Participant recruitment

Studies have shown that the incidence of preeclampsia in the Chinese population is about 4%. The confidence (Z) was set at 1.96, the significance (α) was set at 0.05, the incidence rate (p) was set at 0.04, according to the formula n = [Z2 × p × (1 − p)]/α2, and the minimum sample size calculated was 59. We enrolled 60 pregnant women who underwent regular obstetric check-ups and who delivered at the Obstetrics Department of Fujian Maternity and Child Health Hospital between September 2022 and December 2023. The participants were divided into two groups: the case group (PE group), consisting of 30 pregnant women with preeclampsia, and the control group (CTL), comprising 30 pregnant women without pregnancy complications. The diagnostic criteria for preeclampsia were based on the ninth edition of Obstetrics and Gynecology published by the People’s Health Publishing House [19]. All participants delivered via cesarean section, with those in the PE group undergoing the procedure due to surgical indications. The CTL group had cesarean sections for reasons such as a previous cesarean section, abnormal birth canal, abnormal fetal position, or social factors. Patients with other pregnancies or medical or surgical complications were excluded from the study. Both groups of pregnant women did not have any specific lifestyle habits, such as smoking or alcohol consumption. Informed consent was obtained from all participants through signed consent forms. The experimental procedures were conducted following ethical standards set by local and national human research committees and the principles of the Declaration of Helsinki. Approval for the study protocols was granted by the Ethics Committee of Fujian Maternity and Child Health Hospital (Reference: 2021 KLRD09007) following a review that ensured compliance with relevant regulations, including the “Measures for Ethical Review of Biomedical Research Involving Humans” by the National Health Commission and the Declaration of Helsinki.

Placental sample acquisition

Human placental tissue was collected during cesarean section. Once the placenta had been delivered, we immediately removed a portion of placental villous tissue (1.0 cm3) from the root of the umbilical cord. We ensured that this procedure was carried out under aseptic conditions and made sure that tissue was only removed from areas on the placenta that did not show bleeding, infarction, or calcification.

Generation of trophoblast cells with different p53 expression levels

The BeWo cells, HTR-8/SVneo cells, and HUVEC used in this study were purchased from Wuhan Punosai Life Technology Co., Ltd and cultured at 37℃ in a 5% CO2 cell incubator (Thermo Fisher Scientific). The complete medium was prepared as follows: 80–90% Ham’s F-12 K medium (Wuhan Punosai Life Technology Co., Ltd) + 10–20% common fetal bovine serum (NEWZERUM) + 1% double antibody (penicillin/streptomycin) solution (Dalian Meilun Biotechnology Co., Ltd). 80–90% RPMI (1640) basic medium (Dalian Meilun Biotechnology Co., Ltd) + 10–20% common fetal bovine serum (NEWZERUM) + 1% double antibody (penicillin/streptomycin) solution (Dalian Meilun Biotechnology Co., Ltd); 500 mL ECM basal medium (ScienCell) + 25 mL fetal bovine serum (ScienCell) + 5 mL endothelial growth factor (ScienCell) + 5 mL double antibody (penicillin/streptomycin) solution (ScienCell). Trophoblast cells with different p53 expression levels were constructed as follows:

p53 overexpression group (p53 +/+): Trophoblast cells were co-cultured with the p53 inducer Nutlin-3 for 72 h.

p53 inhibition group (p53 −/−): Trophoblast cells were co-cultured with the p53 inhibitor MG-132 for 48 h.

Control group (CTL): Trophoblast cells were co-cultured with DMSO (dimethyl sulfoxide) for 72 h.

Generation of PE rat models with different p53 expression levels

Female and male Sprague–Dawley rats aged 6–8 weeks and weighing 180 ~ 200 g without a mating history were randomized into four groups of eight rats in each group as follows: (1) p53-overexpressing (p53 +/+) group: before pregnancy, 40 mg/kg of the p53 inducer Nutlin-3 was intraperitoneally injected every 2 days for 14 consecutive days, and 125 mg/kg/day L-NAME was intraperitoneally injected on the 7 th day of pregnancy for 7 consecutive days. (2) The inhibition of p53 expression (p53 −/−) group: before pregnancy, the p53 inhibitor MG-132 (10 μg/kg per day) was intraperitoneally injected for 14 consecutive days, and L-NAME (125 mg/kg/day) was intraperitoneally injected for 7 consecutive days on the 7 th day of pregnancy; (3) The normal pregnant mouse (CTL) group: before pregnancy, 5 mL of saline was injected intraperitoneally every day for 14 consecutive days, and from the 7 th day of pregnancy, 1 mL of normal saline was injected intraperitoneally every day for 7 consecutive days; (4) The preeclampsia pregnant mouse (PE) group: before pregnancy, 5 mL of saline was intraperitoneally injected daily for 14 consecutive days, and 125 mg/kg/day of L-NAME was injected intraperitoneally every day since the 7 th day of pregnancy for 7 consecutive days.

Female and male rats were adaptively fed for 1 week. During estrus, male and female rats were fed in the same cage at a ratio of 1:1 so that the date of the discovery of vaginal suppositories or sperm found in vaginal discharge was set as the first day of pregnancy. Random urine samples were collected on the 6 th and 19 th days of gestation, blood pressure was measured, and placental tissue was collected on the 20 th day of gestation. After inhalation anesthesia with isoflurane was administered to pregnant rats at 20 th day of gestation, cesarean section was performed to remove the placental tissue and then suture the incision. Approval for the study protocols was granted by the Ethics Committee of Fujian Maternity and Child Health Hospital (Reference: 2021 KLRD09007).

Blood pressure measurement in rats

The blood pressure of the rats was measured by a noninvasive tail artery sphygmomanometer (intelligent noninvasive sphygmomanometer, Beijing Soft Long Biotechnology Co., Ltd.). The pressure sensor was placed on the tail root of the rat, and after the pulse was stable, the start/stop key was pressed to start the measurement. The systolic blood pressure measured by the blood pressure meter should be recorded more than 6 times for each rat (valid data: the average difference between the three consecutive systolic blood pressures in the quiet state of the rat should be the least, and the difference between the three results should be less than 10 mmHg). The mean value was calculated as the systolic blood pressure of the rat on that day, and the whole process was completed within 10 min.

Detection of the urinary protein concentration

The 24-h urinary protein concentration of the rats was measured by the CBB method. The reaction mixture was prepared according to Table 6, and the mixture was thoroughly mixed and incubated for 5 min. After the amount of double distilled water was adjusted to zero, the absorbance of each tube was measured at a wavelength of 595 nm and a light diameter of 1 cm. The following formula was used: urinary protein concentration (mg/L) = (measured OD value − blank OD value)/(standard OD value − blank OD value) × standard concentration (563 mg/L).

Table 6 Urinary protein detection reaction systemDetection ROS concentrations in trophoblast cells

The final concentration of DCFH-DA was 10 μM, and the solution was diluted with serum-free medium at 1:1000. The positive control was diluted with serum-free medium at 1:1000 (Rosup, 50 mg/mL) and then added to the cells. DCFH-DA working solution was added, and the cells were incubated at 37 °C for 20 ~ 30 min under light protection. The cells were washed with serum-free medium 3 times to fully remove the DCFH-DA that did not enter the cells. The fluorescence intensity before and after stimulation was detected by flow cytometry (BD LSRFortessa™) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The concentration of ROS in trophoblast cells is expressed as the mean value of the FITC-A channel.

Determination of the concentration of MDA in placental tissue

The samples were prepared as follows: PBS buffer = 1:10 homogenate lysate placental tissue. The supernatant was collected after centrifugation at 10,000 rpm for 15 min at 4 ℃. The MDA probe was fully dissolved in 5 mL of 95 ℃ ultrapure water and mixed with 5 mL of glacial acetic acid to prepare the MDA probe working solution. According to the number of measured samples (including standard products), the MDA working solution was prepared following Table 7.

Table 7 MDA detected working solution

A PBS buffer gradient dilution of the MDA standard (200 μM) was used for data analysis via the standard curve method. According to Table 8, the samples were thoroughly mixed with the MDA detection working solution and incubated at 95 ℃ for 40 min.

Table 8 MDA working solution

After being incubated in an ice bath for 5 min, the mixture was centrifuged at 10,000 rpm for 10 min, after which 200 μL of the supernatant was added to a 96-well plate. The absorbance at 532 nm was detected by a BioTek Epoch. The standard curve was drawn according to the gradient dilution standard group-blank group value data: Y = aX + b (Y is the standard group value − blank group value; X is the MDA concentration of the standard substance; a is the slope of the standard curve; b is the intercept of the standard curve); Unit sample MDA (μmol/gprot) = [(sample group value − blank group value) − b]/a × sample dilution ratio/sample protein concentration (gprot/L).

Determination of the concentration of total iron in placental tissue

Before testing, the reagents in the kit were equilibrated to room temperature. Twenty µl of 10 mmol/L iron standard solution and 1980 μL of double steaming water were mixed evenly. Fresh tissue (0.1 g) was added to 0.9 mL of buffer for homogenization and centrifuged at 10,000 rpm for 10 min, after which the supernatant was collected. Before the formal test, 2 to 3 samples with large expected differences were diluted to different concentrations for the pretest. According to the pretest results, the dilution was carried out in combination with the linear range of the kit (0.4 ~ 50 μmol/L).

Then, 300 μL of the standard solution was added to the corresponding 1.5-mL EP tube as the standard tube. Then, 300 μL of the sample was added to the corresponding 1.5-mL EP tube as the sample tube. Then, 150 μL of chromogenic solution was added and incubated at 37 ℃ for 40 min. After centrifugation at 12,000 rpm for 10 min, 300 μL of the supernatant was added to each corresponding well of the enzyme label plate, and the OD of each well was measured at 593 nm with a BioTek Epoch. Standard fitting curve: y = ax + b; y: standard solution OD value − blank OD value (OD value when standard solution concentration is 0); a: slope of the mark; x: concentration of standard solution; b: intercept of the mark; Tissue total iron content (μmol/kg wet weight) = (△A − b)/a × f/(m/V); △A: the absolute OD value of the sample (OD value of the determination hole − OD value of the control hole); b: intercept of the mark; a: slope of the mark; f: dilution ratio of the sample before adding to the test system; m: sample homogenate quality (g); V: amount of sample homogenate added (mL).

Morphological observation of trophoblast cells in placental tissue

Fresh placental tissue (no more than 1 mm × 1 mm × 1 mm) was fixed in electron microscope fixative solution at 4 ℃ for 2 ~ 4 h and then rinsed with 0.1 M phosphate buffer (pH 7.4) 3 times for 15 min each. After the addition of 1% osmic acid · 0.1 M phosphate buffer, PB (pH 7.4) was fixed at room temperature (20℃) for 2 h and then rinsed with 0.1 M phosphate buffer PB (pH 7.4) 3 times for 15 min each. The samples were dehydrated in 50%–70%–80%–90%–95%–100%–100% alcohol–100%–100% acetone for 15 min each. The samples were transferred to acetone:812 embedding agent = 1:1 for 2 ~ 4 h, acetone:812 embedding agent = 1:2 overnight, and pure 812 embedding agent = 5 ~ 8 h. Pure 812 embedding agent was poured into the embedding plate, which was then placed in an oven at 37 ℃ overnight. The samples were polymerized in an oven at 60 °C for 48 h. Ultrathin Sects. (60 ~ 80 nm) were then prepared using a Leica UC7 ultrathin microtome. Sections were double stained with uranium lead (2% uranium acetate saturated alcohol solution and lead citrate for 15 min each) and dried overnight at room temperature. Images were acquired and analyzed using a transmission electron microscope (HT7700; HITACHI).

Quantitative real-time PCR (qRT‒PCR)

The 50 mg sample was fully ground, mixed with 1 mL of TRIzol and left for 5 min. Then, 200 µL of chloroform was added, and the mixture was shaken for 15 s. The mixture was left for 5 min and centrifuged at 12,000 rpm at 4℃ for 15 min. Then, 500 μL of the supernatant was removed, 500 μL of isopropyl alcohol was added (supernatant:isopropyl alcohol = 1:1), the mixture was allowed to stand for 5 min, and the mixture was centrifuged at 12,000 rpm at 4℃ for 10 min. The supernatant was discarded, 600 μL of 75% ethanol (anhydrous ethanol:DEPC-treated water = 3:1) was added, the precipitate was oscillated, and the precipitate was washed and centrifuged at 12,000 rpm at 4℃ for 5 min (this process was repeated 3 times). The supernatant was discarded, and the total RNA was dissolved in 30 μL of DEPC-treated water. The concentration and purity of the RNA were determined with a Thermo Scientific spectrophotometer.

The reverse transcription kit used in this study was purchased from TaKaRa’s TB Green® Premix Ex Taq™ (Tli RNaseH Plus). According to the reaction system of SYBR® Green analysis, 500 ng of total RNA was needed, and the required RNA volume was calculated as VRNA = 500/CRNA (μL). Total RNA was used as a template, and the reverse transcription reaction solution was prepared on ice according to Table 9. The solution was centrifuged and mixed on a thermal cycler at (1) 37℃ for 15 min, (2) 85℃ for 15 s, and (3) 4℃ for reverse transcription.

Table 9 Reverse transcriptional reaction solution

The primers used in this study were synthesized by Fuzhou Shangya Biological Co., Ltd (Table 10). The PCR kit used in this study was purchased from the PrimeScriptTM RT Reagent Kit (Perfect Real Time) of TaKaRa Company in Japan. The PCR solution was prepared on ice according to Table 11.

Table 10 Primer informationTable 11 PCR reaction solution

The solution was centrifuged and then placed on a Quant Studio 3 fluorescence quantitative PCR instrument. The reaction conditions were as follows: (1) 95℃ for 30 s; (2) 95℃ for 5 s; 60℃ for 34 s for 40 cycles; (3) 95℃ for 15 s; 60℃ for 1 min; and 95℃ for 15 s. The mRNA level of GAPDH was used as the control, and the relative mRNA expression was calculated as 2−△△CT [△△CT = experimental group (objective CT-internal reference CT) − control group (objective CT-internal reference CT)].

Western blot (WB)

One hundred milligrams of sample tissue was added to 1000 μL of freshly prepared protein lysate (RIPA lysis buffer containing the protease inhibitor PMSF = 100:1, ice operation) for cleavage. The samples were centrifuged at 12,000 rpm at 4℃ for 10 min. A BCA protein quantitative/concentration assay kit (Dalian Meilun Biotechnology Co., Ltd.) was used to determine the concentration of each protein sample. Then, 5 × SDS protein loading buffer was added, and the samples were incubated in a 100℃ water bath for 10 min for denaturation. Then, 50 μg of protein sample (Vprotein = 50/Cprotein/0.8) and 5 μL of protein marker were added to the lanes. After 40 min of electrophoresis at a constant voltage of 80 V, the voltage was adjusted to 120 V to continue electrophoresis. The film transfer device was installed, and the film transfer conditions were set at 70 V for 90 min. After membrane transfer, the PVDF membrane was shaken and washed three times with TBST buffer for 10 min each. Fresh 5% skim milk powder sealing solution was added, and the membrane was slowly shaken at room temperature for 2 h. After the membrane was blocked with TBST, the membrane was washed with TBST for 10 min, and this process was repeated three times. The PVDF membrane was placed into a container containing primary antibody solution (Immunoway) (antibody: primary antibody diluent = 1:1000) and incubated in a shaking bed slowly at 4℃ overnight (12 ~ 16 h). After incubation with the primary antibody, the PVDF membrane was washed with TBST for 10 min on a shaker, and this process was repeated three times. The PVDF membrane was placed into a container containing a secondary antibody solution (Immunoway) (antibody: secondary antibody diluent = 1:1000) and incubated in a shaker at room temperature for 1 h. After incubation, the PVDF membrane was washed with TBST buffer for 10 min, and this process was repeated three times. The developing solution was prepared by mixing liquid A and liquid B in a hypersensitive enhanced chemiluminescence (ECL) kit (Dalian Meilun Biotechnology Co., Ltd.) at a 1:1 ratio. The PVDF membrane was immersed in developing solution for 30 s and then photographed with an automatic Western blot imaging analyzer (Fluor Chem M). The gray values of the protein bands were analyzed by Image Lab image analysis software. The relative protein expression was calculated as the gray value of the target protein/gray value of the internal reference protein.

Immunohistochemistry (IHC)

The sections were sequentially washed with 15 min of dewaxing solution I, 15 min of dewaxing solution II, 15 min of dewaxing solution III, 15 min of anhydrous ethanol I, 5 min of anhydrous ethanol II, 5 min of 85% alcohol, 5 min of 75% alcohol, and 5 min of distilled water. The sections were placed in citric acid antigen repair buffer (pH 6.0) in a repair box for antigen repair in a microwave oven, heated for 8 min to boiling, allowed to cool for 8 min to warm and then returned to medium and then to low heat for 7 min. After natural cooling, the sections were placed in PBS (pH 7.4) buffer on a shaker and washed 3 times for 5 min each. The sections were incubated in 3% hydrogen peroxide solution at room temperature for 25 min and then washed. The tissue was uniformly covered with 3% BSA and incubated at room temperature for 30 min. Primary antibody diluent was added to the sections, which were then placed in a wet box at 4 °C overnight incubation before washing. The tissue was covered with secondary antibody diluent, incubated at room temperature for 50 min and then washed. DAB developing solution was added to the sections, and the color development time was controlled under a microscope. The sections were rinsed with tap water to terminate color development. The sections were redyed with hematoxylin for 3 min and then washed with tap water. After differentiation, the sections were washed with hematoxylin differentiation solution and tap water for a few seconds. After the sections were rinsed with blue return water, they were rinsed with hematoxylin blue solution. The sections were incubated in 75% alcohol for 5 min, 85% alcohol for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and n-butanol for 5 min, followed by xylene I for 5 min, after which they were dried and sealed with neutral gum.

Microscopy, image acquisition, and analysisImmunofluorescence and confocal laser technology

The sections were subsequently washed in xylene I 15 min-xylene II 15 min-anhydrous ethanol I 5 min-anhydrous ethanol II 5 min-85% alcohol 5 min-75% alcohol 5 min-distilled water. The sections were placed in EDTA antigen repair buffer (pH 8.0) and subjected to antigen retrieval in a microwave oven. The medium was heated for 8 min, the fire was stopped for 8 min, and the medium was changed to low heat for 7 min. After natural cooling, the sections were placed in PBS (pH 7.4) and shaken 3 times for 5 min each. The sections were placed in 3% hydrogen peroxide solution, incubated for 25 min at room temperature in the dark and then washed. The sections were blocked with BSA for 30 min. Primary antibody diluent was added to the sections, which were then incubated in a wet box at 4 °C overnight and washed. The secondary antibody diluent was added to the sections, which were incubated at room temperature for 50 min and then washed. CY3-TSA was added to the sections, which were incubated at room temperature for 10 min and then washed. The sections were placed in an EDTA antigen repair buffer (pH 8.0) repair box and heated in a microwave oven. The medium was heated for 8 min, the temperature was stopped for 8 min and the medium was changed to low heat for 7 min. The second primary antibody diluent was added to the sections, which were incubated in a wet box at 4 °C overnight and then washed. The secondary antibody diluent was added to the sections, which were incubated at room temperature for 50 min and then washed. The TSA was added to the sections, which were incubated at room temperature for 10 min in the dark and then washed. The sections were placed in an EDTA antigen repair buffer (pH 8.0) repair box and heated in a microwave oven. The medium was heated for 8 min, the temperature was stopped for 8 min, and the medium was changed to low heat for 7 min. The third primary antibody diluent was added to the section, which was incubated in a wet box at 4 °C overnight and then washed. Then, Cy5-labeled fluorescent secondary antibody diluent was added to the sections, which were incubated at room temperature for 50 min in the dark and then washed. DAPI dye solution was added to the sections, which were incubated at room temperature for 10 min in the dark and then washed. The autofluorescence quencher was added to the sections for 5 min, and the sections were rinsed with running water for 10 min and then washed. The sections were sealed with antifluorescence quenching solution. The sections were placed under a scanner to capture images.

ELISA

The sample was centrifuged at 5000 rpm for 5 min, after which the supernatant was collected. The standard product was successively diluted to 1000, 500, 250, 125, 62.5, 31.2, 15.6, and 0 pg/mL. Solution A and solution B were diluted 1:100. The thick washing solution was diluted 30 times. After 100 μL of standard or sample solution was added, the enzyme-labeled plate was coated and incubated at 37℃ for 2 h. The liquid in the hole was discarded, 100 μL of detection solution A was added, and the enzyme-labeled plate was incubated at 37℃ for 1 h. The liquid in the hole was discarded, and the plate was washed 3 times with 350μL of washing liquid. The detection solution B working solution (100 μL) was added, and the enzyme-labeled plate was incubated at 37℃ for 30 min. The liquid in the well was discarded, and the plate was washed 5 times. Next, 90 μL of TMB substrate solution was added, the plate was coated, and the color was developed at 37℃ in the dark. When the gradient of blue was obvious in the first 3 to 4 holes of the standard hole, the gradient of the last 3 to 4 holes was not obvious, and the process could be terminated. Then, 50 μL of termination solution was added to terminate the reaction. The optical density of each well was immediately measured with a BioTek Epoch at a wavelength of 450 nm.

Angiogenesis test

The matrix glue was melted overnight, and the 200 μL pipette head and 48-well plate were precooled in advance. Then, 150 μL of matrix glue was added to the 48-well plate and incubated in a cell incubator at 37℃ for 30 min. The HUVEC suspension was prepared at a density of 1 × 105/mL. According to the groups, 500 μL of cell suspension was added to each of the coated plates and incubated at 37℃ for 3 ~ 4 h. After incubation, endothelial cell canalization was observed under an inverted microscope (Nikon), and angiogenesis was quantified in 3 random fields to analyze the relevant data.