| ObjectiveThe abnormal glucose metabolism model of maternal substitute was established through high-fat diet(HFD)to study the effect of maternal HFD on the expression of intestinal flora,ZO-1 and Occludin proteins in the offspring.By changing the delivery mode(natural delivery or cesarean section)and lactation mode(self-lactation or cross-lactation),the transmission of maternal and infant flora was blocked to explore the effects of adverse intrauterine environment and mother-to-child transmission on the offspring flora and intestinal barrier function.Methods1.A cross-generation C57BL/6J mouse model was established There were 120 female and 40 male C57BL/6J rats aged 7 weeks(7W),and they were fed adaptively for 1 week.Subsequently,female rats were randomly divided into high-fat diet groups(HFD groups,60%fat,5.24 kcal per gram of fat)and normal Chow diet groups(NCD groups,5.3%fat,3.90 kcal per gram of calories).After 8 weeks of intervention with high-fat diet,female rats were subjected to oral glucose tolerance test((oral glucose tolerance test,OGTT)to verify the success of high-fat modeling.After successful modeling,male and female mice mated in a ratio of 3:1.The observation of vaginal plug was recorded on the first day of gestation,and the female mice were fed in a single cage during pregnancy and lactation.To ensure balanced nutrition between cages,the number of mice in each cage was adjusted to 5 after delivery.2.Grouping of progenyThe first step is to study the influence of maternal high-fat diet on progeny in the state of vaginal delivery(VD),and its progeny can be divided into NC/VD/NC group(normal feeding mother rats/natural childbirth/offspring of normal breast milk group)and HF/VD/HF group(progeny of high-fat diet mother rats/natural childbirth/offspring of high-fat breast milk group).The second step is to interrupt the vaginal delivery of the cesarean section(CS)to study the effect of the change of delivery mode on the offspring,and the offspring are divided into NC/CS/NC group(rats and rats fed normal diet/cesarean section/offspring of normal breast milk group)and HF/CS/HF group(rats fed with high fat diet/cesarean section/offspring of high fat breast milk group).Third,cross-lactation was performed to block the transmission of mammalian flora,so as to study the influence of the change of lactation mode on the offspring.The offspring were divided into NC/VD/HF group(normal diet mother rats/natural childbirth/high-fat breast milk offspring)and HF/VD/NC group(high-fat diet mother rats/natural childbirth/normal breast milk offspring).The fourth step was to study the influence of the changes in both delivery and lactation modes on offspring,and divide the offspring into NC/CS/HF group(normal diet mother rats/cesarean section/high-fat breast milk group offspring)and HF/CS/NC group(high-fat diet mother rats/cesarean section/normal breast milk group offspring).The offspring were weaned at 3 weeks and fed with normal diet until 8 weeks.3.Weigh the mother rats and their offspring every week,and record the amount of food and water intake.OGTT was performed at 4 and 8 weeks in offspring4.Fecal collection and sequencing analysis of the 16S rRNA gene V3-V4 region of the intestinal flora in offspring at 4 and 8 weeks of age:After the collected feces were put into the sterilized frozen storage tube of 2ml corresponding rat number and time point,the frozen storage tube was immediately put into liquid nitrogen for quick freezing.Waste collection after intestinal flora α-diversity analysis.5.The expressions of ZO-1 and Occludin were determined by immunohistochemistry in children aged 8 weeksResults1.The maternal high-fat diet resulted in weight gain(P<0.05),and the glycemic values of OGTT at each time point were significantly higher than those of NCD group(P<0.05).2.Influence of maternal high-fat diet on offspring After weaning,the weight of offspring in the high-fat group was significantly higher than that in the normal group.In 4w and 8w offspring,there was no significant difference in the richness and diversity of intestinal flora(P>0.05).OGTT results showed that the blood glucose concentration in the high-fat group at 120min was higher than that in the normal group(P<0.05).Immunohistochemical results showed that the offspring of the high-fat group were lower than those of the normal group(P<0.05).3.Influence of changes in delivery mode(vaginal delivery/Caesarean section)on offspringBefore weaning,there was no significant difference in weight between the two groups.After weaning,the weight of offspring in NC/CS/NC group was higher than that in NC/VD/NC group.OGTT results showed that compared with the HF/VD/HF group,the glucose tolerance of the HF/CS/HF group was decreased(P<0.05).Among 8W offspring mice,the richness and diversity of the HF/CS/HF group were significantly higher than that of the HF/VD/HF group(P<0.05).4.Influence of changes in lactation mode(self-lactation/cross-lactation)on offspringCompared with the VD group,the offspring of the NC/VD/HF group were heavier,and the offspring of HF/VD/NC group were lighter(P<0.05).OGTT results showed that compared with the VD group,the glucose tolerance of the NC/VD/HF group was increased,while the glucose tolerance of the HF/VD/NC group was lower(P<0.05).Among female offspring mice aged 4 weeks,the richness of intestinal flora in the NC/VD/HF group was significantly lower than that in the NC/VD/NC group(P<0.05).In addition,the intestinal flora richness of the offspring mice in the HF/VD/NC group was significantly higher than that of the NC/VD/HF group(P<0.05),and at 8 weeks,the intestinal flora richness of the mice in the NC/VD/NC group,NC/VD/HF group,HF/VD/HF group and HF/NC group tended to be consistent.Immunohistochemical results showed that the expression levels of colonic tight junction proteins ZO-1 and Occludin in cross-lactation offspring were lower than those in the vaginal delivery group(P<0.05).The changes in both production and lactation methods resulted in increased intestinal flora richness in offspring,which was significantly higher than that in HFD group(P<0.05).5.The influence of both delivery and lactation on offspringCompared with the VD group,the weight of the NC/CS/HF offspring was higher,while that of the HF/CS/NC offspring was significantly lower(P<0.05).OGTT results showed that compared with the VD group,the glucose tolerance was increased in the NC/CS/HF group and decreased in the HF/CS/NC group(P<0.05).The changes in both production and lactation methods resulted in increased intestinal flora richness in offspring,which was significantly higher than that in HFD group(P<0.05)Conclusions1.The high-fat diet of the mother generation causes abnormal glucose tolerance of the offspring,which destroys the integrity of the intestinal mucosal barrier of the offspring and increases the intestinal permeability2.Changes in delivery patterns significantly affected the richness and diversity of intestinal flora.It may affect the health of the offspring by blocking the transmission of harmful vaginal bacteria to the offspring in the adverse intrauterine environment group,improving the glucose tolerance of the offspring and blocking the transmission of beneficial vaginal bacteria to the offspring in the normal intrauterine environment group3.The change of lactation mode significantly affected the abundance of ntestinal flora in offspring.Cross-lactation promotes the transmission of beneficial bacteria from the bad intrauterine environment group to the offspring mproves the glucose tolerance of the offspring,and promotes the transmission of harmful bacteria from the normal intrauterine environment group to the offspring,which may affect the health of the offspring.4.Changes in both delivery and lactation modes significantly changed the abundance of intestinal flora in offspring.This method blocked harmful vagina bacteria in the adverse intrauterine environment group,promoted the transmission of beneficial bacteria from milk to offspring,and improved the glucose tolerance of offspring.Blocking the healthy vaginal bacteria in the normal intrauterine environment group promotes the transmission of harmful bacteria from milk to offspring,which may affect the health of offspring. |
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