Paeonol alleviates placental inflammation and apoptosis in preeclampsia by inhibiting the JAK2/STAT3 signaling pathway

Abstract

Preeclampsia (PE) is a multisystemic and placental inflammatory disease that causes maternal and infant health issues. As one of the active components in peony root extract, paeonol (Pae) exerts anti‐apoptosis and anti‐inflammatory effects. Nonetheless, the protective role of Pae in PE has not yet been characterized. A mouse model of PE was constructed through tail vein injection of 1 mg/d phosphatidylserine/dioleoyl‐phosphatidycholine suspension. The levels of inflammatory cytokines in the placenta were examined via enzyme‐linked immunosorbent assay (ELISA). The mRNA levels of inflammatory cytokines (TNF‐α, IL‐6, IFN‐γ, and IL‐4) and apoptosis markers (Bax, Bcl‐2, and caspase‐3) were tested using quantitative reverse transcription‐polymerase chain reaction (qRT–PCR). Western blot analysis was performed to detect the protein levels of apoptosis markers and Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway‐related molecules. Here, Pae repressed the inflammatory response in the placenta of PE‐like mouse models, as demonstrated by the decreased concentrations and mRNA levels of TNF‐α, IL‐6, and IFN‐γ and the increased concentrations and mRNA levels of IL‐4. Apoptosis in the placentas of PE‐like mouse models was attenuated by Pae, as manifested by the downregulated mRNA and protein levels of Bax and cleaved‐caspase‐3 and the upregulated Bcl‐2. Administration of Pae inhibited the phosphorylation of JAK2 and STAT3 in the placental tissues of PE mice. The JAK2/STAT3 pathway agonist (SC‐39100) reversed Pae treatment‐mediated suppression of placental inflammation and apoptosis in PE mice. Overall, Pae inhibits the JAK2/STAT3 signaling pathway to attenuate placental inflammation and apoptosis in PE mice.

1. INTRODUCTION

Preeclampsia (PE) is a kind of multisystemic disease characterized by new onset of hypertension and proteinuria after 20 weeks of gestation. PE and its complications are serious health problems in terms of maternal and infant morbidity and mortality. ¹ Statistically, PE occurs in nearly 3%–5% of pregnant women and affects 5%–8% of all pregnancies. ² The development of fetuses, including growth retardation, abnormalities in oxygenation, and a decrease in amniotic fluid, can be significantly influenced by PE. ³ According to previous research, two major factors in the pathogenesis of PE are the irregularly activated immune system and the systemic dysfunction of vessels in pregnant women and fetuses. ⁴ Although great efforts have been made to fully understand PE, the exact pathological mechanisms and prediction and prevention strategies for PE remain unclear. Therefore, it is urgently required to develop promising drugs or to explore novel therapeutic biological targets for the treatment of PE.

Paeonol (2′‐hydroxy‐4′‐methoxyacetophenone; Pae) is the major phytochemical from Cortex Moutan. ⁵ Increasing applications of Pae in the clinic have been disclosed due to its inhibitory effects on oxidative stress, inflammation, tumors, and cardiovascular diseases. ⁶ The inflammatory signaling pathways and the subsequent release of inflammatory cytokines such as tumor necrosis factor alpha (TNF‐α), interleukin (IL)‐6, interferon‐γ (IFN‐γ), and IL‐4 exert important functions in the pathogenesis of PE. ⁷ As another crucial factor involved in the pathogenesis of PE, apoptosis can bring about an inflammatory response leading to adverse pregnancy outcomes if the inflammatory response cannot be effectively balanced by anti‐inflammatory mediators. ⁸ , ⁹ Pae has been reported to mitigate the inflammatory response and apoptosis in a variety of human diseases. For example, Pae plays anti‐inflammatory and antioxidation roles in a rat model of spinal cord injury. ¹⁰ Another study revealed that Pae prevents methotrexate‐induced nephrotoxicity through antioxidant, anti‐inflammatory, and antiapoptotic mechanisms. ¹¹ Moreover, Pae has been proven to ameliorate epirubicin‐induced heart injury by inhibiting myocardial apoptosis and inflammation as well as improving cardiac dysfunction. ¹² However, whether Pae can alleviate the inflammatory response and apoptosis in PE‐like mouse models is completely unknown.

The Janus kinase signal transducer and activator of transcription (JAK/STAT) signaling pathway is involved in a variety of biological processes, including cell proliferation, apoptosis, and inflammation, as well as metabolic homeostasis. ¹³ Several documents have demonstrated that the JAK/STAT pathway is implicated in the development of PE. ¹⁴ Inhibition of the activation of the JAK2/STAT3 inflammatory signaling pathway has been reported to alleviate disease progression by inhibiting the inflammatory response and cell apoptosis. For instance, matrine alleviates ulcerative colitis by inactivating the JAK2/STAT3 pathway to improve epithelial cell damage and inhibit inflammation and apoptosis. ¹⁵ Curcumin protects against acute kidney injury by attenuating cell apoptosis and inflammation through regulation of the JAK2/STAT3 signaling pathway. ¹⁶ Nonetheless, whether Pae alleviates placental inflammation and cell apoptosis in PE mouse models by inhibiting the JAK2/STAT3 signaling pathway remains unclear.

In our investigation, we aimed to explore the biological functionality of Pae and the mechanisms associated with Pae in the development of PE. We hypothesized that Pae may regulate the JAK2/STAT3 signaling pathway to mitigate placental inflammation and apoptosis in PE, which might provide a novel explanation for the anti‐inflammatory and anti‐apoptosis effects of Pae and reveal the therapeutic potential of Pae for PE treatment.

2. METHODS AND MATERIALS

2.1. Animal studies

Male and female C57BL/6J mice (specific‐pathogen free grade, 6–8 weeks old, weighing 18–22 g) were procured from Vital River Co. Ltd. (Beijing, China). The stable laboratory conditions for all mice were as follows: 50%–70% humidity, 20–25°C room temperature, 12‐h light/dark cycle, ventilation every 8–12 h, noise below 85 db, and free access to food and water. Prior to the animal experiments, all mice were fed for 1 week. The Lianyungang Maternal and Child Health Hospital (Jiangsu, China) approved all animal experimental procedures that were performed strictly according to National Guidelines and the European Directive 2010/63/EU. Healthy male mice and female mice at a ratio of 1:2 were mated overnight after a week of acclimatization. A successful pregnancy was confirmed when sperm was found on a vaginal smear. Based on a method delineated previously, from Day 5 (GD5) to Day 17 (GD17) of gestation, the pregnant mice were injected with 1 mg phosphatidylserine/dioleoyl‐phosphatidylcholine (PS/PC) (Sigma–Aldrich, St. Louis, MO) suspension into the tail vein every day to establish the PE mouse model. ¹⁷ Normal pregnant (control) mice were injected with saline every day. Pae (purity 99%; Sigma–Aldrich) was dissolved in normal saline to achieve various concentrations for oral administration. Afterward, a total of 72 female mice were randomly divided into six groups: control group (n = 12), PE group (n = 12), PE mice administered a low dose (50 mg/kg/d) of Pae (PE + Pae 10 group [n = 12]) from GD0 to GD17, PE mice administered a medium dose (100 mg/kg/d) of Pae (PE + Pae 100 group [n = 12]) from GD0 to GD17, PE mice administered a high dose (150 mg/kg/d) of Pae (PE + Pae 150 group [n = 12]) from GD0 to GD17, and Pae‐treated PE mice intraperitoneally injected with 8 mg/kg of SC‐39100 on GD17 (SC‐39100 + PE + Pae 150 [n = 12]). The dosages of Pae and SC‐39100 were selected in accordance with previous documents. ¹⁸ , ¹⁹ , ²⁰ On GD18, all mice were sacrificed, and the placental tissues were collected and stored in liquid nitrogen at −80°C for later use.

2.2. Examination of systolic blood pressure and urinary protein and evaluation of pregnancy outcomes

The systolic blood pressure of mice was monitored on GD0, GD4, GD8, GD12, and GD16 using a noninvasive volume‐pressure recording blood pressure monitoring system (Visitech Systems, Apex, NC). On GD17, mice were placed in metabolic cages to obtain 24‐h urine, and proteinuria was quantified using a BCA protein assay kit (Beyotime). At the end of the experiment, pregnant mice were euthanized, and the fetal pups and placenta were collected and weighed.

2.3. Enzyme‐linked immunosorbent assay (ELISA)

Before mice were sacrificed, blood samples were collected via the tail vein into tubes containing EDTA and centrifuged at 4000 rpm for 5 min to obtain the supernatant. The plasma thrombin‐antithrombin complex (TAT) levels were measured using Enzygnost, an ELISA commercially available from Dade Behring, Inc. (Marburg, Germany). After the indicated treatment, placental tissues from pregnant mice were harvested, homogenized in lysis buffer, and centrifuged to examine the levels of proinflammatory cytokines. Consistent with the product manuals, the levels of TNF‐α, IL‐6, IFN‐γ, and IL‐4 were tested via a mouse TNF‐α ELISA kit (EK‐M29261, EK‐Bioscience, Shanghai, China), mouse IL‐6 ELISA kit (E03I0006, BlueGene, Shanghai, China), mouse IFN‐γ ELISA kit (MU30038, Bio‐Swamp, Hubei, China), and mouse IL‐4 ELISA kit (E03I0007, BlueGene), respectively.

2.4. RNA extraction and quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR)

TRIzol reagent purchased from Invitrogen was used to extract total RNA from placental tissues. The RNeasy Maxi kit (Qiagen) was employed to purify the extracted RNAs according to the supplier's instructions. Reverse transcription of mRNA into cDNA was achieved using SuperScript III Reverse Transcriptase (Invitrogen). Then, qRT–PCR amplification was carried out with SYBR Green Master Mix (Q131‐02/03, Vazyme, Nanjing, China) on a 7500 Real‐time PCR System (4351151, Thermo Fisher). qPCR was performed according to the following procedures: 95°C for 10 min, 95°C for 15 s, and 72°C for 15 s for a total of 40 cycles. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was utilized as the internal reference. Calculation of the relative RNA levels was achieved using the 2^−ΔΔCt method. ²¹

2.5. Western blot analysis

Placental tissues were incubated on ice with tissue lysis buffer (Beyotime) containing 20 mM Tris–HCl (pH 7.5), 10 mM EGTA, 2 mM EDTA, 0.4% NaF, and protease inhibitors for 10–15 min. Subsequently, the homogenates were centrifuged at 4°C for 10 min at 18,600 × g to collect the supernatant. The separated proteins were subjected to quantification through the bicinchoninic acid (BCA) method. After electrophoresis at a constant pressure of 80 V, the PVDF membrane was soaked in pure methanol, followed by assembly of the transfer device as follows: sponge → layer filter paper → glue → film → layer filter paper → sponge. The transfer tank was placed in an ice bath and then placed on a sandwich deck. After adding the transfer buffer and inserting the electrode, the membranes were sealed in 25 ml 5% nonfat milk powder buffer for 1 h. TBST (15 ml, 5 min each time) was applied to rinse the membranes that were incubated with the following antibodies: rabbit anti‐Bax (dilution ratio of 1:1000, ab182734, Abcam), rabbit anti‐Bcl‐2 (dilution ratio of 1:2000, ab182858, Abcam), rabbit anti‐caspase‐3 (dilution ratio of 1:2000, ab184787, Abcam), rabbit anti‐phospho (p)‐JAK2 (dilution ratio of 1:1000, ab32101, Abcam), rabbit anti‐JAK2 (dilution ratio of 1:5000, ab108596, Abcam), rabbit anti‐STAT3 (dilution ratio of 1:2000, ab76315, Abcam), rabbit anti‐STAT3 (dilution ratio of 1:1000, ab68153, Abcam), rabbit anti‐GAPDH (dilution ratio of 1:10,000, ab181602, Abcam) at 4°C overnight with slow shaking. After being rinsed with 15 ml of TBST three times, the membranes were incubated with secondary antibodies for 1 h at 37°C. The ECL system (Pierce, Rockford) and the ImageJ software were separately utilized for the visualization of the membrane and quantification of band intensity.

2.6. Antifibrin(ogen) immunostaining

Placental tissues were fixed in 4% paraformaldehyde in phosphate‐buffered saline (pH 7.2) at room temperature for 2 h. Heat‐induced antigen retrieval was conducted in citrate buffer (0.1 mol/L, pH 6.0). The sections were incubated with a goat anti‐mouse fibrin (ogen) antibody (Nordic Immunology, Tilburg, The Netherlands), followed by rabbit anti‐goat immunoglobulin (IgG) conjugated with horseradish peroxidase (HRP). The slides were developed with 3‐amino‐9‐ethylcarbazole, followed by a hematoxylin counterstain.

2.7. Statistical analysis

The data from at least three independent trials in formal experiments were presented as the mean ± standard deviation. GraphPad Prism 6.0 (GraphPad, San Diego, CA) was used for statistical analysis. Student's t test was used to compare the data from two groups. One‐way analysis of variance and Tukey's multiple comparison test were employed to compare the data from three or more groups. A p value less than 0.05 was the threshold for statistical significance.

3. RESULTS

3.1. Successful establishment of the PE mouse model

To induce PE in mice, a 1 mg PS/PC suspension was injected into the tail vein every day from GD5 to GD17. The systolic blood pressure was detected every 4 days from GD0‐GD16, which showed no difference in the control and PE groups on GD0‐GD4. Nonetheless, systolic blood pressure displayed a significant elevation after PS/PC injection in the PE group (Figure 1A). The proteinuria level on GD17 was examined and was dramatically increased by PS/PC injection compared with the control group (Figure 1B). TAT levels of mice in the PE group showed notably higher values on GD17 than those of the control group (Figure 1C). Compared with the control group, mice in the PE group showed a marked reduction in fetal weight, placental weight, and fetal/placental weight (Figure 1D–F). In addition, antifibrin immunostaining was performed in placental tissues. Placental tissues of mice in the control group showed scattered fibrin depositions in the labyrinth layer (La), whereas placentas of mice injected with PS/PC suspension showed diffuse fibrin depositions in the labyrinth layer (La) (Figure 1G). In conclusion, PE in mice was successfully induced by PS/PC injection.

FIGURE 1.

The successful establishment of the PE mouse model. Pregnant mice were injected with 1 mg PS/PC suspension into the tail vein every day from GD5 to GD17. (A) The systolic blood pressure was detected every 4 days from GD0‐GD16. (B) Proteinuria levels were measured on GD17. (C) TAT levels of mice in the control and PE groups were examined on GD17. (D–F) On GD 18, mice were sacrificed, and the fetal pups and placenta were collected and weighed. (G) Antifibrin immunostaining was performed in placental tissues to assess fibrin deposition in the labyrinth layer (La) and spongiotrophoblastic layer (Sp). N = 12/group. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control group

3.2. Pae suppresses inflammation in the placenta of a PE‐like mouse model

To explore whether Pae affects placental inflammation in mice with PE, we administered Pae to PE mice and measured the levels of placental inflammatory factors. ELISA revealed that the PE‐like mouse model exhibited higher levels of TNF‐α, IL‐6, and IFN‐γ than control mice. Notably, decreased levels of TNF‐α, IL‐6, and IFN‐γ were identified in PE mice after treatment with 50 mg/kg Pae, which indicated that Pae elicited a protective effect against inflammation in PE mice. Compared with PE mice treated with 50 mg/kg Pae, administration of 100 mg/kg Pae also reduced the levels of TNF‐α, IL‐6, and IFN‐γ. Additionally, treatment with 150 mg/kg Pae further lowered the levels of TNF‐α, IL‐6, and IFN‐γ in PE mice relative to PE mice treated with 100 mg/kg Pae (Figure 2A–C). The level of IL‐4 in the placentas of PE‐like mice models was also examined, which revealed that the concentrations of IL‐4 were decreased in PE mice compared with the control group. After administration of 50, 100, or 150 mg/kg Pae, the concentrations of IL‐4 were elevated in a dose‐dependent manner (Figure 2D). These results showed that a higher dose of Pae presented a better protective effect against inflammation. Subsequently, we explored the influence of Pae on the mRNA expression of TNF‐α, IL‐6, IFN‐γ, and IL‐4 in mouse models. As presented in Figure 2E–H, the mRNA expression of TNF‐α, IL‐6, and IFN‐γ was increased, while that of IL‐4 was decreased in PE mice but not in control mice. After administration of 50, 100, or 150 mg/kg Pae, the mRNA expression of TNF‐α, IL‐6, and IFN‐γ was dose‐dependently downregulated, while that of IL‐4 was dose‐dependently upregulated. Our data showed that Pae mitigated the inflammatory response of PE mice, and therefore, highlighted its potential as a promising anti‐inflammatory drug for PE.

FIGURE 2.

Pae represses the inflammatory response in the placenta of PE‐like mouse models. The mice were divided into five groups: Control group (normal pregnant mice treated with saline), PE group (pregnant mice with PE), PE + Pae 50 group (PE mice received low dose [50 mg/kg] of Pae), PE + Pae 100 group (PE mice received medium dose [100 mg/kg] of Pae), PE + Pae 150 group (PE mice received high dose [150 mg/kg] of Pae). (A–D) Placental levels of TNF‐α, IL‐6, IFN‐γ, and IL‐4 in the indicated groups were analyzed using ELISA. (E–H) The mRNA expression of TNF‐α, IL‐6, IFN‐γ, and IL‐4 in the placental tissues of pregnant mice was analyzed by qRT–PCR. N = 12/group. ***p < 0.001 compared to the control group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 compared to the PE group

3.3. Pae mitigates cell apoptosis in the placentas of PE mice

The effect of Pae on cell apoptosis in the placentas of PE mice was further evaluated via examination of the mRNA levels of apoptosis markers, including Bax, caspase‐3, and Bcl‐2. Compared with the control group, the mRNA expression of Bax and cleaved‐caspase‐3 was increased, while Bcl‐2 expression was downregulated in the PE group. Notably, the promotion of the mRNA expression of Bax and cleaved‐caspase‐3 and suppression of the mRNA expression of Bcl‐2 caused by PE were reversed via administration of 50, 100, or 150 mg/kg Pae (Figure 3A–C). Through western blot analysis, we observed upregulated protein levels of Bax and cleaved‐caspase‐3 and downregulated protein levels of Bcl‐2 in mice with PE relative to healthy pregnant mice. Treatment with Pae resulted in an increase in the protein level of Bcl‐2 and a decrease in the protein levels of Bax and cleaved‐caspase‐3 in PE mice in a dose‐dependent manner (Figure 3D,E). Taken together, Pae alleviates cell apoptosis in the placentas of PE mice.

FIGURE 3.

Pae inhibits cell apoptosis in the placentas of PE mice. The mice were divided into five groups: The control group, PE group, PE + Pae 50 group, PE + Pae 100 group, and PE + Pae 150 group. (A–C) qRT–PCR analysis of the mRNA expression of Bax, Bcl‐2, and caspase‐3 in the placentas of mouse models. (D,E) Protein levels of Bax, Bcl‐2, and caspase‐3 were analyzed by western blot. N = 12/group. ***p < 0.001 compared to the control group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 compared to the PE group

3.4. Pae inactivates the JAK2/STAT3 signaling pathway

Due to the correlation between the activated JAK2/STAT3 signaling pathway and PE development, ¹⁴ , ²² we explored whether Pae regulates the JAK2/STAT3 signaling pathway in our study. The results from western blot analysis delineated that the protein levels of p‐JAK2 and p‐STAT3 were reduced in the placentas of PE mice. Importantly, the increase in dosage (50, 100, and 150 mg/kg) of Pae gradually downregulated the protein levels of p‐JAK2 and p‐STAT3 in the placentas of PE mice (Figure 4A,B). Collectively, Pae inhibits the JAK2/STAT3 signaling pathway in PE mice.

FIGURE 4.

The JAK/STAT3 signaling pathway is inactivated by Pae. (A,B) Placental tissues from PE mice treated with 50, 100, or 150 mg/kg Pae were subjected to western blot analysis for the detection of the protein levels of p‐JAK2, JAK2, p‐STAT3, and STAT3. N = 12/group. ***p < 0.001 compared to the control group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 compared to the PE group

3.5. Pae attenuates placental inflammation by inhibiting the JAK2/STAT3 signaling pathway

To determine whether Pae alleviates placental inflammation in PE mice via inactivation of the JAK2/STAT3 signaling pathway, the JAK2/STAT3 pathway agonist SC‐39100 was used to treat PE mice. Experimental data illuminated that administration of 150 mg/kg Pae notably reduced the placental levels of TNF‐α, IL‐6, and IFN‐γ but increased the levels of IL‐4. These results were all counteracted by SC‐39100 (Figure 5A–D). Consistent with the ELISA results, qRT–PCR analysis revealed that compared to that in the PE group, the mRNA expression of TNF‐α, IL‐6, and IFN‐γ was downregulated, while the mRNA expression of IL‐4 was upregulated in the PE + Pae 150 group. However, injection of SC‐39100 reversed these effects (Figure 5E–H). These findings showed that SC‐39100 partially eliminates Pae‐induced alleviation of placental inflammation in PE mice and that Pae can mitigate placental inflammation by inactivating the JAK2/STAT3 signaling pathway.

FIGURE 5.

Effects of SC‐39100 on inflammation in Pae‐treated placentas of PE mice. PE mice were injected with the JAK/STAT pathway agonist SC‐39100 after administration of 150 mg/kg Pae. Saline‐treated pregnant mice functioned as a control. (A–D) ELISA detected the levels of TNF‐α, IL‐6, IFN‐γ, and IL‐4 in the placentas of mouse models. (E–H) qRT–PCR analysis was used to detect the mRNA expression of TNF‐α, IL‐6, IFN‐γ, and IL‐4 in the placentas of pregnant mice. N = 12/group. ***p < 0.001 compared to the control group; ^## p < 0.01 compared to the PE group; ^& p < 0.05, ^&& p < 0.01 compared to the PE + Pae 150 group

3.6. Pae inhibits apoptosis in the placentas of PE mice via the JAK2/STAT3 signaling pathway

To confirm whether Pae regulates the JAK2/STAT3 signaling pathway to exert an effect on cell apoptosis in the placentas of PE mice, qRT–PCR and western blot analyses were conducted. We found that SC‐39100 treatment counteracted the suppressed mRNA and protein levels of Bax and cleaved‐caspase‐3 and reversed the elevated mRNA and protein levels of Bcl‐2 in 150 mg/kg Pae‐treated PE mice (Figure 6A–E). These results suggested that Pae could inactivate the JAK2/STAT3 signaling pathway to repress cell apoptosis in the placentas of PE mice.

FIGURE 6.

Influence of SC‐39100 on cell apoptosis in the placentas of PE mice treated with Pae. (A–C) The mRNA expression of Bax, Bcl‐2, and caspase‐3 in the control group, PE group, PE + Pae 150 group, and PE + Pae 150 + SC‐39100 group by qRT–PCR analysis. (D,E) The protein levels of Bax, Bcl‐2, and caspase‐3 in the indicated groups were analyzed by western blot. N = 12/group. **p < 0.01 and ***p < 0.001 compared to the control group; ^## p < 0.01 and ^### p < 0.001 compared to the PE group; ^& p < 0.05, ^&& p < 0.01, ^&&& p < 0.001 compared to the PE + Pae 150 group

4. DISCUSSION

Although the protective effects of Pae have been reported in many human diseases, the anti‐inflammatory and antiapoptotic functions of Pae in PE have not yet been investigated. Herein, the mouse model with PE was constructed by tail vein injection of a PS/PC suspension. Not surprisingly, we discovered that Pae effectively alleviated PE‐induced placental inflammation and cell apoptosis in a dose‐dependent manner. Additionally, Pae elicited anti‐inflammatory and antiapoptotic effects via inactivation of the JAK2/STAT3 signaling pathway in PE‐like mouse models.

According to previous studies, the crucial function of the activated immune system characterized by an imbalance between anti‐inflammatory and proinflammatory factors in the pathogenesis of PE has been discovered. ²³ Substantial evidence has shown that the accelerated apoptosis of trophoblasts and retardant intrauterine growth can be caused by imbalanced concentrations of pro‐ and anti‐inflammatory cytokines in the PE placenta. ²⁴ TNFα, IL‐6, and IFN‐γ are pivotal proinflammatory cytokines and mediators in the immune system of pregnant women and are overproduced by immune cells of PE patients. ²⁵ The dysfunction of endothelial cells can be induced by proinflammatory cytokines by enhancing the permeability of vessels and contributing to the apoptosis of trophoblastic cells. ²⁶ , ²⁷ , ²⁸ Maternal inflammation induced by proinflammatory factors through activation and destruction of endothelial cells is also associated with the pathophysiological features of PE. ²⁹ Several studies have verified that preeclamptic symptoms in pregnant rats can be induced by IL‐6 and TNF‐α via activation of the endothelin and renin‐angiotensin systems. ³⁰ Here, ELISA of the maternal placental tissues of mice revealed that the concentrations of TNFα, IL‐6, and IFN‐γ were notably elevated in PE‐like mouse models relative to normal pregnant mice. In addition, the mRNA levels of TNFα, IL‐6, and IFN‐γ were also increased in the placental tissues of PE mice. Importantly, after administration of 50, 100, or 150 mg/kg Pae, the upregulated placental levels and mRNA levels of TNFα, IL‐6, and IFN‐γ were reduced in a dose‐dependent manner. Anti‐inflammatory cytokines such as IL‐4 are crucial for a successful pregnancy. ³¹ Any alteration in the levels of anti‐inflammatory cytokines may influence the function of immune and apoptosis‐associated pathways and thus result in pregnancy‐related syndromes, including PE. ³² In our investigation, we observed significantly increased levels of IL‐4 in the placental tissues of PE mice and upregulated mRNA levels of IL‐4 in PE mouse placental tissues. Moreover, PE‐induced downregulation of the levels of IL‐4 were gradually upregulated via treatment with increased doses of Pae. Therefore, these examined cytokines, including TNFα, IL‐6, IL‐4, and IFN‐γ, may serve as biomarkers to predict and treat PE in the initial stages clinically.

Aberrant apoptosis is regarded as an important pathological characteristic of PE. ³³ The normal apoptosis process maintains the homeostasis of placental trophoblasts, while abnormal apoptosis leads to the pathophysiology of PE, suggesting that modulation of trophoblast apoptosis is essential for normal pregnancy. ³⁴ Bax is a proapoptotic protein contributing to programmed cell death or apoptosis, and the upregulated level of Bax indicates the promotion of cell apoptosis. Bcl‐2 is an antiapoptotic protein, the elevated level of which implies the promotion of cell apoptosis. Therefore, the degree of apoptosis can be directly reflected by the changed levels of Bcl‐2 and Bax. ³⁵ Caspase‐3 belonging to the protease family exerts crucial functions in both extrinsic and intrinsic apoptotic processes. ³⁶ It has been reported that Pae could inhibit cell apoptosis in high glucose and palmitic acid‐induced endothelial cells ³⁷ and Pae activates the class III PI3K/Beclin‐1 pathway to inhibit apoptosis of vascular smooth muscle cells. ³⁸ Our study revealed that the mRNA and protein levels of Bax and cleaved‐caspase‐3 were upregulated, while that of Bcl‐2 was downregulated in PE mice. Pae administration increased the expression levels of Bcl‐2 but decreased the expression levels of Bax and cleaved‐caspase‐3. These results showed that cell apoptosis in PE mice was effectively repressed via administration of Pae.

The pathogenesis of PE is very complicated and involves various molecules and signaling pathways. According to previous reports, JAK/STAT signaling is associated with the progression of PE. ²² Studies have demonstrated that some Chinese medicines, such as matrine and curcumin, can inhibit inflammation and cell apoptosis by inactivating the JAK/STAT signaling pathway. ¹⁵ The role of Pae in the reduction of inflammation and cell apoptosis in PE mouse models was confirmed in our study. We also explored the activity of the JAK/STAT signaling pathway in placental tissues of PE mice. Upregulated protein levels of p‐JAK and p‐STAT3 were identified, which suggested activated JAK/STAT signaling activity in PE mice. However, the phosphorylation levels of JAK and STAT3 were all dose‐dependently downregulated after administration of Pae, implying that the inactivation of JAK/STAT signaling may be associated with Pae‐alleviated inflammation and cell apoptosis in PE. A previous study revealed that the JAK/STAT pathway agonist SC‐39100 partially abrogates microRNA‐210 downregulation‐mediated suppression of the inflammatory response and cell apoptosis in septic rats. ³⁹ Therefore, SC‐39100 may be associated with Pae‐regulated placental inflammation and apoptosis in PE. In our current investigation, we found that the alleviative effects of Pae on inflammation and cell apoptosis in PE mice were reversed after injecting SC‐39100.

In summary, the function and mechanism of Pae have been investigated, and we found that Pae mitigated placental inflammation and apoptosis in PE mice via regulation of the JAK/STAT signaling pathway, which may provide novel therapeutic measures for the treatment of PE as well as a new direction for the analysis of other potential drugs for PE treatment. To be honest, there are some limitations in our study. First, the sample size was relatively small, and expanding the sample size would be better in subsequent experiments to enhance the persuasiveness of our findings. Second, further research on Pae should be conducted to explore other potential signaling pathways associated with PE in future studies. Third, Pae is a promising anti‐inflammatory and antiapoptotic drug for PE, but more research is needed to establish its potential for clinical application.

CONFLICT OF INTEREST

All authors declare no conflict of interest.

Wang H, Liu M‐L, Chu C, Yu S‐J, Li J, Shen H‐C, et al. Paeonol alleviates placental inflammation and apoptosis in preeclampsia by inhibiting the JAK2/STAT3 signaling pathway. Kaohsiung J Med Sci. 2022;38(11):1103–1112. 10.1002/kjm2.12585