| Objective: Based on cell cycle and TGF-?1/Smads/p38 signaling to investigate the effects of Ost on the proliferation of pulmonary artery smooth muscle cells(PASMCs)during the pathogenesis of pulmonary arterial hypertension.Methods:(11)Animals experiment: There were thirty-nine of Sprague-Dawley(SD)male rats,and the body weight was 200~250g.Nine SD rats were randomly divided into control group(Control,n=9)by subcutaneous injection of saline 5 m L/kg.The other thirty of rats were given a single dose of monocrotaline 50 mg/kg subcutaneously to establish the pulmonary hypertension model,which were randomly divided into model group(Model,n=12),low-dose of Osthole treatment group(Ost-10,n=9)and high-dose of Osthole treatment group(Ost-20,n=9).Then the rats in the Ost-treatment groups were gavaged once daily from 1 day to 5 weeks.The other rats in the control and model groups were given the same amount of dd H2 O with tween 80 of 0.5 %.Observing the general status during rats administration.After 5 weeks of administration,the pulmonary arterial pressure was measured by right heart catheterization.The lung and body weingt were weighed after 5 weeks of administration,and the lung weight index was calculated.H&E staining was performed to investigate the pathological alterations of the pulmonary artery.The mRNA expressions of TGF-?1 and Smad2/3 were examined by real time PCR.The protein expressions of PCNA,TGF-?1 and p-Smad2 in lung tissue were detected by Western blotting.(12)Cells experiment: PASMCs were cultured by enzymatic digestion.Immunofluorescence cytochemistry was used to identify PASMCs.CCK-8 assay was used to evaluate the effect of Ost(5 ?M,10 ?M,20 ?M)on the proliferation of PASMCs induced by PDGF-BB(25 ng/m L).The flow cytometry was used to analyse the cell cycle.The protein expressions of PCNA,Cyclin D1,Cyclin E1,CDK4,CDK2,p21,p27,p53,TGF-?1,p-p38,p-Smad2/3 and MMP9 in PASMCs were detected by Western blotting.Results:(11)Animals experiment: In the Model group the rats were in poor spirits,decreased activity and reduced eating;the pulmonary arterial pressure were increased(P<0.05);the weight were lost and the lung weight index were significantly increased which compared with the Control group(P<0.05);pulmonary arterial medial membrane thicker was very serious and lung vascular narrower;the mRNA expressions of TGF-?1 and Smad2/3 mRNA were significantly raised(P<0.05);the protein expressions of PCNA,TGF-?1 and p-Smad2 were significantly up-regulated(P<0.05).In Ost-10,Ost-20 group the rats hair was shiny,activity and eating were increased;the pulmonary arterial pressure were reduced(P<0.05)and BW were rised(P<0.05),and LI were significantly decreased(P<0.05);pulmonary arterial medial thickening of the situation have improved which compared with the Model group.In Ost-10 and Ost-20 group,the mRNA expressions of TGF-?1 and Smad2/3 were significantly down-regulated(P<0.05)and the protein expressions of PCNA,TGF-?1 and p-Smad2 were significantly down-regulated(P<0.05).(12)Cells experiment: In CCK-8 assays,PDGF-BB could significantly increase the proliferation of PASMCs(P<0.05)and Ost-M,Ost-H groups could inhibit the proliferation of PASMCs induced by PDGF-BB(P<0.05).In flow cytometry assays,the percentage of the G2/M+S phase cells were increased by PDGF-BB(P<0.05),but Ost-M,Ost-H groups could decrease the percentage of the G2/M+S phase cells(P<0.05).At the same time,PDGF-BB could up-regulate PCNA,Cyclin D1,Cyclin E1,CDK4,CDK2,p21,TGF-?1,p-p38,p-Smad2/3 and MMP9 protein expressions(P<0.05),down-regulated p27 and p53 protein expressions(P<0.05).However,Ost could down-regulated the protein expressions of PCNA,Cyclin D1,Cyclin E1,CDK4,CDK2,TGF-?1,p-p38,p-Smad2/3 and MMP9(P<0.05),and up-regulate the protein expressions of p21,p27 and p53 in PASMCs(P<0.05).Conclusions: Ost could significantly inhibit the PASMCs proliferation induced by PDGF-BB,the regulation action of it on cell cycle and proliferation may be through TGF-?1/Smads/p38 signal pathway inhibition of PCNA,cyclin D1/CDK4,cyclin E1/CDK2,p21,p27,p53 and MMP9 protein expressions and the prevention of the limiting point that promote cell cycle into S phase to anti-pulmonary arterial hypertension. |
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