Application of alpha-ketoglutaric acid substances in preparation of medicine for treating fetal intrauterine growth limitation

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of an alpha-ketoglutaric acid substance in preparation of a medicine for treating fetal intrauterine growth limitation, and the alpha-ketoglutaric acid substance is alpha-ketoglutaric acid or an alpha-ketoglutaric acid derivative. According to the application disclosed by the invention, by adding alpha-KG into arsenic-induced mouse and human trophoblastic cells, PDGFb hydroxymethylation down-regulation and placental angiogenesis ability disorder caused by arsenic induction can be improved; furthermore, by supplementing alpha-KG, placenta dysplasia and intrauterine growth limitation of fetuses can be prevented. The invention provides sufficient in-vivo and in-vitro evidences which prove that the alpha-KG can be used as an intervention target for treating the harmful influence of the placenta angiogenesis disorder induced by arsenic exposure. The invention shows that arsenic exposure in the gestation period destroys placenta angiogenesis by inhibiting Pdgfb hydroxymethylation, and alpha-KG can be used as an intervention target for relieving the harmful influence of arsenic-induced fetal intrauterine growth limitation in the gestation period.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of an alpha-ketoglutaric acid substance in the preparation of a drug for treating intrauterine growth restriction of a fetus. Background Art

[0002] Placental maldevelopment can lead to intrauterine growth restriction and preeclampsia, and insufficient trophoblast invasion and impaired vascular remodeling are the main causes of placental maldevelopment. Early in vitro experiments found that arsenic inhibited the migration and invasion of human trophoblast cells. Recent studies have reported that blocked invasion of placental trophoblast cells is an important cause of intrauterine growth restriction induced by arsenic exposure during pregnancy. The placental labyrinth is composed of maternal sinusoids and villi. The villi are composed of trophoblasts and the fetal blood vessels they surround, as well as the mesenchymal tissue outside the fetal blood vessels. It is the place where nutrients are exchanged between mother and fetus, and plays an important role in maintaining maternal health and promoting fetal development.

[0003] Existing technologies show that succinic acid promotes the formation of vascular-like features in human umbilical vein endothelial cells and fetal placental artery endothelial cells, but the effect of succinic acid supplementation during pregnancy on placental angiogenesis was not observed in whole animal experiments, and there is still a lack of strong evidence to prove that succinic acid supplementation promotes placental vascular development. Galectin 1 is involved in the establishment of maternal-fetal immune tolerance and placental angiogenesis, but there are still many unknowns about the signaling pathways and expression regulation mechanisms related to the function of galectin 1. Glutathione peroxidase 4, referred to as GPX4, is an important protein for cells to clear lipid peroxides and is involved in regulating multiple signaling pathways; GPX4 supplementation during pregnancy in mice promotes placental vascular development, but the toxic and side effects it produces are far from being clarified. In addition, Bushen Antai Recipe can improve the immune microenvironment and maternal-fetal vascularization of aborted mice, but there is a lack of exploration of the specific mechanism of action of Bushen Antai Recipe and population research.

[0004] In summary, there is an urgent need to provide a new preparation for treating intrauterine growth restriction of the fetus. Summary of the invention

[0005] In order to solve the placental angiogenesis disorder, the present invention provides an application of an α-ketoglutaric acid substance in the preparation of a drug for treating fetal intrauterine growth restriction.

[0006] The technical solution adopted by the present invention is: the present invention provides an application of an α-ketoglutaric acid substance in the preparation of a drug for treating intrauterine growth restriction of a fetus, wherein the α-ketoglutaric acid substance is α-ketoglutaric acid or an α-ketoglutaric acid derivative.

[0007] Preferably, the α-ketoglutaric acid derivative is one or more of α-ketoglutaric acid dimethyl ester, γ-aminobutyric acid, 5-aminolevulinic acid, and 4-hydroxyisoleucine.

[0008] Preferably, the drug has α-ketoglutaric acid substances as active ingredients and is prepared with pharmaceutically acceptable excipients.

[0009] Preferably, the pharmaceutically acceptable excipient is one or more of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative and a stabilizer.

[0010] Preferably, the pharmaceutically acceptable dosage form includes tablets, capsules, granules, injections, pills, powders, pastes or oral liquids.

[0011] Preferably, the intrauterine growth restriction includes insufficient trophoblast cell invasion, placental angiogenesis disorder or placental dysfunction.

[0012] Preferably, the drug is used to reverse angiogenesis disorders or angiogenesis disorders induced by arsenic substances.

[0013] Preferably, the arsenic substance includes any one or more of arsenic trioxide, arsenic pentoxide and sodium arsenite.

[0014] Preferably, the blood vessels comprise mouse placental blood vessels or human placental blood vessels.

[0015] Preferably, the drug is used to increase fetal weight and crown-rump length.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides an application of α-ketoglutaric acid substances in the preparation of drugs for treating intrauterine growth restriction of the fetus. In the present invention, adding α-KG to arsenic-induced mouse and human trophoblast cells can improve arsenic-induced downregulation of PDGFb hydroxymethylation and placental angiogenesis disorder; further, supplementing α-KG can prevent arsenic-induced placental dysplasia and intrauterine growth restriction of the fetus.

[0018] The present invention provides sufficient in vivo and in vitro evidence that α-KG can be used as an intervention target for treating the harmful effects of arsenic exposure-induced placental angiogenesis disorders. The present invention shows that exposure to arsenic can impair angiogenesis capacity. In addition, the effects of reduced TET activity and α-KG content mediate the hypohydroxymethylation of Pdgfb during placental angiogenesis disorders and intrauterine growth restriction induced by arsenic exposure during pregnancy. These new findings indicate that arsenic exposure during pregnancy impairs placental angiogenesis by inhibiting Pdgfb hydroxymethylation. In addition, α-KG can also be used as an intervention target to alleviate the harmful effects of intrauterine growth restriction induced by arsenic exposure during pregnancy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1Effects of sodium arsenite exposure on the placenta of pregnant mice, A-C: The whole picture of the placenta, the largest cross-section of the placenta and the representative H&E pictures of the labyrinth layer of the control group; D-F: The whole picture of the placenta, the largest cross-section of the placenta and the representative H&E pictures of the labyrinth layer of the arsenic exposure group; G: The percentage of the labyrinth layer area to the total placental area; H: The ratio of the labyrinth layer to the trophoblast layer thickness; I: The percentage of blood cell area; J: A representative picture of CD34-positive cells in the placental blood vessels of the control group; K: An enlarged picture of the corresponding local position of the box shown in Figure J; L: Arsenic exposure Representative images of CD34-positive cells in placental blood vessels of the control group; M: Enlarged image of the corresponding local position of the box shown in figure L; N: Number of blood vessels labeled with CD34; O: Diameter of blood vessels labeled with CD34; P: Immunoblotting to detect the expression of CD34 protein in the placenta; Q: Statistics of the expression of CD34 protein in the placenta detected by immunoblotting; R: Representative images of the placental labyrinth layer of the control group labeled with immunofluorescence; S: Representative images of the placental labyrinth layer of the arsenic exposure group labeled with immunofluorescence; T: Number of blood vessels labeled with CD31; U: Diameter of blood vessels labeled with CD31.

[0020] Figure 2 Effects of human chorionic trophoblast cells treated with different concentrations of sodium arsenite on angiogenesis. A~D: Representative images of tubule formation experiments under different concentrations of NaAsO2; E: Total length of tubules corresponding to A~D; F: Number of tubule branch points corresponding to A~D; G~J: Representative images of cell migration experiments under different concentrations of sodium arsenite; K: Number of migrated cells corresponding to G~J.

[0021] Figure 3 The effect of sodium arsenite treatment on angiogenesis of human chorionic trophoblast cells of different generations, A~D: representative images of tubule formation experiment of human chorionic trophoblast cells of different generations; E: total length of tubules corresponding to A~D; F: number of tubule branch points corresponding to A~D; G~J: representative images of cell migration experiment of human chorionic trophoblast cells of different generations; K: number of migrated cells corresponding to G~J.

[0022] Figure 4 The transcriptome sequencing results, A: the number of up-regulated and down-regulated genes; B: KEGG analysis shows the top 20 pathways; C: GSEA analysis shows the differentially expressed genes in the spot adhesion pathway.

[0023] Figure 5To verify the genes involved in the spot adhesion pathway, A: RT-PCR analysis of the relative mRNA expression of genes involved in the spot adhesion pathway; B: Comparison of the change folds of genes involved in the spot adhesion pathway between RNA-seq and RT-PCR; C: Immunoblotting analysis of PDGFb protein expression in HTR8 / SVneo cells; D~I: Representative images of PDGFb-positive cells detected by immunofluorescence.

[0024] Figure 6 Effects of arsenic exposure on PDGFB expression in mouse and human placental trophoblast cells. A and B: PDGFb-positive cells in the placental trophoblast of the control group and the arsenic exposure group were detected by immunohistochemistry at 15 days of gestation; C and D: PDGFb-positive cells in the placental trophoblast of the control group and the arsenic exposure group were detected by immunohistochemistry at 18 days of gestation; E and F: Local magnifications of the areas corresponding to the boxes shown in A and B; G and H: Local magnifications of the areas corresponding to the boxes shown in C and D; I: Number of PDGFb-positive cells; J: Representative picture of PDGFb protein expression in the placenta at 15 days of gestation detected by immunoblotting; K: Quantitative graph of PDGFb gray value.

[0025] Figure 7 To knock down PDGFb, we observed the role of PDGFb in arsenic exposure-induced tubule formation disorder in human placental trophoblast cells. A-D: representative images of tubule formation experiments in different treatment groups; E-H: representative images of cell migration experiments in different treatment groups; I: the number of migrating cells corresponding to E-H; J-K: the total length of tubules and the number of tubule branches corresponding to E-H; a-l: representative images of cell migration detected by wound healing method at different treatment groups at different times; M: e-l scratch area.

[0026] Figure 8 To supplement human PDGFb recombinant protein to observe the role of PDGFb in arsenic exposure-induced tubule formation disorder of human placental trophoblast cells, A-D: representative pictures of tubule formation experiments in different treatment groups; E-H: representative pictures of cell migration experiments in different treatment groups; I: the number of cell migration corresponding to E-H; J-K: the total length of tubules and the number of tubule branches corresponding to E-H; a-l: representative pictures of cell migration detected by wound healing method at different times in different treatment groups; M: the scratch area corresponding to e-l.

[0027] Fig. 9The effect of arsenic treatment on the methylation and hydroxymethylation levels of Pdgfb promoter in human placental trophoblast cells, A: Schematic diagram of methylated fragments of PDGFb promoter in the human genome; B: Methylation value of CpG fragments; C: Volcano plot showing methylation of each CpG site; D: Schematic diagram of hydroxymethylated fragments of PDGFb promoter in the human genome; E: Hydroxymethylation value of CpG fragments; F: Volcano plot showing hydroxymethylation of each CpG site; G: mRNA expression of Dnmt1, Dnmt3a and Dnmt3b. H: mRNA expression of Tet1, Tet2 and Tet3; I: TET activity; J: Intracellular α-KG content.

[0028] Fig.10 Effects of arsenic exposure during pregnancy on methylation and hydroxymethylation levels of Pdgfb promoter in mouse placenta. A: Schematic diagram of methylated fragments of PDGFb promoter in mouse genome. B: Methylation value of CpG fragments. C: Volcano plot showing methylation of each CpG site. D: Schematic diagram of hydroxymethylated fragments of PDGFb promoter in mouse genome. E: Hydroxymethylation of CpG fragments. F: Volcano plot showing hydroxymethylation of each CpG site. G: mRNA changes of Dnmt1, Dnmt3a and Dnmt3b. H: mRNA changes of Tet1, Tet2 and Tet3. I: TET activity. J: α-KG content in placenta.

[0029] Fig.11 To supplement the effects of Dm-αKG on TET activity, Pdgfb methylation and hydroxymethylation in human placental trophoblast cells, A: intracellular α-KG content. B: TET activity. C: methylation value of CpG fragments; D: hydroxymethylation value of CpG fragments.

[0030] Fig.12 To supplement the effect of Dm-αKG on Pdgfb expression and tubule formation in human placental trophoblast cells, A: Relative expression of Pdgfb mRNA. B: PDGFb protein level was analyzed by immunoblotting; C: PDGFb protein detection results by immunoblotting; D-G: Representative images of tubule experiments in different treatment groups. H: Total length of tubules corresponding to D-H. I: Number of tubule branch points corresponding to D-H.

[0031] Fig.13 To supplement the effect of Dm-αKG on the migration of human placental trophoblast cells, A-D: representative images of cell migration experiments in different treatment groups; E: the number of cell migration corresponding to A-D; F-Q: representative images of cell migration detected by wound healing method in different treatment groups at different treatment times; R: scratch area corresponding to J-Q.

[0032] Fig.14To supplement the effects of α-KG on methylation, hydroxymethylation and expression of Pdgfb in mouse placenta, A: methylation value of CpG fragment; B: hydroxymethylation value of CpG fragment; C and E: immunoblotting to detect placental PDGFb protein level; D: ELISA to detect placental PDGFb protein.

[0033] Fig.15 To supplement the effect of α-KG on mouse placental morphology, A-D: representative images of the largest cross-section of the placenta; E: the percentage of the labyrinth area corresponding to A-D to the total placental area; F: the ratio of the labyrinth thickness to the trophoblast thickness corresponding to A-D; G-J: representative images of the labyrinth; K: the blood cell area corresponding to G-J.

[0034] Fig.16 Effects of α-KG supplementation on placental blood vessels and fetal development in mice, A-D: Representative images of blood vessels in different groups labeled with CD34 in the labyrinth; E-H: Local magnified images of the areas corresponding to the boxes shown in A-D; I: The number of blood vessels labeled with CD34 corresponding to A-D; J: The diameter of blood vessels labeled with CD34 corresponding to A-D; K: Immunoblotting to detect the levels of CD34 and VEGF proteins in the placenta; L: Body weight of live fetuses; M: Crown-rump length of live fetuses; N: Correlation analysis between body weight of live fetuses and placenta weight. DETAILED DESCRIPTION

[0035] The present invention is further described below by specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0036] The inventive concept of the present invention is as follows:

[0037] Placental maldevelopment can lead to intrauterine growth restriction and preeclampsia, and impaired vascular remodeling is the main cause of placental maldevelopment. Existing technologies show that current drugs that have the potential to promote placental angiogenesis, such as succinic acid, galectin, GPX4, etc., have the disadvantages of lack of animal experimental evidence and toxic and side effects.

[0038] Based on this, the present invention proposes an application of α-ketoglutarate in the preparation of drugs for treating intrauterine growth restriction of the fetus. α-ketoglutarate, also known as alpha-ketoglutarate or α-KG for short, is an important intermediate product of the tricarboxylic acid cycle. It is produced by decarboxylation of isocitrate under the action of isocitrate dehydrogenase, and further oxidatively decarboxylated to succinyl coenzyme A and NADHH under the action of α-ketoglutarate dehydrogenase. +and CO2. α-KG has multiple biological functions. First, α-KG participates in the oxidation of fatty acids, amino acids and glucose, and is an important source of cellular adenine nucleoside triphosphate; second, α-KG, as a substrate of glutamate dehydrogenase, participates in the biosynthesis of amino acids such as glutamine, proline, arginine and lysine; third, α-KG is a key node in the cell connecting the carbon-nitrogen cycle and participates in regulating the utilization of carbon and nitrogen in the cell; fourth, α-KG has anti-inflammatory and antioxidant effects.

[0039] In order to enable those skilled in the art to better understand the technical solution of the present invention and implement it, the present invention is further described below in conjunction with specific embodiments and drawings. In the description of the present invention, unless otherwise specified, the reagents used are all commercially available, and the methods used are all conventional techniques in the art.

[0040] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0041] 1. Experimental Reagents

[0042]

[0043]

[0044] Note: “ / ” in the table means there is no such item.

[0045] 2. Experimental Methods

[0046] 2.1 Animal experiment design is as follows:

[0047] The experimental animals were 8-week-old female and male CD-1 mice purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. The mice were housed in a specific environment with a temperature of 25°C, a humidity of 55%, and a 12-hour light / 12-hour dark cycle. All mice had free access to food and water. In order to obtain pregnant mice, female and male mice were co-housed in a ratio of 2:1. The presence of vaginal plugs in female mice was considered to be pregnancy day 0, i.e., GD0.

[0048] The animal experiment of the present invention includes two independent contents, namely:

[0049] Experiment 1: To study the effects of arsenic exposure during pregnancy on placental and fetal development, 24 pregnant mice were randomly divided into a control group and an arsenic exposure group, 12 mice per group. The control group drank ultrapure water, and the arsenic exposure group drank ultrapure water containing 15 mg / L NaAsO2 throughout pregnancy.

[0050] Experiment 2: To explore the protective effect of α-KG supplementation during pregnancy on placental and fetal developmental disorders induced by arsenic exposure, 48 pregnant mice were randomly divided into a control group, an α-KG group, an arsenic exposure group, and an α-KG+arsenic exposure group. In the arsenic exposure group and the α-KG+arsenic exposure group, the pregnant mice drank ultrapure water containing 15 mg / L NaAsO2 throughout pregnancy; in the α-KG and α-KG+arsenic exposure groups, the pregnant mice were given 2 g / kg α-KG by gavage throughout pregnancy.

[0051] The pregnant mice were anesthetized and killed, and the placenta was collected to record the weight and crown-rump length of the live fetus. The placenta was used for hematoxylin-eosin staining, immunohistochemistry, immunoblotting and ELISA to detect the content of PDGFb.

[0052] 2.2 Cell culture, as follows:

[0053] HTR8 / SVneo cells are an extravillous trophoblast cell line of early pregnancy. The culture method is to add 5% fetal bovine serum and 1% 100U / mL penicillin / streptomycin to RPM1640 basal medium, maintain at 37°C, 5% carbon dioxide, and grow in an environment with humidity of 95%.

[0054] The cell experiment of the present invention includes three independent contents, namely:

[0055] Experiment 1: To investigate the effect of different concentrations of NaAsO2 treatment on the tubule formation ability of HTR8 / SVneo cells, the cells were treated continuously with 0.02μM, 0.2μM and 2μM NaAsO2 to the 6th generation.

[0056] Experiment 2: To investigate the effect of 2μM NaAsO2 treatment on tubule formation ability of HTR8 / SVneo cells at different generations, the cells were treated with 2μM NaAsO2 to the 2nd, 4th and 6th generations.

[0057] Experiment 3: To observe the protective effect of Dm-αKG supplementation on the tubule formation ability of HTR8 / SVneo cells impaired by arsenic treatment, HTR8 / SVneo cells were co-incubated with 2μM NaAsO2 and 0.5mM Dm-αKG to the 6th generation.

[0058] 2.3 Cell viability assay, as follows:

[0059] Cell viability was detected using the cell counting reagent CCK8 purchased from Biosharp. HTR8 / SVneo cells were seeded into 96-well plates at a density of 4000 cells per well in a volume of 200 μL.

[0060] HTR8 / SVneo cells were treated with different concentrations of Dm-αKG and 2μM NaAsO2 for 24 hours. The concentrations of Dm-αKG were 0.25mM, 0.5mM, 1mM, 2mM, 4mM, and 8mM, respectively. Then, the CCK8 kit was used to detect cell viability.

[0061] HTR8 / SVneo cells were treated with different concentrations of human recombinant PDGFb protein and 2μM NaAsO2 for 24 hours. The concentrations of human recombinant PDGFb protein were 2.5ng / L, 5ng / L, 10ng / L, 20ng / L, 40ng / L, and 80ng / L, respectively. Then, the CCK8 kit was used to detect cell viability.

[0062] 2.4 Cell migration assay, as follows:

[0063] Wound healing assay was used to evaluate cell migration ability. After HTR8 / SVneo cells were exposed to 2μM NaAsO2 for the fifth generation, they were seeded into 6-well plates at a density of 400,000 cells per well in a volume of 500μL and grown for 20 hours. Then, the cells were treated with 2μM NaAsO2 until they were confluent, and then the wound healing assay was performed. Five horizontal lines were evenly drawn perpendicular to the bottom of the 6-well plate, and the cells were gently washed 3 times with PBS to remove the scratched cell debris. The cells were immediately photographed with an inverted microscope and the photographing position was recorded. The cells were then placed in a cell culture incubator and photographed at the same position every 12 hours. Finally, the width of the scratches at different time points was analyzed.

[0064] Transwell migration assay was used to evaluate cell migration ability. First, 250,000 cells were seeded into the upper chamber at a density of 200 μL per well and grown at 37°C, 5% carbon dioxide, and 95% humidity for 20 hours. Then, 200 μL of complete medium containing 2 μM NaAsO2 was added to the upper chamber, and 600 μL of complete medium containing 2 μM NaAsO2 was added to the lower chamber. Subsequently, the cells were incubated at 37°C, 5% carbon dioxide, and 95% humidity for 24 hours. Finally, crystal violet staining was used, and the cells were photographed and counted under a microscope.

[0065] 2.5 Tube formation experiment, as follows:

[0066] The tubule formation assay was used to evaluate the tubule formation ability of HTR8 / SVneo cells. First, 100 μL of matrix gel (Cat. No.: E1270; purchased from Sigma) was added to the bottom of a precooled 48-well plate and incubated at 37°C for 3 h. The cells were seeded into a 48-well plate at a density of 80,000 cells per well in a volume of 400 μL and incubated at 37°C, 5% carbon dioxide, and 95% humidity for 15 h. The 48-well plate was directly observed under a microscope. The total length and number of branch points of the vascular network were analyzed using Angiotool software.

[0067] 2.6 RNA sequencing, as follows:

[0068] Total RNA from HTR8 / SVneo cells was extracted using TRIzol reagent. The quality of total RNA should meet the following conditions: 28s: 18s = 1.8-2.0; RNA integrity value is more than 8. Oligo dT magnetic beads were used to enrich and purify mRNA, and then random hexamer primers and reverse transcriptase were used to synthesize double-stranded cDNA with mRNA as a template. The double-stranded DNA was end-repaired, and the 3' end of the purified and repaired cDNA was added with 'A'. The two ends of the DNA fragment were connected using specific sequencing adapters, and a high-fidelity polymerase was used to amplify and construct a successful sequencing library. Finally, the library was sequenced on the Illumina Hiseq 2000 platform in 2×150bp paired-end sequencing mode to obtain FastQ data. After sequencing, the data were processed using STAR to generate read alignments with the human reference genome hg38. DEseq2 was used for differential gene expression analysis. In this study, genes with fold change>2 and P<0.05 were selected as differential genes.

[0069] 2.7 Real-time fluorescence quantitative PCR, as follows:

[0070] 80 mg of mouse placenta and HTR8 / SVneo cells, each containing 1 million cells, were used to extract RNA using Trizol reagent, 1-bromo-3-chloropropane reagent, and isopropanol. The RNA precipitate was washed with 75% ethanol. RNA was reverse transcribed into cDNA using the Promega Reverse Transcription System Kit. Real-time fluorescence quantitative PCR was performed using the LightCycler 480 SYBR Green I Kit.

[0071] 2.8 Immunoblotting, as follows:

[0072] 30 mg of mouse placenta or 1 million HTR8 / SVneo cells were lysed with RIPA buffer to extract total protein. The total protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then the protein was transferred to a 0.45 μm PVDF membrane. The PVDF membrane was incubated with 5% skim milk for 1 hour, and then incubated with PDGFb antibody for 2.5 hours and β-actin for 1 hour. After washing with PBS containing 0.05% Tween-20, the corresponding secondary antibody was selected according to the source of the primary antibody and incubated at room temperature for 90 minutes; finally, ECL luminescent solution A and solution B were prepared in a volume ratio of 1:1, and the membrane was exposed in an automatic chemiluminescence analysis system. The grayscale value of the target protein and the internal reference protein was analyzed by ImageJ software, and the relative quantification of PDGFb was calculated.

[0073] 2.9 Immunofluorescence, as follows:

[0074] HTR8 / SVneo cells were seeded into 24-well plates at a density of 40,000 cells per well, 800 μL, and treated with 2 μM NaAsO2 for 24 hours. After washing the cells twice with PBS, they were fixed with methanol for 5 minutes, perforated with 0.1% Triton-X-100 for 10 minutes, and blocked with 1% bovine serum albumin for 30 minutes. Then, the cells were incubated with PDGFb antibody at 4°C for 16 hours, incubated with goat anti-rabbit IgG at room temperature for 40 minutes, and Hoechst was used to stain the nucleus for 10 minutes. Finally, the cells were washed 4 times with PBS, and the expression of PDGFb was observed under a fluorescence microscope.

[0075] 2.10 Plasmid transfection, as follows:

[0076] Use of Lipofectamine TM PDGFb shRNA plasmid was transfected into HTR8 / SVneo cells using Lipofectamine 3000 transfection reagent. Transfection was started when the cells grew to 80% confluency. TM 3000 reagents were added to serum-free medium, mixed with plasmids, and allowed to stand for 20 minutes. The mixture was added to cells and cultured for 6 hours at 37°C and 5% carbon dioxide. The medium containing the mixture was then removed and fresh medium was added.

[0077] 2.11 DNA methylation detection, as follows:

[0078] Using Methyltarget TMTechnical analysis of methylation levels of Pdgfb CpG sites. Whole genomic DNA of mouse placenta and HTR8 / SVneo cells was extracted using the Tiangen kit. DNA quality should meet the following conditions: DNA concentration exceeded 20 ng / μL; OD value: 260 / 280 = 1.7-1.9, 260 / 230 ≥ 2.0. Pdgfb CpG fragments should meet the following conditions: length ≥ 200 bp, observed / expected dinucleotide CpG ratio higher than 0.6, GC content ≥ 50%. Three CpG fragments were determined in mice and four CpG fragments were determined in HTR8 / SVneo cells. The methylation levels of CpG fragments were detected on the Illumina Hiseq 2000 platform using a 2×150 bp bidirectional sequencing mode. The methylation level of each CpG fragment is the total average methylation level of all CpG sites in each CpG fragment.

[0079] 2.12 DNA hydroxymethylation detection, as follows:

[0080] APOBEC-coupled epigenetic sequencing was used to analyze the hydroxymethylation level of Pdgfb CpG sites. Whole genome DNA extraction and identification of CpG fragments were described in the DNA methylation detection section. Hydroxylation levels were detected by determining two CpG fragments in mice and three CpG fragments in HTR8 / SVneo cells. Multiplex PCR was used to amplify the deaminated genomic DNA MIX as a template, and the reaction products were then treated with β-GT and APOBEC3A, respectively. The target fragments were enriched and the hydroxymethylation levels of the CpG fragments were detected on the Illumina Hiseq 2000 platform in a 2×150bp bidirectional sequencing mode. The hydroxymethylation level of each CpG fragment is the total average hydroxymethylation level of all CpG sites in each CpG fragment.

[0081] 2.13 TET activity detection, as follows:

[0082] Epizyme TM The 5mC Hydroxylase TET Activity / Inhibition Assay Kit measures TET enzyme activity in mouse placenta and HTR8 / SVneo cells. First, NE-PER TM Nuclear extracts were isolated using the Nuclear and Cytoplasmic Extraction Kit. TM The 5mC Hydroxylase TET Activity / Inhibition Assay Kit measures TET enzyme activity in nuclear extracts. 10 μg of nuclear extracts bind to the substrate and convert the methylated substrate into a hydroxymethylated product. The absorbance of the hydroxymethylated product is measured at a wavelength of 450 nm using a microplate reader, and the absorbance of the hydroxymethylated product is proportional to the TET enzyme activity.

[0083] 2.14α-KG detection, as follows:

[0084] The content of α-KG in mouse placenta and HTR8 / SVneo cells was detected by ExionLC liquid chromatography coupled with 3500QTRAP mass spectrometer. 600 μL of methanol-acetonitrile-water mixture was added to 30 mg of mouse placenta or HTR8 / SVneo cells, each sample containing 1 million cells, and incubated on ice for 10 minutes. The samples were centrifuged at 16000 rpm for 10 minutes at 4 ° C, and the supernatant was injected into the LC-MS / MS system to analyze the content of α-KG.

[0085] 2.15 Enzyme-linked immunosorbent assay, as follows:

[0086] The PDGFb ELISA kit was used to detect the PDGFb content in the mouse placenta. 30 mg of placental tissue was added to 270 μL PBS for homogenization, centrifuged at 3500 g for 10 minutes at 4°C, and the supernatant was collected. The PDGFb protein content in the supernatant was detected according to the ELISA kit.

[0087] 2.16 Statistical analysis, as follows:

[0088] All data are expressed as mean ± standard error. Normally distributed data were analyzed using T test. Data between multiple groups were first analyzed by one-way analysis of variance, and then compared pairwise by LSD method. Spearman was used to analyze the correlation between the two groups of data. *P<0.05, **P<0.01 indicate that the difference is statistically significant. SPSS21.0 and GraphPad Prism8.4.3 software were used for data analysis. Image Pro Plus and AngioTool software were used for image analysis.

[0089] Example 1

[0090] The application of α-ketoglutaric acid substances in the preparation of drugs for treating intrauterine growth restriction of fetuses is as follows:

[0091] (1) Arsenic exposure impairs angiogenesis in mouse placental and human placental trophoblast cells.

[0092] The effects of arsenic exposure during pregnancy on placental morphology were evaluated. The placentas of arsenic-exposed mice showed thicker trophoblasts, reduced labyrinth area, and a significantly reduced ratio of the maximum cross-sectional thickness of the labyrinth to the trophoblast. Figure 1 As shown. The effects of arsenic exposure during pregnancy on placental blood vessels were further evaluated. The blood cell area in the placenta of arsenic-exposed mice was reduced. CD34 and CD31 are endothelial cell markers used to evaluate the development of placental blood vessels. In the placental labyrinth layer of arsenic-exposed mice, the diameter and number of blood vessels positively labeled by CD34 and CD31 were significantly reduced.

[0093] Subsequently, an in vitro model was established to evaluate the effects of arsenic treatment on tubule formation and migration in HTR8 / SVneo cells, and the results are shown in Figure 2 and Figure 3 . HTR8 / SVneo cells were exposed to different concentrations of NaAsO2 and cultured to the 6th generation. Tubule formation experiments were performed to evaluate the tubule formation ability of HTR8 / SVneo cells in the presence of NaAsO2. When cells were exposed to different concentrations of NaAsO2, obvious changes in capillary morphology were observed, and when cells were exposed to 2μM NaAsO2, the total length of the tubule network and the number of branch points decreased. Transwell migration showed that when cells were exposed to different concentrations of NaAsO2, the number of migrating cells decreased in a concentration-dependent manner. Further, HTR8 / SVneo cells were co-incubated with 2μM NaAsO2 to different generations, and the effects on cell tubule formation and cell migration ability were observed. When cells were exposed to 2μM NaAsO2 to different generations, obvious changes in capillary morphology were observed. When cells were exposed to 2μM NaAsO2 to the 4th or 6th generation, the total length of the tubule network and the number of tubule branch points decreased. Transwell migration assay showed that the number of migrating cells decreased when cells were exposed to 2μM NaAsO2 to the 6th generation. In general, human placental trophoblast cells exposed to 2μM NaAsO2 to the 6th generation significantly inhibited tubule formation and cell migration ability, so human placental trophoblast cells exposed to 2μM NaAsO2 to the 6th generation were selected for subsequent experiments.

[0094] (2) Arsenic exposure downregulates PDGFb expression in mouse placenta and human placental trophoblast cells.

[0095] In order to screen the key genes that inhibit placental angiogenesis by arsenic exposure, transcriptome sequencing was performed on HTR8 / SVneo cells. Figures 4 to 6As shown. In arsenic-exposed HTR8 / SVneo cells, a total of 445 differentially expressed genes were identified, of which 301 genes were downregulated and 144 genes were upregulated. KEGG analysis showed the top 20 signaling pathways, among which the spot adhesion pathway was closely related to angiogenesis. Gene set enrichment analysis GSEA analysis showed that the spot adhesion pathway was negatively enriched. The genes in the spot adhesion pathway were verified by real-time fluorescence quantitative PCR and RT-PCR. In arsenic-exposed HTR8 / SVneo cells, type VI collagen a 2 chain, i.e. Col6a2) mRNA expression was upregulated; platelet-derived growth factor, i.e. Pdgfb, integrin subunit 11, i.e. Itga11, Itga2b, protein kinase b, i.e. Prkcb and myosin light chain kinase family member, i.e. 4Mylk4 mRNAs were downregulated. In order to further screen candidate genes, the inventors compared the fold changes of candidate genes between RNA-seq and RT-PCR. The fold changes of Col6a2, Pdgfb, Itga11, and Prkcb mRNAs in RNA-seq and RT-PCR were all greater than 2-fold. Among them, Pdgfb is closely related to angiogenesis. Therefore, Pdgfb expression was further analyzed in HTR8 / SVneo cells. Arsenic exposure resulted in decreased PDGFb protein expression in HTR8 / SVneo cells. Immunofluorescence assay showed that arsenic-exposed HTR8 / SVneo cells had a decrease in PDGFB-positive cells. Next, PDGFb was detected in the placenta of arsenic-exposed mice. Arsenic exposure resulted in a decrease in PDGFb-positive cells in the trophoblast of the GD15 placenta. Immunoblotting experiments showed that arsenic exposure reduced PDGFb protein expression in the GD15 placenta of mice.

[0096] (3) The role of PDGFb in arsenic exposure-induced tubulogenesis disorders in human placental trophoblast cells.

[0097] To investigate the role of PDGFb downregulation in arsenic-induced tubule formation disorders. Figure 7-Figure 8As shown, PDGFb was knocked down using shRNA plasmid. PDGFb knockdown aggravated the destruction of tubule morphology induced by arsenic. The total length and number of branch points of the tubule network in the PDGFb knockdown + arsenic exposure group were lower than those in the arsenic exposure group alone. Transwell migration assay showed that the number of migrating cells in the PDGFb knockdown + arsenic exposure group was lower than that in the arsenic exposure group alone. Wound healing assay showed that the wound area in the PDGFb knockdown + arsenic exposure group was larger than that in the arsenic exposure group alone. To further confirm the inhibitory effect of PDGFb downregulation on As-induced angiogenesis, 10 ng / L human recombinant PDGFb protein was added to the cell culture medium. The addition of human recombinant PDGFb protein attenuated the damage of tubule morphology induced by arsenic exposure. In addition, the addition of human recombinant PDGFb protein restored the reduction of the total length and number of branch points of the tubule network by arsenic exposure. In addition, the addition of human recombinant PDGFb protein improved the inhibition of cell migration by arsenic exposure.

[0098] (4) Arsenic exposure downregulates PDGFb hydroxymethylation by inhibiting the activity of the demethylase TET.

[0099] For HTR8 / SVneo cells, the methylation level of Pdgfb promoter was analyzed. There are 4 methylation-modified fragments in HTR8 / SVneo cells, such as Fig. 9 and Fig.10 As shown. Among them, arsenic exposure led to an increase in the methylation level of the third fragment of Pdgfb. Compared with the control group, the methylation levels of 85 CpG sites in the arsenic exposure group did not change, the methylation levels of 5 CpG sites decreased, and the methylation levels of 4 CpG sites increased. The hydroxymethylation level of the Pdgfb promoter was further analyzed. The second, third, and fourth fragments in the Pdgfb promoter can undergo hydroxymethylation modification. Among them, arsenic exposure led to a decrease in the hydroxymethylation level of the second and third fragments. Compared with the control group, the hydroxymethylation levels of 47 CpG sites in the arsenic exposure group did not change, and the hydroxymethylation levels of 6 CpG sites decreased. In order to explore the mechanism by which arsenic exposure induces changes in methylation and hydroxymethylation patterns in HTR8 / SVneo cells, the inventors analyzed the expression of DNA methyltransferases, including Dnmt1, Dnmt3a, and Dnmt3b; and demethylases, including Tet1, Tet2, and Tet3. The relative expression levels of Dnmt1, Dnmt3a, and Dnmt3b mRNAs and Tet1, Tet2, and Tet3 mRNAs were not significantly different between the arsenic-exposed and control groups. However, arsenic exposure led to a decrease in TET enzyme activity and α-KG content in HTR8 / SVneo cells.

[0100] For mouse placenta, Pdgfb has three methylation-modified fragments. There was no significant difference in the methylation levels of the three fragments of Pdgfb between the control group and the arsenic exposure group. Compared with the control group, the methylation levels of 59 CpG sites in the placenta of mice in the arsenic exposure group did not change, the methylation levels of 3 CpG sites decreased, and the methylation level of 1 CpG site increased. The hydroxymethylation level of Pdgfb was further analyzed. Hydroxymethylation modifications of the first and second fragments in the Pdgfb promoter can be detected. Among them, arsenic exposure caused a decrease in the hydroxymethylation level of the second fragment of the Pdgfb promoter in the mouse placenta. Compared with the control group, the hydroxymethylation levels of 30 CpG sites in the placenta of mice in the arsenic exposure group did not change, the hydroxymethylation levels of 3 CpG sites decreased, and the hydroxymethylation levels of 2 CpG sites increased. Next, the expression levels of DNA methyltransferases and demethylases in the mouse placenta were analyzed. The relative expression of mRNAs for DNA methyltransferases, Dnmt1, Dnmt3a, and Dnmt3b, and demethylases, Tet1, Tet2, and Tet3, showed no significant difference between the arsenic-exposed and control groups. However, arsenic exposure led to a decrease in TET enzyme activity and α-KG content in the mouse placenta.

[0101] (5) In vitro supplementation of Dm-αKG improved arsenic exposure-induced downregulation of Pdgfb promoter hydroxymethylation and weakened tubule formation ability in human placental trophoblast cells.

[0102] Dimethyl α-ketoglutarate is a cell-permeable α-KG analog. Addition of 0.5 mM Dm-αKG to arsenic-exposed cells increased intracellular α-KG and TET activities, such as Figures 11 to 13 . Dm-α-KG supplementation improved the upregulation of methylation level of the third fragment of Pdgfb promoter induced by arsenic exposure. And Dm-α-KG supplementation restored the decreased hydroxymethylation level of the second and third fragments of Pdgfb promoter induced by arsenic exposure. Next, Dm-αKG supplementation reversed the downregulation of Pdgfb mRNA and protein induced by arsenic exposure. The effect of Dm-αKG supplementation on the weakening of tubule formation ability of human placental trophoblast cells induced by arsenic exposure was further analyzed. Dm-αKG supplementation improved the damage of capillary morphology by arsenic exposure. In addition, Dm-αKG supplementation reversed the reduction of total length and number of branch points of tubule network by arsenic exposure. In addition, compared with the group with simple arsenic exposure, the migration ability of HTR8 / SVneo cells supplemented with Dm-αKG was significantly increased.

[0103] (6) In vivo supplementation of α-KG improved placental Pdgfb hydroxymethylation and placental angiogenesis in mice impaired by arsenic exposure during pregnancy.

[0104] Supplementation of α-KG during pregnancy did not significantly change the methylation levels of the three fragments of the Pdgfb promoter. However, supplementation of α-KG during pregnancy reversed the hydroxymethylation level of the second fragment of the placental Pdgfb promoter that was downregulated by arsenic exposure, such as Figure 14 to Figure 16 . α-KG supplementation restored the down-regulation of PDGFb protein in the placenta caused by arsenic exposure. The effects of α-KG supplementation during pregnancy on arsenic exposure-induced placental vascular development disorders and fetal intrauterine growth restriction were further analyzed. The effects of α-KG supplementation on mouse placental morphology were observed. α-KG supplementation improved the reduction in placental labyrinth area and thickness induced by arsenic exposure. The effects of α-KG supplementation on placental blood vessels were further evaluated. Compared with the arsenic-exposed group alone, the area of ​​blood cells in the placental labyrinth of mice supplemented with α-KG was significantly increased, and the diameter and number of blood vessels marked by CD34 positive markers were significantly increased. Immunoblotting results showed that the expression of CD34 and VEGF proteins in the mouse placenta was significantly upregulated after α-KG supplementation. In addition, α-KG supplementation during pregnancy alleviated the decrease in live fetal weight and crown-rump length induced by arsenic exposure. Correlation analysis showed that placental weight was significantly positively correlated with fetal weight.

[0105] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0106] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. The use of α-ketoglutaric acid substances in the preparation of drugs for treating intrauterine growth restriction of fetuses, characterized in that: The α-ketoglutaric acid substance is α-ketoglutaric acid or an α-ketoglutaric acid derivative.

2. The use according to claim 1, characterized in that: The α-ketoglutaric acid derivative is one or more of α-ketoglutaric acid dimethyl ester, γ-aminobutyric acid, 5-aminolevulinic acid, and 4-hydroxyisoleucine.

3. The use according to claim 1, characterized in that: The medicine uses α-ketoglutaric acid substances as active ingredients and is prepared with pharmaceutically acceptable excipients.

4. The use according to claim 3, characterized in that: The pharmaceutically acceptable excipient is one or more of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative and a stabilizer.

5. The use according to claim 1, characterized in that: The pharmaceutically acceptable dosage forms include tablets, capsules, granules, injections, pills, powders, pastes or oral liquids.

6. The use according to claim 1, characterized in that: The intrauterine growth restriction includes insufficient trophoblast cell invasion, placental angiogenesis disorder or placental dysfunction.

7. The use according to claim 1, characterized in that: The drug is used for reversing angiogenesis disorder or angiogenesis disorder induced by arsenic substances.

8. The use according to claim 7, characterized in that: The arsenic substances include any one or more of arsenic trioxide, arsenic pentoxide and sodium arsenite.

9. The use according to claim 7, characterized in that: The blood vessels include mouse placental blood vessels or human placental blood vessels.

10. The use according to claim 1, characterized in that: The drug is used to increase fetal weight and crown-rump length.

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