Use of phosphatidylcholine (16:1e / 16:1) in the preparation of a drug for preventing and treating preeclampsia

Abstract

The application discloses application of phosphatidylcholine (16:1e / 16:1) in preparation of a drug for preventing and treating preeclampsia, and belongs to the field of biological medicines. The application first proposes and verifies that phosphatidylcholine PC (16:1e / 16:1) can improve the blood vessel formation ability, cell migration ability and cell proliferation inhibition ability of a vascular endothelial cell inflammation injury model, realizes effective vascular endothelial function protection, and then improves the preeclampsia-like hypertension and proteinuria phenotype of a mother, improves preeclampsia-related placental injury and abnormal placental development, avoids adverse outcomes of a mother and fetus in a preeclampsia mouse model, and provides a new selection for clinical treatment of preeclampsia.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to the application of a phosphatidylcholine (16:1e / 16:1) in the preparation of a drug for the prevention and treatment of preeclampsia. Background Technology

[0002] Preeclampsia (PE) is a multisystemic disease specific to pregnancy, typically occurring after 20 weeks of gestation. It is characterized by new-onset hypertension accompanied by proteinuria or other maternal organ and placental dysfunction. In severe cases, it can progress to eclampsia, HELLP syndrome, placental abruption, fetal growth restriction, and premature birth, seriously impacting maternal and infant health. Its development is closely related to insufficient invasion of placental trophoblast cells, impaired remodeling of uterine spiral arteries, and inadequate placental perfusion, subsequently inducing placental ischemia and hypoxia, oxidative stress, inflammatory responses, and the release of anti-angiogenic factors, ultimately leading to systemic vascular endothelial dysfunction and multi-organ damage.

[0003] Currently, clinical treatment for preeclampsia mainly includes lowering blood pressure, administering magnesium sulfate to prevent or treat eclamptic seizures, maternal-fetal monitoring, and timely termination of pregnancy based on gestational age and the patient's condition. For higher-risk individuals, low-dose aspirin can be used for prevention. However, existing treatments primarily focus on controlling hypertension, preventing seizures, and managing complications; they cannot directly correct placental abnormalities or reverse the underlying pathological processes that lead to disease progression. Termination of pregnancy remains the fundamental measure for controlling disease progression. Therefore, developing novel interventional substances that can improve placental function, reduce oxidative stress and vascular endothelial damage, and have potential for use during pregnancy remains of great significance.

[0004] Recent studies have shown that abnormal lipid metabolism may be involved in the development of preeclampsia. Lipidomics studies have revealed abnormalities in various lipid compositions and metabolic pathways in the placenta of preeclamps, particularly those related to glycerophospholipid metabolism, membrane lipid homeostasis, and oxidative stress. Phosphatidylcholine (PC) is an important component of mammalian cell membranes, closely related to maintaining membrane structure and fluidity, cell signal transduction, and cell survival. Abnormalities in its metabolic homeostasis can affect cell function, but currently there are no reports of specific PC molecules being used for the prevention or treatment of preeclampsia. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a method for phosphatidylcholine PC (16:1e / 16:1) to alleviate preeclampsia by improving maternal hypertension, relieving placental damage, promoting fetal growth and development, and avoiding vascular endothelial damage, and to apply this method in the preparation of drugs for the prevention and treatment of preeclampsia.

[0006] Technical solution: The application of phosphatidylcholine PC (16:1e / 16:1) described in this invention in the preparation of drugs for the prevention and treatment of preeclampsia.

[0007] Preferably, the CAS number of the phosphatidylcholine PC (16:1e / 16:1) is 56816-00-3.

[0008] Preferably, the drug has one or more of the following pharmacological activities: (1) improving maternal hypertension; (2) preventing and / or treating placental injury; (3) alleviating fetal growth restriction; and (4) preventing and / or treating vascular endothelial injury.

[0009] Preferably, the drug contains phosphatidylcholine PC (16:1e / 16:1) or its pharmaceutically acceptable isomer, solvate or hydrate as an active ingredient.

[0010] Preferably, the drug further contains pharmaceutically acceptable excipients; more preferably, the pharmaceutically acceptable excipients include excipients selected from one or more of diluents, lubricants, flow aids, wetting agents, emulsifiers, or pH buffers.

[0011] Preferably, the dosage form of the drug includes tablets, capsules, granules, oral liquids, syrups, powders, microcapsules, injections, powder injections, infusions, suspensions, microemulsions, liposomes, nanoparticles, oral instant films, and capsules.

[0012] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention is the first to propose and verify that phosphatidylcholine PC (16:1e / 16:1) can improve the angiogenesis ability, cell migration ability and cell proliferation inhibition ability of vascular endothelial cell inflammatory injury model, achieve effective protection of vascular endothelial function, thereby improving the preeclampsia-like hypertension and proteinuria phenotype in the mother, improving preeclampsia-related placental damage and placental developmental abnormalities, avoiding adverse maternal and fetal outcomes in the preeclampsia mouse model, and providing a new option for the clinical treatment of preeclampsia. Attached Figure Description

[0013] Figure 1 The figure shows the effect of phosphatidylcholine on maternal and fetal outcomes in a mouse model of preeclampsia. In the figure, A is a representative image of the fetus and placenta, B is a statistical graph of fetal weight, and C is a statistical graph of fetal-placental weight ratio. Figure 2 The graph shows the effects of phosphatidylcholine on maternal blood pressure and urinary protein levels in a mouse model of preeclampsia. In the graph, A is the curve of systolic blood pressure change and B is the statistical graph of urinary protein content. Figure 3The figure shows the effect of phosphatidylcholine on placental development in a mouse model of preeclampsia. In the figure, A is a representative image of placental H&E staining, and B is a statistical graph of the proportion of placental labyrinth area. Figure 4 The results show the effect of phosphatidylcholine on the angiogenesis capacity of an endothelial cell inflammatory injury model. In this model, A is a representative image of cells from different treatment groups, and B is a statistical graph of total tube length. Figure 5 The figure shows the effect of phosphatidylcholine on the migration ability of endothelial cells in an inflammatory injury model. In the figure, A is a representative image of cells in different treatment groups, and B is a statistical graph of cell migration rate. Figure 6 The figure shows the effect of phosphatidylcholine on the proliferative capacity of endothelial cells in an inflammatory injury model. In this figure, A is a representative image of cells in different treatment groups, and B is a statistical graph of the proportion of EdU-positive cells. Figure 7 The graph shows the effect of phosphatidylcholine on the expression levels of endothelial injury, inflammatory response, or oxidative stress-related biomarkers in an endothelial cell inflammatory injury model. In the graph, A shows the results of the protein expression level measurement of each biomarker, and B shows the statistical results of the protein expression level measurement of each biomarker. Detailed Implementation

[0014] The technical solution of the present invention will be further described below.

[0015] Example 1: Validation of the efficacy of phosphatidylcholine (16:1e / 16:1) (PC(16:1e / 16:1)) in a mouse model of preeclampsia. Female and male C57BL / 6 mice aged 6-8 weeks were purchased from the Experimental Animal Center of Nanjing Medical University. Female and male mice were housed together overnight at a ratio of 2:1. Vaginal plugs were observed the following morning, and mice with vaginal plugs were identified as gestation day 0.5 (E0.5). All pregnant mice were randomly divided into a control group (Ctrl), an LPS model group (LPS), and a phosphatidylcholine treatment group (LPS+PCe), with 8 mice in each group.

[0016] Starting from mid-pregnancy (E7.5), mice in the LPS model group and phosphatidylcholine treatment group were intraperitoneally injected with 1 μg / kg lipopolysaccharide (LPS, Novus Biologicals, NBP2-25295) to establish a preeclampsia-like mouse model; mice in the phosphatidylcholine treatment group were also intraperitoneally injected with 1 mg / kg PC (16:1e / 16:1) (Avanti Research, 850359P); mice in the control group were given an equal volume of PBS.

[0017] 1. Measurement of maternal blood pressure and urine protein levels Blood pressure in mice was monitored using a non-invasive tail artery blood pressure measurement and analysis system before and during pregnancy (E0.5, E4.5, E8.5, E12.5, E16.5). Urine was collected from mice simultaneously, and urinary protein levels were measured using a urinary protein quantification kit (Nanjing Jiancheng Biotechnology, C035-2-1).

[0018] The results are as follows Figure 2 As shown, mice in the LPS group exhibited elevated blood pressure and increased proteinuria, displaying a preeclampsia-like phenotype. Compared with the LPS model group, mice in the PC (16:1e / 16:1) group showed reduced maternal blood pressure and proteinuria levels, indicating that PC (16:1e / 16:1) can improve LPS-induced maternal preeclampsia-like hypertension and proteinuria phenotype.

[0019] 2. Evaluation of maternal-fetal outcomes Mice were euthanized in late pregnancy (E17.5), and placentas and fetuses were collected. The number of fetuses, fetal weight, and placental weight were recorded to evaluate fetal growth.

[0020] The results are as follows Figure 1 As shown, LPS treatment resulted in significant adverse maternal and fetal outcomes in pregnant mice, primarily manifested as decreased fetal weight, abnormal fetal-placental weight ratio, and increased fetal growth restriction, which may be accompanied by an increase in stillbirths or resorption of fetuses. Compared with the LPS group, PC (16:1e / 16:1) intervention improved these abnormal phenotypes, showing increased fetal weight, restored placental weight, reduced fetal growth restriction, and fewer stillbirths or resorption of fetuses. These results suggest that PC (16:1e / 16:1) can improve placental dysfunction and intrauterine fetal growth restriction in an LPS-induced preeclampsia-like mouse model, thereby alleviating adverse maternal and fetal outcomes.

[0021] 3. Pathological evaluation of placental tissue The aforementioned placental tissues were fixed in 4% paraformaldehyde solution for 48 h, then dehydrated in a gradient manner, embedded in paraffin, and cut into 4 μm sections. After dewaxing and hydration, hematoxylin-eosin (H&E) staining was performed, and the sections were mounted with neutral resin. The overall structure of the placenta, the development of the labyrinth region, the structure of blood sinuses, and the degree of tissue damage were observed under a microscope. Images were acquired, and the proportion of the placental labyrinth region area to the total placental area was calculated.

[0022] The results are as follows Figure 3 As shown, the LPS group mice exhibited a reduced proportion of the placental labyrinth layer, structural disorder, and clearly visible local infarcts and necrotic areas (e.g., Figure 3 (As indicated by the red arrows); the PC(16:1e / 16:1) treatment group significantly reduced pathological damage, the labyrinthine structure became more intact, and the infarct area decreased. This indicates that PC(16:1e / 16:1) has a role in improving preeclampsia-related placental damage and placental developmental abnormalities.

[0023] Example 2: Validation of the efficacy of phosphatidylcholine PC (16:1e / 16:1) in an endothelial cell inflammatory injury model 1. Evaluation of angiogenesis capacity HUVEC cells in the logarithmic growth phase were seeded into culture plates. After the cells adhered, the original culture medium was replaced with fresh ECM medium (control group, Ctrl), or ECM medium containing a final concentration of 1 μg / mL LPS (LPS model group, LPS), or ECM medium containing a final concentration of 1 mg / mL PC (16:1e / 16:1) (phosphatidylcholine group, LPS+PCe), and then subjected to routine culture treatment. The ECM medium was purchased from ScienCell, catalog number 1001.

[0024] HUVEC cells from different treatment groups after 24 h of culture were subjected to 1×10⁻⁶ cells per cell. 4 Seeds were planted at a density of 1 cell / well in 96-well plates supplemented with 50 μL M Atrigel (Corning, 356234) and incubated for 30 min. Images were acquired and observed under a microscope after 1 h and 4 h of culture, and the length of the angiogenesis was further calculated.

[0025] The results are as follows Figure 4 As shown, the angiogenesis capacity of HUVECs was significantly reduced after LPS treatment compared to the control group, while the lumen-like structures increased and the total tube length was improved after PC (16:1e / 16:1) intervention; indicating that PC (16:1e / 16:1) can improve angiogenesis impairment in the endothelial cell inflammatory injury model.

[0026] 2. Assessment of transferability HUVEC cells in the logarithmic growth phase were harvested at a concentration of 1×10⁻⁶. 6 Cells were seeded at a density of 1 cell / well in 6-well plates. Once the cell confluence reached approximately 90%, a straight line was drawn in the center of the cell layer using a 200 μL sterile pipette tip. The cells were washed with PBS to remove any detached cells. The original medium was then replaced with fresh ECM medium (control group, Ctrl), or ECM medium containing a final concentration of 1 μg / mL LPS (LPS model group, LPS), or ECM medium containing a final concentration of 1 mg / mL PC (16:1e / 16:1) (phosphatidylcholine group, LPS+PCe). The cells were then cultured using standard methods.

[0027] Images were collected and observed under a microscope at 0 h, 12 h, and 24 h after scratching, and the scratch width was recorded.

[0028] The results are as follows Figure 5As shown, the scratch healing rate of HUVECs after LPS treatment was significantly slower than that of the control group, and the cell migration ability decreased; after PC (16:1e / 16:1) intervention, the scratch healing rate increased and the cell migration rate improved; indicating that PC (16:1e / 16:1) can improve the migration dysfunction in the endothelial cell inflammatory injury model.

[0029] 3. Evaluation of Proliferation Capacity HUVEC cells in logarithmic growth phase, at 1×10 5 The cells were seeded at a density of cells / well in 24-well plates. The original medium was replaced with fresh ECM medium (control group, Ctrl), or ECM medium containing a final concentration of 1 μg / mL LPS (LPS model group, LPS), or ECM medium containing a final concentration of 1 mg / mL PC (16:1e / 16:1) (phosphatidylcholine group, LPS+PCe). The plates were then cultured according to standard procedures.

[0030] After 24 h of treatment, EdU working solution from the EdU cell proliferation assay kit (Beyotime, C0085S) was added to each well for incubation to label proliferating cells. The culture medium was then discarded, and the cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and stained according to the kit instructions. Cell nuclei were stained with DAPI, and images were observed and acquired under a fluorescence microscope. Fields of view were randomly selected from each slide, and EdU-positive cells (green fluorescence) and total cells (blue fluorescence) were counted. The EdU positivity rate (number of EdU-positive cells / total number of cells × 100%) was calculated to assess cellular DNA synthesis activity and proliferation capacity.

[0031] The results are as follows Figure 6 As shown, the proliferation capacity of HUVECs was significantly reduced after LPS treatment compared to the control group, and the proportion of EdU-positive cells decreased; the proportion of EdU-positive cells increased after PC (16:1e / 16:1) intervention, indicating that PC (16:1e / 16:1) can improve the proliferation inhibition of the endothelial cell inflammatory injury model.

[0032] 4. Evaluation of the expression levels of markers related to vascular endothelial injury, inflammatory response, and oxidative stress HUVEC cells in logarithmic growth phase, at 2×10 5 The cells were seeded at a density of cells / well in 6-well plates. After the cells adhered, the original medium was replaced with fresh ECM medium (control group, Ctrl), or ECM medium containing a final concentration of 1 μg / mL LPS (LPS model group, LPS), or ECM medium containing a final concentration of 1 mg / mL PC (16:1e / 16:1) (phosphatidylcholine group, LPS+PCe). The cells were then cultured as usual.

[0033] Cells were collected 24 hours after treatment and lysed using RIPA lysis buffer (Beyotime, P0013B) with a protease inhibitor mixture (Beyotime, P1006). The cells were lysed on ice with shaking for 30 min, centrifuged at 12000 rpm for 30 min at 4°C, and the supernatant was collected. Sample buffer (Beyotime, P0015L) was added proportionally, and the mixture was boiled in a metal bath for 10 min. SDS-PAGE electrophoresis was then performed. After transfer and blocking, eNOS primary antibody (Proteintech, 27120-1-AP) diluted 1:1000, iNOS primary antibody (Proteintech, 2226-1-AP) diluted 1:1000, VCAM-1 primary antibody (Proteintech, 11444-1-AP) diluted 1:1000, ET-1 primary antibody (Proteintech, 12191-1-AP) diluted 1:1000, or diluted 1:1000, were added. Incubate overnight at 4°C with 1000 g of GAPDH primary antibody (Proteintech, 10494-1-AP). After rinsing, incubate at room temperature for 1 h with secondary antibody (Proteintech, RGAR001) diluted 1:5000. Finally, use ECL chemiluminescence kit (Biosharp, BL5965B) for luminescence development, image acquisition, and quantitative analysis.

[0034] The results are as follows Figure 7 As shown, LPS treatment increased the expression of vascular endothelial injury and inflammatory markers such as iNOS, VCAM-1, and ET-1 in HUVECs, while decreasing the expression of eNOS. After PC (16:1e / 16:1) intervention, the expression of the above-mentioned related markers improved. This indicates that PC (16:1e / 16:1) can alleviate LPS-induced human umbilical vein endothelial cell injury, reduce inflammatory response and oxidative stress levels, and has a protective effect on vascular endothelial function.

Claims

1. The application of a phosphatidylcholine PC (16:1e / 16:1) in the preparation of a drug for the prevention and treatment of preeclampsia.

2. The application according to claim 1, characterized in that, The CAS number of the phosphatidylcholine PC (16:1e / 16:1) is 56816-00-3.

3. The application according to claim 1, characterized in that, The drug in question is for improving maternal hypertension.

4. The application according to claim 1, characterized in that, The drug is a drug for the prevention and / or treatment of placental damage.

5. The application according to claim 1, characterized in that, The drug is used to alleviate fetal growth restriction.

6. The application according to claim 1, characterized in that, The drug is a drug for the prevention and / or treatment of vascular endothelial injury.

7. The application according to claim 1, characterized in that, The drug contains phosphatidylcholine PC (16:1e / 16:1) or its pharmaceutically acceptable isomer, solvate or hydrate as the active ingredient.

8. The application according to claim 7, characterized in that, The drug also contains pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that, The pharmaceutically acceptable excipients include excipients selected from one or more of diluents, lubricants, flow aids, wetting agents, emulsifiers, or pH buffers.

10. The application according to claim 1, characterized in that, The dosage forms of the drugs include tablets, capsules, granules, oral liquids, syrups, powders, microcapsules, injections, powder injections, infusions, suspensions, microemulsions, liposomes, nanoparticles, oral instant films, and capsules.

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