| Objective:To investigate the effects of relm-β on pulmonary arterial hypertension(HPH)and the up-regulation of cyclin expression via Ca2+/PI3K/Akt/mTOR pathway,thereby inducing the proliferation of pulmonary arterial smooth muscle cells(PASMCs).Methods:Animal experiments:Hemodynamics,pulmonary vascular morphology and expression of RELM-β,Akt and mTOR in lung tissues of SD rats were detected.There were two types of male SD rats:wild type and RELM-β complete knockout type(RELM-β(-/-)).The rats were divided into 4 groups with 5 rats in each group,which were wild-type hypoxic group,RELM-β(-/-)hypoxic group,wild-type normoxic group and RELM-β(-/-)normoxic group.The HPH model of the hypoxic group was established by intermittent hypoxic method(at atmospheric pressure,the oxygen concentration in the tank was 10%±0.5%,hypoxic for 8 hours a day for 21 days),and the normoxic group was cultivated under atmospheric pressure and normal oxygen conditions.Mean pulmonary arterial pressure(mPAP)of four groups of rats was measured by right cardiac catheter.Right ventricular hypertrophy index(RVHI)was measured by weighing method.The ratio of pulmonary artery wall thickness to vascular diameter(WT%)was calculated to indicate the degree of pulmonary artery remodeling.Western blot(WB)was used to detect the protein expression levels of RELM-β,PI3K,Akt and mTOR in lung tissues.Quantitative Real-time PCR(qPCR)was used to detect the mRNA relative expression of RELM-β in lung tissues.Cell experiment:The expression levels of RELM-β,Ca2+,PI3K,Akt,mTOR,cyclin D1/E and CKD2/4 in PASMCs were detected,and RELM-β up-regulated the expression of cyclin through Ca2+/PI3K/Akt/mTOR signaling pathway,thereby inducing the proliferation of PASMCs.RELM-β(-/-)and wild-type PASMCs of rats were isolated and primary cultured.PASMCs were treated with hypoxia(cells were placed in a 1%O2,94%N2 and 5%CO2 three gas incubator for 24h)and normoxia(cells were placed in a 21%O2,74%N2 and 5%CO2 cell incubator),respectively.The cells were divided into wild-type hypoxic group,RELM-β(-/-)hypoxic group,wild-type normoxic group and RELM-β(-/-)normoxic group.The wild-type hypoxic group was selected to detect the signaling pathway and the regulatory effect on PASMCs proliferation,which was verified by adding Ca2+internal and external chelator(BAPTA-AM+EGTA),PI3K inhibitor(LY294002)and Akt inhibitor(KRK-0401)to detect downstream signaling molecules and PASMCs proliferation,respectively.The expression levels of RELM-β,PI3K,Akt,mTOR,cyclin D1/E and CKD2/4 in PASMCs were detected by WB.The mRNA relative expression of RELM-β in PASMCs was detected by qPCR.Flow cytometry(FCM)was used to detect Ca2+concentration in PASMCs.PASMCs proliferation was detected by EDU.Results:Animal experiments:mPAP,RVHI and WT%of rats in wild-type hypoxia group were significantly higher than those in wild-type normoxia group and RELM-β(-/-)hypoxia group(P<0.05);Compared with RELM-β(-/-)hypoxic group,the mPAP,RVHI and WT%of rats in RELM-β(-/-)hypoxic group were increased(P<0.05);There were no significant differences in mPAP,RVHI and WT%between wild-type and RELM-β(-/-)normoxic groups(P>0.05).RELM-β and downstream pathway protein expression in lung tissues Results:WB and qPCR detection of RELM-β expression indicated that RELM-β(-/-)group in hypoxic and normoxic groups was not expressed at all.Compared with RELM-β(-/-)hypoxic group,RELM-β and downstream phosphorylated protein expression were increased in wild-type hypoxic group(P<0.05).RELM-β and its downstream pathway proteins were significantly increased in wild-type hypoxia group compared with normoxia group(P<0.05).Compared with normoxic group,the expression of downstream phosphorylation pathway protein in RELM-β(-/-)hypoxic group was increased(P<0.05).RELM-β and its downstream pathway proteins were increased in wild-type normoxic group compared with RELM-β(-/-)normoxic group(P<0.05).Cell experiments:Expression of RELM-β,downstream pathway proteins and cyclins in PASMCs:WB and qPCR were used to detect the expression of RELM-β,indicating that RELM-β was not expressed at all in the hypoxic group and the normoxic group;Compared with RELM-β(-/-)hypoxic group,RELM-β,downstream phosphorylated protein and cyclin expression were increased in wild-type hypoxic group(P<0.05).RELM-β,downstream pathway protein and cell cycle protein were significantly increased in wild-type hypoxia group compared with normal oxygen group(P<0.05).Compared with normoxic group,the expressions of downstream phosphorylation pathway proteins and cyclins in RELM-β(-/-)hypoxic group were increased(P<0.05).RELM-β,downstream pathway protein and cyclin were increased in wild-type normoxic group compared with RELM-β(-/-)normoxic group(P<0.05).The proliferation of PASMCs in wild-type hypoxic group was significantly increased compared with RELM-β(-/-)hypoxic group(P<0.05).The proliferation ability of wild-type hypoxic treatment group was increased compared with normal oxygen group(P<0.05).The cell proliferation of RELM-β(-/-)hypoxic group was up-regulated compared with that of RELM-β(-/-)normoxic group(P<0.05).There was no significant difference between wild-type normoxic group and RELM-β(-/-)normoxic group(P>0.05).The proliferation ability of PASMCs was decreased after the addition of inhibitors compared with the control group(P<0.05).The expressions of downstream pathway proteins and cyclins were decreased compared with the control group after addition of all inhibitors(P<0.05).Conclusion:1.In hypoxic conditions,RELM-β up-regulates the expression of cyclin D1/E and CKD2/4 by Ca2+/PI3K/Akt/mTOR signaling pathway,increasing the proliferation ability of PASMCs and playing a role in the formation of HPH... |
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