| Background:Pulmonary artery hypertension(PAH)is a common and serious complication of the left to right shunt congenital heart disease(CHD).Its exact mechanism is not very clear,further research of its pathogenesis is necessary.In 1973,the first WHO meeting on pulmonary hypertension held in Geneva,Switzerland defined PAH as the average arterial pressure greater than or equal to 25mmHg,and/or the pulmonary artery systolic pressure greater than 30mmHg at sea level and at rest,or the average pulmonary artery pressure greater than 30mmHg at exercise.In 2008,the fourth PAH meeting of the WHO in Dana point,California,the United States,redefined PAH as the average pulmonary artery pressure greater than or equal to 25mmHg in the resting state[l]In 2013,the fifth PAH meeting of WHO in Nice,France,defined PAH as pulmonary vascular remodeling,which was a marker of the irreversible development of PAH caused by pulmonary vascular dysfunction.The proliferation of pulmonary artery smooth muscle cells(PASMCs)is the central link of the pathophysiological changes of pulmonary vascular remodeling[2].The incidence of PAH increases year by year,and the resistance and pressure of pulmonary blood vessels gradually increase with the progression of the disease.The condition eventually causes right heart failure,a series of clinical symptoms,even death.Endothelin-1(ET-1)is an important factor regulating the function of the vascular system,which is an active peptide composed of 21 amino acids and one of the strongest vasoconstriction regulators.It is also a kind of mitogen.Many kinds of cells can secrete ET-1,and the main source is vascular endothelial cells.Studies have shown that the production of ET-1 in the plasma and lung tissue of PAH animal models and patients is increased.The binding of ET-1 with its specific receptors mediates the vasoconstriction or diastole,which promotes or inhibits the proliferation and migration of vascular smooth muscle cells[3,4].ET-1 has two major receptors ETA and ETB.In the cardiovascular system,ETA and a small number of ETB receptors present on the surface of vascular smooth muscle cells,while ETB receptor mainly presents on the surface of vascular endothelial cells.The combination of ET-1 with ETA or ETB receptors on the membrane of pulmonary artery smooth muscle cells can promote the proliferation and migration of PASMCs,while the combination with ETB receptors on vascular endothelial cells can reduce the ET-1 in blood circulation,which indirectly relaxes blood vessels and inhibits the proliferation of PASMCs[4].As a dual endothelin receptor antagonist,bosentan(BST)can competitively bind to ETA and ETB receptors on the surface of PASMCs,which prevents the strong effects of ET-1 on promoting vascular contraction,transmission and migration of PASMCs and reduces pulmonary vascular resistance,and finally improves right ventricular function.Bosentan is the first approved oral targeted drugs of endothelin receptor antagonists for the treatment of PAH.Randomized,double-blind,multi-center series of clinical studies confirmed that bosentan was safe and effective in the treatment of PAH in children,and significantly improved symptoms,exercise endurance,and prevented disease progression[5,6].At present,there are few studies on the treatment of congenital heart disease combined with PAH in young infants with bosentan,especially in infants under 1 year old,which is worthy for further study.MicroRNAs(miRNAs)are a class of endogenous non-coding small molecule RNA,which are composed of about 20～24 bases.They can affect the shear of mRNA or cause the direct decomposition of mRNA through the specific binding to the genes encoding protein,which inhibit the translation level of protein and play a role in gene regulation[7].In 2010,a study about pulmonary arterial endothelial cells and PASMCs in PAH patients had confirmed the condition of genomic instability.This provides a theoretical basis to study the role of genomic regulatory dysfunction in pulmonary vascular remodeling.It has been proved that miRNAs play a key role in gene regulation in the occurrence of cardiovascular diseases[8].Through TargetScan software to analyze the target genes of miRNAs,dozens of miRNAs were found to be related to the pathogenesis of PAH.As upstream regulatory molecules,miRNAs are involved in the formation and development of PAH[19].Recent studies have confirmed that microRNA-27a(miR-27a)is up-regulated in the pulmonary tissue in animal hypoxic PAH models,and inhibition of miR-27a expression can reduce the proliferation of pulmonary artery endothelial cells and the generation of PAH[10].However,the effect of miR-27a on human PASMCs proliferation in vitro has not been reported.The peroxisome proliferator-activated receptor y(PPARy)belongs to the superfamily of nuclear hormone receptors,which is a kind of nuclear transcription factors activated by ligand.There are three different subtypes,PPARα、PPAR5 and PPARy,respectively.Each subtype plays a very important role in the celluar growth,celluar differentiation,celluar apoptosis,resistance in inflammatory reaction and in some metabolic diseases[13-16].PPARγ is highly expressed in vascular smooth Imuscle cells and myocardial cells,and regulates gene transcription by binding with ligands[11].Studies have found that PPARy is involved in the formation of hypoxic PAH in rats.The PPARγ ligand rosiglitazone(RSG)can reduce the formation of hypoxic PAH and enhance the expression of PPARγ[12,13].However,the changes and effects of PPARy or its ligands in the left to right shunt PAH have not been reported.Currently,the effect of bosentan on the miR-27a/PPARγ/ET-1 signaling pathway in left to right shunt PAH has not been reported too.Therefore,the following studies were conducted in this experiment:Part Ⅰ The study on the upstream molecular regulation mechanism of bosentan in the treatment of pulmonary artery hypertensionObjectiveThrough the intervention of in vitro cultured human PASMCs,to study the effects of bosentan on endothelin receptors,miR-27a and PPARy of cultured human PASMCs as well as the signal transduction mechanism.And to discuss the role of miR-27a in PAH.Methods1.The effect of bosentan on PASMCs proliferation:human PASMCs(passages 3-7)were inoculated on the 96-well plate(5 ×103 cells/well)for 24h.Then the cultured cells were divided into 3 groups with 6 wells in each group:(1)ET-1 group:1μM ET-1 was added in each well;(2)bosentan group(ET-1+BST):1μM ET-1+50μM bosentan were added per well;PBS was added iinto each well as control group.Continuing to cultivate for 24h,cell proliferation was determined by CCK8 method and BrdU incorporation assay.2.Effect of bosentan on the expression of endothelin receptors,miR-27a and PPARy of PASMCs:The expressions of ETAR mRNA,ETBR mRNA and miR-27a in cultured PASMCs were determined by qRT-PCR.The protein expressions of ETAR,ETBR and PPARy were determined by Western blot.3.Effect of miR-27a overexpression and inhibition on proliferation of PASMCs,expression of endothelin receptor and PPARy of PASMCs:the PASMCs cultured in vitro,after intervention with 0.1 μM ET-1 for 24h,then were transfected with miR-27a mimics(mimics group),miR-27a inhibitors(inhibitor group)and negative controls(miR-27a mimics NC and miR-27a inhibitor NC)using Lipofectarnine 2000.The PASMCs after being transfected with miR-27a mimics or miR-27a inhibitors were divided into two groups,respectively.One group was added 50μM bosentan,respectively(BST+mimics group,BST+inhibitor group).The untransfected group was taken as the mock group.After 48h of continuous culture,qRT-PCR and Western blot were used to detect the effect of miR-27a overexpression and inhibition on the expression of endothelial receptors and PPARy.4.Effect of PPARy inhibitor and ligand on the expression of endothelin receptors and miR-27a of PASMCs:after intervention with ET-1 for 24h,when studying the effect of PPARy inhibitors,1μM GW9662 was added to the GW9662 group,1μM GW9662 and 50μM posentan was added to the GW9662+BST group.To study the effect of PPARy ligand rosiglitazone,10μM rosiglitazone was added to the RSG group;10μM rosiglitazone and 50μM bosentan were added to RSG+BST group.The control groups were added with PBS.After 48h culture,the effect of PPARy on the expression of endothelin receptors and miR-27a of PASMCs was detected by qRT-PCR and Western blot.Results1.The results of CCK8 method and BrdU incorporation assay showed:the cells proliferated significantly after the exogenous application of ET-1.Compared with the control group,the OD value was(1.29±0.09)vs(1.04±0.04),P<0.05;and the number of positive cells BrdU labeled(%)was(59.71±2.52)vs(19.50±1.79),P<0.01.However,after the addition of bosentan,the cell proliferation was reduced.Compared with the ET-1 group,the OD value was(1.05±0.06)vs(1.29±0.09),P<0.05;and the number of positive cells BrdU labeled(%)was(26.83±1.38)vs(59.71±2.52),P<0.01,2.In the ET-1 group,the expressions of ETAR mRNA,ETBR mRNA and miR-27a were significantly increased compared with the control group(P<0.01).The expressions of ETAR mRNA,ETBR mRNA and miR-27a in the ET-1 +BST group were lower than those in the ET-1 group(P<0.01).Compared with the control group,the expressions of ETAR protein and ETBR protein were increased in the ET-1 group(P<0.01),while the expression of PPARy protein was decreased(P<0.01).ETAR and ETBR protein expressions in the ET-1 +BST group were lower than those in the ET-1 group(P<0.01,P<0.05,respectively),while PPARy protein expression was higher than that in the ET-1 group(P<0.01).3.After the transfection of miR-27a mimics,the mRNA and protein expression levels of ETAR and ETBR in the mimics group were significantly higher than those in the mock group(P<0.01),and the expression of PPARy protein was significantly lower than that of mock group(P<0.01).In the BST+mimics group,the mRNA and protein expression levels of STAR and ETBR were significantly lower than those in the mimics group(P<0.01).and the protein expression level of PPARy was significantly higher than that in the mimics group(P<0.01).After the transfection of miR-27a inhibitors,the mRNA and protein expression levels of inhibitor ETAR and ETBR were significantly lower than those of mock group(P<0.01),and the protein expression of PPARy was significantly higher than that of mock group(P<0.01).In BST+inhibitor group the mRNA and protein expression levels of ETAR and ETBR were lower than those in inhibitor group(P<0.05),and PPARγ protein expression was higher than that in inhibitor group(P<0.05).MiR-27a mimics NC and miR-27a inhibitor NC had no significant effect on the mRNA and protein levels of ETAR and ETBR and the expression of PPARy protein(P>0.05).4.After the application of PPARy inhibitor GW9662,the mRNA and protein expression levels of ETAR and ETBR were significantly higher than those of the control group(P<0.01),and the expression of miR-27a was significantly higher than that of the control group(P<0.01).In the GW9662+BST group,the mRNA and protein expression levels of ETAR and ETBR were significantly lower than those in the GW9662 group(P<0.01),and the miR-27a expression was significantly lower than that in the GW9662 group(P<0.01).After the application with PPARy ligand rosiglidone,the mRNA and protein expression levels of ETAR and ETBR in cultured cells were lower than those in the control group(P<0.05),and miR-27a expression was also found to be decreased(P<0.05).The mRNA and protein levels of STAR and ETBR in RSG+BST group were significantly lower than those in RSG group(P<0.01),while miR-27a expression was lower than that in RSG group(P<0.05).Conclusion1.Bosentan inhibited PASMCs proliferation by antagonizing ET-1 and further affected the miR-27a/PPARγ/ET-1 signaling pathway.2.MiR-27a interactively inhibited with PPARy,which regulated ET-1 to promote PASMCs proliferation.So we suggest that miR-27a/PPARy may be the upstream regulatory molecule of the ET-1 signaling pathway.Part Ⅱ The effect of bosentan on the expression of ET-1,miR-27a and PPARy in children with the left to right shunt congenital heart disease complicated with pulmonary artery hypertensionObjectiveThrough the collection of clinical data,to study the effects of bosentan on the ET-1 content in plasma,the expression of miR-27a and PPARγ in children with the left to right shunt congenital heart disease complicated with pulmonary artery hypertension.Methods1.Cases selection and drug use:thirty cases of the left to right shunt congenital heart disease children complicated with PAH,aged 3 months to 14 years,were selected.The children whose pulmonary artery systolic pressure measured by echocardiography higher than or equal to 60mmHg and average pulmonary artery pressure higher than or equal to 45mmHg were enrolled into the group(PAH group).After enrollment,bosentan was taken orally 2mg/kg twice a day for 8 weeks.Twenty healthy children were selected as the control group(normal control group)2.Children with PAH before treatment,after being treated for 4 weeks and 8 weeks were extracted of venous blood 5 ml respectively,including 1 ml blood used in the detection of platelet,liver and kidney functions,1 ml blood putted in EDTA anti coagulation test tube,centrifuging to take plasma in-20 ℃,used for determinating the level of ET-1.Another 3 ml blood was separated for miR-27a determination and keeped in preservation in-20 ℃,and simultaneous extracted of mononuclear cells for PPARy protein assay.3.The EF value of left ventricular and pulmonary artery systolic blood pressure were measured by echocardiography before medication,4 weeks after medication and 8 weeks after medication.4.Determination of ET-1 in plasma:ELISA was used to determine the changes of ET-1 in plasma of children in the normal control group and children with pulmonary artery hypertension before treatment,4 weeks and 8 weeks after the application of bosentan.5.Measurement of miR-27a expression in peripheral blood:qRT-PCR was used to determine the changes of the expression of miR-27a in circulating blood in normal control group and PAH children when before treatment,4 weeks and 8 weeks after the application of bosentan.6.The expression of PPARy in peripheral blood:Western blot was used to determine the PPARγ expression of circulating blood mononuclear cells in children in the normal control group and children with pulmonary artery hypertension before treatment,4 weeks and 8 weeks after the application of bosentan.Results1.Bosentan(2mg/kg)was used twice a day for the treatment of the left to right shunt pulmonary artery hypertension in children.Within 8 weeks,there was no statistically difference in liver and kidney function and platelet count.2.Echocardiography:After oral administration of bosentan for 4 weeks and 8 weeks,compared with before treatment,ejection fraction EF value was observably increased,P<0.01;and pulmonary artery systolic pressure was decreased,P<0.05.3.Compared with that in the healthy control group,the expression of ET-1 in plasma of children with pulmonary artery hypertension was increased,P<0.01.Compared with that before medication,the expression of ET-1 was decreased at 4 weeks and 8 weeks after oral administration of bosentan,P<0.01.4.The expression of miR-27a was increased in children with the left right shunt pulmonary artery hypertension compared with the healthy control group,P<0.01.Compared with those before the administration,the expression of miR-27a in plasma was decreased after oral administration of bosentan for 4 weeks and 8 weeks,P<0.01.5.Compared with healthy control group,PPARy protein level was decreased in children with pulmonary artery hypertension,P<0.01.After 4 weeks and 8 weeks of treatment with bosentan,the protein expression of PPARγ gradually recovered,and compared with that before the treatment,P<0.01.Compared with the control group,the relative protein expression of PPARy after treatment for 8 weeks showed no significant difference(P>0.05).Conclusion1.Bosentan was effective and safe in short-term treatment in children with PAH.2.The expression of miR-27a was enhanced in the circulating blood of children with PAH,and bosentan could reduce miR-27a level in children with PAH.3.The expression of PPARy of mononuclear cells in peripheral blood was decreased in children with PAH,and bosentan could enhance the expression of PPARγ in children with PAH. |
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