Effect Of Erk1/2 Phosphorylation On The Inhibition Of Proliferation Of Pulmonary Artery Smooth Muscle Cells By Treprostinil

ObjectiveOne of the characteristic pathological changes of pulmonary artery hypertension(PAH)is the abnormal proliferation of pulmonary artery smooth muscle cells(PASMCs),leading to progressive stenosis of pulmonary arterioles.Children are also one of the main populations of PAH.Phosphorylation of Erk1/2 is associated with proliferation of a variety of cells.Treprostinil(Trep)has recently been used to treat PAH,but its mechanism needs further investigation.In this study,PASMCs were used to investigate the effect of Trep on the proliferation of PAMSCs and the change of Erk1/2 phosphorylation level,aiming to explore new ideas for the treatment of PAH.MethodsThe PASMCs were resuscitated,the morphology of the cells was observed under microscope,and the cells were identified by immunofluorescence.Different concentrations of platelet-derived growth factor(PDGF)were used to promote the proliferation of PAMSCs,and a suitable concentration of PDGF was found to establish a proliferation model.Firstly,the effect of Trep on the proliferation of PAMSCs was observed and the phosphorylation level of Erk1/2 was detected.Cells were divided into control group,Trep group,PDGF group and PDGF+Trep group.The morphology of each group was observed under microscope.The cell activity of each group was detected by MTT assay.The expression of proliferating cell nuclear antigen(PCNA),t-Erk1/2 and p-Erk1/2 in each group was detected by Western Blot.Secondly,PD98059,the inhibitor of Erk1/2 activation,was used instead of Trep,and the above experiment was repeated to observe the effect of PD98059 on the proliferation of PAMSCs and the phosphorylation level of Erk1/2.Results(1)?-SMA was marked by Fitc with green,which was filamentous and parallel to the longitudinal axis of the cell under the fluorescence microscope.The shape of the cell is fusiform under the inverted phase contrast microscope.(2)Under the treatment of PDGF with a concentration of 20ng/L,PASMCs have the strongest cell activity by MTT method,and the intracellular PCNA protein was the highest by Western Blot.(3)Cell density of PDGF group was higher than that of control group,while cell density of PDGF+ Trep group was lower than that of PDGF group.The MTT assay showed that the cell viability of PDGF group was higher than that of the control group(P<0.05),and the cell viability of PDGF+ Trep group was lower than that of PDGF group(P<0.05).Western Blot showed that the expression of PCNA in PDGF group was higher than that of control group.The expression of PCNA in PDGF+ Trep group was lower than that of PDGF group(P<0.05).The ratio of p-Erk to t-Erk in PDGF group was higher than that of the control group,while the ratio of p-Erk to t-Erk in PDGF+ Trep group was higher than that of the PDGF group(P<0.05).(4)In the experiment of the effect of PD98059 on the proliferation of PAMSCs and the phosphorylation level of Erk1/2,showed that PD98059 had the similar effect with Trep.ConclusionTreprostinil inhibits PDGF-induced proliferation of PASMCs,and its mechanism may be related to altered Erk1/2 phosphorylation.