| Objective:In order to observe the effect of resistin-like molecule-beta on HPH and the expression of signaling pathway proteins(PLC,IP3R,Calcineurin,NF ATc3)in lung tissue of HPH rats.To investigate the role of RELM-β in the formation of hypoxic pulmonary hypertension via PLC/IP3R/Ca2+/C alcineurin/NFATc3 signaling pathway.Methods:In this study,male SD rats with RELM-β gene knockout(RELM-β1-/-)and Wildtype(WT)were used(about 12 weeks of age,weight 260-350 g).Normoxia group and Hypoxia group were set.Normoxia group:21%O2 concentration environment;Hypoxia group:Exposed to hypoxia((10±0.5)%O2 concentration environment)in a ventilated hypoxia chamber for 8 hours a day,uninterrupted for 21 days.The experimental groups were WT+Normoxia,WT+Hypoxia,RELM-β-/-+Hypoxia,RELM-β-/-+Normoxia,with 7 rats in each group.Right heart function was evaluated by ultrasonography.We measured the right ventricle anterior wall thickness(RVAWT)and pulmonary artery acceleration time(PA AT),pulmonary artery ejection time(PAET),ratio of pulmonic acceleration to ejection time(PAAT/PAET),tricuspid annular plane systolic excursion(TAPSE);Hemodynamic test was performed by right heart catheter.Mean pulmonary artery pressure(mPAP)was measured.Weighting method was used to determine the right ventricular hypertrophy index(RVHI%)of rats.HE staining and alpha-smooth muscle actin(α-SMA)immunofluorescence staining were performed on lung tissue.The ratio of wall thickness of small pulmonary vessels to blood vessel diameter(WT%)and the ratio of the wall area of small pulmonary vessels to the total area of blood vessels(WA%)were calculated.According to the level of HE staining andα-SMA staining,pulmonary small arteries were divided into fully muscular(FM),partially muscular(PM),and nonmuscular(NM).Morphological analysis was performed to evaluate the degree of remodeling and muscularization of pulmonary arterioles.The relative expression levels of RELM-β,PLC,IP3R,Calcineurin and NFATc3 in lung tissues of each group were detected by Western-blot.Quantitative Real-time PCR was used to detect the relative expression levels ofRELM-β,PLC,IP3R,Calcineurin and NFATc3 mRNA in lung tissues of each group.The intracellular Ca2+ level of pulmonary artery smooth muscle cells was detected by flow cytometry.Lung tissue sections were stained with PCNA and α-SMA immunofluorescence,and the Positive Rate of PCNA cells in pulmonary artery smooth muscle cells was analyzed.Results:Compared with Normoxia group,RVAWT was significantly thickened(P<0.05),PAAT,PAAT/PAET and TAPSE were significantly decreased(P<0.05),mPAP and RVHI%were significantly increased(P<0.05),and WT%,WA%and FM%were significantly increased(P<0.05)in the Hypoxia group,the relative expression levels of RELM-β,PLC,IP3R,Calcineurin and NFATc3 in lung tissue were significantly increased(P<0.05),the Ca2+ in pulmonary artery smooth muscle cells was significantly increased(P<0.05),and the proliferation level of pulmonary artery smooth muscle cells was significantly increased(P<0.05).Compared with wild-type rats,RVAWT,PAAT,PAAT/PAET and TAPSE were significantly increased(P<0.05),and mPAP and RVHI%were significantly decreased(P<0.05)in RELM-β completely knockout rats under hypoxia condition.WT%,WA%and FM%were significantly decreased(P<0.05),the relative expression levels of RELM-β,PLC,IP3R,Calcineurin and NF ATc3 in lung tissue were significantly decreased(P<0.05),and the Ca2+ in pulmonary artery smooth muscle cells was significantly decreased(P<0.05).The proliferation level of pulmonary artery smooth muscle cells was significantly decreased(P<0.05).Conclusion:Hypoxia upregulates RELM-β in rat lung tissue,RELM-βmay promote PASMCs proliferation and pulmonary vascular remodeling through the PLC/IP3 R/Ca2+/Calcineurin/NF ATc3 signaling pathway,and promote the occurrence and development of HPH. |
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