Anti-pulmonary Artery Hypertension Of Osthole Through CaN/NFATc2 Signaling Pathway

Objective: Based on Calcineurin/Nuclear factor of activated T cells 2(CaN/NFATc2)signaling pathway to study the mechanism of Osthole(Ost)against pulmonary artery hypertension.Methods:(1)In Vivo experiment: 60 male SD rats(280-300 g)were randomly divided into 6 groups: blank control group(n=10),model group(n=10),Ost groups of low dose,middle dose,and high dose(Ost-L,Ost-M,Ost-H,n=10),and cyclosporine A(CsA)group(n=10).After one week of adaptive feeding,all rats,except the blank control group,were subcutaneously injected with MCT 55 mg/kg to establish pulmonary artery hypertension models.After two weeks.Ost(5,10 and 20 mg/kg)were treated by gavage in Ost-L,Ost-M and Ost-H groups.CsA 1mg/kg was treated by gavage in CsA group.The equal volume of double distilled water were given by gavage in the blank control and the MCT group.The weight of lung tissue and body were measured.Then,the lung weight index was calculated as the ratio of lung weight to body weight.HE staining was used to observe the morphology of pulmonary artery.Western Blot was used to detect the protein levels of PCNA,CaN,NFATc2,Kv1.5,Cyt C,Caspase 9,COX-2,IL-6,IL-1?,TNF-a and MCP-1 in lung tissues.(2)In Vitro experiment: Pulmonary artery smooth muscle cells(PASMCs)were isolated from pulmonary arteries using the explant method and immunofluorescence staining was used to identify the PAMSCs with morphology observing,then PASMCs were divided into 5 groups: normal group,platelet-derived growth factor-BB(PDGF-BB)group and PDGF-BB+Ost(5 ?M,10 ?M and 20 ?M)group.All PASMCs,except the normal group,were treated with PDGF-BB(25 ng/mL)to establish the model of proliferation of PASMCs.Culturing PAMSCs 24 hours later,CCK-8 was used to detect the proliferation activity of PASMCs.The protein levels of PCNA,CaN,Kv1.5,Cyt C and Caspase 9 were detected by Western blot in PASMCs groups.Results:(1)In Vivo experiment: Compared with the blank control group,the mean pulmonary artery pressure and lung weight index of the model group were significantly increased(P<0.05);the pulmonary vessel wall was thickened,the lumen was narrow and the squeezed "wavy shape" inner elastic plate was evident in H&E staining;the protein levels of PCNA,CaN,NFATc2,COX-2,IL-6,IL-1?,TNF-a,and MCP-1 were increased significantly in lung(P<0.05),and the protein levels of Kv1.5,Cyt C,and Caspase 9 were significantly decreased in lung(P<0.05).Compared with the MCT model group,the mean pulmonary artery pressure and lung weight index were significantly down-regulated in Ost-M,Ost-H and CsA groups;the thickness of membrane became thinner,and the lumen became wider of the pulmonary artery in Ost-M,Ost-H and CsA groups;the protein levels of PCNA,CaN,NFATc2,COX-2,IL-6,IL-1?,TNF-a and MCP-1 were significantly decreased(P<0.05),and the protein levels of Kv1.5,Cyt C and Caspase 9 were significantly increased(P<0.05)in lung tissue of Ost-M,Ost-H and CsA groups.(2)In Vitro experiment: Compared with the normal group,the proliferation activity of PASMCs was significantly increased,the protein levels of PCNA and CaN were significantly increased and the protein levels of Kv1.5,Cyt C,and Caspase 9 were significantly decreased in PDGF-BB group(P <0.05).However,the proliferation activity of PASMCs was significantly inhibited and the protein levels of PCNA and CaN of PASMCs were significantly decreased and the protein levels of Kv1.5,Cyt C,and Caspase 9 of PASMCs were significantly increased by Ost(P<0.05).Conclusion: Ost can improve the pulmonary artery hypertension,and the mechanisms may be related to the repression of CaN/NFATc2 signaling pathway.