Study On The Mechanism Of ICOS/ICOSL In Neonatal Rats With Bronchopuldysplasia Induced By Intrauterine Infection

Objective:To explore the mechanism of ICOS/ICOSL signaling pathway in Bronchopulmonary dysplasia(BPD)induced by intrauterine infection,and to use the model of neonatal rats with PBD.Methods:1.Establishment of animal model:6-8 weeks of breeding age SD adult rats were randomly selected and caged in a 2:1 ratio of male to female.After natural conception of female rats,some pregnant rats were randomly selected for intrauterine infection induced by intraperitoneal injection of lipopolysaccharide on two consecutive days of gestation 17 and 18,and the newborn mice delivered were the newborn rats in the LPS-induced intrauterine infection group;some pregnant rats were randomly selected for intraperitoneal injection of the same amount of saline at the same time,and the newborn mice delivered were the newborn rats in the saline control group.2.Specimen collection:10 newborn rats in both groups were randomly selected on postnatal day 1(P1)and P3,P7 and P14,anesthetized by intraperitoneal injection of10%chloral hydrate(3.5 ml/kg),and at least 100 ul of peripheral blood was obtained by severing the blood,anticoagulated with sodium heparin anticoagulation tubes,and placed on ice.Afterwards,the lung tissue was taken and sealed in sterile EP tubes and placed in an ultra-low temperature refrigerator at-80°C for biological sample storage.3.Indexes:The lung weight,body weight,lung index(lung weight/body weight)and other general condition indexes were recorded in the two groups of neonatal rats;HE staining was performed to observe the pathological changes of the placenta in the two groups of pregnant rats,the histopathological changes of the lung in the two groups of neonatal rats at each time point,and the radial alveolar count was also calculated;ELISA was used to detect the expression of TNF-αin the lung tissue of the two groups of neonatal rats at each time point,and Western blot was used to detect The expression of NF-k B P65 and ICOSL in the lung tissues of the two groups of neonatal rats at each time point,and the percentage of CD4~+T cells surface ICOS,IL-17A and Fox P3 in the peripheral blood of the two groups of neonatal rats were detected by flow cytometry.Results:1.In the LPS group,HE staining of the placenta showed massive congestion,edema and neutrophil infiltration;in the NS group,no inflammatory reaction was observed in the placenta.The RAC at three time points P3,P7 and P14 was reduced in the LPS group compared with the saline control group(P<0.05).2.The body weight of neonatal rats in the LPS group was lower in P3,P7 and P14 than that in the NS group(P<0.05);the lung weight of neonatal rats in the LPS group was lower than that in the NS group,and the difference between P1,P7 and P14 was statistically significant(P<0.05);the lung index of neonatal rats in both groups tended to decrease with increasing age,and only P7 was statistically different between the two groups(P<0.05).3.TNF-αin the lung tissue of the LPS group was significantly higher than that of the NS group at four time points,P1,P3,P7 and P14,and the difference was statistically significant(P<0.05).4.NF-k B P65 protein expression in the LPS group was higher than that in the saline group at three time points P1,P3 and P7,and the difference was significant(P<0.05);ICOSL protein expression in the LPS group was significantly higher than that in the NS group,with significant differences at three time points P1,P3and P7(P<0.05).5.In the LPS group,NF-KB P65 was positively correlated with the change in ICOSL expression(r=0.697,P<0.001).TNF-αwas positively correlated with the change in ICOSL expression(r=0.941,P<0.001).TNF-αwas positively correlated with the change in NF-KB P65 expression(r=0.575,P<0.001).6.The percentage of CD4~+ICOS~+T lymphocytes in the peripheral blood of newborn rats in the LPS group was significantly higher than that in the NS group,with statistical differences at P1,P3and P7(P<0.05).7.The percentage of CD4~+IL-17A~+T lymphocytes in peripheral blood in the LPS group was significantly higher than that in the NS group at P1,P3,P7 and P14,with statistical differences(P<0.05);the percentage of CD4~+Fox P3~+T lymphocytes in peripheral blood in the LPS group was significantly higher than that in the NS group at P1,P7 and P14,with statistical differences(P<0.05).The percentages of IL-17A~+/Fox P3~+T lymphocytes in peripheral blood of newborn rats in the LPS group were significantly higher than those in the NS group at P3,P7 and P14,and the differences were statistically significant(P<0.05).Conclusion:1.Intrauterine infection-induced BPD in neonatal rats was successfully established by intrauterine injection of LPS in pregnant rats.2.Activation of the ICOS/ICOSL signalling pathway in neonatal rats with BPD may be associated with early lung tissue inflammatory cytokine TNF-αand NF-k B P65 levels.3.Th17/Treg cell imbalance due to activation of the ICOS/ICOSL signalling pathway may play a non-protective role in the pathogenesis of intrauterine infection-induced neonatal BPD in rats.may play a non-protective role in the pathogenesis of BPD induction in neonatal rats.