DHA Antagonize Lipopolysaccharide-induced Fetal Growth Restriction By Activating Placental PPARγ

Objective Inflammatory infection in late pregnancy could induce fetal growth restriction（FGR）,which is an important factor leading to increased fetal morbidity and chronic noninfectious diseases in adulthood.peroxisome proliferators-activated receptor gamma（PPARγ）is highly expressed in mammalian placenta,but the effect and mechanism of PPARγon placenta and fetal growth and development remain unclear.We investigated the antagonistic effect and mechanism of Docosahexaenoic Acid（DHA）,a natural ligand of PPARγ,and rosiglitazone（RSG）,a synthetic ligand of PPARγ,on lipopolysaccharide（LPS）-induced placental inflammation and fetal growth restriction（FGR）in vivo and in vitro.Methods We explored the mechanism of PPARγon LPS-induced FGR by two means.In vivo,the whole experiment was divided into two parts:（1）pregnant mice were divided into four groups-a control group,a DHA group,an LPS group,and a DHA+LPS group-according to body weight Z type,11 mice per group.Mice in the DHA and DHA+LPS groups were given DHA[300mg/(kgd^-1)]by intragastric administration during GD0-17.Meanwhile,mice in the LPS and DHA+LPS groups were administered LPS[100μg/(kgd^-1)]via intraperitoneal injection for 1h after intragastric administration during GD15-17,whereas the other two groups were given the same volume of corn oil or normal saline.All the pregnant mice were sacrificed on GD18.The whole blood and placenta tissues of pregnant mice was recorded.The numbers of live fetuses,stillborn and absorbed fetuses in each litter were recorded.The weight of uterine-fetus,placental diameter and weight,crown-rump length,and fetal weight were measured.（2）Same as above,pregnant mice were divided into four groups,six mice per group.Pregnant mice treated with DHA were intragastric administration orally with DHA（300mg/kg）during GD0-15.The LPS and DHA+LPS groups were intraperitoneally injected with LPS（100ug/kg）1h after DHA treated at GD15,and all pregnant mice were sacrificed 1h after injection.Whole blood,amniotic fluid and placental tissue of pregnant mice were collected for subsequent detection of related indicators.In vitro,we selected human placental chorionic trophoblast（HTR-8）cells to clarify that DHA/RSG antagonized the LPS-induced activation of the NF-κB signaling pathway,which was different in a PPARγ-dependent manner from the aspects of PPARγactivation and silence.Subsequently,cells were treated with cyclohexane（CHX）and proteasome inhibitor MG132.And then,the mechanism of PPARγ-induced p65 degradation was investigated by co-immunoprecipitation and western blot.Results The results showed that LPS exposure in late pregnancy could lead to fetal growth restriction,and damage the normal growth and development of placenta and fetal mouse,which was manifested in the values of all the placental and fetal parameters measured;however,these effects were significantly alleviated by DHA pretreatment.At the same time,there was no significant difference in the placental and fetal weights between the DHA group and the control group.Further results showed that LPS exposure could evoke activation of the NF-κB signaling pathway in placenta,which was manifested in increasing the expression of the NF-κB subunits（p50,p65）in placental nuclear protein fractions and the nuclear translocation of p65 in placental trophoblast cells.Furthermore,DHA intervention activated PPARγexpression and then suppressed the activation of NF-κB signaling pathway.As expected,DHA intervention led to a significant reduction in the levels of pro-inflammatory cytokines（TNF-α,IL-6,IL-1β）and chemokines（MIP-2,KC）induced by the LPS treatment in maternal blood,amniotic fluid and placenta.and we observed that DHA pretreatment further enhanced the effect of LPS on IL-10 expression in maternal serum and amniotic fluid.In HTR-8cells,the expression of p50 and p65 and the levels of the TNF-α、IL-6、IL-1βand MCP-1 were significantly increased after LPS treatment.And there was no significant difference between the DHA/RSG group and the control group.In the DHA/RSG+LPS group,the activation of NF-κB signaling pathway and the increase of inflammatory were inhibited after DHA/RSG pretreatment activated PPARγ.In HTR-8 cells,silencing/blocking of PPARγsignificantly attenuated the antagonistic effect of DHA/RSG on the activation of the NF-κB signaling pathway.After treatment with CHX,the degradation rate of p65 in the DHA+LPS group was significantly faster than that in the LPS treatment group.Then,in the same two groups,MG132 was given during LPS treatment to inhibit the process of ubiquitination.The results showed that the expression level of p65 was upregulated and reached a stable state at 6h.Finally,the results of Co-IP and western blotting showed that the levels of p65 ubiquitination were higher in the DHA+LPS group than in the control group;however,no difference was detected between the LPS and control groups.Conclusion Our results suggested that PPARγ,an important regulator of placental inflammation and fetal growth and development,plays an antagonistic role in LPS-induced FGR by promoting NF-κB p65 ubiquitination and degradation.