Objectives: Necroptosis, a form of programmed cell death, can occur in the placenta of patients with preeclampsia (PE). Hydrogen sulfide (H2S) can inhibit necroptosis of human umbilical vein endothelial cells under the high glucose-induced injury. Whether H2S can protect trophoblasts against necroptosis underlying PE has not been elucidated. This study aimed to explore the protective role of H2S in trophoblast cells against necroptosis underlying PE. Design: This is an in vitro experimental study. Participants: A total of 10 pregnant women with severe PE and 10 matched control normotensive pregnant women were included. The placenta tissues were extracted from participators. The human JEG-3 trophoblasts were commercially available. Methods: The expression and localization of necrotic proteins were assayed in human placenta samples, and the effect of necrotic cell death on the proliferation and apoptosis of human JEG-3 trophoblasts was evaluated. The component expressions of inflammatory cytokine and p38MAPK signaling pathway were measured in samples pretreated with or without NaHS (H2S donor) and SB203580 (p38 inhibitor). Results: RIPA1, RIPA3, and p-p38 levels were significantly higher in PE placental tissue, whereas cystathionine β-synthase expression was decreased. In JEG-3 trophoblasts, necroptosis increased apoptotic cell numbers, suppressed cell proliferation, increased inflammatory cytokine expression, and increased p38MAPK activation, which can be prevented by NaHS. Limitations: In the present study, we did not provide sufficient evidence that necroptosis was a part of the pathogenesis of PE. Conclusions: We proposed the putative role of necroptosis in early-onset PE, reflected by the blockage of caspase-8/3 and increased expression of RIPA1 and RIPA3 in PE placenta tissues. Furthermore, we demonstrated that exogenous H2S protected cytotrophoblasts against ceramide-induced necroptosis via the p38MAPK pathway.

Preeclampsia (PE) presents as maternal high blood pressure (continuously over 140/90 mm Hg) and proteinuria after 20-week gestational age [1, 2]. Worldwide, this disorder occurs in approximately 6% of pregnancies [1]. Once PE is diagnosed, delivery is the only way to resolve the disorder [3]. Premature delivery, accompanied by PE at early gestation, can cause cognitive and physical impairments among infants [4, 5]. PE, particularly a severe one, increases the possibility of adverse maternal outcomes, such as cardiovascular disorders and end-stage renal disease [6, 7]. Nowadays, few drugs have been verified to prevent the progression of PE. Therefore, novel treatments and management strategies for PE are urgently needed.

The placentae of women with PE are characterized by excessive cell death, resulting in hyper-proliferative immature trophoblast cells, particularly in those with early-onset PE [8]. Necroptosis and necrosis, two modes of cell death, share parts of morphological features [9]. However, necroptosis represents an alternative way of cell death and an unconventional of necrosis. Instead of a traditional caspase-dependent passive process, necroptosis is a caspase-independent type of cell death [10]. The formation of necrosome complex, including receptor-interacting protein kinases 1 and 3 (RIPA1 and RIPA3) mixed lineage kinase domain-like protein (MLKL) involves in this caspase-independent type of cell death [11]. Briefly, if caspase is inactivated, RIPA1 recruits RIPA3 to a necroptosis-initiating protein complex along with procaspase-8 and Fas-associated death domain [12]. Meanwhile, RIPA1 can phosphorylate MLKL to oligomerization along with permeabilization of the plasma membrane [13, 14]. Necroptosis may act as a trigger for various vascular and inflammatory disorders. Targeting necroptosis could be a potential therapy for several human diseases generated from animal models [14]. Moreover, necroptotic cell death has been observed in the preeclamptic placenta [15, 16] and therefore may be involved in the pathophysiology of PE. In addition, accumulating studies have reported that necroptosis is activated by cell death-related cytokines [17, 18]. Increasing evidence has proposed the involvement of necroptosis in the pathological process of PE. Targeting necroptosis may offer promising therapeutic options for PE.

Hydrogen sulfide (H2S) is the third endogenous gaseous signaling molecule identified in mammalian tissues [19]. It is synthesized from cystathionine β-synthase (CBS), cystathionine γ-lyase, and 3-mercaptopyurvate-sulfurtransferase. Interestingly, H2S is implicated in numerous physiological and pathological processes, including vasodilation, inflammation, and angiogenesis, in various tissues [20]. We have previously demonstrated that H2S plays a critical role in vascular endothelial growth factor production in the human placenta [21, 22]. In addition, H2S was shown to exert anti-inflammatory cytoprotective effects against reperfusion injury-induced cellular damage [23]. A previous study has reported that exogenous H2S can protect human umbilical vein endothelial cells from high glucose-induced injury by inhibiting necroptosis [24]. However, whether H2S can protect against necroptosis in trophoblast cells underlying PE remains unclear.

The p38 mitogen-activated protein kinase (p38MAPK, p38), an important component of the mitogen-activated protein kinase (MAPK) signaling family, contributes to the inflammatory process and necroptosis [25]. Lin et al. [26] revealed that necroptosis mediated high glucose-induced injury by activating p38MAPK. Wu et al. [25] demonstrated that necroptosis can be inhibited by death-associated protein kinase-1-activated p38MAPK. Accordingly, the relationship between p38MAPK and necroptosis in PE should be further investigated. In a high glucose-induced cell model of cardiomyopathy, treatment with NaHS (a H2S donor) was found to reduce interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α expression via the p38MAPK/NF-κB pathway [27]. However, the role of H2S and necroptosis in trophoblasts during PE-induced injury is unclear. Therefore, in this study, we explored the role of necroptosis in the placenta of patients with PE and the effects of NaHS on p38MAPK and necroptosis in trophoblasts.

Tissue Collection

This study was approved by the Ethics Committee of Changhai Hospital affiliated to Second Military Medical University (PTEC-A-2021-28-1). Written informed consent was obtained from each participator. PE was diagnosed with systolic blood pressure (SBP) ≥ 140 mm Hg and/or diastolic blood pressure ≥ 90 mm Hg, with random urine protein ≥1 + (30 mg/dL) or ≥0.3 g/24 h after 20 weeks of gestation) [28, 29]. Between 2018 and 2019, of the participants, pregnant women with severe PE and matched control normotensive pregnant women were included. Subjects with chronic, infectious diseases, past medical history and preexisting hypertensive disorders, any diabetes-related disorders, any thyroid-related disorders were excluded. There were no significant differences in the maternal age, BMI, and gestational age at delivery between the two groups (p > 0.05) Table 1. The placenta tissues were obtained from pregnant women with severe PE (PE; n = 10) and matched control normotensive pregnant women (control; n = 10). Two small pieces of tissue were randomly collected from separate lobules of each placenta within 1 h of cesarean birth, washed with normal saline, immediately frozen in liquid nitrogen, and stored at −80°C.

Table 1.

Clinical characteristics of the study groups

Clinical characteristicsControl, n = 10PE, n = 10p value
Maternal age 29.30±2.50 30.00±3.46 0.853 
Fetal weight, g 3,427±293.93 2,882.5±237.57 <0.0001* 
Pregnant woman BMI 23.47±1.94 23.41±2.98 0.971 
Gestational age at delivery 37±6 37±4 0.75 
Baseline systolic blood pressure, mm Hg 113.6±9.57 166.0±6.06 <0.001 
Baseline diastolic blood pressure, mm Hg 75.6±6.47 103.6±9.57 <0.001 
Any diabetes-related disorders No No 
Any thyroid-related disorders No No 
Past medical history No No 
Preexisting hypertensive disorders No No 
Clinical characteristicsControl, n = 10PE, n = 10p value
Maternal age 29.30±2.50 30.00±3.46 0.853 
Fetal weight, g 3,427±293.93 2,882.5±237.57 <0.0001* 
Pregnant woman BMI 23.47±1.94 23.41±2.98 0.971 
Gestational age at delivery 37±6 37±4 0.75 
Baseline systolic blood pressure, mm Hg 113.6±9.57 166.0±6.06 <0.001 
Baseline diastolic blood pressure, mm Hg 75.6±6.47 103.6±9.57 <0.001 
Any diabetes-related disorders No No 
Any thyroid-related disorders No No 
Past medical history No No 
Preexisting hypertensive disorders No No 

PE, preeclampsia.

*PE versus Control have significant differences.

Immunohistochemistry

Paraffin sections (5 μm) of the placenta samples were cut and rehydrated, and antigens were extracted using citric acid buffer in a microwave. Nonspecific antibody binding was blocked using 10% rabbit serum after endogenous peroxidases were inhibited using 3% H2O2. The blocked sections were subsequently incubated overnight at 4°C with human RIPA1-specific antibodies (#DF2642, 1:1,000, Affinity Biosciences, Cincinnati, OH, USA). Bound antibodies were stained using a biotin-streptavidin peroxidase system along with UltraSensitive SP kit and visualized with diaminobenzidine, following a counterstain with hematoxylin. IgG replaced the primary antibody, which serves as a negative control.

JEG-3 Cell Culture

The human choriocarcinoma JEG-3 cell line was purchased from the Shanghai Institute of Cell Biology (Shanghai, China) and maintained in the endotoxin-free RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) with 10% (vol/vol) fetal bovine serum in a CO2 incubator at 37°C. JEG-3 cells were seeded into 6-well plates (3 × 105 cells/well) and incubated overnight. Then, JEG-3 cells were treated with 50 μmol/L C16:0 ceramide (CER), in the presence or absence of 50 μmol/L of a potent caspase inhibitor Q-VD-OPh (QVD), 10−3 m of the H2S donor NaHS, 10−3 m of the precursor L-cysteine (L-cys), or 25 μmol/L of the necroptosis inhibitor necrostatin-1 (Nec-1), or treated with 1 × 10-5 mol/L of the P38MAPK pathway inhibitor SB203580 (SB) with CER (50 μmol/L) and QVD (50 μmol/L), followed by a 48 h incubation. All drug concentrations were identified from the literature review and preliminary experiments. CER, NaHS, L-cysteine (purity >98%), and SB203580 were purchased from Sigma Aldrich, while QVD and Nec-1 were from MedChemExpress (Monmouth Junction, NJ, USA).

Western Blot Analysis

Protein was extracted from JEG-3 cells and the placental tissues of control pregnant women or those with PE using RIPA buffer. Total proteins were quantified using a BCA kit (Biosharp, Anhui, China). Total protein (50 μg) were separated in SDS gel electrophoresis before being transferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA). After blocked by 5% fat-free dry milk for 2 h, the membranes were probed with the following primary antibodies: RIPA1 (#3493, 1:1,000), CBS (#14782, 1:1,000), cleaved caspase-3 (#9661, 1:1,000), phospho-p38 (#4511, 1:1,000), total p38 (#8690, 1:1,000), caspase-8 (#9746, 1:1,000), GAPDH (#2118, 1:10,000; Cell Signaling Technology, Danvers, MA, USA), and RIPA3 (ab56164, 1:1,000; purchased from Abcam, Cambridge, UK). After washed with Tris-buffered saline containing 0.1% Tween-20 for three times, the membranes were subjected to one further incubation with horseradish peroxidase-conjugated secondary antibodies (Abcam, 1:5,000). Band densitometry was analyzed using Quantity One imaging software.

Total RNA Extraction and Quantitative Real-Time PCR Analysis

JEG-3 cells were subjected to total RNA extraction using TRIzol reagent (Invitrogen). Then, reverse-transcribed into cDNA using a PrimeScript® RT Reagent Kit (Takara Biotechnology, Shiga, Japan) at 37°C for 15 min, 85°C for 5 s, and maintained at 4°C. Next, cDNA, along with SYBR (Takara Biotechnology) and forward/reverse primers, was loaded on a real-time PCR Detection System (Bio-Rad Laboratories) using the following cycling set: denaturing for 30 s at 95°C, annealing for 1 min at 60°C. Relative expression levels of mRNA were quantified using the 2∆∆Cq method. The following specific primers were used: IL-1β: A: 5′-GTG GCA ATG AGG ATG ACT T-3′; S: 5′-TGG GCT TAT CAT CTT TCA A-3′; IL-6: A: 5′-CCT TCC AAA GAT GGC TGA AA-3′; S: 5′-AGC TCT GGC TTG TTC CTC AC-3′; TNF-α: A: 5′-GCC CCC AGA GGG AAG AGT TCC CCA-3′; S: 5′-GCT TGA GGG TTT GCT ACA ACA TGG GC-3′; CCL-2: A: 5′-CTT CTG TGC CTG CTG CTC AT-3′; S: 5′-GCT TGT CCA GGT GGT CCA T-3′, RIPA1: A: 5′-GGC ATT GAA GAA AAA TTT AGG C-3′; S: 5′-TCA CAA CTG CAT TTT CGT TTG-3′; RIPA3: A: 5′-TGC TGG AAG AGA AGT TGA GTT GC-3′; S: 5′-CTG TTG CAC ACT GCT TCG TAC AC-3′; β-actin: A: 5′-TGT TAC CAA CTG GGA CGA CA-3′; S: 5′-CTG GGT CAT CTT TTC ACG GT-3′.

Cell Proliferation Analysis

Once JEG-3 cells reached 70% confluence, the cells were transferred into 96-well plates (3 × 103 cells/well) for an overnight incubation. The cells were then treated with CER (0, 10, 20, 30, 40, 50, 60, 70, or 80 μmol/L) and QVD (50 μmol/L) in the presence or absence of NaHS (10−3 m), L-cys (10−3 m), or SB (1 × 10−5 mol/L). After incubation for 48 h, proliferation of JEG-3 cells was assessed using cell counting kit-8 (CCK-8, Kumamoto, Japan) and presented as absorbance at 450 nm recorded by a microplate reader.

Flow Cytometry Analysis

CER is a powerful inducer of intrinsic cell death, autophagy, and endothelial dysfunction in PE [30]. Bailey et al. [15] have treated JEG-3 cells with CER+QVD (a potent caspase inhibitor) to induce necroptosis. Accordingly, JEG-3 cells were cultured in 6-well plates and treated with CER+QVD, CER+QVD + NaHS, or CER+QVD + L-cys. Then, the cells were dissociated using TrypLE Express (0.5 mL per well; Invitrogen), washed, and evaluated using an APC Annexin V Apoptosis Detection Kit 1 (62700-80, PeproTech, Rocky Hill, NJ, USA). Briefly, the cells were incubated with APC Annexin V and/or propidium iodide, at a concentration of 1 × 106 cells/mL, thoroughly washed, and counted using a BD FACSCanto flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Unstained cells and those stained only with APC Annexin V or propidium iodide were in the same procedure as controls.

Statistical Analysis

All data was statistically analyzed using SPSS 21.0 software (SPSS, Inc., Chicago, IL, USA) and finally presented as the mean ± SD. Significance tests were made using one-way analysis of variance or the Student-Newman-Keuls test, when appropriate. The relationships between CBS and RIPA1 and RIPA3 levels were assessed by calculating Pearson’s correlation coefficients. p values <0.05 indicated statistically significant results.

PE Placenta Tissues Are Presented with Increased Susceptibility to Necroptosis

First, we examined the expression of necroptosis-associated proteins in PE and control placenta tissues. The results showed that the expression of caspase-8 and caspase-3 were blocked in PE patients compared with controls (p < 0.01, Fig. 1a–c). Considering the blockage of caspase-8 is associated with the occurrence of necroptosis, the key regulatory proteins in the necroptosis were detected. As shown in Figures 1d–f, RIPA1 and RIPA3 protein expression was upregulated in patients with PE (PE groups) compared to those in controls (p < 0.001). Active phospho-p38 levels were significantly higher in the placental tissues of patients with PE (Fig. 1d and g; p < 0.01). However, CBS expression was downregulated in preeclamptic placenta samples (PE groups; Fig. 1d and h; p < 0.01). In addition, RIPA1 and RIPA3 protein levels were negatively correlated with CBS levels in placental tissues from PE women (Fig. 1i; p < 0.01). These findings indicated that RIPA1, RIPA3, and phosphorylated (p) p38 levels were significantly higher in PE placental tissues, whereas CBS expression was decreased compared to control tissues.

Fig. 1.

RIPA1, RIPA3, CBS, p38, and P-p38 protein levels in placenta tissues from control (Nor) pregnant women and those with preeclampsia (PE) and localization of RIPA1 in placenta tissues. a Western blot for the expression of caspase-8, caspase-3 in PE placenta tissues. b Quantitative analysis of caspase-8 expression. c Quantitative analysis of caspase-3 expression. d RIPA1, RIPA3, CBS, and P-p38 protein levels were evaluated by Western blot analysis with GAPDH as a loading control. RIPA1 (e), RIPA3 (f), P–p38 (g), and CBS (h) protein expression were determined by densitometric analysis in Nor and PE placenta biopsies. Comparisons were conducted using a one-way analysis of variance. All data represented the mean ± SD (n = 10). ***p < 0.001, **p < 0.01 versus Nor group. i Correlations between CBS and RIPA1 or RIPA3 expression were analyzed in Nor and PE placenta biopsies obtained during elective cesarean section. j Immunohistochemical analysis shows representative sections with positive staining for RIPA1. Arrows indicate positive staining. Original magnification, ×20. k Statistical analysis of the RIPA1-positive rate.

Fig. 1.

RIPA1, RIPA3, CBS, p38, and P-p38 protein levels in placenta tissues from control (Nor) pregnant women and those with preeclampsia (PE) and localization of RIPA1 in placenta tissues. a Western blot for the expression of caspase-8, caspase-3 in PE placenta tissues. b Quantitative analysis of caspase-8 expression. c Quantitative analysis of caspase-3 expression. d RIPA1, RIPA3, CBS, and P-p38 protein levels were evaluated by Western blot analysis with GAPDH as a loading control. RIPA1 (e), RIPA3 (f), P–p38 (g), and CBS (h) protein expression were determined by densitometric analysis in Nor and PE placenta biopsies. Comparisons were conducted using a one-way analysis of variance. All data represented the mean ± SD (n = 10). ***p < 0.001, **p < 0.01 versus Nor group. i Correlations between CBS and RIPA1 or RIPA3 expression were analyzed in Nor and PE placenta biopsies obtained during elective cesarean section. j Immunohistochemical analysis shows representative sections with positive staining for RIPA1. Arrows indicate positive staining. Original magnification, ×20. k Statistical analysis of the RIPA1-positive rate.

Close modal

Although expressed in human placental tissues, as indicated by positive staining with antibodies, immunoreactivity was eliminated if the antibodies were preabsorbed with excess peptide. Additionally, positive immunostaining of RIPA1 was observed (Fig. 1j), and expression was increased in placental tissue from patients with PE (PE groups; Fig. 1k; p < 0.001).

CER+QVD Treatment Inhibits JEG-3 Cell Viability

Next, we investigated the effect of CER + QVD on JEG-3 cell proliferation using CCK-8 assay. Pretreatment with different doses of CER (0, 10, 20, 30, 40, 50, 60, 70, or 80 μmol/L) following exposure to QVD (50 μmol/L) gradually decreased JEG-3 cell viability, with the lowest levels observed at 40–80 μmol/L CER + 50 μmol/L QVD compared to QVD-treated cells without CER (Fig. 2a; p < 0.01). Based on these results, treatment with 50 μmol/L both CER and QVD was used for the flow cytometry apoptosis experiment. An increase in necroptotic death of JEG-3 cells upon CER+QVD exposure was identified by a fold-increase in APC+/propidium iodide + cells (Fig. 2b).

Fig. 2.

Effects of CER with QVD on JEG-3 cell proliferation. a JEG-3 cell proliferation following exposure to CER (0, 10, 20, 30, 40, 50, 60, 70, or 80 μmol/L) and QVD (50 μmol/L) was determined using a CCK-8 assay. b JEG-3 cells treated with CER (50 μmol/L) with or without QVD (50 μmol/L) were analyzed by flow cytometry. All data represented the mean ± SD (n = 6). **p < 0.01 versus the QVD+CER-treated group.

Fig. 2.

Effects of CER with QVD on JEG-3 cell proliferation. a JEG-3 cell proliferation following exposure to CER (0, 10, 20, 30, 40, 50, 60, 70, or 80 μmol/L) and QVD (50 μmol/L) was determined using a CCK-8 assay. b JEG-3 cells treated with CER (50 μmol/L) with or without QVD (50 μmol/L) were analyzed by flow cytometry. All data represented the mean ± SD (n = 6). **p < 0.01 versus the QVD+CER-treated group.

Close modal

CER Stimulates Necrosome in JEG-3 Cells

CER accumulation can induce excessive trophoblast death and placental dysfunction during PE. Therefore, the effect of CER on the expression of necrosome protein in JEG-3 cells was examined. Exposure to 50 μm CER alone for 24 h significantly increased RIPA1 level in JEG-3 cells (Fig. 3a; p < 0.05), whereas exposure to 50 μm CER along with 50 μm QVD significantly increased both RIPA1 and RIPA3 levels compared to in the control (Fig. 3b; p < 0.01), suggesting that caspase-8 inhibition may initiate the necroptotic machinery in JEG-3 cells.

Fig. 3.

CER stimulates the necrosome in JEG-3 cells. a RIPA1 and RIPA3 protein levels in JEG-3 cells treated with CER (50 μmol/L) alone with GAPDH as a loading control. *p < 0.05, **p < 0.01 versus control group. b RIPA1 and RIPA3 protein levels in JEG-3 cells treated with CER (50 μmol/L) with QVD (50 μmol/L) by Western blot analysis with GAPDH as a loading control. **p < 0.01 versus control group. c Effect of CER on cleaved caspase-3 protein expression by Western blot. *p < 0.05, versus the CER-treated group. d Effect of CER on caspase-8 protein expression by Western blot and densitometry analysis. **p < 0.01 versus the CER-treated group. All data represented the mean ± SD (n = 6).

Fig. 3.

CER stimulates the necrosome in JEG-3 cells. a RIPA1 and RIPA3 protein levels in JEG-3 cells treated with CER (50 μmol/L) alone with GAPDH as a loading control. *p < 0.05, **p < 0.01 versus control group. b RIPA1 and RIPA3 protein levels in JEG-3 cells treated with CER (50 μmol/L) with QVD (50 μmol/L) by Western blot analysis with GAPDH as a loading control. **p < 0.01 versus control group. c Effect of CER on cleaved caspase-3 protein expression by Western blot. *p < 0.05, versus the CER-treated group. d Effect of CER on caspase-8 protein expression by Western blot and densitometry analysis. **p < 0.01 versus the CER-treated group. All data represented the mean ± SD (n = 6).

Close modal

To verify that the absence of active caspase-8 induced necroptosis, we treated the cells with a potent caspase inhibitor QVD. Exposure to QVD blocked cleaved caspase-3 expression in JEG-3 cells (Fig. 3c; p < 0.01), indicating that apoptotic cell death was reduced in favor of necroptosis. Moreover, exposing JEG-3 cells to 50 μm QVD resulted in an active fragment of caspase-8 cleavage (Fig. 3d; p < 0.01), suggesting that cell death may be shunted toward necroptosis. CER + QVD treatment increases the mRNA levels of RIPA1, RIPA3, inflammatory cytokine, and chemokine in JEG-3 cells.

It has been verified that necroptosis is a form of inflammatory cell necrosis. Here, the mRNA expression of RIPA1 and RIPA3 was examined in CER+QVD-treated JEG-3 cells, along with mRNA expression of the inflammatory cytokines (IL-1β, IL-6, and TNF-α). Interestingly, the mRNA expression of the inflammatory cytokines was markedly increased during CER+QVD-induced necroptosis in JEG-3 cells (Fig. 4a–c; all p < 0.05). In addition, quantitative real-time PCR analysis revealed that mRNA expression levels of RIPA1 (Fig. 4d; p < 0.01) and RIPA3 (Fig. 4e; *p < 0.05) were upregulated in CER+QVD-treated JEG-3 cells compared to those in the control groups. The result of caspase-8 mRNA was in line with that represented by caspase-8 protein (Fig. 4f; **p < 0.01).

Fig. 4.

Effects of CER+QVD on inflammatory cytokine expression in JEG-3 cells. IL-1β (a), IL-6 (b), TNF-α (c), RIPA1 (d), RIPA3 (e), and caspase-8 (f) mRNA levels in JEG-3 cells treated with CER (50 μmol/L) with QVD (50 μmol/L) detected by quantitative real-time PCR. All data represented the mean ± SD (n = 6; *p < 0.05, **p < 0.01 vs. control group (a-e); **p < 0.01 vs. CER-treated group (f)).

Fig. 4.

Effects of CER+QVD on inflammatory cytokine expression in JEG-3 cells. IL-1β (a), IL-6 (b), TNF-α (c), RIPA1 (d), RIPA3 (e), and caspase-8 (f) mRNA levels in JEG-3 cells treated with CER (50 μmol/L) with QVD (50 μmol/L) detected by quantitative real-time PCR. All data represented the mean ± SD (n = 6; *p < 0.05, **p < 0.01 vs. control group (a-e); **p < 0.01 vs. CER-treated group (f)).

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Effects of H2S on Proliferation of CER+QVD-Treated JEG-3 Cells

To discover the effect of H2S on the CER+QVD-treated JEG-3 cell proliferation, we performed a CCK-8 assay after pretreatment with the H2S donor NaHS or L-cys for 48 h. Pretreatment NaHS and L-cys can increase CER+QVD-treated JEG-3 cell viability compared to those without pretreatment (Fig. 5a; p < 0.01). Moreover, flow cytometry studies indicated that pretreatment with NaHS or L-cys reversed the promoting effect of CER+QVD on the necroptotic death of JEG-3 cells (Fig. 5b).

Fig. 5.

Protective effect of H2S on CER+QVD-inhibited proliferation of JEG-3 cells. a Effect of NaHS (10−3 m) and L-cysteine (10−3 m) on the proliferation of JEG-3 cells treated with CER (0, 10, 20, 30, 40, 50, 60, 70, or 80 μmol/L) and QVD (50 μmol/L) determined using a CCK-8 assay. b Increased necrotic (propidium iodide+) and decreased early apoptotic (APC+/propidium iodide-) in JEG-3 cells treated with CER (50 μmol/L) with QVD (50 μmol/L) and pretreated with NaHS (10−3 m) or L-cysteine (10−3 m) prior to exposure to CER+QVD analyzed by flow cytometry. **p < 0.01.

Fig. 5.

Protective effect of H2S on CER+QVD-inhibited proliferation of JEG-3 cells. a Effect of NaHS (10−3 m) and L-cysteine (10−3 m) on the proliferation of JEG-3 cells treated with CER (0, 10, 20, 30, 40, 50, 60, 70, or 80 μmol/L) and QVD (50 μmol/L) determined using a CCK-8 assay. b Increased necrotic (propidium iodide+) and decreased early apoptotic (APC+/propidium iodide-) in JEG-3 cells treated with CER (50 μmol/L) with QVD (50 μmol/L) and pretreated with NaHS (10−3 m) or L-cysteine (10−3 m) prior to exposure to CER+QVD analyzed by flow cytometry. **p < 0.01.

Close modal

H2S Protects JEG-3 Cells against CER+QVD-Induced Necroptosis

Previous studies revealed that exposure of JEG-3 cells to CER and QVD significantly increased RIPA1 and RIPA3 levels. In this study, we found that pretreatment with NaHS (Fig. 6a; p < 0.01) or L-cys (Fig. 6b; p < 0.01) significantly decreased RIPA1 and RIPA3 levels in CER+QVD-treated JEG-3 cells. Moreover, pretreatment with the necroptosis inhibitor Nec-1 can eliminate the activation of RIPA1 and RIPA3 kinase induced by CER+QVD in JEG-3 cells (Fig. 6c; p < 0.01).

Fig. 6.

Effect of H2S, L-cysteine, and Nec-1 on RIPA1 and RIPA3 expression induced by CER+QVD. a Effects of NaHS (10−3 m) on RIPA1 and RIPA3 protein expression in JEG-3 cells treated with CER (50 μmol/L) and QVD (50 μmol/L) evaluated by Western blot analysis with GAPDH as a loading control and densitometry analysis. b Effects of L-cysteine (10−3 m) on RIPA1 and RIPA3 protein expression in JEG-3 cells treated with CER (50 μmol/L) and QVD (50 μmol/L) evaluated by Western blot analysis with GAPDH as a loading control and densitometry analysis. c Effects of Nec-1 (25 μmol/L) on RIPA1 and RIPA3 protein expression in JEG-3 cells treated with CER (50 μmol/L) and QVD (50 μmol/L) evaluated by Western blot analysis with GAPDH as a loading control and densitometry analysis. All data represent the mean ± SD (n = 6; *p < 0.05 **p < 0.01 vs. CER+QVD-treated group).

Fig. 6.

Effect of H2S, L-cysteine, and Nec-1 on RIPA1 and RIPA3 expression induced by CER+QVD. a Effects of NaHS (10−3 m) on RIPA1 and RIPA3 protein expression in JEG-3 cells treated with CER (50 μmol/L) and QVD (50 μmol/L) evaluated by Western blot analysis with GAPDH as a loading control and densitometry analysis. b Effects of L-cysteine (10−3 m) on RIPA1 and RIPA3 protein expression in JEG-3 cells treated with CER (50 μmol/L) and QVD (50 μmol/L) evaluated by Western blot analysis with GAPDH as a loading control and densitometry analysis. c Effects of Nec-1 (25 μmol/L) on RIPA1 and RIPA3 protein expression in JEG-3 cells treated with CER (50 μmol/L) and QVD (50 μmol/L) evaluated by Western blot analysis with GAPDH as a loading control and densitometry analysis. All data represent the mean ± SD (n = 6; *p < 0.05 **p < 0.01 vs. CER+QVD-treated group).

Close modal

H2S Suppresses Inflammatory Cytokine Expression in JEG-3 Cell Necroptosis

Pretreatment with NaHS (10−3 m) or L-cys (10−3 m) extinguished the increased IL-1β, IL-6, and TNF-α mRNA expression in CER+QVD-treated JEG-3 cells (Fig. 7a, b). In addition, pretreatment of JEG-3 cells with Nec-1 prevented the CER+QVD-induced increase in inflammatory cytokine expression (Fig. 7c). Together, these findings indicate that exogenous H2S can inhibit inflammatory cytokine expression during JEG-3 cell necroptosis. p38MAPK signaling affects CER+QVD-induced JEG-3 cell necroptosis by modulating necrosome protein and proinflammatory cytokine expression.

Fig. 7.

Effects of H2S on inflammatory cytokine expression induced by CER+QVD in JEG-3 cells. IL-1β, IL-6, TNF-α, RIPA1, and RIPA3 mRNA levels in JEG-3 cells treated with CER (50 μmol/L) and QVD (50 μmol/L) with or without NaHS (10−3 m) (a), L-cysteine (10−3 m) (b), or Nec-1 (25 μmol/L) (c) detected by quantitative real-time PCR. All data represent the mean ± SD (n = 6; #p < 0.01, vs. control group, **p < 0.01 vs. CER+QVD+NaHS group (a); $p < 0.05, #p < 0.01 vs. control group, **p < 0.01 vs. CER+QVD+ L-cys group (b); $p < 0.05, #p < 0.01 vs. control group, **p < 0.01 vs. CER+QVD + Nec-1 group (c)). QVD, Q-VD-OPh, a potent caspase inhibitor.

Fig. 7.

Effects of H2S on inflammatory cytokine expression induced by CER+QVD in JEG-3 cells. IL-1β, IL-6, TNF-α, RIPA1, and RIPA3 mRNA levels in JEG-3 cells treated with CER (50 μmol/L) and QVD (50 μmol/L) with or without NaHS (10−3 m) (a), L-cysteine (10−3 m) (b), or Nec-1 (25 μmol/L) (c) detected by quantitative real-time PCR. All data represent the mean ± SD (n = 6; #p < 0.01, vs. control group, **p < 0.01 vs. CER+QVD+NaHS group (a); $p < 0.05, #p < 0.01 vs. control group, **p < 0.01 vs. CER+QVD+ L-cys group (b); $p < 0.05, #p < 0.01 vs. control group, **p < 0.01 vs. CER+QVD + Nec-1 group (c)). QVD, Q-VD-OPh, a potent caspase inhibitor.

Close modal

The process of necroptosis can activate NF-κB and MAPK signaling pathways in some tissues. Interestingly, we found that p38MAPK signaling was activated in necroptosis-induced JEG-3 cells, which can be suppressed by NaHS (Fig. 8a; #p < 0.01). In addition, CER+QVD-induced RIPA1 and RIPA3 protein expression can be inhibited by pretreatment with the P38MAPK inhibitor SB203580 (Fig. 8b; #p < 0.01, $p < 0.05), but were not influenced by the NF-κB inhibitor BAY-117082 (data not shown). Furthermore, SB203580 pretreatment can increase the cell viability which was inhibited by CER+QVD (Fig. 8c). Furthermore, SB203580 pretreatment inhibited the upregulation of IL-1β mRNA, as well as RIPA1 and RIPA3 protein levels, induced by CER+QVD (Fig. 8d). mRNA levels of IL-6 (Fig. 8e), TNF-α (Fig. 8f), RIPA1 (Fig. 8g), and RIPA3 (Fig. 8h) inhibited by CER+QVD can be restored by SB203580 pretreatment. These findings suggest that the p38MAPK signaling pathway may involve in CER+QVD-induced necroptosis in JEG-3 cells by modulating necrosome protein and proinflammatory cytokine expression.

Fig. 8.

H2S inhibits JEG-3 cell necroptosis via the p38MAPK pathway. a P-p38 protein expression in JEG-3 cells treated with CER (50 μmol/L) and QVD (50 μmol/L) with or without NaHS (10−3 m) evaluated by Western blot analysis with GAPDH as the loading control and densitometric analysis. b RIPA1 and RIPA3 protein levels in JEG-3 cells treated with CER (50 μmol/L) and QVD (50 μmol/L) with or without p38MAPK pathway inhibitor SB203580 (SB 10−5 mol/L) evaluated by Western blot and densitometric analysis. c Effect of SB (10−5 mol/L) on the proliferation of JEG-3 cells treated with CER (0.10, 20, 30, 40, 50, 60, 70, and 80 μmol/L) and QVD (50 μmol/L) determined using a CCK-8 assay. IL-1β (d), IL-6 (e), TNF-α (f), RIPA1 (g), and RIPA3 (h) mRNA levels were detected by quantitative real-time PCR. All data represent the mean ± SD (n = 6; **p < 0.01 vs. control group, #p < 0.01 vs. CER+QVD+NaHS group (a); **p < 0.01 vs. CER+QVD group (b); **p < 0.01, *p < 0.05 vs. control group, $p < 0.05, #p < 0.01 vs. CER+QVD +SB group (c); $p < 0.05, #p < 0.01 vs. control group, **p < 0.01, *p < 0.05 vs. CER+QVD+SB group (d-h)). P-, phosphorylated; SB: p38MAPK pathway inhibitor SB203580; QVD: Q-VD-OPh, a potent caspase inhibitor. H2S, hydrogen sulfide, PE, preeclampsia; RIPA1, receptor-interacting protein kinase-1; RIPA3, receptor-interacting protein kinase-3; CBS, cystathionine β-synthase; CER, ceramide; Q-VD-OPh, quinolyl-valyl-O-methylaspartyl-(2,6-difluorophenoxy)-methyl-ketone.

Fig. 8.

H2S inhibits JEG-3 cell necroptosis via the p38MAPK pathway. a P-p38 protein expression in JEG-3 cells treated with CER (50 μmol/L) and QVD (50 μmol/L) with or without NaHS (10−3 m) evaluated by Western blot analysis with GAPDH as the loading control and densitometric analysis. b RIPA1 and RIPA3 protein levels in JEG-3 cells treated with CER (50 μmol/L) and QVD (50 μmol/L) with or without p38MAPK pathway inhibitor SB203580 (SB 10−5 mol/L) evaluated by Western blot and densitometric analysis. c Effect of SB (10−5 mol/L) on the proliferation of JEG-3 cells treated with CER (0.10, 20, 30, 40, 50, 60, 70, and 80 μmol/L) and QVD (50 μmol/L) determined using a CCK-8 assay. IL-1β (d), IL-6 (e), TNF-α (f), RIPA1 (g), and RIPA3 (h) mRNA levels were detected by quantitative real-time PCR. All data represent the mean ± SD (n = 6; **p < 0.01 vs. control group, #p < 0.01 vs. CER+QVD+NaHS group (a); **p < 0.01 vs. CER+QVD group (b); **p < 0.01, *p < 0.05 vs. control group, $p < 0.05, #p < 0.01 vs. CER+QVD +SB group (c); $p < 0.05, #p < 0.01 vs. control group, **p < 0.01, *p < 0.05 vs. CER+QVD+SB group (d-h)). P-, phosphorylated; SB: p38MAPK pathway inhibitor SB203580; QVD: Q-VD-OPh, a potent caspase inhibitor. H2S, hydrogen sulfide, PE, preeclampsia; RIPA1, receptor-interacting protein kinase-1; RIPA3, receptor-interacting protein kinase-3; CBS, cystathionine β-synthase; CER, ceramide; Q-VD-OPh, quinolyl-valyl-O-methylaspartyl-(2,6-difluorophenoxy)-methyl-ketone.

Close modal

Necroptosis is a newly discovered form of cell death, different from the traditional view that considers necrosis as a passive process [31]. Necroptosis is regulated in a caspase-independent manner and can be reversed [32]. Increasing evidence has indicated that necroptosis is involved in various pathological conditions, such as inflammation [33]. Excessive inflammation is proved to contribute to placental dysfunction in PE [34, 35]. Recent studies suggest that necroptosis is involved in PE development [15, 16]. However, the understanding of molecular mechanisms remains limited. Previous studies showed that H2S affects necroptosis in cardiomyopathies, and its injection decreases the expression of necroptotic markers [36]. Previous studies have shown that H2S protects against high glucose-induced necroptosis in human umbilical vein endothelial cells [24, 26]. It remains unclear whether H2S can inhibit trophoblast necroptosis in PE. Since JEG-3 cells are characterized by highly proliferative ability and absence of syncytial fusion, which is similar with extravillous trophoblasts, JEG-3 has been widely used for modeling PE in vitro [37, 38]. Thus, we conducted a series of studies in vitro with the application of JEG-3 cells and explored whether necroptosis played a crucial role in regulating PE development. First, this study revealed that the expressions of caspase-8 and caspase-3 were significantly blocked in PE placental tissues, in parallel with enhanced RIPA1, RIPA3, and proinflammatory cytokines (IL-1β, IL-6, and TNF-α). In line with this, necroptosis can increase the expression of RIPA1, RIPA3, and proinflammatory cytokines in JEG-3 cells. In addition, necroptosis activated the p38MAPK signaling pathway by regulating the expression of proinflammatory cytokines, RIPA1 and RIPA3 in JEG-3 cells. We also found that pretreatment with the H2S donors NaHS and L-cys reduced the expression of RIPA1, RIPA3, and the proinflammatory cytokines and suppressed p38MAPK activation. Collectively, this study suggests that necroptosis is involved in the pathogenesis of PE.

Healthy placentation relies on successful cytotrophoblast invasion and syncytial trophoblast layer fusion, which require a precise balance between trophoblast proliferation and apoptosis [39, 40]. PE involves the proliferation, adhesion, invasion, transformation, and renewal of extravillous trophoblasts, which results in various types of vascular dysfunction that are harmful to expectant mothers, even life-threatening [41, 42]. In this study, we observed that the necroptosis-associated proteins RIPA1 and RIPA3 were highly expressed in placental tissues from puerpera with PE. Recruitment of RIPA3 by RIPA1 to the necrosome under caspase-8 inhibition is a key step that distinguishes necrosis from apoptosis. A previous study showed that caspase-8 activity was significantly decreased in early-onset PE placental tissue [15]. In this study, we also identified a declined caspase-8 expression in the placental tissues of PE group, which was consistent with the previous one. Moreover, some studies have revealed that excessive trophoblast apoptosis and autophagy can affect their invasion capability, leading to placental vascular recast disorders and PE [43, 44]. Therefore, the dysregulated RIPA3 and RIPA1 in PE placental tissue indicate the involvement of necroptosis in PE.

In necroptosis, RIPA1 is a critical mediator of cell death. In the presence of active caspase-8, RIPA1 is bound to an apoptotic-promoting protein complex with a death domain (Fas-associated death domain). Under conditions of caspase inactivation, RIPA1 recruits RIPA3 to induce necroptosis [12]. RIPA1 has been identified using immunofluorescence in the cytotrophoblast cytoplasm [16]. This enhanced the potential that trophoblast cell death may suppress placental growth, leading to PE. CER is a powerful inducer of intrinsic cell death and induces autophagy and endothelial dysfunction in PE [12]. Bailey et al. [15] treated JEG-3 cells with CER+QVD (a potent caspase inhibitor) to induce necroptosis. This study benefited from them and used the same method in vitro. The results showed that exposure to CER only increased RIPA1 levels in JEG-3 cells, whereas exposure to CER and QVD increased both RIPA1 and RIPA3 levels. Interestingly, necroptosis resulted in a decrease in cell proliferation in a necrosome concentration-dependent manner. However, instead of changes in number of living cells, the number of early apoptotic cells was observed with a significant increase.

Necroptosis is a highly inflammatory pattern of cell death [16]. PE is just a systemic inflammatory reaction resulting from an imbalance between factors produced by the placenta and maternal adaptation [34]. Moreover, excessive release of inflammatory cytokines leads to insufficient invasion HTR8/SVneo cytotrophoblast and damage spiral artery remodeling [45]. Here, we revealed that IL-1β, IL-6, and TNF-α were significantly upregulated in JEG-3 cells treated with CER+QVD. In addition, MAPK signaling pathways including JNK, ERK1/2, and p38, play important roles in activating inflammation. Indeed, elevated JNK, ERK1/2, and p38 phosphorylation have been shown to regulate inflammatory gene expression in lipopolysaccharide-induced RAW264.7 macrophages [46]. Previous research has shown that the p38MAPK inflammatory response is associated with necroptosis pathways in some tissues [25, 26]. MAPK pathway is a key intracellular signal transduction cascade that regulates cell migration and invasion. The trophoblast invasion, represented as expression of MMP-2 and MMP-9, can be suppressed by p38MAPK pathway signaling [47]. Consistent with these previous studies, we observed a significant increase in p38 phosphorylation in human PE placental tissues as well as p38MAPK signaling activation in CER+QVD-induced JEG-3 cells. Therefore, enhanced trophoblast necroptosis may suppress trophoblast invasion via p38MAPK signaling, leading to PE.

In addition, we observed that the H2S synthesis enzyme CBS was expressed in healthy human placental tissue but was downregulated in placental tissue with PE, consistent with our previous research [21]. Importantly, we also found that CBS expression level was negatively correlated with RIPA1 and RIPA3 expression levels in the placenta tissues of pregnant women. Previous studies demonstrated that H2S, an endogenous immune regulator, can exert anti-inflammatory therapeutic effects in various diseases [48]. For instance, in a doxorubicin-induced cardiomyopathy cell model, treatment with the H2S donor NaHS can reduce IL-1β, IL-6, and TNF-α expression, which was mediated by the p38 MAPK/NF-kB pathway [27]. Consistently, we found that NaHS suppressed p38MAPK activation in JEG-3 cells, whereas pretreatment with the p38MAPK inhibitor SB203580 inhibited proinflammatory cytokines (IL-1β, IL-6, TNF-α), RIPA1, and RIPA3 mRNA expression induced by CER+QVD. Moreover, p38MAPK inhibition increased cell viability, demonstrating that p38MAPK signaling affected CER+QVD-induced necroptosis in JEG-3 cells by modulating necrosome protein and proinflammatory cytokine expression. Thus, our results confirm that exogenous H2S protects against necroptosis in trophoblasts.

In conclusion, we proposed the putative role of necroptosis in early-stage PE, reflected by the blockage of caspase-8/3 and increased expression of RIPA1 and RIPA3 in PE placenta tissues. Furthermore, we demonstrated that exogenous H2S protected cytotrophoblasts against CER-induced necroptosis via the p38MAPK pathway. H2S or H2S donors may be the candidate agent for PE by targeting necroptosis of trophoblast cells. Our findings may offer a novel avenue to understand the pathogenesis of PE and guide the effective treatment for PE patients.

This study was approved by the Ethics Committee of Changhai Hospital affiliated to Second Military Medical University (PTEC-A-2021-28-1). Written informed consent was obtained from each participator.

The authors have no conflicts of interest to declare.

The authors wish to thank the nursing and medical staff of the delivery suite and the patients at Changhai Hospital, affiliated to Second Military Medical University (SMMU; Shanghai, China). The authors acknowledge the support of the National Natural Science Foundation of China: Grant No. 31771667 and 31800988, supporting research on the pathogenesis of preeclampsia.

Qianqian Sun and Hang Gu co-designed the study. Huijing Shao and Ziwen Ma conducted the experiment. Rui Guan and Xiaomin Yu collected and processed the data. Caihong Zhang and Zixi Chen wrote the manuscript. All authors read and consented to the publication of this study.

Additional Information

Caihong Zhang and Zixi Chen contributed equally to this work and share first authorship.

Data are not publicly available due to ethical reasons. Further inquiries can be directed to the corresponding author.

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