| Objectives：1.To observe the pathological changes of neonatal rats’ lung tissue which given birth byintrauterine infection of pregnant rats by the model Bell[1]established.2.To study the expression of the ROS、NF-κBp65、TGF-β1in neonatal rats’ lungtissue which have bronchopulmonary dysplasia caused by intrauterine infection andthe role of ROS、NF-κBp65、TGF-β1in BPD.3.To study the effect of ambroxol on expression of the ROS、NF-κBp65、TGF-β1,and to explore its possible mechanism to provide experimental evidence forbecoming drug of early prevention of BPD.Methods:1. Experimental grouping: Totally30pregnant15d rats were randomly dividedinto3groups (n=10in each group):normal saline control group (group NS), lipopoly-saccharide group (group LPS), ambroxol intervention group(group AMB).2. Model building and drug interventing: According to the model Bell[1]established. through injecting the lipopolysaccharide (LPS) into the pregnant ratabdominal cavity. AMB group of pregnant rats were injected of LPS (200μg/kg)through abdominal cavity on the fifteenth,16days for consecutive days,AMB(100mg/kg) was injected to abdominal cavity after injecting of LPS1h,theSeventeenth,18days for3consecutive days; LPS group of pregnant rats wereinjected of LPS (200μg/kg) through abdominal cavity on the fifteenth,16days forconsecutive days,and injected the same volume of normal saline with AMB toabdominal cavity after injecting of LPS1h,the Seventeenth,18days for3consecutivedays; NS group of pregnant rats are injected of the same volume of normal saline atthe same time point.3. Sample collection:Random out of1pregnant rats from three groups aftermodeling,removed the placenta,uterine fixed in4%neutral formalin for HE staining after executed.After the neonatal rats natural birth, weighed the rats and decapitatedin the p1,p3,p7,p14four times,removed lung tissue,washed them with normalsaline,weighed lung tissue after placed them in the filter paper; frozen part of lungtissue in liquid nitrogen to determinate ROS levels in lung tissue; the remaining lungtissue paraffin-embedded for HE and immunohistochemical staining (Each groupeach time point has8rats).4. Observations:Observed normal circumstances of all groups of pregnant ratsafter injection, if there is the phenomenon of death and abortion.observed the placentaand uterus pathological changes of pregnant rats in each group by HEstaining.Weighted the lung weight, body weight of neonatal rats, counted lung index(lung weight/body weight),observed the lung tissue pathological changes by HEstaining.and counted the radial alveolar count(RAC) of P3,P7,P14under themicroscope; Detected the expression of ROS in lung tissue by Elisa method;detected the protein expression of NF-κBp65and TGF-β1in lung tissue byimmunohistochemical staining.Results：1. General conditions and delivery of pregnant rats:Three groups of pregnant ratswere normal activities and eat after injection drugs,There was no significant changesin fur color, one case pregnant rat death,also one case of vaginal bleed occurred inLPS group, no abortion and death occurred.in NS, AMB Group. NS Group get72（72/74）neonatal rats, LPS group get61（61/67）neonatal rats, AMB group get62（62/65）neonatal rats.2. The changes of lung weight and body weight: At p1，p3，p7:the lung weightand body weight were reduced in LPS group compared with the NS group（P<0.05）;AMB group increased compared with the LPS group（P<0.05）,bothreduced compared with the NS group,but no statistical significance(P>0.05). At p14:there was no statistically significant difference between the groups(P>0.05).3. The pathological changes of placenta and uterus:The placenta and the uterinewall of pregnant rats suggested vascular congestion, edema, and a number ofneutrophil infiltration in LPS group; the degree of pathological changes was reduced in AMB group compared with LPS group; the NS group showed no significantinflammatory reaction.4. The pathological changes of lung tissue: The number of vesicle-like structuresin LPS group was fewer than the NS group,AMB group increased compared with theLPS group,but stilly fewer than the NS group at p1. The number of alveoli andalveolar septa in LPS group reduced compared with the NS group, also the alveolarstructure simplification at p7; the number of alveoli also fewer than NS group,thenthe thickness of alveolar septa thicken;the pathological changes of AMB group wassimilar to LPS group,however the degree mitigated.5. The changes of RAC in neonatal rat: There was no statistical significancebetween the groups at p3(P>0.05). The RAC in LPS group was fewer than NS groupat p7,p14（P<0.05）,the RAC in AMB group increased compared with the LPS groupat p7,p14（P<0.05）.6. The expression of ROS, NF-κBp65, TGF-β1in lung tissue in LPS groupincreased compared with NS group at p1,p3,p7(P<0.01); there was no statisticallysignificant difference between the groups at p14(P>0.05); compared ROS, NF-Bp65and TGF-β1pairwise correlation analysis, both there was positive correlation.7. The expression of ROS, NF-κBp65, TGF-β1in lung tissue in AMB groupdecreased compared with LPS group at p1,p3,p7(P<0.01).Conclusion:1. Builded the model of intrauterine infection in pregnant rats by intraperitonealinjection of LPS could lead to the lung weight and body weight decreased, thenumber of alveoli and alveolar septa reduced, the thickness of alveolar septathickened, all the performance prompted that intrauterine infection can result in theoccurrence of BPD.2.The expression of ROS, NF-κBp65and TGF-β1in the lung of neonatal ratsincreased after intrauterine infection, prompted that the oxidative stress、the rise ofNF-κBp65and TGF-β1plays an important role in neonatal rats bronchopulmonarydysplasia caused by intrauterine infection.3. Compared ROS, NF-κBp65and TGF-β1pairwise correlation analysis, both there was positive correlation after intrauterine infection, prompted that the threefactors promoted jointly neonatal rats bronchopulmonary dysplasia caused byintrauterine infection.4. AMB played the protective role in neonatal rats bronchopulmonary dysplasiaafter intrauterine infection by inhibiting the production of ROS, reducing thetranscriptional activity of NF-κBp65and ultimately reducing the expression ofTGF-β1. |
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