| Objective Cysteine-rich protein 61(CCN1,cyr61)is an extracellular matrix protein.Pulmonary artery hypertension(PAH)is a kind of cardiorespiratory disease that causes right heart failure and death due to the continuous increase of pulmonary artery pressure.Astragaloside Ⅳ(ASⅣ)is a natural triterpenoid glycoside extracted from Astragalus.Assess whetherCCN1 participates in PAH,study the potential mechanism of ASⅣ and CCN1 in PAH,and confirm whether the protective effect of ASⅣ on PAH is related to CCN1 and its related ERK1/2signal pathway.Methods In vivo experiments,50 male Sprague-Dawley(SD)rats were placed in a10% oxygen concentration normobaric hypoxia chamber for 12 hours a day for4 weeks to establishPAH model.After that,they were randomly divided into five groups(n =10): Normoxia group(Nor),hypoxia model group(Hyp),hypoxia+low-dose Astragaloside Ⅳ group(ASⅣ 20mg/kg),hypoxia+medium-dose Astragaloside Ⅳ group(ASⅣ 40mg/kg),hypoxia+high-dose Astragaloside Ⅳ group(ASⅣ 80mg/kg).50 male SD rats were randomly selected and 40 rats were intraperitoneally injected with monocrotaline(MCT,60mg/kg)for 4 weeks to establishPAH model.Then they were randomly divided into five groups(n=10): Control group(Con),pulmonary hypertension model group(PAH),MCT+Astragaloside Ⅳ low-dose group(ASⅣ 20mg/kg),MCT+Astragaloside Ⅳ medium-dose group(ASⅣ 40mg/kg),MCD+Astragaloside Ⅳ high-dose group(ASⅣ 80mg/kg).The expression of CCN1 protein in lung tissue of PAH model induced by hypoxia was analyzed by liquid chromatography-mass spectrometry;The right ventricular systolic pressure(RVSP),mean pulmonary artery pressure(mPAP)and right ventricular hypertrophy index(RV/(LV + s)of the two PAH models were measured by hemodynamic method and weighing method respectively;The pathological changes of pulmonary arteriole wall thickness percentage(WT%)and wall area percentage(WA%)in the two PAH models were detected by hematoxylin-eosin(H&E)staining;Detection of pulmonary artery smooth muscle action into PAH models by immunofluorescence(α-SMA);Immunohistochemistry(IHC)was used to detect the expression of CCN1 and Caspase-3 proteins in lung tissue of rats with two PAH models;Tunel fluorescence staining was used to detect the apoptosis of lung tissue cells in the two PAH models;Western blot was used to detect the expression of CCN1,Bax,Bcl-2,Caspase-3,ERK1/2,p-ERK1/2 proteins in the lung tissues of the two PAH models.In vitro experiments,human pulmonary artery endothelial cells(hPAECs)were placed in an anoxic chamber at 3% oxygen concentration for 24 hours to establish the model.Then they were divided into Normoxia group(Nor),hypoxia model group(Hyp),hypoxia+low-dose Astragaloside Ⅳ group(ASⅣ 10μM)Hypoxia+Astragaloside Ⅳ medium dose group(ASⅣ 20μM)and hypoxia+Astragaloside Ⅳ high-dose group(ASⅣ 40μM),hypoxia+ERK1/2agonist(EGF,10 nM);At the same time,monocrotaline pyrrole(MCTP,60μg/ml).Then they were divided into Control group(Con),model group(MCTP),MCTP+Astragaloside Ⅳ low-dose group(ASⅣ 10μM)MCTP+Astragaloside Ⅳ medium dose group(ASⅣ 20μM)and MCTP+high-dose group(ASⅣ 40μM)and MCTP+ERK1/2 agonist(EGF,10 nM).CCK-8 method was used to detect the activity of hPAECs in the two models;The apoptosis of hPAEC in the two models was detected by flow cytometry;The fluorescence expression of CCN1,Caspase-3 and JC-1 in the two models was detected by IF;Western blot was used to detect the expression of CCN1,Bax,Bcl-2,Caspase-3,ERK1/2,p-ERK1/2 proteins in the two models,and the effect of CCN1 knockout virus(sir NACCN1),CCN1 recombinant protein(rCN1)and EGF on the expression of CCN1 and ERK1/2 signal pathways.Results In vivo experiment: proteomic analysis showed that the expression of CCN1 protein in lung tissue was significantly decreased in the fourth week of hypoxia-induced PAH model,but still higher than that in the Nor group;IHC results showed that the expression of CCN1 protein in lung tissue of Hyp/MCT group was higher than that of Nor/Con group;The IF results showed that the expression of CCN1 protein in Hyp/MCTP group was higher than that in Nor/Con group;Western blot showed that the expression of CCN1 protein in lung tissue increased in the early stage of PAH(2-3 weeks after hypoxia induction or intraperitoneal injection of MCT)compared with that in the Nor/Con group;During the progression of PAH(4-5 weeks after hypoxia or intraperitoneal injection of MCT),the expression of CCN1 protein in lung tissue decreased,indicating that CCN1 was expressed in PAH and was dynamic;RVSP,mPAP,RV/(LV+s),WT%,WA% compared with Nor/Con group.The expression of α-SMA protein increased significantly.After ASⅣ treatment,the above indexes were significantly reduced,indicating that ASⅣ has protective effect on PAH;IHC and Western blot results showed that compared with Hyp/MCT group,the expression of CCN1 protein in lung tissue after ASⅣ treatment increased significantly,indicating that ASⅣ can increase the expression of CCN1 protein.The results of Tunel fluorescence analysis and Western blot showed that compared with Hyp/MCT group,the expression of apoptotic protein Caspase-3 and Bax in lung tissue was significantly increased,and the expression of anti-apoptotic protein Bcl-2 was significantly decreased.After ASⅣ treatment,the apoptosis of lung tissue was significantly decreased,the expression of apoptosis-promoting protein Caspase-3 and Bax protein was significantly decreased,and the expression of anti-apoptotic protein Bcl-2 was significantly increased.In vitro experiment: hypoxia/MCTP induced hPAECs at different stages.Western blot results showed that the expression of CCN1 protein increased after 8 and 16 hours of hypoxia or MCTP induction,and the expression of CCN1 protein decreased gradually after 24 and 32 hours of hypoxia or MCTP induction, which was consistent with the experimental results in vivo,indicating that the expression of CCN1 in the model was dynamic;The results of IF detection 24 hours after hypoxia/MCTP induction showed that the expression of CCN1 protein in Hyp/MCTP group was higher than that in Nor/Con group;The results of CCK-8 method showed that compared with Hyp/MCTP group,the activity of hPAECs cells in ASⅣ treatment group and EGF group increased significantly,indicating that ASⅣ has protective effect on PAH,while EGF and ASⅣ have the same effect on polycyclic aromatic hydrocarbons;Compared with Hyp/MCTP group,after treatment with ASⅣ and EGF: 1.Western blot and IF results showed that the expression of CCN1 protein was significantly increased;2.The expression of pro-apoptotic proteins Caspase-3 and Bax decreased significantly,the expression of anti-apoptotic protein Bcl-2 increased significantly,and the apoptosis of hPAECs decreased;3.JC-1 mitochondrial membrane potential increased gradually,the expression of polymer red fluorescence increased,the expression of monomer green fluorescence decreased,and the apoptosis of hPAECs decreased;4.Flow cytometry showed that apoptosis of hPAECs in ASⅣ treatment group decreased;The above results further showed that ASⅣ could inhibit cell apoptosis and regulate the expression of apoptosis-related genes.EGF and ASⅣ have the same effect on PAH;Western blot results showed that compared with the Hyp/MCT group,the effects of rCCN1 and EGF on hPAECs were similar to those of ASⅣ treatment.By increasing the expression of CCN1,activating ERK1/2 signal pathway and increasing the expression of p-ERK protein;The effect of siRNACCN1 on hPAECs is contrary to that of ASⅣ.By reducing the expression of CCN1,inhibiting the ERK1/2 signal pathway and reducing the expression of p-ERK protein,this indicates that there is an interaction between the signal pathway of CCN1 and ERK1/2.Conclusions The results showed that ASⅣ could increase the expression of CCN1,activate ERK1/2 signal pathway,improve PAH and reduce cell apoptosis.(1)CCN1 was expressed in the early to late stage of PAH,showing a dynamic expression of first rising and then decreasing;ASⅣ has a protective effect on PAH by increasing the expression of CCN1 protein,while inhibiting cell apoptosis and the expression of apoptosis-related genes.(2)RCCN1,EGF and ASⅣ have similar therapeutic effects,and activate ERK1/2 signal pathway by up-regulating the expression of CCN1;Contrary to ASⅣ treatment,CCN1 siRNA inhibits the ERK1/2 signal pathway by down-regulating the expression of CCN1. |
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