Use of an agonist of a cAMP-activated exchanger protein for the preparation of a medicament for the treatment of preeclampsia

Abstract

The application discloses an application of an agonist of a cAMP-activated exchange protein in preparation of a drug for treating preeclampsia, and belongs to the technical field of biological medicines, wherein the drug is used for treating preeclampsia by activating a Rap1 signal pathway of human aortic smooth muscle cells inhibited by a RASA3 protein, and the agonist of the cAMP-activated exchange protein is 8-CPT-2'-O-Me-cAMP or a pharmaceutically acceptable salt or solvent mixture thereof. The application proves for the first time that the compound has a function of promoting migration and remodeling in vascular smooth muscle cells, and successfully applies the compound to the treatment of preeclampsia, a pregnancy-related vascular disease, thereby expanding a new use of the compound in the field of clinical treatment of preeclampsia.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. Specifically, it relates to the application of a cAMP-activated exchange protein agonist in the preparation of a drug for treating preeclampsia. Background Technology

[0002] Preeclampsia is a pregnancy-specific disorder characterized by new-onset hypertension and / or proteinuria after 20 weeks of gestation, imposing a heavy psychological, health, and economic burden on individuals, families, and society. Abnormal maternal-fetal interface interaction is central to the development of preeclampsia, and impaired spiral artery remodeling in the uterus is the pathological basis, although the specific pathogenesis remains unclear. Spiral artery remodeling involves multicellular interactions, and trophoblast invasion is closely related to further disintegration of the vascular smooth muscle layer. Escape from vascular smooth muscle cells and reduced apoptosis are important factors mediating impaired spiral artery remodeling and the occurrence of preeclampsia.

[0003] Numerous studies (such as large RCTs like ASPRE) have shown that for pregnant women with high-risk factors (such as a history of preeclampsia, antiphospholipid syndrome, chronic hypertension, diabetes, or multiple pregnancies), starting daily low-dose aspirin (usually 75–150 mg / day) before 16 weeks of gestation can significantly reduce the risk of early-onset preeclampsia (by approximately 50–60%). The American College of Obstetricians and Gynecologists (ACOG), the World Health Organization (WHO), and the UK National Institute for Health and Care Excellence (NICE) all recommend low-dose aspirin for prevention in high-risk pregnant women. In recent years, several small-scale clinical studies and animal experiments have suggested that metformin may have therapeutic potential for existing preeclampsia (especially early-onset). For example, some studies have shown that metformin can prolong gestational age and improve maternal and infant outcomes. Its mechanism of action may go beyond simple glycemic control, targeting the core pathological processes of preeclampsia. Its safety during pregnancy (especially in the mid-to-late stages) has been well supported by data (it is often used to treat gestational diabetes), but more evidence is needed for its use in preventing and treating preeclampsia in non-diabetic pregnant women. Therefore, the development of other drugs is still needed to supplement or combine with preeclampsia treatment. While these approaches have some preventative effects, they cannot reverse placental vascular remodeling defects and have side effects such as fetal growth restriction and liver and kidney damage. Therefore, in-depth research into the molecular regulatory mechanisms of preeclampsia and the development and discovery of substances that improve vascular smooth muscle cell function and spiral artery remodeling are of great significance for the prevention and treatment of preeclampsia.

[0004] 8-CPT-2'-O-Me-cAMP (or 8-pCPT-2'-O-Me-cAMP) is an analogue of cyclic adenosine monophosphate (cAMP). As a specific agonist of the cAMP-activated exchanger protein (Epac), it can effectively activate the downstream Rap1 GTPase signaling pathway. In the field of ophthalmic disease research, this compound has been reported to have a positive effect on maintaining the function of the retinal pigment epithelium (RPE) barrier. For example, existing technologies (such as the article published by Wittchen ES et al.: Rap1 GTPase activation and barrier enhancement in rpe inhibits choroidal neovascularization in vivo, PLoSOne. 2013 Sep 10;8(9):e73070.) show that in a laser-induced choroidal neovascularization (CNV) animal model—which is often used to simulate the pathology of neovascular age-related macular degeneration (AMD)—8-CPT-2'-O-Me-cAMP can promote the recruitment and aggregation of connexins and cortical F-actin at RPE cell junctions by activating Rap1, thereby enhancing the continuity of cell junctions and barrier integrity. In vitro experiments further confirmed that the compound (e.g., treatment at a concentration of 250 µM for hours) can induce Rap1 activation and strengthen the RPE monolayer barrier, ultimately significantly inhibiting the invasion of pathological choroidal endothelial cells and the development of CNV lesions in in vivo models.

[0005] However, to date, no studies have reported or revealed the function, mechanism of action, or potential therapeutic value of 8-CPT-2'-O-Me-cAMP in vascular smooth muscle cell migration and spiral artery remodeling, or in preeclampsia. Summary of the Invention

[0006] This invention provides the application of a cAMP-activated exchange protein (Epac) agonist in the preparation of a drug for treating preeclampsia. Specifically, this invention reveals and verifies for the first time the application potential of the Epac-specific agonist, 8-CPT-2-O-Me-cAMP, in the treatment of preeclampsia. It achieves its therapeutic effect on preeclampsia by targeting and inhibiting RASA3 protein, activating the downstream Rap1 signaling pathway, improving spiral artery remodeling disorders, and restoring placental blood flow perfusion.

[0007] The present invention is achieved through the following technical solution: the application of a cAMP-activated exchange protein agonist in the preparation of a drug for treating preeclampsia, wherein the drug treats preeclampsia by activating the Rap1 signaling pathway in human aortic smooth muscle cells inhibited by RASA3 protein, wherein the cAMP-activated exchange protein agonist is 8-CPT-2'-O-Me-cAMP or a pharmaceutically acceptable salt or solvent mixture thereof.

[0008] Furthermore, the drug promotes actin remodeling in vascular smooth muscle cells and / or enhances the migration ability of vascular smooth muscle cells.

[0009] Furthermore, the drug can partially or completely reverse the inhibition of Rap1 activity and cell migration in human aortic smooth muscle cells caused by RASA3 overexpression.

[0010] Furthermore, the present invention also provides a medicament for treating preeclampsia by blocking the action of RASA3 protein, wherein, as described above, the medicament contains a therapeutically effective amount of a cAMP-activated exchange protein agonist, wherein the cAMP-activated exchange protein agonist is 8-CPT-2'-O-Me-cAMP or a pharmaceutically acceptable salt or solvent mixture thereof, which treats preeclampsia by activating Rap1 to block the inhibitory effect of RASA3 protein on human aortic smooth muscle cell migration and spiral artery remodeling.

[0011] Preferably, the concentration of the cAMP-activated exchange protein agonist in the drug is 10-100 μM.

[0012] Preferably, the drug further comprises a pharmaceutically acceptable carrier or excipient.

[0013] Preferably, the dosage form of the drug is an injection.

[0014] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) This invention utilizes the key role of RASA3 target regulation in the spiral artery remodeling disorder in preeclampsia, demonstrating that the Rap1 agonist 8-CPT-2'-O-Me-cAMP can effectively reverse vascular smooth muscle dysfunction caused by RASA3 overexpression and improve spiral artery remodeling. This mechanism of action differs from existing drugs that primarily rely on antiplatelet aggregation (such as aspirin) or metabolic regulation (such as metformin). This invention directly targets the core pathological link of vascular structural remodeling, intervening in disease progression at the etiological level by promoting the migration and reorganization of vascular smooth muscle cells, thereby providing a new pathologically specific target and strategy for the treatment of preeclampsia.

[0015] (2) This invention is the first to extend the application of a known compound (8-CPT-2'-O-Me-cAMP) to a new disease area. Existing technologies have only reported the use of this compound in ophthalmic disease research (such as macular degeneration), with its effects focused on enhancing the retinal epithelial barrier. This invention is the first to demonstrate that this compound has the function of promoting migration and remodeling in vascular smooth muscle cells, and has successfully applied it to the treatment of preeclampsia, a pregnancy-related vascular disease, thus expanding the new uses of this compound in the clinical treatment of preeclampsia.

[0016] (3) The drug of the present invention enhances the migration ability of vascular smooth muscle cells, helps to repair the damaged spiral artery structure, promotes the recovery of placental blood flow perfusion, and is expected to treat preeclampsia, and even achieve the treatment / reversal of its core pathological link - spiral artery remodeling disorder, filling the gap in the current clinical treatment of direct intervention against the cause. Attached Figure Description

[0017] Figure 1 The effect of different 8-CPT concentrations on the proliferation capacity of vascular smooth muscle cells.

[0018] Figure 2 The effects of altered RASA3 expression on vascular smooth muscle cell migration and Rap1 activity.

[0019] Figure 3 To rescue the inhibitory effect of RASA3 overexpression on vascular smooth muscle cell migration by 8-CPT.

[0020] Figure 4 The effect of 8-CPT on blood pressure measurements in a preeclampsia model mouse.

[0021] Figure 5 The effect of 8-CPT on 24-hour urinary protein levels in a preeclampsia model mouse.

[0022] Figure 6 The effect of 8-CPT on placental pathology in a mouse model of preeclampsia.

[0023] Figure 7 The effects of 8-CPT on liver and kidney pathology in a preeclampsia model rat.

[0024] Figure 8 The effect of 8-CPT on intrauterine development of offspring in a preeclampsia rat model.

[0025] Figure 9 The effect of 8-CPT on spiral artery remodeling in RASA3-overexpressing pregnant mice. Detailed Implementation

[0026] The invention's objective, technical solution, and beneficial effects will be further explained in detail below.

[0027] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the claimed invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0028] This invention reveals for the first time the crucial role of the RASA3 / Rap1 pathway in preeclampsia and proposes a novel strategy for treating preeclampsia by targeting this pathway with Rap1 agonists. Specifically, this invention provides the application of a cAMP-activated exchange protein agonist (i.e., a Rap1 agonist) in the preparation of a therapeutic drug for preeclampsia. This application, through targeted drug intervention, effectively restores vascular smooth muscle cell (HASMC) function and helical artery remodeling impairment, and provides a corresponding therapeutic drug that promotes actin remodeling in vascular smooth muscle cells and / or enhances their migration ability. Furthermore, this drug can partially or even completely reverse the weakened migration function of human aortic smooth muscle cells, helical artery remodeling impairment, and typical clinical features of preeclampsia (hypertension, proteinuria, and multi-organ pathological damage, etc.) caused by RASA3 overexpression. Based on the above mechanism of action, the drug of this invention can achieve the prevention and / or treatment of preeclampsia by improving helical artery remodeling impairment and restoring placental blood perfusion, providing a new solution for the etiological treatment of this disease.

[0029] In this invention, the agonist of the cAMP-activated exchange protein is 8-CPT-2'-O-Me-cAMP (hereinafter referred to as 8-CPT), with CAS number 510774-50-2, and its chemical structure is shown below: .

[0030] This invention systematically investigated the relationship between RASA3 overexpression inhibiting Rap1 activity and weakened HASMC cell migration through in vitro cell experiments and animal model construction. At the cellular level, experiments confirmed that RASA3 overexpression significantly inhibited HASMC migration and led to a decrease in downstream Rap1 activity. Treatment with 8-CPT increased Rap1 activity in a concentration-dependent manner and partially reversed the inhibitory effect of RASA3 overexpression on cell migration. At the animal level, a RASA3-overexpressing preeclampsia mouse model was successfully constructed. This model exhibited a pathological phenotype highly consistent with clinical findings, including persistently elevated blood pressure, significantly increased 24-hour urinary protein, placental developmental disorders, abnormalities in multiple serum biochemical indicators, glomerular structural damage, intrauterine growth restriction in fetuses, and impaired spiral artery remodeling. Intervention with the Rap1 agonist 8-CPT effectively reversed all of the above abnormal pathological indicators. The above results indicate that the Rap1 agonist 8-CPT can correct key pathological aspects of preeclampsia at the cellular and whole-animal levels by targeting and regulating the RASA3 / Rap1 signaling pathway. This suggests its potential for developing drugs to treat preeclampsia, thereby preventing and treating the condition and improving patients' quality of life and survival rate.

[0031] The specific implementation of the present invention will be described below with reference to the embodiments. Of course, the scope of protection of the present invention is not limited to the following embodiments.

[0032] Main reagents and materials: Methyl sulfoxide (DMSO), fetal bovine serum (Hyclone), Lipofectamine 3000 transfection kit (Invitrogen), PBS (Beyotime ST447), RIPA lysis buffer (Strong, P0013B) + PMFS (Beyotime), PVDF membrane, SPF-grade SD rats (Beijing Huafukang Biotechnology Co., Ltd.), RASA3 recombinant adenovirus vector (Shanghai Hanheng Biotechnology Co., Ltd.), 8-CPT-2'-O-Me-cAMP (MedChemExpress), rat PLGF and sFIt-1 kits (Shanghai Zhuocai Biotechnology Co., Ltd.), BCA kit (Beyotime Biotechnology Co., Ltd.), CCK8 detection kit (Abmole), Transwell chamber culture plates (Corning).

[0033] Main instruments: BP-98A Intelligent Non-invasive Blood Pressure Monitor for Rats (Beijing Ruanlong Biotechnology Co., Ltd.), SpectraMAX Microplate Reader (Meigu Molecular Instruments Co., Ltd.), BL-420S Biological Function Experiment System (Chengdu Taimeng Technology Co., Ltd.), PW480 Automatic Plate Washer (Shenzhen Huisong Technology Development Co., Ltd.), Rat Metabolic Cage (Animal Experiment Center, School of Basic Medical Sciences, Sichuan University), BS-200 Fully Automated Biochemical Analyzer (Shenzhen Mindray Bio-Medical Co., Ltd.), Scanning Electron Microscope (Olympus Corporation, Japan).

[0034] Example 1: In vitro cell experiments Cell Culture and Treatment: HASMC cells were routinely cultured in 1640 complete medium (10% FBS + 1% antibiotics). After cell confluence reached 80% or higher, cells were passaged using trypsin digestion. Cells in the logarithmic growth phase were seeded into wells after digestion. siRNA transfection was performed when cells reached approximately 60% confluence, and overexpression plasmid transfection was performed when cells reached approximately 80% confluence. Additionally, 10 μM 8-CPT was used for co-treatment with the RASA3 overexpression plasmid. HASMC cell proliferation was assessed using CCK8 assays; migration was assessed using Transwell culture.

[0035] The specific tests are as follows: (1) Detection of the proliferation of vascular smooth muscle cells by different concentrations of 8-CPT.

[0036] HASMC cells were seeded into 96-well plates with 100 μl of cell suspension (containing 2000 cells) per well. After cell attachment, the culture medium was discarded and replaced with serum-free 1640 medium, followed by starvation synchronization treatment for 12 h. The next day was designated day 0. 100 μl of prepared CCK8 assay solution (10 μl CCK8 reagent + 100 μl 1640 medium) was added to each well, and the cells were incubated for 2 h, until each well turned a different shade of brown. The OD value of each well was measured at 450 nm using a microplate reader. Subsequent assays were performed at 24 h, 48 h, and 72 h. Proliferation capacity was calculated using the formula: (OD experimental group - OD blank group) / (OD control group - OD blank group).

[0037] See test results Figure 1 .

[0038] The results of the CCK-8 experiment showed that different concentrations (1 μM, 10 μM, 50 μM, 100 μM) of 8-CPT treatment had no significant effect on the proliferation activity of vascular smooth muscle cells. However, at the 10 μM concentration, cell proliferation showed a certain upward trend, indicating that this concentration may have a potential promoting effect on cell activity.

[0039] (2) Effects of altered RASA3 expression on vascular smooth muscle cell migration and Rap1 activity After digesting cells in the logarithmic growth phase, count them and divide them into groups of 4 × 10⁴ cells per well. 5 Cells were seeded evenly in a star-shaped pattern in 6-well plates and cultured overnight. The next day, when the cells reached approximately 60% confluence, siRNA transfection was performed, and when they reached approximately 80% confluence, overexpression plasmid transfection was performed. RASA3 siRNA and pcDNA3.1(+) overexpression plasmid transfection were performed using the Lipo3000 transfection kit (ThermoScientific).

[0040] The experimental groups are as follows: Empty-vec (empty vector plasmid control); RASA3-vec (RASA3 overexpression); si-NC (negative control siRNA); si-RASA3 (RASA3 siRNA knockdown); si-RASA3-1# (RASA3 siRNA sequence number 1); si-RASA3-2# (RASA3 siRNA sequence number 2).

[0041] Western blotting was used to detect Rap1 activity in vascular smooth muscle cells (HSMCs), specifically changes in Rap1-GTP expression. First, Rap1 activity was assessed using a Rap1 activation pull-down kit (Thermo Scientific). After cell lysis, GST Ral ​​GDS-RBD fusion protein was added to precipitate activated Rap1. Protein A / G was added to adsorb the antigen-antibody conjugate, and then immunoblotting analysis was performed using a rabbit polyclonal antibody that recognizes Rap1 protein. Proteins from HASMCs were extracted using RIPA lysis buffer containing 1 mM benzyl sulfonyl fluoride (PMSF) (Beyotime, Shanghai, China). Protein concentration was then determined using a BCA protein assay kit (Beyotime), followed by heat denaturation. Each protein sample was separated by 10% SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. After blocking with 5% skim milk solution at room temperature for 1 hour, the membrane was incubated overnight with primary antibody at 4°C. The membrane was then diluted 1:1000 with Rap1 antibody (Thermo Fisher Scientific) and 1:10000 with β-actin antibody (AC004, Abclonal). The next day, the membrane was washed three times with TBST and then incubated with secondary antibody HRP goat anti-rabbit IgG (H+L) (AS014, Abclonal) diluted 1:50000. The antigen-antibody complex was detected using an enhanced ECL chemiluminescent substrate kit. Quantification of the immunoblot was performed using ImageJ software.

[0042] See test results Figure 2 .

[0043] Figure 2 Figure A shows the expression of RASA3 protein, including the Western blot band and its quantitative analysis results. As shown, both RASA3 siRNA sequences (si-RASA3-1# and si-RASA3-2#) can effectively silence the expression level of RASA3 protein in cells, while the overexpression plasmid significantly upregulated its expression level, indicating that the siRNA and plasmid used have good effects in cell transfection efficiency and target protein regulation.

[0044] Figure 2 Figure B shows the effect of RASA3 on the migration ability of HASMC cells. The results showed that, compared with the empty vector control group, RASA3 overexpression significantly inhibited HASMC cell migration, while RASA3 siRNA treatment significantly promoted cell migration.

[0045] Figure 2Tables C and D show the effects of RASA3 on actin remodeling and Rap1 activity in HASMC cells, respectively, and provide the corresponding Western blot bands and their quantitative data. Analysis indicates that RASA3 overexpression significantly inhibited actin remodeling (manifested as a decrease in the F-actin to G-actin ratio) and Rap1 activity, while RASA3 siRNA treatment promoted these processes.

[0046] Figure 2 In the mean squares, *: P<0.05, comparison between RASA3-vec (RASA3 overexpression) group and Empty-vec (empty vector plasmid) group; #: P<0.05, ##: P<0.01, comparison between si-RASA3 (RASA3 low expression) group and si-NC (siRNA negative control) group.

[0047] (3) Effect of RASA3 overexpression plasmid combined with 8-CPT on vascular smooth muscle cell migration.

[0048] Cell culture was performed using 24-well Transwell chambers (8 μm). HASMC cells transfected for 24 h were digested and counted. The cell pellet was resuspended in serum-free medium at a concentration of 8 × 10⁻⁶ cells / well. 3 200 μl of cell suspension was seeded into the upper chamber at a density of cells / well, and 600 μl of complete culture medium was added to the lower chamber. After incubation in an incubator for 24 h, the cells were carefully removed from the upper chamber, fixed with formaldehyde, and stained with 0.1% crystal violet. Microscopic observation was performed, with five different fields of view randomly selected from each well for imaging. ImageJ analysis was used to analyze the average cell number to assess cell migration ability.

[0049] The experimental groups are as follows: Empty-vec (empty vector plasmid control); RASA3-vec (RASA3 overexpression); 8-CPT (8-CPT processing); Vec-RASA3+8-CPT (RASA3 overexpression plasmid and 8-CPT combined treatment).

[0050] Measurement results are shown Figure 3 As shown in the figure, compared with the control group using an empty vector plasmid, RASA3 overexpression significantly inhibited the migration ability of vascular smooth muscle cells; while 8-CPT treatment alone significantly promoted cell migration. It is noteworthy that combining RASA3 overexpression with 8-CPT treatment can partially reverse the inhibitory effect of RASA3 overexpression on vascular smooth muscle cell migration.

[0051] Example 2: Animal Model Experiment Nine-week-old SD rats were selected and housed in a female-to-male ratio of 1:2. After pregnancy was confirmed, the pregnant SD rats were randomly divided into three groups of five rats each: Ad-GFP (vector control group), Ad-sFlt1 (positive control group), Ad-RASA3 (experimental group), and Ad-RASA3 + 8-CPT (drug treatment group). The rats were injected intravenously with 2×10⁻⁶ oz.rw. on day 9.5 of gestation. 9 PFU adenovirus or simultaneous intraperitoneal injection of the Rap1 agonist 8-CPT at 1.04 mg / kd.

[0052] Among them, Ad-GFP (vector control group) was a pregnant mouse injected with adenovirus carrying GFP fluorescence; Ad-sFlt1 (positive control group) was a pregnant mouse injected with adenovirus overexpressing sFlt1; Ad-RASA3 (experimental intervention group) was a pregnant mouse with adenovirus overexpressing RASA3; and Ad-RASA3 + 8-CPT (drug treatment group) was a pregnant mouse with adenovirus treated with a combination of Ad-RASA3 and 8-CPT.

[0053] Blood pressure was measured in pregnant mice on days 6, 9, 12, 15 and 17 of gestation. Urine was collected on days 9, 12, 15 and 17 for 24-hour urinary protein quantification.

[0054] On day 19 of gestation, invasive blood pressure was measured in pregnant mice, and urine was collected again to detect 24-hour urinary protein. Plasma was collected for analysis of biochemical indicators, PlGF, and sFlt-1. After weighing the placenta, placental, liver, and kidney tissues were taken for morphological staining and observation; at the same time, the number, weight, and length of pups were recorded.

[0055] The specific tests are as follows: (1) Changes in blood pressure in pregnant SD rats.

[0056] Blood pressure, including systolic blood pressure (SBP), diastolic blood pressure (DBP), and invasive blood pressure (IBP), was measured at different time points in four groups of pregnant SD mice: Ad-GFP (vector control group), sFlt1 (positive control group), Ad-RASA3 (experimental model group), and Ad-RASA3 + 8-CPT (drug treatment group). The changes in blood pressure were analyzed.

[0057] On days 6, 9, 12, 15, 17, and 19 of gestation, non-invasive blood pressure (including systolic and diastolic pressure) in the tail arteries of rats was monitored daily in the morning using a rat tail cuff system. The instrument was preheated to 36°C, and the rats were secured with a warming sleeve, exposing their tails. The pressure sensor was placed on the tail, in contact with the tail artery. The secured rat was placed in the designated position on the instrument. Inflation began after the pulse signal showed a regular waveform. When the pulse signal became a straight line, it indicated that blood flow in the tail artery was blocked, and deflation began. Blood pressure values ​​were directly obtained from the computer recording system. The measurement experiment was repeated three times, with a 1-minute interval between each measurement, and the mean was calculated.

[0058] Invasive blood pressure monitoring in rats was performed using carotid artery cannulation: Pregnant rats were anesthetized with 2% sodium pentobarbital solution (70 mg / kg) in GD19, fixed in a supine position on a surgical dissection board, and the skin was incised along the midline of the neck, with muscles dissected layer by layer to isolate the right carotid artery with a clear pulsation (avoiding damage to the adjacent vagus nerve). The pressure transducer and the thin tube of the carotid artery cannula were filled with 0.3% heparin, air bubbles were removed, and it was connected to the biomechanical experimental system (BL-420S). The distal end of the carotid artery was ligated with a thin suture, leaving a sliver of suture under the vessel. The arterial clamp was used to clamp the proximal end of the artery, as close to the heart as possible, and the artery was lifted by the forceps handle. A small incision was made in the arterial wall with ophthalmic scissors, and the cannula was inserted about 1 cm proximal through this incision. The cannula was fixed to the artery with silk suture and then fixed again to the upper part of the cannula. The arterial clamp was released, and the blood pressure waveform was observed, indicating successful cannulation. After the blood pressure stabilizes, invasive systolic and diastolic blood pressure are recorded every 10 minutes. If more than 3 records are made, the average value is calculated.

[0059] See measurement results Figure 4 .

[0060] Figure 4 Figure A shows the systolic blood pressure (SBP) measurements of pregnant SD mice on days 6, 9, 12, 15, and 17 of gestation. The results showed that the systolic blood pressure (SBP) of pregnant SD mice in the Ad-sFlt1 and Ad-RASA3 groups was significantly increased compared with that in the Ad-GFP and Ad-RASA3 + 8-CPT groups.

[0061] Figure 4 Figure B shows the diastolic blood pressure (DBP) measurements of pregnant SD mice on days 6, 9, 12, 15, and 17 of gestation. Compared with the Ad-GFP group and the Ad-RASA3 + 8-CPT group, the diastolic blood pressure (DBP) of pregnant SD mice in the Ad-sFlt1 and Ad-RASA3 groups tended to be higher.

[0062] Figure 4Figure C shows the invasive blood pressure (IBP) measurement results of SD pregnant mice on day 19 of gestation. The results showed that the invasive blood pressure (IBP) of pregnant mice in the Ad-sFlt1 and Ad-RASA3 groups was significantly increased; however, the blood pressure of SD pregnant mice in the Ad-RASA3 + 8-CPT group could be significantly reversed.

[0063] Figure 4 In the mean squares, **: P < 0.01, *: P < 0.05, compared with the Ad-GFP group; #: P < 0.05, compared with the Ad-RASA3+8-CPT group.

[0064] (2) 24-hour urine protein levels in SD pregnant mice.

[0065] Urinary protein levels in four groups of pregnant SD rats (Ad-GFP (vector control group), Ad-sFlt1 (positive control group), Ad-RASA3 (experimental model group), and Ad-RASA3 + 8-CPT (drug treatment group)) were measured at different time points, and their changes were analyzed. For the 24-hour urinary protein detection, rats were placed in metabolic cages at 9:00 AM the day before urine collection, allowing them free movement, food, and water. Urine was collected from urine cups at 9:00 AM the following day, and the urine volume was accurately recorded. One ml of urine sample was centrifuged at 1600 rpm for 10 minutes at 4°C, and the supernatant was aliquoted and labeled. Subsequently, the protein content in the urine was quantitatively determined according to the Beyotime BCA kit instructions.

[0066] See test results Figure 5 .

[0067] like Figure 5 As shown, compared with the Ad-GFP control group, the 24-hour urinary protein levels in pregnant mice in the Ad-sFIt1 group and the Ad-RASA3 group were significantly increased. However, after intervention with Ad-RASA3 and 8-CPT (Ad-RASA3 + 8-CPT group), the increase in urinary protein caused by RASA3 overexpression could be partially reversed.

[0068] (3) Pathological changes in multiple organs (placenta, liver, kidney) of pregnant SD mice On day 19 of pregnancy, the placenta, liver, and kidneys were fixed in 4% formaldehyde and embedded in paraffin.

[0069] The placenta and liver were stained with hematoxylin and eosin (HE): dehydrated, sectioned, and dewaxed to water for subsequent operations. After hematoxylin staining for 15 min, rinsed with tap water, separated in 1% hydrochloric acid ethanol until the section color turned light blue, then rinsed with tap water again. The sections were then placed in 0.6% ammonia solution to restore the blue color, rinsed with tap water, and counterstained in 0.5% eosin staining solution for 2 min. Subsequently, the sections were sequentially immersed in 95% ethanol I, 95% ethanol II, anhydrous ethanol I, and anhydrous ethanol II, and allowed to stand for 5 min; the sections were then sequentially immersed in xylene I and xylene II, and allowed to stand for 5 min. The sections were then removed, allowed to air dry thoroughly, and mounted with neutral resin.

[0070] MST staining of placenta: After dewaxing the sections to water, proceed with subsequent operations. Fix in Bouin's solution for 10 min, stain with Harris hematoxylin for 5 min, rinse with tap water for 2 min; differentiate with 0.5% hydrochloric acid alcohol for 30 s, rinse with tap water for 5 min; Masson's compound staining solution for 5 min, rinse briefly with 0.2% acetic acid aqueous solution; differentiate with 5% phosphomolybdic acid for 5 min, rinse briefly with 0.2% acetic acid aqueous solution; 2% aniline blue solution for 30 s, rinse thoroughly with anhydrous ethanol; air dry naturally, and mount with neutral resin.

[0071] Kidney tissue was stained with glycogen (PAS): After dewaxing the sections to water, subsequent procedures were performed. Sections were washed with ddH2O for 2 min; sections were oxidized in periodic acid solution for 10 min; stained with Schiff's reagent for 10 min; sections were washed with ddH2O for 5 min, repeated 3 times; hematoxylin staining was performed for 3 min for nuclear staining; sections were washed with ddH2O for 5 min, repeated 3 times; dehydration, clearing, and mounting were performed as above.

[0072] See the experimental results. Figure 6 .

[0073] like Figure 6 As shown, compared with the Ad-GFP group, the Ad-sFlt1 and Ad-RASA3 groups of pregnant SD mice showed reduced vascular branching in the placental labyrinth at day 19 of gestation, and a decreased area of ​​the labyrinth / sponge trophoblast. MST staining results showed collagen deposition in the placental villi trophoblasts of both the Ad-RASA3 and Ad-sFlt1 groups (indicated by the blue area indicated by the arrows in the figure); after combined treatment with 8-CPT, the aforementioned placental pathological damage was partially restored. HE staining of the liver showed interlobular portal vein necrosis and disordered arrangement of surrounding hepatocytes in the Ad-sFlt1 group, while no significant changes were observed in the Ad-RASA3 group. PAS staining of the kidneys showed renal tubular atrophy, interstitial edema, and endothelial cell swelling in pregnant mice in the Ad-sFlt1 group, which were partially improved after combined treatment with 8-CPT.

[0074] (4) Serum PlGF and sFlt-1 levels in pregnant SD mice.

[0075] When detecting PlGF and sFlt-1, follow the instructions and use ELISA kits (ZC-37237 and ZC-55018) to detect serum sFlt-1 and PlGF levels respectively.

[0076] See test results Figure 7 .

[0077] Figure 7 In the table, A represents serum sFlt-1 level; B represents serum PLGF level; and C represents serum sFlt-1 / PLGF level. ** indicates P < 0.01, and * indicates P < 0.05. These values ​​are compared with the Ad-GFP group.

[0078] like Figure 7 As shown, compared with the Ad-GFP group, the serum PLGF concentration in the Ad-sFlt1 group and the Ad-RASA3 group of pregnant SD mice was significantly reduced, while the serum sFlt-1 concentration and sFlt-1 / PlGF ratio were significantly increased. The PlGF level was partially increased after 8-CPT combined treatment.

[0079] (5) Changes in biochemical indicators in pregnant SD mice.

[0080] Serum biochemical parameters, including ALT (alanine aminotransferase), AST (aspartate aminotransferase), and CRE (lactate dehydrogenase) levels, were measured in four groups of pregnant SD mice: Ad-GFP (vector control group), Ad-sFlt1 (positive control group), Ad-RASA3 (experimental model group), and Ad-RASA3 + 8-CPT (drug treatment group). The data were then compared and analyzed. For testing, samples were first thawed, centrifuged at 1600 rpm for 5 min at 4°C, and more than 200 μl of the supernatant was collected into new centrifuge tubes. ALT, AST, and CRE levels were then measured using an automated veterinary biochemical analyzer.

[0081] The test results are shown in Table 1 below. Compared with the Ad-GFP group, the serum ALT, AST and CRE levels in the Ad-sFlt1 group and the Ad-RASA3 group were significantly increased, while CPT treatment could significantly reverse the damage to liver and kidney function caused by RASA3.

[0082] Table 1. Serum ALT, AST, and LDH levels in pregnant SD mice.

[0083] In Table 1, **: P<0.01, comparing the Ad-sFlt1 group and the Ad-RASA3 group with the Ad-GFP group, and ##: P<0.01, comparing the Ad-RASA3+8-CPT group with the Ad-RASA3 group.

[0084] (6) Determination of intrauterine development of SD pregnant rat pups.

[0085] The fetal weight and placental weight of four groups of pregnant SD mice were measured: Ad-GFP (vector control group), Ad-sFlt1 (positive control group), Ad-RASA3 (experimental model group), and Ad-RASA3 + 8-CPT (drug treatment group). The data of the three groups were compared and analyzed.

[0086] See the test results. Figure 8 .

[0087] Figure 8 In the image, A is an overview picture, B is the length of the fetal mouse, C is the weight of the fetal mouse, and D is the weight of the placenta.

[0088] like Figure 8 As shown, compared with the Ad-GFP group, the Ad-sFIt1 and Ad-RASA3 groups showed significantly reduced fetal weight and body length, along with a significant decrease in placental weight, indicating that both models exhibited significant intrauterine growth restriction and placental dysplasia. The combined intervention of Ad-RASA3 and 8-CPT significantly improved the growth inhibition in fetuses caused by RASA3 overexpression and also had a certain effect on increasing placental weight, suggesting that 8-CPT treatment can partially reverse the adverse effects of RASA3 overexpression on fetal-placental development.

[0089] Figure 8 In the mean squares, * indicates P < 0.05 compared with the Ad-GFP group; # indicates P < 0.05 compared with the Ad-RASA3 group.

[0090] (7) Assessment of spiral artery remodeling in pregnant SD rats To assess the remodeling of the spiral arteries in the uterus of pregnant mice in each group, uterine decidual tissue was collected from each group of SD pregnant mice (Ad-GFP, vector control group, Ad-sFlt1, positive control group, Ad-RASA3, experimental model group, and Ad-RASA3 + 8-CPT, drug treatment group) after euthanasia on day 14 (GD14) and day 19 (GD19). After fixation, embedding, and dewaxing, the tissue was analyzed by immunofluorescence staining.

[0091] Immunofluorescence staining was used to stain endothelial cells (CD31), smooth muscle cells (α-SMA), and trophoblast cells (cytokeratin 7) in the spiral arteries of the uterus to observe changes in spiral artery remodeling. Specific detection methods: After dewaxing the sections to water, endogenous peroxidase was eliminated. Blocking buffer (goat serum) was added to the sections, and incubation was carried out at room temperature for 20 min. Diluted primary antibodies (CK7, α-SMA, and CD31, all diluted 1:200) were added for incubation overnight at 4°C. The next day, the sections were removed, washed with PBS for 5 min, repeated three times, and then incubated with diluted secondary antibody at 37°C for 30 min, followed by washing with PBS for 5 min each time, repeated three times. The sections were mounted with a DAPI-containing anti-fluorescence quenching mounting medium. Microscopic observation was performed, with five randomly selected fields of view photographed, requiring more than five images per group. ImageJ image analysis software was used to analyze optical density values ​​and cell numbers.

[0092] See test results Figure 9 .

[0093] like Figure 9 As shown, on day 14 of pregnancy, the decidual spiral artery in the Ad-GFP group (negative control group) had completed partial remodeling, and some trophoblasts and smooth muscle cells were observed in the spiral artery; while in the Ad-sFlt1 (positive control group) and Ad-RASA3 (experimental model group), there were more endothelial cells and smooth muscle cells residing in the spiral artery; and in the Ad-RASA3 +8-CPT (drug treatment group), there were fewer smooth muscle cells residing.

[0094] On day 19 of pregnancy, the changes in each group were more obvious. The decidual spiral artery in the Ad-GFP group had been completely remodeled (smooth muscle cells had completely migrated out); the remodeling of Ad-sFlt1 (positive control group) and Ad-RASA3 (experimental model group) was incomplete, especially Ad-RASA3 (experimental model group) which led to the retention of a large number of smooth muscle cells; the Ad-RASA3 + 8-CPT (drug treatment group) greatly improved the migration of smooth muscle cells, and the diameter of the spiral artery, like that in the Ad-GFP group, facilitated blood exchange. In summary, RASA3 overexpression leads to impaired spiral artery remodeling in pregnant rats, manifested as obstructed smooth muscle cell migration. Intervention with the Rap1 agonist 8-CPT can effectively reverse this pathological process, promoting smooth muscle cell migration from the vessel wall and restoring normal spiral artery remodeling, thus potentially improving maternal-fetal blood perfusion in preeclampsia.

[0095] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. The use of a cAMP-activated agonist of an exchange protein in the preparation of a drug for treating preeclampsia, characterized in that: The drug treats preeclampsia by activating the Rap1 signaling pathway in human aortic smooth muscle cells, which is inhibited by the RASA3 protein, wherein the agonist of the cAMP-activated exchange protein is 8-CPT-2'-O-Me-cAMP or a pharmaceutically acceptable salt or solvent mixture thereof.

2. The application according to claim 1, characterized in that: The drug promotes actin remodeling in vascular smooth muscle cells and / or enhances the migration ability of vascular smooth muscle cells.

3. The application according to claim 1, characterized in that: The drug can partially or completely reverse the inhibition of Rap1 activity and cell migration in human aortic smooth muscle cells caused by RASA3 overexpression.

4. A drug for treating preeclampsia by blocking the action of RASA3 protein, characterized in that: The drug contains a therapeutically effective amount of a cAMP-activated exchanger agonist, which is 8-CPT-2'-O-Me-cAMP or a pharmaceutically acceptable salt or solvent mixture thereof, which treats preeclampsia by activating Rap1 to block the inhibitory effect of RASA3 protein on human aortic smooth muscle cell migration and spiral artery remodeling.

5. The drug according to claim 4, characterized in that: The concentration of the cAMP-activated exchanger agonist in the drug is 10–100 μM.

6. The drug according to claim 4, characterized in that: The drug also contains a pharmaceutically acceptable carrier or excipient.

7. The drug according to claim 4, characterized in that: The drug is in the form of an injection.

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