The Effect Of Puerarin EnMT On Hypoxic Pulmonary Hypertension By Inhibiting EnMT

This paper studies the puerarin consociation sibeline(Pur)to inhibit the action of the hypoxia happened EnMT bovine pulmonary artery endothelial cells and Pur to hypoxic rats treated by inhibiting the occurrence of EnMT molecular mechanism,provide new means for treatment of pulmonary hypertension.To isolate and cultivate the endothelial cells of bovine pulmonary artery,and to identify them by cell morphology and immunofluorescence assay.Identified as bovine pulmonary artery endothelial cells after training to 2-5 generations can undertake building packet delivery,bovine pulmonary artery endothelial cells respectively in normal incubator and lack of oxygen incubator culture experiments are grouped into:oxygen,oxygen,oxygen+Pur.In vitro experiment using thiazole blue colorimetry(determined by MTT method)determination of oxygen and oxygen under different concentration gradient of Pur influence on bovine pulmonary artery endothelial cell proliferation activity,thus may get Pur best to drug concentration.Scratches and Transwell experimental detection of oxygen for the effects of bovine pulmonary artery endothelial cell proliferation and migration,and after joining Pur for the effects of bovine pulmonary artery endothelial cell proliferation and migration.The changes of endothelial marker protein and mesenchymal marker protein were detected by immunofluorescence,and the effect of Pur on the expression of EnMT in the endothelial cells of cattle pulmonary artery was detected by Western blot method.Raise 18 of SD rats,the experiment group is:oxygen、oxygen,and oxygen+Pur,6 in each group,only 21 days,SD rats in normal separately according to the grouping incubator breeding with lack of oxygen in the incubator.The experimental results showed that the model was successful by ultrasound method,right cardiac catheterization and HE staining.Immunofluorescence test ectomesenchymal sign endothelial marker protein and protein changes,using Western blot method and RT-PCR detection to join at the protein level and mRNA level Pur EnMT related protein expression in bovine pulmonary artery endothelial cells.According to the results of MTT,the optimal concentration of the drug in Pur was 250.The trace and Transwell experiments confirmed that cell proliferation and migration could be caused by hypoxia after adding Pur.In protein level,Western blot experiments testing after joining Pur can make CD31、VE-cadherin expression rise,Vimentin、Fibronection and α-SMA expression down.The results of RT-PCR were consistent with Western blot at mRNA level.In the SD rat experiment,the concentration of puerarin was 20mg/kg.Small animal the size of the ultrasonic detecting method of SD rat aortic diameter changes of the SD rats,right cardiac catheterization method right ventricular systolic blood pressure change and HE staining method to detect the thickness of the blood vessels in the lung tissue were confirmed SD rats hypoxia building success,and puerarin on hypoxic pulmonary hypertension rats may have therapeutic effect.Frozen section method will be double dye,the lung tissue with strong intuitive observed often oxygen groups of CD31 red than α-SMA,CD31 red light green light is weak,lack of oxygen group weak α-SMA green strong,often oxygen CD31 strong vimentin red light weak green light,oxygen groups CD31 weak vimentin green light green light is strong,that hypoxia can make hypoxia pulmonary fibrosis of rat lung tissue,which may lead to pulmonary hypertension,and to join in the hypoxia group puerarin can make CD31 red light relative to the hypoxia group,α-SMA green relatively dim,that puerarin treatment effect,not only to the hypoxic pulmonary hypertension oil also has therapeutic effect on hypoxia caused by pulmonary fibrosis.Westem blot and RT-PCR to determine whether protein levels or mRNA level of oxygen in Pur can make CD31,VE-cadherin protein expression level,Vimentin,Fibronection,a-SMA protein expression.It changes in the related indexes of EnMT in vivo and in vitro show that puerarin can inhibit the occurrence of EnMT,which can have therapeutic effect on pulmonary hypertension.