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

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

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and relates to the use of leptin in preparing a medicine for preventing and / or treating preeclampsia. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Preeclampsia (PE) is a hypertensive disorder of pregnancy that affects 2% to 8% of pregnancies and is a leading cause of maternal and perinatal mortality. It is characterized by new-onset hypertension (≥140 / 90 mmHg) and proteinuria (urine protein ≥0.3 g / 24 h) after 20 weeks of gestation. It often leads to iatrogenic preterm birth and poses a serious threat to maternal and fetal safety. Although it is widely believed that the development of PE is associated with insufficient trophoblast infiltration during placental development, its specific pathogenesis remains unclear. Clinically, effective treatments targeting the cause are lacking, aside from symptomatic treatment with magnesium sulfate and expedited delivery of the fetus and placenta. Therefore, to better inform clinical intervention, it is necessary to screen and identify effective therapeutic targets based on a deeper understanding of the pathogenesis of PE.

[0004] During early human placental development, chorionic cytotrophoblast (CTB) progenitors follow one of two differentiation pathways: (1) differentiation into multinuclear syncytiotrophoblasts (STBs) through cell membrane fusion; and (2) differentiation into extravillous trophoblasts by acquiring invasive ability. Among them, the fusion of mononuclear cytotrophoblasts to form non-proliferative multinuclear trophoblasts (STBs) is crucial for normal placental function, because STBs participate in various processes, including hormone production, nutrient transport, and immune tolerance. Abnormal differentiation and function of STBs lead to abnormal placental function and weight loss, which cannot provide sufficient nutrition to the fetus, resulting in the occurrence of low birth weight babies. It is currently known that preeclampsia is a placental disease. The syncytia of the placenta of patients with preeclampsia are thin, vacuolated, and discontinuous, showing defects in cell fusion function. This indicates that abnormal differentiation and function of STBs in early pregnancy are closely related to the occurrence of PE in mid-to-late pregnancy. Summary of the Invention

[0005] Leptin is a peptide hormone primarily secreted by adipose tissue, and the placenta is the second most leptin-producing tissue in the body. According to the inventors' research, leptin has been identified as a biological marker for preeclampsia, with elevated serum expression in the late pregnancy serum of patients with preeclampsia. However, research on the effects of leptin has primarily focused on its ability to increase metabolic levels, and is believed to increase blood pressure, contributing to the elevated blood pressure phenotype of preeclampsia. Therefore, no studies have demonstrated that elevated leptin expression in late pregnancy may compensate for the early-pregnancy placental STB differentiation and dysfunction, nor have studies demonstrated that its use in the early and mid-pregnancy periods can help prevent and treat the elevated blood pressure and placental dysplasia phenotypes of preeclampsia. The present invention unexpectedly discovered through experiments that the mRNA level of the leptin gene LEP in the placenta of patients with eclampsia in late pregnancy is increased, and the knockdown of LEP reduces the syncytialization process of BeWo cells, a cell line used to study trophoblast syncytial formation. Leptin protein can reduce the blood pressure in late pregnancy when injected intravenously into rats with a preeclampsia model in early and mid-pregnancy. Leptin protein can also increase the weight of the placenta in late pregnancy when injected intravenously into rats with a preeclampsia model in early and mid-pregnancy. This indicates that leptin can prevent and treat preeclampsia.

[0006] Based on the above research results, the present invention proposes the use of leptin in the preparation of a drug for preventing and / or treating preeclampsia.

[0007] Specifically, the present invention proposes the following technical solutions:

[0008] In one aspect, a use of leptin in preparing a medicament for preventing and / or treating preeclampsia.

[0009] In another aspect, a use of leptin in the preparation of a preparation for enhancing the syncytialization process of a trophoblast syncytial cell line.

[0010] The preparation of the present invention can be a medicine or an experimental reagent, and the experimental reagent can be used for basic research.

[0011] In some embodiments, the trophoblast syncytial cell line is a BeWo cell.

[0012] In a third aspect, a leptin is used in the preparation of a drug for preventing hypertension and / or placental maldevelopment in late pregnancy during early or mid-pregnancy in preeclampsia.

[0013] In some embodiments, the administration is intravenous injection.

[0014] In some embodiments, the drug is in the form of a solution, lyophilized powder, or sterilized powder. When the dosage form is a solution, the drug can be administered directly; when the dosage form is a solid preparation such as lyophilized powder or sterilized powder, it can be dissolved in physiological saline or buffer solution before administration.

[0015] The subject of the drug of the present invention can be a human or a non-human mammal, such as a mouse, rat, pig, cow, sheep, orangutan, etc. In some embodiments, the subject of the drug is a rat.

[0016] In the process of preparing leptin as a pharmaceutical, leptin needs to be combined with pharmaceutical excipients (pharmaceutical carriers or excipients) as an active ingredient to prepare a specific drug. Therefore, in some embodiments, the drug is a pharmaceutical composition, and the active ingredient of the pharmaceutical composition is leptin.

[0017] In one or more embodiments, the pharmaceutical composition includes a pharmaceutical carrier, such as liposomes, serum proteins, etc.

[0018] In one or more embodiments, the pharmaceutical composition includes an excipient, such as a solution such as water, physiological saline, or a buffer solution, or a solid preparation such as a lyophilized powder or sterilized powder, such as lactose, glucose, gelatin, or polyvinyl pyrrolidone.

[0019] The beneficial effects of the present invention are:

[0020] The present invention experimentally found that elevated LEP mRNA levels in the placenta of patients with eclampsia and knockdown of LEP reduced syncytialization of trophoblasts, demonstrating that supplementation with Leptin can increase syncytialization of human trophoblasts, improve the cell fusion function defects manifested in the late pregnancy placenta of preeclampsia, rescue the preeclampsia phenotype, and improve maternal and fetal outcomes, which has important value and application prospects for the prevention and treatment of preeclampsia. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0022] Figure 1 This is an analysis of LEP mRNA levels in the placenta of patients with preeclampsia in late pregnancy in the examples of the present invention (N=41 vs 28).

[0023] Figure 2 Figure 5. BeWo cells were transfected with control siRNA or LEP siRNA according to the present invention, and then 10 μM adenylate cyclase activator (Forskolin) or DMSO was added. AC: PCR detection of LEP (A), ERVFRD-1 (B), and CGB3 (C) mRNA levels. LEP, ERVFRD-1, and CGB3 genes encode leptin, syncytin-2, and hCGβ (chorionic gonadotropin β) proteins, respectively. D: Immunofluorescence staining of E-cadherin (cell membrane staining) to analyze the level of cell syncytialization. Scale bar, 50 μm.

[0024] Figure 3 These are the results of animal experiments on the supplementation of recombinant leptin in a preeclampsia rat model according to the examples of the present invention; A: Animal model treatment process, rats were injected with adenovirus overexpressing sFlt-1 (preeclampsia group) or control Fc (control group) through the tail vein on gestational day (G) 8, and recombinant leptin protein (120 μg / kg / day) or PBS was injected into the tail vein on days G12-G19. Rats were killed at G20 for sampling; B: Blood pressure was measured and recorded at G7, G10, G12, G14, G16, and G19, and systolic blood pressure of each group was analyzed; C: Placental weight of each group was measured at G20; quantitative results are expressed as mean ± standard deviation; *p<0.05, **p<0.01, ***p<0.001, statistical method was analysis of variance, ns, no significant difference. DETAILED DESCRIPTION

[0025] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0026] Example 1

[0027] Analysis of leptin (LEP) mRNA levels in the placenta of patients with preeclampsia during late pregnancy

[0028] Placental tissue samples were collected from pregnant women with preeclampsia undergoing cesarean section and from healthy pregnant women. The samples were rinsed with saline, snap-frozen in liquid nitrogen, and then transferred to -80°C for long-term storage. This study was approved by the Ethics Committee of the Medical Integration and Practice Center of Shandong University and adhered to the Declaration of Helsinki. Informed consent was obtained from the patients.

[0029] 1) Tissue RNA extraction

[0030] Weigh an equal amount of tissue sample and add 500μl TRIzol. Grind on a tissue grinder, then add 500μl TRIzol to make up and mix thoroughly. Let it stand for 10 minutes to lyse. Centrifuge at 12000g at 4℃ for 15 minutes, aspirate the supernatant and transfer it to another RNase-free EP tube. Add 200μl chloroform and shake for 15 seconds. Let it stand for 15 minutes, then centrifuge at 12000g at 4℃ for 10 minutes. Transfer the top aqueous phase to a new EP tube, add an equal volume of isopropanol, mix thoroughly, and let it stand at room temperature for 10 minutes. Centrifuge at 12000g at 4℃ for 10 minutes, discard the supernatant, and add 75% ethanol to precipitate RNA. Centrifuge at 8000g at 4℃ for 10 minutes. Dissolve the RNA in 20-50μl DEPC water and measure the RNA concentration and purity using Nanodrop One.

[0031] 2) Reverse transcription PCR

[0032] Use 1 μg RNA as the reaction template and calculate the volumes of RNA and DEPC water in reaction system 1 based on the RNA concentration. Reaction system 1 is shown in Table 1.

[0033] Table 1 Reaction system 1

[0034]

[0035] PCR instrument settings: 42°C, 2 min; 16°C. Then, prepare reaction system 2 for reverse transcription, as shown in Table 2.

[0036] Table 2 Reaction system 2

[0037]

[0038] PCR instrument settings: 37°C, 15 min; 85°C, 5 s; 16°C.

[0039] 3) Quantitative PCR

[0040] Real-time fluorescence quantitative PCR reaction was performed using the TB green kit. The reaction system was shown in Table 3, and the PCR primer sequences were shown in Table 4.

[0041] Table 3 PCR reaction system

[0042]

[0043] Table 4 PCR primer sequences

[0044]

[0045]

[0046] The PCR instrument settings are as in Table 5.

[0047] Table 5 PCR instrument settings

[0048]

[0049] Studies have found that LEP mRNA levels are elevated in placental tissue of patients with late pregnancy eclampsia, suggesting that increased Leptin in the late pregnancy placenta may compensate for early pregnancy placental development disorders caused by preeclampsia.

[0050] Example 2

[0051] Knockdown of LEP reduces syncytialization of BeWo cells

[0052] 1) BeWo cells were transfected with 20 μM control siRNA or LEP siRNA and then treated with 10 μM adenylate cyclase activator (Forskolin) or DMSO for 48 hours.

[0053] 2) Wash the cells after the previous treatment with PBS three times, 5 minutes each time;

[0054] 3) Fix the cells with 4% paraformaldehyde at room temperature for 20 minutes and then wash with PBS three times for 5 minutes each time;

[0055] 4) Permeabilization with 0.3% Triton X-100 (in PBS) at room temperature for 30 min;

[0056] 5) Wash with PBS three times for 5 minutes each time, add normal goat serum, and block at room temperature for 1 hour;

[0057] 6) Discard the blocking solution, add a sufficient amount of diluted primary antibody to each slide, place in a humidified chamber, and incubate overnight at 4°C.

[0058] 7) The next day, add fluorescent secondary antibody and rinse the cells three times with PBST for 5 minutes each time. After removing excess liquid, add diluted fluorescent secondary antibody dropwise. Incubate in a humidified chamber at room temperature in the dark for 1 hour. Then rinse three times with PBST for 5 minutes each time.

[0059] 8) Nuclear restaining: Add mounting solution containing DAPI and anti-fluorescence quencher to stain the nuclei of the cells, and then observe and collect images under a fluorescence microscope.

[0060] The study found that after siRNA transfection into BeWo cells, Figure 2 A shows that the LEP mRNA level is reduced; Figure 2 B. Figure 2 C shows that after LEP knockdown, the expression of syncytin-2 and hCGβ mRNA levels, two markers of BeWo cell syncytialization induced by Forskolin (a drug that induces cell fusion), was reduced; Figure 2 Immunofluorescence results also showed that knockdown of Leptin reduced the degree of syncytialization of BeWo cells. This suggests that Leptin plays a crucial role in trophoblast syncytialization. STBs are involved in various processes, including hormone production, nutrient transport, and immune tolerance, and are closely associated with the development of PE. Recombinant Leptin protein treatment may improve the cell fusion defect in the preeclamptic placenta by promoting trophoblast syncytialization, thereby slowing the progression of preeclampsia.

[0061] Example 3

[0062] Supplementation of recombinant leptin protein improves maternal and fetal outcomes in a rat model of preeclampsia

[0063] 1) Establishment of a preeclampsia rat model

[0064] The rats used in this experiment were Sprague-Dawley rats purchased from Beijing Weitonglihua Laboratory Animal Company. All animals were cared for in accordance with international animal care guidelines and obtained ethical approval from the Animal Care and Research Committee of Shandong University.

[0065] Nine-week-old female SD rats (200-220 g) were mated with male SD rats of similar age and weight. Gestational day 1 (G1) was defined as the presence of a vaginal plug in the female rat's vagina on the morning of the second day of mating. Pregnant rats were randomly divided into four groups: Ad Fc + PBS (n = 3), Ad Fc + Leptin (n = 3), Ad Flt1 + PBS (n = 3), and Ad Flt1 + Leptin (n = 3). Adenovirus overexpressing sFlt-1 or a control Fc (Jikai Bio) was constructed according to previous studies and injected with 1*10 μg of the Fc-containing virus through the tail vein on G8 (early second trimester). 9 PFU adenovirus (Ad Fc or Ad Flt1), human recombinant Leptin protein (120 μg / kg / day) (R&D, #598-LP-05M) or vehicle (PBS) were injected into rats via tail vein every day from G12 to G19.

[0066] 2) Blood pressure measurement in rats

[0067] Systolic blood pressure (SBP) was measured by tail-cuff plethysmography (MRBP, IITC). Three days before the formal measurement, rats were immobilized with a tail-cuff restraint for 30 minutes each day for adaptation training to acclimate them to the blood pressure measurement procedure. After the rats had adapted to the measurement procedure, blood pressure was measured: the rats were immobilized with a restraint and placed in a constant temperature environment of 32°C for 15-20 minutes, and then measured five times in a row, with an interval of 1 minute between each measurement. If another measurement was required, the rats had to rest for 5 minutes before the next measurement. According to this method, blood pressure was measured at G7, G10, G12, G14, G16, and G19.

[0068] 3) Placenta sampling

[0069] At G20, the rats were anesthetized, blood samples were collected, the abdominal cavity was opened, the uterus was removed, the offspring and placenta were separated, and the placenta weight was measured.

[0070] The study found that Figure 3As shown, the group injected with sFlt-1 overexpressing adenovirus (preeclampsia rat model group) showed significantly increased blood pressure and decreased placental weight compared to the group injected with the control Fc virus (control group), demonstrating abnormal placental development in the preeclampsia model rats and elevated maternal blood pressure, consistent with the preeclampsia phenotype, indicating successful model establishment. Following continuous tail vein injections of recombinant Leptin protein from G12 to G19 (mid- to late-gestational age), blood pressure was significantly lower and placental weight increased in the Leptin-treated group compared to the PBS vehicle-only group, reversing the preeclampsia phenotype and improving maternal and fetal outcomes.

[0071] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection 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. The use of leptin according to claim 1 in the preparation of a medicament for preventing hypertension and / or placental dysplasia in late pregnancy during early and mid-pregnancy in preeclampsia, wherein: The dosage form of the drug is solution, lyophilized powder or sterilized powder.

3. Use of the leptin according to 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, characterized in that: The subjects of drug administration were rats.

4. The 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 medicine 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, characterized in that: The pharmaceutical composition includes 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, characterized in that: The pharmaceutical composition includes an excipient.

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