DHA Regulates Placental Inflammation By Inhibiting NLRP3 Inflammasome And NF-κB Signaling Pathway

Objective: JEG3 cells inflammation model was established through LPS-induced in order to explore the effects of DHA on the NLRP3 inflammasome and related inflammatory signal pathway.In animal experiments,we observed that the NLRP3 inflammasome in placental tissue was activated in HFD-induced ICR mice inflammation model.In this study,we further established the inflammation model of JEG3 cells in vitro and used DHA to intervene to investigate the changes of related molecular levels and inflammatory markers.The aim was to investigate the regulation of NDRP3 inflammasome and NF-κB by the inflammatory signaling pathway of DHA.Our findings involving that anti-inflammatory mechanism provide experimental evidence for further clinical DHA supplementation in obese pregnant women.Methods: ICR mice,the age of 8 weeks,were used as experimental animals when adapted feeding and then mated 1 week later.Mice were randomly divided into two groups after conception,normal diet control(C)group and high-fat diet(H)group.Pregnancies were anesthetized with chloral hydrate and placentas were taken out on the 18 th day of gestation(GD18),PCR and WB methods were used to detect the gene and protein expression levels of NLRP3 inflammasome and NF-κB in the placenta.The JEG3 cells were cultured and divided into 6 subgroups: control group,LPS group,LPS+DHA(low concentration group)and LPS+DHA(medium concentration group),LPS+DHA(high concentration group and DHA group).CCK-8 method was used to assess the effects of different concentrations of DHA on JEG3 cell viability.JEG3 cells were stimulated with LPS and ATP,and the inflammatory cytokine IL-1β secretion in supernatant was detected by enzyme-linked immunosorbent assay(ELISA).Western blotting was used to detect the protein expression of NLRP3 and caspase-1 and NF-κB and IL-1β.ROS probe fluorescence quantitative experiments was used to detect the effects of DHA on the production of ROS in LPS+ ATP induced in JEG3 cells.Results:In vivo,the expression of NLRP3,Casepase-1 and IL-1β mRNA in placenta of high-fat diet mice showed a tendency of increasing compared with the control group(P <0.05).There was no statistical difference in the gene expression levels of ASC and Pro-caspase-1.The expression of NLRP3 and Caspase-1 protein levels in HFD group were higher than that in the control group(P <0.01),while there was no significant difference in the expression of Pro-caspase-1 and ASC between the control and treated groups.In vitro experiments,the JEG3 cells induced inflammation of LPS,and then given different concentrations of DHA intervention,the results showed that cell proliferation/toxicity experiments did not reflect the cell viability alteration under different concentrations of DHA intervention.ELISA assay of supernatant showed that there is a dose-dependent relationship that DHA inhibited the secretion of IL-1β in JEG3 cells induced by LPS+ATP.Compared with the control group,the LPS-treated group at the gene level led to an increase in cellular inflammation,the gene expression levels of NLRP3,Caspase-1 and IL-1β presented statistical significance(P<0.01).Compared with LPS-induced group,the DHA+LPS+ATP treatment group had a down-regulation of NLRP3,Caspase-1 and IL-1β gene expression levels,and with the increase of DHA concentration,NLRP3,Caspase-1 and IL-1β gene expression decreased(P<0.05).DHA can inhibit LPS+ATP-induced activation of inflammatory signals such as NF-κB(P65)and inflammasome,and inhibit the activation of NLRP3 inflammasome,the cleavage of Caspase-1 and the secretion of IL-1β in protein levels(P <0.05).According to the fluorescence detection of intracellular reactive oxygen species(ROS)levels,DHA could reduce the inflammation of JEG3 cells by reducing the production of ROS.Conclusions : LPS can induce JEG3 cells inflammation and DHA shows an inhibition function to NF-κB signaling pathway and NLRP3 inflammasome activation when exerting its anti-inflammatory effect.The finding of DHA in the prevention of maternal infection,inflammation,anti-inflammatory effect can provide novel insights for the treatment of inflammatory diseases and some value in the future.DHA may play a important role in improving the fetal birth outcome by inhibiting the activation of inflammatory cytokines NLRP3 and NF-κB signaling pathway,providing some experimental evidence for further clinical DHA supplementation in obese pregnant women.