Five pregnant women with preeclampsia who underwent cesarean section at Jinhua People’s Hospital were enrolled. Venous blood samples (5 mL) were obtained via venipuncture and centrifuged at 3000 revolutions per minute for 10 min to obtain serum. Placental tissues were collected within 60 min after cesarean delivery, immediately frozen in liquid nitrogen, and kept at –80 °C for subsequent analysis. Preeclampsia was diagnosed based on the following criteria: (i) after 20 weeks of gestation, the diastolic ($\ge$90 mmHg) and/or systolic blood pressure ($\ge$140 mmHg) readings were in an abnormal state; (ii) proteinuria level $\ge$0.3 g per day. The normal group (n = 5) was matched with the preeclampsia group by maternal age and gestational week. Individuals with the following complications were excluded from both groups, including renal diseases, primary hypertension, gestational diabetes, alcoholism, smoking, fetal congenital abnormalities and twin pregnancy. All participants gave their written informed consent. The Ethics Committee of Jinhua People’s Hospital granted ethical approvals for this study (approval number: 2023-007). This study adhered to the ethical guidelines outlined in the Declaration of Helsinki. Baseline characteristics of participants are presented in Table 1.

The human immortalized HTR-8-SVneo trophoblast cell line (cat.no. CL-0765), obtained from Pricella Biotechnology (Wuhan, Hubei, China), was maintained in a specialized medium (cat.no. CM-0765; Pricella Biotechnology). The cells tested negative for the presence of mycoplasma and were authenticated by short tandem repeat profiling. Cells in the control group were cultured under standard normoxic conditions (37 °C, 5% CO₂). For hypoxia treatment, cells were exposed to an atmosphere of 93% N₂, 2% O₂, and 5% CO₂ [17, 18].

HTR-8-SVneo cells (1 $\times$ 10⁴ cells per well) were cultured until they reached 90% confluence in 6-well plates. Subsequently, short interfering (siRNA) targeting IP-10 (si-IP-10-1/-2/-3), si-ALX-1/-2/-3 or the negative control (si-NC) (Genomeditech, Shanghai, China) were transfected into HTR-8-SVneo cells for 48 h using GMTrans Liposomal Transfection Reagent (Genomeditech, Shanghai, China). Finally, HTR-8-SVneo cells were collected to detected transfection efficiency through quantitative real-time polymerase chain reaction (qRT-PCR). The relative mRNA expression levels of IP-10/ALX were determined using the 2^{-Δ⁢Δ⁢Ct} method, with si-NC as the negative control. “Highly efficient silencing” was defined as a reduction in mRNA expression of $\ge$60% compared to the si-NC group. Experiments were conducted in triplicate and repeated three times.

HTR-8-SVneo cells and placental tissues were collected for RNA isolation via a Total RNA extraction Kit (cat.no. RE-03011; Foregene Biotechnology, Chengdu, Sichuan, China). cDNA was then synthesized with the SeqHunt® First Strand cDNA Synthesis Kit (cat.no. CA01; Seq-Hunt Biotechnology, Beijing, China). PCR analysis was performed on a Real-time PCR System (version 1.4.1; Applied Biosystems, Foster City, CA, USA) using 2 $\times$ Blue Universal SYBR qPCR Master Mix (cat.no. AF07; Seq-Hunt Biotechnology) per the manufacturer’s instructions. Relative expression was calculated via the 2^{-Δ⁢Δ⁢Ct} values method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene for normalization. Table 2 provided a list of gene primers used in this research. Experiments were conducted in triplicate and repeated three times. Clinical results were performed with five human samples per group.

Protein extracts from HTR-8-SVneo cells and placental tissues were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (cat.no. BL504A; Biosharp Life Sciences, Hefei, Anhui, China). Subsequently, the protein samples were separated by SDS-PAGE and transferred onto a Polyvinylidene Fluoride (PVDF) membrane. Subsequently, the membrane was incubated with 5% non-fat milk (cat.no. BS102; Biosharp Life Sciences) at room temperature for 1 h to block any non-specific binding sites. The membrane was probed with primary antibodies against IP-10 (cat.no. 10937-1-AP; 1:200; Proteintech, Wuhan, Hubei, China), ALX (cat.no. bs-23765R; 1:1000; Bioss Biotechnology, Beijing, China), B-cell lymphoma 2 (Bcl-2) (cat.no. ABP50759; 1:1000; Abbkine Scientific, Wuhan, Hubei, China), B-cell lymphoma 2-associated X protein (Bax) (cat.no. 60267-1-Ig; 1:10,000; Proteintech), Caspase-3 (cat.no. ab32351; 1:5000; Abcam, Cambridge, UK) and GAPDH (cat.no. 60004-1-Ig; 1:50,000; Proteintech) at 4 °C overnight. After three washes with Tris-buffered saline with Tween 20 (TBST, 0.1% Tween 20) for 10 min each, the membrane was further incubated with secondary antibodies (cat.no. A21020 & A21010; 1:10,000; Abbkine Scientific) at 25 °C for 2 h. Protein signals were detected using a SuperKine™ ECL Kit (cat.no. BMU101-CN; Abbkine Scientific), and analyzed with Image Tool software (version 3.0; UTHSCSA, San Antonio, TX, USA). Relative protein expression levels were normalized to GAPDH. Experiments were conducted in triplicate and repeated three times. Clinical results were performed with three human samples per group.

Placental tissues were fixed, paraffin-embedded, deparaffinized, and rehydrated. After antigen retrieval, tissue sections were incubated overnight at 4 °C with primary antibodies IP-10 (1:200; Proteintech), CXC motif chemokine receptor 3 (CXCR3) (1:200; Proteintech) and ALX (1:1000; Bioss Biotechnology). This was followed by a subsequent incubation step using horseradish peroxidase-conjugated secondary antibody (1:3000; Abbkine Scientific) for 1 h at 37 °C. After that, each slice was stained with 3,3’-diaminobenzidine (DAB), and then counterstained with hematoxylin for 1 min. The sections were differentiated with 1% hydrochloric acid ethanol for 30 sec, followed by bluing in 0.5% ammonia water for 1 min. After dehydration through a graded ethanol series and clearing with xylene, the slices were mounted. Microscopic imaging was performed under a light microscope (YONGXIN OPTICS, Ningbo, Zhejiang, China). Clinical results were performed with three human samples per group.

Colony formation assay was performed to assess the proliferative potential. Briefly, HTR-8-SVneo cells, with a density of 1 $\times$ 10³ cells per well, were seeded into 6-well culture plates for one week. Cells were then washed with PBS, fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet for 15 min. Colonies were counted under a light microscope (Olympus, Tokyo, Japan) and analyzed with ImageJ software (version 2.0; National Institutes of Health, Bethesda, MD, USA). Experiments were conducted in triplicate and repeated three times.

For the measurement of cell viability, HTR-8-SVneo cells with the density of 3 $\times$ 10³ cells/mL were seeded in 96-well plates and cultured for 48 h. After that, 10 µL CCK-8 solution (cat.no. CK04; DOJINDO LABORATORIES, Shanghai, China) was added to each well, followed by 2 h of incubation. HTR-8-SVneo cell viability was measured under a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA), where optical density measurements were recorded at a wavelength of 450 nm. Experiments were conducted in triplicate and repeated three times.

Briefly, 100 µL of Matrigel (1:8 diluted in serum-free medium) was uniformly spread onto the polycarbonate membrane surface in upper chamber, and then incubated at 37 °C for 30 min to allow gelation. Complete medium (containing 10% fetal bovine serum) was added to the lower chamber of the 24-well plates, and the transwell chamber was placed in the 24-well plates with tweezers. In the upper chamber, a total of 2 $\times$ 10⁵ HTR-8-SVneo cells was added, and further cultured in an incubator for 24 h. After removing the chamber, Matrigel and non-invading cells in the upper chamber were gently removed. Subsequently, 4% paraformaldehyde was added to a new 24-well plate and fixed for 30 min. Then, 0.1% crystal violet was used to stain the invaded cells for 5 min. After cleaning with PBS for 3 times, the remaining crystal violet was removed, and the upper side of the chamber was wiped with a cotton swab. After air drying, five random fields were selected under a high-power microscope (YONGXIN OPTICS) to observe and count the cells. Experiments were conducted in triplicate and repeated three times.

Horizontal lines were drawn on the back of 6-well plates with a marker pen. HTR-8-SVneo cells (5 $\times$ 10⁵) were seeded and cultured overnight to reach confluence. The following day, a sterilized 20 µL pipette tip was used to scratch perpendicular to the pre-drawn lines. After three washes with PBS, we carefully removed any scratched cells from the medium and replaced them with fresh serum-free culture mediums. Subsequently, HTR-8-SVneo cells were cultured for 48 h at 37 °C in 5% CO₂ incubator and imaged under a microscope (YONGXIN OPTICS). The distance between cells at 0 h and 48 h was calculated using Image J software. Experiments were conducted in triplicate and repeated three times.

Cell apoptosis was assessed using an Annexin V-/Propidium Iodide (PI) Apoptosis Detection kit (cat.no. KTA0002; Abbkine Scientific). HTR-8-SVneo cells were trypsinized, washed three times with PBS, and resuspended in 1$\times$ Annexin V binding buffer containing Fluorescein Isothiocyanate (FITC)-Annexin V (5 µL). Afterward, they were further incubated with PI (5 µL) for 15 min in the dark. The stained cells were analyzed using a flow cytometer (BD Bioscience, San Jose, CA, USA), and the data were processed with Kaluza analysis software (version 2.4; Beckman Coulter, Indianapolis, IN, USA). Experiments were conducted in triplicate and repeated three times.

ROS levels in HTR-8-SVneo cells were measured using a ROS assay Kit (Beyotime, Shanghai, China). Briefly, HTR-8-SVneo cells (1 $\times$ 10⁶) were incubated with 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) for 30 min at 37 °C. Following three washes with PBS, the DCF fluorescence was observed utilizing an Olympus fluorescence microscope.

One-step TUNEL Apoptosis Assay Kit (cat.no. KTA2011; Abbkine Scientific) was used to measure the TUNEL-positive cells. HTR-8-SVneo cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.25% Triton-X 100 for 20 min. Then, 50 µL of TUNEL reaction agent were added and incubated at 37 °C for 1 h. Following washing with PBS, cells were stained with DAPI (5 µg/mL) for 5 min in the dark. Images were captured under a fluorescence microscope (Olympus). Experiments were conducted in triplicate and repeated three times.

Following the manufacturer’s guidelines, malondialdehyde (MDA; cat.no. KTB1050) and superoxide dismutase (SOD; cat.no. KTB1030) concentrations were assessed using kits from Abbkine Scientific. Meanwhile, the levels of IL-1$\beta$ (cat.no. KTE6013; Abbkine Scientific), LXA4 (cat.no. CSB-E09689h; CUSABIO, Wuhan, Hubei, China) and TNF-$\alpha$ (cat.no. KTE6032; Abbkine Scientific) were evaluated using enzyme-linked immunosorbent assays. Experiments were conducted in triplicate and repeated three times. Clinical results were performed with five human samples per group.

Data were presented as mean $\pm$ standard deviation and statistically analyzed with GraphPad Prism 10 software (version 10.4.0, Dotmatics, Boston, MA, USA). All quantitative data were tested for normality using the Shapiro-Wilk test. The data were confirmed to show homogeneity of variance through Levene’s test. Student t-test and ANOVA (one-way) plus Tukey’s post-hoc test were used for comparisons as appropriate. Differences were considered when p 0.05.

A total of 5 patients with preeclampsia and 5 normal pregnancies were enrolled in this study. As illustrated in Fig. 1A, the preeclampsia group exhibited significantly decreased SOD levels (p 0.01) and increased MDA levels (p 0.001). Meanwhile, a notable decrease in the level of LXA4 was observed in both serum and placental tissues of pregnancies with preeclampsia (Fig. 1B, p 0.01). ALX exhibits high affinity for LXA4 and mediates various cellular effects upon binding [19]. Since LXA4 is not a protein, ALX expression can serve as an indirect indicator of LXA4 activity. Therefore, we further assessed LXA4 production in pregnancies with preeclampsia by analyzing the levels of ALX. As shown in Fig. 1C–E, the mRNA and protein levels of ALX were remarkably reduced in the placental tissues of patients with preeclampsia (p 0.05), whereas the expression levels of IP-10 exhibited the contrasting results (p 0.05). It is well known that IP-10 mediates the migration of immune cells (e.g., T cells and natural killer cells) to inflammatory or infectious sites by binding to its specific receptor, CXCR3 [20]. We further observed that CXCR3 expression levels were markedly upregulated in the preeclampsia group compared to the normal group (Fig. 1E, p 0.01).

Fig. 1.

IP-10 expression is upregulated, while LXA4 level is downregulated in patients with preeclampsia. (A,B) SOD and MDA levels as well as LXA4 concentration in patients with preeclampsia were measured via biochemical assay. (C) The mRNA expression of IP-10 and ALX in patients with preeclampsia was examined via qRT-PCR. (D,E) The protein levels of IP-10, ALX and/or CXCR3 in patients with preeclampsia were measured by western blot and immunohistochemistry. Scale bar = 50 µm. The hematoxylin-stained nuclei appear blue (black arrow), and the positive DAB staining is characterized by a brown color (red arrow). *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. For qRT-PCR and biochemical assay, the results were performed with five human samples per group. For western blot and immunohistochemistry, the results were performed with three human samples per group. Student’s t-test was used for comparison. SOD, superoxide dismutase; MDA, malondialdehyde; qRT-PCR, quantitative real-time polymerase chain reaction; CXCR3, Cys-X-Cys motif chemokine receptor 3; DAB, 3,3’-diaminobenzidine.

We subsequently silenced IP-10 and ALX in hypoxia-induced HTR-8-Svneo cells to investigate their effects on cellular behaviors. IP-10 (Fig. 2A, p 0.0001) and ALX (Fig. 2B, p 0.01) expression were markedly reduced after silencing. si-IP-10-3 and si-ALX-2 were selected for subsequent functional experiments due to their relatively high silencing efficiency ($\ge$60% reduction in mRNA expression compared with the si-NC group). Hypoxia treatment significantly inhibited the viability and colony numbers of HTR-8-SVneo cells (Fig. 2C,D, p 0.001). Notably, IP-10 silencing significantly enhanced the proliferative capacity of HTR-8-SVneo cells (Fig. 2C,D, p 0.001), whereas ALX silencing attenuated this capacity (Fig. 2C,D, p 0.01). The subsequent flow cytometry and TUNEL assays showed that silencing of IP-10 exhibited a markedly inhibiting effect on HTR-8-SVneo cell apoptosis (Fig. 2E,F, p 0.01), while silencing of ALX showed the opposite results (Fig. 2E,F, p 0.05). This finding was further confirmed by qRT-PCR and western blot assays. We observed that following transfection of si-IP-10, Bcl-2 expression level was upregulated (Fig. 2G,H, p 0.05), and the expression levels of Caspase-3 and Bax were downregulated (Fig. 2G,H, p 0.05). Concurrently, silencing of ALX exerted the opposite influences on these proteins (Fig. 2G,H, p 0.05). Furthermore, ALX silencing significantly increased IP-10 levels (Fig. 2G,H, p 0.001), and IP-10 silencing promoted ALX expression (Fig. 2G,H, p 0.05). Notably, LXA4 concentration in hypoxia-induced HTR-8-SVneo cells was elevated upon transfection of si-IP-10 (Fig. 2I, p 0.01), but was inhibited upon si-ALX transfection (Fig. 2I, p 0.01). Notably, co-treatment experiments (Fig. 2C–H) showed that ALX silencing significantly reversed the pro-proliferative effect of IP-10 knockdown (p 0.001), and partially attenuated its anti-apoptotic effect (p 0.01). In contrast, silencing of IP-10 remarkably attenuated the inhibiting effects of ALX knocking down on proliferation (p 0.01), and reversed the promoting effects on apoptosis (p 0.05). These results suggested the antagonistic relationship of IP-10 and LXA4 in hypoxia-induced HTR-8-SVneo cells.

Fig. 2.

The antagonistic relationship between IP-10 and LXA4 in the proliferation and apoptosis of hypoxia-induced HTR-8-SVneo cells. (A) IP-10 mRNA expression in hypoxia-induced HTR-8-Svneo cells after transfection of si-IP-10-1/-2/-3 or si-NC. (B) ALX mRNA expression in hypoxia-induced HTR-8-SVneo cells after transfection of si-ALX-1/-2/-3 or si-NC. (C,D) Following transfection, HTR-8-SVneo cell proliferation was assessed by CCK-8 and colony formation assays. (E,F) Flow cytometer and TUNEL staining were conducted to measure the apoptosis of HTR-8-SVneo cells. Scale bar = 100 µm. (G) The mRNA expression of IP-10, ALX, Bcl-2, Bax and Caspase-3 in hypoxia-induced HTR-8-Svneo cells was detected via qRT-PCR. (H) The protein levels of IP-10, ALX, Bcl-2, Bax and Caspase-3 in hypoxia-induced HTR-8-SVneo cells were measured by western blot. (I) LXA4 concentrations in hypoxia-induced HTR-8-SVneo cells were measured via biochemical assay. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. Experiments were conducted in triplicate and repeated three times. ANOVA (one-way) plus Tukey’s post-hoc test was used for comparison. CCK-8, cell counting kit-8; TUNEL, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling.

Subsequently, the effects of IP-10 and LXA4 on the invasion, migration, oxidative stress and inflammation of HTR-8-SVneo cells were further determined. si-IP-10 transfection significantly enhanced the invasion and migration of HTR-8-SVneo cells (Fig. 3A,B, p 0.001). Conversely, ALX silencing significantly inhibited these biological behaviors (Fig. 3A,B, p 0.05). Furthermore, we indicated that the concentrations of IL-1$\beta$ and TNF-$\alpha$ in HTR-8-SVneo cells were significantly reduced following transfection with si-IP-10 (Fig. 3C, p 0.01). In contrast, these indicators exhibited a notable increase when ALX was silenced (Fig. 3C, p 0.001). A similar trend was observed in the mRNA expression of IL-1$\beta$ and TNF-$\alpha$ (Fig. 3D, p 0.05). Additionally, as illustrated in Fig. 3E–H, si-IP-10 transfection in hypoxia-induced HTR-8-SVneo cells resulted in lower ROS and MDA levels, and higher SOD activity (p 0.001), while si-ALX transfection exerted the opposite effects on these oxidative stress indicators. As illustrated in Fig. 3A–H, we found that silencing of ALX significantly reversed the promoting effects of IP-10 knock down on cell invasion and migration (p 0.01), while concurrently partially attenuating the inhibiting effects on oxidative stress and inflammation (p 0.05). Conversely, silencing of IP-10 remarkably attenuated the inhibiting effects of ALX knocking down on cell invasion and migration (p 0.01), and reversed the promoting effects on oxidative stress and inflammation (p 0.05).

Fig. 3.

The antagonistic relationship between IP-10 and LXA4 in the invasion, migration, oxidative stress and inflammation of HTR-8-Svneo cells. (A,B) The invasion (scale bar = 100 µm) and migration (scale bar = 200 µm) of HTR-8-SVneo cells were evaluated through transwell and wound healing assays. (C,D) The concentrations and mRNA expression of IL-1$\beta$ and TNF-$\alpha$ in hypoxia-induced HTR-8-SVneo cells were measured by ELISA and qRT-PCR. (E,F) ROS generation was calculated by DCFH-DA staining in hypoxia-induced HTR-8-SVneo cells. Scale bar = 100 µm. (G,H) The concentrations of SOD and MDA in hypoxia-induced HTR-8-SVneo cells were measured by biochemical assay. *p 0.05, **p 0.01, ***p 0.001. Experiments were conducted in triplicate and repeated three times. ANOVA (one-way) plus Tukey’s post-hoc test was used for comparison. MDA, malondialdehyde; ROS, reactive oxygen species; ELISA, enzyme-linked immunosorbent assay; DCFH-DA, 2^′,7^′-dichlorodihydrofluorescein diacetate.

This study has several notable limitations that should be acknowledged. First, as a pilot study, this research enrolled only 5 patients per group. The small sample size may limit the generalizability of our clinical findings, and larger cohorts are needed in future studies to validate these results. Second, the regulatory mechanisms of IP-10 and LXA4 in HTR-8-Svneo cells—including potential downstream signaling pathways—remain to be fully elucidated. For example, IP-10 activates NF-$\kappa$B signaling pathway by binding to CXCR3, while NF-$\kappa$B pathway serves as the core regulatory mechanism for the transcription of IL-1$\beta$ and TNF-$\alpha$ [10]. The interactions among IP-10, CXCR3, LXA4 and NF-$\kappa$B pathway in preeclampsia may be a valuable focus for future research. Meanwhile, IP-10 mediates immune cell migration to inflammatory sites by targeting CXCR3 [20]. Further exploring whether IP-10 knockdown reduces maternal pro-inflammatory cytokine levels and modulates immune cell polarization would enhance the rigor of our findings. Third, additional experimental data on oxidative stress and inflammation are needed. Additionally, cell line studies also have inherent limitations in recapitulating clinical outcomes. This study only conducted preliminary research at the cellular level, and lacked validation of biological integrity, in vivo microenvironment, and feasibility of transformation. Their roles in animal models also warrant further investigation. Last but not least, HTR-8-SVneo is an immortalized cell line that may not accurately represent the behaviors of primary trophoblasts. Compared to primary trophoblasts, HTR-8-SVneo cells have limitations including: (i) phenotypic alterations induced by immortalization, which modify cell cycle regulation and apoptosis sensitivity; and (ii) limitations of functional features. The core functions of primary trophoblasts, such as spiral artery remodeling and immune tolerance regulation, rely on complex intercellular communication and microenvironment response, while HTR-8-Svneo cells may lose some key functions during long-term passage. We consider validating our findings in primary trophoblast cultures and animal models in future work.