The Mechanism Of Sildenafil Inhibiting Persistent Pulmonary Hypertension In Neonatal Rats Through Notch3/Hes1 Pathway

BackgroundNeonatal persistent pulmonary hypertension(PPHN)was defined that the respiratory system of newborn cannot work normally after birth.It is characterized by abnormally high resistance of pulmonary blood vessels after birth,resulting in the flow of deoxygenated blood from the lung to the systemic circulation.It will lead to serious mortality when PPHN is not treated adequately,.Infants with PPHN may suffer severe respiratory failure and require intensive care and intervention.It is reported that 10%of cases are likely to die.Even the survivor will still suffer from serious complications such as nerve injury,cerebral palsy,and blindness.Structural pulmonary vascular remodeling plays an important role in the pathogenesis of PPHN.Pulmonary vascular remodeling includes thickening of intima,media and adventitia,resulting in stenosis of pulmonary artery lumen and increase of pulmonary artery pressure.The primary pathological basis is the phenotypic transformation of proliferation and migration of pulmonary artery smooth muscle cells(PASMCs),vascular endothelial cells,and fibroblasts.Notch3,one of the four Notch protein families,is a cell membrane receptor that mediates intercellular signaling.It takes a crucial part in intercellular communication.Different Notch receptors can mediate various cell effects.Notch3 mainly locates in VSMCs.The abnormal Notch3 signal is usually related to VSMC hyperproliferation and vascular remodeling induced by pulmonary hypertension(PAH),In addition,Notch3 is considered to be an important mediator of VSMC dedifferentiation and proliferation in pulmonary hypertension.The Hes/Hey family is the key downstream gene of Notch signaling.It has been confirmed that the overexpression of Notch3 leads to a significant increase of PASMCs proliferation of rat,and up-regulation of Hesl transcription factor Hesl transcription factor may be an important transcription target of Notch3 signal in PASMCs.Therefore,it is considered to be one of the valuable ways for treating PPHN by blocking the Notch3/Hes1 pathwaySildenafil is a phosphodiesterase-5(PDE5)inhibitor that induces vasodilation by inhibiting the hydrolysis of cGMP.Inhaled nitric oxide(iNO)is widely used in the treatment of PPHN by non selective dilation of pulmonary vessels,but some patients with PPHN are failure in the treatment of iNO,especially in patients with pulmonary parenchyma disease and pulmonary hypoplasia.However,sildenafil can improve the local microcirculation of neonates,effectively inhibit and prevent lung injury,reduce pulmonary artery pressure,and improve right ventricular function.Therefore,sildenafil may be the best choice for the treatment of PPHN.At present,there is no relevant report on the change and effect of Notch3/Hes1 signal pathway in PPHN rats and PASMCs.And it has not been reported that whether sildenafil’s anti PPHN effect is related to Notch3/Hes1 signal pathway.Therefore,the following research has been carried out in this experiment.Part Ⅰ The effect of sildenafil on PASMC and pulmonary vascular remodeling in PPHN ratsObjective:A rat model of PPHN was established by hypoxia and indomethacin to study the effect of sildenafil on PPHN and the expression of Notch3/Hes1 on the process of PPHN formation.The results showed that sildenafil could inhibit the proliferation of PASMC,reduce the pulmonary artery pressure,and improve the right ventricular function of PPHN rats.Sildenafil could reduce the gene and protein expression of Notch3/Hesl pathway in the lung tissue of PPHN rats.Methods:1.Establishment of PPHN rat model induced by Hypo+Indom and treatment SD rats of 8-weeks old(20 male rats and 46 female rats)were divided into different groups and caged for 24 hours,and the smear of female rats’ Yin thrombus was taken for detection.Under the microscope,the sperm was regarded as pregnant rats,36 pregnant rats in total.After the 11th day of pregnancy,30 rats were randomly divided into control group,model group,sildenafil low dose group(50mg/kg),sildenafil medium dose group(100mg/kg),and sildenafil high dose group(200mg/kg).The pregnant rats in the control group were exposed to normal oxygen and injected with 0.9%NaCl2 solution of the same volume intraperitoneally.The rats in the model group were fed in 12%O2 anoxic incubator after 19 days of gestation.The temperature and humidity in the incubator were the same as those in the outside environment,and the pregnant rats could move freely.Indomethacin 0.5mg/kg was injected intraperitoneally twice a day for 3 days.The treatment of sildenafil in the experimental group was the same as that in the model group.After 11 days of pregnancy,sildenafil with different dosages(50mg/kg,100mg/kg,200mg/kg)was injected intraperitoneally once a day for 10 consecutive days.2.Identification of PPHN rat model(1)Measurement of right ventricular hypertrophy index(RVHI):the rat heart was rapidly dissociated after thoracotomy,the atria tissue was removed,and the right ventricle(RV)was cut off along the ventricular margin.The weight ratio of RV to interventricular septum and left ventricle(LV+IVS)was calculated,and the right ventricular hypertrophy index was estimated.(2)Evaluation of pulmonary vascular remodeling:HE staining was utilized to observe the morphological changes of pulmonary vessels.The pulmonary arteries and pulmonary arterioles with a diameter of 50-200μm were selected to measure the internal and external diameters(ID,ED)of pulmonary vessels.The percentage of PAWT was calculated according to the following formula:PAWT(%)=(ED-ID)/EDx 100%3.Identification of PASMCs PASMCs from newborn rats were cultured by primary culture method,and identified by a-SMA immunofluorescence staining.4.Effects of sildenafil on proliferation,migration and apoptosis of Hypo-PASMC(1)MTT and flow cytometry were used to detect the effect of sildenafil on proliferation and apoptosis of PASMCs;(2)Flow cytometry was used to detect the effect of sildenafil on cycle of PASMCs;(3)Wound healing and Transwell assay were used to detect the effect of sildenafil on migration and invasion of PASMCs.5.Detection of Notch3 and Hesl mRNA expression in lung tissue and PASMCThe expression of Notch 3 and Hesl mRNA in lung tissue and PASMCs was detected by qRT-PCR.6.Detection of Notch3 and Hes1 mRNA protein expression in lung tissue and PASMCThe expression of Notch 3 and Hes1 protein in lung tissue and PASMCs was detected by Western blot.Results:1.Identification of PPHN animal model and effect of sildenafil on Hypoxia-induced pulmonary vascular remodeling and expression of Notch3/Hesl pathway(1)RVHI assay results:Compared with the control group,RVHI in model group was significantly higher than those in model group,(0.23±0.03)vs(0.49±0.05),P<0.001.Compared with the model group,RVHI significantly reduced in sildenafil group,(0.40±0.04)or(0.36±0.03)vs(0.49±0.05),P<0.001.(2)HE lung staining and PAWT results of lung tissue showed that the vascular smooth muscle cells in the control group arranged regularly with smooth intima and large lumen.In the model group,the middle layer of pulmonary artery obviously thickened,the vascular smooth muscle cells hypertrophied and proliferated,and the lumen became narrow.Compared with the model group,the lung tissue morphology in the sildenafil group improved,the middle layer of pulmonary artery obviously thinned,the vascular smooth muscle cells arranged regularly,and the lumen obviously enlarged.The results of PAWT indicated that,compared with the control group,the PAWT in the model group significantly increased,(0.30±0.04)vs(0.52±0.04),P<0.001,while sildenafil could effectively reduce the PAWT(0.42±0.03)or(0.38±0.06)vs(0.52±0.04),P<0.001.(3)The effect of sildenafil on the expression of Notch3 and Hesl protein and mRNA in the lung tissue of PPHN rats:Compared with the control group,the protein and mRNA expression of Notch 3 and Hes1 in the model group significantly increased(P<0.001);after sildenafil administration,the protein and mRNA expression of Notch 3 and Hes1 was lower than those in the model group(P<0.05,P<0.001).2.The results of primary PASMCs identification showed that 97%cells were positive for α-SMA.3.Effects of sildenafil on proliferation,apoptosis,migration,invasion and expression of Notch3/Hesl pathway in cultured hypoxia-induced PASMCs(1)The proliferation and apoptosis of PASMCs:Compared with the control group,the proliferation of PASMCs in the model group enhanced(P<0.001),while sildenafil could significantly reduce the cell proliferation(P<0.001).The proportion of apoptosis in the model group remarkably reduced(P<0.001).After sildenafil was given,the proportion of apoptosis cells significantly increased(P<0.05,P<0.001);(2)Results of PASMCs cycle showed that,compared with the control group,the proportion of S-phase cells in the model group increased(P<0.001).The proportion of S-phase cells decreased significantly after sildenafil treatment(P<0.05,P<0.001);(3)Results of migration and invasion of PASMCs showed that the migration distance and the percentage of migration area in the model group significantly increased when compared with the control group(P<0.001).while the migration distance and the percentage of migration area in the model group decreased after treated with sildenafil(P<0.05,P<0.001).(4)The detection results of the effect of sildenafil on the expression of Notch3 and Hesl protein and mRNA in PASMCs.The results indicated that,compared with the control group,the protein and mRNA expression level of Notch 3 and Hesl in the model group significantly increased(P<0.001).the protein and mRNA expression of Notch3 and Hesl was lower than those in the model group after sildenafil treatment(P<0.05,P<0.001).Conclusion:1.Sildenafil can alleviate RVHI and regress pulmonary vascular remodeling induced by hypo+Indom in PPHN rats.2.Sildenafil can inhibit the proliferation,migration and invasion of PASMCs,and promote the apoptosis of PASMCs.3.Sildenafil can inhibit the increase of Notch 3 and Hesl mRNA and protein in the lung tissue of PPHN in vivo and PASMCs in vitro.Part Ⅱ Sildenafil inhibits the proliferation and invasion of hypoxia-induced PASMCObjective:As above-mentioned,the PASMCs of newborn rats with Hypoxia-induced were primarily cultured to investigate whether sildenafil could inhibit PASMC proliferation and pulmonary vascular remodeling through Notch3/Hes1 pathway.Methods:1.Silence Notch3 Short hairpin RNA(shRNA)was transfected into the cultured Hypoxia-induced PASMCs by lentiviral transfection.The effect of Notch3 on PPHN was detected by knockdown of Notch3.2.Overexpression of Notch3 Notch3 overexpression plasmid was fabricated and transfected into Hypo-PASMCs by lentivirus,and investigated whether sildenafil affects Hypo-PASMC through the Notch3 pathway through the overexpression of Notch3.3.Detection of Notch3 and Hesl mRNA and protein expression qRT-PCR was perfomed to detect the expression of Notch 3 and Hesl mRNA in Hypo-PASMCs;western blot was used to detect the expression of Notch3 and Hesl protein in PASMCs of PPHN rats.4.Detection of Cell proliferation,apoptosis,migration and invasion MTT and flow cytometry were adopted to evaluate cell proliferation and apoptosis;flow cytometry was used to evaluate the effect of cell proliferation cycle;wound healing and Transwell assay were used to evaluate cell migration and invasion.Results:1.Sildenafil play roles through Notch3/Hesl signaling pathway Compared with the control group,the expression level of Notch3/Hesl in the model group significantly increased both at protein and mRNA levels(P<0.001).the expression of Notch3/Hesl protein and mRNA was significantly lower than those in the model group after given sildenafil(P<0.01,P<0.001).The results implied that sildenafil could pass through the Notch3/Hes1 signal pathway.2.Sildenafil inhibits PASMCs proliferation,migration,invasion and increases apoptosis through Notch3/Hesl pathway(1)Proliferation and apoptosis experiment results of cultured PASMCs After transfection of Notch3 shRNA sequence,rat PASMCs proliferation induced by hypoxia was inhibited and apoptosis increased(P<0.001,P<0.001),which exhibits the same effect as the sildenafil group;while Notch3 overexpression reversed sildenafil’s inhibition of cell proliferation and promotion of apoptosis(P<0.001).(2)The results of the flow cytometry experiment of culturing PASMCs Both sildenafil and Notch3 shRNA groups can significantly reduce the increase in the proportion of cells in S phase induced by hypoxia(P<0.001)and keep the cells in G0/G1 phase,while the Notch3 overexpression group can reverse the difference both sildenafil and Notch3 shRNA(P<0.001).(3)The results of migration and invasion experiments indicated that Notch3 silencing group had the same inhibitory effect as sildenafil on PASMCs migration and invasion(P<0.001,P<0.01).While Notch3 overexpression significantly weakened sildenafiul’s inhibitory effect on PASMCs migration and invasion(P<0.05).Conclusion:1.Notch3/Hesl signaling pathway involved in sildenafil inhibiting hypoxia-induced pulmonary artery smooth muscle cell proliferation and pulmonary vascular remodeling.2.Sildenafil inhibits the proliferation,migration and invasion of PASMC by regulating Notch3/Hesl signaling pathway,and promotes PASMC apoptosis,thereby inhibiting pulmonary vascular remodeling in PPHN rats.