Application of choline in preparation of medicine for treating placenta injury induced by flame retardant EHDPP

Abstract

The invention relates to application of choline in preparation of a medicine for treating placental injury induced by a flame retardant EHDPP. The choline has the effects that the activity inhibition, migration inhibition and inflammatory response of the EHDPP on cells can be relieved, and the placenta injury caused by the EHDPP can be improved. The choline comprises choline and derivatives thereof, and the derivatives comprise choline chloride, choline hydroxide, phosphorylcholine and the like. Based on the effects, the invention provides a medicinal preparation containing choline, and the medicinal preparation can be used for treating / relieving EHDPP-induced placenta injury. Choline is a common and relatively safe nutrient substance, and is low in cost and easy to popularize. Through dual verification of in-vitro and in-vivo experiments, the invention discovers and verifies that choline has a positive effect on relieving EHDPP-induced placenta injury for the first time, and provides a brand-new and effective solution for solving placenta-related health problems caused by EHDPP exposure.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, and relates to the application of choline in the preparation of drugs for treating placenta damage induced by the flame retardant EHDPP. Specifically, it relates to the application of choline in the preparation of drugs for treating placenta damage induced by the flame retardant 2-ethylhexyl diphenyl phosphate (EHDPP). Background Art

[0002] With the development of modern industry, the application of various chemical substances in numerous products has become increasingly widespread, and organophosphate esters (OPEs) are one of them. As a class of synthetic organic chemicals, organophosphate esters (OPEs) have the dual functions of flame retardants and plasticizers, gradually replacing traditional brominated flame retardants (BFRs), and are widely present in building materials, household electronic products, textiles, baby products, and various industrial products. And 2-ethylhexyl diphenyl phosphate (EHDPP), as a representative chemical of organophosphate esters (OPEs), is not only the only organophosphate ester approved by the US Food and Drug Administration for use in food packaging materials, but also because the way it is incorporated into polymer materials is through physical mixing rather than chemical bonding, it is easy to be released into the environment and has a high detection rate in various environmental matrices.

[0003] Existing studies have shown that EHDPP poses many hazards to human health. Especially during pregnancy, it will have adverse effects on the placenta and fetus. For example, it can cause placental dysfunction, reduced fetal weight, interfere with placental formation, lead to adverse pregnancy outcomes, affect progesterone secretion, damage follicular development, and have a negative impact on female reproduction. In addition, EHDPP can also induce inflammatory responses and affect the normal physiological functions of cells, such as inhibiting cell viability and reducing cell migration. Currently, there is a lack of effective means to relieve or treat the problem of placenta damage induced by EHDPP, and new countermeasures are urgently needed to protect the health of mothers and infants. Summary of the Invention

[0004] Based on the above-mentioned defects existing in the prior art, the purpose of this application is to provide the application of choline in the preparation of drugs for treating placenta damage induced by the flame retardant EHDPP.

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

[0006] One of the technical solutions of this application provides the application of choline in the preparation of drugs for treating placenta damage induced by the flame retardant 2-ethylhexyl diphenyl phosphate (EHDPP).

[0007] Choline is an important nutrient that plays multiple key roles in the human body. In the nutritional requirements of pregnant women, choline is essential because it is crucial for the brain development and nervous system health of the fetus.

[0008] Furthermore, the present application verified the effect of choline on 2-ethylhexyl diphenyl phosphate (EHDPP)-induced placental injury through experiments. The effects include: choline did not exhibit cytotoxicity and did not significantly interfere with the normal physiological metabolism of the body; choline could relieve the viability inhibition, migration inhibition, and inflammatory response caused by EHDPP to cells, and could improve the placental injury caused by EHDPP.

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

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

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

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

[0013] Furthermore, the present application verified the above effects through in vitro experiments and in vivo experiments.

[0014] Furthermore, the in vitro experiment verification proved that: the choline did not exhibit cytotoxicity and could relieve the viability inhibition, migration inhibition, and inflammatory response caused by EHDPP to cells.

[0015] Even further, through in vitro cell experiments, human chorionic trophoblast cells (HTR-8 / SVneo) and human choriocarcinoma cells (JEG-3) were respectively exposed to EHDPP solutions at different concentrations (0, 10, 20, 30 μM). EHDPP showed a dose-dependent inhibitory effect on cell viability, and the inhibitory effect was most significant at a concentration of 30 μM. Then, choline chloride at a concentration of 100 μM was added to the cells treated with EHDPP for intervention. The experimental results showed that choline chloride at this concentration did not exhibit cytotoxic effects and could relieve the inhibition of the viability of the above two types of cells by EHDPP.

[0016] Furthermore, in terms of cell migration ability, compared with the control group, EHDPP significantly reduced the migration of HTR-8 / SVneo and JEG-3 cells. Supplementing choline chloride alone did not change the original cell migration ability, but supplementing choline chloride in EHDPP-treated cells significantly increased cell migration.

[0017] Furthermore, the detection of inflammation-related indicators found that supplementing choline chloride could also alleviate the inflammatory response induced by EHDPP, specifically manifested as alleviating the gene expression levels of tumor necrosis factor-α (Tnf-α), interleukin-6 (Il-6), and interleukin-1β (Il-1β) in 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 and alleviate the placental damage induced by EHDPP.

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

[0020] Furthermore, the detection of important indicators related to pregnancy found that EHDPP significantly reduced the uterine size, fetal weight, placental weight, and placental diameter. However, compared with the EHDPP-exposed group, supplementing choline chloride could significantly increase these indicator parameters, indicating that choline chloride could improve the adverse effects caused by EHDPP.

[0021] Furthermore, the detection of inflammation-related molecular markers showed that the increased 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 supplementing choline chloride. Moreover, choline chloride also significantly weakened the decrease in the gene expression of the proliferation marker Ki67 and the vascular differentiation marker Cd31 induced by EHDPP. It was proved that choline chloride could alleviate the placental damage induced by EHDPP in vivo.

[0022] Further, based on the above effects of choline, the present application provides a pharmaceutical preparation containing choline.

[0023] Furthermore, the choline-containing pharmaceutical preparation serves as a cell growth promoter, a cell migration promoter, an inflammatory response inhibitor, and a placental injury improver.

[0024] Furthermore, 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 tablets, capsules, granules, oral solutions, or oral suspensions.

[0025] Furthermore, the added dose of choline in the pharmaceutical preparation is generally determined by the specific form of the pharmaceutical preparation, and it is sufficient 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 invention discovers and confirms for the first time that choline has a positive effect in alleviating EHDPP-induced placental injury, providing a new and effective solution for solving placental-related health problems caused by EHDPP exposure. By supplementing choline to relieve a series of adverse effects of EHDPP on the placenta and pregnancy-related aspects, it provides an effective intervention approach for improving the health status of mothers and infants.

[0028] 2. The present invention fully proves the effectiveness and safety of choline application through double verification of in vitro and in vivo experiments. It can not only relieve the damage to the structure and function of the placenta, but also help reduce the inflammatory response caused by EHDPP, which is of great significance for ensuring the normal development of the fetus and the health of the mother.

[0029] 3. The choline involved in the present invention is a common and relatively safe nutrient, with relatively low application costs, easy to promote and implement, and is expected to play an important role in the field of maternal and infant health protection, having good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a data graph showing the dose-dependent inhibitory effect of EHDPP treatment on cell viability in Example 2; in the figure, A is HTR-8 / SVneo cells, and B is JEG–3 cells; in the figure, * indicates 0.01 < p < 0.05, and ** indicates 0.001 < p < 0.01;

[0031] Figure 2 It is the effect of EHDPP, choline chloride, and EHDPP combined with choline chloride treatment on cell viability in Example 3; in the figure, A is HTR-8 / SVneo cells, and B is JEG–3 cells; in the figure, ** indicates 0.001 < p < 0.01;

[0032] Figure 3Effect of EHDPP, choline chloride, and EHDPP combined with choline chloride on cell migration ability in Example 4; in the figure, A is HTR-8 / SVneo cells and B is JEG–3 cells; * in the figure indicates 0.01 < p < 0.05, and ** indicates 0.001 < p < 0.01;

[0033] Figure 4 Effect of EHDPP, choline chloride, and EHDPP combined with choline chloride on the expression levels of cell inflammatory factors Tnf-α, Il-6, and Il-1β detected by real-time fluorescence quantitative PCR in Example 5; in the figure, A is HTR-8 / SVneo cells and B is JEG–3 cells; * in the figure indicates 0.01 < p < 0.05, and ** indicates 0.001 < p < 0.01;

[0034] Figure 5 Treatment process and results of in vivo animal experiments in Example 6; in the figure, A is the flow chart of mouse treatment; B–D are the body weight, food intake, and water intake of mice; E is the tissue samples of mouse uterus, placenta, and fetus; F–H are the statistical results of fetal weight, placental weight, and placental diameter of mice; ** in the figure indicates 0.001 < p < 0.01;

[0035] Figure 6 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 placental tissue in Example 7. Detailed implementation mode

[0036] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be described in detail below with reference to specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made. These all belong to the protection scope of the present invention.

[0037] For all raw materials of the present invention, there is no special limitation on their sources, and those purchased on the market or prepared according to the conventional methods well-known to those skilled in the art are all acceptable.

[0038] The implementation mode provided in this application includes two parts: (i) in vitro cell experiments and (ii) in vivo animal experiments.

[0039] (i) In vitro cell experiments prove the effect of choline on 2-ethylhexyl diphenyl phosphate (EHDPP)-induced placental damage

[0040] Example 1 In vitro cell culture

[0041] Human chorionic 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. At 37°C, 5% CO 2 The cells were cultured in an incubator, and cell medium replacement and subculture were performed regularly to ensure that the cells were in good growth condition. The cell medium replacement and subculture operations are conventional operations well-known to those skilled in the art, and are specifically as follows:

[0042] A) Cell medium replacement:

[0043] a. Stop culturing: Stop culturing when the cell confluence reaches 70% - 80% and prepare for medium replacement.

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

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

[0046] d. Add fresh culture medium: Using a sterile pipette, add pre-warmed (to 37°C) fresh complete culture medium, ensuring that the cells are completely covered, but not too much to prevent cell swelling.

[0047] B) Cell subculture:

[0048] a. Observe and evaluate: Determine the cell growth status and perform subculture when the cells reach an appropriate confluence (usually around 90%).

[0049] b. Prepare in advance: Prepare the required fresh 90% DMEM or MEM medium, trypsin (or other cell digestive solutions), PBS, and sterile instruments.

[0050] c. Digest the cells: Gently drip an appropriate amount of trypsin into the culture flask and gently shake to ensure that the cells are evenly exposed to the trypsin, avoiding strong agitation.

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

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

[0053] f. Resuspend the cells: Add an appropriate amount of fresh medium and gently pipette to fully resuspend the cells.

[0054] g. Subculture inoculation: Aliquot the cell suspension into multiple new culture flasks or culture dishes, inoculate at an appropriate ratio (such as 1:2, 1:3, etc.), and then add pre-warmed culture medium to an appropriate height.

[0055] h. Return to the incubator: Place the culture flasks inoculated with cells back into the incubator at 37°C and 5% CO 2 for continued culture.

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

[0057] Inoculate the human chorionic trophoblast cells (HTR-8 / SVneo) or choriocarcinoma cells (JEG-3) cultured in Example 1 into a 96-well plate at a density of 1.0×104 cells per well and 100 μL, and then place it in an incubator at 37°C and 5% CO 2 for 24 h of culture.

[0058] Then divide the cells in each well above into 4 groups, and respectively give treatments of 0, 10, 20, 30 μM EHDPP. The amount of treatment reagent added to each well is 200 μL, and the treatment time is 24 h.

[0059] After the treatment is completed, use a Spark microplate reader manufactured by TECAN to measure the absorbance at a wavelength of 450 nm.

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

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

[0062] Inoculate the human chorionic trophoblast cells (HTR-8 / SVneo) or choriocarcinoma cells (JEG-3) cultured in Example 1 into a 96-well plate at a density of 1.0×104 cells per well and 100 μL, and then place it in an incubator at 37°C and 5% CO 2 for 24 h of culture.

[0063] Then divide the cells in each well above into 4 groups, and respectively give treatments of blank control solvent (complete culture medium), 30 μM EHDPP, 100 μM choline chloride, and EHDPP (30 μM) combined with choline chloride (100 μM). The amount of treatment reagent added to each well is 200 μL, and the treatment time is 24 h.

[0064] Subsequently, the culture medium in the 96-well plate was replaced with 110 μL of CCK-8 solution (prepared by mixing complete culture medium and 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 detection results are as Figure 2 shown. In the figure, ** indicates 0.001 < p < 0.01. In HTR-8 / SVneo and JEG-3 cells, EHDPP significantly inhibited cell viability. Treatment with choline chloride did not change cell viability, but supplementation with choline chloride alleviated the inhibition of cell viability by EHDPP under EHDPP stimulation.

[0067] Example 4 Scratch assay to detect cell migration

[0068] The 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% CO 2 for 24 h.

[0069] When the cell confluence reached approximately 90% - 100%, a 10 μL pipette tip 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 buffer (PBS), and then the culture medium in the 6-well plate was replaced with fresh low-serum culture medium containing blank control solvent (complete culture medium), 30 μM EHDPP, 100 μM choline chloride, and 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-hour result.

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

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

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

[0075] Scratch width = Scratch width of cells at 0 h - Scratch width of cells at 24 h

[0076] The results are as Figure 3As shown, in the figure observed under the microscope on the left, the two sides of the scratch are marked by black vertical lines; in the statistical chart on the right, * indicates 0.01 < p < 0.05, and ** indicates 0.001 < p < 0.01. In HTR-8 / SVneo and JEG-3 cells, EHDPP significantly inhibited cell migration. Treatment with choline chloride did not change the cell migration ability, but supplementation with choline chloride under EHDPP stimulation alleviated the inhibition of EHDPP on cell migration ability.

[0077] Example 5 Detection of Cell Gene Expression Levels by Real-Time Fluorescent Quantitative PCR

[0078] Inoculate the human chorionic trophoblast cells (HTR-8 / SVneo) or choriocarcinoma cells (JEG-3) cultured in Example 1 into a 6-well plate (the volume of each well is 2 mL), with a total of 4 groups, and at least 3 replicates in each group. Then place them in an incubator at 37 °C and 5% CO 2 for 24 h.

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

[0080] Add 1 mL of ice-cold Trizol reagent (purchased from Nanjing Novozymes Biotech Co., Ltd.), extract total RNA, then perform reverse transcription operation, and then perform real-time fluorescent quantitative PCR to detect inflammation-related factors Tnf-α, Il-6, and Il-1β with the help of SYBR Green fluorescent quantitative PCR mixture (purchased from Nanjing Novozymes Biotech Co., Ltd.).

[0081] The above operations of extracting total RNA, reverse transcription, and real-time fluorescent quantitative PCR are conventional operations well-known to those skilled in the art and can refer to the following steps:

[0082] A) Extract total RNA:

[0083] a. Cell lysis: Use Trizol to break cells and release intracellular RNA.

[0084] b. RNA precipitation: Precipitate RNA by adding RNA precipitant (200 μL of chloroform) and 400 - 500 μL of isopropanol. After thorough mixing, separate RNA by centrifugation (12000 rpm, 10 min).

[0085] c. RNA washing: Wash with 1 mL of 75% ethanol 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 a UV spectrophotometer (A 260 / A 280 ratio) and gel electrophoresis to evaluate the purity and integrity of the RNA. The RNA should show clear 28S and 18S rRNA bands, and the A 260 / A 280 ratio should be close to 1.8 - 2.0.

[0088] f. RNA Dissolution: Dissolve the dried RNA in RNase-free water or TE buffer and store it at -80 °C for later use.

[0089] B) Reverse Transcription Procedure:

[0090] a. Pretreat the RNA Sample: The RNA sample used in this example is from the total RNA extracted above. Remove genomic DNA from the RNA: Add an appropriate amount of template RNA and 4 μL of 4×gDNA wiper Mix to a 200 μL EP tube, and make up the volume to 16 μL with buffer (RNase-free ddH 2 O). After gently mixing, incubate the mixture at 42 °C for 2 min.

[0091] b. Prepare the Enzyme Mixture: Add 4 μL of 5×HiScript II qRT SuperMix II to the above EP tube and mix gently again.

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

[0093] d. Terminate the Reaction: After the reaction is completed, store the sample in a -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 the probe for the target sequence, and avoid non-specific binding through tools such as BLAST.

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

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

[0098] d. Data collection and analysis: During the extension stage of the PCR cycle, the fluorescence signal is monitored in real-time. Using the standard curve, the fluorescence signal is correlated with the initial amount of the template to calculate the copy number of the target sequence in the sample. The analysis software generates the Ct value (cycle threshold) for comparing the relative abundances between samples.

[0099] Using glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as an internal reference control, all primer sequences are shown in Table 1.

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

[0101]

[0102]

[0103] The results are as Figure 4 shown. In the figure, * indicates 0.01 < p < 0.05, ** indicates 0.001 < p < 0.01. In HTR-8 / SVneo and JEG-3 cells, EHDPP treatment upregulated the expression of Tnf-α, Il-6, and Il-1β. Treatment with choline chloride showed a decreasing trend but did not significantly change their expression levels. However, supplementation with choline chloride under EHDPP stimulation significantly reduced the expression levels of inflammatory factors induced by EHDPP. This indicates that choline chloride alleviated the inflammation caused by EHDPP in vitro placental cells.

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

[0105] Example 6 Grouping and treatment of experimental animals in vivo

[0106] Five-week-old ICR female mice (purchased from Xuzhou Medical University) were selected. After one week of environmental adaptation, they were randomly divided into a control group, an EHDPP exposure group, a choline chloride supplementation group, and a choline chloride supplementation + EHDPP exposure group, with 6 mice in each group, as a parallel experiment. 10 mg / kg / day EHDPP was administered by gavage, and 25 mM choline chloride was dissolved in drinking water and administered by drinking for two weeks, and then they were caged with 8-week-old ICR male mice of the same age and continued to be treated until gestational day 17.5 (GD17.5). Figure 5 A).

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

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

[0109] Example 7 Fluorescent quantitative PCR detection of gene expression levels in placental tissue

[0110] Total RNA of the placenta obtained in Example 6 was extracted using ice-cold Trizol reagent. Reverse transcription was performed on the mRNA level, and then real-time fluorescent quantitative PCR was carried out using SYBR Green fluorescent quantitative PCR master mix 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 operations of total RNA extraction, reverse transcription, and real-time fluorescent quantitative PCR described above were the same as those in Example 5; glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was also used as an internal reference control, and the primer sequences are listed in Table 1.

[0111] The results were consistent with the cell trend, asFigure 6 As shown, EHDPP treatment upregulated the expression of inflammatory factors Tnf-α, Il-6, and Il-1β. Choline chloride treatment did not change their expression levels, but supplementation with choline chloride significantly alleviated the induction of inflammatory factors by EHDPP. This indicates that choline chloride indeed alleviated the placental inflammation caused by EHDPP.

[0112] In addition, EHDPP inhibited the gene expression levels of the proliferation marker Ki67 and the vascular differentiation marker Cd31. Treatment with choline chloride alone did not change the expression of Ki67 and Cd31 in the placenta, but choline chloride supplementation attenuated the inhibition of Ki67 and Cd31 expression by EHDPP. The results again showed that choline chloride could improve the damage caused by EHDPP to the placenta.

[0113] More and more studies have shown the accumulation of EHDPP in the human body and its harm to human health. This application demonstrated the damage of EHDPP to the placenta and proposed using choline chloride supplementation to alleviate the placental damage caused by EHDPP. This strategy is novel and has not been reported. On the one hand, the human body can obtain choline from food and can also synthesize choline endogenously. This application proposed additional choline supplementation for preventing and treating placental damage caused by EHDPP, and this invention is also expected to be extended to placental damage caused by other inducing factors.

[0114] The above description of the embodiments is for enabling those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. The use of choline in the preparation of a drug for treating placental damage induced by flame retardant EHDPP, characterized in that: The choline can alleviate the inhibition of cell viability, migration inhibition and inflammatory response caused by EHDPP, and improve the placental damage caused by EHDPP.

2. The use of choline according to claim 1 in the preparation of a drug for treating placental damage induced by flame retardant EHDPP, characterized in that: The choline includes choline and its derivatives, and the derivatives include at least any one of choline chloride, choline hydroxide, and choline phosphate.

3. The use of choline according to claim 1 in the preparation of a drug for treating placental damage induced by flame retardant EHDPP, characterized in that: The cell is any one of a chorionic trophoblast cell, a choriocarcinoma cell, an amniotic cell, and a basal decidual cell.

4. The use of choline according to claim 1 in the preparation of a drug for treating placental damage induced by flame retardant EHDPP, characterized in that: The placental damage included a decrease in uterine size, fetal weight, placental weight, and placental diameter, as well as an inflammatory response.

5. The use of choline according to claim 4 in the preparation of a drug for treating placental damage induced by flame retardant EHDPP, characterized in that: The inflammatory response includes: increased gene expression levels of tumor necrosis factor-α, interleukin-6 and interleukin-1β in cells, and decreased gene expression of proliferation marker Ki67 and vascular differentiation marker Cd31.

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

7. The use of choline according to claim 6 in the preparation of a drug for treating placental damage induced by 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 inflammatory response inhibitor.

8. The use of choline in the preparation of a drug for treating placental damage induced by flame retardant EHDPP according to claim 6, characterized in that: The pharmaceutical preparation is in the form of any one of an injection, an oral preparation, and an external preparation.

9. The use of choline in the preparation of a drug for treating placental damage induced by flame retardant EHDPP according to claim 8, characterized in that: The oral preparation includes any one of tablets, capsules, granules, oral solutions or oral suspensions.

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

Images