CN117982628B

CN117982628B - Use of leptin in preparing medicine for preventing and/or treating preeclampsia

Info

Publication number: CN117982628B
Application number: CN202410037964.7A
Authority: CN
Inventors: 李艳; 魏代敏; 陈子江
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2024-01-09
Filing date: 2024-01-09
Publication date: 2025-09-05
Anticipated expiration: 2044-01-09
Also published as: CN117982628A

Abstract

The present invention belongs to the field of biomedicine and relates to the use of leptin in the preparation of a drug for the prevention and/or treatment of preeclampsia. In vitro experiments demonstrate that leptin can promote trophoblast cell syncytialization. Further in vivo experiments confirm that leptin can lower maternal blood pressure and increase placental weight in a rat model of preeclampsia, suggesting broad application prospects in the drug treatment of preeclampsia.

Description

Application of leptin in preparation of medicine for preventing and/or treating preeclampsia

Technical Field

The invention belongs to the technical field of biological medicines, and relates to application of leptin in preparation of a medicine for preventing and/or treating preeclampsia.

Background

The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.

Preeclampsia (pre-eclampsia, PE) is a hypertensive disease in gestation, affects 2-8% of pregnancy, is a main cause of death of pregnant and parturients and perinatal infants, and is characterized by new hypertension (140/90 mmHg or more) and proteinuria (urine protein 0.3g/24h or more) after 20 weeks of gestation, which often leads to iatrogenic premature birth and seriously threatens maternal-fetal safety. Although the occurrence of preeclampsia is widely believed to be associated with insufficient infiltration of trophoblasts during placental development, its specific pathogenesis is still unclear and there is a lack of effective therapeutic drugs against the etiology, in clinical practice, except for spasmolytic treatment with magnesium sulfate and delivery of fetus and placenta as soon as possible. Thus, for better clinical intervention, it is desirable to screen and identify effective therapeutic targets based on a deep elucidation of the pathogenesis of preeclampsia.

During early human placental development, chorionic Trophoblast (CTB) progenitor cells follow one of two differentiation pathways, namely (1) differentiate into Syngeneic Trophoblasts (STBs) comprising multiple nuclei by cell membrane fusion, and (2) acquire invasiveness and differentiate into extravillous trophoblasts. Among them, the fusion of monocyte trophoblasts to form non-proliferative polynuclear trophoblasts (STBs) is critical to normal placenta function because STBs participate in various processes including hormone production, nutrient transport, and immune tolerance, etc., and their differentiation and dysfunction lead to placenta dysfunction and weight loss, failing to provide sufficient nutrition to the fetus, leading to the occurrence of low weight infants. However, preeclampsia is currently known to be a placenta-derived disease, and the placenta of a preeclampsia patient has thin syncytia, vacuolation and discontinuity, and shows the defect of cell fusion function, which indicates that the differentiation and dysfunction of STBs in early pregnancy are closely related to the occurrence of PE in late pregnancy.

Disclosure of Invention

Leptin (Leptin) is a peptide hormone secreted mainly by adipose tissue, and placenta is the second Leptin-producing tissue of the human body. According to the study of the inventor, leptin has been identified as a biological marker of preeclampsia, and its expression is increased in serum of preeclampsia patients in late pregnancy, however, the study of leptin effects focuses on the direction of increasing metabolic level, and is considered to increase blood pressure, which is the cause of the preeclampsia blood pressure elevation phenotype, so that no study has shown that the increased leptin expression in late pregnancy may be compensating for the differentiation and dysfunction of placental STBs in early pregnancy, and no study has shown that its application in early and mid pregnancy helps to prevent and treat the preeclampsia blood pressure elevation and placenta dysplasia phenotypes. According to experimental accidents, the invention discovers that the mRNA level of a placenta leptin gene LEP of a preeclampsia patient in a gestation period is increased, the knocking-down of the LEP reduces the syncytization process of a nourishing cell syncytization cell line-BeWo cell, leptin protein is intravenously injected into a preeclampsia model rat in a gestation period to reduce the preeclampsia blood pressure, leptin protein is intravenously injected into the preeclampsia model rat in the gestation period to increase the weight of the placenta in the gestation period, so that leptin can prevent and treat the preeclampsia.

Based on the research results, the invention provides application of leptin in preparing medicines for preventing and/or treating preeclampsia.

Specifically, the invention provides the following technical scheme:

in one aspect, the use of leptin in the manufacture of a medicament for the prevention and/or treatment of preeclampsia.

In another aspect, the use of leptin in the preparation of a formulation for increasing the syncytial process of a feeder cell-syncytial cell line.

The preparation can be a drug or an experimental reagent, and the experimental reagent can be used for basic research.

In some embodiments, the feeder cell line is a BeWo cell.

In a third aspect, use of leptin in the manufacture of a medicament for the prevention of late pregnancy hypertension and/or placental dysplasia for early and mid pregnancy in preeclampsia.

In some embodiments, the mode of administration is intravenous injection.

In some embodiments, the pharmaceutical dosage form is a solution, lyophilized powder, sterilized powder, or the like. When the dosage form is a solid preparation such as freeze-dried powder or sterilized powder, the preparation can be added into physiological saline and buffer solution for dissolution and then applied.

The subject to which the medicament of the invention is administered may be a human or a non-human mammal, such as a mouse, rat, pig, cow, sheep, gorilla, etc., and in some embodiments, the subject is a rat.

In the process of preparing leptin, leptin is required to be used as an active ingredient and combined with pharmaceutical excipients (pharmaceutical carriers or excipients) to prepare a specific drug, so that in some embodiments, the drug is a pharmaceutical composition whose active ingredient is leptin.

In one or more embodiments, the pharmaceutical composition includes a pharmaceutically acceptable carrier. The medicinal carrier liposome, serum protein and the like.

In one or more embodiments, the pharmaceutical composition includes an excipient. The excipient, solution such as water, physiological saline, buffer solution, etc., and solid preparation such as lyophilized powder or sterilized powder, such as lactose, glucose, gelatin, polyvinylpyrrolidone, etc.

The beneficial effects of the invention are as follows:

Experiments show that the placenta LEP mRNA level of a eclampsia patient is increased and the level of LEP is knocked down to reduce the syncytization of trophoblasts, and the invention proves that the supplement of Leptin can increase the syncytization of human trophoblasts, improve the defect of cell fusion function expressed by placenta at the late stage of preeclampsia, save the preeclampsia phenotype, improve the maternal-fetal ending, and have important value and application prospect for the prevention and treatment of preeclampsia.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

Fig. 1 shows analysis of placental LEP mRNA levels in pre-term patients (n=41vs 28) in the examples of the present invention.

FIG. 2 shows the mRNA levels of LEP (A), ERVFRD-1 (B) and CGB3 (C) were detected by PCR with 10. Mu.M adenylate cyclase activator (Forskolin) or DMSO added after transfection of BeWo cells with control siRNA or LEP siRNA in the examples of the present invention, wherein the LEP, ERVFRD-1, CGB3 genes encode Leptin, syncytin-2 (syncytin 2) and hCG beta (chorionic gonadotropin beta) proteins, respectively, and the level of cellular syncytia was analyzed by immunofluorescent staining of E-cadherin (stained cell membrane), scale bar, 50. Mu.m.

FIG. 3 shows the results of animal experiments in which pre-eclampsia rats were supplemented with recombinant Leptin according to the examples of the present invention, A, animal model treatment protocol, in which adenovirus overexpressing sFlt-1 (pre-eclampsia group) or control Fc (control group) was used to administer recombinant Leptin protein (120. Mu.g/kg/day) or PBS, to G20, to rats by tail vein injection on days G12-G19, and the samples were sacrificed on day G20, B, G7, G10, G12, G14, G16, and G19, blood pressure was measured and recorded, and systolic blood pressure was analyzed for each group, C, placenta weights were measured at G20, quantitative results were expressed as mean.+ -. Standard deviation, p <0.05, p <0.01, p <0.001, and statistical methods were variance analysis, ns, without significant differences.

Detailed Description

In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail below with reference to specific examples and comparative examples.

Example 1

Analysis of placental leptin LEP mRNA levels in pre-eclampsia patients in late pregnancy

And (3) collecting placenta tissue samples of preeclampsia pregnant women and healthy pregnant women in a caesarean section operation, flushing with normal saline, quickly freezing with liquid nitrogen, and then transferring to-80 ℃ for long-term storage. The study was approved by the Shandong university medical fusion and practice center ethical committee, following the declaration of Helsinki and obtaining informed consent from the patients.

1) Tissue RNA extraction

An equal amount of tissue sample was weighed and then added with 500. Mu.l TRIzol, ground on a tissue grinder, and then added with 500. Mu.l TRIzol for complete mixing, and allowed to stand for 10 minutes for lysis. Centrifugation at 12000g for 15 min at 4℃and the aspirated supernatant transferred to an additional RNase-free EP tube. 200 μl chloroform was added and shaken for 15s, and allowed to stand for 15 minutes, then centrifuged at 4℃and 12000g for 10 minutes, the uppermost aqueous phase was transferred to a new EP tube, and an equal volume of isopropanol was added, mixed well and allowed to stand at room temperature for 10 minutes. Centrifugation at 12000g for 10min at 4℃and removal of supernatant, precipitation of RNA by addition of 75% ethanol. Centrifugation was performed at 8000g for 10min at 4 ℃. RNA was dissolved in 20-50. Mu.l DEPC water and the concentration and purity of RNA were determined by Nanodrop one.

2) Reverse transcription PCR

The volumes of RNA and DEPC water in reaction system 1 were calculated from the concentration of RNA using 1. Mu.g of RNA as a reaction template, and reaction system 1 was as shown in Table 1.

TABLE 1 reaction System 1

The PCR instrument was set at 42℃and 2min, 16 ℃. Then, the reaction system 2 was subsequently configured to carry out reverse transcription as shown in Table 2.

TABLE 2 reaction System 2

The PCR instrument was set at 37℃for 15min, 85℃for 5S, 16 ℃.

3) Quantitative PCR

The real-time fluorescent quantitative PCR reaction was performed using a TB green kit, the reaction system was Table 3, and the PCR primer sequences were Table 4.

TABLE 3PCR reaction System

TABLE 4PCR primer sequences

The PCR instrument was set up as table 5.

Table 5 PCR setting of instrument

The study found that elevated levels of LEP mRNA in placental tissue of patients with preeclampsia suggested that elevated Leptin in the placenta at advanced pregnancy could be a compensatory preeclampsia in early gestational placental dysplasia.

Example 2

Knock-down of LEP reduces syncytization of BeWo cells

1) After 20. Mu.M control siRNA or LEP siRNA transfection of BeWo cells, 10. Mu.M adenylate cyclase activator (Forskolin) or DMSO was added for 48 hours.

2) Immersing the cells treated in the previous step with PBS for 3 times, each time for 5min;

3) Fixing cells with 4% paraformaldehyde at room temperature for 20min, and soaking and washing with PBS for 3 times each for 5min;

4) 0.3% Triton X-100 (PBS) was allowed to permeate for 30min at room temperature;

5) Soaking and washing with PBS for 3 times, each time for 5min, adding normal goat serum, and sealing at room temperature for 1h;

6) Discarding the blocking solution, dropwise adding a sufficient amount of diluted primary antibody to each slide, placing the diluted primary antibody into a wet box, and incubating at 4 ℃ overnight;

7) Adding a fluorescent secondary antibody, soaking cells in PBST for 3 times each for 5min, sucking off redundant liquid, then dripping diluted fluorescent secondary antibody, incubating in a wet box at room temperature in a dark manner for 1h, and soaking cells in PBST for 3 times each for 5min;

8) And (3) counterstaining, namely dripping a sealing liquid containing DAPI and an anti-fluorescence quenching agent, staining the cells, and then observing and collecting images under a fluorescence microscope.

It was found that after siRNA transfected BeWo cells, FIG. 2A shows that LEP mRNA level is reduced, as can be seen from FIGS. 2B and 2C, after LEP is knocked down, forskolin (a drug inducing cell fusion) induces the expression of two markers Syncytin-2 and hCG beta mRNA level of the BeWo cells to be syncytized to be reduced, and FIG. 2D immunofluorescence results also show that the knockdown of LEP reduces the syncytization degree of the BeWo cells. This suggests that Leptin plays an important role in the process of syncytization of trophoblasts, while STBs are involved in various processes such as hormone production, nutrient transport and immune tolerance, and are closely related to the occurrence of PE. Recombinant Leptin protein treatment may improve the defect in cell fusion function of preeclampsia placenta by promoting the process of syncytization of feeder cells, which in turn slows down the progression of preeclampsia.

Example 3

Supplementation with recombinant Leptin (Leptin) protein improves maternal-fetal outcome in pre-eclamptic rat models

1) Establishment of preeclampsia rat model

The rats used in this experiment were Sprague-Dawley rats purchased from Experimental animals Inc. of Toril Hua, beijing, all animals were cared according to the International guidelines for animal care, and were approved by the ethical committee of Shandong university for animal care and research.

Female SD rats (200-220G) of 9 weeks old were mated with male SD rats of similar age and weight, and day 1 of gestation (G1) was defined as vaginal plug detection of female rats in the morning the following day of mating. Pregnant rats were randomly divided into Ad fc+pbs (n=3), ad fc+leptin (n=3), ad flt1+pbs (n=3), and Ad flt1+leptin (n=3) 4 groups. The overexpression of sFlt-1 or control Fc adenoviruses (Ji Kai organisms) was constructed according to previous studies and 1X 10 ⁹ PFU adenoviruses (Ad Fc or Ad Flt 1) were injected by tail vein in G8 (early midgestation). Human recombinant Leptin protein (120 μg/kg/day) (R & D, # 598-LP-05M) or vector (PBS) was injected daily into rats via the tail vein in G12-G19.

2) Rat blood pressure measurement

Systolic Blood Pressure (SBP) (MRBP, IITC) was measured by tail-cuff plethysmography (MRBP, IITC). Rats were fixed with a tail sleeve restraint for 30min daily for adaptation training to accommodate blood pressure measurement operations 3 days before the beginning of the formal measurement. The rats were subjected to blood pressure measurement after adaptation to the measurement procedure by fixing the rats with a restraint, resting in an environment at a constant temperature of 32 ℃ for 15-20min, and then measuring 5 consecutive times, 1 minute apart. If the measurement is needed again, the rats should be allowed to rest for 5 minutes to be measured again. According to this method, blood pressure is measured at G7, G10, G12, G14, G16, G19.

3) Placenta material

Rats were anesthetized at G20, blood samples were collected, the abdomen was opened to remove the uterus, and the mice and placenta were isolated. Placenta weight was measured.

As shown in FIG. 3, the treatment group (pre-eclamptic rat model group) injected with sFlt-1 over-expressed adenovirus has obviously increased blood pressure and decreased placenta weight compared with the treatment group (control group) injected with the control Fc virus, and the abnormal placenta development of the pre-eclamptic rat model is proved, the maternal blood pressure is increased, the pre-eclamptic phenotype is met, and the model establishment is successful. After G12-19, i.e., continuous tail vein injection of recombinant Leptin protein in middle and late gestation, in the model group, leptin-mediated groups had significantly lower blood pressure, increased placenta weight, saved preeclampsia phenotype, and improved maternal-fetal outcome compared to PBS-vehicle-only-mediated groups.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of leptin in the preparation of a medicament for preventing hypertension and/or placental dysplasia in late pregnancy during early or mid-pregnancy in preeclampsia;

The drug is in the form of an intravenous injection.

2. Use of leptin as claimed in claim 1 in the preparation of a medicament for preventing hypertension and/or placental dysplasia in late pregnancy during early or mid-pregnancy in preeclampsia, wherein the medicament is in the form of a solution, a lyophilized powder or a sterilized powder.

3. Use of leptin as claimed in claim 1 in the preparation of a medicament for preventing hypertension and/or placental dysplasia in late pregnancy during early or mid-pregnancy in preeclampsia, wherein the subject of administration is rats.

4. Use of leptin as claimed in claim 1 in the preparation of a medicament for preventing late pregnancy hypertension and/or placental dysplasia in preeclampsia administered in the early or mid-pregnancy period, wherein the medicament is a pharmaceutical composition, and the active ingredient of the pharmaceutical composition is leptin.

5. Use of leptin as claimed in claim 4 in the preparation of a medicament for preventing hypertension and/or placental dysplasia in late pregnancy during early or mid-pregnancy in preeclampsia, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

6. Use of leptin as claimed in claim 4 in the preparation of a medicament for preventing hypertension and/or placental dysplasia in late pregnancy during early or mid-pregnancy in preeclampsia, wherein the pharmaceutical composition comprises an excipient.