The objective of the study was to determine the effect of statins on lipopolysaccharide (LPS)-induced inflammatory response in a mouse model of preterm birth (PTB).
Day 15 CD1 mice were randomly allocated to intraperitoneal LPS injection (100 μg) or control. Mice in the LPS group were pretreated, 16 and 2 hours prior, with pravastatin (10 μg/g), simvastatin (10 μg/g), or vehicle control. Animals were sacrificed 6 hours after LPS. Cytokine messenger ribonucleic acid (mRNA) expression in the uterus and cervix, and concentrations in the maternal serum and amniotic fluid (AF) were determined.
Pravastatin reduced interleukin (IL)-1β and IL-6 mRNA expression in the uterus and cervix, respectively, and serum IL-1β and granulocyte-macrophage colony-stimulating factor (GM-CSF) concentrations. Simvastatin reduced IL-1β and IL-6 mRNA expressions in the uterus, IL-6 and tumor necrosis factor alpha (TNF-α) in the cervix, and IL-1β, IL-2, IL-12p70, IL-13, TNF-α, GM-CSF, and interferon-γ concentrations in the serum and IL-6 in AF.
Statins reduce the LPS-induced inflammatory responses in a mouse model of PTB.
Preterm birth (PTB) complicates more than 12% of pregnancies in the United States. Globally, it is a leading cause of neonatal and infant morbidity and mortality. Additionally, it carries significant socioeconomic consequences, with estimated costs of $26.2 billion in 2005. Despite having multiple etiologies, more than 65% of all PTB follows spontaneous preterm labor (PTL) or preterm premature rupture of membranes. Many of the mechanisms associated with idiopathic PTB involve inflammatory pathways activated through various risk exposures and mechanisms.
During microbial invasion and the establishment of infection, microbial ligands such as lipopolysaccharide (LPS) activate the maternal innate immune response by binding to pattern recognition receptors such as Toll-like receptors (TLRs). This leads to release of inflammatory cytokines that stimulate the secretion of other proinflammatory mediators, metalloproteases, and prostaglandins, which ultimately leads to uterine contractions, cervical ripening, and rupture of membranes resulting in PTB. Fetal inflammatory response associated with microbial invasion of the intraamniotic cavity also contributes to various short- and long-term neonatal morbidities, such as bronchopulmonary dysplasia, necrotizing enterocolitis, periventricular leukomalacia, and cerebral palsy.
Despite advancement in scientific knowledge regarding PTL, management of this syndrome is still limited to the use of tocolytic agents to delay delivery. Tocolytic drugs delay preterm birth through various mechanisms of actions; however, none has been shown to reduce the PTB rate or improve neonatal outcomes. This is partly because these agents do not address the underlying pathways leading to myometrial or cervical activation and are usually administered late in the process when potentially fetal inflammatory damage has occurred. Controlling inflammation should be of interest when focusing on PTL interventions because an inflammatory response is associated with the majority of these cases, irrespective of the etiology.
Although statins, or 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMG CoA reductase) inhibitors, have been mainly used to prevent cardiovascular disease through lipid lowering effects, they also possess additional pleiotropic and antiinflammatory properties that make them potential candidates to modulate the inflammation associated with PTB. We hypothesized that treatment with statins will attenuate the inflammatory response associated with PTB.
The objective of this study was to determine the effects of a hydrophilic (pravastatin) and a lipophilic (simvastatin) HMG CoA reductase inhibitors on the inflammatory response induced by LPS administration in a previously validated PTB animal model.
Materials and Methods
The study protocol and all related procedures were approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch (UTMB), Galveston, TX.
Time pregnant CD1 mice from Charles River (Wilmington, MA) were obtained at days 12-13 of gestation. The mice were maintained in the animal care facility at UTMB, housed separately in temperature- and humidity-controlled quarters, and kept in a 12 hour light/12 hour dark regimen with food and water available ad libitum.
At day 15 of gestation, the mice were randomly allocated to intraperitoneal injection of 100 μg LPS (LPS group) or normal saline as control group. LPS injection time was designated as time zero. To test the effectiveness of statins, animals in the LPS group were pretreated with intraperitoneal injection of pravastatin (10 μg/g of body weight; Pra/LPS), simvastatin (10 μg/g of body weight; Sim/LPS), or vehicle control at 18 and then 2 hours prior to LPS injections (at time zero). Control mice were pretreated with vehicle only. This resulted in 4 study groups: control (n = 5), LPS (n = 7), Pra/LPS (n = 9), and Sim/LPS (n = 9). Six hours after time zero, pregnant mice were euthanized with carbon dioxide and sacrificed ( Figure 1 ). Maternal weight at the time of sacrifice was recorded. We harvested all the pups from each mother and measured the total weight of the litter. Maternal serum, uterus, cervix, and amniotic fluid (AF) were collected. Amniotic fluid was pooled from various gestational sacs randomly. Tissues were flash frozen and stored at −80°C until further analysis.
Real-time polymerase chain reaction (PCR)
Real-time PCR was performed to determine messenger ribonucleic acid (mRNA) expression of various cytokines in the uterus and cervix. Ribonucleic acid (RNA) isolation was done using Direct-Zol RNA MiniPrep (Zymo Research, Irvine, CA) per the manufacturer’s protocol. Complementary deoxyribonucleic acid (DNA) was then made with a high-capacity complementary DNA reverse transcription kit (Applied Biosystems, Foster City, CA). Quantitative PCR was performed using the TaqMan Fast universal PCR master mix (2 times) (Applied Biosystems). Probes and primers used were purchased from Applied Biosystems. The samples were run on a 7500 fast real-time PCR machine according to the manufacturer’s protocol. Real-time PCR was performed, and assays were run in duplicate. Relative quantification (RQ) was used to measure gene expression. RQ was calculated for each animal and defined as the change in expression of the target gene in a test sample (LPS, Pra/LPS, or Sim/LPS) relative to the same gene in the control group. The data were presented as fold change in expression ± SEM, using the ΔΔcycle threshold method for analysis.
Multiplex cytokine assay
Serum cytokines were quantified using the Milliplex mouse cytokine/chemokine 13-plex panel from Millipore (Billerica, MA) per the manufacturer’s instructions. Cytokine-specific wavelength absorbance was determined using a Luminex LX 200 reader using the Xponent Software according to the kit settings (Luminex, Austin, TX). Data were interpreted using the Milliplex Analyst version 3.4.
Enzyme-linked immunosorbent assay
Three cytokines, tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6, were quantified in the AF using commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D, Minneapolis, MN). Samples were run in duplicates according to the manufacturer’s guidelines. The manufacturers report no cross-reactivity with other cytokines for any of the antibodies used in our multiplex assays and ELISA.
LPS from Escherichia coli O55:B5, pravastatin, and simvastatin were obtained from Sigma Aldrich (St. Louis, MO). Multiple protocols and dose regimens of LPS have been studied to model preterm inflammation, and it has been well established that there is significant variability in inflammatory responses in various mice strains and LPS preparations. Thus, we chose the LPS dose of 100 μg (stock 0.1 mg/mL stored at –20°C) based on our unpublished preliminary results of dose response of IL-6 concentrations in maternal serum at various LPS dose regimens. Pravastatin was prepared as 5 mg/mL in isotonic saline and stored at −20°C. Simvastatin was prepared fresh before each experiment as a 5 mg/mL (10 mg/mL in ethanol with a 1:1 dilution in saline). Statins were given in a dose of 10 μg/g body weight as intraperitoneal injection 18 and 2 hours prior to LPS injection. The dose and timing of statin administration were chosen based on previous studies that established significant antiinflammatory action of statins in a murine model of sepsis.
Analysis was performed using SigmaPlot (Systat 11.0; SYSTAT, Chicago, IL) and Prism 4 (GraphPad Software Inc., La Jolla, CA). Normality was assessed using a Shapiro-Wilk test on raw data and/or data that were log transformed. To simplify the tables and graphs, raw untransformed data are presented. One-way analysis of variance (ANOVA) with post-hoc Neuman Keuls testing or nonparametric ANOVA on ranks followed by a post-hoc Dunn test were performed as appropriate. Data are reported as mean ± SEM for ANOVA or median with an interquartile range when nonparametric ANOVA on ranks was used. A 2-tailed P < .05 was considered significant.
There were no differences in either baseline maternal weight prior to injections or litter size between the 4 groups. Total litter weight was significantly lower in the LPS group compared with the control and the statin-treated groups. There was no difference in average pup weight between control and statin-treated groups ( Table 1 ). LPS injection produced a significant increase in expression of inflammatory cytokines in uterine and cervical tissues as well as their concentrations in the maternal serum and amniotic fluid, confirming the validity of our LPS dose and model ( Tables 2 and 3 and Figures 2-4 ).
|Study groups||Control (n = 5)||LPS (n = 7)||Pra/LPS (n = 9)||Sim/LPS (n = 9)||P value|
|Maternal weight, g||43.5 ± 3.7||39.6 ± 1.7||40.6 ± 1.6||39.5 ± 2.2||.104|
|Litter size||14 (11–16)||12 (10–13)||12 (9–14)||13 (11–14)||.497|
|Average pup weight, g||4.2 ± 1.95||2.2 ± 1.15||4.4 ± 1.52||4.6 ± 1.15||.034 a|
|IL-1β||3.05 ± 4.81||117.982 ± 41.25 a||57.527 ± 40.09 a , b||56.39 ± 40.86 b , c||< .001|
|IL-6||0.51 ± 0.38||799.44 ± 1084.57 a||163.72 ± 143.97 a||26.80 ± 28.81 a , b , c||< .001|
|TNF-α||2.19 ± 2.89||115.19 ± 125.40 a||70.57 ± 46.04 a||52.77 ± 43.57 a||< .001|
|IL-1β||2.01 ± 1.3||12.11 ± 4.90 a||15.04 ± 10.87 a||17.87 ± 10.96 a||< .001|
|IL-6||1.86 ± 2.68||127.06 ± 91.96 a||61.85 ± 54.06 b||35.04 ± 26.19 b||.008|
|TNF-α||3.09 ± 1.84||35.09 ± 22.33 a||37.49 ± 18.89 a||16.17 ± 5.93 b , c||.002|
|IL-1β a||6.55 ± 0.97||62.25 ± 13.02 b||36.03 ± 4.29 b , c||18.60 ± 2.75 c , d||< .001|
|IL-2 e||5.32 (5.14–7.57)||10.74 (9.19–18.87) b||8.64 (8.23–10.99)||7.76 (5.61–8.43) c||.004|
|IL-5 e||6.89 (5.87–9.88)||44.72 (24.61–86.1) b||34.91 (29.58–43.04) b||23.76 (12.78–49.80)||.009|
|IL-6 e||22.17 (8.24–27.87)||7998.5 (6125.75–10751.25) b||4793.5 (2487.75–8668.5) b||2648.5 (1399.5–5993.75)||< .001|
|IL-10 a||4.75 ± 1.88||559.28 ± 325.88 b||343.54 ± 293.79 b||310.38 ± 206.66 b||.014|
|IL-12p70 a||7.96 ± 4.22||168.64 ± 60.04 b||116.46 ± 37.98 b||68.16 ± 25.92 b , c , d||< .001|
|IL-13 a||21.73 ± 13.05||48.5 ± 17.97 b||31.71 ± 20.64||20.27 ± 12.45 c||.029|
|TNF-α a||6.47 ± 0.16||98.87 ± 23.01 b||74.68 ± 12.17 b||44.97 ± 4.64 b , c||< .001|
|GM-CSF a||19.95 ± 5.24||120.22 ± 21.68 b||69.44 ± 11.93 b , c||49.93 ± 6.80 c||< .001|
|INF-γ e||3.6 (3.1–5.7)||1548.73 (1239.92–1895.58) b||546.94 (356.19–748.71) b||121.63 (29.12–212.94) c||< 001|
|IL-1β e||1.4 (0–4)||18 (14.45–50.5) b||7.35 (4.72–21.5)||8.1 (5.6–40)||.009|
|IL-6 a||18.88 ± 5.07||1551.92 ± 472.57 b||538.74 ± 205.51 b||495.34 ± 290.94 b , c||.017|