Effects Of Human Amniotic Mesenchymal Stem Cells On TNF-α,IL-8 And NF-κB In Lung Tissue During Amniotic Fluid Embolism In Pregnant Rats

Objective: Utilizing the immunomodulatory effect of h AMSCs and alleviating lung injury,h AMSCs were injected intravenously into the amniotic fluid embolism model of pregnant rats,and the protective mechanism of amniotic fluid embolism leading to lung injury was preliminarily explored from the perspective of inflammation.Methods:1.Forty pregnant SD rats were randomly divided into control group,amniotic fluid meconium group,experimental group1 and experimental group2(10 in each group).The uterus from the pregnant rats to extract amniotic fluid and fetal rat meconium made of amniotic fluid meconium mixed solution of 99: 1.Along the abdominal main vein :control group injected saline;amniotic fluid meconium group inject amniotic fluid meconium mixture;experimental group1 injected amniotic fluid meconium mixture after injection of h AMSCs;experimental group2 was injected h AMSCs after injection of amniotic fluid meconium mixture.Collect 3ml arterial blood and kill the pregnant rats in each group after one hour observation.The left lung was freezed at-80 degrees in low temperature and the right lung was put in ten percent formaldehyde solution,conventionally embedded sections for HE staining and APM staining and immunohistochemistry.2.In order to detect whether the fourth generation of h AMSCs cultured successfully,h AMSCs was detected by flow cytometry and the vimentin expression of h AMSCs was dectected by immunocytochemical.3.The lung coefficient of each group was calculated,right lung tissue were stained with HE,APM staining and immunohistochemistry of CK16 to verify whether the amniotic fluid embolism model was established successfully.4.IHC was used to detect the expression of NF-κB in the right lung tissue in all four groups.The content of NF-κB in the left lung tissue was detected by Western blotting.The concentrations of TNF-α and IL-8 were detected by Enzyme-linked immunosorbent assay.Results:1.Clinical manifestations: control group after infusion of saline did not change much.The amniotic fluid meconium group showed obvious shortness of breath,rapid heartbeat,claw wet and cold,incontinence,convulsions and other symptoms.Experimental group 1 and experimental group 2 accelerated after the gradual smooth breathing,occasional limbs convulsions,claws slightly wet.In control group,experimental group1 and experimental group2,no death occurred in the pregnant rats one hour after the observation.In amniotic fluid meconium group,a pregnant rat died and its amniotic fluid was thick.2.Lung coefficient: control group(3.083±0.256),amniotic fluid meconium group(3.805±0.691),experimental group1(3.043±0.457),experimental group2(3.180±0.436).The amniotic fluid meconium group was significantly different from the control group,experimental group 1,and experimental group 2(P<0.05).HE staining of lung tissue:HE staining of lung tissue: No abnormalities in the control grop.Amniotic fluid meconium group,there were different pulonary edema,hyperemia and increased lung septum.There were macorophages,monocytes,neutrophils and other inflammatory cell infiltration in lung tissues.There were suspected amniotic fluid components and keratinized squamous cell amniotic fluid composition in pulmonary vascular.Experimental group 1 and experimental group 2 also showed pulmonary vascular suspicious amniotic fluid composition and keratinized squamous epithelium,but the pathological changes were lighter than amniotic fluid meconium group,showing a small amount of edema,congestion,a small amount of inflammatory cell infiltration.APM staining of lung tissue: There was no abnormal staining in the pulmonary vascular of the control group;amniotic fluid meconium group,experimental group 1 and experimental group 2 showed fine filamentous keratinized squamous epithelium in the pulmonary vascular,stained blue amniotic fluid mucus composition.Immunohistochemical detection of lung tissue CK16: Brown yellow keratinized squamous epithelium were seen in the amniotic fluid meconium group,experimental group1 and experimental group2.3.Immunocytochemical staining of h AMSCs: The fourth generation of h AMSCs had positive vimentin expression.The immunophenotypes of MSCs were analyzed by flow cytometry.The fourth generation h AMSCs overexpressed CD73,CD90 and CD105,but did not express CD34,CD11 b,CD19,CD45 and HLA-DR.4.IHC of lung tissue NF-κB: control group of alveoli,bronchial,perivascular low expression of light brown particles.amniotic fluid meconium group pulmonary vascular,bronchial,alveolar area has a large number of brown particles,was strongly positive expression.The experimental group1 and the experimental group2 showed a weak positive expression,experimental group2 lighter than experimental group1.The average optical density of each group: control group(0.405±0.059),amniotic fluid meconium group(0.453±0.036),experimental group1(0.376±0.060),experimental group2(0.405±0.047).The amniotic fluid meconium group was significantly different from the control group,experimental group 1,and experimental group 2(P<0.05).Western blotting to detect the concentration of NF-κB:amniotic fluid meconium group,experimental group1,experimental group2,control group,the color of the bands weakened in turn.The gray value of each group: control group(0.539±0.131),amniotic fluid meconium group(0.678±0.228),experimental group1(0.522±0.169)and experimental group2(0.553±0.200).The amniotic fluid meconium group was significantly different from the control group,experimental group 1,and experimental group 2(P<0.05).Enzyme-linked immunosorbent assay: IL-8:control group(123.169±13.615),amniotic fluid meconium group(158.346±47.682),experimental group1(79.316±46.035)and experimental group2(109.637±28.287).The amniotic fluid meconium group was significantly different from the control group,experimental group 1,and experimental group 2(P<0.05).TNF-α:control group(36.169±4.469),amniotic fluid meconium group(9.408±2.406),experimental group1(30.640±4.486)and experimental group2(35.550±2.962).The amniotic fluid meconium group was significantly different from the control group,experimental group 1,and experimental group 2(P<0.05).The above values were statistically analyzed and there was statistically significant difference between amniotic fluid meconium group and control group,experimental group 1,and experimental group2(P<0.05).Conclusion:Through the HE staining and APM staining and immunohistochemical expression of CK16 confirm the amniotic fluid embolism model was successfully prepared;human amniotic mesenchymal stem cells can intervene amniotic fluid embolism in pregnant rats by down-regulating the levels of IL-8 and TNF-α and inhibiting the activation of NF-κB in lung tissue,so as to reduce the amniotic fluid embolism inflammatory reaction and relieve the lung injury.