Effects Of Astragaloside Ⅳ On Vascular Remodeling And POSTN/TGF-β1 Signaling Pathway In Pulmonary Arterial Hypertension

ObjectiveTo investigate the protective effect of astragaloside Ⅳ(ASⅣ)on vascular remodeling in hypoxia-induced pulmonary arterial hypertension(PAH),and to explore its effect on POSTN/TGF-β1 signaling pathway.MethodsIn vivo,sixty healthy male Kunming mice were randomly segmented into normal control group,hypoxia model group,low dose ASⅣ group(40 mg/kg),medium dose ASⅣ group(80 mg/kg)and high dose ASⅣ group(120 mg/kg).The mice in the normal control group were placed in a normoxic environment,and the mice in the other groups were placed in a low oxygen chamber with a10% oxygen concentration invariably for 4 weeks to establish the pulmonary arterial hypertension model.From the first day of modeling,the mice in ASⅣ low,medium and high dose groups were given the corresponding concentration of ASⅣ by gavage once a day.After 4 weeks,mean pulmonary artery pressure(m PAP),right ventricle systolic pressure(RVSP)and right ventricle hypertrophy index(RVHI)were detected.Hematoxylin-eosin staining was used to evaluate the pathological remodeling of pulmonary arterioles,and the vascular wall thickness ratio and vascular wall area ratio were detected.The protein expression of α-smooth muscle actin(α-SMA)was detected by immunofluorescence.Masson staining was used to observe the degree of fibrosis in lung tissue and right heart tissue of mice,and the ratio of collagen deposition area was calculated.The content of hydroxyproline(HYP)in serum and lung tissue homogenate was detected by immunoenzyme-linked immunosorbent assay.Immunohistochemistry was used to detect the positive expression of periostin(POSTN),Col-I,and Col-III in lung tissue and right heart tissue.The expressions of POSTN,matrix metalloproteinase-9(MMP-9),tissue inhibitor of matrix metalloproteinases-1(TIMP-1),Col-I,Col-III,transforming growth factor-β1(TGF-β1)and phosphorylation of Smad2/3 in lung tissue were detected by Western blot.In vitro,primary cultured pulmonary artery smooth muscle cells(PASMCs)from SD rats were placed in a hypoxia incubator(3%O2,97%N2)for 24 hours to establish the hypoxia model.To further verify the inhibitory effect of ASⅣ on POSTN/TGF-β1 signaling pathway,recombinant mouse POSTN(r POSTN)was introduced into cell experiments.PASMCs were divided into normoxia control group,hypoxia model group,hypoxia+recombinant POSTN group(r POSTN,50 ng/m L),hypoxia+ASⅣ group(ASⅣ,40 μM)and hypoxia+r POSTN+ASⅣ group.Ed U staining was used to detect the proliferation of PASMCs.The protein expression levels of POSTN,MMP-9,TIMP-1,TGF-β1 and phosphorylation of Smad2/3 in PASMCs were detected by Western blot.ResultsIn vivo,compared with the normal control group,m PAP,RVSP and RVHI of the hypoxia model group were significantly increased(P<0.01).The wall thickness ratio and wall area ratio of pulmonary arterioles increased significantly(P<0.01).The area ratio of collagen deposition and the protein expression of Col-I and Col-III in lung tissue and right heart tissue were significantly increased(P<0.01).The content of HYP in serum and lung tissue homogenate increased significantly(P<0.01).The protein expression levels ofα-SMA,POSTN,MMP-9,TIMP-1,TGF-β1 and the phosphorylation level of Smad2/3 in lung tissue were significantly increased(P<0.01).Compared with the hypoxia model group,m PAP,RVSP and RVHI of the low,medium and high dose ASⅣ groups were significantly decreased(P<0.01).The wall thickness ratio and wall area ratio of pulmonary arterioles decreased significantly(P<0.01).The area ratio of collagen deposition and the protein expression of Col-I and Col-III in lung tissue and right heart tissue were significantly decreased(P<0.01).The content of HYP in serum and lung tissue homogenate decreased significantly(P<0.05,P<0.01).The protein expression levels of α-SMA,POSTN,MMP-9,TIMP-1,TGF-β1 and the phosphorylation level of Smad2/3 in lung tissue were significantly decreased(P<0.05,P<0.01).In vitro,compared with the normoxia control group,the positive expression of Ed U in PASMCs in the hypoxia model group was significantly increased,and the protein expression levels of POSTN,MMP-9,TIMP-1,TGF-β1,and pSmad2/3 were significantly increased(P<0.01).Compared with the hypoxia model group,the positive expression of Ed U in PASMCs was increased in the hypoxia+r POSTN group,and the protein expression levels of POSTN,MMP-9,TIMP-1,TGF-β1,and p-Smad2/3 were increased(P<0.05).Compared with the hypoxia model group,the positive expression of Ed U in PASMCs in hypoxia+ASⅣ group was significantly decreased,and the protein expression levels of POSTN,MMP-9,TIMP-1,TGF-β1,and p-Smad2/3 were significantly decreased(P<0.01).Compared with the hypoxia+ASⅣ group,the positive expression of Ed U in PASMCs was significantly increased in the hypoxia+r POSTN+ASⅣ group,and the protein expression levels of POSTN,MMP-9,TIMP-1,TGF-β1,and p-Smad2/3 were significantly increased(P<0.01).Conclusions(1)ASⅣ can reduce the fibrosis of lung tissue and right heart tissue in hypoxia-induced PAH mice.(2)ASⅣ can improve pulmonary arterial remodeling in hypoxia-induced PAH mice.(3)ASⅣ can improve hypoxia-induced pulmonary vascular remodeling by inhibiting POSTN/TGF-β1 signaling pathway.