Nicotine protects fetus against LPS-induced fetal growth restriction through ameliorating placental inflammation and vascular development in late pregnancy in rats

Abstract

Our previous work has shown that nicotine suppressed lipopolysaccharide (LPS)-induced placental inflammation by inhibiting cytokine release as well as infiltration of leukocytes into the placenta through the cholinergic anti-inflammatory pathway. Nicotine also increased fetal survival and restored pup weight. In the present study, we aim to further investigate if fetal growth restriction (FGR) occurs with LPS treatment, and evaluate the protective effects of nicotine on fetuses in late gestation of rats. Pregnant Sprague-Dawley rats were divided into control group, nicotine group, LPS group and LPS + nicotine group. Rats were first pretreated with nicotine or vehicle by subcutaneous injection on gestation day (GD)14 and GD15, followed by LPS or vehicle intraperitoneal injection on GD16, and were killed on GD18. Loss of fetuses, number and weights of live fetuses and weights of placentas were recorded. Placentas were collected to evaluate placental pathology and determine inflammatory cytokines and vascular endothelial growth factor (VEGF) levels. We found that LPS treatment increased levels of placental inflammatory cytokines and placental pathological damage, decreased levels of VEGF, reduced number of live fetuses and induced FGR. Pretreatment with nicotine reversed LPS-induced high levels of placental inflammatory cytokines, low levels of placental VEGF and placental pathological damage, then rescued the number and weights of live fetuses. These data demonstrated that activation of the cholinergic anti-inflammatory pathway by nicotine protected fetus against LPS-induced FGR through ameliorating placental inflammation and vascular development in late pregnancy in rats. It may be an alternative therapeutic strategy for inflammation- induced FGR in late pregnancy.

Keywords: fetal growth restriction; lipopolysaccharides; nicotine; pregnancy.

Figure 1. Number of live fetus/litter on GD18 after various treatments

Bars represent means ± SEM of the live fetus number of six rats per group. Treatment with LPS alone significantly decreased the live fetus number compared with normal pregnant control (P<0.001), whereas treatment with LPS + nicotine significantly increased it compared with LPS alone (P=0.001). Significant differences were found between all groups as shown by P-values in the box inside the figure. Abbreviation: N, nicotine.

Figure 2. Weights of live fetuses on GD18 after various treatments

Bars represent means ± SEM of the live fetus weight of six rats per group. Treatment with LPS alone significantly reduced live fetus weight on GD18 (P<0.001), whereas treatment with LPS + nicotine significantly increased live fetus weight on GD18 compared with LPS alone (P<0.001) (data as shown in Figure 1).

Figure 3. Weights of placentas on GD18 in different groups

Bars represent means ± SEM of weight of placentas. Weights of placentas in LPS group and LPS + nicotine group were lower than those in control group (p = 0.019, p = 0.026, respectively). No significant difference was found between nicotine group and control group (p = 0.269), and between LPS + nicotine group and LPS group (p = 0.566).

Figure 4. Nicotine suppressed LPS-induced placental pathological changes

(A) Representative photomicrographs by HE staining of placentas from different groups (40×). (B) HE staining of placentas from LPS group (100×). Left: space of intervillous and minute vessels decreased in villi, solid arrows indicate inflammatory cells infiltrated in dysontogenesis villi; Middle: solid arrows indicate inflammatory cells infiltrated in decidua; Right: solid arrow indicates LPS-induced abscess in fetal membrane, dashed arrow indicates LPS-induced necrosis in fetal membrane.

Figure 5. Placental TNF-α, IL-6, CXCL1 and VEGF levels on GD18 in different groups

Bars represent means ± SEM of concentrations (pg/ml/mg). (A–C) There were increased TNF-α, IL-6 and CXCL1 levels in the LPS treatment group compared with the control group (P<0.001), and decreased TNF-α, IL-6 and CXCL1 levels in the LPS + nicotine group (LPS + N) and nicotine group compared with LPS group (P<0.001). (D) There were decreased VEGF levels in the LPS treatment group compared with the control group (P<0.001), and increased VEGF levels in the LPS + nicotine group (LPS + N) and nicotine group compared with LPS group (P<0.001).