Effect And Mechanism Of TLR4/NF-?B Signaling Pathway On Activation Of NLRP3 Inflammatory Corpuscles During Placental Hypoperfusion In Rats

Objective: Mild intrauterine hypoperfusion hypoperfusion(MIUH)is the common pathological basis of a variety of maternal and fetal diseases in the second trimester of pregnancy,including Intrauterine growth restriction,Twin-to-twin transfusion syndrome,placental hemangioma and eclampsia.Although mild placental hypoperfusion is associated with a lower risk of fetal death,studies have shown that the late learning and memory function of infants affected by MIUH in the second trimester of pregnancy is impaired.Therefore,it is of great clinical significance to study the effects of MIUH exposure in the second trimester of pregnancy on the neurodevelopment of offspring.To elucidate the mechanism of brain injury in offspring induced by mild hypoperfusion may provide a predictive target for fetal neurodevelopmental disorder induced by adverse events during pregnancy,and is of great significance for early diagnosis and intrauterine therapy.Although the exact mechanism of MIUH-induced intrauterine hypoxic-ischemic brain damage is not clear,according to the current findings,it can be confirmed that,neuroinflammation caused by ischemia and hypoxia is an important link in the process of fetal brain injury.Activated neuroinflammation damages neurons in the hippocampus of the developing MIUH fetus during the second trimester of pregnancy and ultimately causes irreversible damage to the learning and memory function of the offspring.Neuronal pyroptosis mediated by NLRP3 inflammatory bodies is one of the newly discovered programmed cell death pathways and plays an important role in the process of neuroinflammation.However,the study of neuronal scorch death in offspring affected by MIUH in the second trimester is still a blank area.Therefore,the MIUH model of SD rats was chosen to study the effect of MIUH on brain cell death in the second trimester of pregnancy.TLR4/NF-?B signaling pathway,as a classical inflammatory initiation pathway,not only activates the secretion of many downstream inflammatory factors,but also activates NLRP3-mediated pyroptosis.Adverse conditions such as ischemia and hypoxia can cause the activation of TLR4 in Pattern recognition receptor.After a series of cascade reactions,NF-?B is activated and leads to the up-regulation of NLRP3 transcription.NLRP3 and ASC,together with activated Caspase-1,form the inflammatory bodies of NLRP3.Therefore,we hypothesized that NLRP3,activated by TLR4/NF-?B signaling pathway,may be involved in neuroinflammation in the hippocampus of fetal rats during the second trimester of pregnancy,furthermore,it leads to the impairment of learning and memory in offspring rats.The aim of this study was to investigate the effect of NLRP3 on learning and memory function of offspring rats by constructing MIUH model of SD rats in the second trimester of pregnancy.Methods: the experimental animals were healthy and clean SD rats at 17.5 days of gestation.Rats were randomly divided into 8 groups(n = 15,120 rats): Sham Group(Sham group);low placental perfusion group(MIUH group);NLRP3 inhibitor group(MCC950 group)NLRP3 inhibitor+MIUH group(MIUH + MCC950 group)NLRP3inhibitor group(MIUH + MCC950 group)TLR4 inhibitor group(TAK242 group);TLR4 inhibitor +MIUH group(TAK242+MIUH group);NF-?B inhibitor group(BAY 11-7082 group);NF-?B inhibitor +MIUH group(MIUH + BAY 11-7082Group).NLRP3 inhibitor MCC950 was injected intraperitoneally into pregnant rats in group MCC950 and group MIUH + MCC950,TLR4 inhibitor TAK242 in group TAK242 and group MIUH + TAK242,NF-?B inhibitor BAY 11-7082 in group BAY11-7082 and group MIUH + BAY 11-7082,the other groups were injected with the same amount of blank solvent.A mild placental hypoperfusion model was established in the second trimester of pregnancy.A 0.24 mm inner diameter metal micro-spring coil was wrapped around the uterine artery and the ovarian uterine artery in the anesthetized and sterilized pregnant rats.After the model was successfully established,the fetal rat brain tissue was harvested at specific time points(12 h after MIUH treatment,24 h after treatment,48 h after treatment,P0 on the day of the rat offspring),and the gene expression of NLRP3 and TLR4/NF-?B in the fetal rat brain tissue was detected by q PCR,the expression of Caspase1 P10 and pro-Caspase1 was detected by Western Blot,and Tunel + Caspase-1 immunofluorescence staining was performed in fetal rat brain to determine the level of neuronal scorch.On the 28 th day after birth,P28,the learning and memory function of the offspring were tested by Morris water maze test.Results: 1.MIUH in the second trimester of pregnancy resulted in focal activation of brain cells,q PCR showed that the expression of NLRP3 was up-regulated,and the expression of NLRP3,ASC,caspase1 p10 and pro-caspase 1 were increased in Western blotting.Immunofluorescence staining showed that TUNEL + caspase-1immunofluorescence double staining cells increased,the results were statistically significant.2.Using the inhibitor of NLRP3 inflammatory body MCC950 can reduce the anxiety death in the fetal rat brain and alleviate the damage of learning and memory function in the MIUH offspring.Compared with MIUH Group,MCC950 +MIUH group decreased the proportion of immunofluorescence double-staining cells,the swimming distance decreased in Morris water maze navigation experiment,the escape latency decreased on the 5th day,and the times of crossing platform increased in space exploration experiment,target quadrant extended duration of stay.3 ?TLR4/nf-?b signaling pathway regulates NLRP3-mediated apoptosis and has long-term effects on the neurodevelopment of offspring rats.The expression of TLR4/NF-?B gene in the fetal rat brain was increased by mild placental hypoperfusion in the second trimester of pregnancy.The TLR4 inhibitor TAK242 and NF-?B inhibitor BAY 11-7082 group were used before MIUH.Compared with the simple MIUH group,the expression of TLR4/NF-?B inhibitor NLRP3,ASC was induced by placental hypoperfusion,the up-regulation of Caspase1 P10 and pro-Caspase1 showed that the ratio of scorched cells decreased,and the offspring rats performed better in the experiment of space navigation and space exploration.Conclusion:Mild Intrauterine Hypoperfusion during the second trimester of pregnancy may activate TLR4/nf-?b pathway and then lead to the activation of NLRP3 inflammatory body pyrogenesis pathway in the fetal rat brain,which may result in the impairment of learning and memory function in the offspring.