Study On The Composition Of Gut Microbiota And The Role Of Acetate In Bronchopulmonary Dysplasia

PART Ⅰ COMPOSITION OF GUT MICROBIOTA IN BRONCHOPULMONARY DYSPLASIAObjective: Bronchopulmonary dysplasia(BPD)is one of the most common complications of preterm infants.With the rapid development of perinatal medicine and Neonatal intensive care unit(NICU)technology,the survival rates of very preterm infants and very low-birth-weight infant has been significantly improved,resulting in the incidence of BPD has increased.In recent years,there has been increasing evidence of the cross-talk between gut microbiota and lung disease.The purpose of our study was to investigate the changes of intestinal microbiota in BPD,which may provide clinical data for the prevention and treatment of BPD.Methods:(1)Inclusion criteria: Premature infants with birth weight<1500g,gestational age <32 weeks and requiring mechanical ventilation(non-invasive or invasive)were sent to the Children’s Hospital of Chongqing Medical University within 24 hours after birth.Informed consent of the subject’s family members is required for inclusion in the study.(2)Exclusion criteria:1)complex congenital heart disease;2)central nervous system malformations;3)respiratory system malformations;4)digestive tract malformations(Anal atresia,Hirschsprung’s disease),necrotizing enterocolitis etc.5)Other malformations: diaphragmatic hernia,chromosomal abnormalities,etc.The diagnostic criteria refer to the diagnostic criteria for BPD adopted by the National Institute of Child Health and Human Development(NICHD)in 2001.According to the diagnostic criteria of BPD,the preterm infants who finally developed BPD were included in the BPD group.The fecal samples in the two groups on Day7 and Day28 were collected.16 S rDNA sequencing technology was used to analyze the composition of the intestinal flora in the two groups.Results:(1)Clinical information: A total of 16 subjects were included in this study,with 8 cases in each of the control and BPD group.There were no differences between the two groups in terms of birth weight,gestational age,sex ratio,model of delivery,feeding,sepsis,Neonatal respiratory distress syndrome(NRDS),hypertensive disorder complicating pregnancy,prenatal antibiotic use and postnatal antibiotic use(p>0.05).(2)Compared with control group,the diversity index of the BPD group was increased on Day7(p<0.05).On Day28,the diversity index of the control group was higher than that of the BPD group,but the difference was not statistically significant.From the Day7 to Day28,the diversity index of the control group increased significantly(p<0.01),and the diversity index of the BPD group increased slightly,and the difference was not statistically significant.At the phylum level,Firmicutes and Proteobacteria were dominant in both groups on Day7.Compared with the control group,the relative abundance of Firmicutes in the BPD group decreased(p<0.05),while the relative abundance of Proteobacteria and Actinobacteria(p<0.05)increased.On Day28,Firmicutes and Proteobacteria were still dominant in the two groups.Compared with control group,the relative abundance of Firmicutes and Bacteroidetes decreased in the BPD group,while the relative abundance of Proteobacteria increased,but the difference was not statistically significant.In conclusion,on Day7 and Day28,the relative abundance of Proteobacteria in the BPD group was always more than that in the control group,while the relative abundance of Firmicutes was always lower than that in the control group.At the genus level,on Day7,the abundance of Klebsiella(p<0.05)and Veillonella(p<0.05)increased in the BPD group compared to control group.On the Day28,the abundance of Escherichia-Shigella(p<0.05)in the BPD group increased compared to control group.Comparing the abundance of gut bacteria on Day7 with that on Day28,there were an increase in Klebsiella(p<0.05)and Enterobacter(p<0.05)in the BPD group.Comparing the abundance of gut bacteria on Day7 with that on Day28,in the control group,the abundance of Clostridium＿sensu＿stricto＿1(p<0.05)and Lachnoclostridium(p<0.05)increased,and the abundance of Staphylococcus(p<0.05)significantly decreased.Conclusion: In the first part,we use 16 S rDNA sequencing technology to analyze the intestinal flora of premature infants in the BPD group and control group.In general,before the occurrence of BPD,the diversity of intestinal flora has changed,and opportunistic pathogens have increased compared with the control group.PART Ⅱ THE ROLE OF ACETATE IN BRONCHOPULMONARY DYSPLASIAObjective: BPD is a common pulmonary complication in preterm infants.Short-chain fatty acids are metabolites produced by the gut microbiota,and their anti-inflammatory functions are well known.The role of short-chain fatty acids in BPD have not been studied.Therefore,we evaluated the gut microbiota and serum levels of short-chain fatty acids in BPD mice,and then intervened in BPD mice with short-chain fatty acids to study the effects of short-chain fatty acids on lung inflammation and lung injury in BPD mice.Methods: 1.The feces and serum samples of hyperoxia and room air mice were collected.Serum levels of short-chain fatty acids were determined by High Performance Liquid Chromatography(HPLC),and intestinal flora of mice in each group was analyzed by 16 S rDNA high-throughput sequencing.2.Animal experiment:C57BL/6 mice were randomly divided into three groups on the 3rd day after birth: room air group,hyperoxia group,and hyperoxia + acetate(250 m M,0.02 ml/g)group.High Performance Liquid Chromatography was used to determine the levels of short-chain fatty acids in the serum of three groups,and 16 S rDNA high-throughput sequencing was used to analyze the intestinal flora of the three groups of mice.HE staining and immunohistochemistry were used to evaluate lung injury and inflammation.The expression levels of IL-1β,IL-18,TNF-α was determined by ELISA and RT-PCR.The expression levels of NLRP3 and caspase-1 were detected by Western blot.3.Cell experiment: A549 cells were stimulated by LPS and ATP to secrete NLRP3 inflammasomes,and then the cells were interfered by acetate and GPR43 antagonist.The expressions of inflammatory cytokines IL-1β and IL-18 were detected by RT-PCR.The expressions of NLRP3 and Caspase-1 were detected by Western blot.Results: 1.Compared with room air mice,the relative abundance of Proteobacteria in intestinal flora of hyperoxia mice was increased(p<0.001),while the relative abundance of Bacteroidetes was decreased(p<0.05),and the level of acetate in serum of hyperoxia mice was also significantly decreased(p<0.05).2.Compared with the room air group,the survival rates of hyperoxia mice were reduced,lung injury was increased(RAC p<0.001;MLI p<0.001)and more macrophage were aggregated in the lung(p<0.001).RT-PCR and ELISA showed increased expressions levels of IL-1β,IL-18 and TNF-α(all p values <0.001)and decreased expression of GPR43(p <0.001)in the hyperoxia mice comparing to room air mice.The expression levels of NLRP3 and Caspase-1 detected by Western blot(all p values <0.01)were increased in BPD mice.After acetate treatment,the lung injury of BPD mice was alleviated(RAC p<0.001;MLI p<0.001)and the aggregation of macrophages in lung was decreased(p<0.05).In hyperoxia mice treated with acetate,the expression levels of inflammatory cytokines TNF-α,IL-1β and IL-18 in lung tissue were decreased(all p values <0.001);The expression levels of NLRP3 and Caspase-1 were also decreased(all p values <0.05),while the expression levels of GPR43 was increased(p<0.001).Moreover,at the phylum level in the BPD mice treated with acetate,the proportion of Proteobacteria was significantly decreased and the relative abundance of Bacteroidetes was significantly increased.At the genus level,there an increase in Bacteroide and a decrease in Escherichia-Shigella in the BPD mice treated with acetate.3.In A549 cells,it was found that the cell inflammation caused by LPS and ATP could be significantly reduced after the intervention of different concentrations of acetate.At the m RNA level,IL-1β and IL-18 expression were decreased in both 5A-LA and 10A-LA groups compared with the LA group(5A-LA: IL-1β p<0.05,IL-18 p<0.01;10A-LA: IL-1β p<0.001;IL-18 p<0.001).At the protein level,the expressions of caspase-1 and NLRP3 were also decreased in 5A-LA and 10A-LA groups compared with the LA group(5A-LA:NLRP3 p<0.05 caspase-1 p<0.05;10A-LA:NLRP3 p<0.01 caspase-1 p<0.01).After adding GPR43 antagonist,compared with the 10A-LA group,the expression levels of IL-1β(p<0.01),IL-18(p<0.05),NLRP3(p<0.05),Caspase-1(p<0.05)were increased.These results suggested that GPR43 antagonist could partially reverse the inhibitory effect of acetate on inflammation.Conclusions: There were significant differences in intestinal flora between BPD mice and room air mice,and serum levels of short-chain fatty acids,especially acetate,were significantly reduced in BPD mice.It is suggested that acetate may play a certain protective role in the occurrence and development of BPD.After treated with acetate in BPD mice,it was found that the lung injury and lung inflammation were reduced,and the expression of NLRP3 inflammasome-related protein was reduced.Moreover,acetate could regulate the intestinal flora in BPD mice.Moreover,in vivo experiments further verified that acetate could inhibit NLRP3 inflammasome by GPR43.Therefore,acetate,one of the important metabolites of intestinal flora,plays a protective role in the progression of BPD by regulating intestinal flora and inhibiting the expression of NLRP3 inflammasomerelated proteins.