CN112807296B - Use of succinic acid for diagnosis or treatment of spontaneous abortion - Google Patents

Use of succinic acid for diagnosis or treatment of spontaneous abortion Download PDF

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CN112807296B
CN112807296B CN202110181710.9A CN202110181710A CN112807296B CN 112807296 B CN112807296 B CN 112807296B CN 202110181710 A CN202110181710 A CN 202110181710A CN 112807296 B CN112807296 B CN 112807296B
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succinic acid
spontaneous abortion
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金莉萍
赵健元
王晓辉
许沙
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Shanghai First Maternity and Infant Hospital
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Abstract

The invention relates to the use of succinic acid for the diagnosis or treatment of spontaneous abortion. The invention compares the glycolysis and citric acid circulating metabolite concentrations in villi and decidua of normal pregnancy and people suffering from recurrent abortion (RSA) through metabolic mass spectrometry, and finds that the low level of succinic acid exists in embryo villus tissues of RSA patients; in vitro succinic acid treatment can promote proliferation and invasion of trophoblasts HTR8 and JEG 3; meanwhile, in vivo experiments prove that the intraperitoneal injection of succinic acid can reduce the embryo absorption rate of a glycine induced spontaneous abortion mouse model and a CBA/JxDBA/2 spontaneous abortion model, thereby providing a new method for clinical diagnosis and treatment of RSA. The invention also reduces the succinic acid level in the mouse body by injecting the glycine into the abdominal cavity, leads to the increase of the embryo absorption rate of the mouse, and establishes a new natural abortion mouse model which is consistent with clinical phenomena and has a definite mechanism.

Description

Use of succinic acid for diagnosis or treatment of spontaneous abortion
Technical Field
The present invention relates to the field of disease diagnosis and treatment, and in particular to the use of succinic acid in the diagnosis or treatment of spontaneous abortion.
Background
Spontaneous abortion, which is continued 3 or more times before 20 weeks of gestation, is called recurrent abortion (RSA), affecting 1-2% of women of childbearing age worldwide. Although etiological studies report various factors affecting RSA, except genetic factors, anatomical factors, endocrine factors, infectious factors, immune dysfunction, prothrombotic state, systemic diseases of pregnant women, environmental factors, etc., most cases of RSA are unexplained. Researchers believe that maternal-fetal interface microenvironment dysbiosis may be the root cause of recurrent abortion for this reason, but the mechanisms involved remain unclear and thus there are no effective targeted interventions and therapeutic strategies.
The maternal-fetal interface is the leading edge of direct contact between the embryo and the mother, the tissue layer comprises two parts of villi and decidua, and the cell layer mainly comprises trophoblasts of fetal origin, decidua stromal cells of maternal origin and decidua immune cells. The cells and the produced cytokines, growth factors, hormones, metabolites and the like form a special microenvironment at the maternal-fetal interface, and the immune tolerance state of the maternal and the growth and development of the embryo are precisely regulated until the birth.
In the early stage of pregnancy, the key to ensure the smooth progress of pregnancy is that the decidua of the mother body is invaded by the extravillous trophoblasts of the embryo. The extravillous trophoblast cells can invade to the deep part of a maternal decidua and replace uterine spiral artery vascular endothelial cells, so that the extravillous trophoblast cells can play a role in fixing the placenta and the fetus; and the uterine vascular system can be recast to provide sufficient nutrition for embryo development, so that the normal exertion of the biological function of the trophoblast is crucial to the implantation of blastocysts, the development of placenta and the establishment of proper maternal-fetal relationship. The extravillous trophoblasts have high proliferation and invasion capacity similar to malignant tumor cells in the early pregnancy, and the proliferation and invasion dysfunction of the trophoblasts are the main reasons of pregnancy-related diseases such as early pregnancy failure (such as spontaneous abortion), mid-late pregnancy complications (such as intrauterine growth limitation of fetus, preeclampsia and the like) and trophoblasts. Therefore, a moderate infiltration of the mother by human early gestation trophoblasts is a necessary condition for normal pregnancy. However, the regulation mechanism of trophoblast invasion at maternal-fetal interfaces is not well understood, so that people still have to be careful in elucidating the nature of physiological pregnancy and exploring the treatment scheme for pathological pregnancy. Therefore, the analysis of the regulation mechanism of maternal-fetal interaction in the invasiveness of the trophoblasts has important theoretical and clinical significance.
At present, the research on the regulation of the function of the trophoblast of the metabolite is still in the initial stage and still has a lot of unsolved puzzles.
Patent document CN109423514A, published japanese patent No. 2019.03.05, discloses 8 microRNA markers that can be used to distinguish between repeated spontaneous abortion tissues and normal tissues, and these 8 microRNA markers can very effectively diagnose and/or predict repeated spontaneous abortion. Patent document CN1440807A, published japanese patent No. 2003.09.10, discloses that cyclosporine a exerts a fetus protection effect by inducing a Th2 type effect and maternal-fetal immune tolerance in a spontaneous abortion model. Patent document CN111686118A, published japanese patent No. 2020.09.22, discloses that oleoyl-L- α -lysophosphatidic acid can maintain the steady state of decidua immune components through decidua macrophages lost due to insufficient enrichment, and significantly reduce the absorption rate of pregnant mouse embryos, thereby improving poor pregnancy outcome, and is expected to be a fetus protection drug for spontaneous abortion.
However, no report on the diagnosis or treatment of spontaneous abortion by succinic acid is available at present.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides application of succinic acid in diagnosis or treatment of spontaneous abortion.
In a first aspect, the present invention provides the use of a substance that increases the level of succinic acid in trophoblast and/or villous tissue in the manufacture of a medicament for promoting embryo implantation or for treating spontaneous abortion.
As a specific embodiment, the substance that increases the level of succinic acid in trophoblast and/or villous tissue is a small molecule or biomacromolecule of a compound.
As a preferred example, the compound small molecule is dimethyl succinate.
In a second aspect, the present invention provides the use of an agent for detecting the level of succinic acid in villus tissue in the manufacture of a kit for the diagnosis of spontaneous abortion.
In a third aspect, the present invention provides a method of constructing an animal model of spontaneous abortion comprising the step of administering to an animal a substance which reduces the level of succinic acid in the villus tissue of the animal.
As a specific embodiment, the substance is a small molecule of a compound or a biological macromolecule.
As a preferred example, the small molecule of the compound is glycine.
As another preferred example, the substance is administered to the animal by intraperitoneal injection.
In a fourth aspect, the invention provides a method for screening potential substances for promoting embryo planting or treating spontaneous abortion, which comprises the step of establishing an animal model of spontaneous abortion by using the establishing method as described in any one of the above.
As a preferred example, the method further comprises the steps of:
a) administering a test agent to an animal model of spontaneous abortion;
b) Comparing the change of the succinic acid level in the villus tissue before and after the administration of the animal model of spontaneous abortion, if the change is increased, the tested substance is a potential substance for promoting embryo planting or treating spontaneous abortion, otherwise, the change is not.
The invention has the advantages that:
1. because the proliferation and invasion capacity of the trophoblast outside villus in the early pregnancy are enhanced, therefore, the hypothesis is that certain metabolites have a regulating effect on the function of the trophoblast, the concentration of glycolysis and citric acid circulating metabolites in villus and decidua of a person with normal pregnancy and a person with RSA is compared through metabolic mass spectrometry, the phenomenon that the low level of succinic acid exists in the embryo villus tissue of a person with natural abortion is found, and the succinic acid level in the embryo villus tissue can be used as a marker for diagnosing the natural abortion, so that a new method is provided for clinically diagnosing the repeated natural abortion.
2. We demonstrate that in vitro succinic acid treatment can promote proliferation and invasion of trophoblasts HTR8 and JEG 3; meanwhile, in vivo experiments prove that the intraperitoneal injection of the succinic acid can reduce the embryo absorption rate of a mouse model of spontaneous abortion caused by glycine and a CBA/JxDBA/2 spontaneous abortion model, thereby providing a new method for treating clinical repeated spontaneous abortion.
3. Glycine is injected into the abdominal cavity, so that the succinic acid level in a mouse body is reduced, the embryo absorption rate of the mouse is increased, and a novel natural abortion mouse model which is consistent with clinical phenomena and has a definite mechanism is established.
Drawings
FIG. 1: succinic acid promotes proliferation of trophoblasts. Wherein, DMS: dimethyl succinate. P <0.05, p <0.01, p < 0.001.
FIG. 2: succinic acid promotes trophoblast invasion.
FIG. 3: glycine reduced succinic acid levels in feeder cell lines HTR8 and JEG 3. Wherein, Glycine: glycine.
FIG. 4: intraperitoneal injection of glycine reduced succinic acid levels in mouse villus tissue.
FIG. 5: the absorptivity of mouse embryo injected with glycine in abdominal cavity is increased, and the absorptivity of mouse embryo injected with DMS in abdominal cavity can be saved. The absorbed embryos are often accompanied by ischemia, hemorrhage and necrosis, the volume is obviously smaller than that of normal surviving embryos, and the absorbed embryos are blackish brown and the surviving embryos are pink in color according to the color of the embryos. The number of surviving embryos and the number of absorbing embryos were counted accordingly. And calculating the embryo absorption rate of the mouse according to the number of dead embryos/(the number of surviving embryos + the number of dead embryos).
FIG. 6: the abdominal cavity injection of succinic acid can reduce the embryo absorption rate of mice model with spontaneous abortion. Wherein, DMS: dimethyl succinate; CBA/J BALB/C: a normal control group; CBA/J DBA/2: and (4) a natural abortion group.
Detailed Description
The following detailed description of the present invention will be made with reference to the accompanying drawings.
EXAMPLE 1 analysis of succinic acid levels in villus and decidua tissues of women with normal pregnancy and patients with spontaneous abortion
Villus and decidua tissues of normal pregnant women and patients with Recurrent Spontaneous Abortion (RSA) are respectively collected 30, and metabolic spectrum analysis is carried out by using Nuclear Magnetic Resonance (NMR), and more than 95% of samples detect various metabolites including metabolic pathways of glucose, amino acids, fatty acids and choline. Of these metabolites, we found that RSA patients had 27.5% lower succinic acid levels in their villi, 22.4% higher fumaric acid levels, and 21.1% lower lactic acid levels than the control (table 1). While there was no statistical difference in the levels of each metabolite in decidua tissues (table 2).
Table 1: metabolite concentrations in villi of patients with normal pregnancy and spontaneous abortion
Figure BDA0002942326170000041
Figure BDA0002942326170000051
Note: normal: villous tissue of normal pregnant women, RSA: villus tissue of patients with recurrent spontaneous abortion. P <0.05 was statistically different.
Table 2: metabolite concentration in decidua of patients with spontaneous abortion and normal pregnancy
Figure BDA0002942326170000052
Note: normal: decidua tissue of normal pregnant women, RSA: decidua tissue of patients with recurrent spontaneous abortion.
EXAMPLE 2 succinic acid promotes trophoblast proliferation and invasion
Upon dimethyl succinate (DMS) treatment, JEG3 and HTR8 cells were treated at 2X 103Cell/well Density in 96-well plates (37 ℃, 5% CO)2) And treating the mixture with 2mM DMS for 24-96 hours. The medium was changed daily until the cells were harvested, then incubated with CCK-8 solution (C0039, Beyotime Biotechnology) for 2h and absorbance was measured at 450 nm. The results show that DMS promotes trophoblast proliferation (FIG. 1).
The invasion of JEG3 and HTR8 cells through the matrigel was evaluated objectively by counting the number of cells transferred through the cell membrane in the invasion chamber. The PET film (pore size 8 μm, diameter 6.5mm) on the upper surface of the filter of a Transwell plate (24-well plate, Corning, New York, USA) was precoated with 15 μ L of matrigel (BD biosciences) and air-dried under sterile conditions. Prior to use, the matrigel was treated with 100. mu.L of hot DMEM/F12 (Gibco; Thermo Fisher) for 2 hours. Will be 1 × 105Each of JEG3 and HTR8 was suspended in 100. mu.l serum-free DMEM/F12 and seeded in the upper chamber, and 600. mu.L of DMEM/F12 containing 10% fetal bovine serum was placed in the lower chamber. To investigate the effect of DMS on cell invasion, JEG3 and HTR8 cells were treated with 0.5mM or 2mM DMS for 2h, respectively, prior to cell invasion experiments. After seeding the cells in the upper chamber, 0.5mM DMS and 2mM DMS were added to the lower chamber medium, respectively. The cells were then incubated at 37 ℃ with 5% CO 2Incubate for 16h in the incubator, and then swab the cells attached to the upper surface of the filter. Cells remaining on the lower surface were fixed in methanol for 15 minutes, stained with DAPI for 5 minutes, and cells that had migrated to the lower surface were counted in random areas under a fluorescent inverted microscope. Each experiment was performed in 3 replicates. The results show that succinic acid can significantly promote invasion of trophoblast lines HTR8 and JEG3 (fig. 2).
Example 3 establishment of Low succinate mouse model
Cell experiments demonstrated that glycine reduced succinate levels in feeder cell lines HTR8 and JEG3 (fig. 3), and intraperitoneal injection of glycine reduced succinate levels in mouse villus tissue (fig. 4). To investigate the effect of low succinic acid levels on embryo implantation and spontaneous abortion, 8-week-old female C57BL/6 mice (body weight 20-30 g, n-24) were randomly divided into three groups, a control group (i.p. saline), a glycine intraperitoneal injection group (glycine: 1500 mg/kg/day) and a glycine + DMS intraperitoneal injection group (glycine: 1500 mg/kg/day + DMS: 3000 mg/kg/day). The term "vaginal embolus" is used in the specification of pregnancy at 0.5 day, intraperitoneal injection from 0.5 to 5.5 days, and embryo absorption rate and implantation number at 14.5 days. We found that intraperitoneal injection of glycine increased embryo uptake and that embryo uptake was rescued by simultaneous intraperitoneal injection of succinic acid (fig. 5).
The spontaneous abortion mouse model selects 8-week-old CBA/J female mice to mate with 8-week-old DBA/2 male mice, and 8-week-old CBA/J female mice and 8-week-old BALB/C male mice are used as a control mating combination. Each group was provided with a control group (i.e., intraperitoneal injection with physiological saline) and a DMS intraperitoneal injection group (i.e., DMS: 3000 mg/kg/day). The results showed that the DMS treated group could rescue the embryo uptake rate of mice in spontaneous abortion (FIG. 6).
Embryo uptake was calculated as follows: the embryo uptake rate is R/(R + V) × 100, where R is the number of embryos absorbed and V is the number of embryos surviving.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

Claims (1)

1. Use of a reagent for detecting succinic acid levels in villus tissue in the preparation of a kit for diagnosing spontaneous abortion.
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US6451840B1 (en) * 1997-12-05 2002-09-17 Medical College Of Georgia Research Institute, Inc. Regulation of T cell-mediated immunity by tryptophan
US9555052B2 (en) * 2003-06-13 2017-01-31 Sumathi Paturu Intrauterine fetal growth restriction—the biochemical rationale of treatment modalities including extraperitoneal transamniotic fetal supplements
US20090226397A1 (en) * 2003-10-24 2009-09-10 Nora Therapeutics, Inc. Compositions and methods for reducing the likelihood of implantation failure or miscarriage in recipients of artificial insemination
RU2282439C2 (en) * 2003-10-31 2006-08-27 Государственное образовательное учреждение высшего профессионального образования "Сибирский государственный медицинский университет Министерства здравоохранения Российской Федерации" (ГОУВПО СибГМУ Минздрава России) Pharmaceutical composition based on substances influencing on energetic metabolism and method for prophylaxis of fetoplacental insufficiency in pregnant of high risk group
CN107636466B (en) * 2015-02-18 2020-07-21 阿斯顿大学 Diagnostic assay and treatment of preeclampsia
CN108387722B (en) * 2018-03-19 2020-07-31 复旦大学附属妇产科医院 Application of 1, 6-fructose diphosphate in preparing medicine for preventing miscarriage

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