HAMP gene as molecular marker of bad pregnancy outcome, therapeutic target and application of HAMP gene

Abstract

The invention discloses an HAMP gene as a molecular marker and a therapeutic target of an adverse pregnancy outcome and application of the HAMP gene, and relates to the technical field of biological medicines. It is found for the first time that placenta iron metabolism instability induced by overexpression of a molecular marker HAMP is an important reason causing damage to placenta angiogenesis. According to the invention, a scientific basis for how the abnormal high expression of HAMP in the placenta causes iron accumulation in placenta tissues and placenta angiogenesis damage by interfering normal metabolism of cell iron is determined from a molecular level, and a new treatment target and an intervention strategy are provided for improving placenta-related pathological conditions; a theoretical basis and a potential medicine research and development direction are provided for healthy development of fetuses.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to HAMP gene as a molecular marker of adverse pregnancy outcomes, a therapeutic target and application thereof. Background Art

[0002] The mother has special physiological characteristics during pregnancy, and these characteristics will continue to change as the pregnancy progresses. An abnormal pregnancy process will increase the risk of adverse pregnancy outcomes. In recent years, the occurrence of pregnancy syndrome induced by a high-fat, high-sugar diet or obesity and related adverse pregnancy outcomes has received widespread attention. The combined factors of increased secretion of proinflammatory factors, increased lipid peroxidation, oxidative stress, etc. caused by obesity or disease may induce significant changes in the maternal internal environment, and even cause abnormal pregnancy progress, ultimately inducing adverse pregnancy outcomes such as miscarriage, IUGR, macrosomia, and stillbirth. As a medium for communication between the mother and the fetus, the normal establishment and development of the placenta is closely related to the maternal body environment. The maternal homeostasis of glucose and lipid metabolism has a huge impact on the normal establishment of placental function. The placenta of obese mothers is in a lipotoxic environment, and the glucose and lipid metabolism, vascular function, inflammation level, and antioxidant capacity of the placenta are abnormal.

[0003] The development of molecular markers and new therapeutic targets for predicting adverse pregnancy can provide new intervention strategies for improving placental-related pathologies, and provide a theoretical basis and potential direction for drug development for the healthy development of the fetus. Summary of the invention

[0004] The purpose of the present invention is to provide HAMP gene as a molecular marker for adverse pregnancy outcomes, a therapeutic target and its application to solve the problems existing in the above-mentioned prior art. The present invention has found that the instability of placental iron metabolism induced by the overexpression of the molecular marker HAMP is an important cause of impaired placental angiogenesis, thereby providing a new therapeutic target and intervention strategy for improving placental-related pathological conditions, and providing a theoretical basis and potential drug development direction for the healthy development of the fetus.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a molecular marker for adverse pregnancy outcomes, wherein the molecular marker is a HAMP gene.

[0007] The present invention also provides the use of a reagent for detecting the expression amount of the above-mentioned molecular marker in preparing a kit for predicting adverse pregnancy outcomes.

[0008] Furthermore, the reagent includes a primer pair for detecting the molecular marker.

[0009] Furthermore, the primer pair includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.2.

[0010] The present invention also provides a kit for predicting adverse pregnancy outcomes, comprising a reagent for detecting the expression amount of the above-mentioned molecular markers.

[0011] The present invention also provides the use of a reagent for inhibiting HAMP gene expression in preparing a medicine for improving adverse pregnancy outcomes.

[0012] Furthermore, the reagent includes siRNA; the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.3, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO.4.

[0013] The present invention also provides a medicine for improving adverse pregnancy outcomes, wherein the active ingredients include an agent for inhibiting HAMP gene expression.

[0014] Furthermore, the active ingredient of the medicine also includes DHA.

[0015] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0016] The present invention discloses the following technical effects:

[0017] (1) The present invention first discovered that the instability of placental iron metabolism induced by the overexpression of the molecular marker HAMP is an important cause of impaired placental angiogenesis. The present invention determined at the molecular level the scientific basis for how abnormally high expression of HAMP in the placenta interferes with the normal metabolism of cellular iron, leading to iron accumulation in placental tissue and impaired placental angiogenesis, providing new therapeutic targets and intervention strategies for improving placental-related pathological conditions, and providing a theoretical basis and potential drug development direction for the healthy development of the fetus.

[0018] (2) The present invention, through the establishment of in vivo and in vitro tests, reveals that the adverse pregnancy outcomes induced by a high-fat and high-sugar diet are closely related to the imbalance of iron metabolism and impaired angiogenesis in the placenta; in addition, it is verified that the iron metabolism imbalance can be alleviated by diet or exogenous intervention with DHA, thereby regulating placental angiogenesis and ultimately improving the adverse pregnancy outcomes, and its regulatory mechanism is related to the expression of the secretory protein HAMP. Compared with the previous research methods that only started from a single model, the present invention combines the two methods of in vivo and in vitro, directly clarifying the damaging effect of iron metabolism imbalance on the placenta, and improving the accuracy and credibility of the results.

[0019] (3) The present invention has important guiding significance for the preparation of products for preventing or improving adverse pregnancy outcomes, and is expected to provide an important theoretical basis and technical support for the development of nutritional strategies or products for preventing or treating adverse pregnancy outcomes induced by a high-fat, high-sugar diet or obesity during pregnancy. It overcomes the deficiencies and shortcomings of traditional detection technologies, has the advantages of greater safety, better effects, and higher specificity, and provides inspiration and possibilities for subsequent research on the preparation of products for preventing and treating adverse pregnancy outcomes induced by a high-fat, high-sugar diet or obesity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The effects of high-fat and high-sugar diet and DHA dietary supplementation during pregnancy on pregnancy outcomes and placental development in maternal mice; a is a result chart recording the proportion of fetal types in the three groups of pregnant mice on the 18.5th day of pregnancy, and fetuses with a weight 2 standard deviations higher or lower than the average of the control group were considered to be macrosomia or IUGR (n=8); e is the morphology of HE staining and Oil Red O staining of the placenta of pregnant mice, the red dotted line part is the maze area, the blue dotted line part is the connection area, and the HE staining was magnified 63 times to observe the morphology of the blood sinusoids; b is a statistical chart of the proportion of the maze area of ​​the placenta of pregnant mice to the total area of ​​the placenta; c is a statistical chart of the proportion of the blood sinusoid area; d is a statistical chart of the proportion of the Oil Red O staining area (n=8); f is a Western blotting of the protein expression levels of CD31, Akt, and p-Akt in the placenta of pregnant mice (n=6) Blot test results and statistical graphs; g is a statistical graph of ferrous ion content in placental tissue; h is a statistical graph of MDA level in placental tissue; i is a statistical graph of GSH content in placental tissue; j is a statistical graph of GSSG content in placental tissue; k is the ratio of GSH to GSSG content (n=6); in ad and gk, C on the horizontal axis represents the control group; H represents the high-fat and high-sugar diet group during pregnancy; HD represents the high-fat and high-sugar diet group supplemented with 2% DHA during pregnancy; the results are expressed as mean ± standard error; *P<0.05, **P<0.01, ***P<0.001;

[0022] Figure 2Figure 2 shows the effects of high palmitic acid and high sugar treatment and DHA intervention on the tube-forming function and iron metabolism of human umbilical vein endothelial cells (HUVECs); AC is the statistical graph of ferrous ion level, ROS level and MDA content of HUVECs under DHA intervention high palmitic acid and high sugar treatment; D is the GSH and GSSG content and the ratio of the two in HUVECs under DHA intervention high palmitic acid and high sugar treatment; E and G are the scratch experiments after adding Ferrostatin-1 (ferroptosis inhibitor) to the cells, respectively. F and H are the results of the scratch test and the statistical graphs of viability, migration and tube formation ability after the cells were added with Erastin (ferroptosis activator), respectively; n=3-4; CON: control group; PG: high palmitic acid and high sugar treatment group; PGD: high palmitic acid and high sugar + DHA treatment group; the results are expressed as mean ± standard error; different lowercase letters in the bar graph indicate significant differences (P<0.05), the same or no letters indicate no significant differences (P>0.05);

[0023] Figure 3 Figure 1 is the analysis result of important core genes affecting placental development; a is the gene GSEA enrichment analysis diagram of the two comparison groups H vs C and HD vs H; b is the GSEA enrichment diagram of the two GO terms Angiogenesis and Intracellular iron ion homeostasis in the two comparison groups H vs C and HD vs H; c is the protein interaction network diagram of DEGs constructed using the STRING database; the Venn diagram shows the common and unique nodes with three centrality parameters (Degree, Betweenness and Eigenvector) greater than the corresponding means, and the nodes with all three centrality parameters greater than the mean are high centrality nodes. The common and unique high centrality nodes between the two comparison groups H vs C and HD vs H were determined, and 6 common high centrality nodes were obtained; d is the relative FPKM value of key genes (Erbb4, Tfrc, Acta1 Hamp Areg and Chrna7) in RNA-seq and their relative expression in placental tissues; eg are Western blotting of HAMP protein in mouse placenta, HUVECs and PIECs, respectively. Blot detection results and expression level statistics; n = 6; in d and e, C on the horizontal axis represents the control group; H represents the high-fat and high-sugar diet group during pregnancy; HD represents the high-fat and high-sugar diet group supplemented with 2% DHA during pregnancy; in f and g, CON on the horizontal axis represents the control group; PG represents the high palmitic acid and high-sugar treatment group; PGD represents the high palmitic acid and high-sugar + DHA treatment group; the results are expressed as mean ± standard error; *P < 0.05, **P < 0.01, ***P < 0.001;

[0024] Figure 4 The results of the mouse chorioallantoic membrane explant test; A is a schematic diagram of the mouse chorioallantoic membrane explant test process; B is the morphological diagram of the mouse chorioallantoic membrane explant before and after treatment and CD31 immunofluorescence staining, bar = 200 μm; CE are the results of statistical analysis of the tube area ratio, node density and average tube length of the chorioallantoic membrane tube area (n = 6); the results are expressed as mean ± standard error; different lowercase letters in the bar graph indicate significant differences (P < 0.05), and the same or no letters indicate no significant differences (P > 0.05);

[0025] Figure 5 is the structure of gene overexpression and silencing experiment in PIECs; wherein, A is the Western Blot detection result and expression level statistical graph of HAMP and GPX4 proteins after PIECs were transfected with HAMP overexpression plasmid in vitro; B is the statistical graph of ferrous ion level after PIECs were transfected with HAMP overexpression plasmid in vitro; C is the statistical graph of ROS level after PIECs were transfected with HAMP overexpression plasmid in vitro; DF is the scratch test graph, cell migration statistical graph and tube formation ability statistical graph after PIECs were transfected with HAMP overexpression plasmid in vitro; G is the Western Blot detection result and expression level statistical graph of HAMP and GPX4 proteins after PIECs were transfected with HAMP siRNA in vitro; H is the statistical graph of ferrous ion level after PIECs were transfected with HAMP siRNA in vitro; I is the statistical graph of ROS level after PIECs were transfected with HAMP siRNA in vitro; JL is the scratch test graph, cell migration statistical graph and tube formation ability statistical graph after PIECs were transfected with HAMP siRNA in vitro. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] The present invention uses C57BL / 6J female mice as the research object. After one week of pre-feeding at three weeks of age, they are fed a high-fat and high-sugar diet with 45% fat and 20% sucrose for energy for 8 weeks, and then the female mice are bred and pregnant, and 2% DHA is added to the high-fat and high-sugar diet during pregnancy. The results showed that the high-fat and high-sugar diet led to adverse pregnancy outcomes in the female mice, including increased embryo absorption rate and abnormal fetal proportion. At the same time, the placental iron metabolism of the female mice on the high-fat and high-sugar diet was unbalanced and angiogenesis was reduced. After the DHA dietary intervention, the above abnormal conditions were alleviated to a certain extent, indicating that the high-fat and high-sugar diet led to obstructed placental function and adverse pregnancy outcomes, and DHA as a nutritional additive can improve these conditions.

[0032] Subsequently, human umbilical vein endothelial cells (HUVECs) and porcine iliac endothelial cells (PIECs) were treated with high palmitic acid and high sugar and exogenously added with DHA to verify the in vivo results in vitro. The results showed that high palmitic acid and high sugar treatment disrupted the intracellular ferrous ion and ROS balance of HUVECs, and the cell's defense against oxidative stress decreased, which was reversed after DHA intervention. In addition, high palmitic acid and high sugar inhibited the cell's ability to form tubes, which was alleviated after DHA intervention, and ferroptosis inhibitors or activators could restore or inhibit the cell's ability to form tubes. Transcriptome analysis and protein interaction network analysis were further performed on the maternal placenta to screen the core genes that regulate placental iron homeostasis and angiogenesis, and combined with mouse chorioallantoic membrane explant culture experiments and in vitro PIECs gene overexpression and silencing techniques, the mechanism of placental iron metabolism balance on placental angiogenesis was explored, and it was found that its molecular mechanism may be achieved by regulating the expression of secretory protein HAMP. The details are as follows:

[0033] Example 1 Regulatory Effects of HAMP on Iron Metabolism and Angiogenesis in Placental Endothelial Cells

[0034] 1. Experimental animals and groups

[0035] Sixty 3-week-old C57BL / 6J female mice were selected and fed with maintenance feed for one week, and then randomly divided into a control diet group (Control, CON) and a high-fat and high-sugar diet group (High-fat-sucrose, HFS). Each group had 10 replicates, 3 female mice per cage, and each cage was 1 replicate. The two groups were fed different feeds, and the energy content and proportion of the feeds are shown in Table 1. Food and water were available ad libitum.

[0036] Table 1 Content and proportion of energy-supplying substances in feed during the modeling period

[0037]

[0038] After 8 weeks of feeding, mating was performed. Male and female mice were co-bred at a ratio of 2:1, mated overnight, and the appearance of vaginal plugs on the next morning was recorded as 0.5 days of pregnancy (Embryonic 0.5, E0.5). Pregnant female mice were selected and raised in individual cages, and female mice that were not pregnant for one week were eliminated. The female mice that successfully became pregnant in the CON group and the HFS group were randomly divided into three groups for treatment, namely, the control diet group during pregnancy (group C, female mice were from the CON group during the modeling period), the high-fat and high-sugar diet group during pregnancy (group H, female mice were from the HFS group during the modeling period), and the isoenergetic high-fat and high-sugar diet group with docosahexaenoic acid (DHA) added to the high-fat and high-sugar diet during pregnancy (group HD, female mice were from the HFS group during the modeling period), with 8 replicates in each group, one pregnant mouse as one replicate, and a total of 24 experimental mice. The three groups of female mice were fed different feeds during pregnancy. The proportion and formula of the energy supply materials in the feed are shown in Table 2. They were free to eat and drink water. The experimental period was 18 days (E0.5-18.5), and the pregnant mice were housed in a single cage and had free access to food and water. The pregnant mice were killed at E18.5, and mouse serum was collected by eye bleeding, and the placenta of the pregnant mice was collected.

[0039] Table 2 Formula and nutritional components of the mouse gestational diet

[0040]

[0041] Note: C: control diet group during pregnancy; H: high-fat and high-sugar diet group during pregnancy; HD: isoenergetic high-fat and high-sugar diet group supplemented with 2% DHA during pregnancy.

[0042] 2. Collection of tissue samples and data recording

[0043] Pregnant mice were killed by cervical dislocation at E18.5 (fasted overnight before sampling), and blood was collected from the eyeballs of pregnant mice. The blood was allowed to stand at room temperature for 1 hour, and then centrifuged to obtain serum, which was stored in aliquots at -80°C. The uterus-placenta-fetus complex was dissected out, the blood was blotted with filter paper, and then weighed. Then it was placed in a culture dish (placed on ice). The uterus was cut open from the vaginal end to the ovarian end along the mesenteric side of the uterus with surgical scissors, and then the embryo wrapped in the decidua was pulled out with forceps, the placenta and fetus were separated, rinsed in pre-cooled PBS solution, blotted with filter paper, and the placental molecular samples were collected and stored at -80°C for subsequent detection and analysis. The collected placenta fixed sample was placed in 4% paraformaldehyde, and the slices were stained for later use. During the sampling period, the weight of the uterus, placenta and fetus was weighed and recorded, and the total number of embryos and the number of absorbed fetuses were recorded to calculate the embryo absorption rate (absorbed fetuses / total number of embryos).

[0044] 3. Cell grouping and treatment

[0045] Human umbilical vein endothelial cells (HUVECs) were pre-cultured in ECM complete medium to allow the cells to adhere to the wall. After the cell density reached 80%, the cells were evenly seeded on the cell culture plate and randomly divided into three groups when the appropriate cell density was reached: (1) control group (CON): basal medium; (2) high palmitic acid and high glucose group (PG): basal medium containing 80μM palmitic acid + 25mM glucose; (3) high palmitic acid and high glucose + DHA group (PGD): high palmitic acid and high glucose medium containing 80μM DHA. After the three groups were treated at 37℃ and 5% CO2 for 24h, the cell viability, migration ability, tube formation ability, protein expression, ferrous ion level, ROS level and GSH / GSSG ratio were detected. In addition, in order to further determine the effect of cellular iron homeostasis on tube formation ability, the ferroptosis-related induction inhibitor (Fer-1) and activator (Erastin) were added to the corresponding culture medium to detect the effect of cellular iron homeostasis on migration and tube formation ability.

[0046] 4. Test methods

[0047] Histomorphology and physiological function indicators: The fixed samples of the placenta of each group of mice were stained with hematoxylin-eosin and Oil Red O. After taking pictures under a microscope, Image J software was used to calculate the proportion of the placental labyrinth area, the proportion of the blood sinus area, and the proportion of the Oil Red O staining area. The molecular samples and cell samples of the placenta of each group of mice were tested and analyzed for indicators such as ferrous ions, malondialdehyde (MDA), ROS, glutathione (GSH), and oxidized glutathione (GSSG).

[0048] Western Blot Test: The collected and preserved placental molecular samples were subjected to Western Blot test to detect the expression levels of CD31, Akt, and p-Akt proteins. CD31 (ab222783) antibody was purchased from Abcam (USA); Akt (10176-2-AP) antibody was purchased from Proteintech (Wuhan, Hubei); p-Akt (4058S), β-actin (4970S) and HRPGoatAnti-Rabbit IgG (7074) antibodies were purchased from Cell Signaling Technology (USA).

[0049] cDNA library construction and data preprocessing: After extracting total RNA from placental samples and digesting DNA with DNase, eukaryotic mRNA was enriched with magnetic beads with Oligo (dT); the mRNA was sheared into short fragments by adding a shearing reagent, and the sheared mRNA was used as a template to synthesize a single-strand cDNA with a six-base random primer, and then a two-strand synthesis reaction system was prepared to synthesize double-stranded cDNA and purify it; the purified double-stranded cDNA was then end-repaired, A-tailed, and connected to the sequencing adapter, and then the fragment size was selected, and finally PCR amplification was performed; the constructed library was quality checked using Agilent 2100 Bioanalyzer. After the library quality inspection was qualified, the constructed library was sequenced on the Illumina HiSeq XTen platform, and 150bp of double-end data was generated. High-quality clean reads were obtained using Trimomatic software. The clean reads were aligned to the reference genome of the corresponding species using HISAT2, and the software parameters were the default parameters. The sample was evaluated by the genome alignment rate. The FPKM expression value of genes was quantified using Cufflinks software. Differential expression analysis was performed using the DESeq (2012) R package, and genes with P-value < 0.05 and |Foldchange| > 1.5 were defined as differentially expressed genes (DEGs).

[0050] Functional enrichment and cluster analysis: GSEA analysis is performed on the sequencing results to evaluate the distribution trend of genes in a predefined gene set in a gene table ranked by phenotype relevance, so as to determine their contribution to the phenotype and the impact of the coordinated changes of genes in the gene set on phenotypic changes.

[0051] Protein interaction network analysis: DEGs obtained from transcriptome sequencing were used to obtain the initial protein-protein interaction network (PPI) through STRING 11.5. The obtained PPI graph was imported into Cytoscape3.9.1 software, and the initial network was analyzed and evaluated for network centrality using the CytoNCA plug-in. Degree, Betweenness and Eigenvector were selected as centrality parameters. Nodes with centrality parameter scores higher than the corresponding parameter mean were calculated and counted, and represented in the form of a Venn diagram to determine the centrality of the nodes and screen core genes.

[0052] RNA extraction and reverse transcription: RNA was extracted from tissue samples using Tissue RNA Purification Kit and cell samples using EZ-Press RNA Purification Kit. After extraction, RNA quality was tested by 1.0% agarose gel electrophoresis. The RNA concentration was measured by ND-1000 UV-visible spectrophotometer and the sample concentration was adjusted to the same. The steps of RNA reverse transcription to synthesize cDNA refer to the instructions of ABScript III RT Master Mix for qPCR with gDNA remover reagent. The reaction procedure is: 37℃2min, 55℃15min, 85℃5min, and stored at 4℃.

[0053] Real-time fluorescence quantitative PCR: cDNA was diluted 20 times with DEPC water and used for subsequent real-time fluorescence quantitative PCR. Primer 6.0 software was used to design the target gene primers. The real-time fluorescence quantitative PCR reaction system was prepared in a 384-well quantitative PCR plate. The reaction procedure was: 95℃ pre-denaturation for 3min; 95℃ for 5s, 60℃ for 30s, and 40 cycles. According to the threshold cycle (Ct) of the target gene and the internal reference, 2 -ΔΔCt The relative expression level of each target gene mRNA in the sample is calculated by the method. The target gene primers include HAMP-F and HAMP-R, the nucleotide sequence of HAMP-F is shown in SEQ ID NO.1, and the nucleotide sequence of HAMP-R is shown in SEQ ID NO.2.

[0054] SEQ ID NO.1: 5'-AGGGCAGACATTGCGATACC-3';

[0055] SEQ ID NO. 2: 5'-GCAACAGATACCACACTGGGA-3'.

[0056] Cell viability test: cells were inoculated in 96-well plates (100 μL / well), and entered the logarithmic phase about 24 hours after inoculation. When the cells were fully grown and attached to the wall, the old culture medium in the plate was discarded, and the treatment was added, and the culture was continued for 24 hours. After the treatment, the basal culture medium containing CCK8 (CCK8 solution:basal culture medium=1:9) was replaced, and the culture was continued for 2 hours. The absorbance of each well was measured at a wavelength of 450nm using an enzyme-linked immunosorbent assay.

[0057] Cell Matrigel matrix tube formation test: Pre-cool the 200μL pipette tip, 96-well plate and Matrigel matrix gel in a 4℃ refrigerator in advance. Prepare an ice operating environment before the experiment, spread 50μL / well liquid matrix gel in a 96-well plate, pay attention to prevent the generation of bubbles, let it stand on ice, keep the liquid level horizontal, and put it in a 37℃ incubator for 30 minutes to solidify the matrix gel. After the matrix gel solidifies, inoculate the cells on the matrix gel at an appropriate density. After culturing in a cell incubator for 6 hours, take pictures under an inverted microscope, and take 3 fields of view for each repeat. Image J software was used to analyze the number of tubes formed.

[0058] Cell scratch test: Digest the cells in the logarithmic growth phase from the culture flask and plant them in a 6-well plate at an appropriate density, with 2 mL of cell suspension per well and 3 replicate wells for each group of cells. After the cells cover the bottom of the well. Use a 200 μL pipette tip to vertically draw a "cross" on the 6-well plate, wash it 3 times with PBS, remove the scratched cells and observe and take pictures under a microscope (0h), and take pictures again after 24h to record the migration of cells. 12-16 fields of view were randomly selected for each well of cells, and statistical analysis was performed using Image J software. Calculation method: migration distance (μm) = 0h scratch distance - 24h scratch distance; migration rate (%) = migration distance / 0h scratch distance.

[0059] 5. Test results

[0060] In order to explore the growth of the fetus and the development of the placenta, a series of indicators of the conceptus tissue were measured and analyzed. After intervention with a high-fat, high-sugar diet and DHA diet, compared with the C group, the embryo absorption rate of the H group increased, and the proportion of normal fetuses decreased significantly, and these negative conditions were reversed in the HD group ( Figure 1 Middle a). HE staining results of pregnant mouse placenta showed that the proportion of the labyrinth area and the proportion of the blood sinus area in the H group placenta were significantly reduced, which was reversed after DHA supplementation; Oil red O staining results showed that the staining area of ​​the H group placenta increased, but decreased after DHA supplementation ( Figure 1 Protein results showed that the expression level of CD31 protein decreased in the H group, but basically recovered to the level of group C in the HD group; at the same time, the phosphorylation level of Akt protein also decreased significantly in the H group, and could be reversed by DHA supplementation ( Figure 1 In addition, the placental ferrous ion level and MDA content in the H group were significantly higher than those in the C and HD groups ( Figure 1 g and h), while the GSH / GSSG ratio of group H was relatively low ( Figure 1 The above results indicate that a high-fat and high-sugar diet can lead to imbalanced placental iron metabolism and insufficient angiogenesis, thereby increasing fetal absorption and reducing normal fetal development in pregnant mice, and supplementing DHA in the diet can improve this situation.

[0061] The iron metabolism of HUVECs was detected and analyzed. The results showed that PG treatment increased the levels of ferrous ions, ROS, and MDA in HUVECs, and reduced the GSH / GSSG ratio, which was reversed to a certain extent after DHA intervention ( Figure 2 In addition, after exogenous addition of ferroptosis-related induction inhibitor (Fer-1) and activator (Erastin), the cell viability, migration and tube formation ability were detected. The results showed that Fer-1 could reverse the inhibitory effect of PG treatment ( Figure 2 E and G), while Erastin could inhibit the mitigating effect of DHA intervention on PG treatment ( Figure 2 F and H). The results showed that high palmitic acid and high sugar intake would affect the iron metabolism balance of endothelial cells, thereby impairing angiogenesis, while DHA could regulate the iron metabolism balance and improve this negative state.

[0062] In the further analysis of the RNA-seq results of placental tissue, the analysis results of the two comparison groups, H vs C and HD vs H, were focused on. Through GSEA functional enrichment analysis of the two comparison groups, it was found that ( Figure 3 In (a) and (b), both comparison groups were significantly enriched in the two GO terms gene sets of angiogenesis and intracellular iron homeostasis, and the enrichment trends of the two gene sets in the two comparison groups were opposite.

[0063] In order to further explore the key genes for DHA to improve placental development under maternal insulin resistance, PPI analysis was performed on the DEGs of the two comparison groups H vs C and HD vs H. The PPI network was obtained using STRING 11.5, and then the network centrality analysis and evaluation of the interaction network were performed using Cytoscape 3.9.1 software and CytoNCA plug-in. The centrality parameters included Degree, Betweenness, and Eigenvector. The average value of each centrality parameter was calculated and set as the threshold. Nodes with three centrality parameters higher than the corresponding mean were considered to have high centrality. Figure 3In the H vs C comparison group, based on the average values ​​of Degree, Betweenness, and Eigenvector of 8, 964, and 0.0328, 81, 76, and 75 nodes with corresponding centrality parameters higher than the average values ​​were identified from the PPI network, respectively, of which 40 nodes showed high centrality; in the HD vs H comparison group, based on the average values ​​of Degree, Betweenness, and Eigenvector of 9.4, 1370, and 0.0211, 115, 107, and 92 nodes with corresponding centrality parameters higher than the average values ​​were identified from the PPI network, respectively, of which 45 nodes showed high centrality. In order to identify the key nodes that play a core role in both comparison groups, the Venn diagram was used to identify 6 common nodes in the high centrality nodes obtained above in the two comparison groups ( Figure 3 c), Erbb4, Tfrc, Acta1, Hamp, Areg and Chrna7. Their gene expression levels in transcriptome sequencing were then determined ( Figure 3 In the figure (d), the expression levels of Erbb4, Acta1, Hamp and Areg genes were downregulated in the H group compared with the C group, and reversed in the HD group, while the expression levels of Tfrc and Chrna7 genes showed the opposite trend. The selected genes were subsequently verified. The results of qPCR detection of pregnant mouse placenta showed that the expression trend of Hamp mRNA in mouse placenta was consistent with the trend of RNA-seq ( Figure 3 (d)

[0064] The protein expression levels of HAMP in mouse placenta, HUVECs and PIECs were subsequently verified. The results showed that the HAMP protein level in the H group mouse placenta was significantly higher than that in the C group, and then decreased in the HD group ( Figure 3 e); The HAMP protein level in HUVECs in the PG group increased significantly, which could be reversed after DHA intervention ( Figure 3 f); similar trends were observed in PIECs treated with high palmitic acid and high sugar ( Figure 3 The above results suggest that HAMP may be the core target for mediating DHA to improve placental development in insulin-resistant mothers.

[0065] In summary, a high-fat, high-sugar diet can disrupt the balance of placental iron metabolism and cause impaired placental angiogenesis, leading to adverse pregnancy outcomes, which is related to the abnormal increase in HAMP expression in placental endothelial cells, and DHA intervention can improve these conditions.

[0066] Example 2 Effect of HAMP on Tube-forming Ability of Mouse Chorioallantoic Membrane Explants

[0067] 1. Experimental materials and methods

[0068] The mouse chorioallantoic membrane explant culture technology was used to simulate the angiogenesis process of the placenta and perform in vitro intervention to further determine the effect of HAMP on tube formation ability.

[0069] Mouse chorioallantoic membrane explant test: Male and female mice mate overnight, and the embolism appears the next morning as E0.5. Pregnant female mice are killed by cervical dislocation at E8.5, and the uterus is dissected and transferred to a culture dish containing sterile pre-cooled PBS. Cut the myometrium and remove the decidua. Under a stereomicroscope, use forceps to cut the anti-medial side of the decidua, peel off the decidua, and expose the embryo. Use fine forceps to separate the embryo from the yolk sac, and then use a pair of fine forceps to cut the complete chorioallantoic membrane from the base of the posterior end of the embryo. Place a cell slide on the bottom of a 24-well plate, add 500μL of complete culture medium, and transfer the chorioallantoic membrane to a 24-well plate. Place at 37℃, 5% CO2, and culture for 8h. Do not move the well plate during this period. After the chorioallantoic membrane was observed under an optical microscope and confirmed to be attached, culture medium with different drugs was added and cultured for 36 hours. The drug treatment was divided into 5 groups: (1) control group (CON): 11mM glucose; (2) high palmitic acid and high glucose group (PG): 150μM palmitic acid + 25mM glucose; (3) high palmitic acid and high glucose + HAMP group (PG+HAMP): 1.5μg / mL HAMP protein was added on the basis of PG; (4) high palmitic acid and high glucose + DHA group (PGD): 150μM DHA was added on the basis of PG; (5) high palmitic acid and high glucose + DHA + HAMP group (PGD+HAMP): 1.5μg / mL HAMP protein was added on the basis of PGD. 5% FBS was added to the culture medium of the above treatment to maintain the growth of chorioallantoic membrane in vitro. After the treatment, the cell slides with attached chorioallantoic membrane were taken out for subsequent CD31 protein immunofluorescence staining.

[0070] 2. Test results

[0071] The test results are as follows Figure 4 As shown. In view of the overexpression of cell secretory protein HAMP under high palmitic acid and high glucose treatment, in order to verify whether HAMP is a key regulatory target for placental angiogenesis, mouse chorioallantoic membrane explants were used to simulate the angiogenesis process of the placenta and treated with exogenous HAMP. The experiment obtained the chorioallantoic membrane that had not yet attached to form the placental labyrinth from E8.5 pregnant mouse embryos and cultured it in a sterile environment in vitro ( Figure 4(A), after the chorioallantoic membrane attached to the cell slide (about 8h), the chorioallantoic membrane was divided into 5 groups for treatment: (1) control group (CON): 11mM glucose; (2) high palmitic acid and high glucose group (PG): 150μM palmitic acid + 25mM glucose; (3) high palmitic acid and high glucose + HAMP group (PG+HAMP): 1.5μg / mL HAMP protein was added on the basis of PG; (4) high palmitic acid and high glucose + DHA group (PGD): 150μM DHA was added on the basis of PG; (5) high palmitic acid and high glucose + DHA + HAMP group (PGD+HAMP): 1.5μg / mL HAMP protein was added on the basis of PGD. 5% FBS was added to the culture medium of the above treatment to maintain the growth of the chorioallantoic membrane in vitro. After 36h of treatment, the cell slide attached with the chorioallantoic membrane was taken out, CD31 immunofluorescence staining was performed, and the slide was placed under an inverted fluorescence microscope for photography, and the tube formation analysis was performed using AngioTool software. The results of mouse chorioallantoic membrane CD31 staining are shown in Figure 4 Middle B, analysis and statistics show that there is no significant difference in the proportion of the middle tube area of ​​the chorioallantoic membrane ( Figure 4 However, the node density and average tube length of the CON and PGD groups were significantly higher than those of the PG, PG+HAMP, and PGD+HAMP groups ( Figure 4 D and E).

[0072] The above results show that: (1) DHA can alleviate the obstruction of chorioallantoic membrane tube formation caused by high palmitic acid and high sugar; (2) HAMP intervention inhibited the improvement of DHA; (3) HAMP could not further inhibit the chorioallantoic membrane tube formation under high palmitic acid and high sugar treatment. The results suggest that excessive presence of HAMP can inhibit the restorative effect of DHA on the chorioallantoic membrane tube formation ability. HAMP is an important target for regulating the tube formation ability of endothelial cells. This result provides a theoretical reference for the subsequent study of strategies to regulate placental vascular development by regulating HAMP expression.

[0073] Example 3 Effect of HAMP on the iron metabolism balance and tube formation ability of PIECs

[0074] 1. Experimental materials and methods

[0075] The effect of HAMP on iron metabolism and tube formation ability was verified by PIECs in vitro target gene overexpression technology.

[0076] PIECs cell culture: PIECs were pre-cultured in complete medium containing 10% FBS to allow cells to adhere. After the cell density reached 80%, it was replaced with serum-free medium containing 25 mM glucose and 150 μM palmitic acid, and cells without any treatment were set as the control group and treated at 37°C, 5% CO2 for 24 h.

[0077] In vitro HAMP overexpression plasmid transfection test in PIECs: PIECs were cultured to an appropriate degree of confluence. TM 3000 liposome transfection reagent instructions, mix the HAMP overexpression plasmid with the transfection reagent to form a complex. Add the complex to the culture medium containing PIECs, mix gently and continue to culture for 24-48h. After transfection, use a fluorescence microscope to observe the expression of the plasmid, and use Western blot to detect the overexpression effect of the HAMP protein. After transfection and culture for 24 or 48h, high palmitic acid, high sugar and DHA treatments are performed according to the test requirements. After the treatment, the cells are collected, and the cell migration and tube formation ability are detected, and the cell ferrous ion level and ROS level are measured.

[0078] In vitro siRNA silencing of HAMP gene expression in PIECs: PIECs were cultured to an appropriate degree of confluence. TM 3000 liposome transfection reagent instructions, mix siRNA with transfection reagent to form a complex. Add the complex to the culture medium containing PIECs, mix gently and continue to culture for 24-48h. After transfection, the silencing of HAMP protein was detected by Western blot. After transfection and culture for 24 or 48h, high palmitic acid and high sugar treatment was performed according to the experimental requirements. After the treatment, the cells were collected, the cell migration and tube formation ability were detected, and the cell ferrous ion level and ROS level were determined. The siRNA sense sequence used to silence HAMP expression is: 5'-GCUGUCGCAAAGCAAUCUGTT-3' (SEQID NO.3); the antisense sequence is: 5'-CAGAUUGCUUUGCGACAGCTT-3' (SEQ ID NO.4). The sense sequence of the negative control siRNA is: 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO. 5); the antisense sequence is: 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO. 6).

[0079] 2. Test results

[0080] The test results are as follows Figure 5 As shown. The HAMP overexpression plasmid transfection experiment in vitro in PIECs was used to verify the regulatory effect of HAMP on cellular iron metabolism and angiogenesis. The results showed that after cellular HAMP was overexpressed, the effect of DHA intervention on the increase of GPX4 in cells under high palmitic acid and high glucose conditions was inhibited ( Figure 5 A), and eliminated the improvement effect of DHA on cellular ferrous ion and ROS levels ( Figure 5B and C), indicating that HAMP overexpression can lead to endothelial cell iron metabolism imbalance and even ferroptosis. Subsequent migration and tube formation results ( Figure 5 DF) further verified that HAMP overexpression inhibited the ability of endothelial cells to form tubes. Conversely, silencing HAMP by siRNA alleviated the reduction of GPX4 expression in cells caused by high palmitic acid and high glucose ( Figure 5 G), and reversed the increase in ferrous ions and ROS levels induced by high palmitic acid and high glucose ( Figure 5 H and I), and the impaired cell migration and tube formation ability caused by high palmitic acid and high glucose were also restored to a certain extent ( Figure 5 The results showed that HAMP, as an important target, regulates the iron metabolism balance and angiogenesis ability of endothelial cells. Specifically, when high palmitic acid and high sugar induce excessive secretion of HAMP, it will lead to cell iron death and damage the tube-forming ability of endothelial cells. DHA has a certain improvement effect on this pathological state. This result provides a theoretical reference for the subsequent research on strategies to regulate the iron metabolism and tube-forming ability of endothelial cells by regulating the expression of HAMP.

[0081] The present invention uses an in vivo model induced by high fat and high sugar and two endothelial cell in vitro models to explore the association between adverse pregnancy outcomes in maternal mice and placental iron metabolism and angiogenesis; on this basis, further transcriptomic analysis is performed, using mouse chorioallantoic membrane explant culture to simulate the in vivo placental cell environment and control experimental variables, as well as PIECs in vitro gene overexpression plasmid transfection experiments, revealing that HAMP overexpression under high fat and high sugar conditions is the key cause of placental endothelial cell iron metabolism imbalance and impaired angiogenesis, and clearly states that HAMP stable expression is an important molecular marker for maintaining placental tissue cell iron homeostasis and normal vascular development. In addition, the results show the potential connection between iron homeostasis and angiogenesis, and provide a dietary nutrition method using DHA to improve placental iron metabolism and angiogenesis. Compared with previous research methods that only started from a single model, the present invention combines the in vivo and in vitro methods to directly clarify the mechanism of placental damage under adverse pregnancy outcomes, improve the accuracy and credibility of the results, and has important guiding significance for the preparation of products and application methods for preventing or improving adverse pregnancy outcomes. It is expected to provide an important theoretical basis and technical support for the development of nutritional strategies or products for the prevention or treatment of adverse pregnancy outcomes during pregnancy.

[0082] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A molecular marker for adverse pregnancy outcomes, characterized in that: The molecular marker is the HAMP gene.

2. Use of a reagent for detecting the expression amount of the molecular marker described in claim 1 in preparing a kit for predicting adverse pregnancy outcomes.

3. The use according to claim 2, characterized in that: The reagents include a primer pair for detecting the molecular marker.

4. The use according to claim 3, characterized in that: The primer pair includes an upstream primer whose nucleotide sequence is shown as SEQ ID NO.1 and a downstream primer whose nucleotide sequence is shown as SEQ ID NO.

2.

5. A kit for predicting adverse pregnancy outcomes, characterized in that: A reagent for detecting the expression level of the molecular marker according to claim 1 is included.

6. Use of reagents that inhibit HAMP gene expression in the preparation of drugs for improving adverse pregnancy outcomes.

7. The use according to claim 6, characterized in that: The reagent includes siRNA; the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.

4.

8. A drug for improving adverse pregnancy outcomes, characterized in that: Active ingredients include agents that inhibit the expression of the HAMP gene.

9. The drug according to claim 8, characterized in that The active ingredient of the drug also includes DHA.

10. The drug according to claim 8, characterized in that The drug also includes pharmaceutically acceptable excipients.

Images