The Effect Of DHA Supplementation Duration Pregnancy On The Expression Of TLR4 And NF-κB In Intrauterine LPS Exposed Immature Rat Brain

Objective In the resent years,epidemiologic and experimental findings showed there wasa close association between proinflammatory cytokines production and intrauterineinfection-induced brain damage.LPS,as a giant molecule on the cell wall of Gram negativebacteria,is one of the important etiological factors during intrauterine infection.LPS cancross through the placenta and induce cytokine precurosor increasing.Monocyte of fetalcirculation product IL- 1β,TNF-α,thus the permeability of the blood brain barrier increase.LPS and cytokine can pass the blood brain barrier and get into fetal central nervous system,evoking LPS mediated gitter cell activation.These inflammatory responses induce nervecell apoptosis and oxidative damage,and finally result in neonatal brain white matterdamage.The present study will establish the intrauterine infection rat model and probe intothe relationship between neonatal brain damage and inflammatory response throughidentifying the expression of TLR4,NF-κB and TNF-αmRNA in fetal and neonatal brain.Methods In the present study,Sprague-Dawley (SD) rats (n=20) at day 17 and 18 ofgestation underwent treatment for two days.We set up the intrauterine infection rat model(LPS-treated group) through intraperitoneal LPS administration (350μg/kg.dX2),while thecontrol group was administrated of the same volume of pyrogen-free saline (NS-treatedgroup).Brain tissues were collected from the fetal rat at pregnant day 20,21 and neonatalrat at postnatal day 1 (P 1),P3,P7 and P 14.Real-time quantitative polymerase chain reaction(RT-PCR) analysis was used to test mRNA expression for TLR4,NF-κB and TNF-αandWestern blotting was used to evaluate the TLR4,NF-κB and TNF-αexpression in thebrain tissues.Results Pregnant rats of each group had normal activity and feeding after intraperitonealinjection.One pregnant rat was dead,four pregnant rats were aborted and one had still birthin the LPS-treated group.The mean birth weight of the rats in the LPS- treated group was significant lower than the control group (p＜0.05).Placental hematoxylin-eosin staining showed remarkable interstitial hyperplasia,narrow capillary andinflammatory infiltration.Brain hematoxylin-eosin staining showed cellular edema,tissueraritas and cell population decreased.Protein express showed TLR4 in LPS-treated groupwas significantly increased compared to that of the control group (F=71.148,p=0.001).NF-κB expression in LPS-treated group was significantly higher than the control group(F=47.844,p=0.002).TNF-αin the LPS-treated group was significantly increased than thatof the control group (F=16.863,p=0.015),and the mean values of the three proteins in LPS-treated group were much higher when compared to the control group.The expression ofTLR4,NF-κB,TNF-αmRNA in LPS-treated group were significantly increased comparedto that of the control group in G20,G21,P1,P3,P7 rat brain with real-time PCRmeasurements (p＜0.05).There was no significant difference on TLR4,NF-κB and TNF-αmRNA levels between the LPS-treated group and the NS-treated group at the P14neonatal rat brain (p＞0.05).Conclusions Intrauterine LPS exposure could induce an inflammatory response of theplacenta and the fetus,then activate the intracephalic immune system.Activation of gittercell and LPS mediated TLR4 signal pathway could induce the releasing of inflammatoryfactor greatly,and eventually result in brain injury.After intraperitoneal injection of LPS,the express of inflammatory factors were remarkably increased in the fetus and neonatalbrain in P1,P3 and P7.But there was no significant difference in the expression ofproinflammatory factors in the neonatal brain in P 14 between the LPS-treated group and theNS-treated group.This suggested that the early inflammatory cascade response played animportant role in the pathogenesis of neonatal brain damage. Objective In the resent years,clinical and animal experimental findings showed therewas a close association between inflammatory reaction,intrauterine infection andneonatal brain damage.Gitter cells in the brain were activated and then LPS signal pathwaywas activated after intrauterine infection.TLR4 played a very important role in the LPSsignal transduction pathway.It induced nuclear factor (NF-κB) activation andproinflammatory cytokines synthesis and release.Inflammation cascade reaction in thebrain resulted in oligodendrocyte progenitor cell apoptosis,horizontal cell proliferation andhypomyelination.These inflammatory response finally resulted in neonatal white matterdamage.DHA is one of theω-3 polyunsaturated fatty acid.It is one of the importantcomponents of cerebral cellular membrane,which has antioxydation function.Intragastricadministration of fish oil to the pregnant rats during the whole pregnancy in this currentstudy.We will probe the effect of supplying DHA on the neonatal rat brain of intrauterineLPS exposure duration of pregnancy.Methods In the current study,Sprague-Dawley (SD) rats (n=20) at day 17 and 18 ofgestation underwent operation continue two days.We set up the intrauterine infection ratmodel (LPS-treated group) through intraperitoneal LPS administration (350μg/kg/d).Pregnant Sprague-Dawley rat were divided into three groups:control group (LPS-treatedgroup,freedom drinking water and eating);DHA1-treated group (intragastricadministration of half pill of fish oil during the whole pregnancy);DHA2-treated group(intragastric administration of one pill of fish oil during the whole pregnancy).Braintissues were collected from the fetal rat at pregnant day 20,21 and neonatal rat at postnatalday 1 (P1),P3,P7 and P14.Real-time quantitative polymerase chain reaction (RT-PCR)analysis was used to test mRNA expression for TLR4,NF-κB and TNF-αand Westernblotting was used to evaluate of TLR4,NF-κB and TNF-αexpression in brain tissues.Results Pregnant rats of each group had normal activity and feeding after intraperitonealinjection.Three pregnant rats were aborted in the LPS-treated group.One pregnant rat was aborted in the low dose DHA-treated group.There was no pregnant rat aborted in the largedose DHA-treated group.There was no pregnant rat died in the three groups.The meanbirth weight of the LPS-treated group neonatal rats was significant lower than the DHA-treated group (p＜0.05).Placental hematoxylin-eosin staining showed obvious inflammatoryresponse in the LPS-treated group.Inflammatory infiltration of the placental in the DHA-treated group was decreased.Brain hematoxylin-eosin staining showed cellular edema,tissue raritas and cell population decreased in the LPS-treated group.Brain cellular edemawas relieved and cell population was not obviously decreased in the DHA-treated group.Protein express showed TLR4 in DHA-treated group was significantly increasing than inLPS-treated group (F=38.944,p＜0.001).NF-κB in DHA-treated group was significantlyincreasing than in LPS-treated group (F=18.997,p=0.003).TNF-αin DHA-treated groupwas significantly increasing than in LPS-treated group (F=22.556,p=0.002).Real-timePCR showed general average of TLR4,NF-κB and TNF-αmRNA in DHA1 and DHA2treated group were significantly decreasing than in LPS-treated group.But there wasdistinction at different time point.Conclusions Intrauterine LPS infection could induce an inflammatory response ofplacenta and fetus,then activate LPS mediated TLR4 signal pathway.The inflammatoryresponse finally resulted in brain injury.DHA was one of the main components of fish oiland indispensable polyunsaturated fatty acid in the central nervous system.It played animportant role in neural development and function.Fetus and neonatal brain cellular edemawas decreased and cell population was increased after supplement of DHA during thewhole pregnancy.Protein of TLR4,NF-κB,TNF-αand TLR4,NF-κB,TNF-αmRNA weredecreased significantly.We proposed that DHA is highly anti-inflammatory by targetingLPS mediated intrauterine infection.DHA could modulate the expression ofproinflammatory cytokines in the brain.