Effect Of Bifidobacterium Triple Viable Bacteria On Improving Maternal Leaky Gut And Protecting Offspring-islet Development And Maturation In Gestational Diabetic Mice

Objective:Gestational diabetes mellitus（GDM）brings serious short-term and long-term adverse outcomes to mothers and offspring,however,the safe and effective intervention measures for patients with mild and moderate GDM are limited in clinic.Intestinal flora disturbance and leaky gut are the important mechanisms that cause metabolic inflammation and aggravate glucolipid metabolic disorders in GDM.Bifidobacterium triple viable bacteria（BiLaEn）is composed of Bifidobacterium longum,Lactobacillus acidophilus and Enterococcus faecalis.It is clinically used to treat acute and chronic diarrhea and constipation caused by intestinal flora imbalance with good effect and no obvious adverse reactions.This study was mainly based on the disturbance of gut microbiota and leaky gut to investigate whether BiLaEn can alleviate metabolic inflammation and glycolipid metabolism in GDM maternal mice by improving intestinal flora and reducing“leaky gut”.At the same time,special attention was paid to the influence of GDM high metabolic stress on the development and maturation of islets in offspring mice,and to clarify whether BiLaEn had a protective effect.Methods:Thirty-six healthy eight-week-old female C57BL/6J mice（17-19g）were divided into three groups according to body weight and fasting blood glucose（FBG）:GDM model group（GDM,n=12）,low concentration of BiLaEn group（BiLaEn＿L,n=12,6.4×10⁶CFU/ml）and high concentration of BiLaEn group（BiLaEn＿H,n=12,1.28×10⁷ CFU/ml）.And female C57BL/6J mice at the same age was selected as normal control（Normal,n=12）.Since the female and male mice were co-caged,mice in BiLaEn＿L and BiLaEn＿H groups were given corresponding doses of BiLaEn,while mice in GDM and Normal groups were given the same amount of sterile distilled water until the maternal mice delivery.During the experiment,the condition of the pregnant mice was closely monitored and the body weight was recorded once a week.On day 15 of pregnancy,fasting blood glucose（FBG）and oral glucose tolerance test（OGTT）was performed to screen pregnant mice with GDM.On the 17^th day of pregnancy,infraorbital vein blood was collected to measure blood lipids（TC,TG,HDL,LDL）,serum insulin level and free fatty acid（FFA）to evaluate the improvement of BiLaEn on glucolipid metabolic disorders in GDM mice.Elisa was used to detect the levels of serum inflammatory factors IL-1β,LPS,and leaky gut marker Zonulin,and immunofluorescence detection of claudin-1（ZO-1）expression and co-localization and expression of macrophage M1-like polarization markers F4/80 and CD11c in maternal ileum of maternal mice to evaluate the improvement effect of BiLaEn on intestinal barrier function.Fresh feces of maternal mice were collected continuously from16 to 18 days of gestation.The composition of the fecal microbial community of pregnant mice were determined by 16Sr RNA gene sequencing,and the concentration of short-chain fatty acids（SCFAs）were determined by GC-MS to evaluate the regulatory effect of BiLaEn on the gut microbiota and SCFAs.After delivery,expression of insulin and glucagon in the islets of offspring mice were detected by immunofluorescence at the day of 0,7,14 and 21 after birth,respectively;and the expression of PDX1（β-cell identity marker）and phosphorylation of ribosomal protein S6（p S6,a downstream target of the m TORC1 kinase）in offspring-islets was detected at the day of 0,7 and 14,respectively;the above results were used to evaluate the effect of BiLaEn on the development and maturation ofβcells in offspring mice.Then,the morphological development of offspring-islets was observed by labeling pancreatic ducts with Dolichos Biflorus Agglutinin（DBA）.After the offspring mice were weaned,the pancreas and ileum of maternal mice were collected,and the expression of insulin and glucagon in islets were detected by immunofluorescence.In order to further observe the glucolipid metabolism of offspring in pregnancy,8-week-old female offspring mice were caged with normal male mice after induction with 60%HFD for one week.OGTT was performed on the 15^th day of pregnancy,and the blood was collected from the infraorbital vein on the 17^th day.FBG,OGTT and blood lipid levels（TC,TG,HDL and LDL）were detected to evaluate the effect of BiLaEn on glucolipid metabolism in pregnant offspring mice.Results:（一）Maternal miceIn terms of glucolipid metabolism in maternal mice,BiLaEn markedly reduced FBG,serum LDL,TC,TG and FFA levels in the late pregnancy,increased the HDL level,relieved oral glucose intolerance and hyperinsulinemia,and significantly improved the disorder of glucolipid metabolism without affecting the labor process.In terms of intestinal microbiota in maternal mice,BiLaEn significantly increased the levels of Lactobacillus and Faecalibaculum in GDM maternal mice,and reduced the numberofnorank＿f＿＿Desulfovibrionaceae,norank＿f＿＿Lachnospiraceae,norank＿f＿＿Oscillospiraceae,Mucispirillum,Colidextribacter.In terms of leaky gut in maternal mice,BiLaEn could reduce the levels of inflammatory factors（LPS,IL-1β）and leaky gut markers（Zonulin）in the serum,inhibit the high expression of M1-like macrophage marker F4/80⁺CD11c⁺in the ileum of GDM pregnant mice,and increased the expression of tight junction protein ZO-1,which confirmed that BiLaEn could enchance the intestinal barrier function of GDM pregnant mice and reduce the degree of metabolic inflammation.Correlation analysis showed that Lactobacillus was negatively correlated with serum levels of FFA,LPS,Zonulin,IL-1β,LDL,but positively correlated with HDL.Similarly,Faecalibaculum was positively correlated with HDL,and negatively correlated with FFA,Zonulin,LPS and LDL.Norank＿f＿Desulfovibrionaceae is positively correlated with FFA,LPS,TG,LDL,and negatively correlated with HDL;norank＿f＿Oscillospiraceae is positively correlated with LPS,TG,LDL,and negatively correlated with HDL;Mucisprillum is positively correlated with LPS.The results of the flora explained the improvement of the glucolipid metabolic disorder and intestinal inflammation of the GDM maternal mice by the BiLaEn.（二）Offspring miceIn the development of offspring islets,it was first found that HFD-induced GDM offspring were born with marked hypoglycemia and obviously high-expression of insulin,PDX1 and glucagon in islets,showing a state of“overdevelopment”and“pre-mature”;Within 21 days after birth,the functional maturation of islets in GDM offspring mice was different from that of normal offspring.The key protein p S6 of m TORC1 pathway was significantly overexpressed at the 7^th day,and the activation state of this pathway was not synchronized with that of normal islets.BiLaEn administration partly inhibited the high expression of insulin,PDX1,glucagon and p S6 in offspring-islets after birth,and made them develop to normalization.The results of offspring pregnancy showed that the offspring mice in the GDM group were prone to glucose intolerance and glucolipid metabolic disorders during pregnancy（induced by HFD）.BiLaEn＿H intervention could reduce the serum levels of FBG,TC,TG and LDL,and increase HDL in offspring mice（female）in late pregnancy.Oral glucose intolerance was also improved to some extent.Therefore,BiLaEn had a certain improvement effect on the glucolipid metabolic disorder of GDM offspring mice during pregnancy.Conclusion:BiLaEn significantly improved the metabolic inflammation and glucolipid metabolism disorder in GDM pregnant mice in late pregnancy by modulating the intestinal flora disturbance and intestinal barrier function.GDM could cause over-development of offspring-islets at birth,showing a state of“quasi-maturity”,and the subsequent functional maturation process at the primary stage was also different from normal.BiLaEn had a certain protective effect on the islet development of offspring,made it develop normally,avoided obvious“overdevelopment”at birth.