A Clinical Research On The FIRS's Influence On The Level Of MMP-9/TIMP-1 In Cord Blood Of Premature Infant And Its Relationship With Heart And Lung Injury

Fetal inflammatory response syndrome?FIRS?is a state of subclinical of fetal immune system that release a large number of inflammatory factors.It is a unique manifestation of fetus and special stage of systemic inflammatory response syndrome?SIRS?.This disease is associated with preterm birth,closely.It is another important viscera damage factors except the suffocation which is involved the brain,heart,lungs,stomach and other important organs.With the development of reproductive medicine condition,the survival rate of premature is greatly increased to 5%-15%.There is a sharp increase in the amount of ultra premature and small premature infant.Therefore survival and outcomes of premature infant become focus of experts' attention.Lung and heart damage becomes one of main reasons which causes the death of preterm infants.The relationship between lung and heart damage and FIRS is more and more cause concern gradually.Bronchopulmonary dysplasia?BPD?and myocardial injury are representative diseases of premature infant's lung and heart damage.They are closely associated with FIRS.BPD is a common disease of respiratory system diseases in ultra premature and small premature infants.It has its own clinical,radiographic,and histologic features.BPD is one of the important causes of death,as well as main cause of infantile chronic respiratory diseases and import inducement of childhood asthma.The data shows,FIRS newborns whose birth weight<2000g have a high level of IL-1? in Alveolar lavage fluid.Study abroad shows,in 70 BPD cases,the occurrence rate of FIRS is 76%.Myocardial injury's modeofonset is hiddenly,clinical feature is various.This disease is lack of specific manifestation and early predicted item.It is easy to miss what lead to delay treatment and poor prognosis.Morbidity ofmyocardial injury is about 30%.However,the morbidity of FIRS cases is rise to 46.8%,the morbidity of arrhythmia is 4.28%,death rate is 0.6%-1%.Although the relationship between FIRS and lung and heart damage is certain,the mechanism is not clear.The situation lead to bottleneck problem of looking for early item of BPD and new treatment of heart and lung damage.Exploration on the mechanism plays an key role on improving the prognosis of premature.The mechanism of FIRS is similar to SIRS.Preliminary study shows: In the process of systemic inflammatory response syndrome?SIRS?,some inflammatory factors,such as IL-6 and IL-8,could mutual promote MMP-9 so that deteriorate inflammatory response as well as the injury of organs.MMP-9is one member of Matrix metalloproteinases?MMPs?.TIMP-1 could highly antagonise MMP-9.MMP-9/TIMP-1 participate in process of multiple organ injury.The system is expression in every stage of lung development.In animal experiments expression of MMP-9 is increased when influenced by hyperoxia and radiation will trigger pulmonary alveoli damage and pulmonary fibrosis.These pathologic change accord with BPD.Otherwise MMP-9 is the most important metalloenzyme of myocardial collagen type I and III.The amount of these two types of myocardial collagen is 90% of myocardial collagen.The rise of expression of MMP-9 may lead to heart damage.This article is aimed at whether MMP-9/TIMP-1 change on the process of FIRS;Whether the change of MMP-9/TIMP-1 is connected with injury of heart and lung.Objective:1 clinical trial1.1 Through the test on the level of IL-6,MMP-9,TIMP-1 in the cord blood of experimental groups and control groups,tried to set an molecular baswas on the mechanwasm that FIRS affect the level of MMP-9/TIMP-1.1.2 Probe into the relationship between IL-6 and MMP-9/TIMP-1,trid to explore whether it was synergwastic effect between IL-6 and MMP-9,which further aggravated organ damage.1.3 Track and revwasit cases to confirm whether these preterm infants fall in bronchopulmonary dysplasi?BPD?or myocardial damage,and to explore FIRS and MMP-9/TIMP-1's influence on the injury of heart and lung.2 Infratest2.1 By establwashing the FIRS mice model to observe pathologic change of models' heart and lung tissue and expression of MMP-9/TIMP-1 to explore its influence on heart and lung tissue injury,to probe into the meachanwasm how FIRS cause heart and lung injury.2.2 By observing the pathologic change of FIRS mice which intervened by MMP-9 inhibitor,to explore the protective function of decline of MMP-9.2.3 Combining with these two parts above,try to expect the function of MMPs inhibitor that improve the condition of heart and lung tissue of premature infants.Method:1 clinical trial1.1 Object of study and its grouping: Collect preterm samples who could exclude nervous system malformation and other systems damage,hereditary metabolic dwasease,and whose mother could exclude lung dwasease,cardiopathy,diabetes,eclampsia and anemia and so on.During May 2014-5 to December 2015-12,a total of 118 preterm samples were obtained at the fourth hospital of Hebei Medical University.According to the FIRS diagnostic criteria and gestational age,these samples were divided into four groups:control group 1?gestational age?32 weeks,undiagnosed FIRS?: N=25;control group 2?32 weeks < gestational age < 37 weeks,undiagnosed FIRS?: N=32;experimental group 1?gestational age?32 weeks,diagnosed FIRS?: N=33;experimental group 2?32 weeks < gestational age < 37 weeks,diagnosed FIRS?: N=28.1.2 Sample collection:Collect cord blood 2-3ml immediately after delivery from umbilical cord of fetal side,gather serum after centrifuge;Collect amniotic fluid 2-3ml in delivery,gather supernate after centrifuge.Both two keep in-80? for testing.Collect tissue of placenta,caul,umbilica as soon as delivery of placenta in gnotobaswas and keep in formalin immediately,and then save in 4? for testing.1.3 Index measurement:1.3.1 Test on level of IL-6 in cord blood by ELISA;level of CRP in amniotic fluid by BN ProSpec;pathological examinations of placenta,caul,umbilica by hematoxylin-eosin staining.1.3.2 Track and revwasit experimental cases?time?1 month?to confirm whether these preterm infants fall in bronchopulmonary dysplasia?BPD?or myocardial damage,calculate the morbidity.2 Infratest2.1 Model building and drug interventing:Totally 30 pregnant 15 d mice were randomly divided into 3 groups?n=10in each group?: normal saline control group?control group?,lipopoly-saccharide group?FIRS group?,FIRS intervention group?RA group?.According to the model Samuli Rounioja establwashed[1]:Intraamniotic injections performed on G15,through injecting the lipopolysaccharide?LPS?into amniotic sac of FIRS group and RA group?0.25ug-2.5ug/sac,total quantity less than 100ug/kg?.Handle NS group by the same way.G17,RA group was began to gavage by RA?50mg/kg/d?till delivery.2.2 Sample collection:Random out of 3 pregnant mice from three groups after modeling,removed the placenta,heart and lung tissue from fetal mice which divided into two parts,one part uterine fixed in 4% neutral formalin for HE stainingafter executed,another frozen in liquid nitrogen and stored in refrigerator?-80??for PCR.After other neonatal mice delivery,decapitated in the p1,3,7,14 four times,removed heart and lung tissue,frozen part of the tissue in liquid nitrogen and stored in refrigerator?-80??for PCR;the remaining tissue paraffin-embedded for HE and immunohwastochemical staining?Each groupeach time point has 10fetus?.2.3 Observations:2.3.1 Observed normal circumstances of all groups of pregnant mice after surgery,record the quantity of death pregnant mice,death newborns and death fetus;2.3.2 Weighted and record the fetus and neonatal mice as well as their lung tissue,calculate the lung index?lung weight/body weight?;2.3.3 Observed the placenta,heart and lung pathological changes of pregnant rats in each group by HE staining;2.3.4 Observed the lung tissue pathological changes by HE staining,and counted the radial alveolar count?RAC?of P3,P7,P14 under themicroscope;2.3.5 Detected the expression of IL-6,MMP-9,TIMP-1 in heart and lung tissue by real-time PCR.3 Statistical analysisAll date calculated by statwastical softwwere13.0.Every date need to be Calculated by normality test.Enumeration data coincided with normal distribution showed by?X ±S?.Enumeration data didn't coincided with normal distribution showed by?M?Q??.Measurement data showed by rate?%?.Two groups of mean value which conform to normal dwastribution werecalculated by t-test.Correlation analys was calculated by Spearman's linear correlation.Three groups of mean value which conform to normal dwastribution and equal variance werecalculated by One-way ANOVA.Multiple comparwason between groups werecalculated by Student-Newman-Keuls test.Three groups of date which conform to skewed dwastribution werecalculated by Kruskal-wall H test,comparwason between any two means werecalculated by Mann-Whitney U test.Correlation analyswas was calculated by Spearman's linear correlation.Occourence rate of diease in different groups was calculated by ?2 test or Fisher exact probability.Result:1 Clinical trial1.1 The level of IL-6,MMP-9,TIMP-1 in cord blood of preterm1.1.1 FIRS's influence on the level of IL-6,MMP-9,TIMP-1 in cord bloodThe level of IL-6,MMP-9,TIMP-1 and M/T?MMP-9/TIMP-1 rate?of experimental group 1 was higher than control group 1?P<0.001?.The level of IL-6,MMP-9,TIMP-1 and M/T of experimental group 2was higher than control group 2?P<0.001?.1.1.2 the level of IL-6,MMP-9,TIMP-1 in cord blood in different gestational ageIn FIRS preterm:gestational age < 32 weeks IL-6 obviously exceed gestational age > 32weeks?P < 0.05?,MMP-9,TIMP-1and M/T no obvious difference with gestational age>32weeks?P>0.05?.In normal preterm:gestational age<32 weeks IL-6 no obvious difference with gestational age > 32weeks?P > 0.05?,MMP-9,TIMP-1 obviously lower than gestational age>32weeks,M/T obviously higher than gestational age>32weeks?P<0.05?.1.1.3 Correlation analyswas about IL-6 and MMP-9,TIMP-1,M/T level in the cord blood.The IL-6 level in cord blood had obvious linear relationship with MMP-9,TIMP-1,M/T level(PMMP-9 < 0.001,rMMP-9=0.875;PTIMP-1 < 0.001,rTIMP-1=0.874;PTIMP-1<0.001,r_M/T=0.745).1.2 Difference of placenta pathology between two groupsCompared with control group:Acute inflammation induced neutrophile granulocyte gathered,organization hyperemia,edema;Chronic inflammation induced neutrophile granulocyte,lymphocyte,monocyte gathered,tissue necrosis and hyperplasia;Chorioamnionitis lesion was focus on chorionic,induced acute and chronic inflammatory cell gathered.Placenta of control group was normal.1.3 Difference of amniotic fluid smear between two groupsCompared with control group:inflammatory cell such as neutrophile granulocyte and lymphocyte.1.4 Occurrence rate of BPD and myocardial damage of prematue1.4.1 FIRS's influence on the occurrence rate of BPD and myocardial damageof prematueThe occurrence rate of BPD and myocardial damage of experimental group 1 was higher than control group 1?P<0.05?.The occurrence rate of myocardial damage of experimental group 2 was higher than control group 2?P < 0.05?.it was no significant statistical differences about occurrence rate of BPD between these two groups?P >0.05?.1.4.2 Occurrence rate of BPD and myocardial damage of prematue in different gestational ageIn FIRS preterm: gestational age < 32 weeks Occurrence rate of BPD was significantly higher than gestational age>32weeks?P<0.05?,occurrence rate of myocardial damage had no difference with gestational age > 32weeks?P>0.05?.In control group: gestational age<32 weeks occurrence rate of BPD and myocardial damage had no difference with gestational age > 32weeks?P >0.05?.2 Infratest2.1 General conditions and delivery of pregnant miceThree groups of pregnant mices were normal,it was no significant changes in nomal activities.After intrathecal injection,there was no pregnant mice death.2 pregnant mice abortion in 48 h after intrathecal injection in FIRS group.1 pregnant mice abortion in 48 h after intrathecal injection in FIRS group.abortion occurrence rate10%?3/30?.Control group get 97?97/98?fetus,64?64/65?neonatal mices,FIRS group get 67?67/79?fetus,33?33/42?neonatal mices,RA group get 82?82/87?fetus,46?46/52?neonatal mice.Pathological examination of placenta showed model success rate is 83.73%?139/166?.2.2 The difference among groups about lung weight,body weight and lung indexAfter FIRS heppened: G19,P1,7,14 d mice lung weight was obvious lower than control group?P< 0.05?;G19,P1 mice body weight was obviouslower than control group?P<0.05?;G19,P1,7d mice lung indes was obvious lower than control group?P<0.05?.After RA intervened:only G19,P1 d mice lung weight was obvious lower than control group?P < 0.01?,obvious higher than FIRS group;only G19 d mice body weight was obvious lower than control group?P < 0.01?,obvious higher than FIRS group;only G19,P1 d mice lung index was obvious lower than control group?P<0.01?,obvious higher than FIRS group.With mice growth,the lung weight,body weight also growed.Those who intervened by RA were better.The lung index was lowest in P14.2.3 The pathological changes of placenta,heart and lung tissue2.3.1 Placenta tissue: Compared with control group: placeta suggested a number of neutrophil infiltration,edema,villi structure dwasappeared and part of tissue fibroswas.2.3.2 Lung tissue: Compared with control group: The number of vesicle-like structures?P1?as well as alveoli and alveolar septa?P7,P14?in FIRS group were fewer,alveolar structure were simpler,the thickness of alveolar septa were thickened,part of lung tissue was congestive.The pathological changes of RA group was similar to FIRS group,however the degree mitigated.2.3.3 heart tissue: Compared with control group: The thickness of ventricular wall in FIRS group was thickened,it were edema and hemorrhagic lesions in tissue.The pathological changes of RA group was similar to FIRS group,however,the degree mitigated.2.4 The changes of RAC in neonatal miceAfter FIRS happened,P3 d mice were no obvious difference with control group,P7,14 d mice were obviously lower than control group?P < 0.01?.After RA intervene P3 d mice were no obvious difference with control group and FIRS group?P < 0.01?,P7,14 d mice were obviously lower than control group?P < 0.01?,higher than FIRS group?P < 0.01?.With mice growth,the RAC also growed.The maximum value was in P14.5 The expression of IL-6,MMP-9,TIMP-1 in heart and lung tissue5.1 Heart tissueAfter FIRS happened: G19 d,P1,7,14 d mice IL-6,MMP-9 expression and M/T rat were obviously higher than control?P<0.05?.After RA intervene,G19 d,P1,7,14 d mice IL-6,MMP-9 expression and M/T rat were obviously higher than control group?P<0.05?,P1,7,14 d mice MMP-9 expression and M/T rat lower than FIRS group?P<0.01?.5.2 Lung tissueAfter FIRS happened: G19 d,P1,7,14 d mice IL-6,MMP-9 expression and M/T rat were obviously higher than control?P<0.05?.After RA intervene,G19 d,P1,7,14 d mice IL-6,MMP-9 expression and M/T rat were obviously higher than control?P < 0.05?,G19dP1,7,14 d mice MMP-9 expression and M/T rat lower than FIRS group?P<0.01?.6 Correlation analys about IL-6 and MMP-9,TIMP-1,M/T level in heart and lung tissue6.1 heart tissue:The IL-6 level in lung and heart tissue had obviously linear relationship with MMP-9,M/T level by Spearman's rank order correlation test(PMMP-9 <0.001,rMMP-9=0.705;PM/T=0.004,r_M/T=0.496);it was no linear relationship between IL-6 level and TIMP-1 level?PTIMP-1=0.207,rTIMP-1=0.229?.6.2 Lung tissue:The IL-6 level in lung and heart tissue had obviously linear relationship with MMP-9,M/T level by Spearman's rank order correlation test(PMMP-9=0.001,rMMP-9=0.551;PM/T=0.038,r_M/T=0.369);it was no linear relationship between IL-6 and TIMP-1?PTIMP-1=0.119,rTIMP-1=0.281?.Conclusion:1 The result showed that FIRS could cause the level of MMP-9 and M/T increased in cord blood,as well as the occurence rate of BPD and myocardial damage in the premature,obviously.Prompt MMP-9 and dwasorder of M/T maybe one of molecular baswas how FIRS induced BPD and myocardial damage.2 The IL-6 level in cord blood had linear relationship with MMP-9,M/T level in the expermental group.Prompt that IL-6 and MMP-9 had synergicrelationship on the process of FIRS,MMP-9 maybe interact with inflammatory medium and participate in injury of organs.3 By building FIRS model of KM mices: FIRS could induce lesion that same to BPD,prompt that FIRS could cause BPD;Otherwase,FIRS was could induce thickness of ventricular wall hickened and cause edema,hemorrhagic lesions in tissue,this may lead to myocardial damage and decreases of cardiac function.RA played an protective role in FIRS neonatal heart and lung injury by inhibiting the production of MMP-9.4 The expression of MMP-9 and MMP-9/TIMP-1 rate in the lung and heart tissue of FIRS KM fetus and newborns,and had liner relationship with IL-6,prompt there maybe synergwastic effect between IL-6 and MMP-9during process that FIRS cause injury of heart and lung.