Regulators of immune function at the maternal-fetal interface

Abstract

The application discloses a maternal-fetal interface immune function regulator and belongs to the technical field of medicines. An effective component of the maternal-fetal interface immune function regulator comprises any one of tarazepide, mifepristone, L-canavanine or butaprost or a pharmaceutically acceptable salt thereof. The maternal-fetal interface immune function regulator is administered during pregnancy. By activating a positive feedback loop COX-2 / PGE2 / PTGER-2, up-regulating placental IDO-1 expression and regulating the steady-state recovery of placental tryptophan metabolism, the maternal-fetal interface immune function regulator promotes the recovery of the maternal-fetal interface immune steady state, improves the maternal-fetal barrier function, improves the development of neurons in the brain of offspring and prevents the neurological development disorder of offspring. The use of the maternal-fetal interface immune function regulator during pregnancy can be applied as a new means for treating the neurological development disorder of offspring, and provides a new experimental basis and treatment idea for the treatment of autism in clinical practice.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a regulator for immune function of the maternal-fetal interface. BACKGROUND

[0002] Neurodevelopmental disorders (NDDs) are caused by abnormal brain development, characterized by cognitive impairment and neurobehavioral abnormalities. Autism spectrum disorders (ASDs) are the most common neurodevelopmental disorders, characterized by social deficits, repetitive stereotyped behaviors and lack of interest. The causes of autism may be related to genetic, developmental and environmental factors. Some high-risk exposure factors during pregnancy can damage the brain development of the fetus and lead to the occurrence of autism, such as high-fat diet, valproate and gestational diabetes mellitus.

[0003] The placental barrier is an important organ for maintaining normal fetal development. The placenta is considered a key hormone and neurotransmitter producer, which can regulate fetal neurodevelopment through the placental-brain axis. A large amount of evidence shows that placental development disorders are often considered to be closely related to the occurrence of autism, such as mitochondrial dysfunction, changes in placental barrier permeability and leakage of trophoblast inclusions. In different environmental factors induced ASDs, maternal-fetal interface homeostasis imbalance is a common complication, usually accompanied by placental immune disorders and maternal-fetal barrier function destruction. Maternal-fetal interface homeostasis is maintained by placenta of fetal origin and decidua of maternal origin. Immune cells in decidua mainly include decidual natural killer cells (dNK), decidual macrophages (dM) and T cells. Placental immune disorders can seriously affect fetal development, such as maternal immune activation mediated by excessive proliferation of effector T lymphocytes (TH17 cells), decidualization defects mediated by dNK cell residence and embryo implantation arrest caused by dM depletion. At present, it is not clear about the correlation between autism and maternal-fetal barrier and the role of placental immune homeostasis in it.

[0004] Studies have shown that imbalance of placental tryptophan metabolism leads to NDDs, and the main pathogenesis is that maternal inflammation activates to promote the overexpression of placental tryptophan hydroxylase 1 (TPH-1), leading to excessive hydroxylation of L-tryptophan (Trp) into 5-hydroxytryptamine (5-HT), and excessive accumulation of placental 5-HT causes neuronal damage. It is not yet clear how excess 5-HT enters the fetal brain. 5-HT cannot freely pass through the normal maternal-fetal barrier (MFB) and blood-brain barrier (BBB), but under the condition of maternal inflammation, the destruction of the maternal-fetal barrier is likely to accelerate the entry of maternal 5-HT into the fetal brain.

[0005] In the prior art, there are few drug and / or gene therapy schemes for neurodevelopmental disorders, and more focus is on the management of behavior education and physical therapy. The Chinese patent document with publication number CN115429782A discloses the application of acetate in the preparation of a drug for preventing or treating neurodevelopmental disorders. The corresponding drug can improve the morphology of neurons and the density of dendritic spines, restore the function of synaptic transmission, improve the electrophysiological function of neurons, and improve cognitive function. Therefore, it is necessary to develop more drug treatment schemes for neurodevelopmental disorders. SUMMARY

[0006] In order to prevent and / or treat neurodevelopmental disorders, the present application provides a maternal-fetal interface immune function regulator.

[0007] The specific technical solutions adopted are as follows:

[0008] The present application provides the use of any one of Taprenepag (Tap), Mifepristone (RU486), L-Kynurenine (Kyn), Butoprost (But) or a pharmaceutically acceptable salt thereof in the preparation of a maternal-fetal interface immune function regulator,

[0009] The structural formula of Taprenepag is shown as formula (I), the structural formula of Mifepristone is shown as formula (II), the structural formula of L-Kynurenine is shown as formula (III), and the structural formula of Butoprost is shown as formula (IV);

[0010]

[0011] The maternal-fetal interface structure includes placenta, decidua and the connection between them.

[0012] The placental interface immune function regulator can promote the recovery of the immune homeostasis of the placental interface, specifically by up-regulating the expression of placental IDO-1, regulating the homeostasis recovery of tryptophan metabolism of the placenta, and recovering the number and ratio of immune cell populations of the placental interface, and the recovery of the immune function of the placental interface can effectively improve the placental barrier function.

[0013] The placental interface immune function regulator can improve the placental barrier function, specifically by up-regulating the expression of tight junction proteins claudin-4 and claudin-5, protecting the tight junction ultrastructure of the amniotic epithelial cells and the placental syncytiotrophoblast cells, to protect the placental barrier function of the high-risk exposure maternal during the pregnancy period.

[0014] The placental interface immune function regulator can improve the development of neurons in the offspring brain and / or prevent the neurological development disorder of the offspring, specifically the curative effect is embodied in improving the development of serotoninergic neurons of the offspring in the valproic acid sodium exposure model during the pregnancy period, promoting the normal development of the thalamocortical axon, and preventing the offspring from having autism and other neurological development disorders.

[0015] The placental interface immune function regulator also comprises at least one of the compounds or pharmaceutically acceptable salts thereof shown in the structural formulae (I)-(IV).

[0016] Preferably, the active ingredient of the placental interface immune function regulator is the compound shown in the structural formula (I) or the compound shown in the structural formula (II).

[0017] The placental interface immune function regulator also comprises a pharmaceutically acceptable carrier, diluent and / or excipient.

[0018] Preferably, the amount of the active ingredient in the placental interface immune function regulator is 0.05 mg / kg body weight to 2.5 mg / kg body weight.

[0019] Further preferably, when the active ingredient of the placental interface immune function regulator is the compound shown in the structural formula (I), (III) or (IV), the amount of the active ingredient in the placental interface immune function regulator is 0.5 mg / kg body weight; and when the active ingredient of the placental interface immune function regulator is the compound shown in the structural formula (II), the amount of the active ingredient in the placental interface immune function regulator is 0.05 mg / kg body weight.

[0020] The administration time of the placental interface immune function regulator is the pregnancy period, and the administration routes include but are not limited to intravenous injection, intraperitoneal injection, subcutaneous injection or oral administration, etc.

[0021] Compared with the prior art, the application has the beneficial effects that:

[0022] (1) The application provides a maternal-fetal interface immune function regulator, an effective component of which comprises any one of tarenfur or mifepristone, L-canavanine or butaprost or a pharmaceutically acceptable salt thereof, the maternal-fetal interface immune function regulator is administered during pregnancy, activates a positive feedback loop COX-2 / PGE2 / PTGER-2, up-regulates placental IDO-1 expression, regulates the steady state recovery of placental tryptophan metabolism, promotes the recovery of maternal-fetal interface immune homeostasis, improves maternal-fetal barrier function, improves the development of neurons in the brain of offspring and prevents the neurological development disorder of offspring.

[0023] (2) The maternal-fetal interface immune function regulator disclosed by the application only needs to be administered in a single dose during pregnancy, can promote the normal development of neurons in the fetal period, and can play a role in preventing autism and other neurological development disorders in the fetal period, the neonatal period and even after adulthood, and has obvious curative effect. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 For the specific regulation site and activity of the maternal-fetal interface immune function regulator; A is the expression level of placental IDO-1 and Arg-1 (marker of dM2 cells) after drug intervention; B is the Western-blot semi-quantitative result of IDO-1 in A (n=6, litters); C is the Western-blot semi-quantitative result of Arg-1 in A (n=7, litters); D is the expression level of placental COX-2 in each group (ungly-Cox-2: inactive low glycosylation COX-2 precursor, 66 and 70 kDa; gly-Cox-2: active high glycosylation mature COX-2, 72 and 74 kDa); E is the expression level of placental PTGER-2 in each group; F is the content of placental PGE2 in each group determined by ELISA method (n=5, litters); G is the semi-quantitative result of gly-COX-2 expression of placenta in each group in D (n=5, litters); H is the semi-quantitative result of ungly-COX-2 precursor expression of placenta in each group in D (n=5, litters); I is the semi-quantitative result of PTGER-2 expression of placenta in each group in E (n=6, litters).

[0025] Figure 2 For the pharmacodynamic evaluation of the recovery of maternal-fetal interface immune homeostasis; A is the quantitative data of placental immune cell populations (T, dM and dNK cells) (n=5, litters); B is the quantitative data of decidual macrophage subpopulation (dM1 and dM2 cells) (n=5, litters).

[0026] Figure 3Pharmacodynamic evaluation of the protection of the maternal-fetal interface by the immunological function regulator on the maternal-fetal barrier; A is the SEM image of the tight junction complex of amniotic epithelial cells in each group, B is the TEM image of the ultrastructure of the SCT tight junction in each group, light-colored triangles mark loose Tjs or cracks, and dark-colored triangles mark normal Tjs; C is the expression level of placental Tjs (claudin-5 and claudin-4) after drug intervention; D is the semi-quantitative result of claudin-5 in C (n = 7, litters); E is the semi-quantitative result of claudin-4 in C (n = 7, litters).

[0027] Figure 4 Pharmacodynamic evaluation of the protection of the maternal-fetal interface by the immunological function regulator on the fetal rat neural development; A is the TPH-2 + neuron cell body in the raphe nucleus, B is the immunofluorescence staining of TPH-2 + neuron cell body, and Netrin-G1a + immunofluorescence staining of thalamocortical axons (TCAs).

[0028] Figure 5 Pharmacodynamic evaluation of the protection of the maternal-fetal interface by the immunological function regulator on the neural development of young rats; A is the TPH-2 + neuron and 5-HT in the raphe nucleus of newborn pups (P10); B and C are the immunofluorescence staining of 5-HT and TPH-2 + in the raphe nucleus of newborn pups, respectively.

[0029] Figure 6 Pharmacodynamic evaluation of the protection of the maternal-fetal interface by the immunological function regulator on the neural development of adult offspring rats; A is the TPH-2 + neuron and 5-HT in the raphe nucleus of adult offspring mice (P55); B and C are the immunofluorescence staining of TPH-2 + and 5-HT in the raphe nucleus of offspring mice, respectively.

[0030] Figure 7Pharmacodynamic evaluation of the effect of the placental interface immune function regulator on improving the autistic-like behavior of the offspring adult mice; A is the separation-induced ultrasonic vocalization test, the total call of the newborn pups within 3 min (n = 12, litter); B is the time spent by the tested mice in the center area of the open field within 10 min; C is the total path length of the tested mice in the open field within 10 min; D is the number of self-grooming behaviors of the tested mice within 10 min; E is the cumulative time spent by the tested mice in self-grooming behavior within 10 min; F is the number of marbles buried by the tested mice within 15 min (n = 12, litter); G is the time spent by the tested mice in the mouse room (Mouse) and the object room (Object) during the social behavior test stage; H is the time spent by the tested mice in the familiar mouse room (Familiar) and the unfamiliar mouse room (Unfamiliar) during the social novelty test stage; I is the time spent by the tested mice in smelling the mouse and the object during the social behavior test stage; J is the time spent by the tested mice in smelling the familiar mouse and the unfamiliar mouse during the social novelty test stage (n = 12, litter).

[0031] The bar chart is expressed as mean ± standard error of the mean, and the individual data values of each sample in the group are marked in the form of scatter points in the bar chart. The "n" of all experiments is represented as the mother mouse or the fetal mouse from different mother mice, i.e. "litter". The difference between two groups of samples is evaluated using a two-tailed Student's t-test; the difference between three or more groups with only one variable is evaluated using one-way ANOVA with Dunnett's post-hoc test; the difference between groups with two or more variables is evaluated using two-way ANOVA with Bonferroni or Tukey post-hoc test. When the P value of the data is less than 0.05, the difference is considered to be significant. ns represents not significant, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. DETAILED DESCRIPTION

[0032] The application will be further illustrated by the following examples and figures. It should be understood that these examples are only used to illustrate the application, and are not used to limit the scope of the application. The operation methods not specified in the following examples are generally carried out according to the conventional conditions, or according to the conditions recommended by the manufacturers.

[0033] Example 1 Placental interface immune function regulator restores immune homeostasis at the placental interface in the VPA exposure model during pregnancy

[0034] 1.1 Modeling of VPA exposure during pregnancy and drug regulation

[0035] Female and male mice were caged together in a 2:1 ratio the previous afternoon. The next morning, the presence of a vaginal plug confirmed successful mating and was recorded as day 0.5 of pregnancy (E0.5). On embryonic day (E12.5), pregnant mice received a subcutaneous injection of 600 mg / kg sodium valproate (VPA) or an equivalent amount of saline (control) in the neck. On E13.5, VPA model pregnant mice in each drug group received a single subcutaneous injection of 0.05 mg / kg RU486 (grouped as VPA+RU486), 0.5 mg / kg Taprenepag (grouped as VPA+Tap), 0.5 mg / kg Kynurenine (grouped as VPA+Kyn), or 0.5 mg / kg Butoprost (grouped as VPA+But) to evaluate the regulatory effect of each drug on placental IDO-1 expression. VPA and control group pregnant mice received an equivalent amount of saline on E13.5. Twenty-four hours after administration, the pregnant mice were sacrificed, and the placentas and fetuses were collected for subsequent experiments.

[0036] 1.2 Maternal-fetal interface immune function regulator upregulates placental IDO-1 expression and regulatory pathways

[0037] Indoleamine 2,3-dioxygenase (IDO-1) is an important immune regulatory site in the placenta. Maternal-fetal interface immune disorders are associated with downregulation of placental IDO-1. The function of IDO-1 is to oxidize L-tryptophan to kynurenine (Kyn), which is involved in the maintenance of immune tolerance in the placenta. Decidual M2 macrophages are an important cell group that plays an immune inhibitory role at the maternal-fetal interface, and arginase-1 is a marker of decidual M2 macrophages (Arg-1). Western blotting was used to determine the expression of IDO-1 and Arg-1 in the placenta of each experimental group to evaluate the effect of the above drugs (RU486, Tap, Kyn, and But) on upregulating placental IDO-1 expression and the total amount of decidual M2 macrophages.

[0038] Western-blotting procedure: placental tissue was extracted, and the samples contained the same concentration of total protein. The samples were separated on an 8-15% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), transferred (U=100 V, 1 h), blocked with 5% skim milk (prepared with 1x PBS) for 1 h, washed, incubated with the corresponding primary and secondary antibodies, and exposed and developed. β-actin was used as an internal reference protein. The primary antibodies used were as follows: β-actin (Multi sciences, ab36861), IDO-1 (Rockland, ME, USA, 200-301-E57S), and Arg-1 (Proteintech, 16001-1-AP).

[0039] Results showed that among all the therapeutic drugs, the placental IDO-1 expression level was most significantly up-regulated in the Tap or RU486 administration groups (A and C in Figure 1 As shown in B in Figure 1 As shown in B in

[0040] 1.3 Modulators of immune function at the maternal-fetal interface activate COX-2 / PGE2 / PTGER-2 pathway to regulate placental IDO-1 expression

[0041] According to the method in 1.2, Western blotting was used to determine the expression of cyclooxygenase-2 (COX-2) and prostaglandin E2 receptor (PTGER-2) in the placenta of each IDO-1 up-regulating drug (RU486, Tap, Kyn and But) group, to evaluate the regulatory effect of VPA modeling and drug treatment on the COX-2 / PGE2 / PTGER-2 pathway. β-actin was used as an internal reference protein. The primary antibodies used included: β-actin (Multi sciences, ab36861), COX-2 (Cell Signaling Technology, 12282) and PTGER-2 (Abeam, ab167171).

[0042] ELISA was used to determine the content of prostaglandin E2 (PGE2) in the placenta of the IDO-1 up-regulating drug (RU486 and Tap) group, to evaluate the regulatory effect of VPA modeling and RU486 and Tap administration on the COX-2 / PGE2 / PTGER-2 pathway. PGE2 ELISA kit was used to determine the PGE2 level of the placental homogenate solution of each experimental group, and BCA protein determination kit was used to quantify the total protein level of the tissue. Placental PGE2 was expressed as the PGE2 content per unit of total protein.

[0043] As shown in D-F in Figure 1 As shown in D-F in Figure 1 As shown in G and Figure 1H) in FIG. 1. Compared with the VPA model group, the expression of placental glycosylated COX-2 was significantly increased in the Tap treatment group Figure 1 G) in FIG. 1. Compared with the VPA model group, the expression of placental glycosylated COX-2 was significantly increased in the Kyn treatment group Figure 1 G) in FIG. 1. Compared with the VPA model group, the expression of placental glycosylated COX-2 was significantly increased in the Kyn treatment group Figure 1 E, F and I) in FIG. 1. The results showed that the positive feedback loop COX-2 / PGE2 / PTGER-2 was significantly inhibited in the placenta of VPA-exposed pregnant mice, leading to the down-regulation of placental IDO-1 and the disorder of the maternal-fetal interface. RU486 and Tap can activate the positive feedback loop COX-2 / PGE2 / PTGER-2 and up-regulate the expression of placental IDO-1, restoring the homeostasis of the maternal-fetal interface. Kyn also has a certain effect on activating the positive feedback loop COX-2 / PGE2 / PTGER-2. Tap is a prostaglandin receptor EP2 (PTGER-2) agonist, while But is a prostaglandin receptor EP4 (PTGER-4) agonist. Therefore, But has a poor effect on up-regulating the pathway and IDO-1. Figure 1 Figure 1 1.4 Maternal-fetal interface immune function regulator restores the immune homeostasis of the maternal-fetal interface

[0044] Flow cytometry was used to analyze the changes in the number of placental immune cell populations in the maternal-fetal interface immune function regulator (RU486 and Tap) groups to evaluate the effect of the maternal-fetal interface immune function regulator (RU486 and Tap) on restoring the immune homeostasis of the maternal-fetal interface. The VPA-exposed pregnant mice model and the administration of RU486 and Tap were completed according to the method in 1.1. Flow cytometry was used to analyze the changes in the number of placental immune cell populations in the VPA-exposed pregnant mice model to evaluate the immune function of the placenta in the VPA-exposed model. The placental immune cells included decidual macrophages dM, decidual natural killer cells dNK and T cells.

[0045] Flow cytometry was used to analyze the changes in the number of placental immune cell populations in the maternal-fetal interface immune function regulator (RU486 and Tap) groups to evaluate the effect of the maternal-fetal interface immune function regulator (RU486 and Tap) on restoring the immune homeostasis of the maternal-fetal interface. The VPA-exposed pregnant mice model and the administration of RU486 and Tap were completed according to the method in 1.1. Flow cytometry was used to analyze the changes in the number of placental immune cell populations in the VPA-exposed pregnant mice model to evaluate the immune function of the placenta in the VPA-exposed model. The placental immune cells included decidual macrophages dM, decidual natural killer cells dNK and T cells.

[0046] ​Placentas from VPA-exposed pregnant rats (E14.5 group) were surgically cut into 2–4 mm fragments, digested in DMEM medium containing Accutase at 37°C for 45 min, filtered through a 300-mesh cell sieve, lysed with erythrocyte lysis buffer for 10 min, centrifuged, and resuspended to obtain single-cell suspensions. The dispersed decidual single-cell suspensions were stained with live / dead distinguishing dyes, fixed with 4% paraformaldehyde (wt. / vol.), and blocked with 10 mg / mL BSA. Flow cytometry was used to determine the population numbers of placental immune cells (dM, dNK, and T cells) in each group. PE-CD45, FITC-CD68, APC-CD3, and Percp / Canine 5.5-CD49a staining were performed, followed by flow cytometry sorting using CytoFLEX LX and analysis using CytExpert software. The antibodies used are as follows: PE-coupled CD45 (Biolegend, Cambridge, UK, 30-F11), APC-coupled CD3 (Biolegend, 145-2C11), PerCP / Cyanine 5.5-coupled CD49a (Biolegend, HMα1) and FITC-coupled CD68 (Biolegend, FA-11).

[0047] The results are as follows Figure 2 As shown in Figure A, compared with the VPA model group (15.10%), the percentage of placental dM cells in the RU486 and Tap groups increased to 26.61% and 25.44%, respectively; compared with the VPA model group (10.82%), the percentage of placental dNK cells in the RU486 and Tap groups increased to 26.50% and 24.01%, respectively; and compared with the VPA model group (9.7%), the percentage of placental T cells in the RU486 and Tap groups recovered to 3.23% and 3.50%, respectively. These results indicate that administration of RU486 or Tap can reverse the maternal-fetal interface immune dysregulation induced by VPA exposure.

[0048] Flow cytometry analysis of the placental decidual macrophage cell types in the maternal-fetal interface immune function regulator (RU486 and Tap) group to evaluate the effect of the maternal-fetal interface immune function regulator (RU486 and Tap) on regulating the population number of placental M2 decidual macrophages (dM2) and M1 decidual macrophages (dM1). According to the method of 1.1, the modeling of VPA-exposed pregnant mice and the administration of each IDO-1 up-regulating drug (RU486 and Tap) were completed. According to the method of 1.2, the dispersed placental single cell suspension was prepared, and the population number of placental M2 decidual macrophages (dM2) and M1 decidual macrophages (dM1) in each group was determined by flow cytometry. PE-CD45, FITC-CD68, APC-Cyanine7 CD86 and Alexa Fluor647 CD206 staining. CytoFLEX LX was used for flow cytometry sorting, and CytExpert software was used for analysis. The antibodies used are as follows: PE-coupled CD45 (Biolegend, Cambridge, UK, 30-F11), FITC-coupled CD68 (Biolegend, FA-11), APC-Cyanine7 CD86 (Biolegend, GL-1) and Alexa Fluor647 CD206 (BD Pharmingen, 565250).

[0049] The results are shown in B of Figure 2 The percentage of dM2 cells in the VPA modeling group was 3.51%, and after administration of RU486 or Tap, it was restored to 15.06% and 10.18%, respectively; the percentage of dM1 cells in the VPA modeling group was 15.26%, and after administration of RU486 or Tap, it was reduced to 4.43% and 4.79%, respectively. The results show that compared with the VPA exposure group, the administration of RU486 or Tap reduces the excessive increase of dM1 cells, restores the number of dM2, and can improve the maternal-fetal interface immune disorder and regulate the recovery of the maternal-fetal interface homeostasis.

[0050] Example 2 Maternal-fetal interface immune function regulator improves maternal-fetal barrier function in VPA exposure model during pregnancy

[0051] 2.1 Maternal-fetal interface immune function regulator improves amniotic epithelial barrier tight junction morphology in VPA exposure model during pregnancy

[0052] Scanning electron microscope (SEM) was used to observe the ultrastructure of the tight junction complex in amnion epithelial cells. The amnion epithelium, which is in direct contact with amniotic fluid, is the first barrier to separate the amniotic fluid from the maternal tissue. The tight junction complex, which is composed of claudin-3, claudin-4 and occludin, is highly expressed in amnion epithelium and involved in the maintenance of amnion barrier function. The pregnant mice were exposed to VPA and the immunomodulators (RU486 and Tap) were administered according to the method 1.1. Fresh amnion (E14.5) was collected and fixed with 2% glutaraldehyde, washed, dehydrated, critical point dried and coated with platinum. The tight junction complex in amnion epithelium was observed by SEM.

[0053] The results are shown in A of FIG. 12. The tight junction complex structure of amnion epithelial cells in the VPA group was severely damaged and gradually decomposed, and the boundary between epithelial cells could not be clearly distinguished. The administration of RU486 or Tap could significantly improve the damage of amnion barrier caused by VPA exposure during pregnancy. Figure 3

[0054] 2.2 Immunomodulators at the maternal-fetal interface improve the morphology of tight junctions in placental syncytiotrophoblast barrier in the model of VPA exposure during pregnancy

[0055] Transmission electron microscope (TEM) was used to observe the ultrastructure of the tight junctions (Tjs) in placental syncytiotrophoblast cells (SCT). The pregnant mice were exposed to VPA and the immunomodulators (RU486 and Tap) were administered according to the method 1.1. Fresh placenta (E14.5) was collected and 2 mm placental tissue was taken from the labyrinth layer, fixed with 2% glutaraldehyde and 1% osmium tetroxide, dehydrated with ethanol, embedded in epoxy resin, and the sections were observed by transmission electron microscope. The blood-placenta barrier in mice is composed of three layers of syncytiotrophoblasts, with the outermost layer of syncytiotrophoblast cells forming the maternal blood sinus (MS), which separates the fetal mouse cells from the maternal blood and is the most critical barrier cell, which can be distinguished by the presence of numerous microvilli on the maternal side; the inner layer is covered by another two layers of syncytiotrophoblast cells. Fetal-derived endothelial cells and nucleated red blood cells are in direct contact with the inner layer of syncytiotrophoblasts.

[0056] The outermost layer of SCT has a large number of microvilli, which are in direct contact with the maternal blood and are the key components of the maternal-fetal barrier. TEM was used to observe the ultrastructure of the Tjs in placental syncytiotrophoblast cells (SCT). The results are shown in FIG. 13. Figure 3 ​Figure 1C-E shows that compared with the VPA model group, the expression levels of placental tight junction proteins claudin-4 and claudin-5 were significantly increased by administration of RU486 or Tap, and the expression level of placental tight junction protein claudin-4 was also increased by administration of But, indicating that the immune function regulators of the maternal-fetal interface can restore the pathological maternal-fetal barrier.

[0057] 2.3 The immune function regulators of the maternal-fetal interface up-regulate the expression of tight junction proteins in placental trophoblast cells in the VPA exposure model during pregnancy

[0058] The VPA exposure model of pregnant mice and the administration of each IDO-1 up-regulating drug (RU486, Tap, Kyn, and But) were completed according to the method of 1.1. The expression of placental tight junction proteins claudin-4 and claudin-5 was determined by Western blotting according to the method of 1.2, and the effect of the IDO-1 up-regulating drug on restoring the function of the maternal-fetal barrier was evaluated. β-actin was used as an internal reference protein. The primary antibodies used were as follows: β-actin (Multi sciences, ab36861), claudin-5 (Invitrogen, 35-2500), and claudin-4 (Abeam, ab53156).

[0059] The results are shown in Figure 1C-E. Figure 3 Figure 1C-E shows that compared with the VPA model group, the expression levels of placental tight junction proteins claudin-4 and claudin-5 were significantly increased by administration of RU486 or Tap, and the expression level of placental tight junction protein claudin-4 was also increased by administration of But, indicating that the immune function regulators of the maternal-fetal interface can restore the pathological maternal-fetal barrier.

[0060] Example 3: The immune function regulators of the maternal-fetal interface improve the neurological developmental disorders of VPA model offspring mice

[0061] E12.5 pregnant mice were injected subcutaneously in the neck with 600 mg / kg of sodium valproate (VPA) or saline (control). E13.5 VPA model pregnant mice were injected subcutaneously with a single dose of each drug, including 0.05 mg / kg RU486 (VPA+RU486) and 0.5 mg / kg Tap (VPA+Tap), to investigate the regulatory effect of each drug on the neurological developmental disorders of the fetal mice. The pregnant mice in the VPA and control groups were injected subcutaneously with an equal amount of saline at E13.5. Twenty-four hours after administration, the pregnant mice were sacrificed and the fetal mice were collected for subsequent experiments.

[0062] Immunofluorescence assays were performed on the TPH-2 nuclei in each drug-treated group. + Number of neurons and Netrin-G1a in the forebrain + Fluorescence positive signal intensity was used to examine the developmental status of serotonergic neurons and thalamocortical axons (TCAs) in the brains of fetal rats in each drug-treated group. Tryptophanhydrolase-2 (TPH-2) is a key enzyme for the synthesis of 5-HT in the brain and a marker of serotonergic neurons. Netrin-G1a is a glycosylphosphatidylinositol connexin and a marker of developing thalamocortical axons. Brain tissue (E14.5) from each group of fetal rats was fixed in 4% paraformaldehyde (wt. / vol.) for 48 h, embedded in paraffin, sectioned (10 μm thick), blocked with 10% BSA, incubated overnight at 4°C with primary antibody, and incubated for 2 h at room temperature with secondary antibody. Confocal microscopy was used for observation and imaging, and Image J semi-quantitative analysis was performed. The primary antibodies used include TPH-2 (Abcam, ab184505) and Netrin-G1a (R&D Systems, AF1166).

[0063] TPH-2 in the raphe nuclei of each drug-treated group was measured by tissue immunofluorescence. + The number of neurons was examined to assess the developmental status of serotonergic neurons in the brains of fetal rats in each drug-treated group. In fetal rats (E14.5) with a VPA exposure model during pregnancy, the number of serotonergic neurons in the dorsal raphe (DR) and median raphe nuclei (MRN) was significantly reduced. Figure 4 (A) Tap administration can upregulate VPA exposure-induced TPH-2. + Downregulation of neuronal number was more effective than upregulation with RU486. The results indicate that Tap administration upregulates TPH-2 in the raphe nucleus. + Reduce the number of neurons and decrease serotonergic neuronal damage caused by VPA exposure.

[0064] Immunofluorescence assay of Netrin-G1a in the forebrain of each drug-treated group + Fluorescence positive signal intensity was used to examine the developmental status of TCAs (thalamic cortical axons) in the thalamus of fetal rats in each treatment group. It has been reported that 5-HT can convert the attraction of netrin-1 to TCAs into a repulsive force, regulating TCA development in the embryonic mouse brain. Compared with the control group, VPA-exposed fetal rats showed reduced immunoreactivity in the dorsal thalamic (DT) region of the Netrin-G1a thalamus and lacked longer Netrin-G1a... + Axon bundle formation ( Figure 4Figure 3.2. The number of TPH-2 positive neurons in the MRN of VPA model offspring was decreased and the 5-HT content was reduced, indicating the damage of serotonergic neurons. Tap treatment increased the number of TPH-2 positive neurons and the 5-HT content in the MRN (A, B and C), indicating that Tap treatment could play a role in protecting the damage of serotonergic neurons. + The number of axon bundles was almost unchanged. Compared with the VPA model group, RU486 partially restored the number of TCAs in the DT, and TCAs in the IC and CP still showed dysplasia, with almost no axon bundle formation. Tap treatment could promote the formation of TCAs in the dorsal thalamus and promote TCAs to project through the internal capsule and reach the caudate nucleus. The results showed that excessive exogenous 5-HT in the fetal rat brain mediated the damage of serotonergic neurons and the degeneration of thalamocortical axons in the offspring of the VPA exposure model during pregnancy. Heptapリン Tap treatment alleviated the VPA-induced neurodevelopmental disorder in fetal rats.

[0065] 3.2 Maternal-fetal interface immune function regulator improves the development of raphe serotonergic neurons in VPA model offspring juvenile mice

[0066] According to the method of 1.1, VPA modeling and administration of heptapリン were completed. That is, E12.5 pregnant mice received a subcutaneous injection of 600 mg / kg valproate water (VPA) or normal saline (control). E13.5 VPA model pregnant mice received a subcutaneous injection of 0.5 mg / kg (VPA+Low Tap) to investigate the effect of heptapリン on promoting the development of neurons in offspring autistic juvenile mice. VPA and control groups of pregnant mice received the same amount of saline at E13.5. Pregnant mice were kept to breed offspring mice. The birth date of the offspring mice was recorded as P0. The offspring mice were used for subsequent behavioral analysis and immunohistochemical examination. According to the method of 3.1, the number of TPH-2 positive neurons and 5-HT content in the raphe nucleus of the offspring juvenile mice (P10) were detected by immunohistochemistry to investigate the development status of serotonergic neurons in the offspring mice of each experimental group. The primary antibodies used are as follows: TPH-2 (Abeam, ab184505) and 5-HT (Sigma-Aldrich, S5545).

[0067] As shown in Figure 5 , the number of TPH-2 positive neurons in the MRN of VPA model juvenile mice was decreased and the 5-HT content was reduced, indicating the damage of serotonergic neurons. Tap treatment increased the number of TPH-2 positive neurons and the 5-HT content in the MRN (A, B and C), indicating that Tap treatment could play a role in protecting the damage of serotonergic neurons. + + Figure 5

[0068] 3.3 Maternal-fetal interface immune function regulator improves the development of raphe serotonergic neurons in VPA model offspring adult mice

[0069] ​​​Following method 1.1, VPA modeling and tarepag administration were performed. Specifically, pregnant mice at E12.5 received a subcutaneous injection in the neck with 600 mg / kg valproate solution (VPA) or saline (control). Pregnant mice at E13.5, also VPA-modeled, received subcutaneous injections of either a low dose (0.5 mg / kg, VPA + Low Tap) or a high dose (2.5 mg / kg, VPA + High Tap) to investigate the effect of different doses of tarepag on neuronal development in the brains of offspring autistic adult mice. Pregnant mice in the VPA and control groups received the same volume of saline at E13.5. The pregnant mice were retained to breed offspring mice. The birth date of the offspring mice was recorded as P0. The offspring mice were used for subsequent behavioral analysis and immunohistochemical examination. Following method 3.1, immunohistochemistry was used to detect the number of TPH-2 positive cells and 5-HT content in the raphe nuclei of offspring pups (P55) to examine the developmental status of serotonin neurons in each experimental group of offspring mice. The primary antibodies used are TPH-2 (Abcam, ab184505) and 5-HT (Sigma-Aldrich, S5545).

[0070] like Figure 6 As shown in A and B, compared with the control group, the VPA group offspring mice had higher levels of TPH-2 in their MRN. + The number of neurons was significantly reduced, consistent with the results measured in the E14.5 fetal rat brain. Figure 5 The presence of C in the C indicates that damage to serotonergic neurons in VPA-induced progeny mice occurs during the embryonic period and persists after birth and into adulthood. TPH-2 is a key enzyme in serotonin synthesis; the damage to serotonergic neurons in VPA-induced progeny mice leads to a significant decrease in 5-HT levels in the MRN. Figure 6 (C in the text). The results showed that ASD-related behavioral abnormalities in VPA-modeled progeny mice were associated with damage to serotonergic neurons and decreased serotonin levels in the raphe nucleus (MRN). Compared to the VPA group, low-dose and high-dose Tap treatment increased the number of serotonergic neurons in the MRN (C in the text). Figure 6 (B in the text). Low doses of Tap more significantly upregulated the 5-HT content in the MRN of VPA-induced progeny mice. Figure 6 (C in the middle).

[0071] Example 4: Treatment of Autism in Offspring with Maternal-Fetal Interface Immune Function Modifiers During Pregnancy 4.1 Improving Communication Abilities in Offspring Rats by Administering Maternal-Fetal Interface Immune Function Modifiers During Pregnancy

[0072] The VPA modeling and Tap administration were performed according to the method of 1.1. That is, E12.5 pregnant mice were injected subcutaneously with 600 mg / kg valproic acid (VPA) or normal saline (control) in the neck. E13.5 VPA modeling pregnant mice were injected subcutaneously with low-dose 0.5 mg / kg (VPA+Low Tap) or high-dose 2.5 mg / kg Taprenepag (VPA+High Tap), respectively, to investigate the therapeutic effect of different doses of Tap on offspring autistic mice. The VPA group and the control group of pregnant mice received the same amount of saline at E13.5. The pregnant mice were kept to breed offspring mice. The birth date of the offspring mice was recorded as P0. The separation-induced ultrasonic vocalization of neonatal pups (male and female) was examined at P3, P5, P7, P9 and P11 to evaluate the communication ability of the pups. The test cage was placed in a soundproof room at 25°C and adapted for 15 min. During the test, the tested pups were taken out of the nest and placed in a 10 cm glass cup wrapped in a soundproof box. The number of ultrasonic vocalizations (USV) of the tested mice within 3 min was detected by UltraSoundGate CM16 / CMPA microphone, and the software AviSoft SASLab Pro (v5.30) was used for analysis. According to the literature report, the acquisition parameters were set as: Hamming window (fast Fourier transform), 256 FFT Length, 100% frame and 50% overlap. The USV recording frequency was 33-125 kHz, and the shortest vocalization duration was 1 ms. All the USV of the pups were counted and analyzed manually by two independent operators who were unaware of the experimental grouping.

[0073] The results are shown in A of Figure 7 Compared with the control group, the number of ultrasonic vocalizations of the VPA group pups decreased significantly during the 5 tests after separation from the mother, indicating that the VPA group pups had communication defects. Compared with the VPA group, the number of ultrasonic vocalizations of the low-dose and high-dose Tap treatment groups of pups increased significantly, indicating that the communication tendency was normalized but not yet restored to normal (lower than the control group). The results showed that low-dose and high-dose Tap treatment could improve the communication behavior of VPA modeling pups.

[0074] Open field test: Adult male offspring (P45) were allowed to freely explore in a 50×50×40 cm 3 for 10 min to evaluate the motor ability and anxiety-like behavior of the tested mice. A camera placed directly above recorded the movement trajectory of the tested mice, and the ANY-maze software (Wood Dale, IL, USA) was used for analysis. The total path length (cm) of the movement of the tested mice was used as an indicator of their motor ability. The central area (17 cm2 Dwell time is used as an indicator to assess anxiety-like behaviors.

[0075] Self-grooming behavior experiment: Self-grooming behavior in rodents is a pathological, spontaneous, repetitive behavior. The self-grooming behavior of adult male offspring (P48) was statistically analyzed to evaluate the repetitive and stereotyped behavior of the tested mice. Self-grooming behavior manifests as stroking, scratching, or licking any part of the body, including the face, head, or upper body, with the forepaws. After a 20-minute adaptation period, the size of each mouse (50×25×20cm) was measured using a stopwatch. 3 The cumulative time spent self-organizing in the box lasts 10 minutes.

[0076] Bead embedding test: The bead embedding test assesses induced repetitive behaviors in rodents. Adult male offspring (P51) were transferred to a new test enclosure (40×20×30cm). 3 The bedding was 4 cm deep; 4×5 marbles were placed at equal intervals in the test area. After the experiment, the mice were gently removed from the test area, and the number of marbles buried within 15 minutes was recorded.

[0077] Three-box social test: The three-box social test evaluates the social behavior of mice. The test consists of three phases. In the first phase, the male mouse (P54) was placed in a black box (20×40.5×22cm) with three interconnected chambers. 3 The mice were acclimatized for 10 minutes in each lateral cavity, with a hollow circular metal cage placed in the corner of each cavity. In the second social phase (Sociability phase), the male mice were allowed to freely explore the testing area for 10 minutes. One metal cage contained a new male, age-matched mouse, while the other contained a new cubic metal object. In the third social novelty phase, the male mice had the opportunity to explore both the familiar and unfamiliar male, age-matched mice from the previous social phase. ANY-maze software recorded the time the mice spent in each room and the time they spent sniffing each cage. The social ability of the mice was measured by the difference in time spent in the mouse and object rooms, or the difference in time spent sniffing mice and objects. The social novelty of the mice was measured by the difference in time spent in the familiar and unfamiliar mouse rooms, or the difference in time spent sniffing familiar and unfamiliar mice.

[0078] Open field assays were used to evaluate the motor function and anxiety-like behavior of offspring mice. Compared with the control group, offspring mice in the VPA group spent significantly less time in the central region of the open field. Figure 7 (B) indicates increased anxiety-like behavior in offspring mice in the VPA group. Compared to the VPA group, the time spent in the open field center region was not significantly different in the low-dose and high-dose Tap groups, indicating that Tap administration was not effective in alleviating anxiety symptoms in VPA-exposed offspring mice. Compared to the control group, there were no significant changes in motor function in offspring mice across all VPA groups. Figure 7 (C in the text). The results showed that low-dose and high-dose Tap treatment did not improve anxiety behavior in VPA-modeled offspring mice.

[0079] The self-grooming behavior test and bead embedding test were used to evaluate repetitive and stereotyped behaviors in offspring mice. Compared with the control group, the cumulative time of self-grooming behavior in the offspring mice of the VPA group ( Figure 7 D in the middle) and number of times ( Figure 7 The significantly increased E in the VPA group indicated that offspring mice exhibited more repetitive and stereotyped behaviors. Both high- and low-dose Tap treatment reduced the cumulative time of self-grooming behavior in VPA-modeled offspring mice, but low-dose Tap was more effective in reducing the frequency of self-grooming behavior in VPA-modeled offspring mice. Compared to the control group, VPA-modeled offspring mice were more engrossed in burying marbles (…). Figure 7 Both high- and low-dose Tap treatments reduced bead-burying behavior in VPA-modeled mice. The results showed that low-dose Tap treatment was more effective in reducing repetitive and stereotyped behaviors in VPA-modeled offspring mice.

[0080] The three-box socialization test assesses the social behavior and social novelty of offspring mice. In the social behavior test, normal test mice generally spent more time in the mouse's "mouse" room than in the object's "object" room. Figure 7 (G in the middle); the normal test mice also spent more time smelling other mice than smelling objects. Figure 7 In the I) group, the offspring mice exhibited a preference for interacting with mice rather than objects. Compared to the control group, VPA-induced offspring mice spent longer time in the object room (“Object”) than in the mouse room (“Mouse”), while the time spent smelling mice was not significantly different from the time spent smelling objects, indicating abnormal social behavior in VPA-induced offspring mice and a tendency towards social avoidance and autism. Both low-dose and high-dose Tap improved the social deficits in VPA-exposed offspring mice and restored the mice's preference for interacting with each other. In the social novelty test, such as Figure 7 H and ​As shown in J, the offspring of the control group or the VPA+Low Tap group were more likely to interact with unfamiliar mice than familiar mice. However, the offspring of the VPA group or the VPA+High Tap group showed ambiguous preference for familiar mice and unfamiliar mice. The results show that low-dose Tap treatment during pregnancy can improve the communication ability, repetitive stereotypy, social behavior and social novelty of the offspring of the VPA model mice.

[0081] The above-described embodiments of the present application are described in detail, it should be understood that the above-described only for the specific embodiments of the present application, and not for limiting the present application, any modification, supplement or similar way of replacement, etc. made within the scope of the principles of the present application, should be included within the scope of the present application.

Claims

1. Use of a compound of structural formula (I) or a pharmaceutically acceptable salt thereof as a prostaglandin receptor PTGER-2 agonist in the preparation of a regulator of immune function at the maternal-fetal interface; the regulator of immune function at the maternal-fetal interface is used for preventing and / or treating autism in offspring. （I）。 2. Use according to claim 1, characterized in that, The regulator of immune function at the maternal-fetal interface can promote the restoration of immune homeostasis at the maternal-fetal interface and / or improve the function of the maternal-fetal barrier.

3. Use according to claim 1, characterized in that, The regulator of immune function at the maternal-fetal interface can improve the development of neurons in the brain of offspring.

4. Use according to claim 1, characterized in that, The regulator of immune function at the maternal-fetal interface further comprises a pharmaceutically acceptable carrier.

5. The use according to claim 1, characterized in that, The regulator of immune function at the maternal-fetal interface is administered during pregnancy.

6. Use according to claim 1, characterized in that, The regulator of immune function at the maternal-fetal interface is administered by intravenous injection, intraperitoneal injection, subcutaneous injection or oral administration.

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