Progesterone Attenuates SIRT1-Deficiency-Mediated Pre-Eclampsia

Pre-eclampsia is a severe hypertensive disorder of pregnancy (HDP), mainly characterized by new-onset hypertension with proteinuria after 20-week gestation. Sirtuin1 (SIRT1), a class III histone deacetylase, is associated with the regulation of various pathophysiological processes, including inflammation, immune response, metabolism, and autophagy. However, the effect of SIRT1 in the pathogenesis of pre-eclampsia remains to be elucidated. In this study, we found that the expression of SIRT1 was relatively lower in the placentas and serum samples of pre-eclampsia patients. Typical pre-eclampsia-like symptoms, such as hypertension, proteinuria, fetal growth restriction, kidney injury, and a narrow placental labyrinth layer, were observed in SIRT1 knockdown (SIRT1+/−) mice. Of note, these performances could be improved after the intraperitoneal injection of SIRT1 agonist SRT2104. More importantly, we found that the efficacy of progesterone on attenuating symptoms of PE was profoundly better than that of metformin in SIRT1+/− mice. In addition, our results suggested that progesterone can promote the invasion and inhibit the apoptosis of trophoblasts. These data suggest that SIRT1 plays an important role in pre-eclampsia and that progesterone alleviates pre-eclampsia-like symptoms mediated by SIRT1 deficiency.


Introduction
Pre-eclampsia (PE) is defined as new-onset hypertension with proteinuria after 20-week gestation. In severe cases, it can also combine with renal, cardiac, pulmonary, hepatic, and neurological dysfunction; hematologic disturbances; fetal growth restriction; stillbirth; and even maternal death [1]. Pre-eclampsia impacts 2 to 8% of all pregnancies worldwide, which is a major cause of maternal and fetal morbidity and mortality, and the only known cure for this complication is delivery [2,3]. Elucidating the pathophysiological mechanisms of pre-eclampsia will help us accurately prevent, manage, and treat it to avoid maternal and fetal losses. SIRT1 (Sirtuin 1) is an NAD+-dependent deacetylase that plays critical roles in many biological events, including metabolism, immune response, cellular inflammation, autophagy, and senescence [4][5][6][7][8][9]. Furthermore, it is reported that SIRT1 can regulate cell migration and invasion in several cancer cells by deacetylating various substrates [10][11][12], and the vascular remodeling of the placenta also depends on the invasive properties of trophoblasts, so we consider whether SIRT1 also affects pre-eclampsia. Additionally, as an important factor causing aging, SIRT1 deficiency can lead to placental senescence [13]. However, whether SIRT1 directly induces pre-eclampsia as well as the comprehensive molecular mechanisms between SIRT1 and pre-eclampsia are still poorly understood. All data values are expressed as mean ± SEM. P values were obtained using the unpaired Student's test on Graph Pad Prism Version 9.2.0. * p < 0.05; **** p < 0.0001. BP: blood pressure; NP: normal pregnancy (n = 95); PE: pre-eclampsia (n = 76); FGR: fetal growth restriction.

Establishment of the Mouse Model
All animals were housed in a temperature-controlled room (23 • C) with a 12:12 light: dark cycle. SIRT1 knockdown mice (SIRT1 +/− ) (HE) were generated by crossbreeding SIRT1 flox/flox (WT) mice with broad-expression Cre transgenic mice. The Cre transgenic mice were provided by the Shanghai Model Organisms Center (Shanghai, China), while the SIRT1 flox/flox mice were purchased from the Jackson Lab (029603, the Jackson Laboratory, Farmington, CT, USA). Successful knockdown of SIRT1 in mice was confirmed with PCR and immunofluorescence.
The PCR cycling conditions for SIRT1 were a primary denaturation at 94 • C for 3 min, followed by 35 cycles of 30 s at 94 • C, annealing temperature at 60 • C for 30 s, and 72 • C for 60 s, with a final extension of 5 min at 72 • C. Primer sequences for PCR were as follows: Cre: forward AGGCGGATTTCTGAGTTCGA and reverse CGTCCCTTGTAATGTTTCCC and floxed SIRT1 gene: forward AGGAATCCCACAGGAGACAG and reverse GGTTAA-GATTAGCCCATTAAAGC. SIRT1 +/− female mice at 8 weeks of age were individually mated with SIRT1 +/− male mice. The initiation of pregnancy was marked by the presence of postcoital vaginal plug at gestation day (GD) 0.5 for early pregnancy study.

Systolic Blood Pressure (SBP) Measurement
The systolic blood pressure of the pregnant mice in each group was measured using a non-invasive blood pressure monitor with a volume pressure-recording sensor and an occlusion tail cuff (BP-2000 Series II, Visitech Systems, Apex, NC, USA). We monitored the systolic blood pressure of mice for 3 consecutive days before officially starting the experiments to allow them to adapt to the environment. Then, SBP was monitored every other day from GD2 to GD17, whether it was the experimental groups or the control groups. It is important to note that SBP should be measured before administration to ensure that the value of SBP is not disturbed.

Mouse Urine Collection and Detection
The 24-h urine of the pregnancy mice in both groups was collected during the 17-18th days of gestation. The concentration of urinary protein was detected using Albuwell M Test Kit (1011, Exocell, Logan Township, NJ, USA), following the protocol.

Placentas, Fetuses, and Kidneys Collection
Mice were euthanized using isoflurane, and their placentas, fetuses, and kidneys were collected on the 18th day of gestation. The placentas and fetuses were weighed and imaged. The kidneys and placental tissues were fixed in 4% paraformaldehyde overnight, embedded in paraffin, and then cut into 5 µm sections for subsequent experiments.

Western Blot Analysis
Total protein was isolated from placental tissues of 7 normal pregnancy women (NP) and 7 women with pre-eclampsia with severe features (sPE). The diagnostic criteria for pre-eclampsia with severe features refer to American College of Obstetricians and Gynecologists' Practice Bulletin on Gestational Hypertension and Pre-eclampsia [28]. The protein samples were separated by electrophoresis, transferred to polyvinylidene difluoride (PVDF) membrane, and then incubated with the following primary antibodies anti-SIRT1 (dilution 1:5000, ab32441, Abcam, UK) overnight at 4 • C. On second day, the PVDF membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (dilution 1:1000, 7074 S, Cell Signaling Technology, USA) for 2 h at room temperature. GAPDH (1:5000, ab181602, Abcam, UK) was used as an internal standard. The signals were visualized with an ECL solution (P0018FS, Beyotime, Shanghai, China). Images were obtained using Amersham Imager 600 (GE Healthcare, Waukesha, WI, USA).

Immunohistochemistry (IHC) and Immunofluorescence (IF)
The human placental sections were deparaffinized and treated with 3% H 2 O 2 to block the endogenous peroxidase activity. Then, the sections were pretreated heat-mediated antigen retrieval using sodium citrate buffer or Tris/EDTA buffer and incubated with primary antibodies against SIRT1 (dilution 1:100, ab32441, Abcam, UK) at 4 • C overnight. Positive signals were visualized by incubation with HRP-conjugated secondary antibodies using microscope (Olympus IX73).
The mice placental sections were used for immunofluorescence, and the process is similar to immunohistochemistry. The sections were incubated with primary antibodies against CK7 (dilution 1:5000, ab181598, Abcam), SIRT1 (dilution 1:100, ab32441, Abcam) at 4 • C overnight. Positive signals were visualized by incubation with fluorophore-conjugated secondary antibodies. The slides were mounted with mounting medium containing DAPI (Abcam) and then viewed and imaged under a fluorescence microscope (Olympus IX73).

Masson Staining and PAS Staining
The Masson staining kit (ab150669, Abcam) and PAS staining kit (ab150680, Abcam) were used for histological analysis of the mice kidney sections. After dehydration with ethyl alcohol and blocking with neutral gum, we used light microscope (Olympus IX73) to observe and evaluate the kidney injury. For Masson staining, the glomerulus is bloodless with pre-eclampsia; for PAS, the capillary lumens are occluded by swollen endothelial cells in typical pre-eclampsia kidney pathology.
The mice placental sections were stained with Masson staining kit (ab150669, Abcam) and imaged under the light microscope (Olympus IX73). Then, we used ImageJ software to measure the area of the labyrinth layer and junctional zone of placentas. The ratio between the labyrinth layer and junctional zone is considered an indicator of placental function [29].

Lentivirus Transfection
Lentiviruses carrying short hairpin RNA (shRNA) targeting human Sirtuin1 (SIRT1) lentivirus vectors (GV248) were purchased from GeneChem (Shanghai, China). HTR8/SVneo cells were transfected with viruses (multiplicity of infection = 20) for 24 h according to the manufacturer's instructions. Then, the cells were transferred to fresh medium containing puromycin (1 µg/mL) for the selection of stable clones after three passages. The shRNA sequence was 5 -GGCTTGATGGTAATCAGTA-3 . Then, the cells were divided into two groups, control and SIRT1KO groups, for P4 treatment.

Transwell Assay
Matrigel (dilution 1:8, BD Biosciences, San Diego, CA, USA) was added to the upper chamber of Transwell chambers, and the Transwell chambers were placed in a 24-well plate and incubated overnight at 4 • C. Then, 200 µL (HTR-8/SVneo, 1 × 10 5 cells/well) DMEM/F12 suspension with or without 10% FBS was added to the upper chamber, and 600 µL DMEM/F12 containing 10% FBS was added to the lower chamber. The cells were cultured for 48 h at 37 • C in a 5% CO 2 incubator. The 24-well plate was removed, and the upper chamber medium and nonpenetrating cells were gently wiped off with a cotton swab, washed three times with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 30 min, and stained with crystal violet for 20 min. Subsequently, random photographs were acquired under an inverted microscope (×200), and 5 visual fields were counted in each chamber. The number of invaded cells was counted using Image J software (National Institutes of Health, Bethesda, MD, USA).
2.6.6. Apoptosis Assay HTR-8/SVneo cells from P4 and vehicle groups were stained with Annexin V and 7-AAD (ab214663, Abcam, UK) for 15 min at RT in the dark. Fluorescence intensities were analyzed by flow cytometry by using the BD Cell Quest Pro software.

Statistical Analysis
All results were analyzed using GraphPad Prism9.2.0 (GraphPad Software, San Diego, CA, USA); Friedman's test was used to analyze the variance. Differences between groups were analyzed by one-way ANOVA (and nonparametric or mixed) and unpaired Student's test (and nonparametric tests), results were presented as the mean ± standard deviation (SD), and values with p < 0.05 were supposed to be statistically significant.

SIRT1 Was Significantly Lower in the Placentas and Serum Samples of Pre-Eclampsia Patients
To study the role of SIRT1 in PE, we evaluated the level of SIRT1 expression in human placental tissues from women with pre-eclampsia with severe features (sPE) (n = 7) and normal pregnant women (NP) (n = 7) ( Figure 1). Compared with controls, the level of SIRT1 expression was significantly lower in the placentas of sPE patients ( Figure 1A), as detected by Western Blotting. Immunohistochemistry results also showed that SIRT1 expression was apparently decreased in the placentas of sPE patients ( Figure 1B).
Next, the levels of SIRT1 in serum samples from 76 PE patients and 95 NP women were detected by ELISA. The result indicated that the SIRT1 expression from serum samples was significantly decreased in the PE patients ( Figure 1C, PE vs. NP: 0.8379 ± 0.3370 vs. 1.089 ± 0.6779 relative absorbance).
To study the role of SIRT1 in PE, we evaluated the level of SIRT1 expression in human placental tissues from women with pre-eclampsia with severe features (sPE) (n = 7) and normal pregnant women (NP) (n = 7) ( Figure 1). Compared with controls, the level of SIRT1 expression was significantly lower in the placentas of sPE patients ( Figure 1A), as detected by Western Blotting. Immunohistochemistry results also showed that SIRT1 expression was apparently decreased in the placentas of sPE patients ( Figure 1B). Figure 1. SIRT1 was significantly lower in the placentas and serum samples of pre-eclampsia patients. (A) Western blot analysis of SIRT1 protein levels in placental tissues from normal pregnancy women (NP, n = 7) and women with pre-eclampsia with severe features (sPE, n = 7). (B) Immunohistochemistry for SIRT1 in placental tissues from NP women (n = 7) and sPE women (n = 7). (C) ELISA analysis of the SIRT1 concentration in the serum of PE women (n = 76) and gestational agematched NP women (n = 95). Error bars, mean ± SD. The data were analyzed by an unpaired Student's test. ** p < 0.01.
Next, the levels of SIRT1 in serum samples from 76 PE patients and 95 NP women were detected by ELISA. The result indicated that the SIRT1 expression from serum samples was significantly decreased in the PE patients (Figure1C, PE vs. NP: 0.8379 ± 0.3370 vs. 1.089 ± 0.6779 relative absorbance).

SIRT1 +/− Mice Demonstrated Pre-Eclampsia-like Symptoms
To investigate the impact of SIRT1 on pre-eclampsia, we developed a SIRT1 knockdown (SIRT1 +/− ) mouse model from SIRT1 flox/flox mice, and the gene knockdown and mice mating strategies are shown in Figure 2A. We found that all SIRT1 −/− mice (HO) died at postnatal 28 days ( Figure 2H), which was consistent with previous research that SIRT1 −/− mice suffered from severe growth retardation and developmental defects [29,[31][32][33]. (A) Western blot analysis of SIRT1 protein levels in placental tissues from normal pregnancy women (NP, n = 7) and women with pre-eclampsia with severe features (sPE, n = 7). (B) Immunohistochemistry for SIRT1 in placental tissues from NP women (n = 7) and sPE women (n = 7). (C) ELISA analysis of the SIRT1 concentration in the serum of PE women (n = 76) and gestational age-matched NP women (n = 95). Error bars, mean ± SD. The data were analyzed by an unpaired Student's test. ** p < 0.01.

SIRT1 +/− Mice Demonstrated Pre-Eclampsia-like Symptoms
To investigate the impact of SIRT1 on pre-eclampsia, we developed a SIRT1 knockdown (SIRT1 +/− ) mouse model from SIRT1 flox/flox mice, and the gene knockdown and mice mating strategies are shown in Figure 2A. We found that all SIRT1 −/− mice (HO) died at postnatal 28 days ( Figure 2H), which was consistent with previous research that SIRT1 −/− mice suffered from severe growth retardation and developmental defects [29,[31][32][33]. Therefore, we selected the SIRT1 +/− mice to finally conduct our research. To confirm that the construction of the SIRT1 +/− mice model was successful, we detected the genotype of the model mice by PCR and agarose gel electrophoresis ( Figure 2B), and the mice with SIRT1 +/− were selected for the following treatment. The expression of SIRT1 in placental tissues of SIRT1 +/− mice was downregulated compared with SIRT1 flox/flox mice by immunofluorescence, as shown in Figure 2C.
Therefore, we selected the SIRT1 +/− mice to finally conduct our research. To confirm that the construction of the SIRT1 +/− mice model was successful, we detected the genotype of the model mice by PCR and agarose gel electrophoresis ( Figure 2B), and the mice with SIRT1 +/− were selected for the following treatment. The expression of SIRT1 in placental tissues of SIRT1 +/− mice was downregulated compared with SIRT1 flox/flox mice by immunofluorescence, as shown in Figure 2C. The labyrinth/junctional zone ratio of each group. Error bars, mean ± SD. The data were analyzed by one-way ANOVA (and nonparametric or mixed) and unpaired Student's test (and nonparametric tests). ** p < 0.01; *** p < 0.001; **** p < 0.0001, ns: no significance.
Subsequently, to verify whether the model mice showed pre-eclampsia-like symptoms, we conducted a series of observations and experiments. We found that the embryoresorption rate was almost normal in SIRT1 +/− mice ( Figure 2E). Additionally, there was no significant difference in placental weight among the two groups ( Figure 2F). Since PE can cause fetal growth restriction (FGR), we analyzed the weight of the live fetus in SIRT1 flox/flox and SIRT1 +/− groups, showing that the weight of the live fetus was dramatically lower in SIRT1 +/− mice ( Figure 2G, SIRT1 +/− vs. WT: 0.7803 ± 0.1651 vs. 0.8559 ± 0.1585 g). Representative images of fetuses and placentas from each group are shown in Figure 2D, and the size of the fetus was smaller compared with WT groups. Subsequently, we analyzed the changes in systolic blood pressure (SBP). Interestingly, the level of SBP increased dramatically at late pregnancy in SIRT1 +/− mice ( Figure 2I, SIRT1 +/− vs. WT: 119.6 ± 9.952 vs. 108 ± 6.340 mmHg). To understand the level of elevated BP, we calculated the difference of SBP between late pregnancy and the basic condition. We found that the ∆BP presented an increasingly high level in SIRT1 +/− groups ( Figure 2J, SIRT1 +/− vs. WT: 12.45 ± 9.186 vs. −1.562 ± 10.47 mmHg). Intriguingly, the level of ∆BP had a positive correlation with the SIRT1 gene value in fetuses ( Figure 2K).
To explore the pathological changes in the placenta, we compared the labyrinth/junction zone ratio in the placentas of each group. The labyrinth layer is the functional layer of the placenta [32]. As a result, we observed that the labyrinth layer was narrow in SIRT1 +/− mice compared with SIRT1 flox/flox (Figure 2N), and the labyrinth/junction zone ratio was significantly decreased in the SIRT1 +/− group compared with the SIRT1 flox/flox group ( Figure 2O).

SRT2104 Significantly Diminished the PE-like Symptoms in SIRT1 +/− Mice
To verify the function of SIRT1 in pre-eclampsia, we intraperitoneally injected the SIRT1 +/− mice with SIRT1 agonist SRT2104 every other day from GD2 to GD17. The process of treating mice is shown in Figure 3A. Before starting the formal experiments, it had been confirmed that the side effect of SRT2104 was mild ( Figure S1A-D). The labyrinth/junctional zone ratio of each group. Error bars, mean ± SD. The data were analyzed by one-way ANOVA (and nonparametric or mixed) and unpaired Student's test (and nonparametric tests). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, ns: no significance.
We found that SRT2104 could reverse the pre-eclampsia-like symptoms driven by SIRT1 knockdown. After treating SIRT1 +/− mice with SRT2104, the embryo-resorption rate had no significant change ( Figure 3C), the weight of the placenta was increased slightly ( Figure 3D, vehicle vs. SRT2104: 0.08519 ± 0.01009 vs. 0.09237 ± 0.01213 g), while the weight of the live fetus was dramatically increased ( Figure 3E, vehicle vs. SRT2104: 0.6808 ± 0.08630 vs. 0.7719 ± 0.1483 g). These results were consistent with the representative images of fetuses and placentas ( Figure 3B). The level of systolic blood pressure at late pregnancy ( Figure 3F were both decreased compared with the vehicle group. The kidney injury was improved with SRT2104, presenting a decreased urinary protein concentration in late pregnancy ( Figure 3H, vehicle vs. SRT2104, 7.397 ± 3.293 vs. 3.392 ± 0.7711 ug/mL) and almost normal glomerulus morphology ( Figure 3I). The placental selectins also showed a widened labyrinth layer after using SRT2104 for intraperitoneal injection ( Figure 3J,K).
However, the effect was not very apparent when we treated SIRT1 +/− mice with SRT2104 via intragastric administration. The embryo-resorption rate had no visible change ( Figure S1E). The weight of the placenta and fetus was significantly increased (Figure S1F,G) when the dose of SRT2104 was 50 mg. Additionally, there was also an increase in the weight of fetus when the dose was 100 mg ( Figure S1G). However, we did not see any difference in the level of systolic blood pressure ( Figure S1H).

P4 Could Alleviate Hypertension in SIRT1 +/− Mice and Promote the Invasion and Inhibit the Apoptosis of Trophoblasts
Previous research reported that P4 and metformin could improve the symptoms of pre-eclampsia [16,31], but the results are still controversial. To determine whether P4 and metformin could be effective drugs for pre-eclampsia, we treated pregnant SIRT1 +/− mice with P4 and metformin. We found that there was no significant difference in the embryoresorption rate ( Figure 4A), and the weight of the placenta was decreased in both groups ( Figure 4B), which may be due to the batch difference caused by the residual decidua in part of the placentae during the initial dissection. However, there was a big difference between the two groups in the weight of the live fetus. P4 could elevate the weight of the live fetus, while the metformin decreased the fetal weight ( Figure 4C, vehicle vs. P4, 0.6808 ± 0.08630 vs. 0.6037 ± 0.1168 g; vehicle vs. metformin, 0.6808 ± 0.08630 vs. 0.8290 ± 0.08013 g). In addition, we found that the increased systolic blood pressure at late gestation was notably attenuated after administration of P4 or metformin ( Figure 4E, vehicle vs. P4, 117.8 ± 8.311 vs. 103.0 ± 7.388 mmHg; vehicle vs. metformin, 117.8 ± 8.311 vs. 105.8 ± 7.001 mmHg). Additionally, ∆BP also presented a downward tendency whether in Met groups and P4 groups ( Figure 4D, vehicle vs. P4, 8.158 ± 9.212 vs. −8.477 ± 7.138 mmHg; vehicle vs. metformin, 8.158 ± 9.212 vs. −9.680 ± 7.041 mmHg). The above were all in the case of sufficient metformin. In the case of half the amount of metformin, there was almost no effect ( Figure 4A-E). Furthermore, we treated HTR8/SVneo cells with P4 and metformin; the results showed that trophoblast invasion was elevated both in Met and P4 groups, and we saw a greater increase in P4 groups ( Figure 4F,G). Therefore, we believed that P4 had better efficacy in diminishing the symptoms of pre-eclampsia.
To further verify the mechanisms of P4 in pre-eclampsia, we knocked down SIRT1 in HTR8/SVneo with lentivirus. The expression of SIRT1 was significantly decreased after transfection ( Figure 4H). The trophoblast invasion was significantly decreased after SIRT1 knockdown, and P4 could reverse this result ( Figure 4I,J). Additionally, there was no significant change in the proliferation of trophoblast when treated with low-dose P4, whether it was treated for 24 h or 48 h ( Figure 4K). However, the apoptosis of trophoblast was dramatically decreased after treatment with P4, which is similar to SRT2104 ( Figure 4L). These results demonstrated that P4 could enhance the invasion and inhibit the apoptosis of trophoblasts in order to prevent the progress of pre-eclampsia.  The apoptosis assay of vehicle, SRT2104 and P4 groups in SIRT1 KD trophoblasts. Error bars, mean ± SD. The data were analyzed by one-way ANOVA (and nonparametric or mixed) and unpaired Student's test (and nonparametric tests). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, ns: no significance.

Discussion
Pre-eclampsia is a severe pregnancy-related complication with the onset of hypertension after week 20 of pregnancy. Pre-eclampsia is mainly characterized by inadequate trophoblasts invasion, which can be caused by autophagy, abnormal metabolism, inflammation, and oxidative stress [34][35][36]. However, the comprehensive mechanisms of PE remain unclear.
In our research, we found that the expression of SIRT1 was significantly lower in placentas and serum samples of pre-eclampsia patients. SIRT1 knockdown (SIRT1 +/− ) mice showed typical pre-eclampsia-like symptoms, such as hypertension, proteinuria, fetal growth restriction, kidney injury, and a narrowed placental labyrinth layer, implying that the construction of the PE-like mice model was successful, and SIRT1 heterozygote knockout is sufficient to drive the symptoms of pre-eclampsia. Interestingly, SRT2104, P4, and metformin all significantly lower blood pressure in SIRT1 +/− mice, while metformin can also cause fetal growth restriction. In addition, our results suggested that P4 can promote the invasion and inhibit the apoptosis of trophoblasts ( Figure 5).

Discussion
Pre-eclampsia is a severe pregnancy-related complication with the onset of hypertension after week 20 of pregnancy. Pre-eclampsia is mainly characterized by inadequate trophoblasts invasion, which can be caused by autophagy, abnormal metabolism, inflammation, and oxidative stress [34][35][36]. However, the comprehensive mechanisms of PE remain unclear.
In our research, we found that the expression of SIRT1 was significantly lower in placentas and serum samples of pre-eclampsia patients. SIRT1 knockdown (SIRT1 +/-) mice showed typical pre-eclampsia-like symptoms, such as hypertension, proteinuria, fetal growth restriction, kidney injury, and a narrowed placental labyrinth layer, implying that the construction of the PE-like mice model was successful, and SIRT1 heterozygote knockout is sufficient to drive the symptoms of pre-eclampsia. Interestingly, SRT2104, P4, and metformin all significantly lower blood pressure in SIRT1 +/− mice, while metformin can also cause fetal growth restriction. In addition, our results suggested that P4 can promote the invasion and inhibit the apoptosis of trophoblasts ( Figure 5). SIRT1 might be a critical mediator in the pathogenesis of pre-eclampsia by multiple pathways. Previous research reported that alteration of autophagy activity in trophoblasts affected the invasion function of trophoblasts and was implicated in the pathophysiology of PE [37][38][39][40][41]. SIRT1 can deacetylate the LIR motif of LC3 to affect cell autophagy [9]. In addition, studies have shown that SIRT3 can regulate trophoblasts' autophagy by SIRT1 might be a critical mediator in the pathogenesis of pre-eclampsia by multiple pathways. Previous research reported that alteration of autophagy activity in trophoblasts affected the invasion function of trophoblasts and was implicated in the pathophysiology of PE [37][38][39][40][41]. SIRT1 can deacetylate the LIR motif of LC3 to affect cell autophagy [9]. In addition, studies have shown that SIRT3 can regulate trophoblasts' autophagy by activating the AMPK-mTOR pathway and reducing GPX4 levels [42]. SIRT1 can module the oxidative stress and apoptosis of trophoblasts by autophagy [34]. We speculated that SIRT1 might regulate trophoblasts' autophagy activity by deacetylating a certain substrate, thereby leading to the occurrence of pre-eclampsia, but further experimental verification is needed. Furthermore, L-NAME (N-nitro-L-arginine methyl ester)-induced pre-eclampsia animal model is a common pre-eclampsia animal model, mainly by inhibiting nitric oxide (NO) synthase activity, as NO is an important vasodilator [43]. It has been reported that SIRT1 induced endothelial NO increase by directly deacetylating eNOs or by activating FOXO and AMPK and is thereby involved in the pathogenesis of pre-eclampsia [44][45][46][47]. However, since SIRT1 has a wide range of functions in vivo and may induce the occurrence of preeclampsia through multiple pathways, the comprehensive mechanisms remain unclear.
SRT2104 can eliminate pre-eclampsia-like symptoms induced by SIRT1 knockdown. SRT2104 is a selected SIRT1 agonist and can elevate the expression of SIRT1 [24]. Studies showed that SRT2104 has positive effects on cardiovascular disease and diabetic nephropathy [48,49], but no studies indicate that SRT2104 has the ability to treat pre-eclampsia. In our study, the pre-eclampsia-like manifestations of SIRT1 +/− mice were significantly improved after intraperitoneal injection of SRT2104. Therefore, we speculated that SRT2104 might correct the manifestations of pre-eclampsia by elevating SIRT1. However, the effect was not very apparent when we treated SIRT1 +/− mice with SRT2104 via intragastric administration ( Figure S1). We believed that the inability to reduce knockdown-SIRT1-induced hypertension via intragastric administration was related to the drug dosage. SRT2104 used as 100 mg/kg/day for 24 weeks could significantly elevate SIRT1 protein in diabetic mice [24,25]. However, our intragastric administration could only use for 18 days during mice pregnancy. Our intraperitoneal results indicated that SRT2104 could be a new therapy for SIRT1 induced pre-eclampsia after more experiments on the safety in pregnant women.
P4 might improve the symptoms of pre-eclampsia. P4 is an essential hormone in the process of reproduction, and circulating P4 is significantly lower in PE women compared to controls [16]. There are pieces of evidence that early P4 treatment in ART improves the incidence and outcome of pre-eclampsia [50][51][52]. In addition, animal experiments also demonstrated that P4 could attenuate hypertension, improve inflammation, and boost fetal weight in a reduced uterine perfusion pressure rat model [16,17,53]. Our results also revealed that P4 could significantly reduce blood pressure and elevate fetal weight to prevent the aggravation of pre-eclampsia. Additionally, P4 treatment can promote trophoblast invasion and inhibit trophoblast apoptosis. This evidence demonstrated that P4 supplementation during pregnancy might play a role in the treatment and prevention of pre-eclampsia.
P4 might interact with SIRT1 to take effect in pre-eclampsia. It has been reported that SIRT1 deacetylated the hinge region of the P4 receptor to regulate the kinetics of PR nucleo-cytoplasmic transport and subsequent transcriptional activity [54,55]. In addition, knockdown of SIRT2, an isoform of the Sirtuins family, stimulated the mRNA abundance of CYP11 A1, which is a rate-limiting enzyme in P4 biosynthesis [56]. Inhibition of SIRT1 expression with nicotinamide (NAM) reduces P4 production in MA-10 cells [57]. This evidence showed that there is an interactive relationship between SIRT1 and P4. However, the direct mechanisms between SIRT1 and P4 in pre-eclampsia need to be further clarified.
Metformin can also reduce the blood pressure in SIRT1 +/− mice but causes fetal intrauterine growth restriction, which increases fetal morbidity and mortality. We concluded that this might be related to the weight-reducing effect of metformin [58]. However, several clinical research papers reported that metformin does not reduce the incidence of large gestational age newborns; it reduces the risk of neonatal intensive care unit admissions [58][59][60]. Related studies on metformin in the treatment of gestational diabetes and PCOS have shown that metformin can improve pregnancy outcomes [61][62][63][64]. However, these results are still controversial.
In our study, we innovatively confirmed that the SIRT1-knockdown mice showed typical pre-eclampsia-like symptoms, suggesting that SIRT1 is profoundly significant in the mechanisms of pre-eclampsia and might be a new therapy marker for pre-eclampsia. In addition, we found that P4 can significantly diminish the symptoms induced by SIRT1 knockdown. We believed that P4 presented a perfect effect on improving the symptoms of pre-eclampsia. However, there were some shortcomings in our research. We used whole-body SIRT1 knockdown mice rather than trophoblast-specific SIRT1 knockout mice. The SIRT1 knockdown is sufficient to present significant pre-eclampsia-like symptoms, and we are convinced that trophoblast-specific SIRT1 knockout mice might show more significant performances. In addition, the mechanisms of P4 in pre-eclampsia still need to be explored.
In conclusion, our study provides a new pre-eclampsia-like mouse model to explore the mechanisms of pre-eclampsia, mainly about SIRT1. In particular, we demonstrated that P4 could improve the symptoms of PE-like mice. Therefore, further studies should conduct basic experiments to conform the comprehensive mechanisms of SIRT1 in preeclampsia based on the mice model and examine relevant clinical evidence to indicate the effectiveness and potential side effects of P4 in the treatment of pre-eclampsia, which will be an innovative finding for the prevention and treatment of pre-eclampsia.

Conclusions
In the present study, we found that the expression of SIRT1 was relatively lower in the placentas and serum samples of pre-eclampsia patients. Additionally, SIRT1 knockdown (SIRT1 +/− ) mice showed typical pre-eclampsia-like symptoms, such as hypertension, proteinuria, fetal growth restriction, kidney injury, and a narrowed placental labyrinth layer. Interestingly, these performances could be improved after the intraperitoneal injection of SIRT1 agonist SRT2104. More importantly, we found that the efficacy of P4 on attenuating PE-like symptoms was profoundly better than that of metformin in SIRT1 +/− mice. In addition, our results suggested that P4 can promote the invasion and inhibit the apoptosis of trophoblasts. These data suggest that SIRT1 plays an important role in pre-eclampsia, and P4 alleviates pre-eclampsia-like symptoms mediated by SIRT1 deficiency.