Model research of circulating Prevotella intermedia inducing stillbirth in pregnant mice via decidual inflammation

Abstract

Objective To construct the model of circulating Prevotella intermedia (Pi) inducing stillbirth in C57BL/6 pregnant mice via decidual inflammation and preliminarily analyze the relevant expression levels of inflammation-related genes in deciduas. Methods C57BL/6 Pregnant mice for 15 or 16 d of gestation were infected with 5 × 106 CFU Pi (ATCC25561) by I.V. injection method. Then, the mice were sacrificed and dissected at 12, 24 and 48 h post infection separately. The placentas (some placentas were separated as decidual tissues and non-decidual tissues) were harvested from each pregnant mouse, weighed, and homogenized in sterile PBS, and subsequently used as the templates of quantitative Real-time PCR assay for detection of expression levels of 10 kinds of cytokines and 7 kinds of signal molecules. In addition, birth outcome of pregnant mice in response to I.V. infection of 5 × 106 CFU Pi (Pi group) was evaluated in comparison to 5 × 106 CFU E.coli JM109 (E.coli group) and PBS (PBS group) as well. Total pups and dead pups were counted for each mouse. The fetal death rate was expressed as the percentage of dead fetuses from the total number of pups born to each mother. Differences of mRNA expression of cytokines and signal molecules between two groups were compared using the Independent Samples t test. Chi-square test was used to evaluate the differences of mortality rates of pups among Pi, E.coli and PBS groups. Results The fetal death rate were calculated as 39.2%, 3.0% and 0% for pregnant mice infected with Pi, E.coli and PBS individually. The rate of Pi group were significantly higher than E.coli group (χ2= 17.54, P<0.001) and PBS group (χ2= 21.49, P<0.001) . compared to PBS group, the mRNA expression of cytokines (IL-1α, IL-1β, TNF-α, IL-6, GCSF and KC) and signal molecules (COX2, NF-κB, MyD88 and TLR4) were significantly enhanced at 24 hours post infection (all P<0.05) . However, the mRNA expression of IFN-γ, IL-12, IL-18, IL-10, PGES, COX1 and Trif didn′t presented significant differences between two groups. Furthermore, the location of placental inflammation was demonstrated to be deciduas after comparison of mRNA expression of IL-1α, IL-1β, TNF-α and COX2 between decidual tissues and non-decudual tissues. Conclusions As one of the common oral conditional anaerobe frequently found in periodontal diseases, the I.V. injection of Pi hold the capability to induce the stillbirth in pregnant mice. The potential mechanism was associated with acute inflammatory response occurred in decidual tissues of placentas. Key words: Prevotella intermedia; Decidua; Pro-inflammatory cytokines; Cyclooxygenase