventive food for improving the maternal and infant outcomes of preeclampsia. Summary of the invention

[0006] The present invention aims to explore the application of Agaricus bisporus polypeptide in preeclampsia.

[0007] In one aspect, the present invention relates to the use of Agaricus bisporus polypeptides in the preparation of a product for promoting the invasion and migration of placental trophoblast cells.

[0008] In another aspect, the present invention relates to the use of Agaricus bisporus polypeptides in the preparation of a product for preventing and treating preeclampsia in a subject.

[0009] In some embodiments, Agaricus bisporus polypeptides promote the invasion and migration of placental trophoblast cells.

[0010] In some embodiments, the Agaricus bisporus polypeptide reverses the inhibition of LPS or L-NAME stimulation on the invasion and migration of placental trophoblast cells.

[0011] In some embodiments, the Agaricus bisporus polypeptide promotes invasion and migration of placental trophoblast cells in a subject.

[0012] In some embodiments, Agaricus bisporus polypeptides promote invasion and migration of placental trophoblast cells in patients with preeclampsia.

[0013] In some embodiments, the products include, but are not limited to, reagents, cell culture media, health products, in-hospital preparations, and traditional Chinese medicine preparations.

[0014] In some embodiments, the placental trophoblast cells comprise human chorionic trophoblast cells.

[0015] In another aspect, the present invention relates to a product for promoting the invasion and migration of placental trophoblastic cells or preventing and treating preeclampsia in a subject, comprising an Agaricus bisporus polypeptide.

[0016] In some embodiments, the products include, but are not limited to, reagents, cell culture media, health products, in-hospital preparations, and traditional Chinese medicine preparations.

[0017] In some embodiments, Agaricus bisporus polypeptides promote the invasion and migration of placental trophoblast cells.

[0018] In some embodiments, the Agaricus bisporus polypeptide reverses the inhibition of LPS or L-NAME stimulation on the invasion and migration of placental trophoblast cells.

[0019] In some embodiments, the Agaricus bisporus polypeptide promotes invasion and migration of placental trophoblast cells in a subject.

[0020] In some embodiments, Agaricus bisporus polypeptides promote invasion and migration of placental trophoblast cells in patients with preeclampsia.

[0021] In some embodiments, the placental trophoblast cells comprise human chorionic trophoblast cells.

[0022] In some embodiments, the concentration of Agaricus bisporus polypeptide in the product is 0.64 μg / mL-2000 μg / mL, such as 16 μg / mL-400 μg / mL, 40 μg / mL-160 μg / mL, preferably 400 μg / mL.

[0023] definition

[0024] As used herein, Agaricus bisporus polypeptides can be commercially available, or can be obtained by methods well known in the art for extracting polypeptides from Agaricus bisporus. For example, a commonly used method for extracting Agaricus bisporus polypeptides includes crushing Agaricus bisporus and then hydrolyzing it with a protease, and then filtering and separating the Agaricus bisporus polypeptides in the hydrolyzate by ultrafiltration. For another example, Agaricus bisporus polypeptides can be prepared as described herein, and the method includes crushing Agaricus bisporus and then sieving, extracting Agaricus bisporus protein by alkali extraction, acid precipitation and centrifugation to collect the Agaricus bisporus protein, drying the collected Agaricus bisporus protein and then hydrolyzing it with a protease, and drying the enzymatic hydrolysis product to obtain the Agaricus bisporus polypeptide.

[0025] As used herein, the term "prevention" refers to methods performed to prevent or delay the occurrence of a disease or disorder or symptom in a subject.

[0026] As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical result. For the purposes of the present invention, a beneficial or desired clinical result includes, but is not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partial or complete), whether detectable or undetectable.

[0027] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human.

[0028] In the product of the present invention, a pharmaceutically acceptable carrier and / or excipient is also included. As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art and includes but is not limited to: diluents, excipients such as water, fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginates, gelatin and polyvinyl pyrrolidone; wetting agents such as glycerol; disintegrants such as agar, calcium carbonate and sodium bicarbonate; absorption promoters quaternary ammonium compounds; surfactants such as hexadecanol; adsorption carriers such as kaolin and bentonite; lubricants such as talc, calcium and magnesium stearate, polyethylene glycol, etc.

[0029] As used herein, a noun without a quantifier may mean one or more. As used herein in the claims, when used in conjunction with the word "comprises", a noun without a quantifier may mean one or more than one.

[0030] Unless explicitly indicated to refer to only alternatives or the alternatives are mutually exclusive, the use of the term "or / or" in the claims is used to mean "and / or", but the present disclosure supports definitions referring to only alternatives and "and / or". "Another" as used herein may mean at least a second or more.

[0031] As used herein, the term "about" is used to indicate that a value includes the inherent error variation for the device, the method being employed to determine the value, or the variation that exists between the subjects.

[0032] Beneficial Effects of the Invention

[0033] Agaricus bisporus is a plant that can be used as both medicine and food, and has health-care effects, including lowering blood pressure. The safety of the drug developed into a drug for treating or preventing preeclampsia is guaranteed. The present invention finds that Agaricus bisporus polypeptide has the effect of promoting the proliferation and migration of human chorionic trophoblast cells, and the promotion of the proliferation and migration of human chorionic trophoblast cells reflects the therapeutic effect of preeclampsia, so Agaricus bisporus polypeptide can be used to treat and prevent preeclampsia in subjects. Without being bound by theory, the Agaricus bisporus polypeptide in the present invention can achieve a therapeutic effect on preeclampsia through a multi-target mechanism of action, which is also consistent with the complex pathogenesis of preeclampsia. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The effects of different concentrations of Agaricus bisporus peptides on the proliferation of human chorionic trophoblast cells are shown;

[0035] Figure 2 The effect of Agaricus bisporus peptides on the proliferation activity of HTR8 was detected by CCK8;

[0036] Figure 3 The effect of Agaricus bisporus peptide on LDH secretion by HTR8 was shown;

[0037] Figure 2 and Figure 3 In the table, the symbol “**” represents p < 0.01 (compared with the control group); the symbol “##” represents p < 0.01 (compared with the LPS group);

[0038] Figure 4 The EDU experimental diagram of different concentrations of Agaricus bisporus peptides is shown. A: blank control; B: low concentration of Agaricus bisporus peptide 16μg / mL; C: medium concentration of Agaricus bisporus peptide 80μg / mL; D: high concentration of Agaricus bisporus peptide 400μg / mL;

[0039] Figure 5The effects of different concentrations of Agaricus bisporus peptide on HTR-8 / Svneo cell migration are shown; A: blank control; B: low concentration of Agaricus bisporus peptide 80 μg / mL; C: medium concentration of Agaricus bisporus peptide 400 μg / mL; D: high concentration of Agaricus bisporus peptide 2000 μg / mL;

[0040] Figure 6 Shows Figure 5 The quantitative bar graph of the effect of different concentrations of Agaricus bisporus peptides on HTR-8 / Svneo cell migration;

[0041] Figure 7 The effect of various concentrations of Agaricus bisporus peptide on cell migration under the action of L-NAME is shown. A: blank control; B: L-NAME 100mg / ml; C: L-NAME + Agaricus bisporus peptide low concentration 80μg / mL; D: L-NAME + Agaricus bisporus peptide high concentration 400μg / mL;

[0042] Figure 8 Shows Figure 7 The quantitative bar graph of the effect of various concentrations of Agaricus bisporus peptide on cell migration under the action of L-NAME, A: blank control B: L-NAME 100mg / ml C: L-NAME + Agaricus bisporus peptide low concentration 80μg / mL D: L-NAME + Agaricus bisporus peptide high concentration 400μg / mL; Note: ***p<0.001 compared with the control group; ###p<0.001 compared with the L-NAME group.

[0043] Fig. 9 The results of the scratch test of HTR-8 / Svneo cells with different concentrations of Agaricus bisporus peptides under LPS stimulation are shown;

[0044] Fig.10 Shows Fig. 9 The quantitative bar graph of the scratch test of HTR-8 / Svneo cells with different concentrations of Agaricus bisporus peptides under LPS stimulation;

[0045] Fig.10 In the table, the symbol “**” represents p<0.01 (compared with the control group); the symbol “#” represents p<0.05 (compared with the LPS group); and the symbol “##” represents p<0.01 (compared with the LPS group). DETAILED DESCRIPTION

[0046] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0047] Example 1: Extraction of Agaricus bisporus polypeptide

[0048] 1. Preprocessing:

[0049] Dry fresh Agaricus bisporus in an oven at 55°C and grind into powder. Sieve the powder through a 60-mesh sieve and collect the dried Agaricus bisporus powder for later use.

[0050] 2. Alkali extraction:

[0051] Weigh 100 g of Agaricus bisporus dry powder and mix them in a ratio of Agaricus bisporus powder: deionized water = 1:12. Adjust the pH of the mixture to 10 with 1 mol / L NaOH, divide the mixture into conical flasks of the same size, seal the conical flasks with plastic wrap and place them in a shaker to ensure that the height of the mixed solution in the conical flask is lower than the height of the water in the shaker. Set the shaker speed to 300 r / min, the time to 3 h, and the temperature to 45°C.

[0052] 3. Acid precipitation:

[0053] The lye solution of Agaricus bisporus protein after reaction was centrifuged at a speed of 5000 r / min, and the supernatant was collected. The pH of the supernatant was adjusted to the isoelectric point of Agaricus bisporus protein, pH 3.5, with 1 mol / L HCl, and then the solution was placed at 4°C for 2 hours. The precipitate was collected by centrifugation at 3000 r / min.

[0054] 4. Drying:

[0055] Agaricus bisporus protein can be obtained after freeze drying.

[0056] 5. Enzymatic hydrolysis: After enzymatic hydrolysis of Agaricus bisporus, Agaricus bisporus peptides are obtained

[0057] (1) Add 0.1 g of Agaricus bisporus protein to 9.9 ml of water to prepare a protein solution with a concentration of 10 mg / ml. Stir the solution on a magnetic stirrer for 12 h at a speed of 280 r / min.

[0058] (2) Weigh 5.6 g of neutral protease, adjust the pH to 8.5 with 1 mol / L sodium bicarbonate, and react at 45.5°C for 5 h.

[0059] (3) The extract is inactivated under high pressure and then freeze-dried to dryness. The polypeptide content in the obtained product is >90% as determined by the biuret method.

[0060] The freeze-dried Agaricus bisporus peptides were reconstituted and desalted, and then analyzed by Easy nLC1200 / QExactiveplus to identify the peptide sequence and molecular weight;

[0061] The chromatographic conditions of Easy nLC1200 / Q Exactive plus are:

[0062] (1) C18 reverse phase analytical column (50 mm × 50 cm monolithic column);

[0063] (2) Mobile phase A was 0.1% formic acid in water, mobile phase B was a mixture of 80% acetonitrile and 0.1% formic acid, and the flow rate was 300 nL / min;

[0064] (3) Gradient elution program: 0-3 min, 2-6% B; 3-42 min, 6-20% B; 42-47 min, 20-35% B; 47-48 min, 35-100% B; 48-60 min, 100% B;

[0065] (4) Mass spectrometry conditions were: ESI+ mode; orbital trap resolution 70000 (AGC 3e6); scanning range 350-1800 m / z;

[0066] The Peaks 10.0 software search engine was used to perform sequence analysis on the test sample data, and a total of 6437 peptides were detected in three parallel samples. The molecular weight of the identified peptides was mainly concentrated in 1000-1500Da. Figure 1 shown.

[0067] Example 2: Verification of the promoting effect of Agaricus bisporus polypeptide on the invasion and migration of placental trophoblast cells at the cellular level.

[0068] This example uses human chorionic trophoblast cell proliferation and migration experiments to study the therapeutic effect and dosage of Agaricus bisporus polypeptide on preeclampsia, providing a basis for the development of drugs to prevent preeclampsia.

[0069] (1) Cell passaging

[0070] Observe the cell growth under a microscope. When the cells grow to about 90%, remove the supernatant and wash twice with PBS.

[0071] Add 500 μL of trypsin for digestion. When the cell morphology changes to a round shape under a microscope, add 2 mL of 1640 complete culture medium to terminate digestion. Use a pipette to gently blow several times until the cells are completely detached from the bottom of the bottle. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 1000 rpm, 4°C for 5 min. After aspirating the supernatant, add 1640 complete culture medium to mix the cells evenly, and transfer them to a new culture bottle at a ratio of 1:3 for continued culture.

[0072] (2) Cell cryopreservation

[0073] When the cells grow to the logarithmic growth phase, the culture medium is aspirated and the cells are washed twice with PBS; 500 μL of trypsin is added for digestion, and when the cell morphology becomes round under the microscope, 2 mL of DMEM complete culture medium is added to terminate the digestion and the cells at the bottom of the bottle are completely blown off with a pipette; the cell suspension is collected, centrifuged at 1000 rpm and 4°C for 5 min; the supernatant is aspirated and 1 mL of cell freezing solution (900 μL FBS + 100 μL DMSO) is added to resuspend the cells; the cell suspension is transferred to a cell freezing tube, placed in a cell gradient cooling freezing box, and after -80°C overnight, transferred to a liquid nitrogen tank for storage.

[0074] (3) Cell proliferation assay

[0075] HTR-8 / SVneo cells in the logarithmic growth phase were digested with 0.25% trypsin and then vibrated into a single cell suspension. The cell density was adjusted to 1×10 4 / mL, inoculated in a 96-well culture plate, 100μL per well, cultured for 24h, when the cells adhered to the wall and grew well, the original culture solution was gently aspirated, the cells were blown evenly, and then spread in a 96-well plate with a uniform density, and allowed to adhere to the wall overnight. Among them, the blank group was a culture medium without cells, the control group used a culture medium without drugs to culture cells, and the experimental group added a newly prepared drug, namely, Agaricus bisporus polypeptide, to the culture medium, which was set to 7 concentrations, 4 replicates for each concentration, and cultured for 24h at 37℃, 5% CO2 saturated humidity, 10μL of MTT solution was added to each well, and incubated for 4h at 37℃, the culture was terminated, and the wavelengths of 490nm and 570nm were selected to measure the absorbance of each well on an enzyme marker.

[0076] (4) EDU experiment

[0077] Dilute the EDU solution with complete cell culture medium at a ratio of 1000:1 to prepare an appropriate amount of 50uM EDU culture medium. Add 200ul 50uM EDU culture medium to each well and incubate for 2h. Discard the culture medium and wash the cells with PBS 1-2 times, 5min each time. Add an appropriate amount of cell fixative to each well and incubate at room temperature for 30min. Add 2mg / ml glycine to each well and incubate on a decolorizing shaker for 5min. Add an appropriate amount of PBS to each well and wash on a decolorizing shaker for 5min. Add an appropriate amount of permeabilization agent (0.5% TritonX-100 in PBS) to each well and incubate on a decolorizing shaker for 10min, wash once with PBS for 5min. Add an appropriate amount of methanol to each well and wash 1-2 times, 5min each time, and wash once with PBS for 5min each time. Add 200ul of 1xApollo staining reaction solution to each well, incubate in a decolorizing shaker for 30min at room temperature and away from light; after removing the staining reaction solution, add an appropriate amount of permeabilization agent (PBS with 0.5% TritonX-100) and wash on a decolorizing shaker 2-3 times, 10min each time; add an appropriate amount of methanol to each well and wash 1-2 times, 5min each time; wash once with PBS, 5min each time. Add 1XHoechst33342 reaction solution to each well, incubate in a decolorizing shaker for 30min at room temperature and away from light. After removing the staining reaction solution, add an appropriate amount of PBS to each well and wash 1-3 times each time to elute the Hoechst33342 reaction solution. Add an appropriate amount of anti-fluorescence quencher to the well and take pictures under a fluorescence microscope as soon as possible.

[0078] (5) Cell migration assay

[0079] The cell migration ability was detected by Transwell chamber method. The cell density was adjusted to 1×10 5 / mL. According to the grouping, 200μL of cell suspension without or with different doses of drug was added to the upper chamber, and 600μL of 1640 culture medium containing 10% serum was added to the lower chamber, and cultured at 37℃, 5% CO2 for 24h; after 24h, PBS was rinsed in the Transwell chamber, and the cells on the upper surface of the upper chamber were gently wiped off with a cotton swab, and inverted to air dry; 4% paraformaldehyde was fixed for 10min, and after air drying, 0.1% crystal violet stain was used, and the chamber was placed in it so that the lower surface of the chamber was just in contact with the surface of the stain, and stained for 10min; the lower surface of the chamber was washed with double pure water, and after drying, 5 fields of view were randomly selected under an inverted microscope (×200 times), and photographed and counted. Three parallel wells were set in each group, and the experiment was repeated 3 times.

[0080] The results are as follows Figure 5As shown, Figure A is the control group, i.e., HTR-8 / Svneo cells that were not treated with drugs, Figure B is HTR-8 / Svneo cells treated with a low concentration of 80 μg / mL of Agaricus bisporus polypeptide, Figure C is HTR-8 / Svneo cells treated with a medium concentration of 400 μg / mL of Agaricus bisporus polypeptide, and Figure D is HTR-8 / Svneo cells treated with a high concentration of 2000 μg / mL of Agaricus bisporus polypeptide. Observed under a 200x microscope, Figures B, C, and D showed a significant increase in the number of cells compared with Figure A, and the number of cells was the largest when treated with Figure B, i.e., a low concentration of 80 μg / mL of Agaricus bisporus polypeptide. Figure 6 A graph showing the number of cell migrations under treatment with different concentrations of Agaricus bisporus peptides.

[0081] Experimental results:

[0082] 1. Agaricus bisporus peptide proliferation experiment by MTT method

[0083] The results are as follows Figure 1 As shown, the cell proliferation rate after treatment with different drug concentrations was measured at 490nm and 570nm. Compared with the blank control group, after treatment with different doses of Agaricus bisporus polypeptide (0.64μg / mL-2000μg / mL), within a certain range (continuously increasing 0.64μg / mL-2000μg / mL), as the concentration of Agaricus bisporus polypeptide increased, the proliferation rate of human chorionic trophoblast cells also increased; when the drug concentration was 16μg / mL-400μg / mL, the proliferation rate of human chorionic trophoblast cells was significantly increased, and when the concentration was 400μg / mL, the proliferation rate of human chorionic trophoblast cells was the highest.

[0084] Figure 2 The results show the effect of Agaricus bisporus peptides on the proliferation activity of HTR8 detected by CCK8. After the model group was stimulated with LPS, the proliferation rate of HTR8 decreased compared with the blank control group. After being treated with different doses of Agaricus bisporus peptides (40μg / mL-160μg / mL), the proliferation rate of human chorionic trophoblast cells was significantly increased.

[0085] Figure 3 The results show the effect of Agaricus bisporus peptide on HTR8 secretion of LDH. After the model group was stimulated with LPS, the level of LDH secreted by HTR8 increased compared with the blank control group. After being treated with different doses of Agaricus bisporus peptide (40μg / mL-160μg / mL), the level of LDH secreted by human chorionic trophoblast cells was significantly reduced.

[0086] 2. EDU method to detect the cell proliferation rate of Agaricus bisporus peptides at different concentrations

[0087] Figure 4The EDU experimental diagram of different concentrations of Agaricus bisporus peptides is shown. HTR-8 / Svneo cells were treated with different concentrations of Agaricus bisporus peptides, followed by edu staining and observation under a confocal microscope. Compared with Figure A, Figures B, C, and D showed a significant increase in the number of cell proliferation, and Figure D, i.e., the high concentration of 400uM, showed the largest number of cell proliferation.

[0088] 3. Effects of different concentrations of Agaricus bisporus peptides on HTR-8 / Svneo cell migration

[0089] Figure 5 The graphs of the migration of HTR-8 / Svneo cells with different concentrations of Agaricus bisporus peptides are shown. Figure A is the control group, i.e., HTR-8 / Svneo cells that were not treated with drugs, Figure B is HTR-8 / Svneo cells treated with Agaricus bisporus peptides at a low concentration of 80 μg / mL, Figure C is HTR-8 / Svneo cells treated with Agaricus bisporus peptides at a medium concentration of 400 μg / mL, and Figure D is HTR-8 / Svneo cells treated with Agaricus bisporus peptides at a high concentration of 2000 μg / mL. Observed under a 200x microscope, the number of cells in Figures B, C, and D increased significantly compared with Figure A, and the number of cells was the highest when treated with Figure B, i.e., Agaricus bisporus peptides at a low concentration of 80 μg / mL. Figure 6 for Figure 5 Quantitative histogram of .

[0090] 4. Differences in the effects of different concentrations of Agaricus bisporus peptides on cell migration under the action of L-NAME

[0091] Figure 7 The figure shows the effect of various concentrations of Agaricus bisporus peptides on cell migration under the action of L-NAME. Figure A is the control group, i.e. HTR-8 / Svneo cells that were not treated with drugs, Figure B is HTR-8 / Svneo cells treated with 100mg / ml L-NAME, Figure C is HTR-8 / Svneo cells treated with a low concentration of 80μg / mL of Agaricus bisporus peptides and L-NAME, and Figure D is HTR-8 / Svneo cells treated with a high concentration of 400μg / mL of Agaricus bisporus peptides and L-NAME. Observed under a 200x microscope, the number of cells in Figure B was significantly reduced compared with Figure A, and the number of cells in Figures C and D was significantly increased compared with Figure B, and the number of cells was the largest when treated with Figure D, i.e. a high concentration of 400μg / mL of Agaricus bisporus peptides. Figure 8 for Figure 7 Quantitative histogram of .

[0092] 5. Differences in the effects of different concentrations of Agaricus bisporus peptides on cell migration under LPS stimulation

[0093] Fig. 9The figure shows the results of the scratch experiment of HTR-8 / Svneo cells intervened by different concentrations of Agaricus bisporus peptides under LPS stimulation. Fig. 9 In the figure, Figure A is the control group, i.e., HTR-8 / Svneo cells that were not treated with drugs, Figure B is HTR-8 / Svneo cells treated with 40 mg / ml LPS, Figure C is HTR-8 / Svneo cells treated with a low concentration of 40 μg / mL of Agaricus bisporus peptide and LPS, Figure D is HTR-8 / Svneo cells treated with a high concentration of 80 μg / mL of Agaricus bisporus peptide and LPS, and Figure E is HTR-8 / Svneo cells treated with a high concentration of 160 μg / mL of Agaricus bisporus peptide and LPS. After 12h and 24h of culture, Figure B shows that cells migrate more slowly at the scratch compared with Figure A, and Figures C, D, and E show a significantly higher migration speed at the scratch compared with Figure B. Fig.10 for Fig. 9 The quantitative bar graph shows that Agaricus bisporus peptides can promote the migration of HTR-8 / Svneo cells after LPS inhibition.

[0094] In the description of the specification, the description with reference to the terms "one embodiment", "specific embodiment", "example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more implementations or examples in a suitable manner.

[0095] It should be noted that the preferred embodiments of the present invention are given in the specification and drawings of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not used as additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all regarded as the scope of the present invention; further, for ordinary technicians in this field, they can be improved or transformed according to the above description, and all these improvements and transformations should belong to the protection scope of the claims attached to the present invention.

Claims

1. Use of Agaricus bisporus polypeptide in the preparation of a product for promoting the invasion and migration of human chorionic trophoblast cells, characterized in that: The Agaricus bisporus polypeptide is prepared by the following steps: Step 1) Preprocessing: Dry fresh Agaricus bisporus in an oven at 55°C and grind into powder, sieve the powder through a 60-mesh sieve, and collect the Agaricus bisporus dry powder for later use; Step 2) Alkaline extraction: Weigh the dried Agaricus bisporus powder and mix them in a ratio of Agaricus bisporus powder to deionized water = 1:

12. Adjust the pH of the mixture to 10 with 1 mol / L NaOH, dispense into conical flasks, seal the conical flasks with plastic wrap and place them in a shaker, ensure that the height of the mixed solution in the conical flask is lower than the height of the water in the shaker, set the shaker speed to 300 r / min, the time to 3 h, and the temperature to 45°C; Step 3) Acid precipitation: The alkali solution of Agaricus bisporus protein after the reaction was centrifuged at a speed of 5000 r / min, and the supernatant was collected; the pH of the supernatant was adjusted to the isoelectric point of Agaricus bisporus protein, pH 3.5, with 1 mol / L HCl, and then the solution was placed at 4°C for 2 hours; and the precipitate was collected by centrifugation at 3000 r / min; Step 4) Drying: After freeze drying, Agaricus bisporus protein can be obtained; Step 5) Enzymatic hydrolysis: After enzymatic hydrolysis of Agaricus bisporus, Agaricus bisporus polypeptides are obtained; Add 9.9 ml of water to every 0.1 g of Agaricus bisporus protein to prepare a protein solution with a concentration of 10 mg / ml, and stir the solution on a magnetic stirrer for 12 hours at a speed of 280 r / min; 5.6 g of neutral protease was weighed, the pH was adjusted to 8.5 with 1 mol / L sodium bicarbonate, and the mixture was reacted at 45.5° C. for 5 h; the obtained extract was inactivated under high pressure and freeze-dried to obtain the Agaricus bisporus polypeptide.

2. The use according to claim 1, characterized in that The product is a cell culture medium.

3. Use of the Agaricus bisporus polypeptide according to claim 1 in preparing a product for treating preeclampsia in a subject.

4. The use according to claim 3, characterized in that The Agaricus bisporus polypeptide promotes the invasion and migration of human chorionic trophoblast cells of a subject.

5. The use according to claim 3, characterized in that: The product described is a traditional Chinese medicine preparation.

6. A product for promoting the invasion and migration of human chorionic trophoblast cells or treating preeclampsia in a subject, characterized in that: The product contains Agaricus bisporus polypeptide; The Agaricus bisporus polypeptide is the Agaricus bisporus polypeptide according to claim 1.

7. The product according to claim 6, characterized in that The product is a cell culture medium or a traditional Chinese medicine preparation.

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