| OBJECTIVE To establish a FGR model of the Nrf2 knockout mouse during pregnancy,and analyze the effect of Nrf2 on offspring body weight and lung development,and explore the genetics mechanism of FGR.METHODS 1.Animal model: 7-weeks SPF C57/BJ6 Nrf2 wild type(Nrf2+/+),heterozygous(Nrf2+/-)and knockout(Nrf2-/-)female and male mice.After a week adaptive breeding,females were grouped into WT,H and KO that according to their genotypes.Females and males were 1:1 matched.The matching methods : WT group: Nrf2+/+ vs.Nrf2+/+;H group: Nrf2+/-vs.? Nrf2-/-;KO group: ? Nrf2-/-vs.? Nrf2+/-.The vagina was examined at 8:00 the next day.If Vaginal plug and/or vaginal secretions were found sperms under a smear microscope,it is records as 0.5 day of pregnancy(E0.5d)and weights its weight as W0.5d.Pregnant mice were randomly divided into a tobacco exposure group(CSE)or a control group(CON)and recorded as: CSE-WT group,CSE-H group,CSE-KO group,CON-WT group,CON-H group,and CON-KO group,then feeds to E17.5d.At 8:00 on E17.5d,pregnant mice were weighed(W17.5d)and sacrificed.The heart blood,fetal lungs,and fetal body weight and length were recorded.2.Testing serum parameter of the pregnant: cotinine,MDA,blood glucose,estradiol,testosterone,TC,TG,LDL and HDL.3.Fetal lung was stained with HE and analyzed the development differences.4.Genome-wide transcriptome sequencing on lung tissues of female fetal mice in CON-H and CSE-H groups and analyze differentially expressed genes.5.The results of sequencing were verified by RT-PCR,and analyzed the differentially expressed genes and explored the possible mechanisms of FGR.RESULTS 1.1 The fetuses weight was decreased which in the Nrf2 knockout rats during pregnant: the fetal weight on E17.5d,the CON-KO and CON-H was significantly lower than the CON-WT(p<0.05),and the CON-KO lower than the CON-H(p<0.05);Similarly,the CSE-KO and CSE-H was significantly lower than the CSE-WT(p<0.01),and the CSE-KO was lower than the CSE-H(p<0.05).This difference is even more pronounced in tobacco exposure.1.2 The incidence of FGR was increased which in the Nrf2 knock-out rats during pregnant:The CON-KO was significantly higher than the CON-WT and CON-H(p<0.05);The CSE-WT was significantly lower than the CSE-KO and CSE-H(p<0.05);The CSE-H significantly higher than the CON-H(p<0.05).The CSE-KO and CSE-WT were higher than those the CON-KO and CON-WT,but the difference was not statistically significant(p>0.05).1.3 The adverse pregnancy outcomes was not increased which exposed to CSE in the Nrf2 knock-out rats during pregnant:Mortality: The CON-KO significantly higher than the CON-WT(p<0.05);The CSE-KO and CSE-H significantly higher than the CSE-WT(p<0.05);There is no significant difference between CON and CSE(p >0.05).Malformation : There was no significant difference between the six groups(p>0.05).1.4 There was no significant difference in weight gain and litters which in the Nrf2 knock-out rats during pregnant:Weight gain: The CON-KO was significantly lower than the CON-WT(p<0.01),the CSE-KO and CSE-H were significantly lower than the CSE-WT(p<0.01).Litters: The CON-KO was significantly lower than the CON-WT(p<0.01),and the CSE-H was significantly lower than the CSE-WT(p<0.01).There was no significant difference between the other groups.1.5 It did not affect the number of live litters which in the Nrf2 knock-out rats during pregnant:There was no significant difference in gender ratio between males and females(p>0.05),but female fetuses were generally lower in body weight than male fetuses,and the offspring Nrf2-/-fetal rats were significantly lower than those of Nrf2+/-fetuses(p<0.05).1.6 Abnormal serum metabolism in pregnant mice which in the Nrf2 knock-out rats during pregnant:The concentration of cotinine in the CSE was significantly higher than the CON.The CSE-H and CSE-KO were significantly higher than the CSE-WT.The MDA level in the CSE was higher than that in the CON,and the CON-KO was significantly higher than the CON-WT.LDL levels were significantly higher in the CON-H than in the CON-WT,and also higher in the CSE-H than the CSE-WT.HDL levels in the CSE-KO were lower than the CSE-WT,and the CON-KO was also lower than the CON-WT;E2 levels in the CSE-H and CSE-WT were higher than those in the control group,CON-KO was higher than CON-WT,and T levels CSE-KO was higher than the CSE-WT.There was no significant difference between serum glucose,TC and TG in groups.2.1 Fetal lung tissue HE staining revealed abnormal lung bronchial development in Nrf2 knock-out fetal mice: CON-H progeny Nrf2-/-female(KO1C)bronchi hyperplasia,CSE-H progeny Nrf2-/-female(KO1T)Hyperplasia and malformation of the bronchi.2.2 Fetal lung tissue transcriptome sequencing revealed differences in Wnt signaling and immune-related gene expression: There were 15 genes significance betweenin fetal lungs(KO1C and KO1T)(p<0.05),among which 13 genes were up-regulated.(fold change >2.0),2 gene down-regulate(fold change < 0.5).Metascape cluster analysis revealed that differentially expressed genes are mainly associated with abnormal expression of subreduction,Wnt signaling,and immune cytokines.2.3 RT-PCR suggested that Nrf2-null mice affected lung development abnormalities: Wnt4 expression was significantly increased after Nrf2 deletion(H1C vs.KO1 C,p<0.001),and maternal exposure to fetal rats was also expressed significantly elevated(H1C vs.H1 T,p<0.01);Wnt5a showed down-regulated expression in maternal tobacco exposed fetuses(H1C vs.H1 T and KO1 C vs.KO1 T,p<0.05);and Wnt5 b,GSK3?,and Fzd were abnormally expressed in Nrf2-deficient fetuses(p<0.05).Ydjc gene expression was up-regulated after Nrf2 deletion(H1C vs.KO1 C,p<0.05),increased in maternal-exposed fetal mice(H1C vs.H1 T,p<0.05),and exposure to maternal tobacco expression was up-regulated in Nrf2-deficient fetuses(KO1C vs.KO1 T,p<0.05).The Scgb1a1 and Foxj-1 genes were up-regulated after Nrf2 deletion(H1C vs.KO1 C,p<0.05)and were significantly increased in maternal-exposed fetal mice(H1C vs.H1 T,p<0.05).The immune-related Tgtp2 and Fgl2 gene expression also showed differences.CONCLUSION 1.Nrf2-/-mice is a viable FGR modeling method.2.There were gender-different that the effect of Nrf2 on fetal weight.The female fetuses had a serious effect on body weight than male,and maternal Nrf2 deletion had more critical effet on fetal weight than paternal Nrf2 deletion.3.Exposed to CSE in the Nrf2 knock-out rats during pregnant can lead serum LDL,estradiol,and testosterone elevated,and HDL reduced.4.Abnormal lung bronchial development in Nrf2 null fetuses and may be related to Wnt signaling differentiation genes. |
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