Gestational LPS Exposure Induces ASD-like Behaviors In Offspring By Reducing DNA Hydroxymethylation In The Cortex

Objective Bacterial lipopolysaccharide（LPS）belongs to Gram-negative bacteria and widely exists in the intestine.Therefore,the human has been exposed to low-dose LPS,LPS enter the blood circulation in the process of drinking,trauma and infection.Some studies suggest that exposure to LPS during gestation leads to embryo loss,growth retardation,disorder of glucose and lipid metabolism,abnormal neurobehavior and so on.This study mainly explored the influence of LPS exposure during gestation on autism like behavior in offspring.Methods Experiment 1: 8 weeks ICR female and male mice.One week of acclimatization in the animal room,female and male mice were mated at 4:2 to produce12 pregnant mice.The newly detected vaginal plug was designated as GD0.12 pregnant mice were divided into two groups,one was the treatment group and the other was the control group according to their body weight in GD13,with 6 pregnant mice in each group.The control group was i.p.with normal saline according to 1% of body weight in GD13,and the treatment group was intraperitoneally injected with LPS（50 μg/kg）according to 1% of body weight in GD13,and the diet and body weight of mice were monitored every day.Wait for the mother rats to give birth naturally.The first day of postnatal day（PND）is recorded as PND 1.Adjust between nests in PND 4 to ensure that there are 8 offspring in each litter,including 4 females and 4 males.Offspring were weaned at PND21,behavioral experiments were performed at PND28.Two mice were randomly selected from each litter of the same sex,and were carried out in the order of stereotype behavior experiment,three-box social experiment,cross elevated experiment,open field experiment,sugar water preference experiment,tail suspension experiment and forced swimming experiment.After two weeks of food and water,the cortex of the mice was collected.Experiment 2: 8 weeks ICR female and male mice.12 pregnant mice were mated according to 4:2.The newly detected vaginal suppository was recorded as GD0.12 pregnant mice were divided into two groups.Cortex of fetal mice were reserved in GD14,GD16 and GD18 respectively.The cortex of GD14,GD16,GD18 and 8 weeks were obtained.The whole genome DNA hydroxymethylation level was detected by LC-MS/MS.The cortex of GD16 were randomly selected for genomewide transcriptome analysis.The cortex of GD16 and 8 weeks were randomly selected.First extract tissue RNA,and the m RNA level was detected by RT-PCR.The cortex of GD16 and 8 weeks were randomly selected.The hydroxymethylation level of specific gene sites was analyzed by APOBEC coupled epigenetic sequencing.Cortex of GD16 were randomly selected,and the hydroxymethylation level of specific gene site was analyzed.In addition,20 mg of GD16 fresh cortex was weighed.The nuclear protein（maintaining enzyme activity）was extracted and quantified with the kit,and the TETs enzyme activity was detected.Results Behavioral studies were performed at four weeks in mice,and marble burying test（MBT）was used to detect whether the mice had stereotyped behavior.The MBT results showed that compared with the control group,the male mice in the treatment group had statistically different stereotyped behaviors.Compared with the control group,the females in the treatment group were smaller than those in the control group.The three-box social experiment was used to test the social ability of the mice.The results of the three-box social experiment showed that compared with the control group,the male mice in the treatment group had lower social preference;compared with the control group,reduced social novelty in female mice in the treatment group.The elevated plus maze（EPM）results showed that compared with the control group,both male and female mice in the treatment group stayed longer in the closed arm and shorter in the open arm,indicating that both male and female mice had anxiety-like behaviors.Open field test（OFT）results showed that compared with the control group,male mice in the treatment group raised both front feet more times.The results of sucrose preference test（SPT）showed that compared with the control group,the female mice in the treatment group consumed lower percentage of sugar water,indicating the existence of depression-like behaviors.The results of forced swim test（FST）showed that compared with the control group,there was no statistically significant difference in the immobility time of male and female mice in the treatment group.The results of genome-wide hydroxymethylation analysis of cortex at GD14,GD16,GD18 and 8 weeks showed that compared with the control group,the GD16 genome-wide hydroxymethylation of the treatment group was statistically different and the level was reduced.The results of genome-wide transcriptome analysis showed that compared with the control group,the transcript levels of Bmp6,Col4a1,Igf2,Mmp2,Lpar1 and Gstp1 were decreased in the treatment group,which was verified by real-time quantitative PCR.By APOBECcoupled epigenetic sequencing,the site hydroxymethylation levels of Bmp6,Col4a1,Igf2,Mmp2,Lpar1 and Gstp1 were detected.Compared with the control group,the cortex of GD16 in the treatment group showed Col4a1,Igf2,Lpar1 and Gstp1 There are statistical differences between the sites.Compared with the control group,the cortex of the treatment group at 8 weeks showed statistical differences in Bmp6,Col4a1 and Igf2 sites.Mechanistic analysis at GD16 showed that compared with the control group,there was no difference in Tets m RNA levels,but alpha-ketoglutarate（alpha-ketoglutarate,AKG）levels decreased,Idh1 m RNA levels decreased,and TETs enzymatic activity decreased.Conclusion In conclusion,LPS exposure during pregnancy reduces the hydroxymethylation of Bmp6、Col4a1、Igf2、Mmp2、Lpar1 and Gstp1 through TETs,and increases the risk of autism in offspring.