阅读说明：本技术 油酰基-L-α-溶血磷脂酸在制备自然流产保胎药物中的应用 (Application of oleoyl-L-alpha-lysophosphatidic acid in preparation of medicament for preventing miscarriage due to spontaneous abortion ) 是由 李明清 杨慧丽 李大金 赖真真 于 2020-07-16 设计创作，主要内容包括：本发明涉及油酰基-L-α-溶血磷脂酸在用于制备治疗、预防自然流产药物中的用途。本发明的油酰基-L-α-溶血磷脂酸可显著促进巨噬细胞对蜕膜基质细胞的黏附。经油酰基-L-α-溶血磷脂酸处理后的巨噬细胞可促进滋养细胞的增殖和侵袭。构建流产孕鼠模型并经体内实验证实,应用油酰基-L-α-溶血磷脂酸可通过其富集不足所致的蜕膜巨噬细胞丢失,从而维持蜕膜免疫成分稳态,并显著降低孕鼠胚胎吸收率,从而改善不良妊娠结局。因此,油酰基-L-α-溶血磷脂酸有望作为自然流产的保胎药物。(The invention relates to application of oleoyl-L-alpha-lysophosphatidic acid in preparing a medicament for treating and preventing spontaneous abortion. The oleoyl-L-alpha-lysophosphatidic acid can remarkably promote the adhesion of macrophages to periostracum stromal cells. Macrophages treated with oleoyl-L- α -lysophosphatidic acid promote the proliferation and invasion of trophoblasts. An abortive pregnant mouse model is constructed and in vivo experiments prove that the application of oleoyl-L-alpha-lysophosphatidic acid can maintain the steady state of an decidua immune component through decidua macrophages lost due to insufficient enrichment, and the absorptivity of a pregnant mouse embryo is obviously reduced, so that the poor pregnancy outcome is improved. Therefore, oleoyl-L-alpha-lysophosphatidic acid is expected to be a fetus-protecting drug for spontaneous abortion.)

1. The application of oleoyl-L-alpha-lysophosphatidic acid in preparing medicine for preventing and treating spontaneous abortion.

2. The use of claim 1, wherein the spontaneous abortion is a recurrent spontaneous abortion.

3. The use of claim 1, wherein the spontaneous abortion is a spontaneous abortion having a low level of an autotoxin of an enzyme metabolizing decidua macrophages.

4. The use according to claim 1, wherein the spontaneous abortion is a spontaneous abortion having a low LPA receptor level.

5. The use according to claim 1, wherein the application of oleoyl-L- α -lysophosphatidic acid significantly promotes the adhesion of macrophages to decidua stromal cells.

6. Use according to claim 1, wherein the application of oleoyl-L- α -lysophosphatidic acid increases decidua macrophage retention locally in a tissue.

7. Use according to claim 1, characterized in that the use of oleoyl-L- α -lysophosphatidic acid increases the promotion of trophoblast proliferation and invasion by decidual macrophages.

8. Use according to claim 1, characterized in that the use of oleoyl-L- α -lysophosphatidic acid improves spontaneous abortion.

Technical Field

The invention belongs to the technical field of medicines, and relates to medicinal application of lysophosphatidic acid (LPA) oleoyl-L-alpha-lysophosphatidic acid, in particular to application of oleoyl-L-alpha-lysophosphatidic acid in preparation of a medicament for treating spontaneous abortion and preventing miscarriage.

Background

Normal pregnancy is similar to successful allografts, in that the mother does not reject embryos carrying paternal antigens, but establishes a unique maternal-fetal interface immune tolerance microenvironment through a fine maternal-fetal conversation, allowing the fetus to develop in utero until delivery. The maternal-fetal interface is mainly composed of trophoblasts derived from embryos, decidua stromal cells derived from mothers and decidua immune cells. The normal maternal-fetal interaction session is key to establishing and maintaining a successful pregnancy. Once abnormal maternal-fetal interaction occurs, trophoblast dysfunction and maternal immune rejection to the embryo are inevitably caused, which are manifested as early pregnancy failure (such as repeated spontaneous abortion) and complications of middle and late pregnancy (such as intrauterine growth restriction of fetus, preeclampsia and the like), and the health of the mother and the infant is seriously damaged; whether the complications of the middle and late pregnancy occur or not depends on the good establishment of the relationship between the early pregnancy and the female fetus. Recent statistics show that the incidence rate of spontaneous abortion is as high as 15%. Even today, the development of assisted reproduction technology is at a rapid pace, and the failure of pregnancy due to the disturbance of maternal-fetal immunoregulation is still not overcome. The causes of this are not well understood, both in the mechanism of development of maternal-fetal immune tolerance and in the mechanism of pathogenesis of maternal-fetal immunoregulatory disorders.

Maternal-fetal immunomodulation is the core content of maternal-fetal interactive sessions. Various immune cells such as natural killer cells (NK cells), macrophages (M phi), T cells, a small number of dendritic cells and the like exist at the maternal-fetal interface. Decidua immune cells play a main role in maternal-fetal immune regulation, and the regulation mechanism of recruitment and residence in the maternal-fetal interface and the molecular mechanism of differentiation and development are the research focus of maternal-fetal immune regulation and tolerance, and have important theoretical and clinical significance. M phi in decidua immune cells at the maternal-fetal interface is nearly 20 percent and is second only to NK cells. Decidua M phi (dM phi) is mainly involved in the physiological processes of pregnancy, such as embryo implantation, spiral artery remodeling, placenta development, cervical ripening and childbirth. The phenotype of the complex M phi of dM phi function regulation has plasticity and participates in various physiological processes of host pathogen resistance, apoptotic cell clearance, angiogenesis and the like.

CD14 in the process of human molting⁺The amount of dM φ has been shown to increase or be relatively constant. The macrophage of the pregnant mouse is exhausted to cause the absorption rate of the embryo of the pregnant mouse to be increased, which indicates that the fetus-mother isAbnormal density and retention of interfacial local decidua macrophages can lead to poor pregnancy outcome. In the event of infection or injury, the process by which lymphocytes are recruited from the blood circulation, adhere to blood vessels, and extravasate into the injured area is a very important link in the inflammatory response. This process is also called lymphocyte homing, i.e. the process by which lymphocytes are enriched in the corresponding organ by interaction with the corresponding vascular addressin on the vascular endothelial cells of the corresponding organ through lymphocyte homing receptors. Many cytokines, chemokines, adhesion molecules (e.g., selectins, integrins), etc., are involved. Although the uterus does not belong to the lymphoid organ, embryo implantation and pregnancy maintenance are processes of proinflammatory and anti-inflammatory dynamic equilibrium, and a large number of decidua immune cells are involved. And decidua and spiral artery have the expression of adhesion molecules such as selectin and the like, which is beneficial to the adhesion and planting of blastocysts. Thus, peripheral blood mononuclear cells may reside locally in the uterus through receptor interaction with ecdysone-responsive ligands, but need further investigation. It has been reported that the content of macrophage colony stimulating factor (M-CSF)/colony stimulating factor 1(CSF1) at the embryo implantation site is significantly higher than that of the non-pregnant uterus. In one aspect, M-CSF stimulates the proliferation of intimal macrophages, and in particular promotes CD209⁺Proliferation of dM phi; on the other hand, it induces macrophages to express the monocyte chemotactic factor CCL2 to promote extravasation of blood-derived monocytes and differentiation into deciduate macrophages.

Lysophosphatidic acid (LPA) is the smallest, structurally simplest phospholipid found to date, an intermediate product of the glycerol phospholipid metabolism of eukaryotic cells. LPA is a simple lipid composed of a glycerol backbone, a single fatty acyl chain and free phosphate groups. LPA is mainly lysophosphatidylcholine decomposed from toxin (ATX/ENPP2) by an enzyme with lysophospholipase D activity. Despite its simple structure, LPA plays an important biological role as a lipid signaling molecule and has become a focus of lipid research in recent years. LPA is secreted by platelets, monocytes-macrophages, fibroblasts, cancer cells and adipocytes, etc., and is a multifunctional "phospholipid messenger" that can activate G-protein coupled receptors, causing growth hormone-like effects, thus producing a wide range of biological effects. It has a plurality of influences on the growth, proliferation, differentiation and migration of cells and the information transmission in the cells, and plays an important role in maintaining the normal physiological function of an organism and participating in the occurrence and development of various pathological processes; also plays an important role in the occurrence and development of various major diseases such as cardiovascular and cerebrovascular diseases, nephropathy and tumors. To date, 6 lysophosphatidic acid receptors (LPARs) have been discovered on biofilms, i.e., LPAR 1-6. In addition, LPA presents an intracellular receptor, the Peroxisome proliferator-activated receptor (PPAR). LPA has been shown to be an intracellular agonist of PPAR γ with higher binding capacity than free fatty acids or other small phospholipids.

LPA plays important biological function in early pregnancy, LPA plays important biological function in early pregnancy through LPAR1 and relies on NF-kB signal path to mediate trophoblast to produce GRO- α, IL-8 and MCP-1 (monocyte chemotactic factor-1), so as to regulate angiogenesis in early pregnancy and chemotaxis of immune cells, LPA/LPAR3 regulates the generation of key molecules (COX-2, IGFBP-1 and IL-10) involved in angiogenesis and decidualization process, creates proper uterine microenvironment for implantation embryo invasion, high expression of LPA in decidualization process of human intimal stromal cells, and LPAR1 signal path is involved in decidualization control, and secondly, it is suggested that LPA downstream signal transduction plays an important role in endometrium deciduation and embryo implantation⁺Monocyte induction was F4/80⁺Macrophages, the process by which LPA activates the mTOR signaling pathway, may play a crucial role. Transcriptome analysis showed that PPAR γ is a key transcription factor in LPA-induced macrophage differentiation. In addition, LPA can mediate the development of human monocytes into a population of CD 206-highly expressing macrophages in a similar way. Finally, LPA can regulate cell adhesion. LPA can activate p38MAPK signal, induce tumor cell migration, invasion and adhesion,such as human liver cancer cells, ovarian cancer cells, and the like. The complex role of LPA in calpain-mediated proteolysis of FAK and other adhesion proteins may play a role in LPA's ability to promote attachment, migration and survival of prostate cancer cells. In addition, LPA induces late expression of adhesion molecule-1 (ICAM-1) and VCAM-1 in human endothelial cells by mediating enhancement of RhoA signaling; promoting CD68 in muscle tissue⁺Infiltration of tissue macrophages.

Chinese patent document CN109646443A discloses the use of lysophosphatidic acid. Lysophosphatidic acid can be used for regulating the expression of inflammatory factors such as IL-1 and/or IL-6, and relieving inflammatory reaction; and can promote fibroblast to secrete collagen, and promote epidermal proliferation and regeneration. Chinese patent document CN109852654A discloses a composition capable of inducing stem cells to secrete cytokines and its application, a composition capable of inducing stem cells to secrete cytokines, characterized in that the composition comprises: 1 mg-5 mg of vitamin A, 50 mg-100 mg of glutamine, 0.5 mg-2 mg of lysophosphatidic acid and 0.01 mg-0.1 mg of icaritin.

However, the application of oleoyl-L-alpha-lysophosphatidic acid in preparing a medicament for treating and preventing spontaneous abortion has not been reported at present.

Disclosure of Invention

The invention aims to provide a new application of oleoyl-L-alpha-lysophosphatidic acid, and particularly relates to an application and a method thereof in fetus protection treatment of spontaneous abortion.

In order to achieve the purpose, the invention adopts the technical scheme that:

the application of oleoyl-L-alpha-lysophosphatidic acid in preparing medicine for preventing and treating spontaneous abortion.

In a further technical scheme, the spontaneous abortion is recurrent spontaneous abortion.

According to a further technical scheme, the spontaneous abortion is spontaneous abortion with low level of self-toxin of decidua macrophage metabolic enzyme.

In a further technical scheme, the spontaneous abortion is spontaneous abortion with low LPA receptor level.

According to a further technical scheme, the adhesion of macrophages to decidua stromal cells can be remarkably promoted by applying the oleoyl-L-alpha-lysophosphatidic acid.

According to a further technical scheme, the application of the oleoyl-L-alpha-lysophosphatidic acid can improve the local residence of decidua macrophages in tissues.

Further, the application of oleoyl-L-alpha-lysophosphatidic acid can improve the promotion effect of decidua macrophages on the proliferation and invasion of trophoblasts.

According to a further technical scheme, the oleoyl-L-alpha-lysophosphatidic acid is applied to improve spontaneous abortion.

The metabolic state and the retention level of LPA of decidua macrophages in natural abortion and normal pregnancy are compared, and the potential value of the metabolite oleoyl-L-alpha-lysophosphatidic acid in regulating decidua macrophages in pregnant mice and improving embryo absorption rate is further evaluated by constructing a natural abortion mouse model; provides a new application of oleoyl-L-alpha-lysophosphatidic acid as a medicament for preventing miscarriage in spontaneous abortion. The results show that: 1) elevated levels of oleoyl-L- α -lysophosphatidic acid in decidua macrophages in normal early-pregnant women compared to peripheral blood mononuclear cells, at elevated levels of both the upstream key metabolic enzyme autophagic toxin ENPP2 and the receptors LPAR1 and PPAR γ; in molting membrane macrophage of a patient with spontaneous abortion, the levels of ENPP2, LPAR1 and PPAR gamma are lower than those of a normal control group; 2) in decidua tissues of patients with spontaneous abortion, the number of decidua resident macrophages is obviously lower than that of normal pregnant women, and adhesion molecules of the macrophages are generally low in expression and in a low adhesion state; 3) macrophages treated in vitro with exogenous oleoyl-L- α -lysophosphatidic acid exhibit strong adhesion to decidual stromal cells; 4) macrophages treated by exogenous oleoyl-L-alpha-lysophosphatidic acid in vitro can remarkably promote the proliferation and invasion of trophoblast JEG3 cells; 5) the in vivo application of oleoyl-L-alpha-lysophosphatidic acid can save embryo loss and placenta dysplasia of a mice model with spontaneous abortion, and reverse the low occupation ratio, the low quantity and the low level of adhesion molecules of macrophages in the uterus of the mice with spontaneous abortion. These results suggest that oleoyl-L- α -lysophosphatidic acid can be used for tocolytic treatment of spontaneous abortion caused by dysmnesia of the uterine decidua macrophage LPA enrichment and poor retention.

Specifically, the invention discloses a medicine which can be used for preparing a medicine for treating spontaneous abortion, wherein the medicine can promote the local adhesion and residence of oleoyl-L-alpha-lysophosphatidic acid in an decidua by acting on a receptor of the lysophosphatidic acid so as to improve the embryo absorption of a pregnant mouse.

The purpose of the invention is realized by the following technical scheme:

collecting peripheral blood and decidua tissue of normal early pregnancy women, separating peripheral blood mononuclear cells and decidua immune cells, and magnetic bead sorting to enrich CD14⁺Peripheral blood mononuclear cells and decidua macrophages, metabonomics detects the metabolite level, the fact that oleoyl-L- α -lysophosphatidic acid is obviously enriched in the decidua macrophages is found, Real-time PCR detects the transcription levels of ENPP2, LPAR1 and PPAR gamma in the peripheral mononuclear cells and the decidua macrophages, the result shows that the transcription level of the genes in the decidua macrophages is obviously higher than that of the peripheral blood mononuclear cells, decidua tissues of patients suffering from natural abortion (the number of times of abortion is more than or equal to 3, no abnormality in postoperative villus chromosome detection and no abnormality in pre-pregnancy endocrine hormone detection) are collected, decidua immune cells are separated and collected, and CD14 is sorted by magnetic beads⁺Macrophages were compared to normal early pregnancy decidua macrophages ENPP2, LPAR1 and PPAR γ transcript levels by Real time-PCR and found to exhibit low levels in decidua macrophages ENPP2, LPAR1 and PPAR γ of spontaneous abortions (as shown in figure 1). After paraffin embedded sections are carried out on decidua of normal early pregnancy groups and spontaneous abortion groups, local CD68 of tissues is detected by means of immunohistochemical technology⁺Macrophage cell counting is carried out under 40 times of microscope, and the number of decidua macrophages in the spontaneous abortion group is found to be lower than that in the normal early group, thus indicating the state of macrophage retention. Real-time PCR detection of CD14⁺The transcription levels of adhesion molecules on decidua macrophages, vascular cell adhesion molecule 1(VCAM-1), L-Selectin (L-Selectin/LSEL), intercellular adhesion molecule-2 (ICAM-2), E-Selectin (E-Selectin/ESEL) and VE-Cadherin (VE-Cadherin/CDH 5). The results showed that the expression of the adhesion molecule was generally decreased in decidua macrophages of spontaneous abortion patients compared to normal pregnant women (as shown in FIG. 2). In vitro on MCSF (50ng/mL) induced macrophagesThe results of experiments on adhesion of macrophages to decidua stromal cell layer by administration of oleoyl-L- α -lysophosphatidic acid (5uM or 10uM) to 48 mice were shown, and as a result, oleoyl-L- α -lysophosphatidic acid significantly promoted adhesion of macrophages to decidua stromal cell layer (as shown in FIG. 3). then, macrophages pretreated with oleoyl-L- α -lysophosphatidic acid (10uM, 48 hours) were co-cultured indirectly with JEG3 cells treated with CFSE dye or with JEG 45 cells inoculated onto glium, flow cytometry or microscopic imaging was used to evaluate the proliferative and invasive abilities of trophoblasts. α -lysophosphatidic acid (as shown in FIG. 4). The macrophages after stimulation with oleoyl-L-lysophosphatidic acid promote proliferation and invasion of JEG3 cells (as shown in FIG. 4). finally, by constructing a natural abortion mouse (CBA/J × A/2. the model, lysophosphatidic acid injection of physiological saline or oleoyl-L-2-lysophosphatidic acid (5. 5, the number of lysophosphatidic acid is shown in the mice after the mice is increased, the number of placental tissue, the mice after the mice is increased, the mice after the mice, the mice are significantly increased in the number of lysophosphatidic acid is increased in the number of sperm injection of lysophosphatidic acid, the mice.

The invention provides the use and method of oleoyl-L-alpha-lysophosphatidic acid for tocolytic treatment of spontaneous abortion; particularly, animal experiments prove that the supplementation of oleoyl-L-alpha-lysophosphatidic acid can improve the local residence of decidua macrophages in tissues, thereby improving the embryo loss of pregnant mice. Therefore, the oleoyl-L-alpha-lysophosphatidic acid can be used for fetus protection treatment of spontaneous abortion.

The invention has the advantages that: the inventor of the application aims to evaluate the potential value of the oleoyl-L-alpha-lysophosphatidic acid in regulating decidua macrophage retention and improving embryo absorption rate by detecting the LPA metabolic state and retention level of decidua macrophages in natural abortion and normal pregnancy and further constructing a natural abortion mouse model, provides the application and the method of the oleoyl-L-alpha-lysophosphatidic acid in the fetus protection treatment of natural abortion, and provides a new method and a new thought for the clinical treatment of natural abortion.

The oleoyl-L-alpha-lysophosphatidic acid can obviously promote the adhesion of macrophages to periostracum stroma cells. Macrophages treated with oleoyl-L- α -lysophosphatidic acid promote the proliferation and invasion of trophoblasts. An abortive pregnant mouse model is constructed and in vivo experiments prove that the application of oleoyl-L-alpha-lysophosphatidic acid can maintain the steady state of an decidua immune component through decidua macrophages lost due to insufficient enrichment, and the absorptivity of a pregnant mouse embryo is obviously reduced, so that the poor pregnancy outcome is improved. Therefore, oleoyl-L-alpha-lysophosphatidic acid is expected to be a fetus-protecting drug for spontaneous abortion.

Drawings

FIG. 1 shows the enrichment and dysfunction of decidua macrophages of a patient with spontaneous abortion due to the enrichment of decidua macrophages oleoyl-L-alpha-lysophosphatidic acid in normal early pregnancy women,

wherein, pMo: peripheral mononuclear cells (PBMC) are obtained,

dM φ: the number of decidua macrophages is increased,

NP: in the normal course of pregnancy,

and SA: the spontaneous abortion is carried out,

LPA (18: 1(9 z)/0: 0): oleoyl-L-alpha-lysophosphatidic acid,

ENPP 2: from the gene encoding the toxin,

LPAR1, PPARG: an LPA receptor-associated gene selected from the group consisting of,

ACTB: an internal reference gene is introduced into the body,

*P<0.05，**P<0.01，***P<0.001，***P<0.0001。

FIG. 2 shows that the decidua macrophage retention of the spontaneous abortion patients is reduced and the adhesion molecule expression is low,

wherein, NP: in the normal course of pregnancy,

and SA: the spontaneous abortion is carried out,

red arrow: CD68⁺The number of decidua macrophages is increased,

VCAM1, LSEL, ICAM2, ESEL, CDH 5: the gene related to the adhesion is obtained by the adhesion,

ACTB: an internal reference gene is introduced into the body,

*P<0.05，**P<0.01，***P<0.001，***P<0.0001。

FIG. 3 is a graph showing that oleoyl-L- α -lysophosphatidic acid promotes macrophage adhesion to decidua stromal cells,

wherein, DSC: decidua stromal cells (green light),

m phi: the number of macrophages (red light),

merge: the overlapping is carried out by overlapping the two parts,

ctrl: in the control group, the concentration of the active ingredients in the active ingredients,

LPA 5uM/10 uM: oleoyl-L-alpha-lysophosphatidic acid group,

*P<0.05，**P<0.01。

FIG. 4 is a graph showing that oleoyl-L-alpha-lysophosphatidic acid-induced macrophages promote the proliferation and invasion of trophoblasts,

wherein, Isotype: the contrast of the same type is that,

Ctrl/Ctrl M0: in the control group, the concentration of the active ingredients in the active ingredients,

LPA/LPA M0: oleoyl-L-alpha-lysophosphatidic acid group,

FITC-CFSE: cell proliferation after CFSE staining is detected by an FITC channel,

MFI: the average fluorescence intensity of the fluorescent light was measured,

*P<0.05，**P<0.01。

FIG. 5 is a schematic representation of the use of oleoyl-L- α -lysophosphatidic acid to rescue embryo absorption and decidua macrophage loss in spontaneously abortive pregnant mice,

wherein Ctrl: in the control group (aborted pregnant mouse model),

LPA: oleoyl-L- α -lysophosphatidic acid group (aborted pregnant mouse model),

embryo reservation rate (%): the rate of absorption of the embryo,

number of implanted albumos: the number of the planted embryos is increased,

weight of embroyos/planta: the weight of the embryo/placenta is determined,

CD11b⁺F4/80⁺m φ of UICs (%): the proportion of macrophages in the immune cells of the uterus,

e-selecting: the amount of E-selectin is such that,

CD 51: the alpha V chain of the integrin,

L-Selectin: the amount of L-selectin is such that,

ICAM-1: the cell-to-cell adhesion factor-1,

p <0.05, P <0.01, NS were not significantly different.

The volume of the absorbed embryo is obviously smaller than that of the normal survival embryo due to ischemia, hemorrhage and necrosis, and the color expression of the absorbed embryo and the survival embryo is also obviously different, wherein the former is dark brown (shown by red arrows in the figure) and the latter is pink. The number of embryos absorbed and the number of surviving embryos were counted accordingly. Embryo absorption rate R: r ═ Re/(Re + F) × 100%. Re is the number of absorbing embryos and F is the number of surviving embryos.

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention.