SIRT1 Alleviates LPS-Induced IL-1β Production by Suppressing NLRP3 Inflammasome Activation and ROS Production in Trophoblasts

Emerging evidence indicates that aberrant maternal inflammation is associated with several pregnancy-related disorders such as preeclampsia, preterm birth, and intrauterine growth restriction. Sirtuin1 (SIRT1), a class III histone deacetylase, is involved in the regulation of various physiopathological processes including cellular inflammation and metabolism. However, the effect of SIRT1 on the placental proinflammatory environment remains to be elucidated. In this study, we investigated the effect of SIRT1 on lipopolysaccharide (LPS)-induced NLRP3 inflammasome activation and its underlying mechanisms in human first-trimester trophoblasts (Sw.71 and HTR-8/SVneo cells). Treatment with LPS elevated SIRT1 expression and induced NLRP3 inflammasome activation in mouse placental tissues and human trophoblasts. Knockdown of SIRT1 enhanced LPS-induced NLRP3 inflammasome activation, inflammatory signaling, and subsequent interleukin (IL)-1β secretion. Furthermore, knockdown of NLRP3 considerably attenuated the increase of IL-1β secretion in SIRT1-knockdown cells treated with LPS. Moreover, SIRT1 inhibited LPS-induced NLRP3 inflammasome activation by reducing oxidative stress. This study revealed a novel mechanism via which SIRT1 exerts anti-inflammatory effects, suggesting that SIRT1 is a potential therapeutic target for the prevention of inflammation-associated pregnancy-related complications.


Introduction
Normal development of the placenta and proper functioning of trophoblasts are important for a healthy pregnancy. An adequate immune system response at the maternal-fetal interface is crucial for implantation and the preliminary stages of placentation [1]. Extravillous trophoblasts are highly proliferative cells that directly invade the uterine spiral arteries through the decidual stroma, and this process increases maternal blood flow to the placenta during early pregnancy [2,3]. Pathological inflammatory conditions such as intrauterine infection cause abnormal placental development, resulting in several pregnancy-related disorders, including preeclampsia, intrauterine growth restriction d-glucose, 10 mM HEPES, 2 mM l-glutamine, and 1 mM sodium pyruvate. All cultures were maintained in a humidified 5% CO 2 atmosphere at 37 • C. Then, the Sw.71 and HTR-8/SVneo cells were incubated in the presence of 100 or 200 ng/mL LPS, or were treated with 100 or 200 ng/mL LPS for 12-48 h and then followed by 5 mM ATP treatment for 45 min. The same volume of phosphate-buffered saline (PBS) was used as the vehicle control.

Plasmids and Viral Production
The plasmid of pcDNA3-myc-SIRT1 was a gift from Dr. Hueng-Sik Choi (Chonnam National University, Gwangju, Korea). HEK293T cells were transfected with the following lentiviral constructs with packaging plasmids, using polyethylenimine reagent: sh-luciferase (sh-Luc), sh-SIRT1, and sh-NLRP3 constructs (Sigma-Aldrich, St. Louis, MO, USA). Lentiviral supernatants were collected and filtered at 48 and 72 h after transfection. The Sw.71 cells and HTR-8/SVneo cells were incubated for 2 days with lentiviral medium in the presence of 4 µg/mL polybrene.

Luciferase Reporter Assay
SIRT1 promoter constructs were generated by PCR amplification of the human SIRT1 promoter region (−1183 to −30 relative to the transcription start site) and were subcloned into the pGL3-basic vector (Promega, Madison, WI, USA) containing a firefly luciferase gene. The Sw.71 cells were transiently transfected with a pGL3-SIRT1 plasmid and a pRL-TK plasmid containing a Renilla luciferase gene as an internal control using polyethylenimine. At 48 h after transfection, the cells were treated with LPS or vehicle for 4 h. The activities of both luciferases were measured using the Dual-Luciferase Reporter System (Promega, Madison, WI, USA) according to the manufacturer's instructions, and luminescent signals were detected using a GloMax 20/20 luminometer (Promega). For each well, the relative luciferase activity was normalized to the firefly luminescence/Renilla luminescence ratio.

Immunoblotting
The Sw.71 cells and HTR-8/SVneo cells were lysed in an ice-cold radioimmunoprecipitation assay buffer containing complete protease inhibitor cocktail (Roche, Basel, Switzerland). The lysates were incubated for 20 min on ice and centrifuged at 18,000 g for 15 min at 4 • C. The protein concentration was measured using the BCA Protein Assay (Pierce). The lysates were boiled in 1 × sodium dodecyl sulfate (SDS) LaemmLi sample buffer for 5 min, resolved using SDS-polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA), and probed with primary antibodies against SIRT1, NLRP3, p-p65, p65, ASC, Caspase-1, c-Myc, α-tubulin, or β-actin. After sample incubation with secondary antibodies conjugated with horseradish peroxidase, chemiluminescence signals were detected using the Fusion Solo System (Vilber Lourmat, Marne-la-Vallée, France). Densitometric analysis of the blots was performed using ImageJ software (National Institutes of Health, NIH, Bethesda, MD, USA), with which the background was removed for each band.

Immunocytochemistry
Sw.71 cells and HTR-8/SVneo cells grown on coverslips were rinsed with PBS and fixed with 4% paraformaldehyde (pH 7.4) for 15 min at room temperature. The cells were blocked in blocking solution for 1 h at room temperature and incubated with an anti-SIRT1 antibody (1:200) or an anti-ASC antibody (1:200) overnight at 4 • C in a humidified chamber. After washing, the cells were incubated with Alexa Fluor-conjugated secondary antibodies (Invitrogen, 1:500, Carlsbad, CA, USA) and mounted with ProLong Gold antifade reagent with 4 ,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA, USA). Fluorescent images were obtained using a laser scanning confocal microscope (LSM 700, Carl Zeiss) or an epifluorescence-equipped microscope (DM2500, Leica, Wetzlar, Germany) and were processed using ImageJ software (NIH). The percentages of cells containing ASC specks relative to the total number of cells was calculated in five randomly chosen fields.

Immunohistochemistry
Mouse placental tissues were fixed in 10% neutral buffered formalin for 24 h, dehydrated, and embedded in paraffin. Paraffin-embedded sections were deparaffinized, rehydrated, and then treated for antigen retrieval. Endogenous peroxidase was quenched with 3% hydrogen peroxide. After blocking nonspecific antigen, the slides were then incubated with anti-SIRT1 antibody overnight at 4 • C, followed by incubation with biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, USA). Antibodies were visualized with Streptavidin-HRP (BD Pharmingen, San Diego, CA, Cells 2020, 9, 728 5 of 18 USA) using diaminobenzidine (Sigma-Aldrich, St. Louis, MO, USA). Hematoxylin was applied to visualize nuclei.

Enzyme-Linked Immunosorbent Assay (ELISA)
Cell supernatants collected from the Sw.71 cells were assayed for levels of the secreted inflammatory cytokine IL-1β using the DuoSet ELISA development kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. The optical density of the final colored reaction product was determined at 450 nm using an Epoch 2 microplate reader (Bio-Tek Instruments, Winooski, VT, USA).

Statistical Analysis
Results are presented as the mean ± standard error of mean (SEM). Unless mentioned otherwise, the data presented in the figure panels are representative of at least three independent experiments. The significance of differences between two experimental groups was determined using a two-tailed Student's t-test. A p value less than 0.05 was considered statistically significant (* p < 0.05; ** p < 0.01; *** p < 0.001).

SIRT1 Is Upregulated in the Placentas and Trophoblast Cells after LPS Exposure
To determine changes in SIRT1 expression in the mouse placenta by LPS-induced maternal inflammatory response, placentas were collected from mice exposed to LPS or saline. Placental SIRT1 mRNA expression was increased 8 h after LPS treatment ( Figure 1A). Immunohistochemistry analysis confirmed that SIRT1 expression was increased in the nuclei of trophoblast in both the junctional and the labyrinth zones of the placentas of LPS-treated mice compared with that of the control placentas of saline-treated mice ( Figure 1B). To investigate changes in the SIRT1 mRNA expression in trophoblasts during maternal inflammation, human first trimester extravillous trophoblasts (Sw.71 and HTR-8/SVneo) were treated with various LPS concentrations at various time points. Immunoblot analysis showed that LPS increased SIRT1 expression in the Sw.71 ( Figure 1C) and HTR-8/SVneo cells ( Figure S1A) in a dose-dependent manner. The time courses of SIRT1 expression were further studied by treating the cells with LPS (200 ng/mL). The results showed that LPS increased the expression of SIRT1 in the Sw.71 ( Figure 1D) and HTR-8/SVneo cells ( Figure S1B) in a time-dependent manner. Immunofluorescence staining was performed to determine the subcellular localization of SIRT1 in the Sw.71 and HTR-8/SVneo cells. The increased SIRT1 was localized exclusively to the nucleus in the Sw.71 cells after treatment with LPS, which differed from findings in control cells ( Figure 1E). To understand the regulation of SIRT1 transcription in the LPS-treated Sw.71 cells, relative luciferase activity was assessed. The results showed that luciferase activity was significantly higher in the LPS-treated cells than in the untreated cells ( Figure 1F).

Inflammation and NLRP3 Inflammasome Activation in the Placentas and Trophoblast Cells after LPS Exposure
To investigate the effects of LPS on the mRNA expression levels of NLRP3 inflammasome-related molecules, placentas from mice exposed to LPS were analyzed by quantitative real-time PCR. The results showed that placental NLRP3 mRNA expression was significantly increased 8 h after LPS treatment (Figure 2A). Proinflammatory gene expression in placentas from mice exposed to LPS was examined. The IL-6, TNF-α, MCP-1, and IL-1β mRNA levels were significantly elevated in the placentas of LPS-treated mice compared with control placentas from saline-treated mice ( Figure 2B). Next, we performed an immunoblot analysis to investigate the effects of LPS on NLRP3 inflammasome expression in Sw.71 and HTR-8/SVneo cells. LPS treatment significantly increased NLRP3 inflammasome expression in Sw.71 ( Figure 2C) and HTR-8/SVneo cells ( Figure S2A) in a dose-dependent manner. When the Sw.71 cells were treated with LPS (200 ng/mL) for various time periods, NLRP3 expression was substantially induced increased in a time-dependent manner ( Figure 2D). Similar to its action on the Sw.71 cells, LPS treatment stimulated NLRP3 protein levels in HTR-8/SVneo cells ( Figure S2B). To investigate the NLRP3 inflammasome activation in Sw.71 and HTR-8/SVneo cells, the expressions of caspase-1 and ASC were evaluated upon stimulation with LPS and ATP by immunoblotting and immunofluorescence. This treatment increased caspase-1 activation but not the expression of the ASC ( Figure 2E and Figure S2C). Immunostaining for endogenous ASC showed that Sw.71 cells contained the ASC pyroptosome after stimulation with LPS and ATP ( Figure 2F). We further determined whether NF-κB, a master regulator of inflammation, was activated in trophoblasts after LPS exposure. Immunoblot analysis demonstrated increased levels of phosphorylated NF-κB in Sw.71 cells ( Figure 2G) and HTR-8/SVneo cells ( Figure S2D) at 1 h after LPS treatment.

SIRT1 Attenuates LPS-Induced Inflammation and NLRP3 Inflammasome Activation in Trophoblast Cells
To evaluate the regulatory effect of SIRT1 on LPS-induced NLRP3 inflammasome activation, lentiviral vectors expressing small interfering RNAs (shRNAs) for human SIRT1 were transduced to Sw.71 and HTR-8/SVneo cells. After the cells were transduced with sh-SIRT1-expression lentivirus, the expression of SIRT1 was remarkably reduced in the Sw.71 ( Figure 3A) and HTR-8/SVneo cells ( Figure S3). Knockdown of SIRT1 obviously enhanced the NLRP3 mRNA and protein levels after LPS treatment ( Figure 3A,B and Figure S3A). To further confirm these results, the IL-1β levels in the culture supernatants were analyzed using ELISA. The results showed that secreted IL-1β levels were significantly increased in LPS-and ATP-treated SIRT1 knockdown cells compared with those in LPSand ATP-treated control knockdown cells ( Figure 3C). Correspondingly, knockdown of SIRT1 also increased pro-IL-1β expression in LPS-treated Sw.71 cells ( Figure 3D). Consistent with its effect on IL-1β production, knockdown of SIRT1 increased caspase-1 activation ( Figure 3E) and ASC pyroptosome formation ( Figure 3F). These data demonstrate that deficiency of SIRT1 augments NLRP3 inflammasome activation and subsequently the maturation of IL-1β. Next, we investigated the effect of overexpression of SIRT1 on LPS-induced NLRP3 inflammasome activation in Sw.71 cells. The results showed that overexpression of SIRT1 reduced the expression of NLRP3 protein and caspase-1 activation ( Figure S3B). These results suggest that SIRT1 attenuates NLRP3 inflammasome activation.   NF-κB mediates the priming signal of NLRP3 inflammasome activation and upregulates the transcriptional expression of NLRP3, as well as of pro-IL-1β [30,31]. To investigate the effects of knockdown of SIRT1 on the NF-κB pathway in Sw.71 and HTR-8/SVneo cells, we measured the phosphorylation levels of NF-κB. Immunoblot analysis of p65 NF-κB showed that the LPS-induced NF-κB mediates the priming signal of NLRP3 inflammasome activation and upregulates the transcriptional expression of NLRP3, as well as of pro-IL-1β [30,31]. To investigate the effects of knockdown of SIRT1 on the NF-κB pathway in Sw.71 and HTR-8/SVneo cells, we measured the phosphorylation levels of NF-κB. Immunoblot analysis of p65 NF-κB showed that the LPS-induced phosphorylation level of p65 NF-κB was higher in the SIRT1 knockdown groups than in the control knockdown groups in the Sw.71 ( Figure 4A) and the HTR-8/SVneo cells ( Figure 4B). Moreover, we examined the expression of proinflammatory cytokine genes, including IL-6, IL-8, and TNF-α in SIRT1 knockdown Sw.71 cells using quantitative real-time PCR. The results showed that the IL-6, IL-8, and TNF-α levels were significantly augmented by the knockdown of SIRT1 in Sw.71 cells ( Figure 4C). Cells 2020, 9, x 11 of 20 phosphorylation level of p65 NF-κB was higher in the SIRT1 knockdown groups than in the control knockdown groups in the Sw.71 ( Figure 4A) and the HTR-8/SVneo cells ( Figure 4B). Moreover, we examined the expression of proinflammatory cytokine genes, including IL-6, IL-8, and TNF-α in SIRT1 knockdown Sw.71 cells using quantitative real-time PCR. The results showed that the IL-6, IL-8, and TNF-α levels were significantly augmented by the knockdown of SIRT1 in Sw.71 cells ( Figure 4C).

SIRT1 Suppresses LPS-Induced IL-1β Secretion in Trophoblast Cells in a NLRP3-Dependent Manner
To examine whether SIRT1 inhibition of IL-1β secretion was dependent on NLRP3 inflammasomes in trophoblasts treated with LPS and ATP, lentiviral vectors expressing shRNAs for human NLRP3 were transduced to Sw.71 cells. After the cells were transduced with sh-NLRP3expression lentivirus, the NLRP3 expression was remarkably decreased in the Sw.71 cells ( Figure  5A). Next, the IL-1β level in the culture supernatants was determined using ELISA. The lentivirusmediated knockdown of NLRP3 significantly abrogated the increase of IL-1β secretion in the SIRT1 knockdown cells treated with LPS and ATP ( Figure 5B). It is known that NLRP3 inflammasome regulates the NF-κB pathway as well as induces multiple cytokine genes [32,33]. We further investigated the effect of NLRP3 inflammasome on the activation of NF-κB in SIRT1 knockdown trophoblasts. Notably, LPS treatment in the SIRT1 knockdown cells elevated the phosphorylation of p65 NF-κB, which was abolished by lentivirus-mediated knockdown of NLRP3 ( Figure 5C). We further examined the effects of the NLRP3 inflammasome on the expression of proinflammatory cytokine genes. LPS treatment in the SIRT1 knockdown cells induced IL-6, IL-8, and TNF-α mRNA

SIRT1 Suppresses LPS-Induced IL-1β Secretion in Trophoblast Cells in a NLRP3-Dependent Manner
To examine whether SIRT1 inhibition of IL-1β secretion was dependent on NLRP3 inflammasomes in trophoblasts treated with LPS and ATP, lentiviral vectors expressing shRNAs for human NLRP3 were transduced to Sw.71 cells. After the cells were transduced with sh-NLRP3-expression lentivirus, the NLRP3 expression was remarkably decreased in the Sw.71 cells ( Figure 5A). Next, the IL-1β level in the culture supernatants was determined using ELISA. The lentivirus-mediated knockdown of NLRP3 significantly abrogated the increase of IL-1β secretion in the SIRT1 knockdown cells treated with LPS and ATP ( Figure 5B). It is known that NLRP3 inflammasome regulates the NF-κB pathway as well as induces multiple cytokine genes [32,33]. We further investigated the effect of NLRP3 inflammasome on the activation of NF-κB in SIRT1 knockdown trophoblasts. Notably, LPS treatment in the SIRT1 knockdown cells elevated the phosphorylation of p65 NF-κB, which was abolished by lentivirus-mediated knockdown of NLRP3 ( Figure 5C). We further examined the effects of the NLRP3 inflammasome on the expression of proinflammatory cytokine genes. LPS treatment in the SIRT1 knockdown cells induced IL-6, IL-8, and TNF-α mRNA expression, which was markedly abrogated by lentivirus-mediated knockdown of NLRP3 ( Figure 5D). These findings suggest that the regulatory effect of SIRT1 on inflammatory response is associated with suppression of the NLRP3 inflammasome activation in trophoblasts.
Cells 2020, 9, x 12 of 20 expression, which was markedly abrogated by lentivirus-mediated knockdown of NLRP3 ( Figure  5D). These findings suggest that the regulatory effect of SIRT1 on inflammatory response is associated with suppression of the NLRP3 inflammasome activation in trophoblasts.

SIRT1 Inhibits LPS-Induced NLRP3 Inflammasome in Trophoblast Cells Via Reducing Oxidative Stress
Various types of cellular stress stimuli trigger the generation of ROS, which may induce activation of the NLRP3 inflammasome through oxidative stress [34][35][36]. We examined whether LPS treatment induced oxidative stress in the Sw.71 cells. The CM-H2DCFDA fluorescence levels showed that LPS treatment stimulated ROS production ( Figure 6A). Next, we tested whether the oxidative stress induced by LPS treatment was augmented by lentivirus-mediated knockdown of SIRT1. The results showed that ROS production induced by LPS treatment was significantly elevated by lentivirus-mediated knockdown of SIRT1 ( Figure 6B). We further investigated the effect of overexpression of SIRT1 on LPS-induced oxidative stress. Relative to control cells, Sw.71 cells overexpressing SIRT1 had a lower level of CM-H2DCFDA fluorescence ( Figure S4). Next, we sought to determine whether the antioxidant NAC could diminish the augmented NLRP3 inflammasome activation in LPS-treated SIRT1 knockdown cells. Immunoblot analysis showed that NAC treatment stopped the LPS-induced increase in NLRP3 levels in the SIRT1 knockdown cells ( Figure 6C).

SIRT1 Inhibits LPS-Induced NLRP3 Inflammasome in Trophoblast Cells Via Reducing Oxidative Stress
Various types of cellular stress stimuli trigger the generation of ROS, which may induce activation of the NLRP3 inflammasome through oxidative stress [34][35][36]. We examined whether LPS treatment induced oxidative stress in the Sw.71 cells. The CM-H 2 DCFDA fluorescence levels showed that LPS treatment stimulated ROS production ( Figure 6A). Next, we tested whether the oxidative stress induced by LPS treatment was augmented by lentivirus-mediated knockdown of SIRT1. The results showed that ROS production induced by LPS treatment was significantly elevated by lentivirus-mediated knockdown of SIRT1 ( Figure 6B). We further investigated the effect of overexpression of SIRT1 on LPS-induced oxidative stress. Relative to control cells, Sw.71 cells overexpressing SIRT1 had a lower level of CM-H 2 DCFDA fluorescence ( Figure S4). Next, we sought to determine whether the antioxidant NAC could diminish the augmented NLRP3 inflammasome activation in LPS-treated SIRT1 knockdown cells. Immunoblot analysis showed that NAC treatment stopped the LPS-induced increase in NLRP3 levels in the SIRT1 knockdown cells ( Figure 6C). Furthermore, the release of IL-1β was measured using ELISA. NAC treatment also decreased the IL-1β secretion in SIRT1 knockdown cells treated with LPS and ATP ( Figure 6D). Subsequently, the effect of NAC on the activation of NF-κB in the SIRT1 knockdown trophoblasts was investigated. As shown in Figure 6E, NAC treatment decreased the phosphorylation of p65 NF-κB. These data suggest that SIRT1 attenuated the NLRP3 inflammasome activation that was induced by oxidative stress. Cells 2020, 9, x 13 of 20 Furthermore, the release of IL-1β was measured using ELISA. NAC treatment also decreased the IL-1β secretion in SIRT1 knockdown cells treated with LPS and ATP ( Figure 6D). Subsequently, the effect of NAC on the activation of NF-κB in the SIRT1 knockdown trophoblasts was investigated. As shown in Figure 6E, NAC treatment decreased the phosphorylation of p65 NF-κB. These data suggest that SIRT1 attenuated the NLRP3 inflammasome activation that was induced by oxidative stress.

Discussion
Placental inflammation due to infectious or noninfectious causes has been linked with various adverse pregnancy outcomes, including preterm delivery, stillbirth, and IUGR. These inflammatory processes result in placental dysfunction and the release of several inflammatory mediators [37]. LPS, the major component of the outer membrane of Gram-negative bacteria, increases the production of various inflammatory cytokines and chemokines and induces innate immunity at the maternal-fetal interface [38,39]. Maternal and fetal adverse outcomes are associated with the release of various proinflammatory mediators such as cytokines and chemokines from the placenta. Elevated IL-1β production might be related to infection-or inflammation-induced spontaneous term parturition and preterm birth [40]. Recent studies have demonstrated a relationship between the maturation of IL-1β and the NLRP3 inflammasome pathway in human trophoblasts [41,42], but the molecular mechanisms underlying LPS-induced NLRP3 inflammasome activation are not completely understood. In the present study, we demonstrated that LPS treatment induces placental SIRT1 expression. The present study demonstrated that LPS treatment induces placental SIRT1 expression. Furthermore, LPS-and ATP-mediated NLRP3 inflammasome activation results in increases in IL-1β production in trophoblasts. Importantly, SIRT1 suppresses NLRP3 inflammasome activation and inflammation via reducing the oxidative stress in trophoblasts (Figure 7). Although SIRT1 is expressed in all trophoblast layers of the placenta and is important for proper trophoblast differentiation and placental development [43], the relationship between LPSassociated placental inflammation and SIRT1 expression has not been studied previously. Recent studies showed that single nucleotide polymorphisms (SNPs) in the SIRT1 gene are associated with carotid atherosclerosis, major depressive disorder, age-related macular degeneration, and severe obesity [44][45][46][47]. However, it is not known whether SNPs in the SIRT1 gene have an influence on the NLRP3 inflammasome-mediated inflammatory diseases. Therefore, further studies are needed to Although SIRT1 is expressed in all trophoblast layers of the placenta and is important for proper trophoblast differentiation and placental development [43], the relationship between LPS-associated placental inflammation and SIRT1 expression has not been studied previously. Recent studies showed that single nucleotide polymorphisms (SNPs) in the SIRT1 gene are associated with carotid atherosclerosis, major depressive disorder, age-related macular degeneration, and severe obesity [44][45][46][47]. However, it is not known whether SNPs in the SIRT1 gene have an influence on the NLRP3 inflammasome-mediated inflammatory diseases. Therefore, further studies are needed to investigate the possibility of genetic association between variants in the SIRT1 loci and inflammatory conditions. Previous studies demonstrated that SIRT1 regulates the inflammatory response in other cell types, such as adipocytes and endothelial cells [29,48,49]. Our study showed that LPS-induced SIRT1 expression is localized predominantly in the nucleus of trophoblasts. We also provided evidence on inhibition of LPS-mediated inflammation with SIRT1 by using a knockdown approach. The immunoblot analysis for p65 NF-κB identified an inhibitory effect of SIRT1 on the activation of NF-κB. We also found that SIRT1 knockdown resulted in a marked increase in the NLRP3 protein level in the Sw.71 and HTR-8/SVneo cells stimulated with LPS. These results suggest that SIRT1 functions in the placenta may not only be involved in the inflammatory response but also affect the NLRP3 inflammasome activation. Consistent with this notion, anti-inflammatory effects of SIRT1 are associated with regulation of the NLRP3 inflammasome in vascular endothelial cells and the epidermis [48,50]. Increasing evidence demonstrates that SIRT1 activators such as resveratrol and SRT1720 can inhibit NF-κB activation as well as inflammatory pathways [51][52][53]. Furthermore, SIRT1 activators inhibited the increase in NLRP3 and IL-1β production in mesenchymal stem cells and vascular endothelial cells [53,54]. Our present study showed that overexpression of SIRT1 inhibited the expression of NLRP3 protein and caspase-1 activation. Therefore, these results indicate that SIRT1 activators might represent suitable therapeutic options for the treatment of inflammation-associated pregnancy complications.
Although SIRT1 has been shown to regulate the NLRP3 inflammasome, its molecular mechanism remains to be identified. NLRP3 inflammasome activation could be induced by ROS [55]. Hypoxia can cause placental tissue damage, including preeclampsia and IUGR, via the production of oxidative stress [56]. In the present study, intracellular ROS amounts were found to be elevated in Sw.71 cells after LPS treatment. In addition, ROS production induced by LPS treatment was aggravated by knockdown of SIRT1, indicating that SIRT1 ameliorates oxidative stress. We have observed that NAC treatment inhibited an increase in NLRP3 levels, as well as the phosphorylation of p65 NF-κB in SIRT1 knockdown cells treated with LPS. These results suggest that SIRT1 inhibits the NLRP3 inflammasome activation through inhibiting oxidative stress. Previous studies have shown bidirectional crosstalk between SIRT1 and nuclear factor erythroid 2-related factor 2 (Nrf2) in human renal proximal tubular and glomerular mesangial cells [57,58]. The upregulation of SIRT1 activates Nrf2 through the inhibition of p53 or activation of AMPK [59][60][61]. In addition, it is known that in ischemic injury Nrf2 suppresses NLRP3 inflammasome activation through regulating the thioredoxin-interacting protein (TXNIP) complex [62]. Thus, Nrf2 may be linked with negative regulation of the NLRP3 inflammasome by SIRT1; however, further experimental studies are needed to explore this concept.

Conclusions
In summary, the results of this study provide evidence for a potential role of SIRT1 in LPS-induced NLRP3 inflammasome activation in trophoblasts by showing that the knockdown of SIRT increased LPS-induced inflammatory signaling, IL-1β secretion, and NLRP3 expression. SIRT1 also ameliorates oxidative stress caused by LPS treatment. Our study elucidates a novel mechanism by which SIRT1 exerts anti-inflammatory effects and defines SIRT1 as a therapeutic target in the prevention of inflammation-associated pregnancy complications.