Silencing Of CRNDE Alleviates Brain Injury In Offspring Rats With Intrauterine Infection Induced By Lipopolysaccharide

Background: Infections during pregnancy can cause intrauterine inflammatory response syndrome in the fetus,which is the main risk factor for spontaneous preterm births and perinatal brain injury.The occurrence of brain injury in the early immature brain implies disruption of multiple developmental programs in a large number of nerve cells during a critical period of neurodevelopment,and its effects are considerable and can lead to a variety of adverse long-term neurodevelopmental outcomes,such as cerebral palsy,motor and mental retardation,cognitive learning disabilities,and emotional disturbances.There are evidences that intrauterine infection with chorioamnionitis can trigger inflammatory responses in the fetus peripherally and cranially,leading to excessive activation of astrocytes and microglia in brain and massive production of pro-inflammatory factors,thus inducing acute and chronic inflammatory responses in the nervous system,leading to brain damage and affecting brain development.Long noncoding RNA(lnc RNA),which can regulate gene expression at multiple levels including transcriptional and post-transcriptional levels,participate in various physiological and pathological processes in the body.lnc RNA has been shown to be essential for brain development and neurological disease formation.Colorectal neoplasia differentially expressed(CRNDE),an lnc RNA located on the long arm of human chromosome 16,plays an oncogenic function in a variety of cancers,including glioma.The expression of CRNDE is tissue and spatio-temporal specific and has been found to be extensively involved in multiple processes such as inflammation,injury,nerve growth and development.Recent studies have shown that CRNDE is highly expressed in traumatic brain injury(TBI)serum,and down-regulation of CRNDE can promote neural repair after TBI.Some in vitro and in vivo studies have confirmed the involvement of CRNDE in the regulation of inflammation,but the exact mechanism remains unclear.The expression of CRNDE in neonatal brain injury induced by intrauterine infection and the effect and mechanism of targeted regulation of CRNDE on intrauterine infection induced brain injury have not been reported.Nuclear factor-κB(NF-κB)is an important intracellular nuclear transcription factor involved in the response of cells to various stimuli such as inflammation and stress.NF-κB/IκB signaling pathway plays a key role in regulating the body’s immune response to inflammation.Nucleotide binding oligomerization domain-like receptors family Pyrin domain containing 3(NLRP3)is an important component of innate immunity.NLRP3 can be initiated by lipopolyside(LPS)induced NF-κB activation and play an important role in neuroinflammation.Inhibition of NLRP3 can reduce hippocampal injury and improve cognitive function.Some studies have speculated the relationship between NLRP3 and preterm birth,suggesting that premature activation of NLRP3 inflammasome in chorionic membrane and placenta is an important mechanism leading to preterm birth.Modulation of the NF-κB/NLRP3 signaling pathway is expected to be an effective means to prevent preterm birth and combat brain injury caused by intrauterine infection.In this study,we detected the expression of CRNDE in brain tissue of LPSinduced intrauterine infected neonatal rats;To investigate the effect of silencing CRNDE on brain injury caused by intrauterine infection.To investigate the role and possible mechanism of NF-κB/IκB/NLRP3 signaling pathway in silencing CRNDE to prevent and alleviates intrauterine infected brain injury.And to provide new ideas and strategies for early detection and prevention of intrauterine infection induced preterm birth and perinatal brain injury,and to improve the quality of life of high-risk infants.Part Ⅰ: Overexpression of CRNDE in brain damage of neonatal rats with intrauterine infection induced by LPSObjective:(1)To investigate the expression of CRNDE in amniotic fluid and serum of pregnant rats with LPS induced intrauterine infection at delivery.(2)To investigate the expression and role of CRNDE in brain injury of neonatal SD rats induced by LPS-induced intrauterine infection.Contents:(1)Established the intrauterine infection model of pregnant SD rats induced by LPS,and detected the expression of CRNDE in peripheral serum and amniotic fluid of pregnant SD rats at deliver.(2)Detected the expression of CRNDE in brain tissues of neonatal rats with intrauterine infection induced by LPS at different ages(postnatal 3,7,14 and 21,hereafter P3,P7,P14 and P21).Methods:(1)32 SPF SD rats were pregnant successfully.The pregnant rats were randomly divided into control group and test group,with 16 rats in each group.(2)On the 10 th day of conception,intrauterine infection was induced by single intraperitoneal injection of LPS(150μg/kg)in pregnant rats of the experimental group.Pregnant rats in the control group were injected intraperitoneally with equal volume of normal saline.(3)On the 21 st day of gestation,5 rats were delivered by cesarean section in the experimental group and the control group respectively.Serum and amniotic fluid samples of pregnant rats were collected,and placentas were taken for pathological section.The remaining pregnant rats delivered naturally.(4)Extract total RNA from peripheral serum and amniotic fluid;The purity and concentration of RNA in serum and amniotic fluid were measured;The expression of CRNDE in serum and amniotic fluid of pregnant rats was measured by quantification real-time quantitative PCR(q RT-PCR).(5)Brain tissue(n=5)of newborn rats of different age(P3,P7,P14,P21)after delivery was collected.Then,extract the whole brain RNA and measure the purity and concentration of RNA.q RT-PCR was used to measure the expression of CRNDE in neonatal rats of different age(P3,P7,P14,P21)after delivery.Results:(1)In the SD rat model of intrauterine infection induced by intrauterine injection of LPS in mid-pregnancy,all pregnant rats in the experimental group showed obvious sepsis-like behavioral changes such as depression,reduced activity,and reduced feeding within 1-2 hours after intraperitoneal injection of LPS,which lasted for 1-2 days.In the experimental group,1 pregnant rat died about 5 hours after LPS administration,1 abortion,1 stillbirth and 2 premature delivery.In the control group,pregnant rats behaved as usual after intraperitoneal injection of saline,and no death,miscarriage,stillbirth or premature births were observed.(2)Pathological sections of placenta of the pregnant rats in the experimental group all showed histopathological chorioamnitis.(3)The average number of litters born in the experimental group was 7 and the average birth weight of newborn rats was(3.0±0.4)g.The average number of litters born in the control group was 10 and the average birth weight of newborn rats was(3.6±0.5)g.The number of litters born in the experimental group decreased and the weight of newborn rats decreased.(4)The expression of CRNDE in serum and amniotic fluid was up-regulated in the experimental group,2.3-fold up-regulated in serum and 3.3-fold up-regulated in amniotic fluid compared with the control group.(5)Compared with the control group,The expression of CRNDE was up-regulated in the brain tissue of the the offspring rats in the experimental group at different ages(P3,P7,P14,P21),and there was an upward trendency with increasing ages.Conclusion:(1)Intrauterine infection model can be induced by single intrauterine injection of LPS(150μg/kg)in the second trimester of pregnancy.(2)CRNDE was highly expressed in amniotic fluid and serum of pregnant mice in intrauterine infection group at delivery.(3)Overexpression of CRNDE maybe aggravate the brain injury caused by intrauterine infection in SD neonatal rats.Part Ⅱ: Silencing of CRNDE alleviates Brain injury in offspring rats with intrauterine infection induced by LPSObjective:(1)To preliminarily investigate the cerebral protective effect of silencing CRNDE in offspring rats with intrauterine infection induced by LPS.(2)To explore the possible mechanism of cerebral protection in intrauterine infected offspring rats by CRNDE silencing.Contents:(1)Established an intrauterine infected rat model induced by LPS with CRNDE silencing.(2)Detected the effect of CRNDE silencing on the activation of astrocytes and microglia in intrauterine infected offspring rats.(3)Detected the brain protective effect and possible mechanism of silent CRNDE on intrauterine infection offspring rats.(1)Methods:(1)On the third day after delivery,18 pregnant rats in the test group were randomly divided into CRNDE shRNA group,Control shRNA group and blank control group(n=6).CRNDE shRNA(250μl/kg),Control shRNA(250μl/kg)and equal volume of saline were injected intraperitoneally once,respectively.(2)q RTPCR was used to measure the expression of CRNDE in the brain of 7-week-old offspring SD rats.(3)Morris water maze(MWM)was performed to test the spatial learning and memory ability of 7-week-old offspring SD rats.Sugar water preference test,open field test and forced swimming test were used to test depression and anxiety.(4)The brain histopathological changes and apoptosis of 7-week-old offspring were detected by hematoxylin and eosin(H&E)staining and terminal deoxynucleotidyl transferase d UTP nick end labeling(TUNEL)staining.(5)Immunohistochemistry was performed to assessed the activation of astrocyte and microglia.(6)m RNA and protein expressions of GFAP,IBA-1,TNF-α,IL-6 and IL-1β were measured by QRTPCR and Western blot.Results:(1)The escape time of MWM in the CRNDE shRNA group was significantly shortened,and the spatial learning and memory ability were improved compared with the control group.(2)Compared with the control group,the offspring of CRNDE silencing group showed no significant difference in sugar water preference,total distance of horizontal movement,retention time in central area,times of independence and duration of motionless swimming.(3)The brain histopathological changes in the offspring of CRNDE shRNA group were less than those in the control group.(4)The apoptosis of cells in brain in the CRNDE shRNA group was less than that in the control group.(5)Immunohistochemistry showed that the expression of GFAP and Iba-1 in hippocampal tissue was decreased,Knocked-down of CRNDE inhibited the activation of astrocyte and microglia.(5)m RNA and protein expressions of GFAP,IBA-1,TNF-α,IL-6 and IL-1β in hippocampal tissue of offspring rats in CRNDE silencing group were inhibited.Conclusion:(1)Silencing CRNDE improved spatial learning and memory ability,alleviated hippocampal injury and inhibited brain cell apoptosis in intrauterine infected offspring rats(2)Silencing CRNDE may exert a cerebroprotective effect by inhibiting the activation of astrocytes and microglia and reducing neuroinflammation.Part Ⅲ: Effect of NF-κB/IκB/NLRP3 signal pathway on brain protection by silencing CRNDE in offspring rats with LPS-induced intrauterine infectionObjective: To investigate the possible mechanism of NF-κB/IκB/NLRP3 signal pathway in brain protection of offspring rats with LPS-induced intrauterine infection by CRNDE silencing.Contents:(1)The m RNA and protein expression of NF-κB/IκB/NLRP3 in Control(LPS)group,LPS+Control shRNA group and LPS+CRNDE shRNA group were detected.(2)The m RNA and protein expression of NF-κB,IκB,NLRP3,TNF-α,IL-6 and IL-1β in hippocampal tissues of offspring rats were detected by introduced NF-κB inhibitor to silence CRNDE and inhibit NF-κB/IκB/NLRP3 signal pathway in harem.Methods:(1)Based on the experiments of Part Ⅱ,The m RNA and protein expressions of NF-κB,IκB and NLRP3 in the hippocampus of rats in the Control(LPS)group,LPS+Control shRNA group and LPS+CRNDE shRNA group were detected by q RT-PCR and Western blot.(2)NF-κB inhibitor NF-κB IN1 was introduced,and 36 offspring SD rats were divided into 6 groups with 6 rats in each group.The subgroups were as follows: NControl(Normal)group,LPS group,LPS+Control shRNA group,LPS+CRNDE shRNA group,LPS+CRNDE shRNA+DMSO group,LPS+CRNDE shRNA+NF-κBIN1 group.(3)LPS-induced Intrauterine infection by intraperitoneal injection LPS in pregnant SD rat on 10 days of gestation.Neonatal rats were intraperitoneally injected with CRNDE shRNA lentivirus 3 days after delivery to silence CRNDE expression.At 21 days of age,intraperitoneal injection of NF-κBIN1inhibited NF-κB.(4)Neurobehavioral Morris water maze test was performed on 7weeks of age.The m RNA and protein expressions of NF-κB,IκB,NLRP3,TNF-α,IL-6 and IL-1β in the hippocampal tissues of rats were detected by q RT-PCR and Western blot.Results:(1)Compared with LPS group,the m RNA and protein expression of NF-κB,IκB and NLRP3 in offspring rats of LPS+CRNDE shRNA group were significantly decreased.(2)Morris water maze test,the escaping time in offspring rats of LPS group and LPS+Control shRNA group were longer than NControl group;LPS+CRNDE shRNA group and LPS+CRNDE shRNA+DMSO group had shorter escaping time than LPS group;offspring rats in LPS+CRNDE shRNA+NF-κB IN1 group had shorter escaping time than that in LPS+CRNDE shRNA group,the difference was statistically significant.(3)The results of q RT-PCR and Western blot showed: Compared with NControl group,m RNA and protein expression of NF-κB,IκB,NLRP3,TNF-α,IL-6 and IL-1β in hippocampus of LPS group and LPS+shRNAControl group were higher than those of NControl group;The m RNA and protein expressions of NF-κB,IκB,NLRP3,TNF-α,IL-6 and IL-1β in hippocampus of LPS+CRNDE shRNA and LPS+CRNDE shRNA+DMSO groups were down-regulated compared with those of LPS group;with NF-κB IN1 introduced after LPS and CRNDE shRNA,in LPS+CRNDE shRNA+NF-κB IN1 group,the m RNA and protein expressions of NF-κB and IκB were not significantly changed,but the m RNA and protein expressions of NLRP3,TNF-α,IL-6 and IL-1β were down-regulated.Conclusion:(1)Silencing CRNDE inhibited the expression of NF-κB/IκB/NLRP3 in the hippocampus of offspring rats intrauterine infected.(2)Silencing CRNDE and inhibition of NF-κB/IκB/NLRP3 signal pathway improved the learning and memory function of offspring rats intrauterine infected.(3)CRNDE silencing down-regulated the expressions of TNF-α,IL-6 and IL-1β and alleviated neuroinflammation possibly by inhibiting NF-κB/IκB/NLRP3 signal pathway.