| Objective To observe the effects of prenatal inflammation induced by lipopolysaccharide(LPS)on ileal tissue development and colonization of intestinal flora neonatal rats within one week of birth,and to explore the possible mechanisms of necrotizing enterocolitis(NEC)caused by prenatal inflammation.Methods Female SD rats with sexual maturity and no reproductive history were selected.Twenty SD pregnant rats were randomly divided into the LPS group(n=15)and the control group(n = 5).0.7 mg / kg LPS solution and an equal volume of sterile saline were injected intraperitoneally at the 15 th day of gestation.1.The postoperative response of pregnant rats were observed closely,and the delivery status of pregnant rats(death,newborns,litter size,birth weight)were recorded.2.At 1d,3d,and 7d,the distal ileum tissue samples of newborn rats were collected for Hematoxylin-Eosin(HE)staining and Zona Occludens-1(ZO-1)immunehistochemistry staining.3.At 3d and7 d,the colonic contents of newborn rats were collected and extracted the total bacterial DNA.The V3-V4 region of the 16 S rRNA gene was amplified by PCR.Illumina Miseq platform was used for double-end sequencing to analyze the composition and diversity of intestinal flora.Results 1.The average body weight of SD pregnant rats in the LPS group was 274.6±22.9g,and the average body weight of SD pregnant rats was278±22.4g in the control group,there was no statistically significant difference(P=0.82).The number of births/litter in the LPS group was 9.6±4.45,and the number of births/litter in the control group was 10±4.42,the difference was no statistically significant(P=0.89).The birth weight of newborn rats in LPS group was 6.5±0.73 g,and the birth weight of babies in control group was 6.7±0.69 g,there was not statistically significant difference(P=0.12).2.HE pathological staining: In all the control groups,the overall structure of the distal ileum was basically normal,the structure of the intestinal villi was intact and the arrangement was neat and without apparent inflammatory cell infiltration.In the LPS group:1d,the intestinal tissue disorder,villous atrophy,epithelial cell edema severely and submucosal edema with infiltration of inflammatory cells;3d,most of the epithelial cells were severely edema,and some intestinal tissue showed a large number of infiltration of inflammatory cells in the submucosa,which was significantly worse than 1d;7d,intestinal epithelial cells were severely edema,and party of intestinal submucosa showed a small amount of inflammatory cell infiltration,which was significantly reduced compared with 1d and 3d.At 1d,the ileal mucosal injury score of the LPS group was slightly higher than that of the control group,but the difference was not statistically significance(P=0.11);At 3d,the ileal mucosal injury score of the LPS group was significantly higher than that of the control group,the difference was statistically significant(P=0.04);At 7d,the ileal mucosal injury score of the LPS group was not difference compare with that of the control group(P=1.00).3.The intestinal epithelium ZO-1 protein in the control groups was brown and uniformly distributed at the junction of ileal epithelial cells,and the ZO-1protein staining in the ileal tissue of the LPS groups was weak and unevenly distributed,and showed discontinuous spots or short bands;compared to control groups,intestinal ZO-1 expression decreased significantly at 1d and 3d(1d P =0.005;3d P = 0.003,respectively).At 7d,we also observed that the expression level of ZO-1 in the LPS group was slightly lower than the control group,but the difference was no statistical significance(P = 0.33).4.(1).All optimized sequences were annotated based on similarity<97%,and a total of 6 phylums,9 classes,16 orders,30 families,59 genus,74 species,and 88 OTUs were obtained.(2).In the control group,the Shannon diversity index at 7d was higher than that at 3d,there was statistically difference(P =0.02);and in the LPS group,the shannon diversity index at 7d was also higher than that at 3d,but the difference was not statistically significant(P=0.06).(3)(1).At the Phylum level,with the change of age,the relative abundance of Firmicute in the control group increased from 49.5% to 91.6%,the difference was statistically significant(P=0.01),while the relative abundance of Proteobacteria reduced from 50.0% to 7.9%,the difference was statistically significant(P=0.01);In the LPS group,the relative abundance of Firmicute and Proteobacteria was not significantly difference with the change of age(both P>0.05);At 7d,the relative abundance of Proteobacteria and Bacteroidetes in the LPS group were significantly higher than those in the control group(27.1%vs 7.9%,P = 0.01;0.8% vs 0.003%,P=0.04,respectively).The relative abundance of Firmicute was significantly lower than that of the control group(71.6% vs 91.6%,P=0.01).(2).At the genus level,the Lactobacillus,Shiga-Escherichia,Streptococcus,and Rodentibacter whom relative abundance≥1%were dominant bacteria in all groups.With the change of age,the relative abundance of Lactobacillus in the control group increased from 39.0% to 89.1%,the difference was statistically significant(P=0.01),the relative abundance of Shiga-Escherichia decreased from 48.1% to 4.4%,the difference was statistically significant(P =0.01);and the relative abundance of Streptococcus decreased form 8.5% to 1.3%,the difference was statistically significant(P =0.01).At 7d,the relative abundance of Shiga-Escherichia in LPS group were significantly higher when contrast to the control group,the difference was statistically significant(24.3% vs 4.4%,P=0.01);The relative abundance of Lactobacillus and Rodentibacter in the LPS group were significantly low while compare to the control group,the differences were all statistically significant(67.3% vs 89.1%,P = 0.01;1.0% vs 3.6%,P = 0.04,respectively).Conclusion1.LPS Exposure during pregnancy can affect the intestinal development of neonatal rats and destroy the integrity of the intestinal epithelium.This effect on the gut can last for more than a week.2.LPS exposure during pregnancy can change the structure of early colonization of intestinal flora of newborn rats,delay colonization of beneficial bacteria,and promote colonization of potential pathogenic bacteria.3.Prenatal inflammation may destroy the intestinal integrity of offspring and causes intestinal bacterial disorders,which may be a possible mechanism of the occurrence of offspring NEC. |
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