Lipopolysaccharide-induced Preterm Birth Research Articles

The Preventive Effects of Quercetin on Preterm Birth Based on Network Pharmacology and Bioinformatics.

Our previous study has shown that quercetin prevented lipopolysaccharide-induced preterm birth. This study aims to clarify the potential targets and biological mechanisms of quercetin in preventing preterm birth. We used bioinformatics databases to collect the candidate targets for quercetin and preterm birth. The biological functions and enriched pathways of the intersecting targets were analyzed by gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. Then, the hub targets were identified by cytoscape plugin cytoHubba from the protein-protein interaction network. We obtained 105 targets for quercetin in preventing preterm birth. The biological processes of the intersecting targets are mainly involved in steroid metabolic process, drug metabolic process, oxidation-reduction process, omega-hydroxylase P450 pathway, positive regulation of cell migration, negative regulation of apoptotic process, and positive regulation of cell proliferation. The highly enriched pathways were steroid hormone biosynthesis, metabolism of xenobiotics by cytochrome P450, proteoglycans in cancer, focal adhesion, and arachidonic acid metabolism. The ten hub targets for quercetin in preventing preterm birth were AKT serine/threonine kinase 1, mitogen-activated protein kinase 3, epidermal growth factor receptor, prostaglandin-endoperoxide synthase 2, mitogen-activated protein kinase 1, estrogen receptor 1, heat shock protein 90 alpha family class A member 1, mitogen-activated protein kinase 8, androgen receptor, and matrix metallopeptidase 9. Molecular docking analysis showed good bindings between these proteins and quercetin. In conclusion, these findings highlight the key targets and molecular mechanisms of quercetin in preventing preterm birth.

The intervention effect of nicotine on cervical fibroblast-myofibroblast differentiation in lipopolysaccharide-induced preterm birth model through activating the TGF-β1/Smad3 pathway

Currently, the clinical treatment of preterm birth, mainly using uterine contraction inhibitors, does not fundamentally reduce the incidence of premature birth (PTB). Premature cervical ripening is an important factor in PTB. We previously found that nicotine-treated pregnant murine had significant cervical resistance to stretch and higher collagen cross-links compared to the control animals, and nicotine prolonged gestation and inhibited cervical ripening. However, the regulatory effects of nicotine on premature cervical ripening and its role in PTB remain unclear. To investigate the effects of nicotine on cervical TGF-β1/Smad3 pathway and fibroblast-myofibroblast differentiation regulated by this pathway in PTB-like models. Intraperitoneal injection with 15 μg lipopolysaccharide (LPS) in 200 μl PBS into pregnant mice was used to induce the PTB-like model. Mice were randomly divided into four groups: control group, LPS-treated group, LPS + Nicotine co-treated group and LPS + Nicotine+α-BGT co-treated group. Pregnancy outcomes were monitored. The collagen content was assessed by Picrosirius red staining. Expressions of genes and proteins in the TGF-β/Smad3 pathway were detected by double immunofluorescence staining and quantitative Real-time PCR (qRT-PCR). myofibroblast differentiation were investigated by double immunofluorescence staining and qRT-PCR. Ultrastructures were analyzed by conventional transmission electron microscopy. The rate of PTB and neonatal mortality at birth was significantly higher in the LPS-treated group than in the control group; collagen content also decreased remarkably; the expression of TGF-β1 in macrophages and p-Smad3 in fibroblasts were reduced; the expression of α-smooth muscle actin (α-SMA, markers for activated fibroblasts) was down-regulated while the expression of calponin and smoothelin (markers for fibroblasts at rest) was up-regulated. Nicotine improved pregnancy outcomes and inhibited collagen degradation, activated the TGF-β1/Smad3 pathway and promoted cervical fibroblast-myofibroblast differentiation in PTB-like mice; such effects could be reversed by α-bungarotoxin (α-BGT). Nicotine inhibited premature cervical ripening in PTB-like models in relation with up-regulating the TGF-β/Smad3 pathway and promoting fibroblast to differentiate into myofibroblasts.

A vaginal nanoformulation of a SphK inhibitor attenuates lipopolysaccharide-induced preterm birth in mice.

Aim: Previously, we have shown that inhibition of SphK by the SphK inhibitor-II (SKI II) prevents lipopolysaccharide-induced preterm birth in mice. The aim of this study was to develop a vaginal self-nanoemulsifying drug-delivery system (SNEDDS) for SKI II. Materials & methods: A SKI II-loaded SNEDDS was characterized and tested in a murine preterm birth model. Results: The SNEDDS immediately formed a gel and then slowly emulsified to nanoglobules with over 500-fold enhancement of SKI II solubility at vaginal pH. Intravaginal administration of the SKI II SNEDDS significantly decreased lipopolysaccharide-induced preterm birth in mice. Conclusion: A vaginal nanoformulation of SKI II represents a novel, noninvasive approach to prevent preterm birth.

Immune tolerance attenuates gut dysbiosis, dysregulated uterine gene expression and high-fat diet potentiated preterm birth in mice

Preterm delivery accounts for 85% of perinatal morbidity and mortality. Although the consumption of a high-fat diet leads to exaggerated proinflammatory responses and, in pregnant women, increased rates of spontaneous preterm birth, the underlying mechanisms remain unclear. We sought to elucidate the mechanisms by which maternal consumption of a high-fat diet leads to a dysregulated immune response and, subsequently, spontaneous preterm birth. We performed 16S ribosomal RNA sequencing of DNA extracted and amplified from stool samples and compared the gut microbiomes of lipopolysaccharide-induced pregnant mice that were maintained on a high-fat diet compared to a normal control diet. Next, we sequenced the uterine transcriptomes of the mice. To test the effect of dampening of the immune response on the microbiome, transcriptome, and risk of spontaneous preterm birth, we induced immune tolerance with repetitive subclinical doses (0.2 mg/kg/week for 8 weeks) of endotoxin and performed 16S ribosomal RNA and uterine transcriptome sequencing on these immunotolerized mice. High-fat diet potentiates lipopolysaccharide-induced preterm birth by affecting the maternal gut microbiome and uterine transcriptome and reduces antioxidant capacity in a murine model. High-fat diet consumption also increases the colonization of the gut by 5 immunogenic bacteria and decreases colonization by Lachnospiraceae_NK4A136_group. Uteri from high-fat diet mice had increased expression of genes that stimulate the inflammatory-oxidative stress axis, autophagy/apoptosis, and smooth muscle contraction. Repetitive endotoxin priming protects high-fat diet dams from spontaneous preterm birth, increases colonization of the gut by Lachnospiraceae_NK4A136_group, decreases levels of immunogenic bacteria in the gut microbiome, and reduces the number of dysregulated genes after high-fat diet consumption from 994 to74. High-fat diet-potentiated spontaneous preterm birth is mediated by increased inflammation, oxidative stress, and gut dysbiosis. The induction of immune tolerance via endotoxin priming reverses these effects and protects high-fat diet dams from spontaneous preterm birth. Based on this work, the role of immunomodulation as a novel therapeutic approach to prevent preterm birth among women who consume high-fat diets should be explored.

Drug delivery to the human and mouse uterus using immunoliposomes targeted to the oxytocin receptor

The ability to provide safe and effective pharmacotherapy during obstetric complications, such as preterm labor or postpartumhemorrhage, is hampered by the systemic toxicity of therapeutic agents leading to adverse side effects in the mother and fetus. Development of novel strategies to target tocolytic and uterotonic agents specifically to uterine myocytes would improve therapeutic efficacy while minimizing the risk of side effects. Ligand-targeted liposomes have emerged as a reliable and versatile platform for targeted drug delivery to specific cell types, tissues or organs. Our objective was to develop a targeted drug delivery system for the uterus utilizing an immunoliposome platform targeting the oxytocin receptor. We conjugated liposomes to an antibody that recognizes an extracellular domain of the oxytocin receptor. We then examined the ability of oxytocin receptor-targeted liposomes to deliver contraction-blocking (nifedipine, salbutamol and rolipram) or contraction-enhancing (dofetilide) agents to strips of spontaneously contracting myometrial tissue invitro (human and mouse). We evaluated the ability of oxytocin receptor-targeted liposomes to localize to uterine tissue invivo, and assessed if targeted liposomes loaded with indomethacin were capable of preventing lipopolysaccharide-induced preterm birth in mice. Oxytocin receptor-targeted liposomes loaded with nifedipine, salbutamol or rolipram consistently abolished human myometrial contractions invitro, while oxytocin receptor-targeted liposomes loaded with dofetilide increased contraction duration. Nontargeted control liposomes loaded with these agents had no effect. Similar results were observed in mouse uterine strips. Following invivo administration to pregnant mice, oxytocin receptor-targeted liposomes localized specifically to the uterine horns and mammary tissue. Targeting increased localization to the uterus 7-fold. Localization was not detected in the maternal brain or fetus. Targeted and nontargeted liposomes also localized to the liver. Oxytocin receptor-targeted liposomes loaded with indomethacin were effective in reducing rates of preterm birth in mice, whereas nontargeted liposomes loaded with indomethacin had no effect. Our results demonstrate that oxytocin receptor-targeted liposomes can be used to either inhibit or enhance human uterine contractions invitro. Invivo, the liposomes localized to the uterine tissue of pregnant mice and were effective in delivering agents for the prevention ofinflammation-induced preterm labor. The potential clinical advantage of targeted liposomal drug delivery to the myometrium is reduced dose and reduced toxicity to both mother and fetus.

Activation of decidual invariant natural killer T cells promotes lipopolysaccharide-induced preterm birth.

Invariant natural killer T (iNKT) cells are crucial for host defense against a variety of microbial pathogens, but the underlying mechanisms of iNKT cells activation by microbes are not fully explained. In this study, we investigated the molecular mechanisms of iNKT cell activation in lipopolysaccharide (LPS)-stimulated preterm birth using an adoptive transfer system and diverse neutralizing antibodies (Abs) and inhibitors. We found that adoptive transfer of decidual iNKT cells to LPS-stimulated iNKT cell deficient Jα18(-/-) mice that lack invariant Vα14Jα281T cell receptor (TCR) expression significantly decreased the time to delivery and increased the percentage of decidual iNKT cells. Neutralizing Abs against Toll-like receptor 4 (TLR-4), CD1d, interleukin (IL)-12 and IL-18, and inhibitors blocking the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinase (MAPK) p38 and extracellular signal-regulated kinase (ERK) significantly reduced in vivo percentages of decidual iNKT cells, their intracellular interferon (IFN)-γ production and surface CD69 expression. In vitro, in the presence of the same Abs and inhibitors used as in vivo, decidual iNKT cells co-cultured with LPS-pulsed dendritic cells (DCs) showed significantly decreased extracellular and intracellular IFN-γ secretion and surface CD69 expression. Our data demonstrate that the activation of decidual iNKT cells plays an important role in inflammation-induced preterm birth. Activation of decidual iNKT cells also requires TLR4-mediated NF-κB, MAPK p38 and ERK pathways, the proinflammatory cytokines IL-12 and IL-18, and endogenous glycolipid antigens presented by CD1d.

Inhibition of Sphingosine Kinase Prevents Lipopolysaccharide-Induced Preterm Birth and Suppresses Proinflammatory Responses in a Murine Model

Premature delivery occurs in 12% of all births, and accounts for nearly half of long-term neurological morbidity, and 60% to 80% of perinatal mortality. Despite advances in obstetrics and neonatology, the rate of premature delivery has increased approximately 12% since 1990. The single most common cause of spontaneous preterm birth is infection. Several lines of evidence have demonstrated the role of endothelin-1 as both a constrictor of uterine myometrial smooth muscle and a proinflammatory mediator. Endothelin-1 activates the phospholipase C pathway, leading to activation of protein kinase C and, in turn, sphingosine kinase (SphK). The inhibition of SphK has been recently shown to control the proinflammatory response associated with sepsis. We show herein, for the first time, that SphK inhibition prevents inflammation-associated preterm birth in a murine model. Rescue of pups from premature abortion with an SphK inhibitor occurs by suppression of the proinflammatory cytokines tumor necrosis factor α, Il-1β, and Il-6 and attenuation of polymorphonuclear inflammatory cells into the placental labyrinth. Moreover, we postulate that inhibition of SphK leads to suppression of endothelin-converting enzyme-1 expression, indicating the presence of an endothelin-converting enzyme 1/endothelin 1-SphK positive feedback loop. This work introduces a novel approach for the control of infection-triggered preterm labor, a condition for which there is no effective treatment.

Probiotic Lactobacillus rhamnosus GR-1 supernatant prevents lipopolysaccharide-induced preterm birth and reduces inflammation in pregnant CD-1 mice

The objective of this study was to determine the effect of probiotic Lactobacillus rhamnosus GR-1 supernatant (GR-1 SN) on lipopolysaccharide-induced preterm birth (PTB) and outputs of cytokines, chemokines, and progesterone in pregnant CD-1 mice. We compared PTB rates after intrauterine injection of lipopolysaccharide with and without previous GR-1 SN treatment. Cytokines and chemokines in the maternal plasma, myometrium, placenta, and amniotic fluid were examined with multiplex assay, and circulating maternal progesterone was measured with enzyme-linked immunoassay. Statistical significance was assessed with 2-tailed 1-way analysis of variance or analysis of variance on ranks. Fetal sex ratios in mice that delivered preterm were compared with those that delivered at term after lipopolysaccharide and GR-1 SN treatments. GR-1 SN reduced lipopolysaccharide-induced PTB by 43%. GR-1 SN significantly decreased the lipopolysaccharide-induced production of interleukin (IL)-1β, -6, and -12p40, tumor necrosis factor-α, CCL4, and CCL5 in maternal plasma; IL-6, -12p70, -17, and -13 and tumor necrosis factor-α in myometrium; IL-6, -12p70, and -17 in placenta; and IL-6, tumor necrosis factor-α, CCL3, and CCL4 in amniotic fluid. Maternal plasma progesterone was reduced significantly after lipopolysaccharide injection with and without GR-1 SN pretreatment. There was no difference in fetal sex ratios between mice that delivered preterm and those that did not after lipopolysaccharide and GR-1 SN treatments. The supernatant of probiotic L rhamnosus GR-1 attenuated lipopolysaccharide-induced inflammation and PTB in vivo. GR-1 SN may confer therapeutic benefits in the prevention of infection-associated PTB by controlling systemic and intrauterine inflammation.

Surfactant protein D modulates levels of IL-10 and TNF-α in intrauterine compartments during lipopolysaccharide-induced preterm birth

Prematurity is the main cause of perinatal mortality and morbidity, and preterm birth is often associated with intrauterine inflammation. Surfactant protein D (SP-D) functions in lung homeostasis and has multiple roles in innate immunity. It is present in amniotic fluid and in gestational tissues. We propose that SP-D may regulate intrauterine inflammatory responses related to preterm labor. Our aim was to investigate the role of SP-D in lipopolysaccharide-induced preterm birth in mice that overexpress rat SP-D (rSP-D) under the human SP-C promoter. SP-D protein in amniotic fluid and in gestational tissues was detected by western analysis. TNF-α, IL-10, and IL-6 concentrations in serum and amniotic fluid and mRNA levels in gestational tissues were quantified using cytometric bead array and ribonuclease protection assay, respectively. Increased levels of SP-D protein were detected in the amniotic fluid and gestational tissues of rSP-D mice. Lipopolysaccharide given at 17days post-coitum to rSP-D dams led to preterm birth of live-born offspring within 18h. Preterm birth of live-born pups was induced with a lower dose of lipopolysaccharide compared to wild-type mice. In rSP-D mice, the lipopolysaccharide-induced levels of TNF-α and IL-10 in amniotic fluid and fetal serum and the expression of IL-10 in placenta and fetal membranes were significantly different from wild-type mice. We conclude that SP-D in fetal and gestational tissues modulates the levels of intrauterine inflammatory mediators involved in preterm birth and may contribute to inflammatory processes related to spontaneous preterm labor.

A New Model for Inflammation-Induced Preterm Birth: The Role of Platelet-Activating Factor and Toll-Like Receptor-4

Preterm birth is a leading cause of neonatal morbidity and mortality. Despite a growing body of evidence correlating inflammation with preterm birth, the signal transduction pathways responsible for the emptying of the uterus in the setting of intrauterine inflammation has not been elucidated. We now report a unique, reproducible mouse model of localized intrauterine inflammation. This model results in 100% preterm delivery with no maternal mortality. Using our model, we also show that platelet-activating factor is a crucial mediator of both inflammation-induced preterm birth and fetal demise. Using C3H/HeJ mice, we demonstrate that toll-like receptor-4 (TLR-4) plays a role in lipopolysaccharide-induced preterm birth but not in inflammation-induced fetal death. Immunohistochemistry studies demonstrate the presence of the platelet-activating factor receptor in both endometrial glands and smooth muscle in uterine tissues. Molecular studies demonstrate the differential expression of platelet-activating factor receptor and TLR-4 in uterine and cervical tissue throughout gestation. Quantitative polymerase chain reaction revealed an up-regulation of TLR-4 in the fundal region of the uterus in response to intrauterine inflammation. The use of this model will increase our understanding of the significant clinical problem of inflammation-induced preterm birth and will elucidate signal transduction pathways involved in an inflammatory state.

Therapeutic effect of cyclo-oxygenase inhibitors with different isoform selectivity in lipopolysaccharide-induced preterm birth in mice

Objective: Cyclo-oxygenase inhibitors have different selectivity for inhibitory action on the isoforms of the enzyme. Our purpose was to compare the tocolytic and maternal toxic effects of various cyclo-oxygenase inhibitors in the lipopolysaccharide-induced preterm birth in pregnant mice. Study Design: We randomly assigned 50 ICR pregnant mice into five groups of 10 mice each and treated them intraperitoneally with either phosphate-buffered saline solution or lipopolysaccharide (50 μg) on day 15 of pregnancy. Of the four groups that received lipopolysaccharide, three groups also were treated either with nonselective cyclo-oxygenase inhibitors (indomethacin [1 mg/kg/d] or diclofenac [2 mg/kg/d]) or with the cyclo-oxygenase-2 preferential inhibitor, meloxicam (2 mg/kg/d), with a gavage tube on days 15 through 18 of pregnancy or until maternal death. The mice were killed immediately after preterm birth or on day 21 of pregnancy. Preterm birth rates and maternal side effect profiles were evaluated. Results: Preterm birth occurred in 90% of mice after intraperitoneal lipopolysaccharide injection and in 20% of mice after phosphate-buffered saline solution injection. Indomethacin and meloxicam, but not diclofenac, significantly decreased the incidence of preterm birth that was induced by lipopolysaccharide (33.3% and 33.3%, respectively; P =.028). Although the overall incidence of maternal gastric and/or renal toxicities was not significantly increased in the indomethacin or meloxicam groups, a significant increase was noticed in the diclofenac group compared with the lipopolysaccharide-treated control group (P =.006). Conclusion: Indomethacin and meloxicam, but not diclofenac, inhibit the lipopolysaccharide-induced preterm birth in an animal model. Meloxicam appears to have no advantage over indomethacin with regard to tocolysis and maternal side effects. (Am J Obstet Gynecol 2003;189:261-6.)