The Effect And Mechanism Of Maternal Obesity On Offspring Respiratory System Were Explored From The Perspective Of Gut-lung Axis

Objectives:1.A retrospective cross-sectional study was conducted to explore the influence of maternal obesity before and during pregnancy on respiratory diseases of their offspring,and to study the risk factors of recurrent respiratory infections in offspring.2.A prospective cohort study was conducted to explore the influence of maternal obesity during pregnancy on respiratory diseases in offspring from the gut microbiota and gut-lung axis.3.Animal experiments were conducted to study the mechanism of maternal obesity affecting the lung injury and repair process in offspring from the gut-lung axis,and antibiotic inhibition experiment was used to verify the role of gut microbiota.4.To explore the modern medical evidence for the TCM theory of "exterior of the lung and large intestine" from the perspective of gut-lung axis.Methods:1.A cross-sectional survey was done in Beijing,utilizing a stratified cluster random sampling strategy,and a total of 7524 preschool-aged children out of 16 districts were selected,aiming to study the influence of maternal obesity and gestational weight gain on respiratory diseases in offspring and to analyze the risk factors of recurrent respiratory infections in offspring.2.A clinical cohort study was done in Beijing,including 30 women who were obese during pregnancy and matched 30 normal pregnant women from the Obstetrics Department of the China-Japan Friendship Hospital,aiming to study the influence of maternal obesity on respiratory diseases in offspring through following up 18 months.Then immunohistochemistry(HIC)staining was used to detect the expression of TNF-α and IL-4 in the placenta,and 16S rRNA gene sequencing was used to detect the gut microbiota of newborns.3.Animal experiments:Obesity models of maternal rats before and during pregnancy were established.After birth,the male offspring were selected and fed with standard diet until 6-week-old.The prepregnancy and pregnancy obesity group and the control group were respectively randomly divided into the model group and the control group.Bleomycin-induced lung injury repair model was established in the model group,and samples were collected from the model group and the control group at 3 and 5 weeks after modeling,and the following items were tested:HE staining and Masson staining were performed on the lung tissue sections of the offspring,and HE staining was performed on the intestinal tissue sections of the offspring;Immunohistochemistry(HIC)staining,Western Blot and real-time quantitative PCR were used to detect the expression of SP-B and SP-D in the lung tissues of the offspring;16S rRNA gene sequencing was used to detect the gut microbiota of offspring;Immunofluorescence assay and Western Blot were used to detect the expression of tight junction proteins ZO-1 and Occludin in intestinal tissues of the offspring;Immunohistochemistry(HIC)staining and enzyme-linked immunosorbent assay(ELISA)were used to detect the expression of TNF-α,IL-6 and IL-4 in lung and intestinal tissues of offspring.4.To carry out antibiotic inhibition verification experiments,the male mice whose mother were pregnancy obesity group and the control group were given a combination of four antibiotics water to establish antibiotic inhibition model.Bleomycin-induced lung injury repair model was established at 6-week-old,and samples were collected at 3 and 5 weeks after modeling,and the following items were tested:HE staining was performed on the lung tissue sections of the offspring with antibiotic inhibition;Immunohistochemical method was used to detect the expression of SP-B in the lung tissues of antibiotic-suppressed mice,and immunofluorescence method was used to detect the expression of ZO-1 in the intestinal tissues of antibiotic-suppressed mice.Results:1.The results of the cross-sectional survey:(1)The incidence of recurrent respiratory tract infection and recurrent upper respiratory tract infection in maternal prepregnant obesity group was significantly higher than that in control group(P<0.05).The incidence of recurrent respiratory tract infection and recurrent lower respiratory tract infection in maternal pregnant obesity group was significantly higher than that in control group(P<0.05).There were no significant differences in the incidence of asthma and allergic rhinitis between the maternal obesity group and the control group(P>0.05).(2)The significant odds of having recurrent respiratory tract infection in offspring were 1.05(95%CI:1.03 to 1.07,P<0.001),1.01(1.00 to 1.02,P=0.035),7.76(5.34 to 11.29,P<0.001),2.32(2.07 to 2.59,P<0.001),1.73(1.50 to 2.00,P<0.001),1.27(1.11 to 1.46,P=0.001)respectively for maternal prepregnant obesity,gestational weight gain,asthma,allergy,initial use of antibiotics<6 m,and breastfeeding duration<6 m.(3)The pairwise interaction analysis was undertaken for the significant risk factors of recurrent respiratory infection.The results showed that when maternal prepregnant obesity was interacted with initial use of antibiotics<6 m,allergy,and breastfeeding duration<6 m,the significant odds of having recurrent respiratory infection in offspring were 2.09,2.46 and 1.75(all P<0.001)2.The results of the clinical cohort study:(1)The incidence of respiratory diseases including recurrent respiratory infection,recurrent upper respiratory infection,recurrent lower respiratory infection and asthma/wheeze in the offspring of maternal pregnant obesity group at 18 months old was higher than that of control group,but the difference was not significant(all P>0.05).(2)The expression of TNF-α and IL-4 in the placenta:The expression of TNF-α in the placenta of the obesity group was significantly higher than that of the control group(P<0.05),and the expression of IL-4 in the placenta of the obesity group was decreased compared with that of the control group,but the difference was not significant(P>0.05).(3)Gut microbiota of newborns:Compared with the control group,the diversity and richness of gut microbiota in newborns of maternal obesity group were higher,and the composition and structure of gut microbiota were changed.And in the obese group the abundance of Acinetobacter,Muribaculaceae,and Lactobacillus was significantly increased.while the abundance of Pseudomonas,Streptococcus,Ralstonia and Alkaliphilus was significantly decreased.3.Similar results were obtained for maternal obesity before and during pregnancy as follows:(1)Pathological morphology:In the lung injury period which was 3 weeks after modeling,in the normal mice model group and obese mice model group,disorder of lung parenchymal structure,collapse of alveolar,pulmonary interstitial edema,deposition of a large number of green collagen fibers,the villi alignment of intestinal mucosa decreased and inflammatory cellular infiltration were observed.In the normal and obese mice control group,normal alveolar structure and intestinal mucosa were observed.In the lung recovery period which was 5 weeks after modeling,the recovery of the obese model group was poor,disorder of lung parenchymal structure,collapse of alveolar,deposition of a large number of green collagen fibers,and inflammatory cellular infiltration were also observed.The alveolar structure and intestinal mucosa were recovered better in the normal model group.These results showed that the model of lung injury repair was successful,and the alveolar structure and intestinal mucosa recovery of obese mice were worse than that of normal mice.(2)The expression of SP-B and SP-D:In the lung injury period,compared with the control group,the expression of SP-B and SP-D in the lung tissues of the normal and obese mice model group was significantly reduced(P<0.05),and there was no significant difference between the two model groups(P>0.05).In the lung recovery period,compared with the control group,the expression of SP-B and SP-D in the obese model group was still significantly reduced(P<0.05),but there was no significant change in the normal model group(P>0.05).(3)The structure and composition of gut microbiota:In the lung injury period,the relative abundance of Lactobacillus was significantly increased in the obese offspring,while the relative abundance of Faecalibacterium and Muribaculaceae was significantly decreased.In the lung recovery period,compared with the normal model group,in the obese model group the abundance of Lactobacillus,Desulfovibrio,and Firmicutes was significantly increased,while the abundance of Faecalibacterium and Muribaculaceae was significantly decreased.(4)The expression of ZO-1 and Occludin:In the lung injury period,compared with the control group,the expression of ZO-1 and Occludin in the intestinal tissues of the normal and obese mice model group was significantly reduced(P<0.05),and there was no significant difference between the two model groups(P>0.05).In the lung recovery period,compared with the control group,the expression of ZO-1 and Occludin in the obese model group was still significantly reduced(P<0.05),but there was no significant change in the normal model group(P>0.05).(5)The expression of LPS,TNF-α,IL-6 and IL-4:In the lung injury period,compared with the control group,the expression of LPS,TNF-α and IL-6 of the normal and obese mice model group was significantly increased(P<0.05),the expression of IL-4 of the model group was significantly reduced(P<0.05),and there was no significant difference between the two model groups(P>0.05).In the lung recovery period,compared with the control group,the expression of LPS,TNF-α and IL-6 in the obese model group was still significantly increased(P<0.05),but there was no significant change in the normal model group(P>0.05).The expression of IL-4 in the lung tissues of the obese model group was still significantly reduced(P<0.05),but the expression of IL-4 in the intestinal tissues was no significant change(P>0.05)4.The results of antibiotic inhibition verification experiments:In the lung injury period,compared with the control group,pathological damage of alveolar,bronchus and intestinal mucosa was observed,and the expression of SP-B and ZO-1 was significantly reduced(P<0.05)in normal and obese model offspring which were inhibited by antibiotics.In the lung recovery period,the alveolar structure and intestinal mucosa of obese and normal model offspring with antibiotic inhibition were recovered well,and the expression of SP-B and ZO-1 was increased.Compared with the control group,there was no significant change(P>0.05).and there was no significant difference between the two model groups(P>0.05)Conclusion:1.Clinical studies have confirmed that maternal obesity before and during pregnancy increased the risk of recurrent respiratory tract infections in offspring and affected the occurrence of respiratory diseases in offspring.The gut microbiota-gut-lung axis may play a role in the process of maternal obesity affecting respiratory diseases in offspring.2.Through animal experiments,it is suggested that the influence of maternal obesity on the repair ability of lung injury in offspring may be caused by the change of the composition and structure of gut microbiota-gut-lung axis.Maternal obesity before and during pregnancy may increase the intestinal permeability of offspring mice by changing the composition and structure of gut microbiota during the repair period of lung injury,leading to high expression of inflammatory factors and low expression of anti-inflammatory factors,gut microbiota and its metabolites and inflammatory factors getting into the body across the damaged intestinal barrier,thus affecting the repair ability of lung injury in offspring mice.3.The gut-lung axis may be one of the mechanisms of the TCM theory of "exterior of the lung and large intestine".