Use of choline in the preparation of a medicament for treating placental damage induced by flame retardant EHDPP

Abstract

The application relates to the application of choline in the preparation of a medicine for treating flame retardant EHDPP-induced placental injury. The functions of the choline include that cholinergic can alleviate the vitality inhibition, migration inhibition and inflammatory reaction of cells caused by EHDPP, and improve the placental injury caused by EHDPP. The choline includes choline and derivatives thereof, and the derivatives include choline chloride, choline hydroxide, choline phosphate and the like. The application provides a medicine preparation containing choline based on the above functions, and the medicine preparation can be used for treating / alleviating EHDPP-induced placental injury. Choline is a common and relatively safe nutrient, is low in cost, and is easy to popularize. Through double verification of in-vitro and in-vivo experiments, it is found and proved for the first time that choline has a positive effect on reducing EHDPP-induced placental injury, and a novel and effective solution is provided for solving the placental health problems caused by EHDPP exposure.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to application of choline in preparation of a medicine for treating placental injury induced by flame retardant EHDPP. BACKGROUND

[0002] With the development of modern industry, various chemical substances are increasingly widely used in many products, and organic phosphates (OPEs) are one of them. As a kind of synthetic organic chemicals, organic phosphates (OPEs) have the dual functions of flame retardants and plasticizers, gradually replacing traditional brominated flame retardants (BFRs), and are widely used in building materials, household electronic products, textiles, baby products and various industrial products. As a representative chemical of organic phosphates (OPEs), 2-ethylhexyldiphenyl phosphate (EHDPP) is not only the only organic phosphate approved by the US Food and Drug Administration for use in food packaging materials, but also is easily released into the environment because it is incorporated into polymer materials through physical mixing rather than chemical bonding, and has a high detection rate in various environmental matrices.

[0003] Existing studies have shown that EHDPP has many hazards to human health, especially during pregnancy, which can have adverse effects on the placenta and fetus, such as causing placental dysfunction, reducing fetal weight, interfering with placenta formation, causing adverse pregnancy outcomes, affecting progesterone secretion, damaging follicle development, and having negative effects on female reproduction. In addition, EHDPP can also induce inflammatory reactions and affect the normal physiological functions of cells, such as inhibiting cell viability and reducing cell migration. At present, there is still a lack of effective relief or treatment methods for EHDPP-induced placental injury, and new coping methods are urgently needed to protect maternal and child health. SUMMARY

[0004] Based on the defects in the prior art described above, the purpose of the present application is to provide the application of choline in the preparation of a medicine for treating placental injury induced by flame retardant EHDPP.

[0005] Based on the above purpose, the present application provides the following technical solutions:

[0006] One of the technical solutions of the present application provides the application of choline in the preparation of a medicine for treating placental injury induced by flame retardant 2-ethylhexyldiphenyl phosphate (EHDPP).

[0007] Choline is an essential nutrient that plays multiple key roles in the human body. It is essential in the nutritional needs of pregnant women, as it is crucial for fetal brain development and nervous system health.

[0008] Further, the application verifies the effect of choline on 2-ethylhexyl diphenyl phosphate (EHDPP)-induced placental injury through experiments. The effect includes that choline does not exhibit cytotoxicity and does not significantly interfere with the normal physiological metabolism of the body; choline can alleviate the inhibition of cell viability, migration inhibition and inflammatory response caused by EHDPP, and can improve the placental injury caused by EHDPP.

[0009] Further, the choline includes choline and derivatives thereof, and the derivatives include at least any one of choline chloride, choline hydroxide and choline phosphate.

[0010] Further, the cell is any one of chorionic trophoblast cells, choriocarcinoma cells, amniotic cells and decidua basalis cells.

[0011] Further, the placental injury includes the reduction of uterine size, fetal weight, placental weight and placental diameter, and inflammatory response.

[0012] Further, the inflammatory response includes the increase of gene expression levels of tumor necrosis factor-α, interleukin-6 and interleukin-1β in cells, and the decrease of gene expression of proliferation marker Ki67 and vascular differentiation marker Cd31.

[0013] Further, the application verifies the above-mentioned effect through in vitro and in vivo experiments.

[0014] Further, the in vitro experiment verification proves that the choline does not exhibit cytotoxicity and can alleviate the inhibition of cell viability, migration inhibition and inflammatory response caused by EHDPP.

[0015] Further, through in vitro cell experiments, human chorionic trophoblast cells (HTR-8 / SVneo) and human choriocarcinoma cells (JEG-3) are respectively exposed to different concentrations (0, 10, 20, 30 μM) of EHDPP solution. EHDPP shows a dose-dependent inhibitory effect on cell viability, and the inhibitory effect is most significant at a concentration of 30 μM. Then, 100 μM choline chloride is added to the cells treated with EHDPP for intervention, and the experimental results show that choline chloride at this concentration does not exhibit cytotoxicity and can alleviate the inhibition of cell viability of the above two cells caused by EHDPP.

[0016] Further, in terms of cell migration ability, compared with the control group, EHDPP significantly reduced the migration of HTR-8 / SVneo and JEG-3 cells, and the supplement of choline chloride alone did not change the migration ability of the cells, but the supplement of choline chloride in the cells treated by EHDPP could significantly increase the migration of the cells.

[0017] Further, the detection of inflammation-related indicators showed that the supplement of choline chloride could also alleviate the inflammatory response induced by EHDPP, which was specifically manifested in the alleviation of the gene expression levels of tumor necrosis factor-α (Tnf-α), interleukin-6 (Il-6) and interleukin-1β (Il-1β) in the cells caused by EHDPP.

[0018] Further, the in vivo experiment verification proved that choline chloride did not significantly interfere with the normal physiological metabolism of the body, and could improve the placental damage (adverse effects) caused by EHDPP.

[0019] Further, the animal experiment model was constructed to verify the effect of choline chloride, and it was found that choline chloride at least had an alleviating effect on the placental damage induced by EHDPP at 10 mg / kg / day. During the experiment, the weight gain, daily food intake and water intake of the mice in each experimental group were monitored, and the results showed that there was no significant difference between the experimental groups in these aspects, indicating that the supplement of choline chloride did not significantly interfere with the normal physiological metabolism of the mice.

[0020] Further, the detection of important indicators related to pregnancy showed that EHDPP significantly reduced the size of the uterus, the weight of the fetus, the weight of the placenta and the diameter of the placenta, but compared with the EHDPP exposure group, the supplement of choline chloride could significantly increase these index parameters, indicating that choline chloride could improve the adverse effects caused by EHDPP.

[0021] Further, the detection of inflammation-related molecular markers showed that the increase in the gene expression of tumor necrosis factor-α (Tnf-α), interleukin-6 (Il-6) and interleukin-1β (Il-1β) in the placenta induced by EHDPP was also significantly alleviated after the supplement of choline chloride. Moreover, choline chloride also significantly reduced the decrease in the gene expression of the proliferation marker Ki67 and the vascular differentiation marker Cd31 induced by EHDPP, proving that choline chloride could alleviate the placental damage induced by EHDPP in vivo.

[0022] Further, based on the above-mentioned effect of choline, the application provides a pharmaceutical preparation containing choline.

[0023] Further, the pharmaceutical preparation containing choline serves as a cell growth promoter, a cell migration promoter, an inflammation reaction inhibitor, and a placental injury improver.

[0024] Further, the pharmaceutical preparation is in the form of any one of an injection, an oral preparation, and a topical preparation; the oral preparation includes any one of a tablet, a capsule, a granule, an oral solution, and an oral suspension.

[0025] Further, the choline additive in the pharmaceutical preparation is generally added in a dose determined by the specific form of the pharmaceutical preparation, so as to ensure that the administration concentration is 1 μM-500 mM and the administration amount is 0.1 mg / kg / day-50 g / kg / day.

[0026] Compared with the prior art, the present application has at least the following advantages:

[0027] 1. The present application first discovers and proves that choline has a positive effect on reducing EHDPP-induced placental injury, and provides a new and effective solution to placental-related health problems caused by EHDPP exposure. By supplementing choline, a series of adverse effects of EHDPP on the placenta and pregnancy can be alleviated, and an effective intervention approach for improving the health of mothers and infants is provided.

[0028] 2. The present application is fully proved by in vitro and in vivo experiments that the effectiveness and safety of choline application are proved, which not only can alleviate the injury of the placenta in structure and function, but also can help to alleviate the inflammation caused by EHDPP, which is of great significance to the normal development of the fetus and the health of the mother.

[0029] 3. The choline involved in the present application is a common and relatively safe nutrient, and its application cost is relatively low, easy to implement, and is expected to play an important role in the field of maternal and infant health protection, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Figure 2 is a data graph of the dose-dependent inhibitory effect of EHDPP treatment on cell viability in Example 2; A is HTR-8 / SVneo cells, and B is JEG-3 cells; * indicates 0.01

[0031] Figure 2 Figure 3 is an effect of EHDPP, choline chloride, and EHDPP combined with choline chloride on cell viability in Example 3; A is HTR-8 / SVneo cells, and B is JEG-3 cells; ** indicates 0.001

[0032] Figure 3Figure 4 shows the effect of EHDPP, choline chloride, and EHDPP combined with choline chloride on the migration ability of cells in Example 4. A represents HTR-8 / SVneo cells, and B represents JEG-3 cells. * indicates 0.01 < p < 0.05, and ** indicates 0.001 < p < 0.01.

[0033] Figure 4 Figure 5 shows the effect of EHDPP, choline chloride, and EHDPP combined with choline chloride on the expression of inflammatory factors Tnf-α, Il-6, and Il-1β in cells in Example 5. A represents HTR-8 / SVneo cells, and B represents JEG-3 cells. * indicates 0.01 < p < 0.05, and ** indicates 0.001 < p < 0.01.

[0034] Figure 5 Figure 6 shows the process flow and results of the in vivo animal experiment in Example 6. A is a flowchart of the process of treating mice. B to D are the weight, food intake, and water intake of mice. E is the tissue sample of the uterus, placenta, and fetus of mice. F to H are the statistical results of the weight of the fetus, the weight of the placenta, and the diameter of the placenta. ** indicates 0.001 < p < 0.01.

[0035] Figure 6 Figure 7 shows the statistical results of the detection of the expression levels of Tnf-α (A), Il-6 (B), Il-1β (C), Ki67 (D), and Cd31 (E) in the placenta tissue in Example 7. DETAILED DESCRIPTION

[0036] To enable those skilled in the art to better understand the technical solutions of the present application, the present application will be described in detail below in conjunction with specific examples. It should be noted that the following examples will help those skilled in the art to further understand the present application, but do not limit the present application in any form. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present application. These all belong to the protection scope of the present application.

[0037] All raw materials of the present application are not particularly limited in source, and can be purchased on the market or prepared according to the conventional method well known to those skilled in the art.

[0038] The embodiments provided in the present application include two parts: (1) in vitro cell experiment and (2) in vivo animal experiment.

[0039] (1) In vitro cell experiment demonstrates the effect of choline on placental injury induced by 2-ethylhexyl diphenyl phosphate (EHDPP)

[0040] Example 1 in vitro cell culture

[0041] Human trophoblast cells (HTR-8 / SVneo) and human choriocarcinoma cells (JEG-3) were selected for culture. The culture medium for HTR-8 / SVneo cells was 90% DMEM and 10% FBS, and the culture medium for JEG-3 cells was 90% MEM and 10% FBS. The cells were cultured in a 37°C, 5% CO2 incubator, and regular cell replacement and subculture operations were performed to ensure that the cells were in good growth condition. The cell replacement and subculture operations are routine operations known to those skilled in the art, and are as follows:

[0042] A) Cell replacement:

[0043] a. Stop culture: Stop the culture when the cells reach 70% to 80% confluence, and prepare for replacement.

[0044] b. Discard old culture medium: Under sterile conditions, gently tilt the culture bottle or dish, and slowly suck off the culture medium, taking care not to disturb the cells.

[0045] c. Wash the cells: Gently rinse the cells 1-2 times with pre-warmed PBS (phosphate buffered solution) at 37°C to remove residual culture medium and metabolic waste.

[0046] d. Add fresh culture medium: Add fresh complete culture medium preheated to 37°C with a sterile pipette, ensuring that the cells are completely covered, but not too much to prevent cell edema.

[0047] B) Cell subculture:

[0048] a. Observation and evaluation: Determine the cell growth condition, and subculture when the cells reach the appropriate confluence (usually about 90%).

[0049] b. Preparation: Prepare the required fresh 90% DMEM or MEM culture medium, trypsin (or other cell digestion solution), PBS, and sterile instruments.

[0050] c. Cell digestion: Add an appropriate amount of trypsin to the culture bottle, gently shake to ensure uniform cell contact with trypsin, and avoid vigorous stirring.

[0051] d. Stop digestion: When the cell connections are broken and single cells or small cell clusters are formed, stop the trypsin digestion with culture medium.

[0052] e. Centrifugal separation: Transfer the cell suspension to a centrifuge tube, and centrifuge gently (usually 300-500g for 5-10 minutes), then discard the supernatant.

[0053] f. Cell resuspension: Add an appropriate amount of fresh culture medium, and gently blow to resuspend the cells.

[0054] g. Passage inoculation: The cell suspension is divided into multiple new culture bottles or culture dishes, inoculated according to the appropriate ratio (such as 1:2, 1:3, etc.), and then preheated culture solution is added to the appropriate height.

[0055] h. Return to the incubator: Put the culture bottle inoculated with cells back into the incubator at 37°C, 5% CO2, and continue to culture.

[0056] Example 2 EHDPP treatment presents a dose-dependent inhibitory effect on cell viability

[0057] The human chorionic trophoblast cells (HTR-8 / SVneo) or choriocarcinoma cells (JEG-3) cultured in Example 1 are inoculated into 96-well plates at a density of 1.0 x 104 cells per well, 100 μL, and then placed in a 37°C, 5% CO2 incubator for 24 h.

[0058] Then the above cells per well are divided into 4 groups, respectively, and given 0, 10, 20, 30 μM EHDPP treatment, and the amount of treatment reagent added per well is 200 μL, and the treatment time is 24 h.

[0059] After the treatment is completed, the absorbance is measured by using the Spark microplate reader manufactured by TECAN Company, and the wavelength is 450 nm.

[0060] The detection results are shown in Figure 1 The figure * indicates 0.01 < p < 0.05, and ** indicates 0.001 < p < 0.01. It can be seen that: EHDPP presents a dose-dependent inhibitory effect on the viability of human chorionic trophoblast cells (HTR-8 / SVneo) or choriocarcinoma cells (JEG-3), and the inhibitory effect is most significant at a concentration of 30 μM.

[0061] Example 3 EHDPP treatment and choline chloride intervention, CCK-8 method for detecting cell viability

[0062] The human chorionic trophoblast cells (HTR-8 / SVneo) or choriocarcinoma cells (JEG-3) cultured in Example 1 are inoculated into 96-well plates at a density of 1.0 x 104 cells per well, 100 μL, and then placed in a 37°C, 5% CO2 incubator for 24 h.

[0063] Then the above cells per well are divided into 4 groups, respectively, and given 0, 10, 20, 30 μM EHDPP treatment, and the amount of treatment reagent added per well is 200 μL, and the treatment time is 24 h.

[0064] Subsequently, the medium in the 96-well plate was replaced with 110 μL of CCK-8 solution (CCK-8 solution was prepared by mixing complete medium with CCK-8 reagent at a ratio of 10:1), and the 96-well plate was returned to the incubator at 37°C for continued incubation for 0.5-4 hours.

[0065] Finally, the absorbance was measured using a Spark microplate reader manufactured by TECAN, at a wavelength of 450 nm.

[0066] The results are shown in Figure 2 The figure shows that 0.001 < p < 0.01, EHDPP significantly inhibited cell viability in HTR-8 / SVneo and JEG-3 cells, and treatment of the cells with choline chloride did not change cell viability, but the addition of choline chloride under the stimulation of EHDPP alleviated the inhibition of cell viability by EHDPP.

[0067] Example 4: Scratch test for detection of cell migration

[0068] Human chorionic trophoblast cells (HTR-8 / SVneo) or choriocarcinoma cells (JEG-3) cultured in Example 1 were seeded into a 6-well plate, and then placed in an incubator at 37°C, 5% CO2for 24 h.

[0069] When the confluence of the cells reached about 90%-100%, a 10 μL pipette gun head was used to make a scratch perpendicular to the surface of the culture plate.

[0070] Next, the cells were washed 3 times with sterile phosphate buffered saline (PBS), and then the medium in the 6-well plate was replaced with fresh low serum medium containing a blank control solvent (complete medium), 30 μM EHDPP, 100 μM choline chloride, EHDPP (30 μM) + choline chloride (100 μM).

[0071] The cells were photographed under an inverted microscope, and the scratch width measured at this time was recorded as the 0 h result.

[0072] Subsequently, the cells were placed in an incubator for continued culture for 24 h, and the scratch width and cell migration were observed and photographed again.

[0073] Finally, the Image J software was used to measure and calculate the scratch width.

[0074] The formula for calculating the scratch width is as follows:

[0075] Scratch width = 0 h cell scratch width - 24 h cell scratch width

[0076] The results are shown in Figure 3The left side of the graph shows the observation under the microscope, and the black vertical lines mark the two edges of the scratch. In the right side of the graph, * indicates 0.01 < p < 0.05, and ** indicates 0.001 < p < 0.01. EHDPP significantly inhibited cell migration in HTR-8 / SVneo and JEG-3 cells. Choline treatment did not change the cell migration ability, but the addition of choline under EHDPP stimulation alleviated the inhibition of cell migration ability by EHDPP.

[0077] Example 5 Real-time fluorescent quantitative PCR detection of cell gene expression level

[0078] Human chorionic trophoblast cells (HTR-8 / SVneo) or choriocarcinoma cells (JEG-3) cultured in Example 1 were inoculated into 6-well plates (2 mL per well), a total of 4 groups, at least 3 replicates per group, and then placed in a 37°C, 5% CO2 incubator for 24 h.

[0079] Then the above 4 groups of cells were respectively given blank control solvent (complete culture medium), 30 μM EHDPP, 100 μM choline, EHDPP (30 μM) combined with choline (100 μM) treatment, and the amount of treatment reagent added per well was 2 mL, and the treatment time was 24 h.

[0080] Add 1 mL of ice Trizol reagent (purchased from Nanjing Novozyme Biotech Co., Ltd.) to extract total RNA, then perform reverse transcription, and then use SYBR Green fluorescent quantitative PCR mixed reagent (purchased from Nanjing Novozyme Biotech Co., Ltd.) to detect inflammatory factors Tnf-α, Il-6, and Il-1β by real-time fluorescent quantitative PCR.

[0081] The above total RNA extraction, reverse transcription, and real-time fluorescent quantitative PCR are routine operations known to those skilled in the art, and the following steps can be referred to:

[0082] A) Extraction of total RNA:

[0083] a. Cell lysis: Trizol is used to break the cells and release the RNA in the cells.

[0084] b. RNA precipitation: RNA is precipitated by adding RNA precipitant (chloroform 200 μL) and isopropanol 400-500 μL. After mixing well, the RNA is separated by centrifugation (12000 rpm, 10 min).

[0085] c. RNA washing: 1 mL of 75% ethanol is used for washing to remove solvents and salts.

[0086] d. RNA drying: Dry the RNA at room temperature to remove ethanol.

[0087] e. RNA purity and quality detection: Use UV spectrophotometer (A 260 / A 280 ratio) and gel electrophoresis to evaluate the purity and integrity of RNA. RNA should present clear 28S and 18S rRNA bands, and A 260 / A 280 ratio close to 1.8-2.0.

[0088] f. RNA dissolution: Dry RNA is dissolved in RNase-free water or TE buffer, stored at -80°C for standby.

[0089] B) Reverse transcription operation:

[0090] a. Pretreatment of RNA sample: The RNA sample used in this example is from the total RNA extracted above. Genomic DNA removal is performed on the RNA: an appropriate amount of template RNA and 4 μL of 4x gDNA wiper Mix are added to a 200 μL EP tube, and the total volume is made up to 16 μL with buffer (RNase-free ddH2O). After gentle mixing, the mixture is reacted at 42°C for 2 min.

[0091] b. Enzyme mixture preparation: 4 μL of 5x HiScript II qRT SuperMix II is added to the above EP tube, and mixed again gently.

[0092] c. Reverse transcription reaction: Incubate the RNA and enzyme mixture, react at 50°C for 15 min, and then hold at 85°C for 5 s. In this step, the reverse transcriptase synthesizes a complementary DNA chain (cDNA) from the mRNA of the RNA molecule as a template according to the base pairing rules.

[0093] d. Reaction termination: After the reaction is completed, the sample is stored in the -20°C refrigerator.

[0094] C) Real-time fluorescent quantitative PCR:

[0095] a. Design primers and probes: Design a pair of fluorescently labeled PCR primers and a fluorescent probe for the target sequence. The probe is usually located between the primers and contains a reporter group and a quencher group. Ensure the specificity of the primers and probes to the target sequence, and avoid non-specific binding by tools such as BLAST.

[0096] b. PCR reaction mixture preparation: Add 5 μL 2x chamQ universal SYBR qRT-PCR Master Mix, 0.2 μL 10 μM designed forward and reverse primers (primer sequences refer to Table 1), 0.2 μL 50x ROX Reference Dye 1 and 1 μL template cDNA in PCR tube, and make up to 10 μL with RNase-free ddH2O.

[0097] c. PCR cycling: Pre-denaturation: heat the sample to about 95 °C to unwind the DNA double strands. Denaturation / annealing / extension cycle: the temperature is usually 95 °C, 55-60 °C (primer binding), 72 °C (DNA polymerase extension). In the annealing stage of each cycle, the probe binds to the target sequence; in the extension stage, the fluorescent signal is released by the reporter group, which is captured by the detector.

[0098] d. Data collection and analysis: The fluorescent signal is monitored in real time during the extension stage of PCR cycling. Using the standard curve, the fluorescent signal is correlated with the initial amount of template, and the copy number of the target sequence in the sample is calculated. The analysis software generates Ct values (cycle threshold) for comparing the relative abundance between samples.

[0099] Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as an internal control, and all primer sequences are shown in Table 1.

[0100] Table 1 Primers used for real-time fluorescence quantitative PCR

[0101]

[0102]

[0103] Results are shown in Figure 4 Figures, where * indicates 0.01 < p < 0.05, and ** indicates 0.001 < p < 0.01. In HTR-8 / SVneo and JEG-3 cells, EHDPP treatment up-regulated the expression of Tnf-α, Il-6 and Il-1β, and choline chloride-treated cells showed a decreasing trend, but did not significantly change their expression levels. However, the addition of choline chloride significantly reduced the expression levels of inflammatory factors induced by EHDPP under EHDPP stimulation. This indicates that choline chloride alleviates the inflammation caused by EHDPP in placental cells in vitro.

[0104] (II) In vivo animal demonstration of the effect of choline chloride on 2- ethylhexyl diphenyl phosphate-induced (EHDPP) placental damage

[0105] Example 6 In vivo experimental animal grouping and treatment

[0106] ICR female mice (purchased from Xuzhou Medical University) were selected at the age of 5 weeks, and after one week of adaptation, they were randomly divided into control group, EHDPP exposure group, choline chloride supplementation, choline chloride supplementation + EHDPP exposure group, 6 in each group, as a parallel experiment. 10 mg / kg / day EHDPP was given by gavage, and 25 mM choline chloride was dissolved in drinking water and given by drinking water. After two weeks of continuous treatment, they were caged with eight-week-old ICR male mice of the same age, and the treatment was continued until the 17.5th day of pregnancy (GD17.5) Figure 5 A).

[0107] During the entire experiment, the body weight (Weight), daily food intake (Diet), and water consumption (Water consumption) of mice in each experimental group were recorded daily, and the corresponding change curves were drawn to observe whether choline chloride supplementation had an impact on normal physiological metabolism of mice. The results showed that there were no differences in body weight ( Figure 5 B), food intake ( Figure 5 C), and water consumption ( Figure 5 D) between the experimental group and the control group.

[0108] On the 17.5th day of pregnancy (GD17.5), the mice in each experimental group were dissected, and tissue samples such as uterus, placenta, and fetus were collected. The weights of the uterus, placenta, and fetus were accurately weighed using an electronic balance, and the placenta diameter was measured. The results showed that EHDPP significantly reduced the size of the uterus ( Figure 5 E), fetal weight, placental weight, and placenta diameter ( Figure 5 F-H), and choline chloride supplementation could attenuate the reduction of these index parameters by EHDPP, but pure choline chloride supplementation had no effect on the above-mentioned indicators, indicating that choline chloride could improve the adverse effects of EHDPP on placenta in vivo.

[0109] Example 7: Fluorescent quantitative PCR detection of placental tissue gene expression level

[0110] The total RNA of the placenta obtained in Example 6 was extracted using Trizol reagent on ice. The mRNA level was reverse transcribed, and then the SYBR Green fluorescent quantitative PCR mixture was used for real-time fluorescent quantitative PCR to detect the gene expression levels of inflammatory factors Tnf-α, Il-6, Il-1β, proliferation marker Ki67, and vascular differentiation marker Cd31 in the placenta. The above-mentioned total RNA extraction operation, reverse transcription operation, and real-time fluorescent quantitative PCR were the same as in Example 5; glycerol-3-phosphate dehydrogenase (Gapdh) was also used as an internal control, and the primer sequences are listed in Table 1.

[0111] The results were consistent with the cell trend, such asFigure 6 As shown, EHDPP treatment up-regulated the expression of inflammatory factors Tnf-a, IL-6, IL-1 b, and choline chloride treatment did not change their expression levels, but supplementing choline chloride under EHDPP stimulation greatly alleviated the induction of inflammatory factors by EHDPP. It is proved that choline chloride indeed alleviates the placental inflammation caused by EHDPP.

[0112] In addition, EHDPP inhibited the gene expression levels of proliferation marker Ki67 and vascular differentiation marker Cd31, and only choline chloride treatment did not change the expression of Ki67 and Cd31 in the placenta, but choline chloride supplementation attenuated the inhibition of EHDPP on the expression of Ki67 and Cd31. The results again show that choline chloride can improve the damage of EHDPP to the placenta.

[0113] More and more studies show that EHDPP accumulates in the human body and causes harm to human health. This application proves the damage of EHDPP to the placenta, and proposes to use choline chloride supplementation to alleviate the placental damage caused by EHDPP, which is a novel strategy and has not been reported. The human body can obtain choline from food and also endogenously synthesize choline. This application proposes to supplement additional choline for the prevention and treatment of placental damage caused by EHDPP, and this invention is also expected to be extended to other causes of placental damage.

[0114] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. Those skilled in the art can easily make various modifications to these embodiments, and apply the general principles described herein to other embodiments without having to go through creative labor. Therefore, the present application is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present application without departing from the scope of the present application should be within the scope of protection of the present application.

Claims

1. Use of choline for the preparation of a medicament for the treatment of flame retardant EHDPP-induced placental damage, characterized in that, The cholinergic alleviates the inhibition of cell viability, migration and inflammation caused by EHDPP, and improves the placental damage caused by EHDPP.

2. Use of choline according to claim 1 for the preparation of a medicament for the treatment of placental damage induced by the flame retardant EHDPP, characterized in that, The choline includes choline and its derivatives, and the derivatives are at least one selected from choline chloride, choline hydroxide and choline phosphate.

3. Use of choline according to claim 1 for the preparation of a medicament for the treatment of placental damage induced by the flame retardant EHDPP, characterized in that, The cell is any one of chorionic trophoblast cells, chorionic carcinoma cells, amniotic cells and decidua basalis cells.

4. Use of choline according to claim 1 for the preparation of a medicament for the treatment of placental damage induced by the flame retardant EHDPP, characterized in that, The placental damage includes the reduction of uterine size, fetal weight, placental weight and placental diameter, and inflammation.

5. Use of choline according to claim 4 for the preparation of a medicament for the treatment of placental damage induced by the flame retardant EHDPP, characterized in that, The inflammation includes the increase of gene expression levels of tumor necrosis factor-α, interleukin-6 and interleukin-1β in cells, and the decrease of gene expression of proliferation marker Ki67 and vascular differentiation marker Cd31.

6. Use of choline according to claim 1 for the preparation of a medicament for the treatment of placental damage induced by the flame retardant EHDPP, characterized in that, The choline is prepared into a pharmaceutical preparation.

7. Use of choline according to claim 6 for the preparation of a medicament for the treatment of placentary damage induced by the flame retardant EHDPP, characterized in that, In the application, the pharmaceutical preparation containing choline is used as a cell growth promoter, a cell migration promoter and an inflammation inhibitor.

8. Use of choline according to claim 6 for the preparation of a medicament for the treatment of placental damage induced by the flame retardant EHDPP, characterized in that, The pharmaceutical preparation is in any one of injection, oral preparation and external preparation.

9. Use of choline according to claim 8 for the preparation of a medicament for the treatment of placentary damage induced by the flame retardant EHDPP, characterized in that, The oral preparation is any one selected from tablets, capsules, granules, oral solutions and oral suspensions.

10. Use of choline according to claim 9 for the preparation of a medicament for the treatment of placental damage induced by the flame retardant EHDPP, characterized in that, The pharmaceutical preparation ensures that the administration concentration of choline is 1 μM-500 mM, and the administration amount is 0.1 mg / kg / day-50 g / kg / day.

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