Endogenous hydrogen sulfide contributes to uterine quiescence during pregnancy

in Reproduction

Recent evidence suggests that uterine activation for labor is associated with inflammation within uterine tissues. Hydrogen sulfide (H2S) plays a critical role in inflammatory responses in various tissues. Our previous study has shown that human myometrium produces H2S via its generating enzymes cystathionine-γ-lyase (CSE) and cystathionine-β-synthetase (CBS) during pregnancy. We therefore explored whether H2S plays a role in the maintenance of uterine quiescence during pregnancy. Human myometrial biopsies were obtained from pregnant women at term. Uterine smooth muscle cells (UMSCs) isolated from myometrial tissues were treated with various reagents including H2S. The protein expression of CSE, CBS and contraction-associated proteins (CAPs) including connexin 43, oxytocin receptor and prostaglandin F receptor determined by Western blot. The levels of cytokines were measured by ELISA. The results showed that CSE and CBS expression inversely correlated to the levels of CAPs and activated NF-κB in pregnant myometrial tissues. H2S inhibited the expression of CAPs, NF-κB activation and the production of interleukin (IL)-1β, IL-6 and tumor necrosis factor α (TNFα) in cultured USMCs. IL-1β treatment reversed H2S inhibition of CAPs. Knockdown of CSE and CBS prevented H2S suppression of inflammation. H2S modulation of inflammation is through KATP channels and phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) signaling pathways. H2S activation of PI3K and ERK signaling is dependent on KATP channels. Our data suggest that H2S suppresses the expression of CAPs via inhibition of inflammation in myometrium. Endogenous H2S is one of the key factors in maintenance of uterine quiescence during pregnancy.

Abstract

Recent evidence suggests that uterine activation for labor is associated with inflammation within uterine tissues. Hydrogen sulfide (H2S) plays a critical role in inflammatory responses in various tissues. Our previous study has shown that human myometrium produces H2S via its generating enzymes cystathionine-γ-lyase (CSE) and cystathionine-β-synthetase (CBS) during pregnancy. We therefore explored whether H2S plays a role in the maintenance of uterine quiescence during pregnancy. Human myometrial biopsies were obtained from pregnant women at term. Uterine smooth muscle cells (UMSCs) isolated from myometrial tissues were treated with various reagents including H2S. The protein expression of CSE, CBS and contraction-associated proteins (CAPs) including connexin 43, oxytocin receptor and prostaglandin F receptor determined by Western blot. The levels of cytokines were measured by ELISA. The results showed that CSE and CBS expression inversely correlated to the levels of CAPs and activated NF-κB in pregnant myometrial tissues. H2S inhibited the expression of CAPs, NF-κB activation and the production of interleukin (IL)-1β, IL-6 and tumor necrosis factor α (TNFα) in cultured USMCs. IL-1β treatment reversed H2S inhibition of CAPs. Knockdown of CSE and CBS prevented H2S suppression of inflammation. H2S modulation of inflammation is through KATP channels and phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) signaling pathways. H2S activation of PI3K and ERK signaling is dependent on KATP channels. Our data suggest that H2S suppresses the expression of CAPs via inhibition of inflammation in myometrium. Endogenous H2S is one of the key factors in maintenance of uterine quiescence during pregnancy.

Introduction

Preterm birth is the prevalent cause of mortality and morbidity in newborn infants. Moreover, the survivors of preterm birth have increased risk of neurodevelopmental impairments, gastrointestinal and respiratory complications (Liu et al. 2012). So far, it lacks effective methods of prediction or prevention for preterm birth. This failure is largely due to our incomplete understanding of the process that maintains uterus in a relatively noncontractile state throughout most of pregnancy and converts it into a procontractile state toward the end of pregnancy.

Human uterine conversion into the procontractile state results from the coordinated expression of a cassette of contraction-associated proteins (CAPs) such as gap junction, ion channels and the receptors of agonists (such as oxytocin and prostaglandins) (Challis et al. 2000), thereby making the uterus to respond to hormonal signals and mechanical forces. In the past decade, increasing body of evidence suggests that uterine activation for labor in women is associated with inflammatory responses within uterine tissues (Romero et al. 2006, Rinaldi et al. 2011, Shynlova et al. 2013). Toward the end of pregnancy, uterine tissues display inflammatory response, which is characterized by an influx of inflammatory cells into the myometrium and cervix with concomitant increases in vascular and leukocyte adhesion molecule expression, activation of NF-κB and increased production of chemokines and proinflammatory cytokines in uterus (Thomson et al. 1999, Osman et al. 2003, Gomez-Lopez et al. 2011). However, the mechanisms underlying modulation of inflammatory response in uterus remain unclear.

Hydrogen sulfide (H2S), the third endogenous gaseous transmitter, has been implicated to be involved in many physiological and pathophysiological processes including angiogenesis (Papapetropoulos et al. 2009), vasodilation (Skovgaard et al. 2011) and inflammation (Wallace et al. 2012). H2S is generated from l-cysteine principally through two pyridoxal-5-phosphate-dependent enzymes: cystathionine-γ-lyase (CSE, EC4.4.1.1) and cystathionine-β-synthetase (CBS, EC 4.2.1.22) (Li et al. 2011). Both CBS and CSE have been identified in gestational tissues including placenta and myometrium (Holwerda et al. 2012, Hu et al. 2016). Our previous study has shown that endogenous H2S produced by CSE and CBS suppresses the spontaneous contractility of pregnant human myoemtrial strips (You et al. 2011). Furthermore, we have also demonstrated that CSE and CBS expression levels and production of H2S in human myometrium are decreased with the onset of labor (You et al. 2011), suggesting that endogenous H2S is involved in the initiation and progress of labor in women.

Based on the above background, we hypothesized that endogenous H2S produced locally may play a role in the maintenance of uterine quiescence via modulation of uterine inflammation during human pregnancy. To test it, we firstly analyzed the correlation between the expression of CAPs and level of CSE and CSE in myometrium obtained from pregnant women at term. We then studied the effect of H2S on the expression of CAPs, investigated whether H2S regulates CAPs via the modulation of inflammatory responses and elucidated molecular mechanisms involved in a model of primary human uterine smooth muscle cells (USMCs). Our data indicate that endogenous H2S contributes to the maintenance of uterine quiescence via inhibition of inflammation during pregnancy.

Materials and methods

Tissue collection

This study was approved by the specialty committee on ethics of biomedicine research, Second Military Medical University, Shanghai, China. Written informed consent was obtained from all patients. Biopsies of human myometrium were obtained at cesarean section from the following group of pregnant women: term no labor (TNL, n = 23) and term labor (TL, n = 21). The average gestational age of these two groups was 38 weeks, with a range of 37–42 weeks. Labor was defined as regular contractions (<5 min apart) plus membrane rupture and cervical dilation (>3 cm) with no augmentation (oxytocin or prostaglandin administration). None of the women included in this study had evidence of underlying disease (e.g. hypertension, diabetes, preeclampsia, intrauterine growth restriction, etc.). Biopsies were excised from the middle portion of upper edge of the incision line in the lower uterine segment. All myometrial samples were dissected free of serosa and immediately placed in Kreb’s solution and maintained at 4°C and transported to the laboratory for cell culture or frozen immediately in liquid nitrogen and stored at −80°C for the subsequent Western blot analysis.

Cultures of USMCs

USMCs were isolated from TNL myometrial tissues as described previously (You et al. 2012). Briefly, myometrial tissue pieces were incubated with DMEM containing 1 mg/mL collagenase type II (Invitrogen) and 1 mg/mL deoxyribonuclease I (Sigma-Aldrich) at 37°C with shaking for 30 min for two times. After filtration, the cell suspension was centrifuged and the cell pellet was resuspended in DMEM containing 10% fetal calf serum (FCS), penicillin (100 U/mL) and streptomycin (100 mg/mL). The cells were then plated into 25 cm2 flasks and kept at 37°C in 5% CO2–95% air-humidified atmosphere until confluent (~2 week). The experiments were performed with the cells at passage 2. The cells were seeded in 12-well plates and then treated with the following reagents for 24 h: NaHS (5–100 × 10−6 M), l-cysteine (1.25–10 × 10−4 M), glibenclamide (10−5 M), PD98059 (10−5 M), LY294002 (10−5 M) and interleukin (IL)-1β (1 ng/mL). The vehicle control was set without additive. In some cases, the cells were treated with NaHS (10−4 M), l-cysteine (10−3 M) or vehicle for 5, 10, 30 and 60 min. All of the previously mentioned reagents were purchased from Sigma-Aldrich. Each treatment was performed in triplicate for each preparation of cells. The concentrations of previously mentioned reagents were determined based on our previous studies (You et al. 2011, Hu et al. 2016) and preliminary data. To assess the purity of myocyte cultures, we routinely performed immunocytochemistry using α-actin monoclonal antibody (Sigma-Aldrich).

Enzyme-linked immunosorbent assay (ELSIA)

The concentrations of IL-6, IL-1β and tumor necrosis factor (TNF)α in culture media of USMCs were determined with specific ELSIA (R&D Systems) according to the manufacturer’s instructions.

Western blot analysis

Myometrial tissues were homogenized in cold T-Per lysis buffer (Pierce), and primary HUSMCs were harvested in the presence of M-Per lysis buffer (Pierce). The amounts of up to 50 µg of protein samples were separated by 10% SDS-PAGE and subsequently transferred to nitrocellulose membranes. After incubation with blocking buffer, the membranes were incubated with specific antibodies: CBS (ab131155, Abcam), CSE (ab133212, Abcam), connexin 43 (CX43) (ab11370, Abcam), oxytocin receptor (OTR) (sc-33209, Santa Cruz), prostaglandin F receptor (PGFR) (sc-67029, Santa Cruz), p65 (ab131485, Abcam), phospho-p65 (ser-529) (ab97726, Abcam), extracellular signal-regulated kinase (ERK)1/2 (4695, Cell signaling), phospho-ERK1/2 (4370, Cell signaling), phosphoinositide 3-kinase (PI3K) (sc-377482, Santa Cruz) and phospho-PI3K (Tyr508) (sc-12929, Santa Cruz) overnight at 4°C at a dilution of 1:1000. Then, the membrane was incubated with a secondary horseradish peroxidase-conjugated antibody for 1 h at room temperature. Immunoreactive proteins were visualized using the enhanced chemiluminescence Western blotting detection system (Santa Cruz). The chemiluminescent signal from the membranes was quantified by a GeneGnome HR scanner using GeneTools software (SynGene). To control sampling errors, the ratio of band intensities to the β-actin was obtained to quantify the relative protein expression level.

Total RNA extraction and quantitative real-time RT-PCR

Total RNA was prepared from myometrial tissues and cells using TRIzol reagent (Invitrogen). Two micrograms RNA were reverse transcribed with oligo(dT)18 primer using the M-MLV reverse transcriptase (Promega). Specific primers for the amplification of IL-1β, IL-6 and TNFα were listed in Table 1. Quantitative real-time PCR was carried out using Rotor-Gene 3000 (Corbett Research, Sydney, Australia). The reaction solution consisted of 2.0 µL diluted cDNA product, 0.2 µmol/L of each paired primer, 200 µmol/L deoxynucleotide triphosphates, 1 U Taq DNA polymerase (Qiagen) and 1× PCR buffer. SYBRGreen (Roche) was used as a detection dye. The annealing temperature was set at 60°C and amplification cycles were set at 40 cycles. The temperature range to detect the melting temperature of the PCR product was set from 60°C to 95°C. To determine the relative quantitation of gene expression for both target and housekeeping genes, the comparative Ct (threshold cycle) method with arithmetic formulae was used (Li et al. 2016). Two reference genes β-actin and GAPDH were measured for each sample as an internal control for sample loading and normalization. Messenger RNA levels were normalized relative to β-actin and GAPDH values respectively. Because very similar results were obtained using these two reference genes, the results were illustrated using β-actin as internal control.

Table 1

List of primers used for the amplification of chemokines and cytokines in USMCs.

NameSense (S) and antisense (A) primer sequence (5′–3′)Genbank/EM BL accession no.Production size (bp)
IL-1βA: 5′-GTGGCAATGAGGATGACTT-3′NM_000576537
S: 5′-TGGGCTTATCATCTTTCAA-3′
IL-6A: 5′-CCTTCCAAAGATGGCTGAAA-3′NM_000600165
S: 5′-AGCTCTGGCTTGTTCCTCAC-3′
TNF-αA: 5′-GCCCCCAGAGGGAAGAGTTCCCCA-3′NM_000594124
S: 5′-GCTTGAGGGTTTGCTACAACATGGGC-3′

RNA interferences

The small interfering RNA (siRNA) for CBS and CSE were desi­gned and synthesized by GenePharma Corporation (Shanghai, China). Control siRNA was scrambled sequence without any specific known target. The sequences for targeting human CSE and CBS are CSE sense: 5′-GGCCUUUGCUUCAGGUUUATT-3′, antisense: 5′-UAAACCUGAAGCAAAGGCCTT-3′; CBS sense: 5′-CGGAACUACAUGACCAAGUTT-3′, antisense: 5′-ACUUGGUCAUGUAGUUCCGTT-3′. Transfection of siRNA was performed by using Lipofectamine TM 2000 as described previously (You et al. 2011, Hu et al. 2016).

Statistical analysis

Data are presented as mean ± s.e.m. In some cases, for illustrative purposes, the results are presented as the mean percent control ± s.e.m. All data were tested for homogeneity of variance by Bartlett’s test before statistical analysis. The data of CAP expression and NFκB activation in TNL and TL tissues were analyzed by t test. Pearson’s correlation was used to explore the relationships of CBS or CSE with CX43, OTR, PGFR and activated NF-κB level in myometrium. The data of cultured USMCs were analyzed by a one-way ANOVA followed by a Student–Newman–Keuls test. A P value <0.05 was considered significant.

Results

The expression levels of CSE and CBS correlate to levels of CAPs and NF-κB in human pregnant myometrium

As expected, the protein levels of CSE and CBS were downregulated in TL myometrial tissues compared with TNL tissues (Fig. 1A and B). Prior studies have shown that the expression of CAPs, such as CX43, OTR and PGFR, and the level of phospho-p65 (p-p65), active form of NF-κB are increased with labor (Condon et al. 2006, Kamel 2010). We therefore examined the protein levels of these proteins and found that the levels of CX43, OTR, PGFR and phospho-p65 were significantly increased in TL myometrial tissues compared with TNL tissues (Fig. 1C, D, E and F). The correlation analysis showed that the level of CSE and CBS inversely correlated to CX43, OTR, PGFR and active NF-κB level (Supplementary Fig. 1, see section on supplementary data given at the end of this article).

Figure 1
Figure 1

The protein levels of CBS, CSE, p-p65, CX43, OTR and PGFR, in pregnant myometrium before and after onset of labor. Myometrial tissues were obtained from pregnant women at term before (TNL) or after the onset of labor (TL). Semi-quantitation of Western blot signals of CBS (A), CSE (B), p-p65 (C), CX43 (D), OTR (E) and PGFR (F) in TNL and TL myometrium biopsies. Representative protein bands were on the top of each histogram. Data were presented as mean ± s.e.m. **P < 0.01 vs TNL.

Citation: Reproduction 153, 5; 10.1530/REP-16-0549

NaHS and l-cysteine inhibit the expression of CX43, OTR and PGFR in USMCs

We then investigated whether H2S modulates CX43, OTR and PGFR expression in primary USMCs. As shown in Fig. 2A, B and C, treatment of USMCs with increasing concentration of NaHS (5 × 10−6 – 10−4 M), the H2S donor, caused a decrease in the expression of CX43, OTR and PGFR in a dose-dependent manner in a 24-h incubation time. l-cysteine (1.25–10 × 10−4 M), the precursor of H2S, treatment also dose-dependently suppressed CX43, OTR and PGFR expression (Fig. 2D, E and F).

Figure 2
Figure 2

The effects of hydrogen sulfide on the expression of CX43, OTR and PGFR in USMCs. USMCs were treated with increasing concentration of NaHS (5 × 10−6 – 10−4 M) or l-cysteine (1.25–10 × 10−4 M) for 24 h. The cells were then collected and protein levels of CX43 (A and D), OTR (B and E) and PGFR (C and F) were determined by Western blot. Values are presented as mean ± s.e.m. for n = 6 subjects performed in triplicate. *P < 0.05, **P < 0.01 vs vehicle control. l-cys, l-cysteine.

Citation: Reproduction 153, 5; 10.1530/REP-16-0549

NaHS and l-cysteine suppresses NFκB activation and production of proinflammatory cytokines in USMCs

Prior studies have shown that H2S suppresses NF-κB activation in macrophages (Du et al. 2014). We therefore examined whether H2S treatment affects NF-κB activation in USMCs. As shown in Fig. 3A and B, treatment of cells with NaHS (10−4 M) and l-cysteine (10−3 M) for 5–60 min decreased p-p65 level in a time-dependent manner.

Figure 3
Figure 3

The effects of hydrogen sulfide on NF-κB activation and the production of cytokines in USMCs. (A, B, C, D, E, F, G and H) USMCs were treated with NaHS (5 × 10−6 – 10−4 M) or l-cysteine (1.25–10 × 10−4 M) for 24 h. The cells were collected for determination of the levels of protein levels of p-p65 (A and B) using Western blot and mRNA levels of IL-1β (C and F), IL-6 (D and G) and TNFα (E and H) by quantitative real-time RT-PCR. The supernatant was collected and levels of IL-1β (C and F), IL-6 (D and G) and TNFα (E and H) were measured by ELISA. Values are presented as mean ± s.e.m. for n = 6 subjects performed in triplicate. Representative protein bands of p-p65 were on the top of each histogram. (I, J and K) USMCs were transfected with siRNA targeting CSE or CBS for 10 h and changed medium with DMEM for 14 h and then followed by l-cysteine (10−4 M) treatment for 24 h. The supernatant was collected and levels of IL-1β (I), IL-6 (J) and TNFα (K) were measured by ELISA. Values are presented as mean ± s.e.m. for n = 3 subjects performed in triplicate. *P < 0.05, **P < 0.01 vs vehicle control.

Citation: Reproduction 153, 5; 10.1530/REP-16-0549

As NF-κB plays a key role in driving cytokine and chemokine production. We explored whether H2S modulates the production of proinflammatory cytokines and chemokines. As shown in Fig. 3C, D and E, NaHS (5 × 10−6 – 10−4 M) treatment dose-dependently inhibited IL-1β, IL-6 and TNFα output in USMCs. Treatment of the cells with l-cysteine also caused a decrease in the output of IL-1β, IL-6 and TNFα in a dose-dependent manner (Fig. 3F, G and H). Both NaHS and l-cysteine significantly inhibited IL-1β, IL-6 and TNFα mRNA expression (Fig. 3C, D, E, F, G and H). However, NaHS and l-cysteine had no effect on the output of chemokines CCL-2 and CXCL-8 (Supplementary Fig. 2).

The role of CSE and CBS in l-cysteine suppression of proinflammatory cytokine production in USMCs

To explore the role of CSE and CBS in the effects of l-cysteine on inflammatory cytokine production, small interfering RNA (siRNA) approach targeting CSE and CBS was applied. The protein level of CSE and CBS was decreased by about 68% and 91% respectively, upon transfection with CSE and CBS siRNA (Supplementary Fig. 3). As shown in Fig. 3G, H and I, knockdown of either CSE or CBS reversed l-cysteine inhibition of the output of proinflammatory cytokines.

IL-1β blocks NaHS and l-cysteine suppression of CX43, OTR and PGFR expression

We then investigated the effects of proinflammatory cytokines on H2S inhibition of the expression of CAPs. As shown in Fig. 4, the inhibitory effect of H2S on CX43, OTR and PGFR expression did not occur in the presence of IL-1β.

Figure 4
Figure 4

The effects of H2S on CX43, OTR and PGFR expression in the presence of IL-1β. USMCs were treated with NaHS (5 × 10−6 – 10−4 M) or l-cysteine (1.25–10 × 10−4 M) in the presence of IL-1β (1 ng/mL) for 24 h. Cells were collected for determination of the protein levels of CX43 (A and D), PGFR (B and E) and OTR (C and F) by Western blot analysis. Representative protein bands are presented on the top of the responding histogram. Values are presented as mean ± s.e.m. for n = 4 subjects performed in triplicate. *P < 0.05, **P < 0.01 vs vehicle control. l-cys, l-cysteine.

Citation: Reproduction 153, 5; 10.1530/REP-16-0549

The role of KATP channels in NaHS and l-cysteine suppression of proinflammatory cytokines

We explored the role of KATP channels in H2S suppression of proinflammatory cytokines because KATP channels have been reported to mediate H2S action in many studies (Jiang et al. 2010, Tang et al. 2010). As shown in Fig. 5A, B and C, KATP channel inhibitor glibenclamide (10−5 M) reversed NaHS and l-cysteine inhibition of IL-1β, IL-6 and TNFα output.

Figure 5
Figure 5

The roles of KATP channels, ERK and PI3K signaling pathways in the effect of H2S on proinflammatory cytokine production. (A, B, C, D, E and F) USMCs were treated with NaHS (10−4 M) or l-cysteine (10−3 M) in combination with KATP channel inhibitor glibenclamide (10−5 M), ERK inhibitor PD98059 (10−5 M) and PI3K inhibitor LY294002 (10−5 M) for 24 h. The supernatant was collected and levels of IL-1β (A and D), IL-6 (B and E) and TNFα (C and F) were measured by ELISA. (G, H, I and J) USMCs were treated with NaHS (10−4 M), l-cysteine (10−3 M) or vehicle for indicated time. Cells were collected and Western blot analysis was used to determine protein levels of ERK1/2 (G and H), p-ERK1/2 (G and H), PI3K (I and J) and p-PI3K (I and J). Representative protein bands are presented on the top of the responding histogram. Values are presented as mean ± s.e.m. for n = 4 subjects performed in triplicate. *P < 0.05, **P < 0.01 vs vehicle control. Gliben, glibenclamide.

Citation: Reproduction 153, 5; 10.1530/REP-16-0549

PI3K and ERK signaling pathways are involved in NaHS and l-cysteine suppression of proinflammatory cytokines

Prior studies have also reported that H2S can activate PI3K and ERK signaling in some tissues (Hu et al. 2008, Tamizhselvi et al. 2009, Ang et al. 2011, Peake et al. 2013). We therefore examined the role of PI3K and ERK signaling in the H2S modulation of proinflammatory cytokine output. It was found that the PI3K inhibitor LY294002 and ERK inhibitor PD98059 could block the inhibitory effects of NaHS and l-cysteine on IL-1β, IL-6 and TNFα production (Fig. 5D, E and F).

As shown in Fig. 5G, H, I and J, treatment of cells with NaHS (10−4 M) and l-cysteine (10−3 M) for 5–60 min increased phospho-PI3K (p-PI3K) and phospho-ERK1/2 (p-ERK1/2) levels in a time-dependent manner.

NaHS and l-cysteine activation of PI3K and ERK signaling is dependent on KATPchannels

We investigated whether H2S activation of PI3K and ERK signaling is associated with activation of KATP channels. As shown in Fig. 6A and B, in the presence of glibenclamide (10−5 M), the effects of NaHS and l-cysteine treatment on PI3K and ERK activation did not occur.

Figure 6
Figure 6

The role of KATP channels in H2S activation of PI3K and ERK signaling and suppression of NF-κB activation. USMCs were treated with NaHS (10−4 M) or l-cysteine (10−3 M) in the presence of glibenclamide (10−5 M) for indicated time. Cells were collected and Western blot analysis was used to determine protein levels of ERK1/2 (A and B), p-ERK1/2 (A and B), PI3K (C and D), p-PI3K (C and D), p-65 (E and F) and p-p65 (E and F). Representative protein bands are presented on the top of the responding histogram. Values are presented as mean ± s.e.m. for n = 4 subjects performed in triplicate. *P < 0.05, **P < 0.01 vs vehicle control.

Citation: Reproduction 153, 5; 10.1530/REP-16-0549

We then examined whether H2S suppression of NF-κB activation is dependent on KATP channels. Glibenclamide (10−5 M) treatment did not affect NaHS and l-cysteine inhibition of p-p65 level (Fig. 6C and D).

Discussion

The present study showed for the first time that both H2S donor NaHS and H2S precursor l-cysteine exhibited suppressive effects on CAP expression and NF-κB activation in cultured USMCs obtained for pregnant myometrial biopsies. NaHS and l-cysteine also inhibited the production of proinflammatory cytokines including IL-1β, IL-6 and TNFα. Knockdown of CSE or CBS reversed suppressive effects of l-cysteine on inflammation, confirming the effects of endogenous H2S produced via CSE and CBS. Moreover, IL-1β blocked the inhibitory effect of H2S on the expression of CAPs in myometrium. Thus, our study indicated that H2S suppresses uterine activation by inhibition of inflammation.

Many previous studies have reported that H2S has anti-inflammatory actions in various tissues, such as inhibition of leukocyte adhesion and the release of inflammatory mediators (Tamizhselvi et al. 2009, Ang et al. 2011, Du et al. 2014, Li et al. 2016). However, some studies also demonstrated that H2S could act as a proinflammatory mediator in the inflammatory responses (Bhatia et al. 2005, 2008, Ang et al. 2011). More recently, Badiei and coworkers (Badiei et al. 2016) reported that knockdown of CSE in these cells can protect the mice against pancreatitis, and therefore, proposed that endogenous H2S produced by CSE is an endogenous inflammatory mediator in monocytes/macrophages. The present study showed that exogenous H2S and H2S produced by CSE and CBS suppressed NF-κB activation and inhibited IL-1β, IL-6 and TNFα production in USMCs. Collectively, it may suggest that the effect of H2S on inflammatory response is dependent on the machinery of the target tissues.

Some studies have demonstrated that H2S affects the expression of proinflammatory cytokines by modulating NF-κB activity (Guo et al. 2013, Du et al. 2014). In consistence with these studies, we also found that H2S suppressed the level of phospho-p65, the active form of NF-κB, in myometrial biopsies. Of note, KATP channels were firstly identified to mediate the dilatory effects of H2S in vasculatures (Tang et al. 2010). Our previous studies have shown that H2S suppresses the spontaneous contraction of human myometrial strips via KATP channels (You et al. 2011). In the present study, we demonstrated that blockage of KATP channels reversed H2S suppression of proinflammatory cytokines. In addition, we showed that the impact of H2S on NF-κB activity was not dependent on KATP channels. A number of studies have implicated that H2S actually exerts its function by modifying l-cysteine in a large number of proteins by S-sulfhydration (Mustafa et al. 2011, Du et al. 2014, Módis et al. 2016, Sun et al. 2016). Moreover, it has been reported that H2S regulates NF-κB activity and KATP channels via sulfhydration of l-cysteine in these proteins (Mustafa et al. 2011, Du et al. 2014). Taken together, it suggests that H2S suppresses the production of proinflammatory cytokines by regulating the activity of NF-κB and KATP channels in pregnant USMCs.

The present study also showed that PI3K and ERK signaling pathways were involved in H2S suppression of inflammation. Many studies have demonstrated that PI3K signaling is a negative regulator during inflammatory responses, such as lipopolysaccharide (LPS)-induced inflammatory response (Schabbauer et al. 2004, Luyendyk et al. 2007, Zhang et al. 2007, Bi et al. 2016). For instance, Zhang and coworkers (Zhang et al. 2007) reported that α-lipoic acid attenuates LPS-induced inflammation via activation of PI3K signaling. Moreover, in pancreatic cells, it has been shown that H2S suppresses caerulein-induced IL-1β and TNFα production via PI3K signaling (Bi et al. 2016). Many studies have demonstrated that ERK signaling also promotes inflammatory responses in many tissues (Guha & Mackman 2001, Maeng et al. 2006, Mandrekar & Szabo 2009, Youn et al. 2016). However, some other studies reported that ERK signaling can serve as an anti-inflammation signaling in some tissues. Maeng and coworkers (Maeng et al. 2006) showed that activation of ERK suppresses NF-κB-dependent genes in endothelial cells. More recently, Subedi and coworkers (Subedi et al. 2016) demonstrated that the extract of Lindera neesiana, an herb medicine, decreased the production of proinflammatory cytokines and nitric oxide via ERK signaling pathway in neural cell lines. With regard to the mechanism by which H2S induces PI3K and ERK signaling pathways, we found that H2S activation of PI3K and ERK signaling is dependent on KATP channels, which is consistent with the study by Hu and coworkers (Hu et al. 2008) in which they showed that H2S activates PI3K and ERK signaling via KATP channels in cardiomyocytes.

Human parturition is an inflammatory process, which includes increased the expression and secretion of inflammatory cytokines and chemokines in uterus. Our previous study has demonstrated that human parturition is associated with the activation of inflammatory processes in uterus as evidenced by the activation of NFκB leading to increased expression of CAPs in myometrium (You et al. 2014). In the present study, we showed that downregulation of H2S-producing enzymes CSE and CBS is associated with increased expression of CAPs and level of active NF-κB in myometrium during pregnancy. In vitro study, we found that H2S suppression of CAP expression, NF-κB activation and the production of proinflammatory cytokines. Collectively, our data show that endogenous H2S produced locally play a critical role in the maintenance of uterine quiescence, and this effect is associated with suppression of inflammation in uterus.

In conclusion, H2S produced by CSE and CBS suppresses the expression of CAPs via inhibition of NF-κB and proinflammatory cytokines in pregnant human myometrial cells. The inhibitory effect of H2S on proinflammatory cytokines is dependent on activation of KATP channels. Our data indicate that H2S plays a critical role in the maintenance of uterine quiescence during pregnancy.

Supplementary data

This is linked to the online version of the paper at http://dx.doi.org/10.1530/REP-16-0549.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

National Natural Science Foundation of China (Nos. 81620108013, 81370734, 31371175 and 81471479) and the Global Alliance for the Prevention of Prematurity and Stillbirth (GAPPS) Preventing Preterm Birth Initiative, an initiative of Seattle Children’s Hospital.

Acknowledgement

The authors wish to thank the nursing and medical staff of the delivery suite and the patients at Changhai Hospital and Maternity for their participation.

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    The protein levels of CBS, CSE, p-p65, CX43, OTR and PGFR, in pregnant myometrium before and after onset of labor. Myometrial tissues were obtained from pregnant women at term before (TNL) or after the onset of labor (TL). Semi-quantitation of Western blot signals of CBS (A), CSE (B), p-p65 (C), CX43 (D), OTR (E) and PGFR (F) in TNL and TL myometrium biopsies. Representative protein bands were on the top of each histogram. Data were presented as mean ± s.e.m. **P < 0.01 vs TNL.

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    The effects of hydrogen sulfide on the expression of CX43, OTR and PGFR in USMCs. USMCs were treated with increasing concentration of NaHS (5 × 10−6 – 10−4 M) or l-cysteine (1.25–10 × 10−4 M) for 24 h. The cells were then collected and protein levels of CX43 (A and D), OTR (B and E) and PGFR (C and F) were determined by Western blot. Values are presented as mean ± s.e.m. for n = 6 subjects performed in triplicate. *P < 0.05, **P < 0.01 vs vehicle control. l-cys, l-cysteine.

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    The effects of hydrogen sulfide on NF-κB activation and the production of cytokines in USMCs. (A, B, C, D, E, F, G and H) USMCs were treated with NaHS (5 × 10−6 – 10−4 M) or l-cysteine (1.25–10 × 10−4 M) for 24 h. The cells were collected for determination of the levels of protein levels of p-p65 (A and B) using Western blot and mRNA levels of IL-1β (C and F), IL-6 (D and G) and TNFα (E and H) by quantitative real-time RT-PCR. The supernatant was collected and levels of IL-1β (C and F), IL-6 (D and G) and TNFα (E and H) were measured by ELISA. Values are presented as mean ± s.e.m. for n = 6 subjects performed in triplicate. Representative protein bands of p-p65 were on the top of each histogram. (I, J and K) USMCs were transfected with siRNA targeting CSE or CBS for 10 h and changed medium with DMEM for 14 h and then followed by l-cysteine (10−4 M) treatment for 24 h. The supernatant was collected and levels of IL-1β (I), IL-6 (J) and TNFα (K) were measured by ELISA. Values are presented as mean ± s.e.m. for n = 3 subjects performed in triplicate. *P < 0.05, **P < 0.01 vs vehicle control.

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    The effects of H2S on CX43, OTR and PGFR expression in the presence of IL-1β. USMCs were treated with NaHS (5 × 10−6 – 10−4 M) or l-cysteine (1.25–10 × 10−4 M) in the presence of IL-1β (1 ng/mL) for 24 h. Cells were collected for determination of the protein levels of CX43 (A and D), PGFR (B and E) and OTR (C and F) by Western blot analysis. Representative protein bands are presented on the top of the responding histogram. Values are presented as mean ± s.e.m. for n = 4 subjects performed in triplicate. *P < 0.05, **P < 0.01 vs vehicle control. l-cys, l-cysteine.

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    The roles of KATP channels, ERK and PI3K signaling pathways in the effect of H2S on proinflammatory cytokine production. (A, B, C, D, E and F) USMCs were treated with NaHS (10−4 M) or l-cysteine (10−3 M) in combination with KATP channel inhibitor glibenclamide (10−5 M), ERK inhibitor PD98059 (10−5 M) and PI3K inhibitor LY294002 (10−5 M) for 24 h. The supernatant was collected and levels of IL-1β (A and D), IL-6 (B and E) and TNFα (C and F) were measured by ELISA. (G, H, I and J) USMCs were treated with NaHS (10−4 M), l-cysteine (10−3 M) or vehicle for indicated time. Cells were collected and Western blot analysis was used to determine protein levels of ERK1/2 (G and H), p-ERK1/2 (G and H), PI3K (I and J) and p-PI3K (I and J). Representative protein bands are presented on the top of the responding histogram. Values are presented as mean ± s.e.m. for n = 4 subjects performed in triplicate. *P < 0.05, **P < 0.01 vs vehicle control. Gliben, glibenclamide.

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    The role of KATP channels in H2S activation of PI3K and ERK signaling and suppression of NF-κB activation. USMCs were treated with NaHS (10−4 M) or l-cysteine (10−3 M) in the presence of glibenclamide (10−5 M) for indicated time. Cells were collected and Western blot analysis was used to determine protein levels of ERK1/2 (A and B), p-ERK1/2 (A and B), PI3K (C and D), p-PI3K (C and D), p-65 (E and F) and p-p65 (E and F). Representative protein bands are presented on the top of the responding histogram. Values are presented as mean ± s.e.m. for n = 4 subjects performed in triplicate. *P < 0.05, **P < 0.01 vs vehicle control.

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