A total of 20 male and 20 female Sprague-Dawley rats (weight, 250-300 g; age, 7-9 weeks; Guangdong Medical Laboratory Animal Center, Guangzhou, China) were kept in cages at a temperature set between 23 and 26°C (50-70% humidity and 12-h light/dark cycle). The rats had free access to food and water, and the ratio of male to female was 1:1. After mating of the rats, in the following morning, small wet cotton swabs with saline were used to collect vaginal secretions of female rats, and this was recorded as the day 0 of pregnancy (vaginal plugs were used to determine if the rats were pregnant). A hypertensive model group was established by gavaging 6 pregnant rats using 80 mg/kg/day Nω-nitro-L-arginine methyl ester (L-NAME; Sigma-Aldrich; Merck KGaA, Darmstadt Germany) for 8 days starting on day 12 of the pregnancy, and this was termed the L-NAME group. In the control group, 6 pregnant rats were gavaged using 80 mg/kg/day normal saline for 8 days. All animal tests conducted in the present study were approved by the Ethics Committee of Hebei General Hospital (Shijiazhuang, China).

The systolic blood pressure (SBP), diastolic blood pressure (DBB) and mean arterial pressure (MAP) of rats were detected on days 12, 16 and 20 of pregnancy, using a non-invasive rat tail arterial blood pressure monitor (Tail Cuff Blood Pressure Systems; IITC Life Science, Woodland Hills, CA, USA). The method was as follows: The pregnant rats were placed in a fixator, which was adjusted in terms of the size of the pregnant rat. An air bag was sleeved in the middle of the tail, and the measuring instrument was well-connected. The blood pressure of the rat tail artery was detected when the rat was calm and the pulse wave of the blood pressure measuring instrument was stable. The mean value of each rat was obtained based on five measured values.

In addition, the rats were kept in standard metabolic cages on days 12, 16 and 20 of their pregnancy, and their urine volume was collected for 24 h. The 24-h urinary protein (UP) level was tested with an automatic biochemical analyzer (AU5800; Beckman Coulter, Inc., Brea, CA, USA).

Prior to placental tissue collection, urine was obtained from the rats on day 20 of pregnancy. Under anesthesia by an intraperitoneal injection of chloral hydrate (350 mg/kg), the animals were sacrificed by rapid cervical dislocation (21). The uterus was exposed and cut open, and the placenta was removed. The placenta was fixed using 4% polymethylene for 24 h. Next, the placenta was embedded in paraffin and prepared for hematoxylin and eosin (H&E) and immunohistochemical staining.

Following the collection of placental tissue, the abdominal organs and the abdominal viscera were pushed to one side in order to expose the inferior vena cava and the abdominal aorta. An index finger was used to touch the blood vessel, and the abdominal aorta was more evident. The blood of the rats was collected from the bifurcation of the abdominal aorta and centrifuged at 1,500 × g for 10 min at 4°C. The serum was then used for enzyme-linked immunosorbent assay (ELISA).

Paraffin-embedded placental tissue was sliced, and the thickness of each slice was ~4 µm. The slices were routinely dewaxed with dimethylbenzene for 15 min and 95% ethanol for 3 min at room temperature, and then washed three times with distilled water. Next, the slices were stained by hematoxylin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 15 min at room temperature. The slices were then washed three times with distilled water, followed by treatment with 1% hydrochloric acid alcohol. The slices were then washed three times with distilled water and stained with eosin staining solution (Beijing Solarbio Science & Technology Co., Ltd.). Subsequently, the slices were dehydrated using gradient alcohol (80% ethanol for 2 sec, 95% ethanol for 3 min and absolute alcohol for 6 min), and neutral gum (Beijing Solarbio Science & Technology Co., Ltd.) was used for mounting. Finally, the slices were observed under an inverted fluorescence microscope (MF53; Micro-shot Technology Co., Ltd., Guangzhou, China).

The tissue slices were routinely dewaxed, washed with distilled water, soaked in 0.01 M citrate buffer (pH=6.0) and heated to 95°C using a microwave. Subsequent to cooling, the slices were washed using PBS (pH=7.4), following which 3% hydrogen peroxide was added to the slices and maintained for 20 min. The slices were then incubated with anti-ANXA1 antibody (cat. no. ab214486; 1:1,000; Abcam, Cambridge, MA, USA), at 4°C for 24 h. On the following day, goat anti-mouse IgG secondary antibodies (cat. no. ab6708; 1:8,000; Abcam) were added to the slices at 37°C and maintained for 30 min. The slices were then stained with 3,3′-diaminobenzidine solution (Leica Microsystems, Shanghai, China) for 10 min. After washing three times with distilled water, the slices were dyed using hematoxylin for 30 sec at room temperature. The slices were finally observed under an inverted fluorescence microscope.

The levels of the inflammatory factors-tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and IL-8 were measured using TNF-α ELISA kit (cat. no. RTA00; R&D Systems, Inc., Minneapolis, MN, USA), IL-1β ELISA kit (cat. no. RLB00; R&D Systems, Inc.), IL-6 ELISA kit (cat. no. ESK6029; Sangon Biotech Co., Ltd., Shanghai, China) and respectively. All the regents used for the ELISA assay were provided by the kits and the determination was performed according to the manufacture’s protocols. The optical density value at 450 nm was measured using SMR16.1 multimode reader (USCN Business Co., Ltd., Wuhan, China).

The placental trophoblasts were obtained from the placenta of pregnant rats with hypertension. In brief, the villi tissues were cut into −1 mm³ pieces and digested with 0.25% trypsin (Beijing Solarbio Science & Technology Co., Ltd.) and 0.1% collagenase (Beijing Solarbio Science & Technology Co., Ltd.) at 37°C for 25 h. The reaction was stopped by addition of the serum. The solution was centrifuged at 1,200 × g for 30 min at 4°C. The collected trophoblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in an incubator that contained 5% CO₂. The cells were identified under an inverted microscope.

For ANXA1 knockdown, ANXA1 small interfering RNA (siRNA) and empty vectors (30 nM) were purchased from the Genomeditech (Shanghai, China). The vectors were transfected into trophoblasts by Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.).

The trophoblasts were isolated from placental tissues and then the expression of vimentin was examined by immunofluorescence staining to identify trophoblasts. Briefly, the villi tissues were homogenized and soaked in Hank’s balanced salt solution supplemented with HEPES (25 mmol), DNase1 and collagenase (15 U/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37°C with agitation for 30 min. The dispersed cells were separated by filtering through a screen mesh (40-mm diameter per pore). The cells in Ficoll suspension were centrifuged at 1,200 × g for 30 min at 4°C, and the obtained cells were maintained in DMEM with 10% FBS. Subsequent to culturing, the trophoblasts were washed using PBS and fixed with 4% paraformaldehyde at 4°C for 20 min. Goat serum (Beyotime Institute of Biotechnology, Shanghai, China) was then added to block the cells (at room temperature for 30 min), followed by incubation with the anti-vimentin primary antibody (cat. no. ab8978; 1:1,000; Abcam) 4°C for 24 h. PBS was used as the negative control for the anti-vimentin primary antibody. Next, the cells were incubated using a fluorescence-conjugated secondary antibody (Alexa-Fluor^® 594 goat anti-rabbit antibody IgG; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 1.5 h at room temperature. Nuclei were counterstained by Hoechst 33258 (Beyotime, Shanghai, China), and images were obtained using an inverted fluorescence microscope.

Cell apoptosis was analyzed using an Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (Beijing Solarbio Science & Technology Co., Ltd.). The trophoblasts were first seeded in a 6-well plate (5×10⁴ cells/well) for 24 h, and then treated with PBS, siRNA-ANXA1 or empty vector using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) and incubated at 37°C for further 24 h. The cells were digested by trypsin (Beijing Solarbio Science & Technology Co., Ltd.) and re-suspended with a 1X binding buffer. Next, the cells were double-stained with Annexin V-FITC/PI for 20 min, and the cell apoptosis was determined using flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).

The total RNA of cells was isolated using a TRIzol reagent (Promega Corporation, Beijing, China). The concentration of RNA was measured by ultra-micro protein nucleic acid analyzer (NanoDrop; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). In total, 1 µg RNA was used for cDNA synthesis by an ABScript II cDNA First Strand synthesis kit (ABclonal Biotech Co., Ltd., Wuhan, China) following the manufacturer’s protocols. A SYBR Premix Taq™ II kit (Dalian Meilun Biology Technology Co., Ltd, Dalian, China) was then used to amplify cDNA, according to the instructions of the kit. The primers used were purchased from BersinBio Co., Ltd. (Guangzhou, China), and are listed in Table I. The data were analyzed with the 2^−ΔΔCq calculation (22), and the relative mRNA expression levels were normalized to that of GAPDH.

The cells were lysed by radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.). The protein concentrations of samples were measured using a BCA protein assay kit (Qiyi Biological Technology Co., Ltd., Shanghai, China). Next, the proteins (25 µg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). Tris-buffered saline/Tween 20 buffer containing 5% skimmed milk was added to block the membranes at room temperature for 2 h. The corresponding primary antibodies were subsequently used to incubate the membranes overnight at 4°C, including anti-ANXA1 (cat. no. ab214486; 1:1,000; Abcam), anti-pro-caspase-3 (cat. no. ab32499; 1:1,000; Abcam), anti-cleaved-caspase-3 (cat. no. ab2302; 1:700; Abcam), anti-B-cell lymphoma-2 (Bcl-2; cat. no. ab692; 1:800; Abcam), anti-Bcl-2-associated X protein (Bax; cat. no. 2774; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-JAK2 (cat. no. ab108596; 1:600; Abcam), anti-phosphorylated (p)-JAK2 (cat. no. ab195055; 1:800; Abcam; anti-STAT3 (cat. no. ab119352; 1:1,000; Abcam), anti-p-STAT3 (cat. no. ab32143; 1:800; Abcam) and anti-GAPDH (cat. no. ab8245; 1:600; Abcam). On the following day, the membranes were incubated with the following secondary antibodies at room temperature for 1 h: Rabbit anti-mouse IgG (cat. no. ab6709; 1:6,000; Abcam), mouse anti-rabbit IgG (cat. no. 93702; 1:8,000, Cell Signaling Technology, Inc.), and goat anti-rabbit (cat. no. ab205718; 1:6,000; Abcam). The blots were subsequently developed using a chemiluminescence substrate kit (BeyoECL Star; Beyotime Institute of Biotechnology, Haimen, China). The results were analyzed by the ECL system (Amersham; GE Healthcare, Chicago, IL, USA).

Statistical analysis was conducted using SPSS version 20.0 software (IBM Corporation, Armonk, NY, USA). Data are expressed as the mean ± standard deviation. Student’s t-test was used to compare the differences between two groups, while one-way analysis of variance and the post-hoc Dunnett’s test were performed to compare the differences among more than two groups. P<0.05 was considered to denote a statistically significant difference.

The uterus and placenta were examined, and serum was obtained (Fig. 1A). The results revealed that the SBP, DBP, MAP and UP levels remained stable on day 12 of the rat pregnancy in the L-NAME group, as compared with those in the control group. However, the SBP, DBP, MAP and UP levels in the L-NAME group were significantly increased in comparison with those in the control group on day 20 of the pregnancy (P<0.05; Fig. 1B-E). These results demonstrated that the PE model was successfully established in the L-NAME-treated rats.

H&E staining, ELISA and immunohistochemical assay were performed to investigate the histopathological changes, inflammatory reaction and ANXA1 expression in placental tissues. The H&E staining images displayed that, in comparison with the control group, the trophoblast proliferation and inflammatory cell infiltration were evidently increased in the L-NAME group (Fig. 2A). The ELISA data revealed that the levels of TNF-α, IL-1β, IL-6 and IL-8 in L-NAME group were higher as compared with those in the control group (Fig. 2B-E; P<0.01). The immunohistochemical assay results also revealed that there was a considerable amount of brown particles in the L-NAME group, which represented positive expression of ANXA1 (Fig. 2F and G). These results indicated that the expression of inflammatory factors and ANXA1 was upregulated during PE.

The trophoblasts were identified by staining with vimentin antibody (Fig. 3). The RT-qPCR and western blotting data demonstrated that the mRNA and protein levels of ANXA1 were reduced in cells transfected with siRNA-ANXA1 as compared with those transfected with empty vector (Fig. 4A and B; P<0.01). The flow cytometry results also observed that the apoptosis rate was significantly decreased when cells were transfected with siRNA-ANXA1 (Fig. 4C; P<0.01). As displayed by the ELISA data, siRNA-ANXA1 markedly suppressed the levels of TNF-α, IL-1β, IL-6 and IL-8 (Fig. 4D-G; P<0.05). Therefore, downregulation of ANXA1 decreased the apoptosis during PE.

Bcl-2 and Bax are known to regulate the cell apoptosis (23), while caspase-3 is activated by multiple apoptotic signals by cleaving pro-caspase-3 into cleaved-caspase-3 (24). Therefore, RT-qPCR and western blot analysis were performed to explore the effect of ANXA1 on these apoptosis-associated factors in trophoblasts. As demonstrated by the RT-qPCR data, siRNA-ANXA1 transfection significantly decreased Bax mRNA expression and increased Bcl-2 mRNA expression (Fig. 5A and B; P<0.05). Furthermore, the western blot analysis data revealed that siRNA-ANXA1 promoted the expression levels of pro-caspase-3 and Bcl-2 protein, whereas it reduced the expression levels of cleaved-caspase-3 and Bax protein (Fig. 5C; P<0.01). Therefore, downregulation of ANXA1 inhibited the apoptosis during PE through regulating the expression of apoptosis-related genes.

JAK2/STAT3 pathway was examined using western blot analysis to help investigate the pathway of ANXA1 in trophoblasts. The data revealed that the phosphorylation of JAK2 and STAT3 was downregulated in siRNA-ANXA1-treated cells as compared with the empty vector group. The levels of JAK2 and STAT3 remained stable in all groups (Fig. 6; P<0.01). Therefore, the inhibition of JAK2/STAT3 signaling pathway may be related to the effect produced by si-ANXA1.